Decreased IL-12 production and Th1 cell development by acetyl salicylic acid-mediated inhibition of NF-kappaB. 
IL-12 is a 75-kDa heterodimeric cytokine composed of two covalently linked p35 and p40 chains. This pro-inflammatory cytokine plays a prominent role in the development of Th1 cell-mediated immune responses. Th1 cell-mediated immune responses have been implicated in the pathogenesis of chronic inflammatory autoimmune diseases. Thus, IL-12 appears to be a critical factor in the generation and maintenance of chronic inflammatory conditions. In this study, we investigated the effects of a commonly prescribed anti-inflammatory drug, acetyl salicylic acid (ASA), on IL-12 production and Th1 cell development. ASA was found to inhibit secretion of the IL-12 heterodimer as well as p40 monomer by human monocytic cells. This was associated with the down-regulation of IL-12p40 mRNA expression. Analysis of the regulation of the p40 gene promoter revealed that ASA inhibited NF-kappaB activation and binding to the p40-kappaB site in the p40 promoter, leading to transcriptional repression of the p40 gene. Addition of ASA to an in vitro T helper cell differentiation system, at concentrations compatible with plasma levels reached during anti-inflammatory therapy, resulted in reduced development of Th1 cells. These results suggest that the inhibition of NF-kappaB activation by ASA leads to down-regulation of IL-12 production and inhibition of Th1 cell development. 


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NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. 
The eukaryotic transcription factor nuclear factor-kappa B (NF-kappa B) participates in many parts of the genetic program mediating T lymphocyte activation and growth. Nuclear expression of NF-kappa B occurs after its induced dissociation from its cytoplasmic inhibitor I kappa B alpha. Phorbol ester and tumor necrosis factor-alpha induction of nuclear NF-kappa B is associated with both the degradation of performed I kappa B alpha and the activation of I kappa B alpha gene expression. Transfection studies indicate that the I kappa B alpha gene is specifically induced by the 65-kilodalton transactivating subunit of NF-kappa B. Association of the newly synthesized I kappa B alpha with p65 restores intracellular inhibition of NF-kappa B DNA binding activity and prolongs the survival of this labile inhibitor. Together, these results show that NF-kappa B controls the expression of I kappa B alpha by means of an inducible autoregulatory pathway. 


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Activation of the NF-kappaB transcription factor in a T-lymphocytic cell line by hypochlorous acid. 
Reactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-kappaB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-kappaB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-kappaB inhibitory subunits (IkappaB) demonstrated that both IkappaB-alpha proteolysis and p105 processing were induced by the treatment. NF-kappaB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-kappaB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-alpha (TNF-alpha) in the supernatants. The importance of TNF-alpha release in NF-kappaB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-kappaB was promoted in untreated cells; and (2) synergism between TNF-alpha and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-kappaB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-alpha release. 


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Human interferon regulatory factor 2 gene. Intron-exon organization and functional analysis of 5'-flanking region. 
Interferon regulatory factor 2 (IRF-2) is a transcriptional regulatory protein that terminates interferon beta expression initiated by interferon regulatory factor 1. In this study, we isolated the genomic DNA for human IRF-2 gene, determined the intron-exon structure of the human IRF-2 gene, mapped the major transcription initiation site, identified a number of potential regulatory elements in the 5'-flanking region, and localized the IRF-2 gene on human chromosome 4. The IRF-2 promoter region contains a CpG island, with several GC boxes, a putative NF-kappa B-binding site, and a CAAT box, but no TATA box. When the promoter region was linked with a heterologous reporter gene, we found that the promoter region is inducible by both interferons (interferon-alpha and -gamma) and interferon regulatory factor 1. The region which induced these inductions was identified as being confined to 40 nucleotides 5' to the major transcriptional initiation site by testing a series of clones with truncated promoter of IRF-2. This region contains elements which are shared with the transcriptional enhancers of other genes including interferon regulatory factor 1, interferon beta, and interferon-inducible genes. These data suggest that interferon regulatory factor 1 not only triggers the activation of the interferon signal transduction pathway, but also may play a role in limiting the duration of this response by activating the transcription of IRF-2. 


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IL-10 inhibits nuclear factor-kappa B/Rel nuclear activity in CD3-stimulated human peripheral T lymphocytes. 
IL-10 markedly reduces nuclear factor (NF)-kappa B/Rel nuclear activity induced in PBMC by stimulation with the anti-CD3 mAb OKT3. The inhibition is exerted specifically on the NF-kappa B/Rel activation induced by mAb OKT3, and not that produced by PMA. As judged by supershifting the DNA-protein complexes with Abs recognizing specific components of the NF-kappa B/Rel protein family, the p50/p65 (Rel A) heterodimeric form of NF-kappa B is primarily affected. The maximal effect is observed at the IL-10 concentration of 20 U/ml. IL-10 inhibitory activity is exerted on T lymphocytes and is mediated by monocytes. Indeed, monocytes pretreated with IL-10 are able so inhibit NF-kappa B nuclear activity in purified T lymphocytes stimulated with OKT3. Soluble factors do not appear to be involved in the mechanism of inhibition. On the other hand, the up-regulation of CD80 Ag, found on monocytes obtained from PBMC incubated with OKT3, is not detected after addition of IL-10, and the anti-CD28 mAb CLB-CD28/1 restores the NF-kappa B/Rel nuclear activity in IL-10-inhibited lymphocytes. Therefore, the NF-kappa B/Rel inhibition might be ascribed to a lack of cooperation between accessory cells and T lymphocytes, resulting from down-regulation of a costimulatory molecule, such as CD80, produced by IL-10 on activated monocytes. Our results demonstrate that IL-10 can inhibit the induction of NF-kappa B/Rel nuclear activity in CD3-stimulated T lymphocytes. Since inappropriate activation of kappa B-driven genes has a physiopathologic role in a number of diseases, such as HIV infection, our findings support the possibility of using this cytokine to suppress an undesirable activation of these transcription factors. 


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TNFalpha cooperates with the protein kinase A pathway to synergistically increase HIV-1 LTR transcription via downstream TRE-like cAMP response elements. 
Activating protein-1 (AP-1) binding TPA responsive elements (TRE) are located downstream of the transcription initiation site in the U5 region of the HIV-1 long terminal repeat (LTR). These downstream sequence elements, termed DSE, can bind both AP-1 and CREB/ATF transcription factors. Recently, we demonstrated that the DSE are also cAMP-responsive elements (CRE), since they mediated activation signals elicited by cholera toxin (Ctx), a potent activator of the cAMP-dependent protein kinase A (PKA) signal transduction pathway. In the present study, we demonstrate that the HIV-1 DSE can mediate the transcriptional synergy elicited by the combination of Ctx and TNFalpha. Ctx combined with TNFalpha or IL-1beta to produce a synergistic increase in p24 antigen production in U1 promonocytic cells. Transfection studies of LTR reporter constructs indicated that mutation of the DSE sites abrogated the LTR-mediated synergy induced by Ctx and TNFalpha, whereas the synergy induced by Ctx and IL-1beta was unaffected, suggesting TNFalpha and IL-1beta cooperate differently with the cAMP/PKA activation pathway to induce HIV-1 expression in U1 cells. Because the DSE are also TRE sites, we assessed the effect of the agonist combinations on AP-1-dependent transcription. TNFalpha as well as IL-1beta cooperated with Ctx to produce a synergistic activation of AP-1-mediated transcription. These data indicate that the TRE-like cAMP-responsive DSE sites within the 5'-untranslated leader can mediate the transcriptional cooperativity between TNFalpha and the cAMP/PKA pathway. Since the DSE and TRE sites cannot bind CREB/ATF homodimers, we propose a mechanism in which the HIV-1 DSE bind heterodimers composed of both AP-1 and CREB/ATF proteins. Copyright 1997 Academic Press. 


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Signaling events induced by lipopolysaccharide-activated toll-like receptor 2. 
Human Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-kappaB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-kappaB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-kappaB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants. 


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The role of p16 in the E2F-dependent thymidine kinase regulation. 
The role of alterations of the MTS1 tumor suppressor gene on chromosome 9p21, which encodes p16, the inhibitor of cyclin-dependent-kinase-4 and 6, in tumorigenesis is not yet clear. Phosphorylation of the retinoblastoma protein by cyclin-dependent kinases 4 and 6 prevents its interaction with the transcription factor E2F, which subsequently promotes the expression of S phase regulated genes, such as thymidine kinase. Although a role of p16 in this regulation has been presumed, there is no proof so far that loss of this tumor suppressor gene really affects E2F-mediated regulations. We investigated the regulation of thymidine kinase in phytohemagglutinin-stimulated normal human lymphocytes and in the p16-negative human acute lymphoblastic leukemia cell lines, MOLT-4 and CEM. Compared to normal lymphocytes, MOLT-4 and CEM cells exhibited an altered cell cycle regulation of thymidine kinase, a much higher intracellular activity of this enzyme, and higher thymidine kinase mRNA expression. Transient expression of p16 in normal human lymphocytes caused arrest in G1, but was without effect on the cell growth of MOLT-4 and CEM cells, although all of them express functional retinoblastoma protein. Nevertheless, in the two leukemia cell lines transient overexpression of p16 reestablished the normal regulation of thymidine kinase, paralleled by an increase of the underphosphorylated form of retinoblastoma protein and decrease of free E2F bound to its motif in the thymidine kinase promoter. We demonstrate that loss of p16 causes upregulation of this DNA precursor pathway enzyme via activation of E2F by a mechanism involving retinoblastoma protein. 


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Modulation of endogenous IL-1 beta and IL-1 receptor antagonist results in opposing effects on HIV expression in chronically infected monocytic cells. 
A proportion of HIV-infected individuals experience episodes of localized or systemic bacterial infections caused by Gram-negative bacteria. Many of the clinical side effects of these infections are associated with the production of proinflammatory cytokines, which are induced primarily by LPS, a constituent of the bacterial cell wall of Gram-negative bacteria. The present study examines the mechanisms involved in LPS-mediated induction of HIV expression in U1 cells, a promonocytic cell line chronically infected with HIV. Stimulation of U1 cells by LPS alone induced minimal levels of HIV expression, which was significantly enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF). Costimulation of U1 cells with LPS plus GM-CSF resulted in the accumulation of steady-state levels of HIV RNA; however, only a weak induction of HIV long terminal repeat-driven transcription, which was not associated with the activation of the cellular transcription factor nuclear factor-kappa B, was noted. Costimulation of cells with LPS plus GM-CSF induced the production of proinflammatory cytokines, IL-8, IL-1 beta and IL-6, but not TNF-alpha. IL-1 receptor antagonist (ra) inhibited LPS enhancement of HIV expression in GM-CSF-stimulated cells, suggesting that endogenous IL-1 was involved in LPS-mediated viral production. In this regard, anti-inflammatory cytokines inhibited LPS plus GM-CSF-stimulated HIV expression, and this effect closely correlated with inhibition of IL-1 beta release and, in particular, with up-regulation of endogenous IL-1ra production. Thus, the balance between an endogenously produced viral inducer (IL-1 beta ) and an inhibitor (IL-1ra) may represent an important pathway leading to modulation of HIV expression from monocytic cells. 


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Characterization of peripheral blood T-lymphocytes transduced with HTLV-I Tax mutants with different trans-activating phenotypes. 
Tax1, a transcriptional trans-activator of the Human T-cell leukemia virus type I (HTLV-I), induces the expression of many cellular genes through interaction with at least three distinct cellular transcription factors; CREB/ATF, NF-kappaB, and SRF. This Tax1-induced activation of cellular genes is considered to be a critical event in T-cell transformation by HTLV-I. To elucidate the role of each Tax1-inducible transcriptional pathway in T-cell transformation, we introduced Tax1 mutants with different trans-activating phenotypes into peripheral blood lymphocytes (PBL) by retroviral vectors. Analysis of these PBLs revealed that activation of the NF-kappaB pathway is sufficient to promote the growth response to IL-2. However, for the clonal expansion of CD4+ T-cells, which is a characteristic result of HTLV-I infection, activation of the CREB/ATF and SRF pathways is also required. 


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Activation of the Janus kinase 3-STAT5a pathway after CD40 triggering of human monocytes but not of resting B cells. 
CD40/CD40 ligand interactions play a key role in the immune responses of B lymphocytes, monocytes, and dendritic cells. The signal transduction events triggered by cross-linking of the CD40 receptor have been widely studied in B cell lines, but little is known about signaling following CD40 stimulation of monocytes and resting tonsillar B cells. Therefore, we studied the CD40 pathway in highly purified human monocytes and resting B cells. After CD40 triggering, a similar activation of the NF-kappaB (but not of the AP-1) transcription factor complex occurred in both cell preparations. However, the components of the NF-kappaB complexes were different in monocytes and B cells, because p50 is part of the NF-kappaB complex induced by CD40 triggering in both monocytes and B cells, whereas p65 was only induced in B cells. In contrast, although the Janus kinase 3 tyrosine kinase was associated with CD40 molecules in both monocytes and resting B cells, Janus kinase 3 phosphorylation induction was observed only in CD40-activated monocytes, with subsequent induction of STAT5a DNA binding activity in the nucleus. These results suggest that the activation signals in human B cells and monocytes differ following CD40 stimulation. This observation is consistent with the detection of normal CD40-induced monocyte activation in patients with CD40 ligand+ hyper IgM syndrome in whom a defect in CD40-induced B cell activation has been reported. 


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Identification of an I kappa B alpha-associated protein kinase in a human monocytic cell line and determination of its phosphorylation sites on I kappa B alpha. 
Nuclear factor kappa B (NF-kappa B) is stored in the cytoplasm as an inactive form through interaction with I kappa B. Stimulation of cells leads to a rapid phosphorylation of I kappa B alpha, which is presumed to be important for the subsequent degradation. We have recently reported the establishment of a lipopolysaccharide (LPS)-dependent cell-free activation system of NF-kappa B in association with the induction of I kappa B alpha phosphorylation. In this study, we have identified a kinase in cell extracts from the LPS-stimulated human monocytic cell line, THP-1, that specifically binds and phosphorylates I kappa B alpha. LPS stimulation transiently enhanced the I kappa B alpha-bound kinase activity in THP-1 cells. Mutational analyses of I kappa B alpha and competition experiments with the synthetic peptides identified major phosphorylation sites by the bound kinase as Ser and Thr residues in the C-terminal acidic domain of I kappa B alpha. Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of I kappa B alpha, blocked the LPS-induced NF-kappa B activation as well as inducible phosphorylation of endogenous I kappa B alpha in a cell-free system using THP-1 cells. These results suggested that the bound kinase is involved in the signaling pathway of LPS by inducing the phosphorylation of the C-terminal region of I kappa B alpha and subsequent dissociation of the NF-kappa B.I kappa B alpha complex. 


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MIP1 alpha nuclear protein (MNP), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 alpha gene. 
Murine macrophage inflammatory protein 1 alpha (MIP-1 alpha) and its human equivalent (GOS19, LD78, or AT464) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived MIP-1 alpha/GOS19 inhibits primitive hematopoietic stem cells and appears to be involved in the homeostatic control of stem cell proliferation. It also induces chemotaxis and inflammatory responses in mature cell types. Therefore, it is important to understand the mechanisms which control the expression of MIP-1 alpha/GOS19. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the GOS19 promoter. One member, ICK-1A, behaves as a strong negative regulator. In this communication, we provide evidence that the pathway of induction in the macrophage cell line U937 is different from that in Jurkat cells. Furthermore, we show that the ICK-1 binding site does not confer negative regulation in U937 cells. We provide evidence for an additional binding site, the MIP-1 alpha nuclear protein (MNP) site, which overlaps the ICK-1 site. Interaction of nuclear extracts from various cell lines and tissue with the MNP site leads to the formation of fast-migrating protein-DNA complexes with similar but distinct electrophoretic mobilities. A mutation of the MNP site which does not abrogate ICK-1 binding inactivates the GOS19.1 promoter in U937 cells and reduces its activity by fourfold in Jurkat cells. We propose that the MNP protein(s) binding at the MNP site constitutes a novel transcription factor(s) expressed in hematopoietic cells. 


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Interferons induce normal and aberrant retinoic-acid receptors type alpha in acute promyelocytic leukemia cells: potentiation of the induction of retinoid-dependent differentiation markers. 
Treatment of the acute promyelocytic (APL) cell line NB4 with interferon alpha (IFN(alpha)), as well as IFN(beta) and gamma, results in an increased expression of the transcripts coding for retinoic-acid receptor type alpha (RAR(alpha)) and the leukemia-specific retinoic acid receptor PML-RAR. Transcriptional induction of the RAR(alpha) and PML-RAR mRNAs is rapid and it is parallelled by an increase in the corresponding proteins. Up-regulation of RAR(alpha) and PML-RAR gene expression by IFN(alpha) is accompanied by a strong potentiation in the induction of 2 retinoid-dependent granulocytic markers, i.e., granulocyte-colony-stimulating factor receptor mRNA and leukocyte alkaline phosphatase. However, IFN(alpha) does not have any effects on the retinoid-dependent regulation of the myeloid surface markers CD11b and CD33. The IFN-dependent increase in RAR(alpha) levels and the enhancing effect of the cytokine on retinoid-dependent granulocytic markers expression may be a characteristic of PML-RAR positive cells, since the phenomena are not observed in HL-60 promyelocytes. Interferons as well as retinoids inhibit the growth of NB4 cells, although the 2 classes of compounds do not significantly interact in terms of anti-proliferative activity. These results suggest the possible use of combinations between IFNs and retinoic acid in the cyto-differentiating treatment of APL patients. 


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Nuclear factor kappa B activates proenkephalin transcription in T lymphocytes. 
Upon activation, T lymphocytes accumulate high levels of the neuropeptide enkephalin which correlate with high levels of proenkephalin mRNA in the cells. Here we investigated the transcriptional basis for these changes. The proenkephalin promoter contains a sequence GGGGACGTCCCC, named B2, which is similar to the kappa B sequence GGGGACTTTCC, the binding site of the transcription factor nuclear factor (NF)-kappa B. Activation of T lymphocytes induces an NF-kappa B-like binding activity to the B2 site, concomitant with activation of the proenkephalin promoter. Mutations at the B2 site abolish this transcriptional activation. The purified homodimer (two p50s) of the DNA-binding subunit of NF-kappa B binds the B2 site of proenkephalin relatively better than does the heterotetramer (two p65s plus two p50s) form of the factor. Thus, it appears that the T-cell-specific activation of the proenkephalin promoter is mediated by NF-kappa B. However, as NF-kappa B is ubiquitous and the transcriptional activation through the B2 site is T cell specific, yet another T-cell-specific factor which synergizes with NF-kappa B should be considered. 


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A hydrophobic domain of Ca2+-modulating cyclophilin ligand modulates calcium influx signaling in T lymphocytes. 
Ca2+-modulating cyclophilin ligand (CAML) was originally described as a cyclophilin B-binding protein whose overexpression in T cells causes a rise in intracellular calcium, thus activating transcription factors responsible for the early immune response. As reported here, structure-function analysis of the CAML gene in Jurkat T cells indicates that two of CAML's putative membrane-spanning domains are necessary and sufficient for the modulation of intracellular calcium. We propose that the hydrophobic C-terminal tail of CAML forms its effector domain, thus implicating the N-terminal hydrophilic domain in a regulatory role. These findings define a novel protein motif that functions in intracellular calcium signaling. 


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Regulation of interleukin-1beta transcription by Epstein-Barr virus involves a number of latent proteins via their interaction with RBP. 
Epstein-Barr virus (EBV) infects B cells, resulting in the outgrowth of immortalised lymphoblastoid cell lines (LCLs). Here, we demonstrate through the use of intracellular staining that interleukin-1beta (IL-1beta) is expressed in LCLs and investigate the influence of the individual latent proteins on the expression of IL-1beta. Using RT-PCR, IL-1beta was shown to be up-regulated in EBV-transformed LCLs as well as in group III Burkitt's lymphoma (BL) cell lines, compared with group I BL cell lines. The up-regulation of IL-1beta message could be mediated by the latent membrane protein-1, EBV nuclear proteins 2, 3, 4, and 6 genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that the -300 region of the IL-1beta promoter, which contains a nuclear factor-kappaB (NF-kappaB) binding site, contained a functional RBP binding site. Binding of RBP to this site could be inhibited by addition of EBV nuclear proteins 3 and 6, suggesting that these proteins displace RBP from its recognition sequence, removing transcriptional repression and allowing gene transcription to occur. In group I BL cells, containing low levels of NF-kappaB, only RBP binding was observed in EMSAs, whereas NF-kappaB binding could be demonstrated in EBV-transformed B cell lines containing high levels of activated NF-kappaB. In addition, the expression of latent membrane protein-1 led to activation of NF-kappaB that was capable of binding the IL-1beta promoter. The study demonstrates that EBV can up-regulate IL-1beta expression, possibly by using RBP, NF-kappaB, or both. Copyright 1998 Academic Press. 


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Essential role of alveolar macrophages in intrapulmonary activation of NF-kappaB. 
Acute inflammatory injury in rat lung induced by deposition of immunoglobulin G immune complexes requires expression of cytokines and chemokines as well as activation of the transcription factor nuclear factor (NF)-kappaB. There is little direct evidence regarding the role of alveolar macrophages in these activation events. In the present studies, rat lungs were depleted of alveolar macrophages by airway instillation of liposome-encapsulated dichloromethylene diphosphonate. These procedures, which greatly reduced the number of retrievable alveolar macrophages, suppressed activation of lung NF-kappaB in the inflammatory model. In addition, bronchoalveolar lavage levels of tumor necrosis factor-alpha (TNF-alpha) and the CXC chemokine, macrophage inflammatory protein-2, were substantially reduced. In parallel, upregulation of the lung vascular adhesion molecule, intercellular adhesion molecule-1, was greatly reduced by intrapulmonary instillation of phosphonate-containing liposomes. Neutrophil accumulation and development of lung injury were also substantially diminished. Lung instillation of TNF-alpha in alveolar macrophage-depleted rats restored the NF-kappaB activation response in whole lung. These data suggest that, in this inflammatory model, initial activation of NF-kappaB occurs in alveolar macrophages and the ensuing production of TNF-alpha may propagate NF-kappaB activation to other cell types in the lung. 


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Suppression of MHC class II expression by human class II trans-activator constructs lacking the N-terminal domain. 
The class II trans-activator (CIITA) is a bi- or multi-functional domain protein which plays a critical role in the expression of MHC class II genes. We report that removal of the N-terminal 151 amino acids, encompassing all of the acidic domain but leaving intact the proline/serine/threonine-rich domain, results in a mutant protein with potent suppressive properties for MHC class II expression. HeLa cells stably or transiently transfected with mutant CIITA constructs showed up to 99% suppression of MHC class II antigen induction by IFN-gamma and marked suppression of HLA-DRA mRNA expression. Transient transfection of a B lymphoma line resulted in up to 89% reduction of constitutive MHC class II expression within 5 days and suppression of HLA-DRA mRNA synthesis. 


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Regulation of jun and fos gene expression in human monocytes by the macrophage colony-stimulating factor. 
The macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. However, the signaling events responsible for these effects remain unclear. The present studies have examined the effects of M-CSF on potential signaling pathways involving expression of the jun and fos early response genes. Low levels of c-jun transcripts were detectable in resting human peripheral blood monocytes. Treatment of these cells with 10(3) units/ml human recombinant M-CSF was associated with rapid and transient increases in c-jun mRNA levels. Nuclear run-on assays and mRNA stability studies demonstrated that M-CSF regulates c-jun expression by both an increase in transcription rate and a prolongation in the half-life of c-jun transcripts. M-CSF treatment was also associated with a rapid induction of the jun-B gene, although expression of this gene was prolonged compared to that of c-jun. We further demonstrate that M-CSF increases c-fos mRNA levels in human monocytes through control at both the transcriptional and posttranscriptional levels. Maximal induction of the c-fos gene was followed by that for the fos-B gene. Moreover, M-CSF-induced expression of the fos-related gene, fra-1, was delayed compared to that for both c-fos and fos-B. Taken together, the results indicate that M-CSF treatment is associated with differential activation of multiple members of the jun/fos family and that expression of these genes could contribute to nuclear signaling mechanisms that regulate a specific program of monocyte differentiation. 


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Uncoupling activation-dependent HS1 phosphorylation from nuclear factor of activated T cells transcriptional activation in Jurkat T cells: differential signaling through CD3 and the costimulatory receptors CD2 and CD28. 
CD3, CD2, and CD28 are functionally distinct receptors on T lymphocytes. Engagement of any of these receptors induces the rapid tyrosine phosphorylation of a shared group of intracellular signaling proteins, including Vav, Cbl, p85 phosphoinositide 3-kinase, and the Src family kinases Lck and Fyn. Ligation of CD3 also induces the tyrosine phosphorylation of HS1, a 75-kDa hematopoietic cell-specific intracellular signaling protein of unknown function. We have examined changes in HS1 phosphorylation after differential stimulation of CD3, CD2, and CD28 to elucidate its role in T cells and to further delineate the signaling pathways recruited by these receptors. Unlike ligation of CD3, stimulation with anti-CD28 mAb or CHO cells expressing the CD28 ligands CD80 or CD86 did not lead to tyrosine phosphorylation of HS1 in Jurkat T cells. Additionally, no tyrosine phosphorylation of HS1 was induced by mitogenic pairs of anti-CD2 mAbs capable of activating the transcription factor NFAT (nuclear factor of activated T cells). Costimulation through CD28 and/or CD2 did not modulate the CD3-dependent phosphorylation of HS1. In vivo studies indicated that CD3-induced HSI phosphorylation was dependent upon both the Src family tyrosine kinase Lck and the tyrosine phosphatase CD45, did not require MEK1 kinase activity, and was regulated by protein kinase C activation. Thus, although CD3, CD28, and CD2 activate many of the same signaling molecules, they differed in their capacity to induce the tyrosine phosphorylation of HSI. Furthermore, activation-dependent tyrosine phosphorylation of HS1 was not required for NFAT transcriptional activation. 


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Cytokine rescue from glucocorticoid induced apoptosis in T cells is mediated through inhibition of IkappaBalpha. 
We previously reported that dexamethasone (DEX), a synthetic glucocorticoid, causes apoptosis in mature Th cell lines, and that this induction of cell death is prevented by specific cytokines, namely, by IL-2 in Th1 cells and by IL-4 in Th2 cells. We now show that this differential rescue by specific cytokines in Th cells correlates with the level of IkappaBalpha that is regulated by DEX and cytokines. In both cell types the cellular levels of IkappaBalpha mRNA and protein were evaluated by DEX treatment. Interestingly, the DEX-mediated IkappaBalpha induction was completely inhibited by IL-2, but not IL-4, in Th1 cells, while the reverse profile was seen in Th2 cells. In both cell types, the cytokine that inhibits the induction of IkappaBalpha by DEX, also rescues these cells from DEX-induced apoptosis, although the rescue cytokine is different in Th1 and Th2 cells. Our results imply that T cells need to maintain a certain level of NF-kappaB transcriptional activity in order to survive; up- or down-regulation of nuclear NF kappaB through modulation of IkappaBalpha expression by cytokines or DEX may lead to cell survival or cell death, respectively. 


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Tissue-specific regulation of the rabbit 15-lipoxygenase gene in erythroid cells by a transcriptional silencer. 
The 15-lipoxygenase (lox) gene is expressed in a tissue-specific manner, predominantly in erythroid cells but also in airway epithelial cells and eosinophils. We demonstrate in this report that the 5' flanking DNA of the 15-lox gene contains sequences which down-regulate its activity in a variety of non-erythroid cell lines but not in two erythroid cell lines. The element has characteristics of a transcriptional 'silencer' since it functions in both orientations. The main activity of the silencer has been mapped to the first 900 bp of 5' flanking DNA, which contains nine binding sites for a nuclear factor present in non-erythroid cells but not in erythroid cells. These binding sites have similar sequences and multiple copies of the binding sites confer tissue-specific down-regulation when attached to a minimal lox promoter fragment. The 5' flanking DNA also contains a cluster of three binding sites for the GATA family of transcription factors. 


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Nuclear appearance of a factor that binds the CD28 response element within the interleukin-2 enhancer correlates with interleukin-2 production. 
Activation of T lymphocytes requires the combined signaling of the T cell receptor and costimulatory molecules such as CD28. The ability of T cells to produce interleukin-2 (IL-2) is a critical control point in T lymphocyte activation. The IL-2 enhancer contains a functional motif named CD28 response element (CD28RE) that serves a role as a target for mitogenic T cell activation signals. The CD28RE sequence reveals similarity to the consensus kappaB binding motif. Here we demonstrate that CD28RE binds an inducible protein with a molecular mass of approximately 35 kDa called nuclear factor of mitogenic-activated T cells (NF-MATp35) that is clearly different from the known NF- kappaB/Rel family members. Induction of NF-MATp35 was shown to depend on de novo protein synthesis and was restricted to T cells that received a mitogenic combination of T cell stimuli, not necessarily including CD28 signaling. Nonmitogenic T cell stimulation did not result in appearance of NF-MATp35. These results indicate that mitogenic combinations of T cell activation signals are integrated at the level of NF-MATp35 induction. Similar to its effect on IL-2 production, cyclosporin A inhibited the induction of NF-MATp35. Taken together, these data demonstrate that the nuclear appearance of NF-MATp35 shows excellent correlation with IL-2 production, which is a unique characteristic among nuclear factors implicated in the control of IL-2 gene expression. 


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Regulation of Id3 cell cycle function by Cdk-2-dependent phosphorylation. 
The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro (E.Hara, M.Hall, and G.Peters, EMBO J.16:332-342, 1997). We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or heterodimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Furthermore, compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. Cdk2-dependent phosphorylation therefore provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation. 


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Molecular and cellular analysis of human immunodeficiency virus-induced apoptosis in lymphoblastoid T-cell-line-expressing wild-type and mutated CD4 receptors. 
We have previously shown that the presence of the CD4 cytoplasmic tail is critical for human immunodeficiency virus (HIV)-induced apoptosis (J.Corbeil, M.Tremblay, and D.D.Richman, J.Exp.Med.183:39-48, 1996). We have pursued our investigation of the role of the CD4 transduction pathway in HIV-induced apoptosis. To do this, wild-type and mutant forms of the CD4 cytoplasmic tail were stably expressed in the lymphoblastoid T-cell line A2.01. Apoptosis was prevented when CD4 truncated at residue 402 was expressed; however, cells expressing mutated receptors that do not associate with p56(lck) (mutated at the dicysteine motif and truncated at residue 418) but which conserved proximal domains of the cytoplasmic tail underwent apoptosis like wild- type CD4. The differences between wild-type and mutated receptors in the induction of apoptosis were not related to levels of p56(lck) or NF- kappaB activation. Initial signaling through the CD4 receptor played a major role in the sensitization of HIV-infected T cells to undergo apoptosis. Incubation of HIV-infected cells with monoclonal antibody (MAb) 13B8-2, which binds to CD4 in a region critical for dimerization of the receptor, prevented apoptosis without inhibiting HIV replication. Moreover, the apoptotic process was not related to Fas-Fas ligand interaction; however, an antagonistic anti-Fas MAb (ZB-4) enhanced apoptosis in HIV-infected cells without inducing apoptosis in uninfected cells. These observations demonstrate that CD4 signaling mediates HIV-induced apoptosis by a mechanism independent of Fas-Fas ligand interaction, does not require p56(lck) signaling, and may involve a critical region for CD4 dimerization. 


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Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes. 
To characterize the human elk-1 promoter, we mapped the transcriptional start site and isolated elk-1-specific genomic phage clones that contained extensive upstream and downstream sequences. A TATA-like motif was identified immediately upstream of the transcriptional start site. Functional analyses of DNA fragments containing the TATA element and the identification of a DNase I-hypersensitive chromatin site (HS 1) in close proximity to the TATA box suggest that the identified TATA motif is important for elk-1 transcription in vivo. Sequences upstream and downstream from the TATA box were found to contribute to elk-1 promoter activity. A second hypersensitive site (HS 2) was identified within the first intron in pre-monocytic cells, which express Elk-1 only when differentiating to monocytes. In a variety of other cell types, which display a constitutive Elk-1 expression, HS 2 did not exist, suggesting that inducibility of elk-1 expression is associated with the presence of HS 2. Egr-1 and the serum response factor were found to interact specifically with the intronic sequence at +265 and +448, respectively. Because Egr-1 mRNA and protein levels were observed to increase significantly before induction of elk-1 expression, we propose that Egr-1 is important for the regulation of elk-1 transcription in differentiating monocytes. 


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Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets. 
BACKGROUND: Activated platelets tether and activate myeloid leukocytes. To investigate the potential relevance of this mechanism in acute myocardial infarction (AMI), we examined cytokine induction by leukocyte-platelet adhesion and the occurrence of leukocyte-platelet conjugates in patients with AMI. METHODS AND RESULTS: We obtained peripheral venous blood samples in 20 patients with AMI before and daily for 5 days after direct percutaneous transluminal coronary angioplasty (PTCA) and in 20 patients undergoing elective PTCA. Throughout the study period, CD41 immunofluorescence of leukocytes (flow cytometry) revealed increased leukocyte-platelet adhesion in patients with AMI compared with control patients (mean +/- SE of fluorescence [channels] before PTCA: 77 +/- 16 versus 35 +/- 9; P = .003). In vitro, thrombin-stimulated fixed platelets bound to neutrophils and monocytes. Within 2 hours, this resulted in increased mRNA for interleukin (IL),1 beta, IL-8, and monocyte chemoattractant protein (MCP)-1 in unfractionated leukocytes. After 4 hours, IL-1 beta and IL-8 concentration of the cell-free supernatant had increased by 268 +/- 36% and 210 +/- 7%, respectively, and cellular MCP-1 content had increased by 170 +/- 8%. Addition of activated platelets to adherent monocytes had a similar effect and was associated with nuclear factor-kappa B activation. Inhibition of binding by anti-P selectin antibodies reduced the effect of activated platelets on cytokine production. CONCLUSIONS: In patients with AMI, leukocyte-platelet adhesion is increased. Binding of activated platelets induces IL-1 beta, IL-8, and MCP-1 in leukocytes. Our findings suggest that leukocyte-platelet adhesion contributes to the regulation of inflammatory responses in AMI. 


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Paternal expression of WT1 in human fibroblasts and lymphocytes. 
The Wilms' tumor suppressor gene ( WT1 ) was previously identified as being imprinted, with frequent maternal expression in human placentae and fetal brains. We examined the allele-specific expression of WT1 in cultured human fibroblasts from 15 individuals. Seven of 15 fibroblast lines were heterozygous for polymorphic alleles, and the expression patterns were variable, i.e., equal, unequal or monoallelic paternal expression in three, two and two cases, respectively. Exclusive paternal expression of WT1 was also shown in non-cultured peripheral lymphocytes from the latter two individuals. The allele-specific expression profiles of other imprinted genes, IGF2 and H19, on human chromosome 11 were constant and consistent with those in other tissues. Our unexpected observations of paternal or biallelic expression of WT1 in fibroblasts and lymphocytes, together with the previous findings of maternal or biallelic expression in placentae and brains, suggest that the allele-specific regulatory system of WT1 is unique and may be controlled by a putative tissue- and individual-specific modifier. 


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Activation of primary human T-lymphocytes through CD2 plus CD28 adhesion molecules induces long-term nuclear expression of NF-kappa B. 
Stimulation of highly purified human T-cells via CD2 and CD28 adhesion molecules induces and maintains proliferation for more than 3 weeks. This potent interleukin 2 (IL-2)-dependent activation does not require monocytes or accessory cells. Long-lasting IL-2 receptivity is associated with high-level expression of the inducible IL-2 receptor alpha chain (IL-2R alpha) gene that is regulated at both transcriptional and posttranscriptional levels. Increase of IL-2R alpha gene transcription involves the enhanced binding of the transcription factor NF-kappa B to its consensus sequence in the 5'-regulatory region of the IL-2R alpha gene. To dissect the molecular basis for the unusually persistent transcription of the IL-2R alpha gene, we analyzed nuclear NF-kappa B binding to a radiolabeled IL-2R alpha kappa B-specific oligonucleotide probe during the time course of CD2 + CD28 activation. Resting T-cell nuclear extracts contained KBF1/p50 homodimer. After stimulation, two new kappa B-specific complexes were identified as NF-kappa B p50-p65 heterodimer and putative c-Rel homodimer or c-Rel-p65 heterodimer. Both inducible complexes persisted for at least 3 weeks. Their relative levels were very similar for the duration of proliferation. In parallel, CD2 + CD28 activation triggered a significant intracellular thiol decrease, suggesting that oxygen radicals are involved in the signaling pathway of adhesion molecules. Finally, micromolar amounts of pyrrolidine dithiocarbamate, an oxygen radical scavenger that efficiently blocked the nuclear appearance of NF-kappa B in T-lymphocytes, also inhibited IL-2 secretion, IL-2R alpha cell surface expression, and T-cell proliferation. Together, these results suggest that NF-kappa B plays an important role in long-term activation of human primary T-lymphocytes via CD2 + CD28. 


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Overproduction of NFKB2 (lyt-10) and c-Rel: a mechanism for HTLV-I Tax-mediated trans-activation via the NF-kappa B signalling pathway. 
Molecular, biochemical and epidemiological evidence implicate HTLV-I as an etiologic agent of adult T cell leukemia (ATL). The Tax protein of HTLV-I, a positive transcriptional activator of HTLV-I gene expression, is a viral oncogene that also increases transcription of cellular genes including GM-CSF, IL-2R alpha and IL-2. One of the cellular targets of the trans-activating effects of Tax is the NF-kappa B/Rel family of transcription factors, pleiotropic regulators of immunoregulatory, cytokine and viral gene expression. In this report, we demonstrate that NFKB2 (lyt-10) and c-Rel are overexpressed in HTLV-I infected and Tax-expressing cells and, together, account for the majority of the constitutive NF-kappa B binding activity in these cells before and after PMA stimulation. Most importantly, we show a Tax-dependent correlation between expression of NFKB2(p100) and processing to the DNA binding NFKB2(p52) form, induction of c-Rel, and trans-activation of NF-kappa B-mediated gene expression. Furthermore, the NFKB2 precursor is physically associated with c-Rel and with Tax in HTLV-I infected cells. We propose that NFKB2 synthesis and processing allows continuous nuclear expression of an otherwise cytoplasmic protein and, in conjunction with overexpression of c-Rel, NFKB2 alters the NF-kappa B signalling pathway and contributes to leukemic transformation of T cells by HTLV-I. 


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Abnormal regulation of the IL-2 promoter in lpr CD4-CD8- T lymphocytes results in constitutive expression of a novel nuclear factor of activated T cells-binding factor. 
The inert quality of MRL-Ipr/Ipr (Ipr) peripheral CD4-CD8- (CD4-8-) T cells manifests primarily as an inability to proliferate or produce IL-2 in response to TCR or mitogenic stimulation. Yet these same cells do initiate early TCR-mediated signaling events, such as generation of inositol phosphates and increased intracellular calcium. They also display constitutively high levels of p59fyn and CD3 zeta tyrosine phosphorylation. The generation of second messengers in T cells normally leads to downstream signaling that results in transcriptional activation of the IL-2 gene. We, therefore, compared the activation state of the IL-2 gene promoter region in freshly isolated and stimulated Ipr CD4-8- T cells with that of normal T lymphocytes. Levels of the octamer, NF-kappa B (p50-p65 heterodimer), and AP-1 transcriptional factors are constitutively elevated in freshly isolated Ipr CD4-8- T cells, consistent with the activated phenotype of these cells. Upon stimulation with mitogens, formation of the transactivating complex, nuclear factor of activated T cells (NF-AT), occurs with normal kinetics in Ipr CD4-8- T cells. Yet, the levels of the activating NF-AT complex never reach those observed in similarly stimulated normal T cells. Furthermore, nuclear extracts from Ipr CD4-8- T cells display high levels of a novel specific binding activity at the NF-AT site, which is present at much lower levels in freshly isolated normal T lymphocytes. Upon mitogenic stimulation, the binding activity of the novel NF-AT-binding factor is rapidly down-regulated in normal T cells, but persists at high levels in Ipr CD4-8- T cells. These two abnormalities at the NF-AT site provide a potential mechanism to account for the defect in IL-2 production from Ipr CD4-8- T cells. 


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IL-2-induced growth of CD8+ T cell prolymphocytic leukemia cells mediated by NF-kappaB induction and IL-2 receptor alpha expression. 
The binding of interleukin-2 (IL-2) to its receptor on normal T cells induces nuclear expression of nuclear factor kappaB (NF-kappaB), activation of the IL-2 receptor (IL-2R) alpha chain gene, and cell proliferation. In the present study, the role of IL-2R signaling in the growth of CD8+ T cell prolymphocytic leukemia (T-PLL) cells has been investigated. Flow cytometry revealed that primary leukemia cells from a patient with CD8+ T-PLL expressed IL-2Ralpha and beta chains, and the cells showed a proliferative response and an increase in IL-2Ralpha expression on culture with exogeneous IL-2. Northern blot analysis failed to detect IL-2 mRNA, suggesting that IL-2 may act in a paracrine manner in vivo. Electrophoretic mobility-shift assays revealed that recombinant IL-2 increased NF-kappaB binding activity in nuclear extracts of the leukemia cells, and Northern blot analysis showed that IL-2 increased the abundance of mRNAs encoding the NF-kappaB components c-Rel and KBF1 in these cells. IL-2 binding analysis demonstrated that IL-2 markedly increased the number of low affinity IL-2Rs on the leukemia cells, without an effect on the number of high-affinity IL-2Rs. These results show that IL-2 is capable of inducing the nuclear expression of NF-kappaB in primary CD8+ T-PLL cells, and that this effect is mediated, at least in part, at a pretranslational level. 


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Impaired fetal thymocyte development after efficient adenovirus-mediated inhibition of NF-kappa B activation. 
We introduce a new experimental system combining adenovirus-mediated gene transfer and fetal thymic organ culture (FTOC). This system allowed us to efficiently express in developing thymocytes a mutant form of the NF-kappa B inhibitor I kappa B alpha (mut-I kappa B) and to study the maturation defects occurring when NF-kappa B activation is inhibited during fetal development. Fetal thymocytes infected with adenovirus containing mut-I kappa B were found to develop normally until the CD44-CD25+, CD4-CD8- double-negative stage, while production of more mature double-positive and single-positive populations was strongly decreased. Proliferation, as measured by the percentage of cells in cycle appeared normal, as did rearrangement and expression of the TCR beta-chain. However, apoptosis was much higher in FTOC infected with adenovirus containing mut-I kappa B than in FTOC infected with a control virus. Taken together, these results suggest that NF-kappa B plays a crucial role in ensuring the differentiation and survival of thymocytes in the early stages of their development. 


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Positive and negative regulation of granulocyte-macrophage colony-stimulating factor promoter activity by AML1-related transcription factor, PEBP2. 
The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter contains a consensus sequence for the polyomavirus enhancer binding-protein 2 (PEBP2) transcription factor, which consists of alpha and beta subunits. There are at least two genes, alpha A and alpha B, encoding the alpha subunit. alpha B is the mouse homologue of human AML1 gene detected at the breakpoints of t(8;21) and t(3;21) myeloid leukemias. We examined alpha A1 (an alpha A-gene product) and alpha B1 and alpha B2 (two alpha B-encoded isomers) for their effects on the GM-CSF promoter. PEBP2 alpha A1, alpha B1, and alpha B2 proteins bound the PEBP2 site within the mouse GM-CSF promoter. PEBP2 alpha A1 and alpha B1 enhanced the expression of the GM-CSF promoter-driven reporter plasmid in unstimulated and 12-O-tetradecanoylphorbol 13-acetate/phytohemagglutinin-stimulated human Jurkat T cells. In contrast, the promoter activity was suppressed by alpha B2. Coexpression of alpha B1 and alpha B2 showed that the promoter activity could be determined by the alpha B1/alpha B2 ratio. Jurkat cell extract contained PEBP2 site-binding protein(s) that cross-reacted with antimouse alpha A1 antibodies. Northern blot analysis indicated the expression of human PEBP2 alpha A, alpha B (AML1), and beta genes in Jurkat cells. Although further studies are required to determine the precise role of PEBP2 in the GM-CSF promoter activity, the present findings suggested the importance of the relative ratio of different PEBP2 isoforms in regulating the levels of the promoter activity. 


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Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2. 
Human lysosomal acid lipase (LAL) is a hydrolase required for the cleavage of cholesteryl esters and triglycerides derived from plasma lipoproteins. It is shown here that during monocyte to macrophage differentiation, the expression of LAL-mRNA is induced. This induction is dependent on protein kinase C activity and protein synthesis. The cell type-specific increase in LAL expression is further investigated in the THP-1 cell line with respect to transcriptional regulation. The human monocytic leukemia cell line THP-1 differentiates into macrophage-like cells when treated with phorbol esters. In order to determine the cis-acting elements necessary for both basal and phorbol 12-myristate-13 acetate (PMA)-enhanced promoter activity, we performed deletion analysis and reporter gene assays. A PMA responsive element has been identified between -182 bp and -107 bp upstream of the major transcription start site. Gel mobility shift assays demonstrated that binding of Sp1 and AP-2 to the LAL promoter is increased by PMA in THP-1 cells. Co-transfections with expression plasmids for Sp1 and AP-2 further emphasized the important role of these transcription factors in both basal and PMA-enhanced LAL expression. Our data suggest that differentiation dependent increase of lysosomal acid lipase (LAL) expression in THP-1 cells is mediated by a concerted action of Sp1 and AP-2. 


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NF-kappaB regulates Fas/APO-1/CD95- and TCR- mediated apoptosis of T lymphocytes. 
The maintenance of lymphocyte homeostasis by apoptosis is a critical regulatory mechanism in the normal immune system. The transcription factor NF-kappaB has been shown to play a role in protecting cells against death mediated by TNF We show here that NF-kappaB also has a role in regulating Fas/APO-1/CD95-mediated death, a major pathway of peripheral T cell death. Transfection of Jurkat cells with the NF-kappaB subunits p50 and p65 confers resistance against Fas-mediated apoptosis. Reciprocally, inhibition of NF-kappaB activation by a soluble peptide inhibitor or a dominant form of the NF-kappaB inhibitor, IkappaB, makes the cells more susceptible to Fas-mediated apoptosis. Furthermore, inhibition of NF-kappaB activation by a soluble peptide inhibitor rendered a T cell hybridoma more susceptible to TCR-mediated apoptosis. Correspondingly, transfection of p50 and p65 provided considerable protection from TCR-mediated apoptosis. These observations were corroborated by studies on Fas-mediated death in primary T cells. Concanavalin A-activated cycling T cell blasts from mice that are transgenic for the dominant IkappaB molecule have increased sensitivity to Fas-mediated apoptosis, associated with a down-regulation of NF-kappaB complexes in the nucleus. In addition, blocking TNF, itself a positive regulator of NF-kappaB, with neutralizing antibodies renders the cells more susceptible to anti-Fas-mediated apoptosis. In summary, our results provide compelling evidence that NF-kappaB protects against Fas-mediated death and is likely to be an important regulator of T cell homeostasis and tolerance. 


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An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein. 
Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors. 


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Genomic organization, sequence, and transcriptional regulation of the human eotaxin gene. 
Eotaxin is an eosinophil specific beta-chemokine assumed to be involved in eosinophilic inflammatory diseases such as atopic dermatitis, allergic rhinitis, asthma and parasitic infections. Its expression is stimulus- and cell-specific. We here describe the genomic organisation (3 exons of 132, 112 and 542 bp and 2 introns of 1211 and 378 bp) and sequence including 3 kb of DNA from the immediate 5' upstream region of the human eotaxin gene. Among the regulatory promoter elements potentially regulating eotaxin gene expression and/or mediating the effects of anti-inflammatory drugs we identified consensus sequences known to interact with nuclear factors like NF-IL6, AP-1, a NF-kappa-B like consensus sequence and gamma-interferon- as well as glucocorticoid response elements. 


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Calcium-dependent immediate-early gene induction in lymphocytes is negatively regulated by p21Ha-ras. 
The induction of immediate-early (IE) response genes, such as egr-1, c-fos, and c-jun, occurs rapidly after the activation of T lymphocytes. The process of activation involves calcium mobilization, activation of protein kinase C (PKC), and phosphorylation of tyrosine kinases. p21(ras), a guanine nucleotide binding factor, mediates T-cell signal transduction through PKC-dependent and PKC-independent pathways. The involvement of p21(ras) in the regulation of calcium-dependent signals has been suggested through analysis of its role in the activation of NF-AT. We have investigated the inductions of the IE genes in response to calcium signals in Jurkat cells (in the presence of activated p21(ras)) and their correlated consequences. The expression of activated p21(ras) negatively regulated the induction of IE genes by calcium ionophore. This inhibition of calcium-activated IE gene induction was reversed by treatment with cyclosporin A, suggesting the involvement of calcineurin in this regulation. A later result of inhibition of this activation pathway by p21(ras) was down-regulation of the activity of the transcription factor AP-1 and subsequent coordinate reductions in IL-2 gene expression and protein production. These results suggest that p2l(ras) is an essential mediator in generating not only positive but also negative modulatory mechanisms controlling the competence of T cells in response to inductive stimulations. 


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NF-X2 that binds to the DRA X2-box is activator protein 1. Expression cloning of c-Jun. 
Human class II MHC Ag are a family of cell surface glycoproteins. Their constitutive expression is limited to B lymphocytes and thymic epithelial cells. In many other cells their expression can be induced by IFN-gamma. Conserved upstream promoter sequences regulate this tissue-specific expression of class II genes. In the DRA promoter, one of these cis-acting regulatory motifs is the X2-box to which nuclear factor X2 (NF-X2) binds. Here, we present the isolation and characterization of the full-length cDNA clone encoding NF-X2. This cDNA clone was isolated by expression cDNA cloning, and encodes the human c-Jun protein, which together with c-Fos forms the heterodimeric activator protein-1 transcription complex. Whereas c-Fos/c-Jun heterodimers do not exist in B cells, they form and bind to the X2-box in class II nonexpressing cells. Thus, c-Fos/c-Jun heterodimers might contribute to the repression of DRA gene expression. 


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The lymphotoxin promoter is stimulated by HTLV-I tax activation of NF-kappa B in human T-cell lines. 
The HTLV-I transcriptional activator tax was used to gain insight into the mechanism of lymphotoxin (LT; TNF-beta) gene induction. Tax-expressing cell lines produce LT biologic activity. An LT promoter (LT-293) CAT construct that contained an NF-kappa B site was active in the LT-producing C81-66-45 cell line, which contains defective HTLV-I but expresses tax. The observation that a mutated LT-kappa B construct (M1-CAT) was inactive in C81-66-45, confirmed the importance of NF-kappa B in LT gene expression. Tax was transfected into HTLV-I-negative human T-cell lines. Jurkat T cells stably expressing tax contained elevated levels of NF-kappa B that directly bound to the LT-kappa B site. Tax co-transfected with reporter constructs into Jurkat cells maximally activated HTLV-I-LTR-CAT and kappa B-fos-CAT and also activated LT-293 to a lesser extent. In JM T cells, tax induced LT-293 activity by two- to four-fold, though there was no induction of M1-CAT. The increase in LT-293 CAT activity mirrored the increase in LT biologic activity seen under these conditions. These studies, the first to demonstrate induction of LT promoter activity over basal levels, indicate that HTLV-I tax causes low-level activation of both endogenous LT and the LT promoter, at least in part through activation of NF-kappa B. 


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Triggering of HLA-DR antigens differentially modulates tumor necrosis factor alpha release by B cells at distinct stage of maturation. 
Triggering of HLA class II antigens by the anti-HLA-DR monoclonal antibody (mAb) L243 significantly (P < 0.05) and differentially enhanced the release of tumor necrosis factor alpha (TNF-alpha) by the non-Hodgkin's lymphoma cells Ri-I, Ci-I, and Sc-I, which are at a distinct stage of B-cell differentiation, and by the more mature Burkitt lymphoma cell Raji; in contrast, it did not induce TNF-alpha release by the pre-B leukemia cells Nalm-6 and BV173. TNF-alpha release peaked at 24 h and decreased thereafter, and it was dose dependent and preceded by an increase of TNF-alpha mRNA detectable after 3 h of stimulation with mAb L243. Secreted TNF-alpha mediated the enhancement of nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) binding activity; in fact, the triggering of HLA-DR antigens in the presence of antihuman TNF-alpha-neutralizing antibodies did not upregulate NF-kappa B and AP-1. In contrast, released TNF-alpha was not responsible for the homotypic aggregation of Ri-I, Ci-I, Sc-I, and Raji cells induced by mAb L243, and it did not affect the proliferation of B cells investigated. Altogether, our data demonstrate that: (a) the ability of B cells to release TNF-alpha after triggering of HLA-DR antigens depends on their stage of differentiation; (b) levels of released TNF-alpha seem to correlate with the stage of B-cell maturation but do not correlate with the amounts of cell surface HLA-DR antigens; (c) secreted TNF-alpha regulates the levels of expression of NF-kappa B and AP-1 by an autocrine loop; and (d) intracellular signals mediating TNF-alpha release by B cells are distinct from those regulating homotypic aggregation and proliferation. 


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The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-kappaB activation. 
A site in the Epstein-Barr virus (EBV) transforming protein LMP1 that constitutively associates with the tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein TRADD to mediate NF-kappaB and c-Jun N-terminal kinase activation is critical for long-term lymphoblastoid cell proliferation. We now find that LMP1 signaling through TRADD differs from TNFR1 signaling through TRADD. LMP1 needs only 11 amino acids to activate NF-kappaB or synergize with TRADD in NF-kappaB activation, while TNFR1 requires approximately 70 residues. Further, LMP1 does not require TRADD residues 294 to 312 for NF-kappaB activation, while TNFR1 requires TRADD residues 296 to 302. LMP1 is partially blocked for NF-kappaB activation by a TRADD mutant consisting of residues 122 to 293. Unlike TNFR1, LMP1 can interact directly with receptor-interacting protein (RIP) and stably associates with RIP in EBV-transformed lymphoblastoid cell lines. Surprisingly, LMP1 does not require RIP for NF-kappaB activation. Despite constitutive association with TRADD or RIP, LMP1 does not induce apoptosis in EBV-negative Burkitt lymphoma or human embryonic kidney 293 cells. These results add a different perspective to the molecular interactions through which LMP1, TRADD, and RIP participate in B-lymphocyte activation and growth. 


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Inhibition of NF-kappa B activity in human T lymphocytes induces caspase-dependent apoptosis without detectable activation of caspase-1 and -3. 
NF-kappa B is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Our findings show that suppression of NF-kappa B activity with cell-permeable SN50 peptide, which masks the nuclear localization sequence of NF-kappa B1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-kappa B resulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apoptosis. DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by Ac-DEVD-fmk, suggesting that SN50-mediated apoptosis is caspase-dependent. Interestingly, apoptosis induced by NF-kappa B suppression, in contrast to that induced by TPEN (N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in the absence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like and without cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and DNA fragmentation factor-45. These findings suggest either low level of activation is required or that different caspases are involved. Preactivation of T cells resulting in NF-kappa B nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential role of NF-kappa B in survival of naive PBL. 


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Dysregulation of monocytic nuclear factor-kappa B by oxidized low-density lipoprotein. 
Nuclear factor-kappa B (NF-kappa B)/Rel transcription factors may be involved in atherosclerosis, as is suggested by the presence of activated NF-kappa B in human atherosclerotic lesions. The aim of the present study was to investigate the effects of oxidized LDL (oxLDL) on the NF-kappa B system in human THP-1 monocytic cells as well as adherent monocytes. Our results demonstrate that short-term incubation of these cells with oxLDL activated p50/p65 containing NF-kappa B dimers and induced the expression of the target gene IL-8. This activation of NF-kappa B was inhibited by the antioxidant and H2O2 scavenger pyrrolidine dithiocarbamate and the proteasome inhibitor PSI. The oxLDL-induced NF-kappa B activation was accompanied by an initial depletion of I kappa B-alpha followed by a slight transient increase in the level of this inhibitor protein. In contrast, long-term treatment with oxLDL prevented the lipopolysaccharide-induced depletion of I kappa B-alpha, accompanied by an inhibition of both NF-kappa B activation and the expression of tumor necrosis factor-alpha and interleukin-1 beta genes. These observations provide additional evidence that oxLDL is a potent modulator of gene expression and suggest that (dys)regulation of NF-kappa B/Rel is likely to play an important role in atherogenesis. 


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The interleukin-8 AP-1 and kappa B-like sites are genetic end targets of FK506-sensitive pathway accompanied by calcium mobilization. 
FK506, an immunosuppressant, inhibits the production of several cytokines in T lymphocytes. We observed that FK506 suppressed the transcription of a chemotactic cytokine, interleukin-8 (IL-8) in a human T cell line, Jurkat cells, activated by phorbol 12-myristate 13-acetate (PMA) and calcium (Ca2+) ionophore (ionomycin). By deleted and mutated analysis of the IL-8 promoters, the AP-1 and kappa B-like sites were identified as the responsive elements for PMA and ionomycin. FK506 suppressed the transcriptions through the AP-1 or kappa B-like sites induced by PMA plus Ca(2+)-mobilizing agents, but not those induced by Ca(2+)-independent stimuli. In gel retardation analysis, FK506 had little effect on the binding to the AP-1 site of PMA/ionomycin-induced nuclear factors, which were recognized with anti-JunD or c-Fos antibody. In contrast, FK506 or EGTA (Ca2+ chelator) similarly affected the formation of kappa B-like site binding complexes, which were not recognized by any antibodies against the human Rel family proteins (c-Rel, p65, p50, and p49). Furthermore, we confirmed the previous report that FK506 suppressed the PMA/ionomycin-induced activation through authentic kappa B site of immunoglobulin (Ig) gene, to which NF-kappa B binding was also decreased by FK506, indicating that both IL-8 kappa B-like site and Ig kappa B site are FK506-sensitive in spite of the difference of binding factors. Our results indicate that not only the reported IL-2 NF-AT and NFIL-2A sites and Ig kappa B site, but also the IL-8 AP-1 and kappa B-like sites are terminals of FK506-sensitive pathway involving Ca2+ mobilization. 


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Interactions of a transcriptional activator in the env gene of the mouse mammary tumor virus with activation-dependent, T cell-specific transacting factors. 
The mouse mammary tumor virus env gene contains a transcriptional activator (META) that can control transcription of the adjacent long terminal repeat region. Transcriptional control by META parallels that of several lymphokine genes, being specific to T cells, dependent on their activation, and inhibited by the immunosuppressive drug cyclosporine (CsA). DNase I footprinting indicated that nuclear factors from activated T lymphocytes bound a promoter-proximal site, META(P), and a promoter-distal site, META(D+), within the 400-base pair META region. Nuclear factors from unstimulated, but not from activated cells, bound a site, META(D-), adjacent to META(D+). META(D+) directed transcription of a linked luciferase gene, and gel shift analysis revealed binding of inducible, CsA-sensitive T cell factors, in parallel with transfection results. Authentic NFAT and NF-kappaB targets did not compete for the META(D+) binding factor(s). The SV40 core sequence competed for META(D+) binding factors, but META(D+) failed to compete for the complexes obtained with the SV40 probe. Our results, taken together, indicate that META(D+) is a novel transcriptional enhancer element that is similar in its cell-type specificity, activation dependence, and CsA sensitivity to the NFAT element. It may be relevant to the role of MMTV in expression of Mls antigens or the induction of T cell lymphomas. 


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Cytomegalovirus immediate early genes upregulate interleukin-6 gene expression. 
BACKGROUND: The immediate early genes (IE) of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Interleukin-6 (IL-6) plays a central role in numerous inflammatory and immune processes. Interleukin-6 levels are increased in lung transplant patients clinically diagnosed with CMV pneumonitis. The regulation of IL-6 is dependent on various stimuli that include lipopolysaccharide (LPS), viruses, and other cytokines. These studies examined the ability of CMV IE gene products to modulate IL-6 production. METHODS: THP-1 cells, a monocytic cell line, were transfected with the CMV IE genes. Interleukin-6 protein and IL-6 mRNA were measured in control and CMV immediate early transfected cells. Cotransfection of CMV IE genes and IL-6 chloramphenicol acetyl transferase (CAT) or IL-6 luciferase constructs were used to study IL-6 promoter activity. RESULTS: Interleukin-6 protein and mRNA production were significantly increased in cells transfected with the CMV IE genes and stimulated with LPS compared to LPS-stimulated control cells. Cytomegalovirus IE gene products significantly enhanced LPS stimulation of IL-6 promoter activity in both IL-6 CAT and IL-6 luciferase assays. A deletion construct that contains a NF-kappa B site but is missing the multiple response region demonstrated a continued increase in IL-6 luciferase activity in LPS-stimulated CMV transfected cells. CONCLUSION: Cytomegalovirus immediate early gene products significantly enhanced expression of IL-6 in LPS-stimulated cells. The increase in IL-6 luciferase activity occurs in the absence of the multiple response region, the area of the IL-6 promoter responsive to IL-1, TNF alpha, cyclic amp, and phorbol 12-myristate 13-acetate. The ability of CMV IE gene products to enhance IL-6 production may play an important role in immune inflammatory states associated with CMV infection. 


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Costimulation requirement for AP-1 and NF-kappa B transcription factor activation in T cells. 
The transcriptional activity of the IL-2 promoter requires T-cell costimulation delivered by the TCR and the auxiliary receptor CD28. Several transcription factors participate in IL-2 promoter activation, among which are AP-1-like factors and NF-kappa B. Protein phosphorylation has an important role in the regulation of these two factors: (1) it induces the transactivating capacity of the AP-1 protein c-Jun; and (2) it is involved in the release of the cytoplasmic inhibitor, I kappa B, from NF-kappa B, allowing translocation of the latter into the nucleus. We have recently shown that both phosphorylation processes require T-cell costimulation. Furthermore, in activated T cells, the kinetics of the two phosphorylation events are essentially similar. According to our results, however, the kinases responsible for the two processes are distinct entities. Whereas TPCK inhibits phosphorylation of I kappa B and, consequently, activation of NF-kappa B, it markedly enhances the activity of JNK, the MAP kinase-related kinase that phosphorylates the transactivation domain of c-Jun. We, therefore, propose the activation scheme presented in FIGURE 3 for T-cell costimulation. Costimulation results in the activation of a signaling pathway that leads to the simultaneous induction of the two transcription factors, AP-1 and NF-kappa B. Integration of the signals generated by TCR and CD28 engagement occurs along this pathway, which then bifurcates to induce I kappa B phosphorylation and NF-kappa B activation on the one hand, and JNK activation and c-Jun phosphorylation on the other. We are currently engaged in defining where the two signals integrate along the AP-1/NF-kappa B pathway. 


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The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB. 
The Epstein-Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-kappaB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-kappaB activation also appears to be a critical component of long-term outgrowth. 


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Antisense oligonucleotides to the p65 subunit of NF-kappa B block CD11b expression and alter adhesion properties of differentiated HL-60 granulocytes. 
NF-kappa B is a pleiotropic regulator of a variety of genes implicated in the cellular response to injury. This function has been attributed to the coordinated binding of subunits of NF-kappa B to distinct regions of the promoter elements of numerous genes, including cytokines, growth factor receptors, and adhesion molecules. Antisense phosphorothioate oligonucleotides to the p50 and p65 subunits of the NF-kappa B complex were used to define the physiologic role of this transcription factor in resting and stimulated granulocytes. A reduction in the expression of p65 was produced by treatment with the phosphorothioate antisense oligodeoxynucleotide. This reduction was accompanied by rapid changes in the cellular adhesion of dimethyl sulfoxide-differentiated HL-60 leukemia cells stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These effects were characterized by a marked reduction in CD11b integrin expression on the surface of treated cells. Furthermore, the p65 antisense oligomer effectively abolished an upregulation of CD11b that was produced by formyl-met-leu-phe and TPA. However, the p65 antisense phosphorothioate oligodeoxynucleotide had no significant effect on the production of reactive oxygen intermediates or on phagocytosis by these cells. These findings indicate that antisense oligomers to p65 can be used to define the role of NF-kappa B in the activation pathways of neutrophils. 


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ETS transcription factors regulate an enhancer activity in the third intron of TNF-alpha. 
We describe an enhancer site in the third intron of tumor necrosis factor alpha (TNF-alpha). A reporter construct containing the 5'-flanking region of the mouse TNF-alpha gene displayed weak activity when transfected into RAW264.7 macrophage-like cells. The addition of the third intron of TNF-alpha to this construct resulted in an enhancement of CAT protein. This enhancement was eliminated if a conserved 20-bp sequence was removed from the intron or if a dominant-negative ets-binding factor was co-transfected with the reporter gene. Mutations of this site that destroyed potential ets transcription factor binding sites had reduced transcriptional activity. The major transcription factor that bound to the oligonucleotide was confirmed to be GABP by supershift and competition analysis. In RAW264.7 cells, the binding was constitutive, however, in bone marrow-derived macrophages binding activity was shown to be interferon-gamma inducible. This may imply a role for ets transcription factors in the production of TNF-alpha. 


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Xenogeneic human serum promotes leukocyte adhesion to porcine endothelium under flow conditions, possibly through the activation of the transcription factor NF-kappa B. 
Endothelial cell activation and leukocyte infiltration are a consistent feature of discordant xenograft rejection. Here we evaluated whether xenogeneic serum, as a source of xenoreactive natural antibodies and complement, induced endothelial cell activation with consequent leukocyte adhesion under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porcine serum (control) or 10% xenogeneic human serum and then perfused with total human leukocytes in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2). Adherent cells were counted by digital image analysis. Xenogeneic human serum significantly (P < 0.01) increased the number of adherent leukocytes as compared with porcine serum. A similar adhesive response was elicited by TNF alpha (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here used as positive control. In order to elucidate possible mechanisms underlying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NF-kappa B, a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. By confocal fluorescence microscopy, we observed a positive staining for NF-kappa B (p65 subunit) in the nuclei of PAEC exposed for 1 hr 30 min to human serum, which indicated NF-kappa B activation in this setting. At variance, in PAEC incubated with the homologous serum, NF-kappa B was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NF-kappa B inhibitors pyrrolidinedithiocarbamate (PDTC, 25 microM) and tosyl-phechloromethylketone (TPCK, 25 microM) significantly (P < 0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic serum promotes leukocyte-endothelium interaction possibly through NF-kappa B activation might be relevant for designing future therapeutic strategies aimed at prolonging xenograft survival. 


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Epstein-Barr virus replicative gene transcription during de novo infection of human thymocytes: simultaneous early expression of BZLF-1 and its repressor RAZ. 
Epstein-Barr virus (EBV) is known to infect B cells and epithelial cells. We and others have shown that EBV can also infect a subset of thymocytes. Infection of thymocytes was accompanied by the appearance of linear EBV genome within 8 hr of infection. Circularization of the EBV genome was not detected. This is in contrast to the infection in B cells where the genome can circularize within 24 hr of infection. The appearance of the BamHI ZLF-1 gene product, ZEBRA, by RT-PCR, was observed within 8 hr of infection. The appearance of a novel fusion transcript (RAZ), which comprised regions of the BZLF-1 locus and the adjacent BRLF-1 locus, was detected by RT-PCR. ZEBRA protein was also identified in infected thymocytes by immunoprecipitation. In addition, we demonstrated that the EBNA-1 gene in infected thymocytes was transcribed from the Fp promoter, rather than from the Cp/Wp promoter which is used in latently infected B cells. Transcripts encoding gp350/220, the major coat protein of EBV, were identified, but we did not find any evidence of transcription from the LMP-2A or EBER-1 loci in infected thymocytes. These observations suggest that de novo EBV infection of thymocytes differs from infection of B cells. The main difference is that with thymocytes, no evidence could be found that the virus ever circularizes. Rather, EBV remains in a linear configuration from which replicative genes are transcribed. 


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Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145. 
A theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (-508 to -501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and compatible protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer. 


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Evidence for distinct intracellular signaling pathways in CD34+ progenitor to dendritic cell differentiation from a human cell line model. 
Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment. 


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Induction of tyrosine phosphorylation and T-cell activation by vanadate peroxide, an inhibitor of protein tyrosine phosphatases. 
Rapid tyrosine phosphorylation of key cellular proteins is a crucial event in the transduction of activation signals to T-lymphocytes. The regulatory role of protein tyrosine phosphatases (PTPases) in this process was explored by studying the effects of a powerful PTPase inhibitor, vanadate peroxide (pervanadate), on the activation cascade of Jurkat human leukaemic T-cells. Pervanadate induced activation of the tyrosine kinases lck and fyn (4- and 3-fold respectively) and a dramatic increase in tyrosine phosphorylation of cellular proteins, notably phospholipase C gamma 1. After this event, we observed a rise in intracellular Ca2+ concentration, corresponding to an influx. This effect required surface expression of the CD45 PTPase and was not observed in CD45-deficient variants of Jurkat cells. In the CD45-negative variant, the effect of pervanadate on tyrosine phosphorylation was globally decreased and some phosphorylated substrates were specifically missing. Pervanadate also stimulated transcription of the c-fos gene and accumulation of its mRNA as well as several other hallmarks of T-lymphocyte activation such as surface expression of the CD69 antigen and the interleukin 2 receptor alpha-chain (CD25). Pervanadate synergized with signals delivered by T-cell antigen receptor engagement or by a phorbol ester to induce interleukin 2 production. Pervanadate activated NF-kappa B, as shown by an increase in DNA-binding activity of this transcription factor. We thus conclude that PTPases play a crucial role in the negative regulation of signal transduction culminating in T-lymphocyte activation. Moreover, induction of tyrosine phosphorylation appears sufficient per se to initiate a complete activation programme. 


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Relationship between IkappaBalpha constitutive expression, TNFalpha synthesis, and apoptosis in EBV-infected lymphoblastoid cells. 
In order to understand the role of NF-kappaB in EBV transformation we have established stably transfected IkappaBalpha into lymphoblastoid cells. Two clones were obtained in which the loss of NF-kappaB binding activity correlated with the constitutive expression of the transgenic IkappaBalpha. Protein latency expression was determined by immunocytochemistry. Expression of surface markers, intracytoplasmic content of cytokines cell cycle analysis after BrdU incorporation and DNA staining with propidium iodide were studied by flow cytometry. Percentage of apoptotic cells was determined by in-situ labelling of DNA strand breaks. No significative changes in EBV latency nor in cell surface marker expression was found. In contrast, intracytoplasmic TNFalpha levels were strongly reduced in transfected clones. Furthermore, 30% of IkappaBalpha transfected cells were apoptotic after 8 h of TNFalpha treatment. This correlated with a strong reduction of BrdU incorporation after 24 h of TNFalpha treatment. No effect was seen with non transfected cells or with cells transfected with a control plasmid. Our results suggest that the TNFalpha gene could be one of the targets of NF-kappaB in EBV infected cells and that NF-kappaB protects EBV-infected cells from apoptosis induced by TNFalpha, which may favour the proliferative effect of this cytokine. 


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Involvement of the N-terminal region of the human mineralocorticoid receptor hormone-binding domain in agonist and antagonist binding as revealed by a new monoclonal antibody. 
To gain a better understanding of the mechanism of binding to the human mineralocorticoid receptor (hMR), we developed a new monoclonal antibody (mAb) raised against the hormone-binding domain (HBD). For this purpose, mice were immunized with a fusion protein including the sequence Thr729-Lys984 of hMR. After ELISA screening, mAb 18C7 was selected for its specificity towards the HBD. This antibody recognized both the denatured and native MR forms, as well as the hetero-oligomeric MR form and the transformed MR state. By using several HBD subfragments, the mAb 18C7 epitope was located in the N-terminal region of the HBD from Thr729 to Leu765. We then studied the effect of the antibody on aldosterone and progesterone binding to the hMR. When 18C7 was incubated with liganded MR, it was able to partly displace (20%) the hormone from its binding site. When 18C7 was incubated with MR before aldosterone or progesterone, the antibody inhibited 75-80% of the binding. The effect of 18C7 on the binding was similar with both hormones. A sucrose gradient analysis indicated the simultaneous presence of two kinds of receptor complexes: the steroid-MR complex and the antibody-MR complex. After its associated proteins, especially the heat-shock protein hsp90, had been cross-linked with the hMR by dimethylpimelimidate, 18C7 was still able to react with the receptor. Our results indicated that the epitope recognized by 18C7 was directly implicated in hormone binding. The lack of steroid binding of HBD mutants with the Thr729-Leu765 sequence deleted [Jalaguier, Mesnier, Leger and Auzou (1996) J.Steroid Biochem.Mol.Biol.57, 43-50] supports this hypothesis. Because of the similar behaviours of aldosterone and progesterone, we conclude that the N-terminal Thr729-Leu765 region of the HBD is similarly involved in the binding of both hormones. 


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High molecular weight dextran sulfate increases the activity of NF-kappaB-regulated promoter in monocyte-derived macrophages. 
It is known that sulfated polysaccharides can mimic the action of common T-cell mitogens. To investigate the molecular basis of the mitogenic effect of high molecular weight dextran sulfate (HMDS), monocyte-derived macrophages were transfected with recombinant plasmid containing chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 long terminal repeat (LTR) promoter, which is regulated by transcription factor NF-kappaB. We observed that HMDS, similar to bacterial lipopolysaccharide (LPS), increases the expression of CAT reporter gene suggesting increased activity of NF-kappaB. The activation of NF-kappaB correlated with the increased expression of B7.1 molecules. It was postulated that this NF-kappaB-regulated promoter might play a role in the activation of the accessory cells as well as the rate of replication of HIV-1 in monocyte-derived macrophages. 


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ETS1 transactivates the human GM-CSF promoter in Jurkat T cells stimulated with PMA and ionomycin. 
Activation of T helper cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one such cytokine whose increased expression results partly from increases in transcription. Cis-acting elements with NF kappa B, AP-1 and ETS-like motifs have been identified in the promoter region of the GM-CSF gene, which are important for transcriptional activity following PMA and ionomycin stimulation. A number of the ETS family of transcription factors are expressed in T cells, including ETS1 and ELF1. Here we describe the ability of these factors to interact with a site (GM5), located within the CLE0 element, -47 to -40 upstream of the GM-CSF transcription initiation site. Exogenous ETS1, but not ELF1, can transactivate GM-CSF, through the GM5 site, in a PMA/ionomycin dependent manner. Other unidentified ETS-like factors present in Jurkat cells are also capable of binding GM5. Mutation of the core ETS binding site from -GGAA- to -GGAT- prevents the binding of ETS-like factors with the exception of ETS1. The GM-CSF promoter, modified in this way to be ETS1 specific, is fully responsive to PMA/ionomycin induction, in addition to ETS1 transactivation in the presence of PMA and ionomycin. Together these data suggest that ETS1 may be involved in mediating the increased GM-CSF production associated with T cell activation. 


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Monoclonal anti-endothelial cell antibodies from a patient with Takayasu arteritis activate endothelial cells from large vessels. 
OBJECTIVE: To create monoclonal anti-endothelial cell antibodies (mAECA) from a patient with Takayasu arteritis to evaluate their ability to activate human umbilical vein endothelial cells (HUVEC), and to characterize the mechanism of EC activation. METHODS: A panel of mAECA was generated from peripheral blood lymphocytes of a patient with Takayasu arteritis, using Epstein-Barr virus transformation. Activity against macrovascular EC (HUVEC) and microvascular EC (human bone marrow EC immortalized by SV40) antigens was detected by enzyme-linked immunosorbent assay. Inhibition studies were used to select the monoclonal antibodies (mAECA) which share the same EC epitope binding specificity as the total IgG-AECA from the Takayasu arteritis patient. The binding of the mAECA to human aortic EC was studied by immunohistochemistry. The secretion levels of interleukin-6 (IL-6) and von Willebrand factor (vWF) were determined, to serve as markers for EC activation. The activated EC were examined for the adherence of a monocytic cell line (U937), as well as for expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and E-selectin. In addition, nuclear extracts of the mAECA-treated EC were analyzed for the induction of translocation of nuclear factor kappaB (NF-kappaB), using a specific NF-kappaB oligoprobe in an electrophoretic mobility shift assay. RESULTS: Six mAECA were selected, the mixture of which produced 100% inhibition of binding of the original IgG (from the patient with Takayasu arteritis) to HUVEC. All mAECA possessed high activity against macrovascular EC, but none had significant antimicrovascular EC activity. The mAECA, but not normal human IgG, had anti-human aortic EC activity. Four of the 6 mAECA activated EC, manifested by increased IL-6 and vWF secretion. The 4 mAECA induced EC expression of adhesion molecules and increased adhesion of U937 monocytic cells to EC. In addition, these mAECA stimulated the nuclear translocation of the NF-kappaB transcription factor. CONCLUSION: Our findings suggest that AECA may directly stimulate EC in Takayasu arteritis through elevation of adhesion molecule expression associated with NF-kappaB activation and adhesion of monocytes, and may therefore play a pathogenic role in the development of the vasculopathy in Takayasu arteritis. 


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Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia. 
Productive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia lack activated cells as determined by an absence of staining for Ki-67 cell cycle antigen. The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells lack active NF-kappa B but express Sp1 as expected. DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication without ostensible immune stimulation. 


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Possible differences in the mechanism(s) of action of different glucocorticoid hormone compounds. 
Different glucocorticoid hormones (GCH) show differences in the intensity and in the kinetics of their immunomodulating activity. The mechanism(s) of action of GCH is under investigation, but is has been noted that they exert immune activity via the genomic pathway. We have studied the effects of prednisone (PDN), deflazacort (DFC), and dexamethasone (DXM) on the production of cytokines (IL-2, IL-6, TNF-alpha, IL-10) by peripheral T lymphocytes, and the effects on the inhibition of NF-kB DNA binding activity by activated Jurkat cell line. The data obtained show that the three GCH molecules exert an immunosuppression on cytokine production by T lymphocytes and a strong decrease in the nuclear translocation of NF-kB in Jurkat cells; moreover, (a) not all the cytokines investigated were affected, and not with the same intensity, by the three GCH and (b) DXM inhibited the binding activity of NF-kB less than that of DFC and PDN. These data are in agreement with the concept that different GCH compounds might differ in their binding and affinity properties, tissue-specific metabolism, and interaction with transcription factor. 


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Abnormal NF-kappa B activity in T lymphocytes from patients with systemic lupus erythematosus is associated with decreased p65-RelA protein expression. 
Numerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-kappa B contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-kappa B protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-kappa B binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-kappa B activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-kappa B binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-kappa B activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-kappa B activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients. 


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Apoptosis-resistant T cells have a deficiency in NF-kappaB-mediated induction of Fas ligand transcription. 
Apoptosis induced through the TCR in CD4+ T cells is mostly mediated by the inducible expression of Fas ligand (FasL) as a primary event leading to the commitment to death. To gain a better understanding of the transcriptional events that regulate this expression, we took advantage of our previously described mutant Jurkat cells. These cells are deficient in FasL expression and apoptosis induced upon TCR triggering, although their cytokine (IL-2 and IFN-gamma) production is normal. Here we show that both a FasL- and a consensus NF-kappaB- reporter construct are inefficiently induced in these cells compared to wild-type cells. In addition, we demonstrate that the inducible transcriptional activity of the FasL reporter is abolished by specific inhibitors of NF-kappaB activation. Thus, we could trace the deficit of the mutant cells to an inefficient NF-kappaB activation, evidencing a relevant role for NF-kappaB in the regulation of FasL expression in activated T cells. Furthermore, our results suggest that the induction of FasL versus cytokine gene expression is differentially sensitive to NF-kappaB deprivation. 


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cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins. 
We demonstrate that JunD, a component of the AP-1 transcription factor complex, activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase, protein kinase A. Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP-, phorbol ester-, and Ca(2+)-inducible enhancer, and JunD is shown to bind the enhancer as a homodimer. Another component of the AP-1 transcription complex, JunB, is shown to inhibit activation mediated by JunD. As a homodimer JunB is unable to bind the enhancer; however in the presence of c-Fos, high-affinity binding is observed. Furthermore, JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion, further blurring the distinction between these response elements. These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second-messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways. 


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Triggering of the human interleukin-6 gene by interferon-gamma and tumor necrosis factor-alpha in monocytic cells involves cooperation between interferon regulatory factor-1, NF kappa B, and Sp1 transcription factors. 
We investigated the molecular basis of the synergistic induction by interferon-gamma (IFN-gamma)/tumor necrosis factor-alpha (TNF-alpha) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS). Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-gamma and/or TNF-alpha action, whereas only one of these regions seemed to be implicated in LPS activation. The three regions concerned are: 1) a region between -73 and -36, which is the minimal element inducible by LPS or TNF-alpha; 2) an element located between -181 and -73, which appeared to regulate the response to IFN-gamma and TNF-alpha negatively; and 3) a distal element upstream of -224, which was inducible by IFN-gamma alone. LPS signaling was found to involve NF kappa B activation by the p50/p65 heterodimers. Synergistic induction of the IL-6 gene by IFN-gamma and TNF-alpha, in monocytic cells, involved cooperation between the IRF-1 and NF kappa B p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter. This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-gamma. 


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Preassociation of STAT1 with STAT2 and STAT3 in separate signalling complexes prior to cytokine stimulation. 
A variety of cytokines and growth factors act through an induction of gene expression mediated by a family of latent transcription factors called STAT (signal transducers and activators of transcription) proteins. Ligand-induced tyrosine phosphorylation of the STATs promotes their homodimer and heterodimer formation and subsequent nuclear translocation. We demonstrate here that STAT protein heterocomplexes exist prior to cytokine treatment. When unstimulated HeLa cells are ruptured in hypotonic buffer without salt or detergent, immunoadsorption of either STAT1 or STAT2 from the resulting cytosol yields coimmunoadsorption of the other STAT protein. Similarly, STAT1-STAT3 heterocomplexes are coimmunoadsorbed from hypotonic cytosol. STAT1 and STAT2 or STAT1 and STAT3 translated in reticulocyte lysate spontaneously form heterocomplexes when the translation lysates are mixed at 0 degrees C. Our data suggest that interferon-alpha /beta-induced tyrosine phosphorylation increases the stability of a preexisting, latent, STAT1-STAT2 signaling complex. Newly translated STAT1 binds in equilibrium fashion to STAT2 and STAT3, but we show that STAT2 and STAT3 exist in separate heterocomplexes with STAT1, consistent with a model in which STAT1 contains a common binding site for other STAT proteins. 


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The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-kappaB, a member of the TNFR superfamily. 
Receptor activator of NF-kappaB (RANK) is a recently identified member of the tumor necrosis factor receptor superfamily and is expressed on activated T cells and dendritic cells. Its cognate ligand (RANKL) plays significant roles in the activation of dendritic cell function and osteoclast differentiation. We demonstrate here the interaction of RANK with tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 both in vitro and in cells. Mapping of the structural requirements for TRAF/RANK interaction revealed multiple TRAF binding sites clustered in two distinct domains in the RANK cytoplasmic tail. These TRAF binding domains were shown to be functionally important for the RANK-dependent induction of NF-kappaB and c-Jun NH2-terminal kinase activities. Site-directed mutagenesis demonstrated that these TRAF binding sites exhibited selective binding for different TRAF proteins. In particular, TRAF6 interacted with membrane-proximal determinants distinct from those binding TRAFs 1, 2, 3, and 5. When this membrane-proximal TRAF6 interaction domain was deleted, RANK-mediated NF-kappaB signaling was completely inhibited while c-Jun NH2-terminal kinase activation was partially inhibited. An NH2-terminal truncation mutant of TRAF6 inhibited RANKL-mediated NF-kappaB activation, but failed to affect constitutive signaling induced by receptor overexpression, revealing a selective role for TRAF6 in ligand-induced activation events. 


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The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter. 
Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at -150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter. 


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Lipopolysaccharide induction of the tumor necrosis factor-alpha promoter in human monocytic cells. Regulation by Egr-1, c-Jun, and NF-kappaB transcription factors. 
Biosynthesis of tumor necrosis factor-alpha (TNF-alpha) is predominantly by cells of the monocytic lineage. This study examined the role of various cis-acting regulatory elements in the lipopolysaccharide (LPS) induction of the human TNF-alpha promoter in cells of monocytic lineage. Functional analysis of monocytic THP-1 cells transfected with plasmids containing various lengths of TNF-alpha promoter localized enhancer elements in a region (-182 to -37 base pairs (bp)) that were required for optimal transcription of the TNF-alpha gene in response to LPS. Two regions were identified: region I (-182 to -162 bp) contained an overlapping Sp1/Egr-1 site, and region II (-119 to -88) contained CRE and NF-kappaB (designated kappaB3) sites. In unstimulated THP-1, CRE-binding protein and, to a lesser extent, c-Jun complexes were found to bind to the CRE site. LPS stimulation increased the binding of c-Jun-containing complexes. In addition, LPS stimulation induced the binding of cognate nuclear factors to the Egr-1 and kappaB3 sites, which were identified as Egr-1 and p50/p65, respectively. The CRE and kappaB3 sites in region II together conferred strong LPS responsiveness to a heterologous promoter, whereas individually they failed to provide transcriptional activation. Furthermore, increasing the spacing between the CRE and the kappaB3 sites completely abolished LPS induction, suggesting a cooperative interaction between c-Jun complexes and p50/p65. These studies indicate that maximal LPS induction of the TNF-alpha promoter is mediated by concerted participation of at least two separate cis-acting regulatory elements. 


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Synergism between the CD3 antigen- and CD2 antigen-derived signals. Exploration at the level of induction of DNA-binding proteins and characterization of the inhibitory activity of cyclosporine. 
We have demonstrated earlier that the crosslinkage of the CD3/TCR complex with the CD2 antigen results in the proliferation of normal human T cells. The effect of this synergism was perceptible at the level of induction of the IL-2 gene, a process critical for T cell growth. To further understand the molecular and nuclear basis for this synergism, we have explored the induction of DNA-binding proteins in highly purified normal human T cells signaled via the CD3 and/or CD2 proteins. The effect of transmembrane signaling of T cells with ionomycin, and/or sn-1,2 dioctanoyl glycerol, was also determined. The emergence of nuclear binding proteins was investigated using interleukin-2 sequence specific oligonucleotide probes in the electrophoretic mobility shift assay. Our studies demonstrate for the first time that CD3 antigen-derived signals and CD2 antigen-derived signals are synergistic in inducing the emergence of transcription factors that bind to the NF-AT1, AP-1, and NF-kB sites located in the promoter/enhancer region of the IL-2 gene. Moreover, cyclosporine, at concentrations readily accomplished in clinical practice, was found to inhibit the emergence of these DNA-binding proteins in normal human T cells signaled via cell surface proteins implicated in antigen-dependent T cell activation and in T cells stimulated by mobilization of cellular calcium and activation of protein kinase C. 


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Monocytic cell type-specific transcriptional induction of collagenase. 
Interstitial collagenase (MMP-1), a metalloproteinase produced by resident and inflammatory cells during connective tissue turnover, cleaves type I collagen fibrils. This catalytic event is rate limiting in remodeling of tissues rich in fibrillar collagen such as the skin and lungs. The regulation of collagenase expression is cell-type specific; bacterial LPS and zymosan, a yeast cell wall derivative, are potent inducers of collagenase expression in macrophages, but do not alter fibroblast collagenase expression. Since promoter elements controlling collagenase transcription in monocytic cells have not been previously defined, we sought to delineate responsive cis-acting elements of the collagenase promoter in transiently transfected human (U937) and murine (J774) monocytic cell lines. Deletion constructs containing as little as 72 bp of 5' -flanking sequence of the collagenase promoter were sufficient for LPS- or zymosan-mediated transcriptional induction, whereas phorbol inducibility exhibited an absolute requirement for upstream elements including the polyoma enhancer A-binding protein-3 site (-83 to -91) and TTCA sequence (-102 to -105) in both monocytic cells and fibroblasts. Mutagenesis of the activator protein-1 [AP-1] site at -72 abolished basal promoter activity and LPS/zymosan inducibility, while mutagenesis of an NF-kappaB-like site at -20 to -10 had no effect. Nuclear extracts from LPS- and zymosan-treated cells showed strong AP-1 activity by gel-shift analysis, and supershift analysis showed the AP-1 complexes contained specific members of both the jun and fos gene families. These data indicate that, in contrast to most LPS effects, AP-1, but not nuclear factor-kappaB, mediates LPS induction of collagenase transcription in macrophagelike cells. Furthermore, as compared to regulation by phorbol ester, collagenase induction in monocytic cells by cell wall derivatives of bacteria or yeast is largely independent of upstream promoter sequences. 


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Salicylates inhibit lipopolysaccharide-induced transcriptional activation of the tissue factor gene in human monocytic cells. 
Binding of plasma Factor VII/VIIa to the tissue factor (TF) receptor initiates the coagulation protease cascades. TF expression by circulating monocytes is associated with thrombotic and inflammatory complications in a variety of diseases. Transcriptional activation of the human TF gene in monocytic cells exposed to bacterial lipopolysaccharide (LPS) is mediated by binding of c-Rel/p65 heterodimers to a kappa B site in the TF promoter. Here, we report that a family of anti-inflammatory agents, known as the salicylates, inhibited LPS induction of TF activity and TF gene transcription in human monocytes and monocytic THP-1 cells at clinically relevant doses. Furthermore, sodium salicylate blocked the LPS-induced proteolytic degradation of I kappa B alpha, which prevented the nuclear translocation of c-Rel/p65 heterodimers. In contrast, two other nonsteroidal anti-inflammatory drugs, ibuprofen and indomethacin, did not inhibit LPS induction of the TF gene. These results indicated that salicylates inhibited LPS induction of TF gene transcription in monocytic cells by preventing nuclear translocation of c-Rel/p65 heterodimers. The clinical benefits of salicylates in the treatment of several diseases, including atherosclerosis and rheumatoid arthritis, may be related to their ability to reduce monocyte gene expression. 


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CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival. 
CD30 is a cell-surface receptor that can augment lymphocyte activation and survival through its ability to induce the transcription factor NF-kappaB. CD30, however, has also been implicated in the induction of apoptotic cell death of lymphocytes. Here we show that one of the effects of CD30 signal transduction is to render cells sensitive to apoptosis induced by the type 1 tumor necrosis factor receptor (TNFR1). This sensitization is dependent on the TRAF-binding sites within the CD30 cytoplasmic domain. One of the proteins that binds to these sites is TRAF2, a signal transduction molecule that is also utilized by TNFR1 to mediate the activation of several downstream kinases and transcription factors. During CD30 signal transduction, we found that binding of TRAF2 to the cytoplasmic domain of CD30 results in the rapid depletion of TRAF2 and the associated protein TRAF1 by proteolysis. These data suggest a model in which CD30 limits its own ability to transduce cell survival signals through signal-coupled depletion of TRAF2. Depletion of intracellular TRAF2 and its coassociated proteins also increased the sensitivity of the cell to undergoing apoptosis during activation of death-inducing receptors such as TNFR1. Consistent with this hypothesis, expression of a dominant-negative form of TRAF2 was found to potentiate TNFR1-mediated death. These studies provide a potential mechanism through which CD30, as well as other TRAF-binding members of the TNFR superfamily, can negatively regulate cell survival. 


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Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B. 
The interleukin-2 (IL-2) promoter consists of several independent T cell receptor (TcR) responsive elements. The induction of promoters dependent on these elements is inhibitable by the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK-506). Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the FK-506- and CsA-sensitive enzyme required for TcR mediated activation of the IL-2 promoter. We report that a constitutively active form of calcineurin partially substitutes for the Ca2+ co-stimulus required to activate the IL-2 promoter elements IL-2A (which binds the factors OAP and Oct-1) and IL-2E (which binds NF-AT), and completely substitutes for the Ca2+ co-stimulus required to stimulate an NF-kappa B-dependent element. Calcineurin stimulates the NF-kappa B element by enhancing inactivation of I kappa B/MAD3, an inhibitor of NF-kappa B, thereby increasing the amount of nuclear NF-kappa B DNA binding activity. These data provide the first demonstration in vivo that activation of a protein phosphatase can inactivate I kappa B, and suggest one possible explanation for mechanism-based toxicities associated with FK-506 and CsA by demonstrating that these drugs can inhibit the calcineurin-dependent activation of a virtually ubiquitous transcription factor. 


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Active suppression of the class II transactivator-encoding AIR-1 locus is responsible for the lack of major histocompatibility complex class II gene expression observed during differentiation from B cells to plasma cells. 
In this study the genetic control of major histocompatibility complex (MHC) class II gene expression during the transition from B cell to plasma cell has been analyzed. Class II molecules are not expressed in plasma cells because of an active suppression resulting in the abrogation of class II gene transcription. We show here that the plasma cell-specific repressor function, designated SIR (suppressor of immune response genes), does not act directly on the transcription of class II genes, but instead on the transcription of the AIR-1 gene, whose product, the class II transactivator (CIITA), is fundamental for the regulation of the constitutive and inducible expression of MHC class II genes. This was unambiguously demonstrated by the fact that plasmacytoma x B cell hybrids carrying an AIR-1 locus derived from CIITA-expressing cells do not express CIITA-specific transcripts. Transfection of a cDNA containing the human CIITA coding sequence under the control of an heterologous promoter restores expression of human MHC class II genes in the hybrids and is responsible for de novo expression of mouse MHC class II genes in both the mouse plasmacytoma cell line and the hybrids. These results confirm and extend the notion of the functional conservation of the AIR-1 gene product across species barriers. Interestingly, in CIITA-transfected cell hybrids, cell surface expression of the human HLA-DQ heterodimer was not observed. This result was not attributable to lack of HLA-DQ alpha or -DQ beta transcription, because both transcripts were present in the CIITA-transfected hybrids, although at reduced levels. These findings further support our previous observations on the distinct regulation of expression of the human HLA-DQ class II subset, which may be thus controlled at the posttranscriptional level by a CIITA-independent mechanism. 


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Suppression of NF-kappaB activation in normal T cells by supernatant fluid from human renal cell carcinomas. 
T lymphocytes from patients with renal cell carcinoma (RCC) show reduced immune function and impaired activation of the transcription factor, NF-kappaB. We determined the mechanism of NF-kappaB suppression in T cells of RCC patient and determined whether supernatant fluid from RCC explants (RCC-S) induced the same phenotype of NF-kappaB suppression in normal T cells that is observed in patient T cells. The pattern of kappaB-binding activity in T cells of RCC patient was altered as compared to that seen in T cells obtained from normal volunteers. In some patients, no activation of RelA/NFkappaB1-binding activity was detectable, while in others kappaB-binding activity was modestly induced but the duration was reduced. IkappaBalpha was degraded normally following stimulation in both normal controls and T cells from RCC patients. RCC-S did not alter the cytoplasmic levels of RelA and NF-kappaB1 but did suppress their nuclear localization and inhibited the activation of RelA/NF-kappaB1 binding complexes. These results show that RCC-S can induce in normal T cells the same phenotype of impaired NF-kappaB activation that is detected in T cells of RCC patient. It also appears that NF-kappaB suppression by RCC-S may contribute to the immunosuppression of host immunity. 


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An isotype-specific activator of major histocompatibility complex (MHC) class II genes that is independent of class II transactivator. 
Patients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the absence of HLA-DR and HLA-DP, despite its mapping by complementation analysis to this group. It was possible that one of the clone 13 CIITA alleles bore a mutation that allowed HLA-DQ, but not HLA-DR or -DP transcription. Alternatively, another factor, distinct from CIITA, might control HLA-DQ expression. We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do not restore expression of HLA-DQ in another CIITA-deficient cell line, RJ2.2.5. In addition, no CIITA protein is detectable in clone 13 nuclear extracts. In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA. 


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CIITA activates the expression of MHC class II genes in mouse T cells. 
It has long been a puzzle that MHC class II molecules are expressed in human T cells after activation but not in mouse T cells; this expression is believed to play a role in the cell mediated immune response. Recently the MHC class II transactivator (CIITA) has been reported to be a major regulatory factor for both the constitutive and IFN inducible expression of MHC class II genes. Here we show that human T cells expressing MHC class II have CIITA transcripts while MHC class II-negative human T cells and mouse T cells do not. The expression of MHC class II genes in mouse T cells can be reconstituted upon transfection with the human CIITA cDNA. These data indicate that the expression of CIITA explains the expression or lack of expression of MHC class II in human and mouse T cells respectively. 


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Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF. 
Globin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal (gamma)-to adult (beta)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Kruppel-like factor (EKLF), is required for expression of the beta-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human beta-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human beta-globin expression is dramatically reduced, gamma-globin transcripts are elevated approximately 5-fold. Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene. 


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Membrane-associated lymphotoxin on natural killer cells activates endothelial cells via an NF-kappaB-dependent pathway. 
BACKGROUND: Inhibition of complement in small animal models of xenotransplantation has demonstrated graft infiltration with natural killer (NK) cells and monocytes associated with endothelial cell (EC) activation. We have previously demonstrated that human NK cells activate porcine EC in vitro, which results in adhesion molecule expression and cytokine secretion. In this study, we used the NK cell line NK92 to define the molecular and cellular basis of NK cell-mediated EC activation. METHODS: EC were transfected with either reporter constructs containing the luciferase gene driven either by E-selectin or interleukin (IL)-8 promoters or a synthetic NF-kappaB-dependent promoter. In addition, a dominant-negative mutant tumor necrosis factor receptor I (TNFRI) expression vector was co-transfected in inhibition studies. Forty-eight hours after transfection, EC were stimulated with NK cells or NK cell membrane extracts for 7 hr and activation was measured by a luciferase assay. RESULTS: Co-culture of NK cells with transfected EC enhanced E-selectin, IL-8, and NF-kappaB-dependent promoter activity. NK cell membrane extracts retained the capacity to activate EC and induced nuclear translocation of NF-kappaB (p50 and p65). Western blotting of NK cell and membrane extracts detected the presence of Lymphotoxin-alpha (LTalpha) but not tumor necrosis factor-alpha. Furthermore, LTalpha was secreted in NK:EC co-cultures. Co-transfection with dominant-negative mutant TNFRI inhibited EC activation by NK cell membrane extracts and by NK cells by 80% and 47%, respectively. The same pattern of inhibition was observed using anti-human LT sera. CONCLUSIONS: Human NK cell membrane-bound LT signals across species via TNFRI, leading to NF-kappaB nuclear translocation and transcription of E-selectin and IL-8, which results in EC activation. The discrepancy in the degree of inhibition by membrane extracts and NK cells with mutant TNFRI suggests that additional pathways are utilized by NK cells to activate EC. 


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NF-kappaB only partially mediates Epstein-Barr virus latent membrane protein 1 activation of B cells. 
The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is required for EBV-induced immortalization of human B cells and causes tumorigenic transformation of cell lines. LMP1 expression induces phenotypic changes resembling B cell activation, such as cell size increase and up-regulation of cell surface activation markers. LMP1 contains two domains that activate the transcription factor NF-kappaB, one through interactions with TRAF proteins and the other with the TRADD protein. The purpose of the present study was to investigate the importance of NF-kappaB induction in the up-regulation of the B cell activation markers ICAM-1 and CD71 by LMP1. This study shows that expression of LMP1 activates transcription from p50/p65- and c-Rel- responsive promoters, and that this activity can be completely inhibited by expression of a dominant inhibitory IkappaB mutant. ICAM-1 and CD71 are nevertheless up-regulated by LMP1 in primary B cells and cell lines expressing the dominant IkappaB. Furthermore, LMP1-induced cell size increase of primary B cells was unaffected by IkappaB expression. It was concluded that even when LMP1 is unable to activate NF-kappaB, it is still capable of inducing certain characteristics of activated B cells, strongly suggesting that LMP1 can also activate cells independently of NF-kappaB. 


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Potent gene regulatory and antiproliferative activities of 20-methyl analogues of 1,25 dihydroxyvitamin D3. 
The biological active form of vitamin D3, 1,25-dihydroxyvitamin D3 (VD), regulates cellular growth and differentiation. This provides the hormone with an interesting therapeutic potential. However, hypercalcemia is a side effect, which is caused by VD's classical action, the regulation of calcium homeostasis. This made the need for VD analogues with selectively increased cell regulatory properties. Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD. In this report the biological properties of the compounds ZK161422 and ZK157202, which are 20-methyl- and 20-methyl-23-eneanalogues, respectively, have been analyzed in comparison with VD. Both compounds show about 2-fold lower affinity to the VD receptor (VDR) than VD. However, compared to VD, their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells. Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold, ZK161422 has the same calcium-mobilizing potency as VD. Moreover, ZK161422, but not ZK157202, showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides (IP9) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides (DR3). This observation supports a model, in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues. 


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Cell-specific bifunctional role of Jun oncogene family members on glucocorticoid receptor-dependent transcription. 
Interaction between protein kinase C (PKC)- and glucocorticoid receptor (GR)-mediated signaling is suggested by the ability of the PKC activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to inhibit GR-dependent transcription of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Here we report that this interference is cell specific, as TPA augmented dexamethasone-induced transcriptional activation of the MMTV LTR in several T cell lines but was inhibitory in NIH-3T3 fibroblasts. TPA-GR synergism was determined to have occurred at the GR-responsive element (GRE) level by functional analysis of deletion mutants or synthetic GRE oligonucleotides driving chloramphenicol acetyl-transferase expression. Synergism required an intact GR DNA-binding domain, whereas amino- or carboxyl-terminal domains were dispensable. The effect was abrogated by the PKC inhibitor staurosporine, suggesting a role for PKC. Increased c-jun, jun-B, and jun-D expression above basal levels and increased transcriptional activity of AP-1/TPA responsive elements fused to chloramphenicol acetyl-transferase vectors were observed in T cells treated with TPA alone or in combination with dexamethasone. The ability of Jun proteins to cooperate with GR in T cells has been investigated after transfection of c-jun, jun-B, or jun-D expression vectors, which augmented GR-dependent transcription from either MMTV LTR or GRE. Conversely, c-jun and jun-B transfection blunted GR-dependent transcription in HeLa cells. The presence of c-fos had a negative influence on GR function and correlated with the cell-specific synergistic or antagonistic activity of Jun with respect to GR; high basal expression of c-fos as well as AP-1 DNA binding and transcriptional activity were observed in HeLa cells, but not in T cells. Furthermore overexpression of exogenous c-fos has an inhibitory effect on GR-dependent transcription from GRE in T cells. We propose that Jun plays a bifunctional role on GR-dependent transcriptional activation of GRE, selecting either synergistic or antagonistic activity depending on the cell-specific microenvironment. In this regard, intracellular levels of c-fos appear to be influential. 


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Cupric ion blocks NF kappa B activation through inhibiting the signal-induced phosphorylation of I kappa B alpha. 
A transcription factor NF kappa B, which regulates expression of various cellular genes involved in immune responses and viral genes including HIV, is sequestered in the cytoplasm as a complex with an inhibitory protein I kappa B. Various extracellular signals induce phosphorylation and rapid degradation of I kappa B alpha to release NF kappa B. Cu2+ was found to inhibit the activation of NF kappa B induced by TNF-alpha, TPA, or H2O2. Deoxycholate treatment of the cytoplasmic extract prepared from cells stimulated by TNF-alpha in the presence of Cu2+ resulted in the release of NF kappa B from I kappa B alpha, indicating that Cu2+ interferes with the dissociation of the NF kappa B-I kappa B complex. Neither phosphorylation nor degradation of I kappa B alpha was observed upon TNF-alpha stimulation in the presence of Cu2+. These results indicate that Cu2+ inhibits the release of NF kappa B by blockade of a signal leading to the phosphorylation of I kappa B alpha. 


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Vitamin D3- and retinoic acid-induced monocytic differentiation: interactions between the endogenous vitamin D3 receptor, retinoic acid receptors, and retinoid X receptors in U-937 cells. 
Retinoic acid (RA) and 1,25 alpha-dihydroxycholecalciferol (VitD3) are potent regulators of hematopoletic differentiation. Yet, little is known as to how the RA and VitD3 receptor network operates in hematopoietic cells, and whether receptor interactions can explain the interplay between the RA- and VitD3-signaling pathways during differentiation. Therefore, we analyzed the expression, DNA binding, and transcriptional activity of the endogenous RA and VitD3 receptors [retinoic acid receptors (RARs), retinoid X receptors (RXRs), and VitD3 receptor (VDR)] in the U-937 cell line, in which RA and VitD3 induce distinct monocytic differentiation pathways. VitD3 induction resulted in the formation of VDR/RXR DNA-binding complexes on both VitD3 response elements and RA response elements (RAREs). However, transcriptional activation was only observed from a VitD3 response element-driven reporter construct. Several DNA-binding complexes were detected on RAREs in undifferentiated cells. Stimulation by RA resulted in increased RAR beta/RXR DNA binding, activated RARE-dependent transcription, and increased expression of RAR-beta. Concomitant stimulation by VitD3 inhibited the RA-stimulated formation of RAR beta/RXR heterodimers, favoring VDR/RXR binding to the RARE. Also, VitD3 inhibited the expression of CD23 and CD49f, characteristic markers of retinoid-induced U-937 cell differentiation. In contrast, neither the RA-stimulated, RARE-mediated transcription nor the induced RAR-beta expression was suppressed by VitD3, suggesting that VitD3 selectively inhibited the retinoid-induced differentiation program but not the RARE-mediated signal. These results demonstrate a complex role for VitD3 in modifying the retinoid differentiation pathway and may have implications for differentiation-inducing therapy of hematopoietic tumors. 


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Effects of overexpression of IL-1 receptor-associated kinase on NFkappaB activation, IL-2 production and stress-activated protein kinases in the murine T cell line EL4. 
The association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms. 


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Inhibition of p105 processing by NF-kappaB proteins in transiently transfected cells. 
Regulation of the transcription factor NF-kappaB involves proteasome-mediated processing of the NF-kappaB1 p105 precursor protein, which generates the p50 subunit of NF-kappaB. The processing of p105 occurs constitutively in vivo but can be markedly enhanced by various cellular activation agents, although the underlying regulatory mechanism is not yet clear. In the present study, we demonstrate that signal-mediated induction of p105 processing in human T cells is associated with de novo synthesis of this precursor protein. Transient transfection studies performed in COS7 cells revealed that the newly synthesized p105 protein appears to be more rapidly processed compared to its accumulated form that is already associated with the processed product p50. Interestingly, the processing rate of p105 is markedly inhibited in cells co-transfected with p50 or other NF-kappaB subunits, including RelA and c-Rel, that physically interact with p105. These findings suggest that the processing of p105 is subject to negative regulation by the various NF-kappaB subunits. We further demonstrate that p105 undergoes degradation in lipopolysaccharide-stimulated human monocytic cells. However, the inducible degradation of p105 is not coupled with the generation of p50. Together, these studies demonstrate that the processing and inducible degradation of p105 are differentially regulated. 


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The role of jun and fos gene family members in 12-O-tetradecanoylphorbol-13-acetate induced hemopoietic differentiation. 
Terminal differentiation of the leukemic cell lines U-937 and HL-60 by 12-O-tetradecanoylphorbol-13-acetate is accompanied by marked changes in gene expression. In this study, we demonstrate that the expression of jun and fos gene family members is induced with variable kinetics during 12-O-tetradecanoylphorbol-13-acetate induced differentiation, with c-jun expression best paralleling differentiation. The generation of AP-1 complexes, as measured by DNA binding activity, closely parallels morphological differentiation. Furthermore, the ability of these complexes to regulate gene expression is demonstrated by increased transcription from an AP-1 driven reporter construct and marked increases in the expression of endogenous AP-1 regulated genes. Differentiation assays using water soluble phorbol esters reveal that differentiation becomes irreversible soon after AP-1 appears. This tight correlation between c-jun expression, the generation of AP-1 activity, and differentiation suggests a critical role for this gene and transcriptional complex during this process. 


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p21ras initiates Rac-1 but not phosphatidyl inositol 3 kinase/PKB, mediated signaling pathways in T lymphocytes. 
p21ras is activated by the T cell antigen receptor (TCR) and then co-ordinates important signaling pathways for T lymphocyte activation. Effector pathways for this guanine nucleotide binding protein in T cells are mediated by the serine/threonine kinase Raf-1 and the Ras-related GTPase Rac-1. In fibroblasts, an important effector for the Ras oncogene is Phosphatidylinositol 3-kinase (PtdIns 3-kinase). Activation of this lipid kinase is able to induce critical Rac-1 signaling pathways and can couple p21ras to cell survival mechanisms via the serine/threonine kinase Akt/PKB. The role of PtdIns 3-kinase in Ras signaling in T cells has not been explored. In the present study, we examined the ability of PtdIns 3-kinase to initiate the Rac-1 signaling pathways important for T cell activation. We also examined the possibility that Akt/PKB is regulated by Ras signaling pathways in T lymphocytes. The results show that Ras can initiate a Rac-1 mediated pathway that regulates the transcriptional function of AP-1 complexes. PtdIns 3-kinase signals cannot mimic p21ras and induce the Rac mediated responses of AP-1 transcriptional activation. Moreover, neither TCR or Ras activation of AP-1 is dependent on PtdIns 3-kinase. PKB is activated in response to triggering of the T cell antigen receptor; PtdIns 3-kinase activity is both required and sufficient for this TCR response. In contrast, p21ras signals are unable to induce Akt/PKB activity in T cell nor is Ras function required for Akt/PKB activation in response to the TCR. The present data thus highlight that PtdIns 3-kinase and Akt/PKB are not universal Ras effector molecules. Ras can initiate Rac-1 regulated signaling pathways in the context of T cell antigen receptor function independently of PtdIns 3-kinase activity. 


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Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-kappa B/AP-1 in T cells. 
Two cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12-myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)- expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+- signaling pathways downstream of T cell activation. 


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Nasal NK- and T-cell lymphomas share the same type of Epstein-Barr virus latency as nasopharyngeal carcinoma and Hodgkin's disease. 
Nasal T/NK-cell lymphomas can be further separated into those of natural killer (NK) cell lineage or of T-cell lineage, with differences in cellular phenotype, T-cell receptor (TcR) gene rearrangement and TcR transcript expression. Both NK- and T-cell subtypes are closely associated with Epstein-Barr virus (EBV). In this study, EBV gene expression was determined in 23 cases of nasal lymphoma (NL) by in situ hybridisation (ISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IH). Of the 23 cases, 19 were classified as NK-cell and 4 as T-cell tumours. ISH for EBV-encoded small non-polyadenylated RNAs showed that all cases, whether NK or T, harboured EBV in virtually all tumour cells. RT-PCR demonstrated that NL of both subtypes expressed EBNAI of the QUK splice pattern, the latent membrane proteins, LMP1 and 2 and the BamHI A rightward transcripts in the absence of EBNA2 mRNAs, compatible with the latency type II pattern. In addition, analysis of EBV protein expression by IH revealed a heterogeneous pattern of EBV gene expression at the single-cell level consisting of both LMP1+ and LMP1- tumour cells, suggesting a mixture of latency I and II. Although 2 early lytic transcripts, BZLF1 and BHRF1, were also detected in 13 and 10 cases, respectively, the lack of ZEBRA staining in any case indicates that these lytic transcripts are most likely expressed by rare cells in the biopsies entering lytic cycle. The viral transcriptional pattern similar to that of nasopharyngeal carcinoma and Hodgkin's disease suggests that EBV can exploit common regulatory mechanisms for gene transcription in diverse host cell types. Down-regulation of immunogenic proteins (EBNA2-EBNA6) in nasal lymphoma may enable tumour cells to evade host cytotoxic T-cell surveillance. 


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Stimulation of CD40 on immunogenic human malignant melanomas augments their cytotoxic T lymphocyte-mediated lysis and induces apoptosis. 
Here, we report the functional expression of CD40 on human malignant melanomas (MMs). Comparison of tumor specimen from MM precursor lesions, primary tumors, and metastases revealed that CD40 surface expression is down-regulated during tumor progression. CD40 expression was confirmed in 7 human MM cell lines established from immunogenic primary tumors or metastases, whereas 11 cell lines established from advanced stages were CD40 negative. CD40 expression could be enhanced in CD40-positive MM by stimulation with IFN-gamma and tumor necrosis factor-alpha but not by interleukin (IL)-1beta or CD40 triggering. CD40 ligation on MM by CD40L-transfected murine L-cells or by a soluble CD40L fusion protein up-regulated their expression of intercellular adhesion molecule-1 and MHC class I and class II molecules and their secretion of IL-6, IL-8, tumor necrosis factor-a, and granulocyte macrophage colony-stimulating factor and also induced a rapid activation of the transcription factor nuclear factor kappaB. Furthermore, CD40 ligation of a HLA-A2+, MelanA/MART1+ MM cell line enhanced its susceptibility to specific lysis by a HLA-A2-restricted, MelanA/MART-1-specific CTL clone. Finally, CD40 ligation induced growth inhibition and apoptosis in MM. These results indicate that CD40-CD40L interactions may play an important role in augmenting antitumor immunity and inducing apoptosis in some CD40-positive immunogenic human MMs. 


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Involvement of adenylate cyclase and p70(S6)-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1. 
Our previous results show that recombinant gp41 (aa565-647), the extracellular domain of HIV-1 transmembrane glycoprotein, stimulates interleukin-10 (IL-10) production in human monocytes. The signal cascade transducing this effect is not yet clear. In this study, we examined whether gp41-induced IL-10 up-regulation is mediated by the previously described synergistic activation of cAMP and NF-kappaB pathways. gp41 induced cAMP accumulation in monocytes in a time- and concentration-dependent manner and the adenylate cyclase inhibitor SQ 22536 suppressed gp41-induced IL-10 production in monocytes. In contrast, gp41 failed to stimulate NF-kappaB binding activity in as much as no NF-kappaB bound to the main NF-kappaB-binding site 2 of the IL-10 promoter after addition of gp41. We also examined the involvement of other signal transduction pathways. Specific inhibitors of p70(S6)-kinase (rapamycin), and Gi protein (pertussis toxin), prevented induction of IL-10 production by gp41 in monocytes, while inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin) and mitogen-activated protein kinase (MAPK) pathway (PD 98059) did not. Thus HIV-1 gp41-induced IL-10 up-regulation in monocytes may not involve NF-kappaB, MAPK, or PI 3-kinase activation, but rather may operate through activation of adenylate cyclase and pertussis-toxin-sensitive Gi/Go protein to effect p70(S6)-kinase activation. 


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Role of IKK1 and IKK2 in lipopolysaccharide signaling in human monocytic cells. 
Mononuclear phagocytes play a major role in immune and inflammatory responses. Bacterial lipopolysaccharide (LPS) induces monocytes to express a variety of genes by activating the NF-kappaB/Rel transcription factor family. Recently, we have reported that the tumor necrosis factor and interleukin 1 signaling pathways activate two kinases, IKK1 and IKK2. Phosphorylation of the IkappaB cytoplasmic inhibitors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, by these kinases triggers proteolytic degradation and the release of NF-kappaB/Rel proteins into the nucleus. At present, the role of the IKKs in LPS signaling has not been investigated. Here, we report that LPS induces IKK activity in human monocytes and THP-1 monocytic cells. The kinetics of activation of kinase activity in monocytic cells are relatively slow with maximal activity observed at 60 min, which coincides with the degradation of IkappaBs and the nuclear translocation of NF-kappaB. In transfection experiments, overexpression of wild type IKK1, a dominant negative mutant IKK1 (K44M), or wild type IKK2 did not affect LPS-induced kappaB-dependent transcription in monocytic cells. In contrast, a dominant negative mutant of IKK2 inhibited LPS induction of kappaB-dependent transcription in a dose-dependent manner. These results indicate that LPS induction of kappaB-dependent gene expression in human monocytic cells requires activation of IKK2. 


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GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction. 
Triggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E.Flory, A. Hoffmeyer, U.Smola, U.R.Rapp, and J.T.Bruder, J.Virol.70:2260- 2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation. 


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Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein. 
Dimerization among transcription factors has become a recurrent theme in the regulation of eukaryotic gene expression. Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is a homeodomain-containing protein that functions as a dimer. A dimerization cofactor of HNF-1 alpha (DCoH) was identified that displayed a restricted tissue distribution and did not bind to DNA, but, rather, selectively stabilized HNF-1 alpha dimers. The formation of a stable tetrameric DCoH-HNF-1 alpha complex, which required the dimerization domain of HNF-1 alpha, did not change the DNA binding characteristics of HNF-1 alpha, but enhanced its transcriptional activity. However, DCoH did not confer transcriptional activation to the GAL4 DNA binding domain. These results indicate that DCoH regulates formation of transcriptionally active tetrameric complexes and may contribute to the developmental specificity of the complex. 


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Inhibitory effect of E3330, a novel quinone derivative able to suppress tumor necrosis factor-alpha generation, on activation of nuclear factor-kappa B. 
(2E)-3-[5-(2,3-Dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2- propenoic acid (E3330), is a novel agent with hepatoprotective activity. We report the effect of E3330 on transcriptional activation of tumor necrosis factor (TNF)-alpha gene and on nuclear factor (NF)-kappa B activation. Nuclear run-on experiments showed that E3330 decreases transcriptional activation of TNF-alpha gene induced by lipopolysaccharide (LPS) stimulation in human peripheral monocytes. To investigate the inhibitory mechanisms, we constructed a secreted-type placental alkaline phosphatase (PLAP) reporter gene whose transcription is controlled by a 1.4-kb human TNF-alpha promoter. A stable transformant of the PLAP reporter gene derived from human monocytic cell line showed very little activity on the promoter before stimulation, whereas LPS stimulation led to a dramatic increase in PLAP activity. E3330 inhibited this induced promoter activity in a dose-dependent manner. There are four putative NF-kappa B binding sites (kappa B-1, kappa B-2, kappa B-3, kappa B-4) in human TNF-alpha promoter. By using mutated promoter-PLAP plasmids, we established that these NF-kappa B sites were necessary for induction of TNF-alpha transcription on stimulation with LPS. A gel retardation experiment with synthetic double-stranded oligonucleotides showed that activated NF-kappa B consisting of p50/p65 heterodimer bound to all four putative NF-kappa B DNA probes, suggesting that all four putative NF-kappa B recognition sites play an important role in inducible TNF-alpha expression. E3330 decreased activated NF-kappa B in nuclei, suggesting that E3330 inhibits NF-kappa B activation and/or translocation of the nuclei. Western blotting analysis with anti-I kappa B-alpha antibody indicated that E3330 inhibited degradation of I kappa B-alpha, which is an inhibitory protein of NF-kappa B, in LPS-stimulated monocytes. E3330 may suppress the production of active oxygen species serving as common messengers to activate NF-kappa B. 


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Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NF-IL6 and NF-kappa B [published erratum appears in Proc Natl Acad Sci U S A 1995 Apr 11;92(8):3632] 
The host response to Mycobacterium tuberculosis includes granuloma formation at sites of infection and systemic symptoms. Cytokines have been identified by immunohistochemistry in granulomas in animal models of bacillus Calmette-Guerin (BCG) infection and are released by mononuclear phagocytes upon stimulation by mycobacterial proteins. In this regard, the cytokine interleukin 6 (IL-6) may play a role in the clinical manifestations and pathological events of tuberculosis infection. We have demonstrated that lipoarabinomannan (LAM) from the mycobacterial cell wall, which was virtually devoid of lipopolysaccharide (LPS), stimulated mononuclear phagocytes to release IL-6 in a dose-response manner. LAM and LPS were potent inducers of IL-6 gene expression in peripheral blood monocytes. Both LAM- and LPS-inducible IL-6 promoter activity was localized to a DNA fragment, positions -158 to -49, by deletion analysis and chloramphenicol acetyltransferase assay. Two nuclear factor NF-IL6 (positions -153 to -145 and -83 to -75) and one nuclear factor NF-kappa B (positions -72 to -63) motifs are present within this fragment. Site-directed mutagenesis of one or more of these motifs within the IL-6 promoter demonstrated that each has positive regulatory activity and that they could act in a function- and orientation-independent manner. Deletion of all three elements abolished inducibility of IL-6 promoter activity by both LAM and LPS. We conclude that the NF-IL6 and NF-kappa B sites mediate IL-6 induction in response to both LPS and LAM, acting as bacterial or mycobacterial response elements. 


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Homodimerization of the human interleukin 4 receptor alpha chain induces Cepsilon germline transcripts in B cells in the absence of the interleukin 2 receptor gamma chain. 
The cytokines interleukin (IL)-4 and IL-13 play a critical role in inducing Cepsilon germline transcripts and IgE isotype switching in human B cells. The IL-4 receptor (IL-4R) in B cells is composed of two chains, the IL-4-binding IL-4Ralpha chain, which is shared with the IL-13R, and the IL-2Rgamma (gammac) chain, which is shared with IL-7R, IL-9R, and IL-15R. IL-4 induces Cepsilon germline transcripts and IgE isotype switching in B cells from patients with gammac chain deficiency. Induction of Cepsilon germline transcripts by IL-4 in B cells that lack the gammac chain may involve signaling via the IL-13R. Alternatively, the IL-4Ralpha chain may transduce intracellular signals that lead to Cepsilon gene transcription independently of its association with other chains. We show that ligand-induced homodimerization of chimeric surface receptors consisting of the extracellular and transmembrane domains of the erythropoietin receptor and of the intracellular domain of IL-4Ralpha induces Janus kinase 1 (Jak1) activation, STAT6 activation, and Cepsilon germline transcripts in human B cell line BJAB. Disruption of the Jak1-binding proline-rich Box1 region of IL-4Ralpha abolished signaling by this chimeric receptor. Furthermore, B cells transfected with a chimeric CD8alpha/IL-4Ralpha receptor, which is expressed on the cell surface as a homodimer, constitutively expressed Cepsilon germline transcripts. These results suggest that homodimerization of the IL-4Ralpha chain is sufficient to transduce Jak1-dependent intracellular signals that lead to IgE isotype switching. 


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The class II trans-activator CIITA interacts with the TBP-associated factor TAFII32. 
The class II trans- activator (CIITA) is the main transcriptional co-activator for the expression of MHC class II proteins. Its N-terminal 125 amino acids function as an independent transcriptional activation domain. Analyses of the primary amino acid sequence of the activation domain predict the presence of three alpha-helices, each with a high proportion of acidic residues. Using site-directed mutagenesis, we found that two of these predicted alpha-helices are required for full transcriptional activation by CIITA. Moreover, a CIITA protein in which both functional alpha-helices have been deleted displays a dominant negative phenotype. This activation domain of CIITA interacts with the 32 kDa subunit of the general transcription complex TFIID, TAFII32. Decreased transcriptional activation by N-terminal deletions of CIITA is correlated directly with their reduced binding to TAFII32. We conclude that interactions between TAFII32 and CIITA are responsible for activation of class II genes. 


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Transcriptional regulation of the interleukin-2 gene in normal human peripheral blood T cells. Convergence of costimulatory signals and differences from transformed T cells. 
To study transcriptional regulation in normal human T cells, we have optimized conditions for transient transfection. Interleukin-2 (IL-2) promoter-reporter gene behavior closely parallels the endogenous gene in response to T cell receptor and costimulatory signals. As assessed with mutagenized promoters, the most important IL-2 cis-regulatory elements in normal T cells are the proximal AP-1 site and the NF- kappaB site. Both primary activation, with phytohemagglutinin or antibodies to CD3, and costimulation, provided by pairs of CD2 antibodies or B7-positive (B cells) or B7-negative (endothelial) accessory cells, are mediated through the same cis-elements. Interestingly, the nuclear factor of activated T cell sites are much less important in normal T cells than in Jurkat T cells. We conclude that IL-2 transcriptional regulation differs in tumor cell lines compared with normal T cells and that different costimulatory signals converge on the same cis-elements in the IL-2 promoter. 


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IL-2-mediated cell cycle progression and inhibition of apoptosis does not require NF-kappa B or activating protein-1 activation in primary human T cells. 
The IL-2 growth hormone is the major growth factor of activated T lymphocytes during a developing immune response. IL-2 is required not only for cell cycle progression but also to protect Ag-activated T cells from programmed cell death. In several cell types, activation of NF-kappa B and/or activating protein-1 (AP-1) has been demonstrated to be extremely important in blocking apoptosis. To determine whether either or both of these transcription factors are involved in cell survival or cell cycle progression in response to IL-2, primary human T cells responsive to the growth factor were analyzed for NF-kappa B and AP-1 activation. The current study clearly demonstrates that IL-2 does not induce I kappa B alpha degradation or NF-kappa B activation in primary human T cells that respond to IL-2 by entering the cell cycle and avoiding apoptosis. Similarly, IL-2 neither activates JNK nor increases AP-1 binding activity to a consensus o-tetradecanoylphorbol 13-acetate (TPA) response element. On the other hand, the growth factor does induce the activation of STAT3 and STAT5 in these cells, as has been previously demonstrated. These data show that neither NF-kappa B nor AP-1 activation is required for IL-2-mediated survival or cell cycle progression in activated primary human T cells. 


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Transcription factor NF-kappaB regulates inducible Oct-2 gene expression in precursor B lymphocytes. 
The POU transcription factors Oct-1 and Oct-2 regulate the activity of octamer-dependent promoters, including those that direct transcription from rearranged immunoglobulin genes. Unlike Oct-1, which is constitutively expressed in many cell types, Oct-2 expression is restricted primarily to B lymphocytes and can be induced in precursor B cells by stimulation with bacterial lipopolysaccharide (LPS). However, the precise factors that mediate this induction mechanism remain unknown. In the present study, we monitored Oct-2 expression in cells arrested for the activation of NF-kappaB, an LPS-responsive member of the Rel transcription factor family. Despite stimulation with LPS, disruption of the NF-kappaB signaling pathway in precursor B cells led to the loss of inducible Oct-2 DNA binding activity in vitro and the suppression of Oct-2-directed transcription in vivo. This biochemical defect correlated with a specific block to Oct-2 gene expression at the level of transcription, whereas the expression of Oct-1 was unaffected. The finding that Oct-2 is under NF-kappaB control highlights an important cross-talk mechanism involving two distinct transcription factor families that regulate B lymphocyte function. 


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Interleukin 10 induced c-fos expression in human B cells by activation of divergent protein kinases. 
IL-10 is a potent mediator of human B cell growth and plasma cell formation. However, signal transduction of IL-10 in B cells is poorly understood. In this study the effect of IL-10 on the expression of the protooncogene c-fos was investigated, because Fos plays a potential role in the regulation of B cell proliferation and differentiation. B cells were purified from buffy coat preparations of healthy blood donors by positive selection using an anti CD20 monoclonal antibody and a MiniMACS separation unit. B cells were prestimulated with SAC for 48 hrs. Then, cells were incubated with medium or IL-10 (100 ng/ml) for 10 to 120 min. RNA was extracted by phenol/chloroform and c-fos expression was analyzed by PCR assisted mRNA assay. A significant 2-4 fold increase of c-fos expression was observed within 30 min of stimulation with IL-10 (p < 0.01). After 2 hrs c-fos expression declined to basal levels. The effect of IL-10 was dose-dependent with a maximum stimulation using 100 ng/ml of IL-10. The IL-10 effect on c-fos expression was not blocked by polymyxin B. Using the tyrosine kinase inhibitor genistein (10 microM) a complete inhibition of IL-10 induced c-fos expression was observed. In addition, H-7 (10 microM), a specific inhibitor of serine/threonine kinases, significantly blocked IL-10 mediated c-fos expression (p < 0.05). In conclusion, these data show that IL-10 induces c-fos expression in human B-cells by activation of tyrosine and serine/threonine kinases. Since this is the first report on IL-10 induced signal transduction, these data may help to identify the intracellular mechanisms by which IL-10 stimulates human B-cells. 


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GM-CSF and IL-2 share common control mechanisms in response to costimulatory signals in T cells. 
Antigen complexed with major histocompatibility complex class I or II molecules on the surface of antigen presenting cells interacts with the T cell receptor (TCR) on the surface of T cells and initiates an activation cascade. So called costimulatory signals, mediated by other cell surface interactions or soluble cytokines produced by antigen presenting cells, are also required for complete T cell activation. High levels of cytokine gene expression in T cells also required both TCR and costimulatory signals. The granulocyte-macrophage colony-stimulating factor requires sequences in the promoter as well as a powerful enhancer located 3kb upstream to respond to TCR-like signals. These promoter and enhancer regions are mainly activated by the transcription factor nuclear factor of activated T cells (NFAT). The activation of NFAT by TCR signals has been well described for interleukin-2 (IL-2) and IL-4 gene transcription in T cells. Costimulatory signals, such as activation of the CD28 cell surface molecule on T cells, lead to activation through a distinct region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter. This region is termed the CK-1 or CD28RE and appears to bind specific members of the NF-kappa B family of transcription factors. Human T leukemia virus type 1 (HTLV-1) infects T cells and can lead to increase GM-CSF expression. We have found that the HTLV-1 transactivator protein, tax, acts as a costimulatory signal for GM-CSF and IL-2 gene transcription, in that it can cooperate with TCR signals to mediate high level gene expression. Tax activates the GM-CSF promoter through the CK-1/CD28RE region and also activates nuclear factor-kappa B binding to this region. However, other transcription factors or coactivators of NF-kappa B are required for tax activation but these remain to be identified. The CK-1/CD28RE of GM-CSF shows a high degree of similarity to the IL-2 CD28RE and the IL-3 gene also contains a related region. This observation, together with the fact that both GM-CSF and IL-2 respond to TCR signals via NFAT, implies a high degree of conservation in the regulation of cytokine gene expression in T cells. 


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IL-7 reconstitutes multiple aspects of v-Abl-mediated signaling. 
The mechanism by which early lymphoid cells are selectively transformed by v-Abl is currently unknown. Previous studies have shown constitutive activation of IL-4 and IL-7 signaling pathways, as measured by activation of Janus protein kinase (JAK)1, JAK3, STAT5, and STAT6, in pre-B cells transformed by v-Abl. To determine whether activation of these cytokine signaling pathways by v-Abl is important in the cellular events induced by the Abelson murine leukemia virus, the effects of IL-4 and IL-7 on pre-B cells transformed with a temperature-sensitive v-Abl mutant were examined. Whereas IL-4 had little or no effect, IL-7 delayed both the apoptosis and cell cycle arrest that occur upon v-Abl kinase inactivation. IL-7 also delayed the decreases in the levels of c-Myc, Bcl-2, and Bcl-xL that occur upon loss of v-Abl kinase activity. IL-7 did not maintain v-Abl-mediated differentiation arrest of the pre-B cells, as activation of NF-kappaB and RAG gene transcription was unaffected by IL-7. These results identify a potential role for IL-7 signaling pathways in transformation by v-Abl while demonstrating that a combination of IL-4 and IL-7 signaling cannot substitute for an active v-Abl kinase in transformed pre-B cells. 


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Constitutive activation of NF-kappaB in primary adult T-cell leukemia cells. 
Human T-cell leukemia virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia (ATL). The viral protein Tax induces the activation and nuclear translocalization of transcription factor NF-kappaB, which is proposed to play a crucial role in the transformation of T cells by HTLV-I. However, the HTLV-I genes including Tax are not expressed significantly in primary leukemic cells from ATL patients. In this study, we examined the basis for NF-kappaB activation in freshly isolated leukemic cells from ATL patients. We found that leukemic cells from ATL patients, like HTLV-I-infected T-cell lines, display constitutive NF-kappaB DNA binding activity and increased degradation of IkappaBalpha (an inhibitor of NF-kappaB). Whereas the NF-kappaB binding activity in Tax-expressing T-cell lines consisted mostly of p50/c-Rel, fresh ATL samples contained p50/p50 and p50/p65 heterodimers. One T-cell line derived from ATL leukemic cells, TL-Om1, displayed constitutive NF-kappaB activity, as well as enhanced degradation of IkappaBalpha, despite the lack of detectable Tax expression. Interestingly, the NF-kappaB in TL-Om1 consists of p50/p50 and p50/p65 like that in fresh primary leukemic cells. Our results suggest that activation of NF-kappaB occurs through a Tax-independent mechanism in leukemic cells of ATL patients, possibly due to differential NF-kappaB subunit activation. 


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The retinoblastoma gene product negatively regulates transcriptional activation mediated by the human cytomegalovirus IE2 protein. 
The IE2 gene product of human cytomegalovirus (HCMV) is one of a few viral regulatory proteins expressed immediately upon infection of the host cell. It is a potent transcriptional activator of many viral and cellular promoters. We found that the retinoblastoma susceptibility gene product (Rb) dramatically suppressed this IE2 transactivation of various promoters. However, unlike another tumor suppressor protein, p53, Rb did not have any significant effect on basal levels of transcription, suggesting that Rb specifically interacts with IE2 rather than other cellular factors involved in the general transcription machinery. We found by protein-affinity chromatography that Rb in nuclear extracts or produced by in vitro translation directly bound to IE2. Our results suggest that Rb may regulate the life cycle of HCMV, which is endemic in the human population. Furthermore, these data may provide new insights into the slow rate of HCMV DNA replication in cells and the possible involvement of HCMV in tumorigenesis. 


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Regulation of Fas ligand expression and cell death by apoptosis-linked gene 4. 
Programmed cell death is a process required for the normal development of an organism. One of the best understood apoptotic pathways occurs in T lymphocytes and is mediated by Fas/Fas ligand (FasL) interaction. During studies of apoptosis induced by T cell-receptor engagement, we identified ALG-4F, a truncated transcript that prevents T cell-receptor-induced FasL upregulation and cell death. Overexpression of full-length ALG-4 induced transcription of FasL and, consequently, apoptosis. These results indicate that ALG-4 is necessary and sufficient for FasL expression. Fas/FasL interaction initiates cell death in many other systems, and its dysregulation is a mechanism by which several pathologic conditions arise. Understanding the molecular mechanisms of FasL regulation could be very useful in elucidating how these diseases develop and in identifying potential therapeutic targets. 


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Inhibition of NF-AT signal transduction events by a dominant-negative form of calcineurin. 
An inhibitory, "dominant-negative," form of the calcineurin catalytic (A) subunit was prepared, which lacks the calmodulin-binding domain, autoinhibitory domain and most of its catalytic core but possesses the regulatory (B) subunit binding domain. When tested for its ability to block calcineurin-dependent signaling in Jurkat cells, expression of this "B-subunit knock-out" (BKO) construct suppressed reporter gene activity driven by NF-AT, the pivotal promoter element for interleukin (IL)-2 gene induction. Immunoprecipitation of epitope-labeled BKO demonstrated for the formation of a tight complex with endogenous B subunit in Jurkat cells, consistent with an inhibitory mechanism that involves the sequestration of the B subunit. Furthermore, the sharply reduced NF-AT activity produced by co-transfecting BKO could be "rescued" by overexpression of transfected B subunit, suggesting that depletion of this subunit was responsible for the inhibition. These data suggest the potential utility of agents that disrupt calcineurin-mediated signal transduction pathways by blocking formation of the catalytically active dimer of calcineurin A and B subunits. 


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Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor. 
The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF. 


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Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor. 
High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Anti-CD2 receptor antibodies activate the HIV long terminal repeat in T lymphocytes. 
The CD2 T lymphocyte glycoprotein surface molecule mediates both cell to cell adhesion and T cell activation, two processes that are involved in the spread of HIV infection. Treatment of chronically HIV-infected PBMC with anti-CD2 mAb has been shown to induce the expression of infectious virus from these cultures. In this study we investigated the mechanisms whereby anti-CD2 antibodies stimulate viral production. We demonstrate that treatment of transiently transfected T lymphocytes with anti-CD2 antibodies results in activation of the HIV long terminal repeat. Furthermore, CAT assays using mutated HIV long terminal repeat-CAT constructs and gel shift assays demonstrate that this activation is dependent on the NF-kappa B enhancer. These studies suggest that interaction of CD2 with its natural ligand, LFA-3, may play a role in regulation of HIV expression. 


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Defective translocation of protein kinase C in multidrug-resistant HL-60 cells confers a reversible loss of phorbol ester-induced monocytic differentiation. 
Previous studies have demonstrated that human HL-60 myeloid leukemia cells differentiate in response to phorbol esters. This event is associated with induction of the c-jun early response gene and appearance of a monocytic phenotype. The present studies have examined the effects of vincristine-selected, multidrug resistance on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 cell differentiation. The results demonstrate that multidrug-resistant HL-60 cells, designated HL-60/vinc, fail to respond to TPA with an increase in c-jun transcripts or other phenotypic characteristics of monocytic differentiation. By contrast, treatment of HL-60/vinc cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases, induces c-jun transcription, growth arrest, and expression of the c-fms gene. Studies were also performed with an HL-60/vinc revertant (HL-60/vinc/R) line that has regained partial sensitivity to vincristine. The finding that HL-60/vinc/R cells respond to TPA with induction of a monocytic phenotype, but not c-jun expression, suggests that c-jun induction is not obligatory for monocytic differentiation. Other studies further demonstrate that the jun-B and fra-1 genes are induced by TPA in both HL-60/vinc and HL-60/vinc/R cells, whereas c-fos expression is attenuated in the HL-60/vinc line. Since TPA activates protein kinase C (PKC), we examined translocation of PKC from the cytosol to the membrane fraction. Although HL-60 and HL-60/vinc/R cells demonstrated translocation of PKC activity, this subcellular redistribution was undetectable in HL-60/vinc cells. Activity of the mitogen-activated protein kinase family with associated phosphorylation of c-Jun Y-peptide was markedly diminished in TPA-treated HL-60/vinc cells, but not in response to okadaic acid. Taken together, these findings suggest that vincristine resistance confers insensitivity to TPA-induced differentiation and can include defects in PKC-mediated signaling events and induction of jun/fos early response gene expression. 


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HMG-I binds to GATA motifs: implications for an HPFH syndrome. 
We have examined binding of the nuclear protein HMG-I to the human gamma-globin promoter. We find that HMG-I binds preferentially to the more 3' of a pair of GATA motifs in the gamma-globin promoter; this paired motif is bound by the erythroid factor GATA-1. A naturally occurring mutation (-175 T-C) in the area bound by HMG-I results in overexpression of gamma-globin in adult red blood cells (HPFH) and up-regulation of the gamma-globin promoter in in vitro expression assays; HMG-I does not bind to this mutant sequence. A survey of GATA motifs from other globin cis-elements demonstrates HMG-I binding to most of them. These findings implicate HMG-I in the HPFH phenotype; we speculate that it may participate in the formation of multiprotein complexes that regulate globin gene expression. 


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A nongenomic mechanism for progesterone-mediated immunosuppression: inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes. 
The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin-resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl- channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression. 


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Involvement of Rel, Fos, and Jun proteins in binding activity to the IL-2 promoter CD28 response element/AP-1 sequence in human T cells. 
CD28 is an important costimulatory molecule in the activation of human T cells. Costimulation of T cells through both the Ag receptor and CD28 leads to high level IL-2 production, which is vital to the development of an immune response in vivo. Previous reports have suggested the CD28 stimulation contributes to the activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including AP-1 and nuclear factor-kappaB (NF-kappaB)/Rel family members as well as an uncharacterized transcription factor called CD28 response complex. While several lines of investigation have suggested that NF-kappaB/Rel family members make up the CD28 response complex transcription factor, other work has not supported this conclusion. Recent studies suggest that the CD28 response element (CD28RE) does not function independently but works instead in conjunction with the adjacent promoter proximal AP-1-binding site and this hypothesis is confirmed here. Also in the current study, binding activity to the CD28RE/AP-1 sequence of the IL-2 promoter is evaluated. Although four specific complexes can be detected binding to this sequence, only one of these complexes is specific for both the CD28RE and the adjacent AP-1 site. Of the NF-kappaB/Rel family members tested, this CD28RE/AP-1-specific complex contains predominantly c-Rel, despite the fact that both p50 and RelA can efficiently bind to the CD28RE. c-Fos and c-Jun are also found in this CD28RE/AP-1-specific complex. These data indicate that functional complexes encompassing both the CD28RE and the AP-1-binding sites influence IL-2 promoter activity in CD28-costimulated T cells. 


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Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation [see comments] 
Adhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to P-selectin, the most rapidly expressed endothelial tethering factor, increased the secretion of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) by the leukocytes when they were stimulated with platelet-activating factor. Increased cytokine secretion was specifically inhibited by G1, an anti-P-selectin mAb that prevents P-selectin from binding to its ligand (P-selectin glycoprotein ligand-1) on myeloid cells. Moreover, tethering by P-selectin specifically enhanced nuclear translocation of nuclear factor-kappa B (NF-kappa B), a transcription factor required for expression of MCP-1, TNF-alpha, and other immediate-early genes. These results demonstrate that P-selectin, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues. 


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Role of cellular tumor necrosis factor receptor-associated factors in NF-kappaB activation and lymphocyte transformation by herpesvirus Saimiri STP. 
The STP oncoproteins of the herpesvirus saimiri (HVS) subgroup A strain 11 and subgroup C strain 488 are now found to be stably associated with tumor necrosis factor receptor-associated factor (TRAF) 1, 2, or 3. Mutational analyses identified residues of PXQXT/S in STP-A11 as critical for TRAF association. In addition, a somewhat divergent region of STP-C488 is critical for TRAF association. Mutational analysis also revealed that STP-C488 induced NF-kappaB activation that was correlated with its ability to associate with TRAFs. The HVS STP-C488 P10-->R mutant was deficient in human T-lymphocyte transformation to interleukin-2-independent growth but showed wild-type phenotype for marmoset T-lymphocyte transformation in vitro and in vivo. The STP-C488 P10-->R mutant was also defective in Rat-1 fibroblast transformation, and fibroblast cell transformation was blocked by a TRAF2 dominant-negative mutant. These data implicate TRAFs in STP-C488-mediated transformation of human lymphocytes and rodent fibroblasts. Other factors are implicated in immortalization of common marmoset T lymphocytes and may also be critical in the transformation of human lymphocytes and rodent fibroblasts. 


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Signal transduction abnormalities in T lymphocytes from patients with advanced renal carcinoma: clinical relevance and effects of cytokine therapy. 
Studies have demonstrated abnormalities of the CD3/T-cell antigen receptor (TCR) and pathways of signal transduction in T lymphocytes from animals and patients with advanced malignancy. Diminished expression of TCRzeta and p56(lck) that are associated with the TCR and reduced nuclear localization of RelA containing nuclear factor kappaB (NFkappaB) complexes have been noted. These defects have been described in T cells from patients with malignant melanoma, renal cell carcinoma (RCC), ovarian cancer, and colorectal cancer. Preliminary observations also indicate possible correlation with clinical variables such as stage in selected instances. To further characterize altered expression of TCRzeta, p56(lck), and impaired activation of NFkappaB, T lymphocytes were obtained from 65 patients with RCC, the majority of whom were receiving combination cytokine therapy [interleukin (IL)-2, IFN alpha-containing regimens] and 37 control individuals. In 29 of these patients, levels of TCRzeta and p56(lck) were determined by Western blots of T-cell lysates and semiquantitated using densitometry. Relative levels were then correlated with a series of clinical variables including response to therapy, performance status, survival, disease sites, age, and others. In another group of 28 patients (three individuals from the first group), the frequency of abnormal NFkappaB activation was studied using electrophoretic mobility shift assays after activation of T cells with phorbol myristate acetate/ionomycin or anti-CD3 monoclonal antibody. Changes in these signaling molecules during cytokine treatment were also investigated. TCRzeta and p56(lck) were detected in the peripheral blood T cells in 27 of 29 patients, and overall, reduced levels were noted visually in 12 of 29 (41%) and 13 of 29 (45%) individuals, respectively. When levels were semiquantitated using densitometry, significant decreases of TCRzeta (P = 0.029) and p56(lck) (P = 0.029) but not CD3epsilon (P = 0.131), compared with control levels, were found. In patients treated with IL-2/IFN alpha-based therapy, relative levels of TCRzeta increased significantly (P = 0.002) on day 15 of cycle one compared with the baseline. Correlations of TCRzeta or p56(lck) levels with response or disease variables, except for lower TCRzeta levels (P < 0.001) in the presence of bone metastases, were not found. Abnormal NFkappaB activation after stimulation with phorbol myristate acetate/ionomycin and/or anti-CD3 monoclonal antibody was found in 59% of patients (17 of 28) and was not accounted for by the advanced age of the study cohort. Activation of NFkappaB in peripheral blood T cells was inducible during cytokine therapy in four of six individuals who displayed impaired NFkappaB activity prior to therapy. Moreover, impaired activation of NFkappaB does not appear linked to a reduction of TCRzeta expression, because in five patients, normal TCRzeta levels were present although kappaB binding was not inducible. In the majority of patients with advanced RCC, peripheral blood T cells express TCRzeta and p56(lck), and in a subset, reduced levels of these TCRzeta associated molecules are seen that may increase during cytokine-based therapy. Abnormal activation of NFkappaB is also present in >50% of patients and may also revert to normal during IL-2/IFN alpha-based treatment. This alteration in NFkappaB activation occurred in the presence of normal expression of TCRzeta-associated signaling elements. The clinical significance of these findings remains unclear. 


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Activation of protein kinase C and elevation of cAMP interact synergistically to raise c-Fos and AP-1 activity in Jurkat cells. 
We have earlier found that in Jurkat cells activation of protein kinase C (PKC) enhances the cyclic adenosine monophosphate (cAMP) accumulation induced by adenosine receptor stimulation or activation of Gs. Here we have therefore examined the effect of the phorbol ester PMA (phorbol 12-myristate 13-acetate) which stimulates PKC and a combination of the adenosine receptor agonist NECA (5'-(N-ethyl)-carboxamido adenosine) and forskolin to raise cAMP, on the levels of c-Fos and Jun and on the binding and transcriptional activity of the transcription factor, activator protein-1 (AP-1). PMA treatment caused a concentration- and time-dependent increase in both c-Fos and Jun immunoreactivity in contrast to cAMP elevation that had only a slight effect. Both PMA and the combination of NECA and forskolin acted together either to increase (c-Fos) or decrease (Jun) protein levels as well as increasing AP-1 binding, as judged by gel-shift assay, and AP-1 transcriptional activity. Furthermore there was a clear-cut synergy between the PKC stimulator and the cAMP elevating agents. The results demonstrate that the simultaneous activation of PKC and elevation of cAMP leads to an enhanced AP-1 transcriptional activity in a T-leukemia cell line, suggesting that the previously observed interaction between the parallel signal transduction pathways may have functional consequences at the level of gene transcription. 


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c-Myb and Ets proteins synergize to overcome transcriptional repression by ZEB. 
The Zfh family of zinc finger/homeodomain proteins was first identified in Drosophila where it is required for differentiation of tissues such as the central nervous system and muscle. ZEB, a vertebrate homolog of Zfh-1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. We present evidence here that ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c-Myb and Ets. These factors act synergistically to activate transcription, and this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c-Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c-Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c-Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. We demonstrate that one target of this positive/negative regulation in vivo is the alpha4 integrin, which play a key role in normal hematopoiesis and function of mature leukocytes. 


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Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IkappaB kinase signalsome activation and IkappaB proteolysis. 
The inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor (TNF) are potent activators of NF-kappaB. This study compared the effect of these stimuli on endogenous IkappaB kinase (IKK) signalsome activation and IkappaB phosphorylation/proteolysis in human monocytic cells and investigated the role of the signalsome proteins IKK-alpha, IKK-beta, NF-kappaB-inducing kinase (NIK), IKK-gamma (NF-kappaB essential modulator), and IKK complex-associated protein. Kinase assays showed that TNF elicited a rapid but short-lived induction of IKK activity with a 3-fold greater effect on IKK-alpha than on IKK-beta, peaking at 5 min. In contrast, LPS predominantly stimulated IKK-beta activity, which slowly increased, peaking at 30 min. A second peak was observed at a later time point following LPS stimulation, which consisted of both IKK-alpha and -beta activity. The endogenous levels of the signalsome components were unaffected by stimulation. Furthermore, our studies showed association of the IKK-alpha/beta heterodimer with NIK, IkappaB-alpha and -epsilon in unstimulated cells. Exposure to LPS or TNF led to differential patterns of IkappaB-alpha and IkappaB-epsilon disappearance from and reassembly with the signalsome, whereas IKK-alpha, IKK-beta, and NIK remained complex-associated. NIK cannot phosphorylate IkappaB-alpha directly, but it appears to be a functionally important subunit, because mutated NIK inhibited stimulus-induced kappaB-dependent transcription more effectively than mutated IKK-alpha or -beta. Overexpression of IKK complex-associated protein inhibited stimulus-mediated transcription, whereas NF-kappaB essential modulator enhanced it. The understanding of LPS- and TNF-induced signaling may allow the development of specific strategies to treat sepsis-associated disease. 


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Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner. 
When immunoglobulin (Ig) expressing B cells are fused with non-B cells, Ig expression is rapidly suppressed at the level of transcription, a phenomenon termed extinction. Here we demonstrate that fusion of HeLa cells with either diploid or tetraploid B cells (Daudi) results in widespread extinction of several other B cell-encoded genes that are expressed in a B cell-specific manner. In contrast, expression of B cell-expressed genes that are not dependent on cell-specific controls is unaffected. We show that the molecular mechanism(s) underlying Ig gene extinction can be explained, at least in part, by a lack of transcription factors that are essential for Ig gene transcription. These transcription factors are either not produced due to block of transcription of their respective genes (Oct-2, OBF-1, PU.1), or are rendered inactive posttranslationally (NF-kappa B, E47). By isolating Daudi x HeLa heterokaryons a few hours after fusion, we have studied the initial fate of two B cell-specific transcription factors involved in Ig gene transcription, Oct-2 and NF-kappa B. This report provides the first demonstration that upon fusion with HeLa cells, the nuclear contents of B cell-expressed transcription factors are depleted within a few hours with kinetics that are as fast or faster than that of Ig gene extinction. Thus, the extinguishing mechanism is effective very early after fusion. We suggest that extinction of Ig genes is part of a global mechanism that suppresses the differentiation program foreign to the HeLa phenotype. 


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Sequential development of structural and functional alterations in T cells from tumor-bearing mice. 
The TCR alpha beta or -gamma delta chains bind the peptide ligand, whereas the associated CD3 deltaepsilongamma and TCR zeta subunits couple the TCR to intracellular signal transduction components. Recently, several groups have described marked alterations in signal transduction elements in T cells from cancer patients or in mice bearing tumor for a few weeks (>26 days). The sequence in which these alterations develop is unknown. The aim of this study was to explore the kinetics of the development of alterations in signal transduction molecules (TCR zeta chain, NF kappaB family proteins, and tyrosine kinase p56(lck)) in mice bearing MC38 colon adenocarcinoma. The results demonstrate that alterations in NF kappaB family proteins, specifically the failure of p65 translocation to the nucleus, occur earlier and more frequently than the decrease in zeta-chain. These defects are paralleled by an impaired ability to produce Th1 cytokines (IL-2 and IFN-gamma). These initial changes are followed by the eventual loss of TCR zeta chain and p56(lck) and a marked decrease in cytotoxic function. An increased rate of lysosomal degradation is one of the mechanisms responsible for the loss of zeta-chain. 


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Thymocytes control the CD4 gene differently from mature T lymphocytes. 
We analyzed the activity of the enhancer, the promoter and the silencer of the human CD4 gene during T cell development using transgenic mice. Immunofluorescence studies on thymic populations of mice carrying transgenes in various combinations of these regulatory DNA elements revealed that thymocytes control the CD4 gene in a different manner than mature peripheral T lymphocytes. The 5'-positive regulatory unit, consisting of the promoter and the 5' enhancer, is already active at the CD4-CD8-double-negative (DN) stage of development. However, its activity becomes lower in the double-positive and a fraction of the CD4+ CD8int/- cell population, indicating that an additional enhancer, located in either the first or the third intron of the CD4 gene, is required for CD4 gene expression in this population. The other studied regulatory element is the minimal CD4 silencer which inhibits CD4 gene expression in peripheral CD8 T lymphocytes. This silencer is inactive in the most immature DN thymocytes, which probably use a distinct silencer mechanism to down-regulate CD4 gene expression. Unexpectedly, the CD4 silencer is also active in CD4+ CD8int/- cells of the thymus, implying that an anti-silencer may be required to resume CD4 expression in this cell population. Altogether, the CD4 gene is regulated by several positive and negative regulatory mechanisms which come into play in a developmentally coordinated manner. 


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Globin gene switching. In vivo protein-DNA interactions of the human beta-globin locus in erythroid cells expressing the fetal or the adult globin gene program. 
To characterize the protein-DNA interactions important for the developmental control of the human beta-globin locus, we analyzed by in vivo dimethyl sulfate footprinting erythroid cells expressing either the fetal or the adult globin developmental program. In the locus control region (LCR) of the beta-globin locus, in vivo footprints on NF-E2 (or AP-1) and GATA-1 motifs remained the same regardless of whether the fetal or the adult globin genes are expressed. In contrast, in vivo footprints on GT (CACCC) motifs differed between the cells expressing the fetal or the adult globin program. In promoter regions, the actively transcribed genes demonstrated extensive and consistent footprints over the canonical elements, such as CACCC and CCAAT motifs. The adult globin expressing cells displayed more extensive footprints than the fetal globin expressing cells in the 3' regulatory sequences of both the Agamma- and the beta-globin genes, suggesting a role of these 3' elements in beta-globin gene expression. Our results suggest that the bulk of protein-DNA interactions that underlies the developmental control of globin genes takes place in the gamma- and beta-globin gene promoters, and that GT motifs of the beta-globin locus LCR may play a role in the developmental regulation of human beta-globin gene expression, perhaps by increasing the probability of interaction of the LCR holocomplex with the fetal or the adult globin gene. 


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Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation. 
Human myeloid differentiation is accompanied by a decrease in cell proliferation. Because the translation rate is an important determinant of cell proliferation, we have investigated translation initiation during human myeloid cell differentiation using the HL-60 promyelocytic leukemia cell line and the U-937 monoblastic cell line. A decrease in the translation rate is observed when the cells are induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. The inhibition in protein synthesis correlates with specific regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages by IFN-gamma or PMA results in a dephosphorylation and consequent activation of 4E-BP1. Dephosphorylation of 4E-BP1 was also observed when U-937 cells were induced to differentiate into monocytes/macrophages following treatment with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with retinoic acid or DMSO, which results in a granulocytic differentiation of these cells, decreases 4E-BP1 amount without affecting its phosphorylation and strongly increases 4E-BP2 amount. Taken together, these data provide evidence for differential regulation of the translational machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway. 


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Regulatory effects of interleukin-11 during acute lung inflammatory injury. 
The role of interleukin-11 (IL-11) was evaluated in the IgG immune complex model of acute lung injury in rats. IL-11 mRNA and protein were both up-regulated during the course of this inflammatory response. Exogenously administered IL-11 substantially reduced, in a dose-dependent manner, the intrapulmonary accumulation of neutrophils and the lung vascular leak of albumin. These in vivo anti-inflammatory effects of IL-11 were associated with reduced NF-kappaB activation in lung, reduced levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage (BAL) fluids, and diminished up-regulation of lung vascular ICAM-1. It is interesting that IL-11 did not affect BAL fluid content of the CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-inducible neutrophil chemoattractant (CINC); the presence of IL-11 did not affect these chemokines. However, BAL content of C5a was reduced by IL-11. These data indicate that IL-11 is a regulatory cytokine in the lung and that, like other members of this family, its anti-inflammatory properties appear to be linked to its suppression of NF-kappaB activation, diminished production of TNF-alpha, and reduced up-regulation of lung vascular ICAM-1. 


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v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII. 
The v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone (T3/T4) receptor c-erbA (type alpha). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes. Here, we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II (CAII), a gene whose transcription is efficiently suppressed by v-erbA. This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein. In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA/T3 receptor at an approximately equimolar ratio, c-erbA activity is dominant over v-erbA. T3 efficiently induced erythroid differentiation in these cells, thus overcoming the v-erbA-mediated differentiation arrest. Likewise, T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site. The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein. 


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Differential induction of the NF-AT complex during restimulation and the induction of T-cell anergy. 
Stimulation of human CD4+ T-cell clones through the T-cell receptor (TcR) by high doses of specific peptide results in the induction of a long-lived state of nonresponsiveness that has been called anergy. During the induction of anergy, T cells are phenotypically similar to cells responding to an immunogenic stimulus. The amount of TcR at the cell surface is downmodulated, whereas the CD2 and CD25 receptors are increased. When restimulated, however, anergic T cells fail to up-regulate transcription of the IL-2 gene and in consequence do not produce IL-2. In this study, we have compared the ability of various transcription factors to bind to their appropriate site on DNA. Factors were isolated from the nuclei of T cells that were in the induction phase of anergy or were undergoing activation. The pattern of binding activity in restimulated T cells is consistent with the pattern that has previously been shown to regulate T-cell-specific expression of the IL-2 and the beta chain of the TcR genes. The measured binding to a TCF-1 site is the same in the nuclei of resting, activated, and anergized cells. The inducible factors NK-kappa B, beta E2, CD28RC, and AP-1 are not expressed in resting cells and are twofold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT, a member of the class of inducible factors that regulates IL-2 gene transcription. (ABSTRACT TRUNCATED AT 250 WORDS) 


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The p53 paradox in the pathogenesis of tumor progression. 
Recent evidence suggests that the p53 molecule appears in two different forms: the mutant p53 that stimulates tumor progression, and wild type p53 that inhibits tumor progression. In addition, it has been established that tumor necrosis factor-alpha (TNF-alpha) can activate the expression of wild type p53 in concert with the nuclear transcription factor, NF-kappa B. Both TNF-alpha and NF-kappa B are also involved in the stimulation of the pathway that leads to the expression of major histocompatibility complex (MHC) class I molecules and, hence, antigen presentation to the T cells. In this paper we shall advance the hypothesis that: (i) TNF-alpha indirectly controls immune surveillance; and (ii) TNF-alpha controls DNA repair and tumor suppression through the regulation of wild type p53. Thus, it is hypothesized that elevated TNF-alpha is primarily responsible for promoting tumor progression. 


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Human T lymphotropic virus-I infection of human T lymphocytes induces expression of the beta-galactoside-binding lectin, galectin-3. 
Animal lectins play important roles in a variety of biological processes via their recognition of glycoconjugates. Galectin-3 is a beta-galactoside-binding lectin previously designated as epsilon BP (IgE-binding protein), CBP35, Mac-2, L-29, and L-34, and its expression has been associated with various physiological and pathological processes, including cell growth, tumor transformation, and metastasis. Galectin-3 is widely distributed in various tissues and cell types and is expressed in many leukocytes, with the notable exception of B and T lymphocytes. We now report that galectin-3 is abundantly expressed in a number of human T lymphotropic virus (HTLV)-I-infected human T cell lines, including F6T, HUT 102, K3T, MT-2, and SLB-I, but is not expressed in non-HTLV-I-infected T cell lines such as Jurkat, CEM, and MOLT-4. In addition, the galectin-3 level was markedly increased in human thymocytes after infection with HTLV-I as compared with uninfected thymocytes. The up-regulation of galectin-3 expression appeared to correlate well with HTLV-I gene expression, as undetectable or very low levels of galectin-3 were found in the S1T and ATL-1K cell lines, which are nonproductively infected with HTLV-I. In co-transfection experiments, the galectin-3 promoter was significantly up-regulated by expression vectors encoding the 40-kd Tax protein, a potent transactivator in HTLV-I. Analysis of various Tax mutants suggested that galectin-3 promoter induction is dependent on activation of the cyclic-AMP-responsive element binding protein/activation transcription factor family of transcription factors and, to a lesser extent, nuclear factor-kappa B/Rel induction. Transfection of human promonocytic U-937 cells with an HTLV-I Tax expression vector induced galectin-3 expression in this cell line. Functionally, galectin-3 was shown to activate interleukin-2 production in Jurkat T cells. Together, these findings raise the possibility that HTLV-I Tax production induces the transcription and subsequent synthesis and secretion of galectin-3, which in turn may further activate these T cells and contribute to the altered properties of cell growth found in adult T cell leukemia induced by HTLV-I. 


Document 003002562 ends.


Initiation binding repressor, a factor that binds to the transcription initiation site of the histone h5 gene, is a glycosylated member of a family of cell growth regulators [corrected] [published erratum appears in Mol Cell Biol 1996 Feb;16(2):735] 
Initiation binding repressor [corrected] (IBR) is a chicken erythrocyte factor (apparent molecular mass, 70 to 73 kDa) that binds to the sequences spanning the transcription initiation site of the histone h5 gene, repressing its transcription. A variety of other cells, including transformed erythroid precursors, do not have IBR but a factor referred to as IBF (68 to 70 kDa) that recognizes the same IBR sites. We have cloned the IBR cDNA and studied the relationship of IBR and IBF. IBR is a 503-amino-acid-long acidic protein which is 99.0% identical to the recently reported human NRF-1/alpha-Pal factor and highly related to the invertebrate transcription factors P3A2 and erected wing gene product (EWG). We present evidence that IBR and IBF are most likely identical proteins, differing in their degree of glycosylation. We have analyzed several molecular aspects of IBR/F and shown that the factor associates as stable homodimers and that the dimer is the relevant DNA-binding species. The evolutionarily conserved N-terminal half of IBR/F harbors the DNA-binding/dimerization domain (outer limits, 127 to 283), one or several casein kinase II sites (37 to 67), and a bipartite nuclear localization signal (89 to 106) which appears to be necessary for nuclear targeting. Binding site selection revealed that the alternating RCGCRYGCGY consensus constitutes high-affinity IBR/F binding sites and that the direct-repeat palindrome TGCGCATGCGCA is the optimal site. A survey of genes potentially regulated by this family of factors primarily revealed genes involved in growth-related metabolism. 


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Apoptosis signaling pathways in normal T cells: differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity. 
Fas-mediated apoptosis plays an important role in regulating the immune response in peripheral T cells. Restimulation of T cell blasts up-regulates Fas and Fas ligand expression, with subsequent interaction leading to cell death. Overexpression of Bcl-2 in tumor cells blocks apoptosis induced by many stimuli, but inhibition of Fas-mediated killing has not been consistently observed. To examine the behavior of Bcl-2 in normal cells, T cell blasts were transiently transfected with Bcl-2 and related gene products to determine the effect on apoptotic signaling. Transient overexpression of Bcl-2 in mouse and human T cell blasts did not block Fas-mediated apoptosis, whereas etoposide- and glucocorticoid-induced cytotoxicity was potently inhibited. Expression of Bcl-xL and adenovirus E1B 19K did not interfere with anti-Fas killing. In contrast, interleukin-1beta-converting enzyme family protease inhibitors Ac-DEVD-CHO and CrmA blocked Fas-mediated apoptosis. These results suggest that peripheral T cells use distinct apoptosis signaling pathways with differential sensitivity to Bcl-2 and interleukin-1beta-converting enzyme family protease inhibitors. Since T cells normally express Bcl-2 and Bcl-xL following activation, their inability to block Fas-mediated apoptosis may allow for the elimination of self-reactive cells and the appropriate regulation of immune responses. 


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Induction of monocytic differentiation and NF-kappa B-like activities by human immunodeficiency virus 1 infection of myelomonoblastic cells. 
The effects of human immunodeficiency virus 1 (HIV-1) infection on cellular differentiation and NF-kappa B DNA binding activity have been investigated in a new model of myeloid differentiation. PLB-985 cells represent a bipotential myelomonoblastic cell population capable of either granulocytic or monocytic differentiation after induction with appropriate inducers. By virtue of the presence of CD4 on the cell surface, PLB-985 cells were chronically infected with HIV-1 strain IIIB. PLB-IIIB cells clearly possessed a more monocytic phenotype than the parental myeloblasts, as determined by differential staining, increased expression of the myeloid-specific surface markers, and transcription of the c-fms proto-oncogene. NF-kappa B binding activity was inducible by tumor necrosis factor and phorbol myristate acetate in PLB-985. However, in PLB-IIIB cells, constitutive expression of a novel NF-kappa B complex was detected, composed of proteins ranging between 70 and 110 kD. These proteins interacted specifically with the symmetric NF-kappa B site from the interferon beta (IFN-beta) promoter. Mutations affecting the 5' guanine residues of the kappa B site were unable to compete for these NF-kappa B-related proteins. Inducibility of endogenous IFN-beta and IFN-alpha RNA was also increased in PLB-IIIB cells. These studies indicate that HIV-1 infection of myelomonoblastic cells may select for a more mature monocytic phenotype and that unique subunit associations of NF-kappa B DNA binding proteins may contribute to differential NF-kappa B-mediated gene expression. 


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NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I. 
The effect of constitutive Tax expression on the interaction of NF-kappa B with its recognition sequence and on NF-kappa B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-kappa B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-kappa B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters. 


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Identification and characterization of a leukocyte-specific component of the nuclear body. 
The nuclear body (NB) is a cellular organelle that is involved in the pathogenesis of acute promyelocytic leukemia and viral infection. The NB is also a target of antibodies in the serum of patients with the autoimmune disease primary biliary cirrhosis. In this study, serum from a patient with primary biliary cirrhosis was used to identify a cDNA encoding a novel component of the NB, a 140-kDa protein designated Sp140. The predicted amino acid sequence of the amino-terminal portion of Sp140 was similar to Sp100, a previously identified NB protein. The carboxyl portion of Sp140 contained a zinc-finger domain and a bromodomain, motifs that are present in proteins regulating gene transcription. High levels of Sp140 mRNA were detected in human spleen and peripheral blood leukocytes, but not other human tissues. The level of SP140 mRNA in myeloid precursor cell lines HL60 and NB4 markedly increased in response to chemically induced cellular differentiation. Immunohistochemical techniques were used to demonstrate that SP140 localized to the NB in differentiated HL60 and NB4 cells. The location of Sp140 in the NB, and expression of this gene in cells involved in host defense, suggest that Sp140 may be involved in the pathogenesis of acute promyelocytic leukemia and viral infection. 


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Activation of pp90rsk and early growth response-1 gene expression by pokeweed mitogen in human B cells. 
The present studies have examined the effects of pokeweed mitogen (PWM) on the induction of early growth response-1 gene (EGR-1) in normal human B cells. PWM regulates EGR-1 gene expression by both transcriptional and post-transcriptional mechanisms. Transient transfection assays with EGR-1 promoter fragments linked to the chloramphenicol acetyltransferase (CAT) gene demonstrated that PWM induced EGR-1 transcription is conferred by the CArG motif (C C[AT]6GG) in the EGR-1 promoter. The results further demonstrated the activation of S6 kinase (pp90rsk), evidenced by phosphorylation of S6 and serum response factor (SRF) peptides, in PWM treated B cells. Taken together, these findings suggest that PWM is able to initiate an intracytoplasmic signalling cascade and EGR-1 induction in normal human B cells. 


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Autoregulation of the NF-kappa B transactivator RelA (p65) by multiple cytoplasmic inhibitors containing ankyrin motifs. 
RelA (p65) functions as the critical transactivating component of the heterodimeric p50-p65 NF-kappa B complex and contains a high-affinity binding site for its cytoplasmic inhibitor, I kappa B alpha. After cellular activation, I kappa B alpha is rapidly degraded in concert with the induced nuclear translocation of NF-kappa B. The present study demonstrates that tumor necrosis factor alpha-induced degradation of I kappa B alpha in human T cells is preceded by its rapid phosphorylation in vivo. However, these effects on I kappa B alpha result in nuclear mobilization of only a fraction of the entire cytoplasmic pool of RelA. Subsequent studies have revealed that (i) cytoplasmic RelA is stably associated not only with I kappa B alpha but also with other ankyrin motif-rich proteins including the products of the NF-kappa B2 (p100) and NF-kappa B1 (p105) genes; (ii) in contrast to RelA-I kappa B alpha, RelA-p100 cytoplasmic complexes are not dissociated following tumor necrosis factor alpha activation; (iii) p100 functions as a potent inhibitor of RelA-mediated transcription in vivo; (iv) the interaction of RelA and p100 involves the conserved Rel homology domain of both proteins but not the nuclear localization signal of RelA, which is required for I kappa B alpha binding; (v) p100 inhibition of RelA function requires the C-terminal ankyrin motif domain, which mediates cytoplasmic retention of RelA; and (vi) as observed with I kappa B alpha, nuclear RelA stimulates p100 mRNA and protein expression. These findings thus reveal the presence of a second inducible autoregulated inhibitory pathway that helps ensure the rapid but transient action of nuclear NF-kappa B. 


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Functional interaction between the two zinc finger domains of the v-erb A oncoprotein. 
The v-erb A oncogene of avian erythroblastosis virus is a mutated and virally transduced copy of a host cell gene encoding a thyroid hormone receptor. The protein expressed by the v-erb A oncogene binds to DNA and acts as a dominant negative inhibitor of both the thyroid hormone receptor and the closely related retinoic acid receptor. The v-erb A protein has sustained two amino acid alterations within its DNA-binding domain relative to that of c-erb A, one of which, at serine 61, is known to be important for v-erb A function in the neoplastic cell. We report here that the second alteration, at threonine 78, also plays an important, although more indirect, role: alteration of the sequence at threonine 78 such that it resembles that of c-erb A can act as an intragenic suppressor and can partially restore function to a v-erb A protein rendered defective due to a mutation at position 61. Threonine 78 lies within the D-box of the v-erb A protein, a region thought to mediate receptor-receptor dimerizations, and is not in physical proximity to the serine at position 61. It therefore appears that an indirect interaction occurs between these two sites and that this interaction is crucial for v-erb A function. 


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Abnormal T lymphocyte development induced by targeted overexpression of IkappaB alpha. 
A role in thymic maturation for factors of the NF-kappaB family has long been suspected, but not yet proven. Transgenic mice with a lymphocyte-specific defect in NF-kappaB activation were produced by targeted expression of human IkappaB alpha. The thymic cellularity of these mice was significantly decreased. The proportion of mature, TCRhigh thymocytes of the alphabeta lineage was reduced, and the remaining TCRhigh population contained an unusually high proportion of double-positive cells. This defect in maturation resulted in a transgene dose-dependent reduction in peripheral T lymphocytes, with the CD8 lineage being more severely affected. These data provide direct evidence for the involvement of NF-kappaB/Rel family proteins in late stages of T lymphocyte development, coincident with positive and negative selection. 


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Inhibition of transcription factors belonging to the rel/NF-kappa B family by a transdominant negative mutant. 
The KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c-rel and v-rel (proto)oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 (delta SP), unable to bind to DNA but able to form homo- or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family (KBF1/p50, c- and v-rel). This mutant also functions in vivo as a trans-acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF-kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF-kappa B family. 


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Human MHC class II gene transcription directed by the carboxyl terminus of CIITA, one of the defective genes in type II MHC combined immune deficiency. 
Type II major histocompatibility complex combined immune deficiency (type II MHC CID or bare lymphocyte syndrome) is a congenital immunodeficiency disease characterized by absent MHC class II expression. Four distinct complementation groups have been identified. Recently, the defective gene in group II type II MHC CID has been isolated and termed CIITA. Here, we demonstrate that CIITA is an MHC class II gene-specific transcription activator. The transcription activation function is provided by the N-terminal acidic domain (amino acids 26-137), which is experimentally exchangeable with a heterologous viral transcription-activating domain. The specificity of CIITA for three major MHC class II genes, DR, DQ and DP, is mediated by its remaining C-terminal residues (amino acids 317-1130). The transactivation of multiple cis elements, especially S and X2, of the DR alpha proximal promoter in group II CID cells is CIITA dependent. Since CIITA overexpression in normal cells did not increase class II expression, we propose that initiation of CIITA expression serves as the on-off switch, while availability of downstream interactor(s) limits transcription. 


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Induction of Sp1 phosphorylation and NF-kappa B-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid. 
In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes. 


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Functional association of Nmi with Stat5 and Stat1 in IL-2- and IFNgamma-mediated signaling. 
Using the coiled-coil region of Stat5b as the bait in a yeast two-hybrid screen, we identified the association of Nmi, a protein of unknown function previously reported as an N-Myc interactor. We further show that Nmi interacts with all STATs except Stat2. We evaluated two cytokine systems, IL-2 and IFNgamma, and demonstrate that Nmi augments STAT-mediated transcription in response to these cytokines. Interestingly, Nmi lacks an intrinsic transcriptional activation domain; instead, Nmi enhances the association of CBP/p300 coactivator proteins with Stat1 and Stat5, and together with CBP/p300 can augment IL-2- and IFNgamma-dependent transcription. Therefore, our data not only reveal that Nmi can potentiate STAT-dependent transcription, but also suggest that it can augment coactivator protein recruitment to at least some members of a group of sequence-specific transcription factors. 


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Vitamin D receptor 3'-untranslated region polymorphisms: lack of effect on mRNA stability. 
Allelic variation at the 3'-end of the vitamin D receptor (VDR) gene has been associated with a 3-5-fold increased risk of developing prostate cancer and with differences in bone mineralization. This genetic diversity does not alter the VDR protein structurally, but instead may be a marker(s) of other, nearby polymorphisms that influence message stability or translation. The work reported here was instigated to identify additional VDR 3'-UTR polymorphisms that may have functional significance and to then test whether these genetic variants alter message stability. Initially, four novel, frequently occurring sequence variants were identified that associated with two common haplotypes that were described previously. These common sequence variants were not found within three message-destabilizing elements that we mapped within the 3'-UTR of the vitamin D receptor mRNA. Furthermore, the two VDR 3'-UTR haplotypes conferred an identical half-life on a heterologous beta-globin reporter gene, in an in vitro assay. We therefore conclude that common polymorphisms within the VDR 3'-UTR do not influence message stability. 


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Circadian rhythm of glucocorticoid receptors in human peripheral leukocytes and their reactivity to glucocorticoids. 
1) There exists a CR of GR in human leukocytes, PMN, and monocytes with the peak values from 0400 to 0800 hr and the trough values between 2300 and 0000 hr. The difference between them was significant statistically. 2) The FI of the chemotactic migration rate of PMN by cortisol also showed diurnal changes which were synchronous with that of GR. This indicates that the CR of GR may be of functional significance. 3) In Cushing's syndrome, the CR of GR was normal in spite of the fact that the CR of plasma cortisol was disturbed. This indicates the independency of the CR of GR from that of cortisol. 4) In apoplexy caused by brain ischemia, the CR of GR was abolished in patients with basal lesions but preserved when the lesions were located in the cerebral cortex. These results strongly suggest that the main "circadian pacemaker" of GR is located in the basal brain, most probably in the suprachiasmatic nuclei as has been suggested for rodents. 


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Progesterone suppression of pregnancy lymphocytes is not mediated by glucocorticoid effect. 
This study investigated whether the suppressive effect of progesterone on pregnancy lymphocytes is mediated by specific progesterone receptors. The effects of a competitive progesterone antagonist (RU486) and a specific glucocorticoid receptor blocker (RU43044) were tested on the release of a blocking factor by progesterone-treated pregnancy lymphocytes. RU 486 tested at an equal concentration as progesterone significantly inhibited the production of the blocking factor, while RU 43044 was without effect. These data suggest that in pregnancy, lymphocyte progesterone acts on specific progesterone receptors and glucocorticoid binding sites are not involved. 


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Infection with Theileria annulata induces expression of matrix metalloproteinase 9 and transcription factor AP-1 in bovine leucocytes. 
Theileria annulata infects bovine leucocytes and results in their reversible transformation such that they become immortalised and metastatic. The present study describes parasite-induced changes in host cell gene expression which have a direct bearing on this transformation process. T. annulata-infected leucocytes produce a number of novel metalloproteinase activities. One of these, previously called B1, is a 97-kDa protein which is secreted in large amounts and has been purified from protein-free, conditioned medium. An antiserum to this enzyme was used to isolate a cDNA clone. The predicted protein sequence of B1 is 81% identical to human matrix metalloproteinase 9 (MMP9), demonstrating that it is the bovine homologue of this enzyme. RNAase protection assays demonstrated that the MMP9 activity, unique to infected cells, is due to increased MMP9 mRNA levels. We also assayed the levels of transcription factor AP-1 and demonstrated that it was constitutively present in increased amounts in Theileria-infected cells. In addition we assayed the level of mRNA encoding c-Fos, a common component of AP-1 and observed that it was indeed up-regulated in infected cells. Since AP-1 is implicated in the control of the cell cycle, and MMP9 can confer metastatic properties, these results are of considerable significance with respect to the transformed phenotype induced by Theileria infection. 


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Tyloxapol inhibits NF-kappa B and cytokine release, scavenges HOCI, and reduces viscosity of cystic fibrosis sputum. 
Cystic fibrosis (CF) patients develop progressive cytokine-mediated inflammatory lung disease, with abundant production of thick, tenacious, protease- and oxidant-rich purulent airway secretions that are difficult to clear even with physiotherapy. In the search for a potential treatment, we have tested tyloxapol, an alkylaryl polyether alcohol polymer detergent previously used as a mucolytic agent in adult chronic bronchitis. Tyloxapol inhibits activation of the transcription factor nuclear factor-kappa B (NK-kappa B), reduces resting secretion of the cytokine interleukin-8 (IL-8) in cultured human monocytes, and inhibits lipopolysaccharide (LPS)-stimulated release of tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and the eiconsanoids thromboxane A2 and leukotriene B4 (LTB4). We have previously shown that tyloxapol is a potent antioxidant for hydroxyl radicals ( OH). Tyloxapol (0.05 to 0.1% wt/vol) effectively scavenges the oxidant hypochlorous acid (HOCl; 1 to 7.5 mM) in vitro, and protects from HOCl-mediated lung injury in rats. Tyloxapol also reduces the viscosity of CF sputum (from 463 +/- 133 to 128 +/- 52 centipoise). We conclude that tyloxapol is potentially useful as a new antiinflammatory therapy for CF lung disease, and could possibly promote clearance of secretions in the CF airway. 


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The murine BCL6 gene is induced in activated lymphocytes as an immediate early gene. 
The chromosomal translocation involving 3q27 is often detected in human B-cell lymphomas, especially diffuse lymphomas with a large-cell component. The BCL6 gene has been isolated from the chromosomal breakpoint in these lymphomas. Here we cloned the murine BCL6 (mBCL6) cDNA from the muscle cDNA library using the human BCL6 (hBCL6) cDNA as a probe. The predicted amino acid sequence was 95% identical to that of hBCL6. It contains six repeats of the Kruppel-like zinc-finger motif that are completely identical to those of hBCL6, indicating that the BCL6 gene is well conserved between humans and mice. Expression of the mBCL6 gene was ubiquitously detected in adult mouse tissues including lymphatic organs. Furthermore, it was induced in lymphocytes activated with phorbol ester and Ca2+ ionophore within 30 min after stimulation. This induction was not inhibited by treatment of the cells with a protein synthesis inhibitor, cycloheximide. These results suggest that BCL6 plays a role in activated lymphocytes as an immediate early gene. 


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Differences in phosphorylation of the IL-2R associated JAK/STAT proteins between HTLV-I(+), IL-2-independent and IL-2-dependent cell lines and uncultured leukemic cells from patients with adult T-cell lymphoma/leukemia. 
To determine activation status of the IL-2R-associated (Jak/STAT) pathway in the HTLV-I infected cells, we examined tyrosine phosphorylation of Jak3, STAT3, and STAT5 in several HTLV-I(+) T-cell lines and in uncultured leukemic T cells isolated from patients with adult T-cell lymphoma/leukemia (ATLL). Constitutive basal phosphorylation of Jak3 and, usually, STAT3 and STAT5 was detected in all four IL-2-independent cell lines tested, but in none of the three IL-2-dependent cell lines. Similarly, there was no detectable basal phosphorylation of Jak3 and STAT5 in the leukemic cells from ATLL patients (0/8 and 0/3, respectively). However, stimulation with IL-2 resulted in Jak3 and STAT5 phosphorylation in both leukemic ATLL cells and IL-2-dependent lines. Furthermore, expression of SHP-1 phosphatase which is a negative regulator of cytokine receptor signaling, was lost in most IL-2 independent cell lines (3/4) but not in the leukemic ATLL cells (0/3). Finally, the HTLV-I(+) T-cell lines (313) but not the control, HTLV-I(-) T-cell lines were resistant to rapamycin and its novel analog RAD. We conclude that (1) HTLV-I infection per se does not result in a constitutive phosphorylation of the Jak3, STAT3, and STAT5 proteins; (2) malignant transformation in at least some cases of ATLL does not require the constitutive, but may require IL-2-induced, activation of the IL-2R Jak/STAT pathway; and (3) there are major differences in T-cell immortalization mechanism(s) which appear to involve SHP-1 and target molecules for rapamycin and RAD. 


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Glucocorticoid-induced cell death requires autoinduction of glucocorticoid receptor expression in human leukemic T cells. 
In contrast to the negative autoregulation of glucocorticoid receptor (GR) expression seen in most cells and tissues, GR expression is positively autoregulated in human leukemic T cells and in other cells sensitive to glucocorticoid-induced cell death. To determine whether positive autoregulation is a necessary component of glucocorticoid-induced cell death, a wild-type GR gene under the control of a tetracycline-regulated promoter was stably transfected into glucocorticoid-resistant cells lacking endogenous functional receptor. Transfectants grown in the presence of tetracycline contained about 15,000 receptors/cell, a value approximately equal to basal level GR expression in glucocorticoid-sensitive 6TG1.1 cells before steroid treatment. Under these conditions, dexamethasone had a minimal effect on cell growth, elicited little internucleosomal DNA fragmentation, and induced no cell cycle perturbation. In the absence of tetracycline, GR mRNA and protein expression increased 2-3-fold, and cells expressed 48,000 receptors, a level nearly equivalent to that present in 6TG1.1 cells after 18 h of autoinduction. Under these conditions, dexamethasone markedly inhibited cell growth, caused G1 arrest, and induced significant internucleosomal DNA fragmentation. These studies therefore suggest that basal level GR expression is inadequate to mediate glucocorticoid-induced apoptosis in glucocorticoid-sensitive T cells and that positive autoregulation is a necessary component of this process. 


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Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors: a possible target for v-erbA oncogene action. 
The v-erbA oncogene, a mutated version of the thyroid hormone receptor alpha (c-erbA/TR-alpha), inhibits erythroid differentiation and constitutively represses transcription of certain erythrocyte genes, suggesting a normal function of the proto-oncogene c-erbA in erythropoiesis. Here we demonstrate that the endogenous thyroid hormone receptor alpha (c-erbA/TR-alpha) and the closely related retinoic acid receptor alpha (RAR-alpha) play a role in the regulation of normal erythroid differentiation. Retinoic acid (RA) distinctly modulated the erythroid differentiation program of normal erythroid progenitors and erythroblasts reversibly transformed by a conditional tyrosine kinase oncogene. When added pulsewise to immature cells, differentiation was accelerated while more mature cells underwent premature cell death. Thyroid hormone (T3) alone caused similar but weaker effects. Interestingly, T3 strongly enhanced the action of RA, suggesting cooperative action of the two receptors in modulating erythroid differentiation. Expression of the human RAR-alpha in receptor-negative erythroblasts conferred RA-induced regulation of differentiation to the otherwise unresponsive cells, thus showing that the RAR-alpha is essential for the RA effect. Likewise, enhanced expression of exogenous c-erbA/TR-alpha in erythroblasts rendered them susceptible to modulation of differentiation by T3, suggesting a similar function of both receptors. 


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The internal methionine codons of human T-cell leukemia virus type II rex gene are not required for p24rex production or virus replication and transformation. 
Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) have two nonstructural trans-acting regulatory genes, tax and rex, located in the 3' region of the viral genome. The tax gene product (HTLV-I p40tax and HTLV-II p37tax) is the transcriptional activator of the viral long terminal repeat. The rex gene encodes two protein products, p27rex/p21rex and p26rex/p24rex in HTLV-I and HTLV-II, respectively. Rex acts posttranscriptionally to facilitate accumulation of full-length gag/pol and singly spliced env mRNA in the cytoplasm of HTLV-infected cells. Previous studies showed that the first ATG of the rex gene is critical for Rex production and function. The importance of the internal ATGs to Rex function is not known. However, in vitro mutagenesis of the HTLV-I rex gene has provided indirect evidence which suggests that p21rex, and by analogy HTLV-II p24rex, results from initiation at an internal AUG of the tax/rex mRNA. By using an infectious molecular clone of HTLV-II, we investigated the importance of the internal ATGs of the rex gene on Rex protein production and function. Our results indicate that p24rex of HTLV-II is not initiated at an internal AUG and that the internal methionine codons are not crucial to the function of the rex gene and, ultimately, the transforming properties of the virus. 


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[Induction of apoptosis in lymphocytes by glucocorticoids: between physiology and pharmacology] 
Glucocorticoids are physiological molecules that are also extensively used in clinics as anti-inflammatory, immunosuppressive or anti-tumoral agents. Glucocorticoids can induce apoptosis on normal lymphoid cells and play a key role in the physiology of thymic selection. In clinics these molecules are also used for their potencies in inducing apoptosis of malignant lymphoid cells. Glucocorticoids are mediating their effects after binding to an intracellular receptor belonging to the steroid receptor superfamily: the glucocorticoid receptor (GR). Once activated, the GR, can mediate his effects through direct binding on the DNA or via protein/protein interactions with transcription factors. Depending on the type of lymphocytes, the mechanism of apoptosis induced by glucocorticoids fall roughly in two categories: induction of "death genes" by the activated GR (I kappa B, c-jun) or repression of survival factors (AP-1, c-Myc). In the case of thymic selection the mechanism is more subtle depending on the mutual repression of Nur77 and GR. 


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Phosphorylation of the transcription factor NFATp inhibits its DNA binding activity in cyclosporin A-treated human B and T cells. 
Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding. 


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A mutation of the glucocorticoid receptor in primary cortisol resistance. 
The precise molecular abnormalities that cause primary cortisol resistance have not been completely described. In a subject with primary cortisol resistance we have observed glucocorticoid receptors (hGR) with a decreased affinity for dexamethasone. We hypothesize that a mutation of the hGR glucocorticoid-binding domain is the cause of cortisol resistance. Total RNA isolated from the index subject's mononuclear leukocytes was used to produce first strand hGR cDNAs, and the entire hGR cDNA was amplified in segments and sequenced. At nucleotide 2,317 we identified a homozygous A for G point mutation that predicts an isoleucine (ATT) for valine (GTT) substitution at amino acid 729. When the wild-type hGR and hGR-Ile 729 were expressed in COS-1 cells and assayed for [3H]-Dexamethasone binding, the dissociation constants were 0.799 +/- 0.068 and 1.54 +/- 0.06 nM (mean +/- SEM) (P < 0.01), respectively. When the wild-type hGR and hGR-Ile 729 were expressed in CV-1 cells that were cotransfected with the mouse mammary tumor virus long terminal repeat fused to the chloramphenicol acetyl transferase (CAT) gene, the hGR-Ile 729 conferred a fourfold decrease in apparent potency on dexamethasone stimulation of CAT activity. The isoleucine for valine substitution at amino acid 729 impairs the function of the hGR and is the likely cause of primary cortisol resistance in this subject. 


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Expression and role of PML gene in normal adult hematopoiesis: functional interaction between PML and Rb proteins in erythropoiesis. 
The expression of the PML gene was investigated in purified early hematopoietic progenitor cells (HPCs) induced to unilineage erythroid or granulocytic differentiation. PML mRNA and protein, while barely detectable in quiescent HPCs, are consistently induced by growth factor stimulation through the erythroid or granulocytic lineage. Thereafter, PML is downmodulated in late granulocytic maturation, whereas it is sustainably expressed through the erythroid pathway. In functional studies, PML expression was inhibited by addition of antisense oligomers targeting PML mRNA (alpha-PML). Interestingly, early treatment (day 0 HPCs) with alpha-PML reduced the number of both erythroid and granulocytic colonies, whereas late treatment (day 5 culture) reduced erythroid, but not granulocytic, clonogenesis. These findings suggest that PML is required for early hematopoiesis and erythroid, but not granulocytic maturation. The pattern of PML expression in normal hematopoiesis mimics that of retinoblastoma pRb 105. Combined treatment of HPCs with alpha-PML and alpha-Rb oligomers inhibited both PML and Rb protein expression and completely blocked erythroid colony development. Furthermore, PML and pRb 105 were co-immunoprecipitated in cellular lysates derived from erythroid precursors indicating that this functional interaction may have a biochemical basis. These results suggest a key functional role of PML in early hematopoiesis and late erythropoiesis: the latter phenomenon may be related to the molecular and functional interaction of PML with pRb 105. 


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Glucocorticoid receptors in anorexia nervosa and Cushing's disease. 
BACKGROUND: Patients with anorexia nervosa do not display cushingoid features in spite of elevated cortisol plasma levels. Whether a cortisol resistance or a reduced availability of the metabolic substrates necessary to develop the effect of glucocorticoids is responsible for this has not been established. METHODS: Twenty-two patients with severe restrictive anorexia nervosa, 10 patients with active Cushing's disease, and 24 healthy volunteers without psychiatric disorders or mood alterations were investigated. Glucocorticoid receptor characteristics were examined on mononuclear leukocytes by measuring [3H]dexamethasone binding and the effect of dexamethasone on [3H]thymidine incorporation, which represents an index of DNA synthesis. RESULTS: The number of glucocorticoid receptors on mononuclear leukocytes (MNL) was comparable in patients with anorexia nervosa, patients with active Cushing's disease, and normal subjects (binding capacity 3.3 +/- 0.23 vs. 3.7 +/- 0.30 and 3.5 +/- 0.20 fmol/10(6) cells). Conversely, glucocorticoid receptor affinity was significantly decreased in anorexia nervosa as well as in Cushing's patients compared to control subjects (dissociation constant 4.0 +/- 0.31 and 4.1 +/- 0.34 vs. 2.9 +/- 0.29 nmol/L, p < .001) and inversely correlated with the levels of urinary free cortisol in both groups of patients. Basal [3H]thymidine incorporation in MNL was significantly reduced in anorexia nervosa as well as in Cushing's patients compared to control subjects (p < .001) and was diminished by dexamethasone to an extent similar to control subjects in patients with anorexia nervosa, but significantly (p < .001) less in those with Cushing's disease. In patients with anorexia nervosa, the incorporation of [3H]thymidine into the MNL was inversely correlated with urinary free cortisol levels. CONCLUSIONS: These data indicate that the lack of cushingoid features in patients with anorexia nervosa is not ascribable to a reduced sensitivity to glucocorticoids but is more likely due to the paucity of metabolic substrates. 


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Genes that regulate interleukin-4 expression in T cells. 
Interleukin-4 is an immunomodulatory cytokine which plays a central role in the regulation of allergic and atopic immune responses. Significant progress has been made in gaining a detailed understanding of the transcriptional regulation of the interleukin-4 gene. The recent identification and characterization of several key transcription factors has helped to elucidate the molecular mechanisms of T helper cell cytokine gene expression. 


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Extracellular signal-regulated protein kinase (ERK)-dependent and ERK-independent pathways target STAT3 on serine-727 in human neutrophils stimulated by chemotactic factors and cytokines. 
STAT3 (signal transducer and activator of transcription 3) is a latent transcription factor that is activated by tyrosine phosphorylation (Tyr-705) in cells stimulated with cytokines or growth factors. Recent studies suggest that one or more cytoplasmic serine kinases also phosphorylate STAT3 and are necessary for maximal gene activation. Here we demonstrate, with a site-specific antibody, that STAT3 is phosphorylated on Ser-727 in human neutrophils stimulated with chemotactic factors (N-formyl-methionyl-leucyl-phenylalanine and complement C5a), cytokines [granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF)], or a protein kinase C activator (PMA). (2-Amino-3'-methoxyphenyl)oxanaphthalen-4-one (PD 98059), an inhibitor of extracellular signal-regulated protein kinase (ERK) activation, blocked the serine phosphorylation of STAT3 induced by chemotactic factors or PMA. The drug was less effective on cytokines: it virtually abolished the response to GM-CSF that occurred 5 min after stimulation but only partly decreased those at 15-30 min and did not appreciably alter responses to G-CSF regardless of incubation time. 1-(5-Isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride (H7), an inhibitor of a putative STAT3 serine kinase, and 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) 1H-imidazole (SB 203580), an inhibitor of p38 mitogen-activated protein (MAP) kinase, did not dampen any of these serine phosphorylation responses. We propose that neutrophils use both ERK-dependent and ERK-independent pathways to phosphorylate Ser-727 on STAT3. The former pathway is recruited by all ERK-activating stimuli, whereas the latter pathway uses an undefined serine kinase and is recruited selectively by cytokines. 


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Effects of IL-10 and IL-4 on LPS-induced transcription factors (AP-1, NF-IL6 and NF-kappa B) which are involved in IL-6 regulation. 
Interleukin-10 (IL-10), like IL-4, is known to inhibit cytokine expression in activated human monocytes. We showed that both IL-10 and IL-4 inhibit LPS-induced IL-6 mRNA and protein expression by inhibiting the transcription rate of the IL-6 gene. The strong inhibition of the IL-6 transcription rate prompted us to study the effect of IL-10 and IL-4 on the expression of transcription factors. We questioned whether or not IL-10 and IL-4 affected the expression of transcription factors that are known to be involved in the control of the IL-6 transcription rate, namely activator protein-1 (AP-1), nuclear factor IL-6 (NF-IL6), and nuclear factor kappa B (NF-kappaB). In electrophoretic mobility shift assays (EMSAs) we showed that IL-10 and IL-4 inhibited LPS-induced AP-1 binding activity. The inhibiting effect of IL-4 was slightly more pronounced than that of IL-10. Downregulation of LPS-induced AP-1 was accompanied, and thus possibly explained, by a reduced expression at mRNA level of the two major components of the AP-1 complex, namely c-fos and c-jun as determined by Northern experiments. Binding activity of NF-IL6 was also strongly inhibited by IL-4 whereas IL-10 showed no effect. NF-IL6 mRNA levels were not affected by IL-10 or IL-4, suggesting that IL-4 affects binding activity of preexisting NF-IL6. Neither IL-10 nor IL-4 inhibited LPS-induced NF-kappa B binding activity. In agreement with this finding, Northern experiments where p65 and p105 mRNA levels were determined, demonstrated that expression of these components of the NF-kappa B transcription factor were not affected by IL-10 or IL-4. Furthermore, neither IL-10 nor IL-4 showed any effect on I-kappa B mRNA expression as determined by Northern experiments. Thus, IL-10 and IL-4 similarly affect IL-6 expression. However, for IL-4 this was accompanied with a reduction of AP-1 and NF-IL6 binding activity whereas IL-10 only inhibited AP-1 binding activity. 


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Thyroid hormone receptors form distinct nuclear protein-dependent and independent complexes with a thyroid hormone response element. 
We have examined the binding of nuclear proteins and recombinant thyroid hormone receptors (TRs) to the palindromic thyroid hormone responsive element AGGTCATGACCT (TREp) using a gel electrophoretic mobility shift assay. Four specific protein-DNA complexes were detected after incubation of nuclear extracts (NE) from T3-responsive pituitary (GH3) cells with a TREp-containing DNA fragment. This was compared with the TREp binding of reticulocyte lysate-synthesized TRs. TR alpha 1 and TR beta 2 each formed a single major TR:TREp complex which comigrated with the least retarded complex formed by GH3 NE, while TR beta 1 formed multiple complexes suggesting that it can bind to TREp as an oligomer. Interestingly, coincubation of 35S-TR alpha 1, GH3 NE, and unlabeled TREp resulted in not only the 35S-TR:TREp complex, but in two additional more greatly retarded complexes containing 35S-TR alpha 1 and comigrating with those formed by GH3 extract alone. Incubation of each of the TRs with NE from COS-7 cells, which do not possess sufficient endogenous TRs to mediate T3-responses, resulted in formation of a new, more greatly shifted complex. A similar, heat labile activity which altered mobility of the TR:TRE complex was also present in NE from T3-unresponsive JEG-3 cells. At high concentration of NE, all of the TR bound to TREp was more greatly retarded than in the absence of NE. Truncation of TR alpha 1 at amino acid 210 prevented additional complex formation in the presence of NE without affecting DNA binding, suggesting that the carboxyl-terminus of the TRs is essential for interaction with nuclear proteins. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Megakaryocytic and erythrocytic lineages share specific transcription factors. 
Erythroid-specific genes contain binding sites for NF-E1 (also called GF-1 and Eryf-1; refs 1-3 respectively), the principal DNA-binding protein of the erythrocytic lineage. NF-E1 expression seems to be restricted to the erythrocytic lineage. A closely related (if not identical) protein is found in both a human megakaryocytic cell line and purified human megakaryocytes; it binds to promoter regions of two megakaryocytic-specific genes. The binding sites and partial proteolysis profile of this protein are indistinguishable from those of the erythroid protein; also, NF-E1 messenger RNA is the same size in both the megakaryocytic and erythroid cell lines. Furthermore, point mutations that abolish binding of NF-E1 result in a 70% decrease in the transcriptional activity of a megakaryocytic-specific promoter. We also find that NF-E2, another trans-acting factor of the erythrocytic lineage, is present in megakaryocytes. Transcriptional effects in both lineages might then be mediated in part by the same specific trans-acting factors. Our data strengthen the idea of a close association between the erythrocytic and the megakaryocytic lineages and could also explain the expression of markers specific to the erythrocytic and megakaryocytic lineages in most erythroblastic and megakaryoblastic permanent cell lines. 


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Tumor necrosis factor alpha decreases, and interleukin-10 increases, the sensitivity of human monocytes to dexamethasone: potential regulation of the glucocorticoid receptor. 
Resistance to glucocorticoid therapy has been observed in patients with autoimmune/inflammatory diseases and may be related to the inflammatory process itself. The aim of this study was to examine the ability of tumor necrosis factor alpha (TNFalpha, a proinflammatory cytokine) and interleukin (IL)-10 (an anti-inflammatory cytokine) to differentially regulate the sensitivity of human monocytes/macrophages to glucocorticoids. To accomplish this, we first analyzed the pattern of TNFalpha and IL-10 inhibition by dexamethasone in LPS-stimulated whole-blood cell cultures. Second, we studied the modulation of the sensitivity of these cells to dexamethasone by preincubation with TNFalpha or IL-10 and measurement of LPS-stimulated IL-6 secretion. In addition, we evaluated the effect of dexamethasone on phorbolmyristate-acetate-stimulated IL-1 receptor antagonist secretion by the human monocytic cell line U937. Finally, we investigated whether the modulation of corticosensitivity in TNFalpha- and IL-10-pretreated U937 cells was related to a change of the glucocorticoid receptor concentration and affinity. Dexamethasone had different effects on LPS-induced TNFalpha and IL-10 secretion; whereas it suppressed TNFalpha in a dose-dependent fashion, its effect on IL-10 secretion was biphasic, producing stimulation at lower, and inhibition at higher doses. The concentration of LPS employed influenced the effect of dexamethasone on IL-10 secretion (P < 0.001). Pretreatment with TNFalpha diminished, and with IL-10 improved, the ability of dexamethasone to suppress IL-6 secretion in whole-blood cell cultures (P < 0.01 for both) and to enhance IL-1 receptor antagonist secretion by U937 cells (P < 0.05 for both). TNFalpha decreased (P < 0.001), while IL-10 increased (P < 0.001), the concentration of dexamethasone binding sites in these cells, with no discernible effect on their binding affinity. We conclude that glucocorticoids differentially modulate TNFalpha and IL-10 secretion by human monocytes in a LPS dose-dependent fashion and that the sensitivity of these cells to glucocorticoids is altered by TNFalpha or IL-10 pretreatment; TNFalpha blocks their effects, whereas IL-10 acts synergistically with glucocorticoids. This is accompanied by opposite glucocorticoid receptor changes, respectively opposing and favoring glucocorticoid actions. This study suggests that the pattern of pro-/antiinflammatory cytokine secretion may alter the response of patients to glucocorticoid therapy. 


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CD14-mediated signal pathway of Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts. 
Lipopolysaccharide (LPS) induces expression of inflammatory cytokines in monocytes/macrophages via CD14, one of the LPS receptors, which is expressed predominantly in these cells. It has been demonstrated that Porphyromonas gingivalis LPS (P-LPS) also is able to induce inflammatory cytokines in human gingival fibroblasts. Therefore, it is important to determine whether CD14 is expressed in gingival fibroblasts and to define the P-LPS-mediated signal-transducing mechanism in the cells. In this study, we observed unexpectedly by immunohistochemical, Western blotting (immunoblotting), and Northern (RNA) blotting assays that CD14 is expressed at high density in human gingival fibroblasts. P-LPS-induced expression of the monocyte chemoattractant protein 1 (MCP-1) gene in the cells was inhibited markedly by treatment with anti-human CD14 antibody and was completely inhibited by herbimycin A, a potent inhibitor of tyrosine kinase. The inhibitor also dramatically inhibited monocyte chemotactic activity of and MCP-1 production by the cells. Furthermore, P-LPS-induced expression of the MCP-1 gene in the cells also was blocked by inhibitors of two transcription factors, i.e., curcumin, an inhibitor of AP-1, and pyrolidine dithiocarbamate, an inhibitor of NF-kappaB. Both inhibitors inhibited monocyte chemotactic activity in the culture supernatant of P-LPS-treated cells. Gel shift mobility assay showed stimulation of the AP-1 and NF-kappaB contents in P-LPS-treated cells. This study is the first to demonstrate the expression of CD14 in human gingival fibroblasts and to show that the signal-transducing pathway of P-LPS in the cells is mediated by CD14. 


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UV-induced CYP1A1 gene expression in human cells is mediated by tryptophan. 
Induction of cytochrome P-4501A1 (CYP1A1) activity by UV has been observed earlier in animal studies via a mechanism that has not yet been resolved. Our previous data have indicated that formylated indolocarbazoles which are formed by UV irradiation of tryptophan solutions are very potent Ah-receptor agonists. To evaluate the effect of UV light on cytochrome P4501A1 gene expression, we studied the induction of CYP1A1 mRNA by UV irradiation of cultured human keratinocytes (HaCaT cell line), primary human blood lymphocytes and mouse Hepa-1 cells. The cells were exposed to UV light delivered by a bank of 6 Philips TL20/12RS sun lamps emitting primarily in the UVB range in the absence and presence of tryptophan. A semiquantitative reverse transcriptase-linked polymerase chain reaction (RT-PCR) was used for analysis of gene expression in the treated cells. The results show that the CYP1A1 mRNA level induced by UV in the presence of tryptophan was higher than that induced by UV alone in both HaCaT cells and lymphocytes after 3 h of incubation post-UV irradiation. To find out if the induction by UV light is caused by the formation of an Ah receptor ligand, Hepa-1 wild-type and Ah receptor deficient c12 cell lines were applied. Wild-type (wt) cells were inducible either by the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) or by UV-irradiation but very low or undetectable levels were observed in the c12 cells. This shows that the induction of gene expression by FICZ and UV is Ah receptor dependent. Together, these results indicate that UV-induced CYP1A1 gene expression in mammalian cells is mediated by an Ah receptor ligand formed from tryptophan. Thus, the photoproducts of tryptophan are suggested to be mediators of light via binding to the Ah receptor and as such also could have a role in light-regulated biological rhythms. 


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The Ah receptor recognizes DNA binding sites for the B cell transcription factor, BSAP: a possible mechanism for dioxin-mediated alteration of CD19 gene expression in human B lymphocytes. 
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits murine and human B lymphocyte immunoglobulin production through an unknown mechanism. This study investigated the effect of TCDD on expression of the CD19 gene in a human B lymphocyte cell line. Northern blot analysis showed that TCDD treatment decreased steady state levels of CD19 mRNA by 67% in the IM-9 cell line. Using a gel mobility shift assay, we identified a DNA-binding complex in IM-9 nuclear extracts that by several criteria appears to be the Ah receptor. In addition, the Ah receptor complex recognized a DNA binding site for B cell lineage-specific activator protein (BSAP) in the promoter region of the human CD19 gene which is similar to the consensus Ah receptor DNA binding site. These results suggest that the AhR could interfere with BSAP-stimulated CD19 gene transcription by competition for a common DNA binding site. 


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Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations [see comments] 
BACKGROUND: Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia via induction of differentiation and programmed cell death (apoptosis). We investigated the effects of As2O3 on a panel of malignant lymphocytes to determine whether growth-inhibitory and apoptotic effects of As2O3 can be observed in these cells at clinically achievable concentrations. METHODS: Eight malignant lymphocytic cell lines and primary cultures of lymphocytic leukemia and lymphoma cells were treated with As2O3, with or without dithiothreitol (DTT) or buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis). Apoptosis was assessed by cell morphology, flow cytometry, annexin V protein level, and terminal deoxynucleotidyl transferase labeling of DNA fragments. Cellular proliferation was determined by 5-bromo-2'-deoxyuridine incorporation into DNA and flow cytometry and by use of a mitotic arrest assay. Mitochondrial transmembrane potential (delta psi(m)) was measured by means of rhodamine 123 staining and flow cytometry. Protein expression was assessed by western blot analysis or immunofluorescence. RESULTS: Therapeutic concentrations of As2O3 (1-2 microM) had dual effects on malignant lymphocytes: 1) inhibition of growth through adenosine triphosphate (ATP) depletion and prolongation of cell cycle time and 2) induction of apoptosis. As2O3-induced apoptosis was preceded by delta psi(m) collapse. DTT antagonized and BSO enhanced As2O3-induced ATP depletion, delta psi(m) collapse, and apoptosis. Caspase-3 activation, usually resulting from delta psi(m) collapse, was not always associated with As2O3-induced apoptosis. As2O3 induced PML (promyelocytic leukemia) protein degradation but did not modulate expression of cell cycle-related proteins, including c-myc, retinoblastoma protein, cyclin-dependent kinase 4, cyclin D1, and p53, or expression of differentiation-related antigens. CONCLUSIONS: Substantial growth inhibition and apoptosis without evidence of differentiation were induced in most malignant lymphocytic cells treated with 1-2 microM As2O3. As2O3 may prove useful in the treatment of malignant lymphoproliferative disorders. 


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The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii. 
To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoS mutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in the rpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS in L. pneumophila is very different from what has previously been reported for E. coli rpoS. 


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Modulation of CD28 expression: distinct regulatory pathways during activation and replicative senescence. 
The costimulatory molecule CD28 has a restricted tissue distribution and is expressed on T cells and some plasmacytoma cells. Although CD28 is constitutively expressed, its expression is transiently down-regulated following T cell activation and declines progressively with in vitro senescence. In vivo, CD8+ T cells and, less frequently, CD4+ T cells may completely lose CD28 surface expression during chronic infections and with aging. This correlates with changes of nuclear protein-binding activities to two motifs, site alpha and beta, within the CD28 minimal promoter. Both alpha- and beta-bound complexes are found only in lymphoid tissues, in CD28+ T cells, and in some transformed B cells. These complexes are coordinately expressed except during replicative senescence, which is characterized by the down-modulation of site beta- but not site alpha-binding activities. In contrast, T cell activation induces a parallel decline in both site alpha- and beta-binding activities. CD4+ and CD8+ T cells differ in their beta-binding profiles, which may explain the more pronounced down-regulation of CD28 in senescent CD8+ T cells. In vivo expanded CD4+CD28null and CD8+CD28null T cells uniformly lack alpha- and beta- bound complexes, resembling the pattern seen in chronically activated cells and not of senescent cells. 


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The DNA-binding properties of two heat shock factors, HSF1 and HSF3, are induced in the avian erythroblast cell line HD6. 
Avian cells express three heat shock transcription factor (HSF) genes corresponding to a novel factor, HSF3, and homologs of mouse and human HSF1 and HSF2. Analysis of the biochemical and cell biological properties of these HSFs reveals that HSF3 has properties in common with both HSF1 and HSF2 and yet has features which are distinct from both. HSF3 is constitutively expressed in the erythroblast cell line HD6, the lymphoblast cell line MSB, and embryo fibroblasts, and yet its DNA-binding activity is induced only upon exposure of HD6 cells to heat shock. Acquisition of HSF3 DNA-binding activity in HD6 cells is accompanied by oligomerization from a non-DNA-binding dimer to a DNA-binding trimer, whereas the effect of heat shock on HSF1 is oligomerization of an inert monomer to a DNA-binding trimer. Induction of HSF3 DNA-binding activity is delayed compared with that of HSF1. As occurs for HSF1, heat shock leads to the translocation of HSF3 to the nucleus. HSF exhibits the properties of a transcriptional activator, as judged from the stimulatory activity of transiently overexpressed HSF3 measured by using a heat shock element-containing reporter construct and as independently assayed by the activity of a chimeric GAL4-HSF3 protein on a GAL4 reporter construct. These results reveal that HSF3 is negatively regulated in avian cells and acquires DNA-binding activity in certain cells upon heat shock. 


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Increased glucocorticoid receptor beta in airway cells of glucocorticoid-insensitive asthma. 
Glucocorticoid (GC)-insensitive asthma is a challenging clinical problem that can be associated with life-threatening disease progression. The molecular basis of GC insensitivity is unknown. Alternative splicing of the GC receptor (GCR) pre-mRNA generates a second GCR, termed GCRbeta, which does not bind GC but antagonizes the transactivating activity of the classic GCR. Thus increased expression of GCRbeta could account for glucocorticoid insensitivity. Bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMC) were examined for GCRbeta immunoreactivity using a GCRbeta-specific antibody by immunohistochemical staining. Cell localization of GCRbeta expression was performed using a double immunostaining technique. Patients with GC-insensitive asthma expressed a significantly higher number of GCRbeta-immunoreactive cells in their BAL and peripheral blood than GC-sensitive asthmatics or normal control subjects. Furthermore, GCRbeta expression in GC-insensitive asthma was particularly high in airway T cells, which are thought to play a major role in the pathogenesis of asthma. We also examined the expression of GCRbeta in specimens from the airways of patients with chronic bronchitis. In chronic bronchitis, few cells were GCRbeta-positive and their numbers did not differ significantly from normal control subjects. We conclude that GC-insensitive asthma is associated with increased expression of GCRbeta in airway T cells. 


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Tyrphostin AG-490 inhibits cytokine-mediated JAK3/STAT5a/b signal transduction and cellular proliferation of antigen-activated human T cells. 
Janus kinase 3 (JAK3) is a cytoplasmic tyrosine kinase required for T cell development and activated by cytokines that utilize the interleukin-2 (IL-2) receptor common gamma chain (gamma(c)). Genetic inactivation of JAK3 is manifested as severe combined immunodeficiency disease (SCID) in humans and mice. These findings have suggested that JAK3 represents a pharmacological target to control certain lymphoid-derived diseases. Here we provide novel evidence that AG-490 potently inhibits the autokinase activity of JAK3 and tyrosine phosphorylation and DNA binding of signal transducer and activator of transcription 5a and 5b (STAT5a/b). Similar inhibitory effects were observed with other cytokines that use gamma(c). AG-490 also inhibited IL-2-mediated proliferative growth in human T cells with an IC50) = 25 microM that was partially recoverable. Moreover, we demonstrate that this inhibitor prevented tetanus toxoid antigen-specific T cell proliferation and expansion but failed to block activation of Zap70 or p56Lck after anti-CD3 stimulation of human T cells. Taken together, these findings suggest that AG-490 inhibits the JAK3-mediated Type II signaling pathway but not the T cell receptor-derived Type I pathway and possesses therapeutic potential for T cell-derived pathologies such as graft-versus-host disease, allergy, and autoimmune disorders. 


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Inhibition of T cell activation by the extracellular matrix protein tenascin. 
Tenascin (TN) is an extracellular matrix protein that is expressed widely in the fetus and sparingly in the adult, but reappears at high levels in certain areas of tissue insult such as tumor matrices and sites of wound healing. We show here that soluble TN inhibits proliferation of human T cells in response to alpha CD3 Ab co-immobilized with the extracellular matrix protein fibronectin (FN). TN also inhibits proliferation driven by alpha CD3/IL-2 or by phorbol ester/IL-2, and it prevents high level induction of IL-2R. The presence of TN in culture medium does not detectably alter the pattern of tyrosine phosphorylation resulting from T cell triggering with alpha CD3, but at later time points prevents the appearance of functional NF-AT1 transcription factor complexes in T cell nuclear extracts. These findings are consistent with the postulated role for TN as a natural antagonist to FN action, and suggest that T cell responses occurring at tissue sites in which TN is expressed could be influenced by its presence. 


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CBP/p300 integrates Raf/Rac-signaling pathways in the transcriptional induction of NF-ATc during T cell activation. 
NF-ATc, an inducibly expressed transcription factor, controls gene expression in T lymphocytes and cardiomyocytes. We show here that the transcriptional co-activators CBP/p300 bind to and control the activity of the inducible N-terminal transactivation domain of NF-ATc, TAD-A. Similar to the N terminal transactivation domain of c-Jun, TAD-A is inducibly phosphorylated, but this phosphorylation is dispensable for the interaction with CBP/p300. Constitutive active versions of c-Raf and Rac synergistically enhance the CBP/p300-mediated increase of TAD-A activity, indicating the important role CBP/p300 plays in the integration of T cell activation signals. Since a mutation of CBP abolishing HAT activity is almost as active as wild-type CBP in T cells, functions of CBP/p300 other than histone acetylation appear to control the NF-AT-dependent transcription in T cells. 


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Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins. 
The survival-promoting activity of the Bcl-2 family of proteins appears to be modulated by interactions between various cellular proteins. We have identified a novel cellular protein, Bik, that interacts with the cellular survival-promoting proteins, Bcl-2 and Bcl-xL, as well as the viral survival-promoting proteins, Epstein Barr virus-BHRF1 and adenovirus E1B-19 kDa. In transient transfection assays, Bik promotes cell death in a manner similar to the death-promoting members of the Bcl-2 family, Bax and Bak. This death-promoting activity of Bik can be suppressed by coexpression of Bcl-2, Bcl-XL, EBV-BHRF1 and E1B-19 kDa proteins suggesting that Bik may be a common target for both cellular and viral anti-apoptotic proteins. While Bik does not show overt homology to the BH1 and BH2 conserved domains characteristic of the Bcl-2 family, it does share a 9 amino acid domain (BH3) with Bax and Bak which may be a critical determinant for the death-promoting activity of these proteins. 


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Cell type- and stage-specific expression of the CD20/B1 antigen correlates with the activity of a diverged octamer DNA motif present in its promoter. 
The CD20(B1) gene encodes a B cell-specific protein involved in the regulation of human B cell proliferation and differentiation. Studies with 5' deletion CD20 promoter-CAT constructs have previously revealed two regions of the promoter between bases -186 and -280 and between bases -280 and -454 which contained positive regulatory elements. In this study we identified a sequence element present in the most proximal region located between bases -214 and -201, TTCTTCTAATTAA, which is important in the high constitutive expression of CD20 in mature B cells and the induction of CD20 in pre-B cells. This sequence element was referred to as the BAT box and its deletion significantly reduced the activity of a CD20 promoter-CAT construct in B cells. Mobility shift assays with various mutant probes and B cell nuclear extracts demonstrated that the core sequence TAAT was essential for binding to this site. Cross competition experiments with an octamer sequence from the Ig heavy chain promoter, the BAT box, and a TA-rich sequence present in the CD21 promoter revealed that all three sequences bound the same nuclear proteins suggesting that the BAT box binding proteins were Oct-1 and Oct-2. Southwestern blotting and UV cross-linking studies confirmed that the BAT box binding proteins were Oct-1 and Oct-2. The affinity of the BAT box binding proteins for the BAT box was approximately 25-fold less than for the octamer sequence and the BAT box binding proteins dissociated from the BAT box 10-fold more rapidly than from the octamer sequence. Despite this lower affinity, a trimer of the BAT box sequence was as efficiently transactivated by an Oct-2 expression vector as was a trimer of the octamer sequence in HeLa cells. The BAT box and Oct-2 were also implicated in the induction of CD20 in the pre-B cell line, PB-697, via phorbol esters. The induction of CD20 mRNA was temporally associated with induction of Oct-2 mRNA and a BAT box-deleted CD20-CAT construct, in contrast to the wild type, was poorly induced by phorbol esters. Together these results suggest that the BAT box binding proteins are important in the B cell specific expression of CD20 and perhaps CD21. 


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Retinoic acid-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid receptor-alpha. 
CD38 is a leukocyte differentiation antigen that has been thought to be a phenotypic marker of different subpopulations of T- and B- lymphocytes. In myeloid cells, CD38 is expressed during early stages of differentiation. Virtually no information is available on regulation and functions of CD38. Recently we reported that all-trans-retinoic acid (ATRA) is a potent and highly specific inducer of CD38 expression in human promyelocytic leukemia cells. Here we report that ATRA-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid-alpha receptor (RAR alpha). ATRA failed to induce CD38 expression in a mutant subclone of the HL-60 myeloid leukemia cell line (designated HL-60R) that is relatively resistant to ATRA-induced granulocytic differentiation. Retroviral vector-mediated transduction of RA receptor (RAR alpha) into this HL-60R subclone completely restored the sensitivity of these cells to ATRA in terms of their ability to express CD38. In contrast, CD38 expression was not inducible by ATRA in HL-60R cells, transfected with a functional RAR beta, RAR gamma, or RXR alpha receptor. Induction of CD38 in acute promyelocytic and acute myeloblastic leukemia cells was independent of ATRA-induced cytodifferentiation. Following culture with ATRA, increased CD38 protein levels were also observed in normal CD34+ bone marrow cells, but not on normal circulating granulocytes. From these results, we conclude that CD38 is ATRA inducible in myeloid leukemia cells and normal CD34+ bone marrow cells. This effect is independent of differentiation and is mediated by RAR alpha in HL-60 cells, suggesting a similar role for RAR alpha in CD38 expression in other hematopoietic cells. 


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Altered memory T cell differentiation in patients with early rheumatoid arthritis. 
The chronic immune response in rheumatoid arthritis (RA) might be driven by activated Th1 cells without sufficient Th2 cell differentiation to down-modulate inflammation. To test whether disordered memory T cell differentiation contributes to the typical Th1-dominated chronic inflammation in RA we investigated differentiation of resting CD4+ memory T cells in patients with early (6 wk to 12 mo) untreated RA and in age- and sex-matched healthy controls in vitro. No difference in cytokine secretion profiles of freshly isolated memory T cells was detected between patients and controls. A cell culture system was then employed that permitted the differentiation of Th effectors from resting memory T cells by short term priming. Marked differences were found in response to priming. Th2 cells could be induced in all healthy controls by priming with anti-CD28 in the absence of TCR ligation. By contrast, priming under those conditions resulted in Th2 differentiation in only 9 of 24 RA patients. Exogenous IL-4 could overcome the apparent Th2 differentiation defect in seven patients but was without effect in the remaining eight patients. In all patients a marked decrease in IL-2-producing cells and a significant increase in well-differentiated Th1 cells that produced IFN-gamma but not IL-2 were evident after priming with anti-CD3 and anti-CD28. The data suggest that CD4+ memory T cells from patients with early untreated RA manifest an intrinsic abnormality in their ability to differentiate into specific cytokine-producing effector cells that might contribute to the characteristic Th1-dominated chronic (auto)immune inflammation in RA. 


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c-Myc and E1A induced cellular sensitivity to activated NK cells involves cytotoxic granules as death effectors. 
The contact of natural killer (NK) cells with foreign cells and with certain virus-infected or tumor cells triggers the cytolytic machinery of NK cells. This triggering leads to exocytosis of the cytotoxic NK cell granules. The oncoproteins c-Myc and E1A render cells vulnerable to NK cell mediated cytolysis yet the mechanisms of sensitization are not well understood. In a model where foreign cells (rat fibroblasts) were cocultured with human IL-2 activated NK cells, we observed that NK cells were capable of efficiently killing their targets only if the cells overexpressed the oncogene c-Myc or E1A. Both the parental and the oncogene expressing fibroblasts similarly triggered phosphoinositide hydrolysis in the bound NK cells, demonstrating that NK cells were cytolytically activated in contact with both resistant parental and oncogene expressing sensitive target fibroblasts. The cell death was independent of wild-type p53 and was not inhibited by an anti-apoptotic protein EIB19K. These results provided evidence that c-Myc and E1A activated the NK cell induced cytolysis at a post-triggering stage of NK cell-target cell interaction. In consistence, the c-Myc and E1A overexpressing fibroblasts were more sensitive to the cytolytic effects of isolated NK cell-derived granules than parental cells. The data indicate that oncogenes activate the cytotoxicity of NK cell granules. This mechanism can have a role in directing the cytolytic action of NK cells towards the virus-infected and cancer cells. 


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Lymphocyte glucocorticoid receptor: predictor of sertraline response in adolescent major depressive disorder (MDD). 
Major depressive disorder (MDD) in adolescents demonstrates resistance to tricyclic antidepressants and absence of hypercortisolemia. The efficacy of serotonin reuptake inhibitors (SRIs) is uncertain, and response predictors are unavailable. Abnormal fast feedback and negative feedback of the hypothalamic-pituitary-adrenal axis implicates a dampened limbic-hippocampal glucocorticoid type II receptor (GCII). We hypothesized that lymphocyte GCII is altered in adolescent MDD and could serve as a marker for response to SRIs. In an open-label study, adolescents (n = 20) meeting DSM-III-R criteria for MDD showed baseline lymphocyte GCII sites per cell (sites/cell) values of 793 +/- 106 versus 2,563 +/- 499 (+/- SEM) for matched controls (n = 18) (t = 3.5; df = 36; p < .001). GCII was bimodally distributed, with SRI responders differing from nonresponders (t = 3.9; df = 14; p < .001). GCII accurately classified 90 percent of sertraline responders and 80 percent of nonresponders. Only SRI responders showed GCII sites/cell upregulated after 6 weeks of treatment (t = 2.1, df = 10; p < .05). 


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Transcriptional activity of core binding factor-alpha (AML1) and beta subunits on murine leukemia virus enhancer cores. 
Core binding factor (CBF), also known as polyomavirus enhancer-binding protein 2 and SL3 enhancer factor 1, is a mammalian transcription factor that binds to an element termed the core within the enhancers of the murine leukemia virus family of retroviruses. The core elements of the SL3 virus are important genetic determinants of the ability of this virus to induce T-cell lymphomas and the transcriptional activity of the viral long terminal repeat in T lymphocytes. CBF consists of two subunits, a DNA binding subunit, CBF alpha, and a second subunit, CBF beta, that stimulates the DNA binding activity of CBF alpha. One of the genes that encodes a CBF alpha subunit is AML1, also called Cbf alpha 2. This locus is rearranged by chromosomal translocations in human myeloproliferative disorders and leukemias. An exogenously expressed Cbf alpha 2-encoded subunit (CBF alpha 2-451) stimulated transcription from the SL3 enhancer in P19 and HeLa cells. Activity was mediated through the core elements. Three different isoforms of CBF beta were also tested for transcriptional activity on the SL3 enhancer. The longest form, CBF beta-187, increased the transcriptional stimulation by CBF alpha 2-451 twofold in HeLa cells, although it had no effect in P19 cells. Transcriptional activation by CBF beta required binding to the CBF alpha subunit, as a form of CBF beta that lacked binding ability, CBF beta-148, failed to increase activity. These results indicated that at least in certain cell types, the maximum activity of CBF required both subunits. They also provided support for the hypothesis that CBF is a factor in T lymphocytes that is responsible for recognition of the SL3 cores. We also examined whether CBF could distinguish a 1-bp difference between the enhancer core of SL3 and the core of the nonleukemogenic virus, Akv. This difference strongly affects transcription in T cells and leukemogenicity of SL3. However, no combination of CBF alpha and CBF beta subunits that we tested was able to distinguish the 1-bp difference in transcription assays. Thus, a complete understanding of how T cells recognize the SL3 core remains to be elucidated. 


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Selective DNA-binding activity of interleukin-10-stimulated STAT molecules in human monocytes. 
It has been demonstrated that interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) have various reverse effects on macrophages; however, the molecular mechanism of this difference has not been fully understood. In this study, we analyzed the binding activity of IL-10- and IFN-gamma-activated STAT molecules to two kinds of GAS-motif sequences. IL-10-activated STAT1 could bind to the GAS-motif sequence in the promoter region of the Fcgamma receptor, but not to that in the promoter region of the COX-2 gene, whereas IFN-gamma-activated STAT1 and STAT5 could bind to both sequences. IL-10 inhibited IFN-gamma-induced STAT activation without newly synthesized protein. We further demonstrated that aspirin, but not dexamethasone, suppressed IFN-gamma-induced STAT activation. Taken together, these results suggest that IL-10-activated STAT1 has a specificity in binding to the GAS-motif sequences, whereas IFN-gamma-activated STAT1 and STAT5 have a broader spectrum in binding to the GAS-motif sequences. This may explain the difference between IL-10 and IFN-gamma in biological activity, and the inhibitory effect of IL-10 on IFN-gamma activities. 


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Protein kinase inhibitor H-7 blocks accumulation of unspliced mRNA of human T-cell leukemia virus type I (HTLV-I). 
Rex, the post-transcriptional regulator of human T-cell leukemia virus type I (HTLV-I), is known to induce accumulation of the unspliced viral gag-pol mRNA. Rex is a phosphoprotein found in the cell nucleolus, whose function may be regulated by its localization and phosphorylation. We have examined the role of phosphorylation on Rex function by using a protein kinase inhibitor, H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine]. Treatment of an HTLV-I infected human T-cell line with H-7 blocked specifically accumulation of the unspliced gag-pol mRNA, resulting in the decreased Gag protein synthesis that corresponds with the decreased in vivo phosphorylation of Rex. In contrast, other viral and cellular products have not been influenced by the level of H-7 used. Therefore, the phosphorylation of Rex is required for the viral RNA partition of HTLV-I. 


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G(Anh)MTetra, a natural bacterial cell wall breakdown product, induces interleukin-1 beta and interleukin-6 expression in human monocytes. A study of the molecular mechanisms involved in inflammatory cytokine expression [published erratum appears in J Biol Chem 1994 Jun 17;269(24):16983] 
It is believed that induction of cytokine expression by bacterial cell wall components plays a role in the development and course of sepsis. However, most attention has been focused on lipopolysaccharide (LPS). We studied the ability of N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D- isoglutamyl-m-diaminopimelyl-D-alanine (G(Anh)MTetra), a naturally occurring breakdown product of peptidoglycan that is produced by soluble lytic transglycosylase of Escherichia coli, to induce cytokine expression in human monocytes. G(Anh)MTetra was found to strongly induce interleukin (IL)-1 beta and IL-6 mRNA expression after 2 h and IL-1 beta and IL-6 protein secretion after 48 h of activation. The increase in mRNA accumulation was at least partly due to an increase in the transcription rates of the respective genes and was accompanied by a strong induction of nuclear factor-kappa B and activator protein-1 transcription factor expression. Experiments using inhibitors of protein kinase C, protein kinase A, and tyrosine kinase-dependent pathways revealed that G(Anh)MTetra-induced IL-1 beta and IL-6 mRNA expression involves activation of an H7-inhibitable pathway. By using the protein synthesis inhibitor cycloheximide, it was shown that G(Anh)MTetra-induced IL-6 mRNA expression depends on the synthesis of new protein, whereas G(Anh)MTetra-induced IL-1 beta mRNA accumulation does not. When responses to G(Anh)MTetra were compared with those to LPS and muramyldipeptide (MDP), it was found that the optimal response to G(Anh)MTetra induction was similar to that of LPS but significantly higher than the response to MDP. Furthermore, maximal G(Anh)MTetra-induced IL-1 beta and IL-6 mRNA expression could be enhanced by co-stimulation with LPS or MDP, suggesting that different receptors and/or transduction pathways were involved. These results indicate that G(Anh)MTetra induces IL-1 beta and IL-6 expression in human monocytes suggesting a possible role for G(Anh)MTetra in the release of cytokines during sepsis. 


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C/EBP beta in rheumatoid arthritis: correlation with inflammation, not disease specificity. 
Rheumatoid arthritis synovial tissue was examined and compared with osteoarthritis tissue for the presence of the nuclear transcription factor C/EBP beta (NF-IL-6). The region (lining or sublining), cell type, and subcellular distribution (cytoplasmic or nuclear) of the expression of C/EBP beta was characterized. Rheumatoid arthritis synovial fluid and blood and normal peripheral blood were also examined. C/EBP beta was detected in the synovial lining and in sublining cells of synovial tissue from patients with both rheumatoid and osteoarthritis. A significant (P < 0.001 and < 0.05, respectively) increase in the percentage of cells with nuclear staining was seen in the lining layer, compared to cells in the sublining region, in rheumatoid and osteoarthritis. In both diseases a strong correlation (r = 0.79, P < 0.001) was observed between the percentage of cells in the synovial lining that were positive for nuclear C/EBP beta and lining cell depth. Two-color immunohistochemistry demonstrated that both macrophages and fibroblast-like synoviocytes were positive for nuclear C/EBP beta. The presence of C/EBP beta was confirmed by immunohistochemistry and Western blot analysis with isolated synovial fibroblasts. Nuclear C/EBP beta was also detected in rheumatoid synovial fluid monocytes/macrophages, but not in lymphocytes or neutrophils. Western blot analysis confirmed the presence of C/EBP beta in these cells. The intensity of C/EBP beta staining was greater (P < 0.001) in synovial fluid monocytes than in those from normal or rheumatoid peripheral blood. In conclusion, the enhanced nuclear staining for C/EBP beta in the synovial lining, compared to the sublining, suggesting activation in the lining, and the positive correlation of lining layer depth with the percentage of cells in the lining positive for nuclear C/EBP beta, suggest a potential role for C/EBP beta in chronic inflammation. The regulation of the production or activity of C/EBP beta, to inhibit inflammatory mediator expression by synovial macrophages and fibroblasts, offers a novel approach to therapeutic intervention. 


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The activation of the Jak-STAT 1 signaling pathway by IL-5 in eosinophils. 
The intracellular signal transduction of IL-5 in eosinophils is unknown. The objective of this study was to investigate the involvement of the newly discovered Jak-STAT pathway in the IL-5 signal transduction mechanism. Eosinophils were purified from peripheral blood by discontinuous Percoll gradients and stimulated with IL-5. The involvement of Jak 2 was investigated by immunoprecipitation followed by immunoblotting for tyrosine phosphorylation. The activation of Jak 2 was studied by autophosphorylation of the immunoprecipitated kinase. Jak 2 was tyrosine phosphorylated within 1 to 3 min after stimulation of eosinophils with IL-5. Further, the immunoprecipitated Jak 2 obtained from IL-5-stimulated cells underwent autophosphorylation. Jak 2 coprecipitated with the beta-subunit of the IL-5 receptor, suggesting a physical association of the kinase with the receptor. The nuclear factor STAT-1 (p91) was investigated by immunoprecipitation followed by immunoblotting for tyrosine phosphorylation. STAT-1 was tyrosine phosphorylated within 15 min of IL-5 stimulation. The presence of STAT-1 in the nuclear extract was studied by electrophoretic mobility shift assay. IL-5 induced two proteins that bound to the gamma-activating sequence. In the presence of an anti-STAT-1 Ab, the band was supershifted. Thus, we demonstrated that IL-5 activated the Jak 2-STAT 1 signaling pathway in eosinophils. We speculate that the Jak 2-STAT 1 pathway may be involved in the activation of IL-5-inducible genes in eosinophils. 


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Retinoid X receptor (RXR) agonist-induced activation of dominant-negative RXR-retinoic acid receptor alpha403 heterodimers is developmentally regulated during myeloid differentiation. 
The multiple biologic activities of retinoic acid (RA) are mediated through RAR and retinoid X receptor (RXR) nuclear receptors that interact with specific DNA target sequences as heterodimers (RXR-RAR) or homodimers (RXR-RXR). RA receptor activation appears critical to regulating important aspects of hematopoiesis, since transducing a COOH-terminally truncated RARalpha exhibiting dominant-negative activity (RARalpha403) into normal mouse bone marrow generates hematopoietic growth factor-dependent cell lines frozen at the multipotent progenitor (EML) or committed promyelocyte (MPRO) stages. Nevertheless, relatively high, pharmacological concentrations of RA (1 to 10 &mgr;M) overcome these differentiation blocks and induce terminal granulocytic differentiation of the MPRO promyelocytes while potentiating interleukin-3 (IL-3)-induced commitment of EML cells to the granulocyte/monocyte lineage. In the present study, we utilized RXR- and RAR-specific agonists and antagonists to determine how RA overcomes the dominant-negative activity of the truncated RARalpha in these different myeloid developmental stages. Unexpectedly, we observed that an RXR-specific, rather than an RAR-specific, agonist induces terminal granulocytic differentiation of MPRO promyelocytes, and this differentiation is associated with activation of DNA response elements corresponding to RAR-RXR heterodimers rather than RXR-RXR homodimers. This RXR agonist activity is blocked by RAR-specific antagonists, suggesting extensive cross-talk between the partners of the RXR -RARalpha403 heterodimer. In contrast, in the more immature, multipotent EML cells we observed that this RXR-specific agonist is inactive either in potentiating IL-3-mediated commitment of EML cells to the granulocyte lineage or in transactivating RAR-RXR response elements. RA- triggered GALdbd-RARalpha hybrid activity in these cells indicates that the multipotent EML cells harbor substantial nuclear hormone receptor coactivator activity. However, the histone deacetylase (HDAC) inhibitor trichostatin A readily activates an RXR-RAR reporter construct in the multipotent EML cells but not in the committed MPRO promyelocytes, indicating that differences in HDAC-containing repressor complexes in these two closely related but distinct hematopoietic lineages might account for the differential activation of the RXR-RARalpha403 heterodimers that we observed at these different stages of myeloid development. 


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Decreased concentration of 1,25-dihydroxyvitamin D3 receptors in peripheral mononuclear cells of patients with X-linked hypophosphatemic rickets: effect of phosphate supplementation. 
Abnormal renal tubular phosphate transport is considered to be the primary defect in X-linked hypophosphatemic rickets (XLH). However, the resistance to vitamin D treatment in XLH cannot be explained by hypophosphatemia alone. Since most of the actions of vitamin D are mediated by its receptors (VDR), abnormalities of VDR have been postulated in XLH. In order to investigate this possibility, we measured the concentration of VDR in PHA-activated peripheral mononuclear cells from 10 XLH patients. Patients without phosphate supplementation showed significantly lower concentration (21.7 +/- 5.1 fmol/mg protein, mean +/- SEM) compared to the normal controls (60.7 +/- 4.0). On the contrary, there was no significant difference between the phosphate-supplemented patients (58.3 +/- 2.7) and controls. There was a significant positive correlation between VDR concentration and serum phosphate (P less than 0.05). In two patients, VDR was increased after daily phosphate supplementation was started. These results indicate that a decreased concentration of VDR secondary to persistent hypophosphatemia is one of the causes of vitamin D resistance in XLH. 


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Spi-C, a novel Ets protein that is temporally regulated during B lymphocyte development. 
A novel Ets protein was isolated by yeast one-hybrid screening of a cDNA library made from lipopolysaccharide-stimulated mouse splenic B cells, using the SP6 kappa promoter kappaY element as a bait. The novel Ets protein was most closely related to PU.1 and Spi-B within the DNA binding Ets domain and was therefore named Spi-C. However, Spi-C may represent a novel subgroup within the Ets protein family, as it differed significantly from Spi-B and PU.1 within helix 1 of the Ets domain. Spi-C was encoded by a single-copy gene that was mapped to chromosome 10, region C. Spi-C interacted with DNA similarly to PU.1 as judged by methylation interference, band-shift and site selection analysis, and activated transcription of a kappaY element reporter gene upon co-transfection of HeLa cells. Spi-C RNA was expressed in mature B lymphocytes and at lower levels in macrophages. Furthermore, pre-B cell and plasma cell lines were Spi-C-negative, suggesting that Spi-C might be a regulatory molecule during a specific phase of B lymphoid development. 


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Expression of the nucleoside diphosphate kinase in human skin cancers: an immunohistochemical study. 
Expression of nucleoside diphosphate(NDP) kinase, which is homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential. However, the relationship remains controversial according to the tumor cell types and experimental system, with conflicting results from different research groups. In order to determine whether NDP kinase expression serves as a marker for metastatic potential in human skin cancer, we assessed the levels of NDP kinase expression in 9 keratoacanthomas (KAs), 26 squamous cell carcinomas (SCCs), and 25 basal cell carcinomas (BCCs) using immunohistochemistry. The expression of NDP kinase was intense in KA and SCC compared with BCC. However, the difference of NDP kinase expression between KA and SCC was not statistically significant. And there was no statistically significant difference in NDP kinase expression between SCC with metastasis and SCC without metastasis. Our results contradict the hypothesis concerning the possible role of nm23 gene as a metastatic suppressor gene in human skin cancer. The mechanism of overexpression in various tumor cell types and its biological significance in cutaneous carcinogenesis remain to be determined. 


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Glucocorticoid receptors are down-regulated in inflamed colonic mucosa but not in peripheral blood mononuclear cells from patients with inflammatory bowel disease [see comments] 
BACKGROUND: Growing evidence indicates that the immune system and the hypothalamic-pituitary-adrenal system are linked by several mechanisms, for example intracellular glucocorticoid receptors (hGR). Glucocorticoids are the standard treatment of acute attacks of inflammatory bowel disease (IBD). Binding of glucocorticoids to hGR down-regulates the transcription of inflammatory genes that can propagate IBD. PATIENTS AND METHODS: IBD patients were either treated with 5-60 mg of prednisolone for more than 1 week or were without glucocorticoid treatment for more than 4 weeks. hGR levels were determined from isolated cytosol of peripheral blood mononuclear cells (PBMCs) or mucosal biopsies using a radioassay with [3H]-dexamethasone. Interleukin (IL) 6 levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The systemic (PBMC) hGR levels of corticosteroid-treated IBD patients were significantly lower than those of control subjects (59.6 +/- 57.1 dpm mg-1 cytosol protein vs. 227.0 +/- 90.8 dpm mg-1 cytosol protein, P = 0.007) and IBD patients not receiving glucocorticoid treatment (179.7 +/- 171.3 dpm mg-1 cytosol protein, P = 0.002). Systemic hGR levels in untreated IBD patients did not differ significantly from those in control subjects. In patients with connective tissue diseases, systemic hGR levels were also found to be decreased in the absence of glucocorticoid treatment. Systemic hGR levels in patients with Crohn's disease (CD) treated with steroids (66.6 +/- 61.0 dpm mg-1 cytosol protein) were not different from those in patients with ulcerative colitis (UC) (56.1 +/- 51.6 dpm mg-1 cytosol protein). In contrast to these findings, mucosal hGR levels were significantly decreased in both steroid-treated (18.0 +/- 15.5) and not steroid-treated (37.8 +/- 30.5) patients compared with control subjects ( 125.6 +/- 97.1; P = 0.00009 and P = 0.0008 respectively ). IL-6 levels in all IBD groups with and without steroids were significantly different from those in control subjects. CONCLUSION: In IBD there is no difference in systemic hGR levels between not steroid-treated patients and control subjects, in spite of inflammatory activity (IL-6). Mucosal hGR levels were decreased independently of treatment, probably leading to a decreased protection against NF-kappaB action in the intestinal mucosa. 


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Expression of tal-1 and GATA-binding proteins during human hematopoiesis. 
Tal-1 rearrangements are associated with nearly 30% of human T acute lymphoblastic leukemia. Tal-1 gene encodes a putative transcription factor with a basic helix-loop-helix domain and is known to be predominantly expressed in hematopoietic cells. We investigated the pattern of tal-1 expression in purified human hematopoietic cells by in situ hybridization and reverse transcriptase polymerase chain reaction analysis. Both methods demonstrated that the tal-1 gene is expressed in megakaryocytes and erythroblasts as well as in basophilic granulocytes. In addition, our results indicate that the tal-1 1A promoter, which contains two consensus GATA-binding sites, is active mainly in these lineages. Because the GATA-1 gene is known to transactivate several genes specific for the erythroid, megakaryocytic, and mastocytic/basophilic lineages, we studied GATA-1 expression in these purified hematopoietic cells. We found that GATA-1 and tal-1 genes are coexpressed in these three lineages. Remarkably, the expression of both genes is downmodulated during erythroid and megakaryocytic terminal maturation. In immature hematopoietic cells, tal-1 and GATA-1 genes are coexpressed in committed progenitors cells (CD34+/CD38(2+)), whereas they are not detectable in the most primitive cells (CD34(2+)/CD38-). In contrast, GATA-2 is strongly expressed in both most primitive and committed progenitors cells, whereas GATA-3 is mostly detected in most primitive ones. Altogether our results strongly suggest that GATA-1 modulates the transcription of tal-1 during the differentiation of the erythroid, megakaryocytic, and basosophilic lineages. 


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Activation of human CD4 T lymphocytes. Interaction of fibronectin with VLA-5 receptor on CD4 cells induces the AP-1 transcription factor. 
Fibronectin synergized with anti-CD3 antibody to promote CD4 cell proliferation in a serum-free culture system whereas no proliferation was observed when CD4 cells were cultured with anti-CD3 alone or fibronectin alone. In addition, anti-CD29 (integrin beta 1) as well as anti-VLA-5 (human fibronectin receptor) antibodies blocked this CD4 cell activation in this system. Although anti-CD3 alone or fibronectin alone cannot induce IL-2 message by CD4 cells, the combination of anti-CD3 plus fibronectin induced IL-2 message by CD4 cells. In an analysis of the molecular mechanism by which IL-2 message was generated, we showed that a fibronectin-VLA-5 fibronectin receptor interaction may contribute an independent signal distinct from the CD3 pathway of activation by the induction of an AP-1 transcriptional factor. Thus the VLA-5 fibronectin receptor on CD4 cells can play a complementary role in CD3-TCR-mediated signal transduction through its interaction with fibronectin. 


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Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells at nutritionally relevant concentrations. 
Daidzein and genistein glucuronides (DG and GG), major isoflavone metabolites, may be partly responsible for biological effects of isoflavones, such as estrogen receptor binding and natural killer cell (NK) activation or inhibition. DG and GG were synthesized using 3-methylcholanthrene-induced rat liver microsomes. The Km and Vmax for daidzein and genistein were 9.0 and 7.7 micromol/L, and 0.7 and 1.6 micromol/(mg protein. min), respectively. The absence of ultraviolet absorbance maxima shifts in the presence of sodium acetate confirmed that the synthesized products were 7-O-glucuronides. DG and GG were further purified by a Sephadex LH-20 column. DG and GG competed with the binding of 17beta-(3H) estradiol to estrogen receptors of B6D2F1 mouse uterine cytosol. The concentrations required for 50% displacement of 17beta-(3H) estradiol (CB50) were: 17beta-estradiol, 1.34 nmol/L; diethylstilbestrol, 1.46 nmol/L; daidzein, 1.6 micromol/L; DG, 14.7 micromol/L; genistein, 0.154 micromol/L; GG, 7.27 micromol/L. In human peripheral blood NK cells, genistein at <0.5 micromol/L and DG and GG at 0.1-10 micromol/L enhanced NK cell-mediated K562 cancer cell killing significantly (P < 0.05). At > 0.5 micromol/L, genistein inhibited NK cytotoxicity significantly (P < 0.05). The glucuronides only inhibited NK cytotoxicity at 50 micromol/L. Isoflavones, and especially the isoflavone glucuronides, enhanced activation of NK cells by interleukin-2 (IL-2), additively. At physiological concentrations, DG and GG were weakly estrogenic, and they activated human NK cells in nutritionally relevant concentrations in vitro, probably at a site different from IL-2 action. 


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The human TCF-1 gene encodes a nuclear DNA-binding protein uniquely expressed in normal and neoplastic T-lineage lymphocytes. 
The TCF-1 gene encodes a putative transcription factor with affinity for a sequence motif occurring in a number of T-cell enhancers. TCF-1 mRNA was originally found to be expressed in a T cell-specific fashion within a set of human and mouse cell lines. In contrast, expression reportedly occurs in multiple nonlymphoid tissues during murine embryogenesis. We have now raised a monoclonal antibody to document expression and biochemistry of the human TCF-1 protein. As expected, the TCF-1 protein was detectable only in cell lines of T lineage. Its expression was always restricted to the nucleus. Immunohistochemistry on a panel of human tissues revealed that the TCF-1 protein was found exclusively in thymocytes and in CD3+ T cells in peripheral lymphoid tissues. Western blotting yielded a set of bands ranging from 25 kD to 55 kD, resulting from extensive alternative splicing. The TCF-1 protein was detectable in all samples of a set of 22 T-cell malignancies of various stages of maturation, but was absent from a large number of other hematologic neoplasms. These observations imply a T cell-specific function for TCF-1, a notion corroborated by recent observations on Tcf-1 knock-out mice. In addition, these results indicate that nuclear TCF-1 expression can serve as a pan-T-lineage marker in the diagnosis of lymphoid malignancies. 


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Transient pseudo-hypoaldosteronism following resection of the ileum: normal level of lymphocytic aldosterone receptors outside the acute phase. 
Pseudo-hypoaldosteronism (PHA) is due to mineralocorticoid resistance and manifests as hyponatremia and hyperkalemia with increased plasma aldosterone levels. It may be familial or secondary to abnormal renal sodium handling. We report the case of a 54-year-old woman with multifocal cancer of the colon, who developed PHA after subtotal colectomy, ileal resection and jejunostomy. She was treated with 6 g of salt daily to prevent dehydration, which she stopped herself because of reduced fecal losses. One month later she was admitted with signs of acute adrenal failure, i.e. fatigue, severe nausea, blood pressure of 80/60 mmHg, extracellular dehydration, hyponatremia (118 mmol/l); hyperkalemia (7.6 mmol/l), increased blood urea nitrogen (BUN) (200 mg/dl) and creatininemia (2.5 mg/dl), and decreased plasma bicarbonates level (HCO3-: 16 mmol/l; N: 27-30). However, the plasma cortisol was high (66 microg/100 ml at 10:00 h; N: 8-15) and the ACTH was normal (13 pg/ml, N: 10-60); there was a marked increase in plasma renin activity (>37 ng/ml/h; N supine <3), active renin (869 pg/ml; N supine: 1.120), aldosterone (>2000 pg/ml; N supine <150) and plasma AVP (20 pmol/l; N: 0.5-2.5). The plasma ANH level was 38 pmol/l (N supine: 5-25). A urinary steroidogram resulted in highly elevated tetrahydrocortisol (THF: 13.3 mg/24h; N: 1.4+/-0.8) with no increase in tetrahydrocortisone (THE: 3.16 mg/24h; N: 2.7+/-2.0) excretion, and with low THE/THF (0.24; N: 1.87+/-0.36) and alpha THF/THF (0.35; N: 0.92+/-0.42) ratios. The number of mineralocorticoid receptors in mononuclear leukocytes was in the lower normal range for age, while the number of glucocorticoid receptors was reduced. Small-bowel resection in ileostomized patients causes excessive fecal sodium losses and results in chronic sodium depletion with contraction of the plasma volume and severe secondary hyperaldosteronism. Nevertheless, this hyperaldosteronism may be associated with hyponatremia and hyperkalemia suggesting PHA related to the major importance of the colon for the absorption of sodium. In conclusion, this case report emphasizes 1) the possibility of a syndrome of acquired PHA with severe hyperkalemia after resection of the ileum and colon responding to oral salt supplementation; 2) the major increase in AVP and the small increase in ANH; 3) the strong increase in urinary THF with low THE/THF and alpha THF/THF ratios; 4) the normal number of lymphocytic mineralocorticoid receptors outside the acute episode. 


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Genetic evidence for an additional factor required for erythropoietin-induced signal transduction. 
Erythropoietin (EPO) and its receptor (EPOR) are required for the development of mature erythrocytes. After binding of ligand, the EPOR activates a variety of signaling pathways that ultimately control cellular proliferation, survival, and specific gene expression. Although erythroid progenitors appear to be the principal EPO-responsive cell type in vivo due to the restricted expression of the EPOR, many growth factor-dependent cell lines expressing the EPOR can respond to EPO by activating many or all of these pathways. In the present study, we have identified a cellular context (the interleukin-2 [IL-2]-dependent HT-2 line) in which the EPO stimulation of the EPOR fails to support cellular proliferation, STAT-5 induction, or MAPK activation, despite efficient phosphorylation of the EPOR and JAK2 and inhibition of apoptosis after withdrawal of IL-2. Interestingly, when we fused HT-2 cells expressing the EPOR with Ba/F3 cells in a complementation assay, the resulting hybridomas proliferated and potently activated STAT-5 and MAPK in response to EPO. These data indicate that an unidentified cellular factor is needed to mediate signaling by the EPOR. Moreover, Ba/F3 cells apparently express this factor(s) and somatic fusions can, therefore, confer EPO-responsiveness to HT-2 cells that lack this factor. 


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Interleukin-7 can induce the activation of Jak 1, Jak 3 and STAT 5 proteins in murine T cells. 
The activation of Janus protein tyrosine kinases (Jak) and STAT (signal transducer and activator of transcription) proteins has recently been linked to the signal transduction mechanism of several cytokines. IL-7 was observed to induce a rapid and dose-dependent tyrosine phosphorylation of Jak 1 and Jak 3 and concomitantly, the tyrosine phosphorylation and DNA binding activity of multiple STAT proteins. The STAT proteins utilized by IL-7 were identical to those induced by IL-2 and could be identified as various STAT 5 isoforms. Moreover, the induction of both Jak 1 and 3, and STAT 5 activity strongly correlated with the growth-promoting effects of IL-7, suggesting that this signal transduction mechanism may play a key role in IL-7-induced proliferation. 


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Tyrosine kinase and cAMP-dependent protein kinase activities in CD40-activated human B lymphocytes. 
In vitro, human B lymphocytes undergo long-term proliferation when activated through CD40, a protein expressed on their cell surface. The nature of CD40-dependent signals in proliferating fresh human Epstein-Barr virus-negative B lymphocytes is currently unknown. In this study, a CD40-dependent B cell culture system was used to examine the role of different signal transduction elements. Protein kinase C (PKC) depletion generated by a long-term phorbol 12 myristate 13-acetate treatment had weak effects on proliferation. Rather, tyrosine phosphorylation was shown to be directly involved in mediating CD40-dependent signals. The use of the protein tyrosine kinase (PTK)-specific inhibitor herbimycin A dramatically decreased cellular proliferation without altering the activity of the human immunodeficiency virus-1 long terminal repeat (HIV-1 LTR), a promoter largely dependent on the binding of nuclear factor kappa B (NF- kappa B). In contrast, the cAMP-dependent protein kinase specific inhibitor H-89 totally inhibited HIV-1 LTR activity at a concentration as low as 100 nM without affecting cellular proliferation. Electrophoretic mobility shift assay (EMSA) and supershift assay using an NF-kappa B binding sequence from the kappa light chain as a probe, revealed that both p65 (RelA) and c-Rel were present in CD40-stimulated B cells. While PKC depletion did not alter the NF-kappa B level, treatment of B lymphocytes with H-89 or herbimycin A provoked a decrease in the NF-kappa B level. These observations establish the importance of different signal transducing pathways leading to CD40 activation of B lymphocytes. 


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Effects of 1 alpha, 25-dihydroxyvitamin D3 on the human chronic myelogenous leukemia cell line RWLeu-4. 
The effects of 1 alpha, 25-dihydroxyvitamin D3 (VD3) on proliferation, differentiation, and macromolecular synthesis in the new Philadelphia chromosome-positive chronic myelogenous leukemia cell line, RWLeu-4, were investigated. Binding of [3H]VD3 was saturable, with approximately 2000-3000 sites/cell, and half-maximal binding occurring at 0.21-0.33 nM. Treatment of RWLeu-4 cells with VD3 induced 24R-hydroxylase activity, a marker of vitamin D3 responsiveness in many tissues. Exposure of RWLeu-4 cells to VD3 also inhibited proliferation and DNA synthesis with a 50% effective dose of 3.5-10 nM within 72 h; in addition, protein and RNA synthesis were inhibited by VD3 treatment. Exposure of RWLeu-4 cells to 5 nM VD3 for 72 h caused 50% of the cells to differentiate into macrophage/monocyte type cells as judged by nitroblue tetrazolium staining and adherence to plastic. Progressive expression of cell surface maturation-specific antigens of the monocyte/macrophage lineage was induced by treatment of RWLeu-4 cells with VD3 for 24 to 72 h at doses that inhibited cellular proliferation. c-myc RNA, which is constitutively expressed in RWLeu-4 cells, increased after 0.5 h of treatment with 50 nM VD3 and then rapidly decreased to barely detectable levels after 4 h of treatment. Finally, the in vitro tyrosine kinase activity associated with the p210bcr-abl oncogene product was decreased approximately 50% by VD3 treatment. Because of the presence of a functional receptor-effector system for VD3 and multiple biological responses to the hormone, these cells provide a unique model system with which to probe the specific effects of VD3 on cell growth and differentiation in chronic myelogenous leukemia. 


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Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors. 
The programmed activation/repression of transcription factors in early hematopoietic differentiation has not yet been explored. The DNA-binding protein GATA-1 is required for normal erythroid development and regulates erythroid-expressed genes in maturing erythroblasts. We analyzed GATA-1 expression in early human adult hematopoiesis by using an in vitro system in which "pure" early hematopoietic progenitors are induced to gradual and synchronized differentiation selectively along the erythroid or granulocyte-macrophage pathway by differential treatment with hematopoietic growth factors. The GATA-1 gene, though virtually silent in quiescent progenitors, is activated after entrance into the cell cycle upon stimulation with hematopoietic growth factors. Subsequently, increasing expression along the erythroid pathway contrasts with an abrupt downregulation in the granulocyte-macrophage lineage. These results suggest a microenvironment-directed, two-step model for GATA-1 expression in differentiating hematopoietic progenitors that involves (i) cycle-dependent initiation and (ii) lineage-dependent maintenance or suppression. Hypothetically, on/off switches of lineage-restricted transactivators may underlie the binary fate decisions of hematopoietic progenitors. 


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HIV-1 envelope glycoproteins induce activation of activated protein-1 in CD4+ T cells [published erratum appears in J Biol Chem 1995 Dec 1;270(48):29038] 
Activation of CD4 positive T cells is a primary requirement for human immunodeficiency virus (HIV) entry, efficient HIV replication, and progression to AIDS, Utilizing CD4 positive T cell lines and purified T cells from normal individuals, we have demonstrated that native envelope glycoproteins of HIV, gp 160, can induce activation of transcription factor, activated protein-1 (AP-1). The stimulatory effects of gp160 are mediated through the CD4 molecule, since treatment of gp160 with soluble CD4-IgG abrogates its activity, and CD4 negative T cell lines fail to be stimulated with gp160. Immunoprecipitation of the gp 160-induced nuclear extracts with polyclonal antibodies to Fos and Jun proteins indicates that AP-1 complex is comprised of members of these family of proteins. The gp160-induced AP-1 complex is dependent upon protein tyrosine phosphorylation and is protein synthesis-independent. This stimulation can also be abolished by inhibitors of protein kinase C, but it is unaffected by calcium channel blocker or cyclosporine A. This gp160 treatment adversely affects the functional capabilities of T cells: pre-treatment of CD4+ T cells with gp160 for 4 h at 37 degrees C inhibited anti-CD3-induced interleukin-2 secretion. Effects similar to gp160 were seen with anti-CD4 mAb. The aberrant activation of AP-1 by gp160 in CD4 positive T cells could result in up-regulation of cytokines containing AP-1 sites, e.g. interleukin-3 and granulocyte macrophage colony-stimulating factor, and concurrently lead to T cell unresponsiveness by inhibiting interleukin-2 secretion. 


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[The role of glucocorticoid receptors and HLA antigens in the pathogenesis of Cushing's syndrome] 
Lymphocytic levels of glucocorticoid receptors were evaluated in 114 patients suffering from Icenko-Cushing's syndrome. Incidence of HLA antigens was determined in 94 of them. A significant rise of A10 and B27 antigen incidence compared to that in normal subjects allows these antigens to be considered genetic markers of Icenko-Cushing's syndrome. The levels of glucocorticoid receptors in lymphocytes of the patients are lower than in normal subjects both in the active stage of the disease and following bilateral total adrenalectomy. The patients carrying B27 antigen had lymphocytic receptor concentrations under the levels of such in patients free of the antigen carriage. Antigen B27 seems to be a cause of lower levels of glucocorticoid receptors in blood lymphocytes. 


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Human immunodeficiency virus-associated Hodgkin's disease derives from post-germinal center B cells. 
Human immunodeficiency virus-associated Hodgkin's disease (HIV-HD) displays several peculiarities when compared with HD of the general population. These include overrepresentation of clinically aggressive histologic types and frequent infection of Reed-Sternberg (RS) cells by Epstein-Barr virus (EBV). Recently, we have reported that the histogenesis of HD of the general population may be assessed by monitoring the expression pattern of BCL-6, a transcription factor expressed in germinal center (GC) B cells, and of CD138/syndecan-1 (syn-1), a proteoglycan associated with post-GC, terminal B-cell differentiation. In this study, we have applied these two markers to the study of HIV-HD histogenesis and correlated their expression status to the virologic features of this disease. We have found that RS cells of all histologic categories of HIV-HD consistently display the BCL-6(-)/syn-1(+) phenotype and thus reflect post-GC B cells. Although BCL-6(-)/syn-1(+)RS cells of HIV-HD express CD40, they are not surrounded by CD40 ligand-positive (CD40L+) reactive T lymphocytes, which, in HD of the general population, are thought to regulate the disease phenotype through CD40/CD40L interactions. Conversely, RS cells of virtually all HIV-HD express the EBV-encoded latent membrane protein 1 (LMP1), which, being functionally homologous to CD40, may contribute, at least in part, to the modulation of the HIV-HD phenotype. 


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Inhibition of nuclear factor kappa B subunit p65 mRNA accumulation in lipopolysaccharide-stimulated human monocytic cells treated with sodium salicylate. 
Lipopolysaccharide is one of the most potent trigger substances for monocytes and macrophages causing secretion of inflammatory mediators such as tumor necrosis factor and interleukin-1. The nature of the nuclear factors involved in regulation of these cytokine genes is still unknown. Nuclear factor kappa B (NF-kappa B; heterodimer of p50 and p65) proteins have been suggested to play an important role in gene transcription of inflammatory mediators when monocytes are stimulated with lipopolysaccharide. Nonsteroidal anti-inflammatory drugs such as salicylates have been used to treat symptoms of inflammation, and a new mechanism of drug action was suggested recently. Salicylates have been shown to inhibit lipopolysaccharide-induced gene transcription via inhibition of NF-kappa B activation by preventing the degradation of NF-kappa B inhibitor "I kappa B", blocking the translocation of NF-kappa B into the nuclear compartment. However, the nature of the subunit involved in this mechanism has not been defined. To examine the mechanisms by which salicylates affect cytokine gene transcription, the amount of active and inactive NF-kappa B and NF-kappa B mRNA, in Porphyromonas gingivalis lipopolysaccharide-stimulated human monocytic cells was assessed. High doses of sodium salicylate suppressed NF-kappa B p65 mRNA accumulation, resulting in suppression of total NF-kappa B, p50 on tissue oligonucleotide had no effects on lipopolysaccharide-induced NF-kappa B activation. The data demonstrate that the p65 subunit of NF-kappa B is inhibited by salicylate treatment and highlight the role of salicylate in the control of gene expression of inflammatory mediators. 


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ICSAT overexpression is not sufficient to cause adult T-cell leukemia or multiple myeloma. 
ICSAT (Interferon Consensus Sequence binding protein for Activated T cells) is a lymphocyte-specific member of the interferon regulatory factor (IRF) family of transcription factors, originally identified through Southwestern screening of the ATL(Adult T-cell leukemia)-16T expression library. In this study, we created transgenic mice overexpressing ICSAT in lymphocytes. Although spontaneous tumorigenesis was not observed, IL-2 production with Concanavalin A stimulation was significantly increased in transgenic mice overexpressing ICSAT. ICSAT overexpression in lymphocytes seems insufficient for the leukemogenesis of ATL or multiple myeloma (MM), however, it may regulate T cell activation and its overexpression may lead to leukemogenesis via controlling IL-2 production. Copyright 1999 Academic Press. 


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The DNA and steroid binding domains of the glucocorticoid receptor are not altered in mononuclear cells of treated CLL patients. 
The aim of this study was to investigate whether mutations in the glucocorticoid receptor could account for the increasing unresponsiveness of patients with chronic lymphatic leukemia (CLL) to combination chemotherapy. The receptor was tested immunocytochemically, in steroid binding assays, and by a mutation screening (denaturing gradient gel electrophoresis) of the receptor-cDNA. The receptor concentration, as measured by staining and steroid binding test, varied considerably but showed no clear correlation to clinical response. Using a highly sensitive mutation screening assay of the DNA- and the steroid-binding region, none of the treated patients revealed any mutation, suggesting that the glucocorticoid receptor in the CLL patients tested is not altered in these domains. In one individual who had not been treated before analysis a silent mutation was found in one receptor allele. The results suggest that mechanisms other than altered ligand or DNA binding of the receptor may be responsible for the lack of response to chemotherapy. This conclusion is discussed in relation to the mechanism of corticoid resistance in mouse and human lymphoma cells in culture. 


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Block of granulocytic differentiation of 32Dcl3 cells by AML1/ETO(MTG8) but not by highly expressed Bcl-2. 
The chimeric gene, AML1/ETO (MTG8), generated in t(8;21) acute myeloid leukemia enhances the expression of Bcl-2. To evaluate whether this enhancement is the primary role of AML1/ETO in leukemogenesis, effects of over-expression of Bcl-2 in the murine myeloid precursor cell line, 32Dcl3, were examined. When 32Dcl3 cells expressing exogenous Bcl-2 were induced to differentiate, the onset of morphological differentiation was delayed. However, even the cells expressing very high levels of exogenous Bcl-2 eventually underwent differentiation without a significant decrease in the synthesis of Bcl-2. On the contrary, 32Dcl3 cells stably expressing AML1/ETO were completely resistant to differentiation and continued to grow in the presence of G-CSF. These results are consistent with the interpretation that stimulation of Bcl-2 expression is not the primary target of AML1/ETO. 


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Cloning of murine TCF-1, a T cell-specific transcription factor interacting with functional motifs in the CD3-epsilon and T cell receptor alpha enhancers. 
CD3-epsilon gene expression is confined to the T cell lineage. We have recently identified and cloned a human transcription factor, TCF-1, that binds to a functional element in the T lymphocyte-specific enhancer of CD3-epsilon. In a panel of human cell lines, TCF-1 expression was restricted to T lineage cells. TCF-1 belonged to a novel family of genes that contain the so-called high mobility group 1 (HMG) box. Here we report the cloning of murine TCF-1. Two splice alternatives were identified that were not previously observed in human TCF-1. Murine and human TCF-1 displayed a 95.5% overall amino acid homology. Recombinant murine and human TCF-1 recognized the same sequence motif in the CD3-epsilon enhancer as judged by gel retardation and methylation interference assays. With the murine cDNA clones several aspects of TCF-1 were analyzed. First, deletion analysis revealed that a region of TCF-1 containing the HMG box was sufficient for sequence-specific binding. Second, by high stringency Northern blotting and in situ hybridization, TCF-1 expression was shown to be confined to the thymus and to the T cell areas of the spleen. Third, TCF-1 bound specifically to a functional T cell-specific element in the T cell receptor alpha (TCR-alpha) enhancer. The T lineage-specific expression and the affinity for functional motifs in the TCR-alpha and CD3-epsilon enhancers imply an important role for TCF-1 in the establishment of the mature T cell phenotype. 


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Constitutive expression of HIV-1 tat protein in human Jurkat T cells using a BK virus vector. 
The production and characterization of Jurkat cell lines that constitutively express functional human immune deficiency virus type 1 (HIV-1) tat protein, using a BK virus plasmid expression vector and HIV-1 tat cDNA, is described. An increased growth rate of these Jurkat-tat cell lines as compared with control cell lines was observed. 


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RB and a novel E2F-1 binding protein in MHC class II deficient B-cell lines and normal IFN-gamma induction of the class IL transactivator CIITA in class II non-inducible RB-defective tumor lines. 
The major histocompatibility (MHC) class II genes encode cell surface proteins that bind antigenic peptide for presentation to T-cells. The class II proteins are expressed constitutively on B-cells and EBV-transformed B-cells, and are inducible by IFN-gamma on a wide variety of cell types. Retinoblastoma protein (RB) is a tumor suppressor and functions as a transcriptional repressor by binding and inactivating the transactivator E2F-I. RB-defective tumor lines are non-inducible for MHC class II by IFN-gamma, or very weakly inducible, but transfection of 2 different lines with RB expression vectors re-establishes or substantially enhances class II inducibility. Therefore, we examined the RB status of a series of B-cell mutants that are defective in class II expression, generated either in vitro or derived from Bare Lymphocyte Syndrome (BLS) patients. Nuclear matrix-bound RB was detectable in all cases, indicating that loss of RB is not responsible for decreased class II expression in these lines. A second E2F-I binding protein, most likely DP-I, was also apparently normal in both class II-positive and -negative B-cell lines. We also examined the IFN-gamma induction of CIITA in RB-defective lines. CIITA is a class II gene transactivator known to be defective in one form of BLS and to be required for the induction of MHC class II by IFN-gamma. CIITA mRNA is normally inducible by IFN-gamma in class II non-inducible, RB-defective lines, and in one line, re-expression of RB has no effect on CIITA mRNA induction levels. Thus, the block in MHC class II inducibility in RB-defective cells is not due to a block in CIITA inducibility. 


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The rhombotin family of cysteine-rich LIM-domain oncogenes: distinct members are involved in T-cell translocations to human chromosomes 11p15 and 11p13. 
A chromosomal translocation in a T-cell leukemia involving the short arm of human chromosome 11 at band 11p15 disrupts the rhombotin gene. This gene encodes a protein with duplicated cysteine-rich regions called LIM domains, which show homology to zinc-binding proteins and to iron-sulfur centers of ferredoxins. Two homologues of the rhombotin gene have now been isolated. One of these, designated Rhom-2, is located on human chromosome 11 at band 11p13, where a cluster of T-cell leukemia-specific translocations occur; all translocation breakpoints at 11p13 are upstream of the Rhom-2 gene. Human and mouse Rhom-2 are highly conserved and, like rhombotin, encode two tandem cysteine-rich LIM domains. Rhom-2 mRNA is expressed in early mouse development in central nervous system, lung, kidney, liver, and spleen but only very low levels occur in thymus. The other gene, designated Rhom-3, is not on chromosome 11 but also retains homology to the LIM domain of rhombotin. Since the Rhom-2 gene is such a common site of chromosomal damage in T-cell tumors, the consistency of translocations near the rhombotin gene was further examined. A second translocation adjacent to rhombotin was found and at the same position as in the previous example. Therefore, chromosome bands 11p15 (rhombotin) and 11p13 (Rhom-2) are consistent sites of chromosome translocation in T-cell leukemia, with the 11p15 target more rarely involved. The results define the rhombotin gene family as a class of T-cell oncogenes with duplicated cysteine-rich LIM domains. 


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Aiolos transcription factor controls cell death in T cells by regulating Bcl-2 expression and its cellular localization. 
We searched for proteins that interact with Ras in interleukin (IL)-2-stimulated or IL-2-deprived cells, and found that the transcription factor Aiolos interacts with Ras. The Ras-Aiolos interaction was confirmed in vitro and in vivo by co-immunoprecipitation. Indirect immunofluorescence shows that IL-2 controls the cellular distribution of Aiolos and induces its tyrosine phosphorylation, required for dissociation from Ras. We also identified functional Aiolos-binding sites in the Bcl-2 promoter, which are able to activate the luciferase reporter gene. Mutation of Aiolos-binding sites within the Bcl-2 promoter inhibits transactivation of the reporter gene luciferase, suggesting direct control of Bcl-2 expression by Aiolos. Co-transfection experiments confirm that Aiolos induces Bcl-2 expression and prevents apoptosis in IL-2-deprived cells. We propose a model for the regulation of Bcl-2 expression via Aiolos. 


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Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes. 
Phagocyte recognition, uptake, and nonphlogistic degradation of neutrophils and other leukocytes undergoing apoptosis promote the resolution of inflammation. This study assessed the effects of anti-inflammatory glucocorticoids on this leukocyte clearance mechanism. Pretreatment of "semimature" 5-day human monocyte-derived macrophages (M phi) for 24 h with methylprednisolone, dexamethasone, and hydrocortisone, but not the nonglucocorticoid steroids aldosterone, estradiol, and progesterone, potentiated phagocytosis of apoptotic neutrophils. These effects were specific in that the potentiated phagocytosis of apoptotic neutrophils was completely blocked by the glucocorticoid receptor antagonist RU38486, and glucocorticoids did not promote 5-day M phi ingestion of opsonized erythrocytes. Similar glucocorticoid-mediated potentiation was observed with 5-day M phi uptake of alternative apoptotic "targets" (eosinophils and Jurkat T cells) and in uptake of apoptotic neutrophils by alternative phagocytes (human glomerular mesangial cells and murine M phi elicited into the peritoneum or derived from bone marrow). Importantly, methylprednisolone-mediated enhancement of the uptake of apoptotic neutrophils did not trigger the release of the chemokines IL-8 and monocyte chemoattractant protein-1. Furthermore, longer-term potentiation by methylprednisolone was observed in maturing human monocyte-derived M phi, with greater increases in 5-day M phi uptake of apoptotic cells being observed the earlier glucocorticoids were added during monocyte maturation into M phi. We conclude that potentiation of nonphlogistic clearance of apoptotic leukocytes by phagocytes is a hitherto unrecognized property of glucocorticoids that has potential implications for therapies aimed at promoting the resolution of inflammatory diseases. 


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Tumor necrosis factor receptor expression and signal transduction in HIV-1-infected cells. 
OBJECTIVE: To examine the inter-relationship between HIV-1 infection and the cell surface receptors for tumor necrosis factor (TNF)-alpha, an immunoregulatory cytokine that can enhance HIV-1 replication. DESIGN: Infected promyelocytic and promonocytic cells were examined because they normally express both types of TNF receptors. METHODS: TNF receptor surface expression was determined by specific monoclonal antibody recognition and flow cytometry, and signal transduction was detected by gel shift analysis. HIV-1 activation and expression was quantitated by reverse transcriptase assay. RESULTS: In the OM-10.1 promyelocytic model of chronic infection, TNF-alpha-induced HIV-1 expression also resulted in a substantial increase in 75 kd TNF receptor (TR75) expression although 55 kD TNF receptor (TR55) levels were not dramatically altered. A series of uninfected parental HL-60 subclones all reduced TR75 surface expression in response to TNF-alpha treatment. Enhanced TR75 expression on OM-10.1 cells followed the same TNF-alpha-dose dependency as that observed for HIV-1 production. An increase in TR75 expression was also evident during the peak of an acute HIV-1 infection of HL-60 promyelocytes. Although TR55 expression was unaltered during TNF-alpha-induced HIV activation, this receptor was still involved in the viral activation process. Antibody cross-linking of TR55, in the absence of exogenous TNF-alpha, induced maximal HIV-1 expression, an up-modulation of surface TR75, and nuclear NF-kappa B activity in OM-10.1 cultures. Surprisingly, this was the case even when an antagonistic anti-TR55 antibody was used. Anti-TR55 antibody cross-linking in chronically infected U1 promonocytic cultures could only partially substitute for TNF-alpha-induced HIV-1 expression. CONCLUSIONS: Our results demonstrated that HIV-1 infection can selectively influence the surface expression of TNF receptors, potentially influencing its own expression and altering normal immunoregulatory signal transduction. 


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A chimeric type II/type I interleukin-1 receptor can mediate interleukin-1 induction of gene expression in T cells. 
The type I interleukin-1 receptor (IL-1R) is capable of transducing a signal resulting in promoter activation in T cells. This signal transduction is dependent on the cytoplasmic domain, which consists of 213 amino acids. In contrast to the type I receptor, the type II IL-1R has a small cytoplasmic tail, and it is not clear whether this receptor is a signal-transducing or a regulatory molecule. Here we report that the type II IL-1R does not mediate gene activation in Jurkat cells. However, a hybrid receptor composed of the extracellular and transmembrane regions of the human type II interleukin-1 fused to the cytoplasmic domain of the human type I IL-1R was capable of transducing a signal across the membrane resulting in a pattern of gene activation identical to that mediated by the type I IL-1R. Our results indicated that the extracellular domain of the type II IL-1R was capable of functionally interacting with interleukin-1 and transmitting the resulting signal to a heterologous cytoplasmic domain. 


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Evidence for normal vitamin D receptor messenger ribonucleic acid and genotype in absorptive hypercalciuria. 
Absorptive hypercalciuria (a stone-forming condition) is characterized by gut hyperabsorption of calcium, hypercalciuria, and reduced bone density. Inasmuch as these features implicate enhanced calcitriol action in gut and bone, we analyzed the vitamin D receptor (VDR) gene to ascertain whether an abnormality of this gene marks patients with intestinal hyperabsorption of calcium. We have compared the frequency of a restriction fragment length polymorphism (Bsm I) associated with different alleles of the VDR gene in a group of 33 well characterized absorptive hypercalciuric patients and a group of 36 normal race- and age-matched control subjects. There was no difference between the distribution of the VDR alleles in the patient population when compared with the normal population. The coding region of VDR messenger RNA was also normal, as determined by both DNA sequence analysis and chemical mismatch cleavage analysis of copy DNA from 11 index absorptive hypercalciuric patients. On the basis of these results, we propose that the enhanced intestinal calcium absorption invariably seen in absorptive hypercalciuria and attendant symptoms of this disorder are not attributable to mutations of the VDR and are not linked to a common VDR genotype. 


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Abnormalities of cyclic adenosine monophosphate signaling in platelets from untreated patients with bipolar disorder. 
BACKGROUND: Abnormalities in the cyclic adenosine monophosphate (cAMP)-dependent phosphorylation system have been recently reported in patients with bipolar disorder. We evaluated the immunoreactivity of the regulatory and catalytic subunits of cAMP-dependent protein kinase (protein kinase A) and 1 of its substrates, Rap1, in platelets from untreated euthymic, manic, and depressed patients with bipolar disorder and healthy subjects. METHODS: Platelets were collected from 112 drug-free patients with bipolar disorder (52 euthymic, 29 depressed, and 31 manic) and 62 healthy subjects. The levels of cAMP-dependent protein kinase and Rap1 were assessed by Western blot analysis, immunostaining, and computer-assisted imaging. RESULTS: The immunolabeling of the catalytic subunit of cAMP-dependent protein kinase was significantly different among groups (P<.001), with higher values in untreated depressed and manic patients with bipolar disorder compared with untreated euthymic patients with bipolar disorder and healthy subjects. No significant differences were found in the immunolabeling of the regulatory subunits (type I and type II) of cAMP-dependent protein kinase. The immunolabeling of Rap1 was significantly higher (P<.001) in untreated euthymic, depressed, and manic patients than in healthy persons. CONCLUSIONS: Levels of Rap1 and the catalytic subunit of cAMP-dependent protein kinase are altered in the platelets of bipolar patients. These findings may provide clues toward understanding the involvement of cAMP signaling in the pathogenesis of bipolar disorder. 


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Phorbol ester-induced mononuclear cell differentiation is blocked by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059. 
The purpose of this study was to evaluate whether the mitogen-activated protein kinase (MAPK) signaling pathway contributes to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mononuclear differentiation in the human myeloblastic leukemia ML-1 cells. Upon TPA treatment, the activity of ERK1 and ERK2 rapidly increased, with maximal induction between 1 and 3 h, while ERK2 protein levels remained constant. The activity of JNK1 was also significantly induced, with JNK1 protein levels increasing moderately during exposure to TPA. Treatment of cells with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK), inhibited TPA-induced ERK2 activity. Furthermore, PD98059 completely blocked the TPA-induced differentiation of ML-1 cells, as assessed by a number of features associated with mononuclear differentiation including changes in morphology, nonspecific esterase activity, phagocytic ability, NADPH oxidase activity, mitochondrial respiration, and c-jun mRNA inducibility. We conclude that activation of the MEK/ERK signaling pathway is necessary for TPA-induced mononuclear cell differentiation. 


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Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-alpha-induced apoptosis. 
Induction of apoptosis of mononucleated cells is a physiological process for regulating the intensity of the immune response. The female steroid hormones estrogen (E2) and progesterone (Prog) are known to modulate the reactivity of the immune system; recently it has been demonstrated that they can regulate induction of apoptosis of endothelial cells and osteoblasts. TNF-alpha-mediated induction of apoptosis has been well characterized in myeloid cells. We investigated whether E2 and Prog could interfere with TNF-alpha-induced apoptosis of the monoblastoid U937 cell line. Treatment with E2 or Prog increased survival and prevented apoptosis induced by TNF-alpha in both undifferentiated and macrophage-like PMA-differentiated U937 cells, as assessed by trypan blue exclusion cell counting, thymidine incorporation, AnnexinV labeling, followed by flow cytometry and DNA fragmentation studies. This effect can be associated with the activation of specific hormone receptors, since we observed the expression of the estrogen receptor alpha (ER-alpha), ER-beta, and progesterone receptor (PR) mRNAs; the ER-alpha protein expression was confirmed by immunocytochemical analysis. In addition, hormone-mediated survival against apoptosis was concentration dependent, reaching the half-maximal effect at 10 nM and blocked by the ER antagonist ICI 182,780 in undifferentiated cells, further supporting a receptor-mediated mechanism of cell survival. Other steroid receptor drugs such as Raloxifene, RU486, or the ICI 182,780 in PMA-differentiated cells displayed agonist activity by preventing TNF-alpha-induced apoptosis as efficiently as the hormones alone, providing further evidence to the notion that steroid receptor drugs may manifest agonist or antagonist activities depending on the cellular context in which they are studied. Treatment with E2 was also associated with a time-dependent decrease in the mRNA level of the proapoptotic Nip-2 protein, supporting the hypothesis that hormone responsiveness of U937 cells is mediated by target gene transcription. Together, these results demonstrate that ER and PR can be activated by endogenous or exogenous ligands to induce a genetic response that impairs TNF-alpha-induced apoptosis in U937 cells. The data presented here suggest that the female steroid receptors play a role in regulation of the immune response by preventing apoptosis of monoblastoid cells; this effect might have important consequences in the clinical use of steroid receptor drugs. --Vegeto, E., Pollio, G., Pellicciari, C., Maggi, A. Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-alpha-inuced apoptosis. 


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Diminished responses to IL-13 by human monocytes differentiated in vitro: role of the IL-13Ralpha1 chain and STAT6. 
The primary IL-13 receptor complex on human monocytes is believed to be a heterodimer comprised of the IL-4R alpha chain and the IL-2R gamma chain (gamma(c))-like molecule, IL-13R alpha1. mRNA levels for IL-13R alpha1, but not IL-4R alpha, were markedly decreased in in vitro monocyte-derived macrophages (MDMac), and with increasing time of monocytes in culture correlated with the loss of IL-13 regulation of lipopolysaccharide-induced TNF-alpha production. Analysis of cell lines Daudi and THP-1 that differentially express gamma(c) and IL-13R alpha1 showed that IL-13 can activate STAT6 in IL-13R alpha1-positive THP-1 cells but not in gamma(c)-positive, IL-13R alpha1-negative Daudi cells. IL-13 activation of STAT6 was reduced in MDMac which was associated with diminished IL-13-induced expression of CD23 and MHC class II. However, with reduced IL-13R alpha1 expression and low nuclear STAT6 activity, some IL-13-induced responses were unaltered in magnitude in MDMac. In the absence of functional IL-13R alpha1 and gamma(c), IL-13 must signal through an alternative receptor complex on MDMac. Experiments with a blocking antibody to IL-4R alpha showed that this chain remains an essential component of the IL-13 receptor complex on MDMac. 


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A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export. 
We recently developed an assay in which nuclear export of the shuttling transcription factor NFAT (nuclear factor of activated T cells) can be reconstituted in permeabilized cells with the GTPase Ran and the nuclear export receptor CRM1. We have now used this assay to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot hydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alone do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cytoplasmic periphery of the nuclear pore complex (NPC) in association with the p62 complex and Can/Nup214. RanGTP-dependent association of CRM1 with these nucleoporins was reconstituted in vitro. By biochemical fractionation and reconstitution, we showed that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC. Taken together, our results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that RanBP1 and probably RanBP2 are involved in the dissociation of nuclear export complexes from the NPC in a terminal step of transport. 


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1,25-Dihydroxyvitamin D3 receptor RNA: expression in hematopoietic cells. 
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] induces differentiation and inhibits proliferation of myeloid leukemic cells from various lines and patients; these effects are probably mediated through the 1,25(OH)2D3 receptor. Little is known of expression of 1,25(OH)2D3 receptor RNA in hematopoietic cells. We examined the expression and modulation of expression of 1,25(OH)2D3 receptor RNA in various proliferating and nonproliferating hematopoietic cells. Constitutive expression of 1,25(OH)2D3 receptor RNA was detected in various kinds of hematopoietic cells, including macrophages and activated T lymphocytes, as well as in cell lines KG-1 (myeloblasts), HL-60 (promyelocytes), ML-3 (myelomonoblasts), U937, THP-1 (monoblasts), K562 (erythroblasts), and S-LB1 (HTLV-1-transfected T lymphocytes). Receptor transcripts were 4.6 kilobases (kb), and no variant sizes were observed. All cell lines examined in this group also expressed 1,25(OH)2D3 receptors. Most B lymphocyte lines expressed negligible levels of 1,25(OH)2D3 receptor RNA and protein; however; analysis of a lymphoid/myeloid somatic hybrid suggested that suppression of expression of 1,25(OH)2D3 receptor RNA in B lymphocytes may be a dominant characteristic. HL-60 cells were cultured with 10(-7) mol/L 1,25(OH)2D3 for 24 to 72 hours, and levels of expression of 1,25(OH)2D3 receptor and its RNA were examined. Levels of RNA coding for the receptor were not modulated by exposure to high levels of ligand. Levels of occupied 1,25(OH)2D3 receptor protein increased in these HL-60 cells; but the total number of 1,25(OH)2D3 receptors decreased about 50% at 24 hours and returned toward normal at 72 hours. Steady-state levels of 1,25(OH)2D3 receptor RNA were not affected by terminal differentiation of HL-60 toward either granulocytes or macrophages. Nondividing macrophages from normal individuals also expressed 1,25(OH)2D3 receptor RNA. In contrast, nondividing peripheral blood lymphocytes from normal individuals did not express 1,25(OH)2D3 receptor RNA; with stimulation of proliferation of these cells, accumulation of 1,25(OH)2D3 receptor RNA increased markedly. Half-life (t1/2) of 1,25(OH)2D3 receptor RNA in T lymphocytes was short (1 hour) as determined by measuring decay of the message after addition of actinomycin D. Consistent with this short t1/2, accumulation of 1,25(OH)2D3 receptor RNA increased in cells as their protein synthesis was inhibited. Further studies are required to understand the physiologic role of 1,25(OH)2D3 receptors in myeloid cells and proliferating T lymphocytes. 


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Interleukin-2 induces tyrosine phosphorylation and nuclear translocation of stat3 in human T lymphocytes. 
An early biochemical event associated with T cell activation through the interleukin-2 receptor (IL-2R) is tyrosine phosphorylation of several intracellular substrates. The exact mechanism by which IL-2 regulates transcription of different genes is presently unknown. Here, we report that stimulation through the IL-2R induced tyrosine phosphorylation and subsequent nuclear translocation of stat3, a newly identified member of the signal transducers and activators of transcription (STAT) family of proteins. In contrast, stat1 proteins were not tyrosine phosphorylated after IL-2 ligation, whereas tyrosine-phosphorylated stat1 proteins (91 and 84 kDa proteins) were translocated to the nucleus following interferon-gamma treatment of HeLa cells. Apart from stat3, another cytoplasmic protein was tyrosine phosphorylated and subsequently translocated to the nucleus in response to IL-2. This protein had an apparent molecular mass of 84 kDa and was not recognized by stat3 or stat1 mAb or antisera. Since IL-2 induced nuclear translocation of the 84 kDa protein and stat3 followed identical kinetics, p84 is a candidate for a new, yet undefined, member of the STAT family. Taken together, we report that IL-2 induces tyrosine phosphorylation and subsequent nuclear translocation of stat3 and an as yet undefined 84-kDa protein in antigen-specific human T cell lines. 


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Activation of nuclear factor-kappaB via T cell receptor requires a Raf kinase and Ca2+ influx. Functional synergy between Raf and calcineurin. 
Signals transduced via the TCR activate the transcription factor nuclear factor-kappaB (NF-kappaB), which, in turn, is critical to the transcriptional induction of many genes important for the proliferation and expression of a differentiated phenotype. Treatment of T cells with the protein kinase C activator PMA in combination with Ca2+ ionophores mimics this process, and the two agents are often substituted for TCR stimulation, bypassing the TCR. Here we identify intracellular signaling components involved in activation of NF-kappaB following TCR stimulation. TCR signaling was triggered by treating Jurkat T cells with PHA or anti-CD3 Abs, and NF-kappaB activation was monitored by electrophoretic mobility shift assays and/or by kappaB-dependent reporter assays. Contrary to the idea that protein kinase C is involved in TCR-mediated activation of NF-kappaB, high doses of staurosporine did not interfere with activation of NF-kappaB by PHA, while the same dose of staurosporine completely blocked activation by PMA. PHA-induced kappaB-dependent reporter activity was, however, effectively blocked by a dominant negative form of Raf-1, suggesting a critical role for a Raf kinase. The TCR-mediated activation of NF-kappaB was also dependent on a Ca2+ influx, because the Ca2+ channel blocker, SK&F 96365, as well as other agents that prevented the Ca2+ influx, inhibited NF-kappaB activation. Cotransfection of a constitutively active form of calcineurin largely substituted for the Ca2+ requirement and reversed the blockade by SK&F 96365. Consistent with these observations, coexpression of constitutively active forms of Raf-1 and calcineurin synergistically induced kappaB-dependent reporter activity, suggesting a physiologically relevant functional interaction between the kinase and the phosphatase. 


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Increased proliferation, cytotoxicity, and gene expression after stimulation of human peripheral blood T lymphocytes through a surface ganglioside (GD3) [published erratum appears in J Immunol 1994 Jul 15;153(2):910] 
Previous studies have suggested that gangliosides have an important role in cell signaling and recognition. However, their specific function in these processes has not been clearly defined. A mAb, R24, that reacts specifically with a cell surface ganglioside (GD3) has been demonstrated to stimulate proliferation of T cells derived from human peripheral blood. In this study, we have investigated the mechanisms by which the R24 mAb affects T cell functions. We have observed that the R24 mAb stimulates GD3+ T cell proliferation, cytotoxicity, and surface marker expression of IL-2R alpha-chain, IL-2R beta-chain, HLA-DR, CD11a, and CD11c. Additionally, IFN-gamma activity but not IL-1, IL-2, or IL-4 activity was present in culture supernatants 72 h after R24 stimulation. In some donors, increased IL-6 and TNF-alpha activity also was detected after R24 treatment. Furthermore, R24 treatment resulted in translocation of c-rel, but little or no NF kappa B p50 or p65, from the cytoplasm to the nucleus and an increase of NF kappa B binding complexes containing c-rel and p50. This treatment also caused increased tyrosine phosphorylation of specific protein substrates. R24-stimulated increases in proliferation, cytotoxicity, and cell surface protein expression could be blocked by cyclosporin and staurosporin, indicating that cyclophilin/calcineurin and protein kinase C may be involved in the R24 signaling pathway. Additionally, herbimycin A, a tyrosine kinase inhibitor, blocked the R24-stimulated increase in proliferation but not cytotoxicity at concentrations consistent with specificity for tyrosine kinases. These results suggest that multiple biochemical pathways are involved in the activation of human T cells by R24. 


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Nuclear NF-ATp is a hallmark of unstimulated B cells from B-CLL patients. 
B lymphocytes from the peripheral blood of patients with chronic lymphocytic leukaemia (CLL) were analysed for the nuclear presence and DNA binding of a panel of transcription factors which are involved in the gene control of lymphoid cells. The following transcription factors were studied: the Octamer factors Oct-1 and Oct-2, members of the AP-1 factor family, NF-AT factors, in particular NF-ATp, and members of the Rel/NF-kB family. We show that the constitutive nuclear translocation of NF-ATp, a member of the growing family of NF-AT factors, is a hallmark of nonstimulated B cells from CLL patients that distinguishes B-CLL cells from 'normal' B lymphocytes. Constitutive nuclear appearance was also observed for NF-kB2/p52. Constitutive binding of further factor proteins to DNA, such as JunD, c-Fos and FosB, was detected in several patients whereas the localisation and DNA binding of other factors such as c-Jun, RelA/p65 and c-Rel was unaltered. It is remarkable that in B-CLL cells the nuclear appearance and DNA binding of specific transcription factors is dramatically affected whereas other members of the same factor family remained unaltered in these leukemic cells. It remains to be shown which molecular events lead to the specific 'pre-activation', i.e. constitutive nuclear translocation and DNA binding, of these members of NF-AT, NF-kB and AP-1 factor families. 


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MDS1/EVI1 enhances TGF-beta1 signaling and strengthens its growth-inhibitory effect but the leukemia-associated fusion protein AML1/MDS1/EVI1, product of the t(3;21), abrogates growth-inhibition in response to TGF-beta1. 
MDS1/EVI1, located on chromosome 3 band q26, encodes a zinc-finger DNA-binding transcription activator not detected in normal hematopoietic cells but expressed in several normal tissues. MDS1/EVI1 is inappropriately activated in myeloid leukemias following chromosomal rearrangements involving band 3q26. The rearrangements lead either to gene truncation, and to expression of the transcription repressor EVI1, as seen in the t(3;3)(q21;q26) and inv(3)(q21q26), or to gene fusion, as seen in the t(3;21)(q26;q22) which results in the fusion protein AML1/MDS1/EVI1. This fusion protein contains the DNA-binding domain of the transcription factor AML1 fused in-frame to the entire MDS1/EVI1 with the exclusion of its first 12 amino acids. In this report, we have analyzed the response of the hematopoietic precursor cell line 32Dcl3, expressing either the normal protein MDS1/EVI1 or the fusion protein AML1/MDS1/EVI1, to factors that control cell differentiation or cell replication. The 32Dcl3 cells are IL-3-dependent for growth and they differentiate into granulocytes when exposed to G-CSF. They are growth-inhibited by TGF-beta1. We show that whereas the expression of MDS1/EVI1 has no effect on granulocytic differentiation induced by G-CSF, expression of AML1/MDS1/EVI1 blocks differentiation resulting in cell death. This effect is similar to that previously described by others for 32Dcl3 cells that express transgenic Evil. Furthermore, we show that whereas the expression of the fusion protein AML1/MDS1/EVI1 completely abrogates the growth-inhibitory effect of TGF-beta1 and allows 32Dcl3 cells to proliferate, expression of the normal protein MDS1/EVI1 has the opposite effect, and it strengthens the response of cells to the growth-inhibitory effect of TGF-beta1. By using the yeast two-hybrid system, we also show that EVI1 (contained in its entirety in MDS1/EVI1 and AML1/MDS1/EVI1) physically interacts with SMAD3, which is an intracellular mediator of TGF-beta1 signaling. Finally, we have correlated the response of the cells to G-CSF or TGF-beta1 with the ability of the normal and fusion proteins to activate or repress promoters which they can directly regulate by binding to the promoter site. We propose that mutations of MDS1/EVI1 either by gene truncation resulting in the transcription repressor EVI1 or by gene fusion to AML1 lead to an altered cellular response to growth and differentiation factors that could result in leukemic transformation. The different response of myeloid cells ectopically expressing the normal or the fusion protein to G-CSF and TGF-beta1 could depend on the different transactivation properties of these proteins resulting in divergent expression of downstream genes regulated by the two proteins. 


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[Glucocorticoid receptors in peripheral blood lymphocytes of patients with bronchial asthma] 
Quantitation of glucocorticoid receptors (GCR) and the study of their affinity for glucocorticosteroids (GCS) were made in peripheral blood lymphocytes of bronchial asthma (BA) patients in consideration of GCR treatment and serum levels of endogenous cortisol. It is stated that GCR of healthy controls and GCS-untreated patients outnumbered those of cortisol-dependent BA patients on hormone therapy. Following discontinuation of glucocorticoid drugs GCR count in cortisol-dependent BA tends to rise. Endogenous cortisol has no effect on GCR level estimated by 3H-triamcinolone acetonide. 


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Does activation of the TAL1 gene occur in a majority of patients with T-cell acute lymphoblastic leukemia? A pediatric oncology group study. 
Almost 25% of patients with T-cell acute lymphoblastic leukemia (T-ALL) have tumor-specific rearrangements of the TAL1 gene. Although TAL1 expression has not been observed in normal lymphocytes, TAL1 gene products are readily detected in leukemic cells that harbor a rearranged TAL1 allele. Hence, it has been proposed that ectopic expression of TAL1 promotes the development of T-ALL. In this report, we show that TAL1 is expressed in the leukemic cells of most patients with T-ALL, including many that do not display an apparent TAL1 gene alteration. A polymorphic dinucleotide repeat in the transcribed sequences of TAL1 was used to determine the allele specificity of TAL1 transcription in primary T-ALL cells. Monoallelic expression of TAL1 was observed in the leukemic cells of all patients (8 of 8) bearing a TAL1 gene rearrangement. In the leukemic cells of patients without detectable TAL1 rearrangements, TAL1 transcription occurred in either a monoallelic (3 of 7 patients) or a biallelic (4 of 7 patients) fashion. Thus, TAL1 activation in these patients may result from subtle alterations in cis-acting regulatory sequences (affecting expression of a single TAL1 allele) or changes in trans-acting factors that control TAL1 transcription (affecting expression of both TAL1 alleles). 


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BCL-6 and the molecular pathogenesis of B-cell lymphoma. 
The results presented identify the first genetic lesion associated with DLCL, the most clinically relevant form of NHL. Although no proof yet exists of a role for these lesions in DLCL pathogenesis, the feature of the BCL-6 gene product, its specific pattern of expression in B cells, and the clustering of lesions disrupting its regulatory domain strongly suggest that deregulation of BCL-6 expression may contribute to DLCL development. A more precise definition of the role of BCL-6 in normal and neoplastic B-cell development is the goal of ongoing study of transgenic mice engineered either to express BCL-6 under heterologous promoters or lacking BCL-6 function due to targeted deletions. In addition to contributing to the understanding of DLCL pathogenesis, the identification of BCL-6 lesions may have relevant clinical implications. DLCL represent a heterogeneous group of neoplasms which are treated homogeneously despite the fact that only 50% of patients experience long-term disease-free survival (Schneider et al. 1990). The fact that BCL-6 rearrangements identify biologically and clinically distinct subsets of DLCL suggests that these lesions may be useful as markers in selection of differential therapeutic strategies based on different risk groups. Furthermore, the BCL-6 rearrangements can be used to identify and monitor the malignant clone with sensitive PCR-based techniques. Since clinical remission has been observed in a significant fraction of DLCL cases, these markers may serve as critical tools for sensitive monitoring of minimal residual disease and early diagnosis of relapse (Gribben et al. 1993). 


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Attenuation of gamma interferon-induced tyrosine phosphorylation in mononuclear phagocytes infected with Leishmania donovani: selective inhibition of signaling through Janus kinases and Stat1. 
The induction of gene transcription in response to gamma interferon is impaired in mononuclear phagocytes infected with Leishmania donovani, and the mechanisms involved are not fully understood. The changes in gene expression brought about by gamma interferon are thought to involve transient increases in the activities of cellular protein tyrosine kinases, including the Janus kinases Jak1 and Jak2, leading to tyrosine phosphorylation of the transcription factor Stat1. To investigate the mechanisms accounting for the impaired responses to gamma interferon, a model system for examining overall changes in protein tyrosine phosphorylation, activation of Jak1 and Jak2 and phosphorylation of Stat1 was developed in phorbol 12-myristate 13-acetate-differentiated U-937 cells. Analysis of whole-cell lysates by antiphosphotyrosine immunoblotting showed that incubation with gamma interferon brought about specific increases in phosphotyrosine labeling of several proteins. Increased labeling of these proteins occurred to similar extents in control cells and in cells that had been infected with L. donovani for 16 h. Jak1, Jak2, and Stat1 were immunoprecipitated from control and interferon-treated cells, and tyrosine phosphorylation of these proteins, detected by antiphosphotyrosine immunoblotting was used to measured their activation. Tyrosine phosphorylation of Jak1, Jak2, and Stat1 increased markedly, in a dose-dependent manner, in U-937 cells incubated with gamma interferon. In contrast, in cells infected with L. donovani, tyrosine phosphorylation of Jak1, Jak2, and Stat1 was markedly impaired. This effect was dependent upon the duration of exposure to L. donovani and was maximal and complete at 16 h. Results similar to those observed with U-937 cells were also obtained with human peripheral blood monocytes. These findings indicate that infection of human mononuclear phagocytes with L. donovani leads to impaired gamma interferon-mediated tyrosine phosphorylation and selective effects on the Jak-Stat1 pathway. Unresponsiveness to gamma interferon for activation of this pathway may explain impaired transcriptional responses in leishmania-infected cells. 


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Multiple proteins interact with the nuclear inhibitory protein repressor element in the human interleukin-3 promoter. 
T cell expression of interleukin 3 (IL-3) is directed by positive and negative cis-acting DNA elements clustered within 300 base pairs of the transcriptional start site. A strong repressor element, termed nuclear inhibitory protein (NIP), was previously mapped to a segment of the IL-3 promoter between nucleotides -271 and -250. Functional characterization of this element demonstrates that it can mediate repression when linked in cis to a heterologous promoter. DNA binding experiments were carried out to characterize the repressor activity. Using varying conditions, three distinct complexes were shown to interact specifically with the NIP region, although only one correlates with repressor activity. Complex 1 results from binding of a ubiquitous polypeptide that recognizes the 3' portion of this sequence and is not required for repression. Complex 2 corresponds to binding of transcription factor (upstream stimulatory factor) to an E-box motif in the 5' portion of the NIP region. DNA binding specificity of complex 3 overlaps with that of upstream stimulatory factor but is clearly distinct. To determine which of the latter two complexes represents NIP activity, we incorporated small alterations into the NIP site of an IL-3 promoter-linked reporter construct and examined their effects on NIP-mediated repression. Functional specificity for repression matches the DNA binding specificity of complex 3; both repressor activity and complex 3 binding require the consensus sequence CTCACNTNC. 


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Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes. 
Interleukin 12 (IL-12) is an important immunoregulatory cytokine whose receptor is a member of the hematopoietin receptor superfamily. We have recently demonstrated that stimulation of human T and natural killer cells with IL-12 induces tyrosine phosphorylation of the Janus family tyrosine kinase JAK2 and Tyk2, implicating these kinases in the immediate biochemical response to IL-12. Recently, transcription factors known as STATs (signal transducers and activators of transcription) have been shown to be tyrosine phosphorylated and activated in response to a number of cytokines that bind hematopoietin receptors and activate JAK kinases. In this report we demonstrate that IL-12 induces tyrosine phosphorylation of a recently identified STAT family member, STAT4, and show that STAT4 expression is regulated by T-cell activation. Furthermore, we show that IL-12 stimulates formation of a DNA-binding complex that recognizes a DNA sequence previously shown to bind STAT proteins and that this complex contains STAT4. These data, and the recent demonstration of JAK phosphorylation by IL-12, identify a rapid signal-transduction pathway likely to mediate IL-12-induced gene expression. 


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Heterogeneous expression of Epstein-Barr virus latent proteins in endemic Burkitt's lymphoma. 
Epstein-Barr virus (EBV)-infected cells may sustain three distinct forms of virus latency. In lymphoblastoid cell lines, six EBV-encoded nuclear antigens (EBNA1, 2, 3A, 3B, 3C, -LP), three latent membrane proteins (LMP1, 2A, 2B), and two nuclear RNAs (EBERs) are expressed. This form of latency, termed latency III, is also encountered in some posttransplant lymphoproliferative disorders. In EBV-positive cases of Hodgkin's disease, the EBERs, EBNA1, and the LMPs are expressed (latency II), whereas in Burkitt's lymphoma (BL) only the EBERs and EBNA1 have been detected (latency I). We have studied the expression of EBV proteins in 17 cases of EBV-positive endemic BL by immunohistology. Expression of LMP1 was seen in variable proportions of tumor cells in two cases and EBNA2 was detected in some tumor cells in three other cases. Also, the BZLF1 trans-activator protein was expressed in a few tumor cells in 6 cases, indicating entry into the lytic cycle. A phenotypic drift from latency I to latency III has been observed previously in some BL cell lines. Our results suggest that a similar phenomenon may occur in BL in vivo and indicate that the operational definition of EBV latencies is not easily applied to human tumors. 


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Expression of erythroid-specific genes in acute megakaryoblastic leukaemia and transient myeloproliferative disorder in Down's syndrome. 
Acute megakaryoblastic leukaemia (M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. To clarify properties of the blast cells in M7 and TMD cases, we examined erythroid markers expression in blasts from six cases with M7 and seven cases with TMD in this study. Erythroid-specific mRNAs encoding gamma-globin and erythroid delta-aminolevulinate synthase were found to be expressed in blasts from most of these cases, indicating that majorities of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. We also found that mRNAs encoding GATA-1 and GATA-2 are expressed in all these cases. These results suggest that M7 blasts and TMD blasts correspond to the erythroid/megakaryocytic bipotential progenitor cells. 


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Growth regulation and cellular changes during differentiation of human prostatic cancer LNCaP cells as induced by T lymphocyte-conditioned medium. 
Human prostatic epithelial cells from an androgen-dependent LNCaP cell line were examined in response to conditioned medium (CM) derived from phytohemagglutinin (PHA)-stimulated lymphocytes. Addition of CM caused a greater than 70% reduction of cell proliferation by cell counting and cell cycle. These cells showed G1 phase arrest and the clonogenicity was reduced. The growth-modulating effect was dose-dependent and not due to cell lysis or apoptosis. The binding of androgen to androgen receptor on these cells showed approximately 50% reduction, underlining a proliferation reduction mechanism. The prostate-specific antigen (PSA) was downregulated to approximately 75% during the process. Cell morphology showed dendritic processes extending from cytoplasm and other neuroendocrine cell characteristics. The expression of several cytoskeleton and intracellular proteins increased as determined by immunostaining on slides and by ELISA procedures. These included vimentin, correlating to cell shape changes, cytokeratins 8 and 18, associated with differentiated cell types of prostate epithelia, and neuron-specific enolase and serotonin, associated with neuroendocrine cells. From these cellular changes, we can infer that the cell growth was modulated along with induction of terminal differentiation. Activated T cells were demonstrated to be important in providing the modulating activity. This growth modulator was semipurified and had an estimated molecular weight 13,000 to 24,000 Da. The activity was determined to be distinct from TGF, TNF, and some commonly known lymphokines. The interaction between lymphoid and prostatic cells in growth and development is described. 


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Two distinct forms of active transcription factor CREB (cAMP response element binding protein). 
Mammalian cells express two distinct forms of transcription factor CREB (cAMP response element binding protein) that are apparently the products of alternative splicing of the CREB gene transcript. The two proteins differ by a 14-amino acid serine-rich insertion present in one of the CREB isoforms. We show that both CREB isoforms are expressed in many cell types and mammalian species. Both encode proteins that bind specifically to a cAMP response element in vitro. As expected for proteins of this class, the CREB proteins bind DNA as dimers. Both proteins impart cAMP-regulated transcriptional activity to a heterologous DNA-binding domain, showing that cAMP directly modulates the transcriptional stimulatory activity of CREB. The presence of multiple CREB isoforms with identical DNA-binding specificities but differences in the presumed regulatory domain raises the possibility that CREB proteins may be able to integrate distinct regulatory signals at the level of gene transcription. 


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Transcription factors as targets for oxidative signalling during lymphocyte activation. 
We previously have demonstrated a requirement for oxidative events during cell cycle entry in T lymphocytes and have hypothesised that reactive oxygen species may act as intracellular signalling agents during lymphocyte activation. In the current study, cysteamine, an aminothiol compound with antioxidant activity, has been used to further investigate the role of oxidative signalling during lymphocyte activation. Treatment of normal human peripheral blood lymphocytes with cysteamine in vitro was found to inhibit proliferation in a dose-dependent manner, with essentially complete inhibition occurring at a dose of 400 microM. This inhibitory effect was limited to the first 2 h after mitogenic activation, localizing the time-frame of action of cysteamine to within the commitment period. It therefore was of interest to establish which, if any, commitment events were affected by oxidative signalling during cell cycle entry. Taking the IL-2 gene as a candidate, we examined the effect of cysteamine treatment on early gene expression during lymphocyte activation, and on the activity of transcription factors AP-1, NF-kappa B, NF-AT and Oct1, whose functions are required for expression of the IL-2 mRNA. Cysteamine treatment inhibited both expression of the IL-2 mRNA and secretion of IL-2 into the culture medium. The inhibitory effect of cysteamine may be mediated at least in part by an effect on transcription factor function, as the DNA binding activities of AP-1 and NF-kappa B extracted from mitogen-stimulated cells were significantly inhibited by cysteamine treatment. Interestingly, Oct1 and NF-AT DNA binding activity were not affected by cysteamine treatment, suggesting that oxidative signalling processes operate in a selective manner. The identification of regulatory proteins, such as transcription factors, as molecular targets for oxidative signalling provides further evidence to implicate oxidative signalling as being intimately involved in the G0 to G1 phase transition in T lymphocytes. 


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Constitutively activated Jak-STAT pathway in T cells transformed with HTLV-I. 
Human T cell lymphotropic virus I (HTLV-I) is the etiological agent for adult T cell leukemia and tropical spastic paraparesis (also termed HTLV-I-associated myelopathy). HTLV-I-infected peripheral blood T cells exhibit an initial phase of interleukin-2 (IL-2)-dependent growth; over time, by an unknown mechanism, the cells become IL-2-independent. Whereas the Jak kinases Jak1 and Jak3 and the signal transducer and activator of transcription proteins Stat3 and Stat5 are activated in normal T cells in response to IL-2, this signaling pathway was constitutively activated in HTLV-I-transformed cells. In HTLV-I-infected cord blood lymphocytes, the transition from IL-2-dependent to IL-2-independent growth correlated with the acquisition of a constitutively activated Jak-STAT pathway, which suggests that this pathway participates in HTLV-I-mediated T cell transformation. 


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Characterization of a CD43/leukosialin-mediated pathway for inducing apoptosis in human T-lymphoblastoid cells. 
The monoclonal antibody (mAb) J393 induces apoptosis in Jurkat T-cells. NH2-terminal amino acid sequence analysis identified the 140-kDa surface antigen for mAb J393 as CD43/leukosialin, the major sialoglycoprotein of leukocytes. While Jurkat cells co-expressed two discrete cell-surface isoforms of CD43, recognized by mAb J393 and mAb G10-2, respectively, only J393/CD43 signaled apoptosis. J393/CD43 was found to be hyposialylated, bearing predominantly O-linked monosaccharide glycans, whereas G10-2/CD43 bore complex sialylated tetra- and hexasaccharide chains. Treatment with soluble, bivalent mAb J393 killed 25-50% of the cell population, while concomitant engagement of either the CD3.TcR complex or the integrins CD18 and CD29 significantly potentiated this effect. Treatment of Jurkat cells with mAb J393 induced tyrosine phosphorylation of specific protein substrates that underwent hyperphosphorylation upon antigen receptor costimulation. Tyrosine kinase inhibition by herbimycin A diminished J393/CD43-mediated apoptosis, whereas inhibition of phosphotyrosine phosphatase activity by bis(maltolato)oxovanadium-IV enhanced cell death. Signal transduction through tyrosine kinase activation may lead to altered gene expression, as J393/CD43 ligation prompted decreases in the nuclear localization of the transcriptional regulatory protein NF-kappaB and proteins binding the interferon-inducible regulatory element. Since peripheral blood T-lymphocytes express cryptic epitopes for mAb J393, these findings demonstrate the existence of a tightly regulated CD43-mediated pathway for inducing apoptosis in human T-cell lineages. 


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1,25-Dihydroxyvitamin D3 induces differentiation of a retinoic acid-resistant acute promyelocytic leukemia cell line (UF-1) associated with expression of p21(WAF1/CIP1) and p27(KIP1). 
Retinoic acid (RA) resistance is a serious problem for patients with acute promyelocytic leukemia (APL) who are receiving all-trans RA. However, the mechanisms and strategies to overcome RA resistance by APL cells are still unclear. The biologic effects of RA are mediated by two distinct families of transcriptional factors: RA receptors (RARs) and retinoid X receptors (RXRs). RXRs heterodimerize with 1, 25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor (VDR), enabling their efficient transcriptional activation. The cyclin-dependent kinase (cdk) inhibitor p21(WAF1/CIP1) has a vitamin D3-responsive element (VDRE) in its promoter, and 1,25(OH)2D3 enhances the expression of p21(WAF1/CIP1) and induces differentiation of selected myeloid leukemic cell lines. We have recently established a novel APL cell line (UF-1) with features of RA resistance. 1,25(OH)2D3 can induce growth inhibition and G1 arrest of UF-1 cells, resulting in differentiation of these cells toward granulocytes. This 1, 25(OH)2D3-induced G1 arrest is enhanced by all-trans RA. Also, 1, 25(OH)2D3 (10(-10) to 10(-7) mol/L) in combination with RA markedly inhibits cellular proliferation in a dose- and time-dependent manner. Associated with these findings, the levels of p21(WAF1/CIP1) and p27(KIP1) mRNA and protein increased in these cells. Northern blot analysis showed that p21(WAF1/CIP1) and p27(KIP1) mRNA and protein increased in these cells. Northern blot analysis showed that p21(WAF1/CIP1) and p27(KIP1) transcripts were induced after 6 hours' exposure to 1,25(OH)2D3 and then decreased to basal levels over 48 hours. Western blot experiments showed that p21(WAF1/CIP1) protein levels increased and became detectable after 12 hours of 1,25(OH)2D3 treatment and induction of p27(KIP1) protein was much more gradual and sustained in UF-1 cells. Interestingly, the combination of 1, 25(OH)2D3 and RA markedly enhanced the levels of p27(KIP1) transcript and protein as compared with levels induced by 1, 25(OH)2D3 alone. In addition, exogenous p27(KIP1) expression can enhance the level of CD11b antigen in myeloid leukemic cells. In contrast, RA alone can induce G1 arrest of UF-1 cells; however, it did not result in an increase of p21(WAF1/CIP1) and p27(KIP1) transcript and protein expression in RA-resistant cells. Taken together, we conclude that 1,25(OH)2D3 induces increased expression of cdk inhibitors, which mediates a G1 arrest, and this may be associated with differentiation of RA-resistant UF-1 cells toward mature granulocytes. 


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Effects of interleukin-10 on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide. 
Deactivation of mononuclear phagocytes is critical to limit the inflammatory response but can be detrimental in the face of progressive infection. We compared the effects of the deactivating cytokine interleukin 10 (IL-10) on human peripheral blood mononuclear cell (PBMC) responses to lipopolysaccharide (LPS), Cryptococcus neoformans, and Candida albicans. IL-10 effected dose-dependent inhibition of tumor necrosis factor alpha (TNF-alpha) release in PBMC stimulated by LPS and C. neoformans, with significant inhibition seen with 0.1 U/ml and greater than 90% inhibition noted with 10 U/ml. In contrast, even at doses as high as 100 U/ml, IL-10 inhibited TNF-alpha release in response to C. albicans by only 50%. IL-10 profoundly inhibited release of IL-1beta from PBMC stimulated by all three stimuli. TNF-alpha mRNA and release was inhibited even if IL-10 was added up to 8 h after cryptococcal stimulation. In contrast, inhibition of IL-1 beta mRNA was of lesser magnitude and occurred only when IL-10 was added within 2 h of cryptococcal stimulation. IL-10 inhibited translocation of NF-kappaB in response to LPS but not the fungal stimuli. All three stimuli induced IL-10 production in PBMC, although over 10-fold less IL-10 was released in response to C. neoformans compared with LPS and C. albicans. Thus, while IL-10 has deactivating effects on PBMC responses to all three stimuli, disparate stimulus- and response-specific patterns of deactivation are seen. Inhibition by IL-10 of proinflammatory cytokine release appears to occur at the level of gene transcription for TNF-alpha and both transcriptionally and posttranscriptionally for IL-1beta. 


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The zinc finger transcription factor Egr-1 is essential for and restricts differentiation along the macrophage lineage. 
We have isolated cDNA clones of myeloid differentiation primary response (MyD) genes, activated in the absence of de novo protein synthesis following induction for differentiation along either the macrophage or granulocyte lineage in human myeloblastic leukemia HL-60 cells. One cDNA clone of a primary response gene, expressed upon macrophage differentiation, encoded for Egr-1, a zinc finger transcription factor. The Egr-1 gene was observed to be transcriptionally silent in HL-60 cells, but active in U-937 and M1 cells, the latter two being predetermined for macrophage differentiation. Egr-1 antisense oligomers in the culture media blocked macrophage differentiation in both myeloid leukemia cell lines and normal myeloblasts. HL-60 cells constitutively expressing an Egr-1 transgene (HL-60Egr-1) could be induced for macrophage, but not granulocyte, differentiation. These observations indicate that expression of Egr-1 is essential for and restricts differentiation of myeloblasts along the macrophage lineage. 


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[Glucocorticoid receptors on human peripheral mononuclear and polymorphonuclear leucocytes: changes in patients with yang-deficiency] 
It was found that, in former works, the glucocorticoid receptors (GCR) on peripheral mixed leucocytes in patients with Yang-deficiency were decreased. In this work, the mixed leucocytes were further separated into mononuclear (MNL) and polymorphonuclear (PML) leucocytes, and GCR were determined in each part of leucocytes. GCR on MNL and PML in 6 Yang deficient patients were 3473 +/- 413 and 4433 +/- 651 sites/cell respectively, statistically significant from the normal control group (4462 +/- 962 and 5622 +/- 782 sites/cell respectively, P less than 0.05). GCR on MNL, PML and mixed leucocytes in 5 patients were determined simultaneously, and all lowered from the control group. The results were 3369 +/- 370, 4986 +/- 419 and 4524 +/- 852 sites/cell respectively, with the lowest GCR on MNL and highest on PML. 


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p70(s6k) integrates phosphatidylinositol 3-kinase and rapamycin-regulated signals for E2F regulation in T lymphocytes. 
In T lymphocytes, the hematopoietic cytokine interleukin-2 (IL-2) uses phosphatidylinositol 3-kinase (PI 3-kinase)-induced signaling pathways to regulate E2F transcriptional activity, a critical cell cycle checkpoint. PI 3-kinase also regulates the activity of p70(s6k), the 40S ribosomal protein S6 kinase, a response that is abrogated by the macrolide rapamycin. This immunosuppressive drug is known to prevent T-cell proliferation, but the precise point at which rapamycin regulates T-cell cycle progression has yet to be elucidated. Moreover, the effects of rapamycin on, and the role of p70(s6k) in, IL-2 and PI 3-kinase activation of E2Fs have not been characterized. Our present results show that IL-2- and PI 3-kinase-induced pathways for the regulation of E2F transcriptional activity include both rapamycin-resistant and rapamycin-sensitive components. Expression of a rapamycin-resistant mutant of p70(s6k) in T cells could restore rapamycin-suppressed E2F responses. Thus, the rapamycin-controlled processes involved in E2F regulation appear to be mediated by p70(s6k). However, the rapamycin-resistant p70(s6k) could not rescue rapamycin inhibition of T-cell cycle entry, consistent with the involvement of additional, rapamycin-sensitive pathways in the control of T-cell cycle progression. The present results thus show that p70(s6k) is able to regulate E2F transcriptional activity and provide direct evidence for the first time for a link between IL-2 receptors, PI 3-kinase, and p70(s6k) that regulates a crucial G1 checkpoint in T lymphocytes. 


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USF/c-Myc enhances, while Yin-Yang 1 suppresses, the promoter activity of CXCR4, a coreceptor for HIV-1 entry. 
Transcription factors USF1 and USF2 up-regulate gene expression (i.e., HIV-1 long terminal repeats) via interaction with an E box on their target promoters, which is also a binding site for c-Myc. The c-Myc oncoprotein is important in control of cellular proliferation and differentiation, while Yin-Yang 1 (YY1) has been shown to control the expression of a number of cellular and viral genes. These two proteins physically interact with each other and mutually inhibit their respective biological functions. In this study, we show that USF/c-Myc up-regulates, while YY1 down-regulates the promoter activity of CXCR4, a coreceptor for T cell-tropic HIV-1 entry. We have identified an E box around -260 and a YY1 binding site around -300 relative to the transcription start site. Mutation of the E box abolished USF/c-Myc- mediated up-regulation of CXCR4 promoter activity, and mutation of the YY1 binding site was associated with unresponsiveness to YY1-mediated inhibition. These data suggest that USF/c-Myc and YY1 may play an important role in the HIV-1-replicative cycle, by modulating both the viral fusion/entry process and viral expression. 


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The transcriptionally active factors mediating the effect of the HTLV-I Tax transactivator on the IL-2R alpha kappa B enhancer include the product of the c-rel proto-oncogene. 
The transactivator HTLV-I Tax activates the promoter of the gene coding for the interleukin 2 alpha-chain receptor (IL-2R alpha) via a kappa B site that can bind several protein species of the rel family. Tax1 strongly activates the enhancer activity of this motif, in both epithelial HeLa and lymphoid Jurkat cells. This activation was not observed in undifferentiated embryocarcinoma F9 cells. Overexpression of the p50, p65 and Rel proteins in these cells showed that significant activation of the IL-2R alpha kappa B site was observed only with Rel and Rel plus p65. Moreover, whereas both Tax and phorbol 12-myristate 13-acetate (PMA) are able to efficiently induce the binding of NF-kappa B to the IL-2R alpha kappa B site, PMA is functionally inactive. Using the DNA affinity precipitation assay, we observed that Tax1 is able to efficiently induce the binding of Rel, whereas PMA is not. This established a clear difference between both stimuli, indicating that Rel is the functionally active factor. We conclude from these results that the functional activity of members of the rel family is regulated by their interaction with DNA and that Rel can be a potent transcriptional activator on specific kappa B sites. 


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Phenotypic and functional studies of leukocytes in human endometrium and endometriosis. 
The aetiology of endometriosis, a common and disabling disorder, is presently unknown, although immune dysfunction could allow ectopic endometrial fragments to survive outside the uterine cavity. These studies investigate the relationship between leukocyte populations, steroid hormone receptor expression, proliferative activity, bcl-2 expression and apoptosis in eutopic and ectopic endometrium from women with endometriosis or adenomyosis at different phases of the menstrual cycle. Significantly increased oestrogen receptor expression, bcl-2 expression and numbers of CD8+ leukocytes were found in ectopic compared with eutopic endometrium in endometriosis, and CD56+ endometrial granulated lymphocytes (eGLs) were significantly reduced in ectopic endometrium. Apoptotic cells were rarely found in control and subject endometria. In contrast with endometriosis, adenomyotic lesions showed identical steroid hormone receptor expression, proliferative activity, bcl-2 expression and leukocyte subpopulations to eutopic endometrium, indicating different aetiologies for these disorders. The unusual CD56+ CD16- eGLs present in large numbers in late secretory phase eutopic endometrium were highly purified (>98%) by immunomagnetic separation. Except for a negligible cytotoxic activity of eGLs from early proliferative samples, cytotoxic activity of eGLs from non-pregnant endometrium during the menstrual cycle was comparable with those in peripheral blood, predominantly CD56+ CD16+ natural killer cells. eGLs from non-pregnant endometrium and early pregnancy showed a variable proliferative response to 5 and 100 U/ml interleukin-2 over 48-h and 120-h time courses. eGLs are evidently functionally important in the eutopic endometrium. Their absence in endometriotic lesions together with increased CD+8 T-cell numbers and increased oestrogen receptor and bcl-2 expression may have significant effects on the development and progression of endometriosis. 


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Constitutive activation of an epithelial signal transducer and activator of transcription (STAT) pathway in asthma. 
Cytokine effects on immunity and inflammation often depend on the transcription factors termed signal transducers and activators of transcription (STATs), so STAT signaling pathways are candidates for influencing inflammatory disease. We reasoned that selective IFN responsiveness of the first STAT family member (Stat1) and Stat1-dependent immune-response genes such as intercellular adhesion molecule-1 (ICAM-1), IFN regulatory factor-1 (IRF-1), and Stat1 itself in airway epithelial cells provides a basis for detecting cytokine signaling abnormalities in inflammatory airway disease. On the basis of nuclear localization and phosphorylation, we found that epithelial Stat1 (but not other control transcription factors) was invariably activated in asthmatic compared with normal control or chronic bronchitis subjects. Furthermore, epithelial levels of activated Stat1 correlated with levels of expression for epithelial ICAM-1, IRF-1, and Stat1, and in turn, ICAM-1 levels correlated with T-cell accumulation in tissue. However, only low levels of IFN-gamma or IFN-gamma-producing cells were detected in airway tissue in all subjects. The results therefore provide initial evidence linking abnormal behavior of STAT pathways for cytokine signaling to the development of an inflammatory disease. In that context, the results also change the current scheme for asthma pathogenesis to one that must include a localized gain in transcriptional signal ordinarily used for a T helper 1-type cytokine (IFN-gamma) in combination with allergy-driven overproduction of T helper 2-type cytokines. 


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Protein kinase B (c-Akt), phosphatidylinositol 3-kinase, and STAT5 are activated by erythropoietin (EPO) in HCD57 erythroid cells but are constitutively active in an EPO-independent, apoptosis-resistant subclone (HCD57-SREI cells). 
We found that erythropoietin (EPO) and stem cell factor (SCF) activated protein kinase B (PKB/Akt) in EPO-dependent HCD57 erythroid cells. To better understand signals controlling proliferation and viability, erythroid cells that resist apoptosis in the absence of EPO were subcloned and characterized (HCD57-SREI cells). Constitutive activations of PKB/Akt, STAT5a, and STAT5b were noted in these EPO-independent cells. PI3-kinase activity was an upstream activator of PKB/Akt because the PI3-kinase inhibitor LY294002 blocked both constitutive PKB/Akt and factor-dependent PKB/Akt activity. The LY294002 study showed that proliferation and viability of both HCD57-SREI and HCD57 cells correlated with the activity of PKB/Akt; however, PKB/Akt activity alone did not protect these cells from apoptosis. Treatment of HCD57 cells with SCF also activated PKB/Akt, but did not protect from apoptosis. This result suggested that PKB/PI3-kinase activity is necessary but not sufficient to promote viability and/or proliferation. Constitutive STAT5 activity, activated through an unknown pathway not including JAK2 or EPOR, may act in concert with the constitutive PI3-kinase/PKB/Akt pathway to protect the EPO-independent HCD57-SREI cells from apoptosis and promote limited proliferation. 


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Clonality of isolated eosinophils in the hypereosinophilic syndrome. 
The idiopathic hypereosinophilic syndrome (IHES) is a rare disorder characterized by unexplained, persistent eosinophilia associated with multiple organ dysfunction due to eosinophilic tissue infiltration. In the absence of karyotypic abnormalities, there is no specific test to detect clonal eosinophilia in IHES. Analysis of X-chromosome inactivation patterns can be used to determine whether proliferative disorders are clonal in origin. Methylation of HpaII and Hha I sites near the polymorphic trinucleotide repeat of the human androgen receptor gene (HUMARA) has been shown to correlate with X-inactivation. In this study, we have used the polymerase chain reaction (PCR) with nested primers to analyze X-inactivation patterns of the HUMARA loci in purified eosinophils from female patients with eosinophilia. Peripheral blood eosinophils were isolated by their autofluoresence using flow cytometric sorting. Eosinophils purified from a female patient presenting with IHES were found to show a clonal pattern of X-inactivation. Eosinophil-depleted leukocytes from this patient were polyclonal by HUMARA analysis, thus excluding skewedness of random X-inactivation. After corticosteroid suppression of her blood eosinophilia, a clonal population of eosinophils could no longer be detected in purified eosinophils. In contrast, eosinophils purified from a patient with Churg-Strauss syndrome and from six patients with reactive eosinophilias attributed to allergy, parasitic infection, or drug reaction showed a polyclonal pattern of X-inactivation by HUMARA analysis. The finding of clonal eosinophilia in a patient presenting with IHES indicates that such patients may have, in reality, a low-grade clonal disorder that can be distinguished from reactive eosinophilias by HUMARA analysis. Further, the method described can be used to monitor disease progression. 


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Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. 
The multiple biological activities of tumor necrosis factor (TNF) are mediated by two distinct cell surface receptors of 55 kd (TNFRp55) and 75 kd (TNFRp75). Using gene targeting, we generated a TNFRp55-deficient mouse strain. Cells from TNFRp55-/-mutant mice lack expression of TNFRp55 but display normal numbers of high affinity TNFRp75 molecules. Thymocyte development and lymphocyte populations are unaltered, and clonal deletion of potentially self-reactive T cells is not impaired. However, TNF signaling is largely abolished, as judged by the failure of TNF to induce NF-kappa B in T lymphocytes from TNFRp55-deficient mice. The loss of TNFRp55 function renders mice resistant to lethal dosages of either lipopolysaccharides or S. aureus enterotoxin B. In contrast, TNFRp55-deficient mice are severely impaired to clear L. monocytogenes and readily succumb to infection. Thus, the 55 kd TNFR plays a decisive role in the host's defense against microorganisms and their pathogenic factors. 


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T helper differentiation proceeds through Stat1-dependent, Stat4-dependent and Stat4-independent phases. 
Much of our focus in understanding Th1/Th2 development has been on the signals delivered by IL-12 and IL-4 as final determinants of terminal T cell differentiation. Because extinction of IL-12 signaling in early Th2 development could potentially be important in imprinting a more permanent Th2 phenotype on a population of T cells, we have also examined various parameters regulating the IL-12 signaling pathway. Whereas IL-4 appears to repress functional IL-12 signaling through inhibition of IL-12R beta 2 expression, IFN-gamma in the mouse, and IFN-alpha in the human appear to induce IL-12R beta 2 expression and promote IL-12 responsiveness. We propose that Th1 development can be considered in two stages, capacitance and development. Capacitance would simply involve expression of IL-12R beta 1 and beta 2 subunits, regulated by TCR, IL-4 and IFNs. The second stage, development, we propose is the true IL-12 induced developmental stage, involving expression of Stat4 inducible proteins. In the human, this may also occur via IFN-alpha, which is able to activate Stat4. It is perhaps possible that all of Stat4 actions on Th1 development may be exert directly by Stat4 at the IFN-gamma gene, however we suggest that, more likely, Stat4 may act to induce Th1 development through the induction of other non-cytokine genes, whose stable expression maintains the transcriptional state of a Th1 cell. 


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Differential inhibition of Smad6 and Smad7 on bone morphogenetic protein- and activin-mediated growth arrest and apoptosis in B cells. 
Smad6 and Smad7 prevent ligand-induced activation of signal-transducing Smad proteins in the transforming growth factor-beta family. Here we demonstrate that both Smad6 and Smad7 are human bone morphogenetic protein-2 (hBMP-2)-inducible antagonists of hBMP-2-induced growth arrest and apoptosis in mouse B cell hybridoma HS-72 cells. Moreover, we confirmed that the ectopic expressions of Smad6 and Smad7 inhibited the hBMP-2-induced Smad1/Smad5 phosphorylation. We previously reported that Smad7 is an activin A-inducible antagonist of activin A-induced growth arrest and apoptosis in HS-72 cells. Interestingly, although mRNA expression of Smad6 was induced by activin A in HS-72 cells, Smad6 showed no antagonistic effect on activin A-induced growth arrest and apoptosis. Moreover, we found that the ectopic expression of Smad7, but not Smad6, inhibited the activin A-induced Smad2 phosphorylation in HS-72 cells. Thus, Smad6 and Smad7 exhibit differential inhibitory effects in bone morphogenetic protein-2- and activin A-mediated signaling in B lineage cells. 


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The interleukin-5/receptor interaction activates Lyn and Jak2 tyrosine kinases and propagates signals via the Ras-Raf-1-MAP kinase and the Jak-STAT pathways in eosinophils. 
We have shown that the interaction of interleukin (IL)-5 with the receptor activates Lyn tyrosine kinase within 1 min and Jak2 tyrosine kinase within 1-3 min. IL-5 also stimulates GTP binding to p21ras. The signal is subsequently propagated through the activation of Raf-1, MEK, and MAP kinases as shown by their increased autophosphorylation in vitro and phosphorylation in situ. Jak2 kinase has been shown to phosphorylate STAT nuclear proteins. The activation of STAT nuclear factors was studied by electrophoretic mobility shift assay using a gamma activation site (GAS) probe. We found that IL-5 induces two GAS-binding proteins in eosinophils, one of which is STAT1. We conclude that IL-5 induced signals are propagated through two distinct pathways: (1) Lyn-->Ras-->Raf-1-->MEK-->MAP kinase and (2) Jak2-->STAT1. 


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Analysis of the modulation of transcriptional activity in myelopoiesis and leukemogenesis. 
Acute myeloid leukemia (AML) is still associated with a mortality of 60 to 80%. AML is characterized by a block in myeloid differentiation. The transcription factors PU.1 and C/EBPalpha are responsible for normal myeloid differentiation from stem cells to monocytes or granulocytes. In particular, PU.1 induces expression of the macrophage colony-stimulating factor (M-CSF) receptor and the development of monocytes, whereas C/EBPalpha increases the expression of the granulocyte colony-stimulating factor (G-CSF) receptor and leads to mature granulocytes. In AML, chromosomal aberrations result in oncoproteins such as AML1/ETO, PML/RARalpha, or activated Ras, which can deregulate genes important for normal myelopoiesis. Thus, AML1/ETO can bind to the transcription factor C/EBPalpha, inhibit C/EBPalpha-dependent transcription, and block granulocytic differentiation. However, AML1/ETO can also synergize with the transcription factor AML1 to enhance the activity of the M-CSF receptor promoter. On the other hand, the PML/RARalpha fusion protein causes transcriptional repression by recruiting the nuclear corepressor (N-CoR) histone deacetylase complex to the DNA, which results in decreased histone acetylation and a repressive chromatin organization. Here we describe methods to investigate whether and how signaling agonists induce myeloid differentiation and how oncoproteins might cause AML by modulating the activity of transcription factors that are pivotal for normal myeloid development. Copyright 1999 Academic Press. 


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Interferon-beta mediates stromal cell rescue of T cells from apoptosis. 
The resolution of immune responses is characterized by extensive apoptosis of activated T cells. However, to generate and maintain immunological memory, some antigen-specific T cells must survive and revert to a resting G0/G1 state. Cytokines that bind to the common gamma chain of the IL-2 receptor promote the survival of T cell blasts, but also induce proliferation. In contrast, soluble factors secreted by stromal cells induce Tcell survival in a resting G0/G1 state. We now report that interferon-beta is the principal mediator of stromal cell-mediated Tcell rescue from apoptosis. Interferon-alpha and -beta promote the reversion of blast Tcells to a resting G0/G1 configuration with all the characteristic features of stromal cell rescue; such as high Bcl-XL expression and low Bcl-2. Type I interferons and stromal cells stimulate apparently identical signaling pathways, leading to STAT-1 activation. We also show that this mechanism may play a fundamental role in the persistence of T cells at sites of chronic inflammation; suggesting that chronic inflammation is an aberrant consequence of immunological memory. 


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Effects of intranasal glucocorticoids on endogenous glucocorticoid peripheral and central function. 
Glucocorticoids are among the most potent antiinflammatory agents that can be used in the treatment of rhinitis. Their mechanisms of action are multiple and complex and a number of reports describe significant systemic effects of locally administered glucocorticoids. In order to evaluate the short-term systemic effects of intranasally administered glucocorticoids, 14 normal healthy subjects were treated with two doses of either budesonide (BUD) or fluticasone propionate (FP) for 2 weeks. Before treatment, at regular intervals during the treatment, 1 week and finally 6 weeks after termination of treatment, the effects on glucocorticoid receptor (GR) and methallothionein (MTIIa) mRNA expression levels were examined in peripheral lymphocytes using a solution hybridization assay. Serum cortisol, osteocalcin and urinary cortisol levels were also determined. An insulin tolerance test (ITT) was performed at the end of the second week of treatment and at the end of the 6-week washout period with no statistically significant change in cortisol response. In peripheral lymphocytes, GR mRNA levels were significantly down-regulated. MTIIa mRNA levels increased significantly. Serum osteocalcin decreased significantly during treatment with both BUD and FP. Serum cortisol decreased after 1 week of treatment whereas urinary cortisol was not affected until the second week of treatment. In conclusion, intranasal glucocorticoids at clinically recommended doses have not only significant systemic effects on adrenal function, but also have an effect on specific gene expression in peripheral lymphocytes. These effects are receptor-dependent, reversible, and according to serum and urinary cortisol levels and ITT, leave the hypothalamic-pituitary-adrenal function intact. Finally, these short-term systemic effects were not associated with any of the noticeable side-effects usually observed during long-term treatment with glucocorticoids. 


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Association of the interleukin-4 receptor alpha chain with p47phox, an activator of the phagocyte NADPH oxidase in B cells. 
Interleukin (IL)-4 plays an important role in IgE synthesis in B cells and in Th2 differentiation in T cells. IL-4 conducts its biological activities through binding to the IL-4 receptor (IL-4R) on the surface of target cells. IL-4R are thought to be composed of the IL-4R alpha chain (IL-4R alpha) and either the IL-2R gamma chain or the IL-13R alpha chain. We have previously shown that the membrane-proximal portion in the cytoplasmic domain of the human IL-4R alpha (hIL-4R alpha) is critical for proliferation, generation of germline epsilon transcript, and activation of STAT6, based on analyses of truncated hIL-4R alphas. In this study, we found that p47phox, an activator of the phagocyte NADPH oxidase, binds to this portion by the two-hybrid system. Furthermore, we observed the association of p47phox with the hIL-4R alpha in B cells derived from a normal donor. These results suggest that p47phox is involved in the signal transduction of IL-4 in B cells. However, activation of STAT6, CD23 expression, and IgE synthesis induced by IL-4 were not affected in p47phox-deficient patients, which raises the possibility that p47phox may be important in other signaling activities as well in B cells. 


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PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene. 
Growth factor receptors play an important role in hematopoiesis. In order to further understand the mechanisms directing the expression of these key regulators of hematopoiesis, we initiated a study investigating the transcription factors activating the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha gene. Here, we demonstrate that the human GM-CSF receptor alpha promoter directs reporter gene activity in a tissue-specific fashion in myelomonocytic cells, which correlates with its expression pattern as analyzed by reverse transcription PCR. The GM-CSF receptor alpha promoter contains an important functional site between positions -53 and -41 as identified by deletion analysis of reporter constructs. We show that the myeloid and B cell transcription factor PU.1 binds specifically to this site. Furthermore, we demonstrate that a CCAAT site located upstream of the PU.1 site between positions -70 and -54 is involved in positive-negative regulation of the GM-CSF receptor alpha promoter activity. C/EBP alpha is the major CCAAT/enhancer-binding protein (C/EBP) form binding to this site in nuclear extracts of U937 cells. Point mutations of either the PU.1 site or the C/EBP site that abolish the binding of the respective factors result in a significant decrease of GM-CSF receptor alpha promoter activity in myelomonocytic cells only. Furthermore, we demonstrate that in myeloid and B cell extracts, PU.1 forms a novel, specific, more slowly migrating complex (PU-SF) when binding the GM-CSF receptor alpha promoter PU.1 site. This is the first demonstration of a specific interaction with PU.1 on a myeloid PU.1 binding site. The novel complex is distinct from that described previously as binding to B cell enhancer sites and can be formed by addition of PU.1 to extracts from certain nonmyeloid cell types which do not express PU.1, including T cells and epithelial cells, but not from erythroid cells. Furthermore, we demonstrate that the PU-SF complex binds to PU.1 sites found on a number of myeloid promoters, and its formation requires an intact PU.1 site adjacent to a single-stranded region. Expression of PU.1 in nonmyeloid cells can activate the GM-CSF receptor alpha promoter. Deletion of the amino-terminal region of PU.1 results in a failure to form the PU-SF complex and in a concomitant loss of transactivation, suggesting that formation of the PU-SF complex is of functional importance for the activity of the GM-CSF receptor alpha promoter. Finally, we demonstrate that C/EBP alpha can also active the GM-CSF receptor alpha promoter in nonmyeloid cells. (ABSTRACT TRUNCATED AT 400 WORDS) 


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The NF-kappa B inhibitor, tepoxalin, suppresses surface expression of the cell adhesion molecules CD62E, CD11b/CD18 and CD106. 
Tepoxalin, a dual enzyme inhibitor of cyclooxygenase and 5-lipoxygenase has been shown to inhibit T-cell activation. Its immunosuppressive property is distinct from cyclosporin because only tepoxalin, but not cyclosporin, suppresses NF-kappa B activation. Here we report that tepoxalin selectively inhibits intercellular adhesion molecule-1 (ICAM-1, CD54)/MAC-1 (CD11b/CD18) dependent adhesion of polymorphonuclear cells to IL-1 activated human umbilical vein endothelial cells. The mechanism of inhibition is related to the surface expression of several cell adhesion molecules. Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65/p50 subunit of NF-kappa B, and then stimulated with PMA, revealed a reduced expression of CD11b/CD18 on monocytic HL60 cells, and endothelial adhesion molecule-1 (CD62E) and vascular adhesion molecule-1 (CD106) on human umbilical vein endothelial cells. Expression of other adhesion molecules such as lymphocyte function associated-antigen-1 (CD11a/CD18) and CD54 were unaffected. Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine, IL-8, a known inducer of CD11b/CD18 expression. Thus the suppression of CD11b/CD18 expression by tepoxalin may involve IL-8. Our results suggest that by inhibiting NF-kappa B activation, surface expression of several adhesion molecules can be modulated and that tepoxalin may be useful in treating selected adhesion mediated events such as leukocyte migration or atherosclerotic plaque formation. 


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Chronic human immunodeficiency virus type 1 infection of myeloid cells disrupts the autoregulatory control of the NF-kappaB/Rel pathway via enhanced IkappaBalpha degradation. 
Productive human immunodeficiency virus type 1 (HIV-1) infection causes sustained NF-kappaB DNA-binding activity in chronically infected monocytic cells. A direct temporal correlation exists between HIV infection and the appearance of NF-kappaB DNA-binding activity in myelomonoblastic PLB-985 cells. To examine the molecular basis of constitutive NF-kappaB DNA-binding activity in HIV1 -infected cells, we analyzed the phosphorylation and turnover of IkappaBalpha protein, the activity of the double-stranded RNA-dependent protein kinase (PKR) and the intracellular levels of NF-kappaB subunits in the PLB-985 and U937 myeloid cell models. HIV-1 infection resulted in constitutive, low-level expression of type 1 interferon (IFN) at the mRNA level. Constitutive PKR activity was also detected in HIV-1-infected cells as a result of low-level IFN production, since the addition of anti-IFN-alpha/beta antibody to the cells decreased PKR expression. Furthermore, the analysis of IkappaBalpha turnover demonstrated an increased degradation of IkappaBalpha in HIV-1-infected cells that may account for the constitutive DNA binding activity. A dramatic increase in the intracellular levels of NF-kappaB subunits c-Rel and NF-kappaB2 p100 and a moderate increase in NF-kappaB2 p52 and RelA(p65) were detected in HIV-1-infected cells, whereas NF-kappaB1 p105/p50 levels were not altered relative to the levels in uninfected cells. We suggest that HIV-1 infection of myeloid cells induces IFN production and PKR activity, which in turn contribute to enhanced IkappaBalpha phosphorylation and subsequent degradation. Nuclear translocation of NF-kappaB subunits may ultimately increase the intracellular pool of NF-kappaB/IkappaBalpha by an autoregulatory mechanism. Enhanced turnover of IkappaBalpha and the accumulation of NF-kappaB/Rel proteins may contribute to the chronically activated state of HIV-1-infected cells. 


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The role of gamma/delta T cell receptor positive cells in pregnancy. 
PROBLEM: Due to the lack of classical HLA antigens on the trophoblast, fetal antigens are possibly presented in a non major histocompatibility complex (MHC) restricted way. Decidual gammadelta T cells, which significantly increase in number during pregnancy, might play a role in recognition of fetal antigens and also in determining the quality of the response to these antigens. Our study was aimed at investigating the role of this cell population in progesterone-dependent immunomodulation. METHOD OF STUDY: Peripheral lymphocytes from healthy pregnant women and from habitual aborters were tested by immunocytochemistry for the presence of gamma/delta T cell receptor (TCR) and progesterone receptor. To investigate the effect of treatment with a pan anti gamma/delta antibody, lymphocytes were incubated for 3 hr with the antibody, and then interleukin (IL)-10, IL-12 and progesterone-induced blocking factor (PIBF) expression (by immuno-cytochemistry) as well as natural killer (NK) cell activity were determined. RESULTS: In peripheral blood of healthy pregnant women the percentage of gamma/delta TCR+ cells was significantly higher (P < 0.001) than in that of recurrent aborters or of non-pregnant individuals. Ninety-seven percent of gamma/delta TCR+ pregnancy lymphocytes expressed progesterone receptor. Binding of a specific antibody to the gamma/delta TCR inhibited PIBF- as well as IL-10 production, whereas it increased NK activity and IL-12 expression. CONCLUSIONS: These data suggest the role of gamma/delta TCR-bearing lymphocytes in progesterone-dependent immunomodulation. 


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The functional synergy between IL-12 and IL-2 involves p38 mitogen-activated protein kinase and is associated with the augmentation of STAT serine phosphorylation. 
IL-12 and IL-2 can stimulate mitogen- or CD3-activated T cells to proliferate, produce IFN-gamma, and kill tumor cells. The magnitude of these functional responses is greatly augmented when T cells are activated by the combination of IL-12 and IL-2. Although peripheral blood T cells are largely unresponsive to these cytokines without prior activation, a small subset of CD8+ T cells (CD8+CD18bright) is strongly activated by the combination of IL-12 and IL-2. In this report we show that the functional synergy between IL-12 and IL-2 in CD8+CD18bright T cells correlates with the activation of the stress kinases, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/Jun N-terminal kinase, but not with the activation of the extracellular signal-regulated kinases. The functional synergy between IL-2 and IL-12 is also associated with a prominent increase in STAT1 and STAT3 serine phosphorylation over that observed with IL-12 or IL-2 alone. By contrast, STAT tyrosine phosphorylation is not augmented over that seen with either cytokine alone. A specific inhibitor of p38 MAP kinase completely inhibits the serine phosphorylation of STAT1 and STAT3 induced by IL-12 and IL-2 and abrogates the functional synergy between IL-12 and IL-2 without affecting STAT tyrosine phosphorylation. This suggests that p38 MAP kinase may play an important role in regulating STAT serine phosphorylation in response to the combination of IL-12 and IL-2. Furthermore, these findings indicate that the optimal activation of T cells by IL-12 and IL-2 may depend on an interaction between the p38 MAP kinase and Janus kinase/STAT signaling pathways. 


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Interleukin-6 production in hemorrhagic shock is accompanied by neutrophil recruitment and lung injury. 
Hemorrhagic shock (HS) initiates an inflammatory cascade that includes the production of cytokines and recruitment of neutrophils (PMN) and may progress to organ failure, inducing acute respiratory distress syndrome (ARDS). To examine the hypothesis that interleukin-6 (IL-6) contributes to PMN infiltration and lung damage in HS, we examined the lungs of rats subjected to unresuscitated and resuscitated HS for the production of IL-6 and activation of Stat3. Using semiquantitative RT-PCR, we found a striking increase in IL-6 mRNA levels only in resuscitated HS, with peak levels observed 1 h after initiation of resuscitation. Increased IL-6 protein expression was localized to bronchial and alveolar cells. Electrophoretic mobility shift assay of protein extracts from shock lungs exhibited an increase in Stat3 activation with kinetics similar to IL-6 mRNA. In situ DNA binding assay determined Stat3 activation predominantly within alveoli. Intratracheal instillation of IL-6 alone into normal rats resulted in PMN infiltration into lung interstitium and alveoli, marked elevation of bronchoalveolar lavage cellularity, and increased wet-to-dry ratio. These findings indicate that IL-6 production and Stat3 activation occur early in HS and may contribute to PMN-mediated lung injury, including ARDS after HS. 


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A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors. 
A novel B cell-restricted activity, required for high levels of octamer/Oct-dependent transcription from an immunoglobulin heavy chain (IgH) promoter, was detected in an in vitro system consisting of HeLa cell-derived extracts complemented with fractionated B cell nuclear proteins. The factor responsible for this activity was designated Oct coactivator from B cells (OCA-B). OCA-B stimulates the transcription from an IgH promoter in conjunction with either Oct-1 or Oct-2 but shows no significant effect on the octamer/Oct-dependent transcription of the ubiquitously expressed histone H2B promoter and the transcription of USF- and Sp1-regulated promoters. Taken together, our results suggest that OCA-B is a tissue-, promoter-, and factor-specific coactivator and that OCA-B may be a major determinant for B cell-specific activation of immunoglobulin promoters. In light of the evidence showing physical and functional interactions between Oct factors and OCA-B, we propose a mechanism of action for OCA-B and discuss the implications of OCA-B for the transcriptional regulation of other tissue-specific promoters. 


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Heterogeneity of antigen molecules recognized by anti-tax1 monoclonal antibody Lt-4 in cell lines bearing human T cell leukemia virus type I and related retroviruses. 
Using a monoclonal antibody, Lt-4, directed against human T cell leukemia virus type I (HTLV-I) trans-activator (tax1) antigen, we examined the expression of tax1 and related antigens in a variety of T cell lines bearing HTLV-I and related retroviruses, simian T cell leukemia virus type I (STLV-I) and HTLV-II, by immunofluorescence and immunoblot assays. Lt-4 reacted with all HTLV-I-bearing cell lines tested and five out of eight simian cell lines bearing STLV-I, but not with an HTLV-II-bearing cell line. Lt-4 detected 40 kd tax1 antigen molecules in most HTLV-I-bearing cell lines except one cell line that expressed 39 kd tax1 antigen. In the STLV-I-bearing T cell lines, tax1-related antigen molecules detected by Lt-4 were heterogeneous, having molecular weights in the range of 36-41 kd. 


Document 003003375 ends.


Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells. 
Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways. 


Document 003003378 ends.


Anti-immunoglobulin M activates nuclear calcium/calmodulin-dependent protein kinase II in human B lymphocytes. 
We and others have previously shown that the nuclear protein, Ets-1, is phosphorylated in a calcium-dependent manner after ligation of immunoglobulin (Ig) M on B lymphocytes. As this phosphorylation was independent of protein kinase C activity, we tested whether a calcium/calmodulin-dependent protein kinase (CaM kinase) might phosphorylate the Ets-1 protein after elevation of intracellular free calcium concentrations. The dephosphorylated form of Ets-1 has been shown to bind to chromatin, suggesting that the operative kinase should be detectable in the nucleus. We prepared nuclear extracts from two human B cell lines in which increased intracellular free calcium levels correlated with increased phosphorylation of the Ets-1 protein. Activity of the CaM kinases was determined using a synthetic peptide substrate both in the absence and presence of an inhibitor specific for the CaM kinase family, KN-62. Stimulation of cells with anti-IgM led to increased activity of a nuclear kinase that could phosphorylate the peptide, and this activity was reduced by 10 microM KN-62. Kinase activity was reduced in lysates preadsorbed using an antibody specific for CaM kinase II. Two-dimensional phosphopeptide maps of the Ets-1 protein from cells incubated with ionomycin or anti-IgM contained two unique phosphopeptides that were absent in untreated cells. Incubation of isolated Ets-1 protein with purified CaM kinase II produced phosphorylation of peptides that migrated identically to those found in cells incubated with either anti-IgM or ionomycin. These data suggest a model of signal transduction by the antigen receptor on B lymphocytes in which increased intracellular free calcium can rapidly activate nuclear CaM kinase II, potentially resulting in phosphorylation and regulation of DNA-binding proteins. 


Document 003003384 ends.


Regulation of the balance of cytokine production and the signal transducer and activator of transcription (STAT) transcription factor activity by cytokines and inflammatory synovial fluids. 
The balance between type 1 and 2 T helper cell cytokine production plays an important role in several animal models of autoimmunity, and skewed patterns of cytokine expression have been described in human inflammatory diseases. Many cytokines activate signal transducer and activation of transcription (STAT) transcription factors, which, in turn, activate transcription of inflammatory effector genes. We used mononuclear cell priming cultures and inflammatory synovial fluids (SFs) derived from arthritis patients to examine the regulation of cytokine production and STAT activity by an inflammatory synovial microenvironment. Exposure to SFs during priming resulted in an 81% inhibition of interferon (IFN)-gamma, but not interleukin (IL) 4, production by effector cells generated in priming cultures. SF suppression was mediated by IL-4 and IL-10 and inhibition of IL-12 expression, and it was reversed in a dominant fashion by exogenous IL-12. SFs blocked the sustained activity of transcription factor Stat1, but not Stat3, during the priming period, and Stat1 activity was differentially regulated by cytokines in parallel with their positive or negative regulation of IFN-gamma production. Active Stat3, but not Stat1, was detected in cells from inflamed joints. These results suggest a role for altered balance of Stat1 and Stat3 transcriptional activity in the regulation of T cell differentiation and in the pathogenesis of inflammatory synovitis. 


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CD40, but not lipopolysaccharide and anti-IgM stimulation of primary B lymphocytes, leads to a persistent nuclear accumulation of RelB. 
In this study we analyzed the effect of CD40 stimulation on the activity and nuclear appearance of Rel/nuclear factor kappaB (NF-kappaB) factors in primary murine B lymphocytes. We show that triggering of CD40 signaling pathway(s) by CD40 ligands expressed on L cells led to strong activation of an NF-kappaB-controlled beta-globin reporter gene in primary B lymphocytes from transgenic mice. Analyses of nuclear translocation of individual members of Rel proteins after CD40 induction of primary B cells showed a strong and long-lasting accumulation of RelB and, less pronounced, of c-Rel. LPS stimulation did not give rise to a persistent nuclear accumulation of RelB and c-Rel, whereas nuclear c-Rel, but not RelB, accumulated after B cell receptor stimulation. CD40 induced not only nuclear translocation but also de novo synthesis of RelB RNA and protein. S107 plasmacytoma cells, which express CD40 but are defective for the nuclear appearance of p50/p65-NF-kappaB, do not express RelB after CD40 stimulation. In S107 cells stably transfected with relB genes, stimulation of nuclear RelB translocation by CD40 was observed. These results indicate that stimulation of CD40 signaling pathways exerts a long-lasting stimulatory effect on both the transcription and nuclear translocation of RelB. Since LPS and anti-IgM were unable to activate RelB, CD40 appears to trigger a special program of gene expression involved in the proliferation and/or differentiation of B lymphocytes. 


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Temperature-induced down-regulation of the glucocorticoid receptor in peripheral blood mononuclear leucocyte in patients with sepsis or septic shock. 
OBJECTIVE: Activation of the hypothalamic-pituitary-adrenal axis is of vital importance during critical illness. We have studied the adaptive mechanisms which occur at the level of the glucocorticoid receptor in glucocorticoid target tissues in patients with sepsis or septic shock. DESIGN: The effects of hypercortisolaemia, hyperthermia and cellular composition on number of glucocorticoid receptors per cell and their affinity were evaluated, both in vitro and in vivo, in peripheral blood mononuclear leucocytes of control subjects and in patients with sepsis or septic shock. SUBJECTS: Fifteen patients (age 25-79) with sepsis or septic shock who were admitted to an intensive care unit were studied. The control group consisted of 24 healthy laboratory employees. MEASUREMENTS: The binding capacity and affinity of the glucocorticoid receptors were measured and compared to clinical data and the plasma cortisol concentrations. RESULTS: Hypercortisolaemia, in vitro, resulted in a decreased affinity and a decreased binding capacity of the glucocorticoid receptor. In vitro, hyperthermia as well as variations in the cellular composition did not influence the glucocorticoid receptor. In vivo, there was no change in the number of receptors per cell in patients with sepsis or septic shock as compared to healthy controls. However, a decreased affinity of the glucocorticoid receptor was observed. There was a weak but significant negative correlation between body temperature and the number of glucocorticoid receptors in the patient group. There was no relation between circulating cortisol concentrations and glucocorticoid receptor affinity and number. CONCLUSIONS: There is no obvious regulation of the number of glucocorticoid receptors by plasma cortisol concentrations in vivo. The decreased affinity of the glucocorticoid receptor together with the negative correlation between hyperthermia and the number of glucocorticoid receptors in patients with sepsis or septic shock suggest that hypothalamic-pituitary-adrenal axis activation during critical illness is accompanied by peripheral adaptation in glucocorticoid receptor number and affinity. 


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Bacterial peptidoglycan induces CD14-dependent activation of transcription factors CREB/ATF and AP-1. 
Peptidoglycan (PGN), the major cell wall component of Gram-positive bacteria, induces secretion of cytokines in macrophages through CD14, the pattern recognition receptor that binds lipopolysaccharide and other microbial products. To begin to elucidate the mechanisms that regulate the transcription of cytokine genes, we wanted to determine which transcription factors are activated by PGN in mouse RAW264.7 and human THP-1 macrophage cells. Our results demonstrated that: (i) PGN induced phosphorylation of the transcription factors ATF-1 and CREB; (ii) ATF-1 and CREB bound DNA as a dimer and induced transcriptional activation of a CRE reporter plasmid, which was inhibited by dominant negative CREB and ATF-1; (iii) PGN induced phosphorylation of c-Jun, protein synthesis of JunB and c-Fos, and transcriptional activation of the AP-1 reporter plasmid, which was inhibited by dominant negative c-Fos; and (iv) PGN-induced activation of CREB/ATF and AP-1 was mediated through CD14. This is the first study to demonstrate activation of CREB/ATF and AP-1 transcription factors by PGN or by any other component of Gram-positive bacteria. 


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Nonradioactive quantification of glucocorticoid receptor expression during differentiation of human monocytic cells (U937). 
We describe a method for relative quantification of specific mRNA using a nonradioactive assay based on DNA strand competition between identical sequences of biotin- and fluorescein-labeled amplicon (probe) and unlabeled amplicon (target) during hybridization. As the target quantity increased, that of the double-labeled probe decreased in accordance with the mass action law. This technique was successfully applied to evaluate differences in glucocorticoid receptor expression in U937 cells before and after the addition of potent differentiation inducers: 12-O-tetradecanoylphorbol 13-acetate (TPA) and a combination of all-trans retinoic acid (RA) and 1,25-dihydroxyvitamin D2 (VD). We observed that TPA treatment was associated with an increase in specific binding of [3H]dexamethasone and up-regulation of GR mRNA while no enhanced GR expression was perceived with RA/VD treatment. 


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Alternative polyadenylation events contribute to the induction of NF-ATc in effector T cells. 
The transcription factor NF-ATc is synthesized in three prominent isoforms. These differ in the length of their C terminal peptides and mode of synthesis. Due to a switch from the use of a 3' polyA site to a more proximal polyA site, NF-ATc expression switches from the synthesis of the two longer isoforms in naive T cells to that of short isoform A in T effector cells. The relative low binding affinity of cleavage stimulation factor CstF-64 to the proximal polyA site seems to contribute to its neglect in naive T cells. These alternative polyadenylation events ensure the rapid accumulation of high concentrations of NF-ATc necessary to exceed critical threshold levels of NF-ATc for gene induction in effector T cells. 


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Direct demonstration of NFATp dephosphorylation and nuclear localization in activated HT-2 cells using a specific NFATp polyclonal antibody. 
Nuclear factor of activated T cells (NFAT) regulates transcription of a number of cytokine genes, and NFAT DNA binding activity is stimulated following T cell activation. Several lines of evidence have suggested that NFAT is a substrate for calcineurin, a serine/threonine phosphatase. Using a polyclonal antibody to murine NFATp, Western blot analysis of various mouse tissues demonstrated that the 110-130-kDa NFATp protein was highly expressed in thymus and spleen. Treatment of immunoprecipitated NFATp from untreated HT-2 cells with calcineurin resulted in the dephosphorylation of NFATp, demonstrating that NFATp is an in vitro substrate for calcineurin. NFATp immunoprecipitated from 32P-labeled HT-2 cells migrated as an approximately 120-kDa protein that was localized to the cytosol of the cells. Treatment of the cells with ionomycin resulted in a decrease in the molecular weight of NFATp and a loss of 32P, consistent with NFATp dephosphorylation. The dephosphorylation of NFATp was accompanied by localization of the protein to the nuclear fraction. Both of these events were blocked by preincubation of the cells with FK506, a calcineurin inhibitor, consistent with the hypothesis that NFATp is a calcineurin substrate in cells. 


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p105 and p98 precursor proteins play an active role in NF-kappa B-mediated signal transduction. 
The Rel/NF-kappa B family of transcription factors is composed of two distinct subgroups, proteins that undergo proteolytic processing and contain SWI6/ankyrin repeats in their carboxyl termini (p105, p98), and those without such repeats that do not require processing (p65, c-Rel, RelB, and Dorsal). We demonstrate that the p105 and p98 precursors share functional properties with the I kappa B proteins, which also contain SWI6/ankyrin repeats. Both p105 and p98 were found to form stable complexes with other Rel/NF-kappa B family members, including p65 and c-Rel. Association with the precursors is sufficient for cytoplasmic retention of either p65 or c-Rel, both of which are otherwise nuclear. These complexes undergo stimulus-responsive processing to produce active p50/c-Rel and p55/c-Rel complexes. These observations suggest a second pathway leading to NF-kappa B induction, in which processing of the precursors rather than phosphorylation of I kappa B plays a major role. 


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IL-10 induces the tyrosine phosphorylation of tyk2 and Jak1 and the differential assembly of STAT1 alpha and STAT3 complexes in human T cells and monocytes. 
IL-10 affects monocytes and T cells by driving the progression of immune responsiveness such that Th2 lymphocyte-mediated effects predominate. In this report, we show that in monocytes and T cells IL-10 stimulates tyrosine phosphorylation of the signal transducers and activators of transcription, STAT1 alpha and STAT3, in a differential manner such that the relative formation of homo- and heterodimers varies between the two cell types. Moreover, monocytes express a novel IL-10-stimulated STAT protein with an M(r) of 70 kDa that is recognized by the anti-STAT3 Ab but is not observed in T cells. IL-10 treatment of both T cells and monocytes results in the ligand-induced tyrosine phosphorylation of tyk2 and Jak1, but not Jak2 or Jak3. Selective modulation of immune responsiveness by IL-10 in cells such as monocytes and T cells may result in part from the differential activation of STAT protein pairs. 


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Impaired binding of a DQ2 and DQ8-binding HSV VP16 peptide to a DQA1*0501/DQB1*0302 trans class II heterodimer. 
DQalpha and DQbeta trans heterodimeric HLA-DQ molecules form in individuals heterozygous for the DQ2 and DQ8 specificities. Unique functions and disease associations have been postulated for such trans-dimers, which may be different from cis-encoded DQ molecules encoded by the corresponding haplotypes. We analyzed the ability of the trans-dimer encoded by HLA-DQA1*0501/DQB1*0302 to bind a peptide antigen which interacts with DQ molecules encoded by both parental haplotypes. Markedly impaired binding was observed, consistent with both the use of different anchor residues and with changes in levels of DQ cis-dimer availability for peptide binding interactions. 


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Constitutive overexpression of the L-selectin gene in fresh leukemic cells of adult T-cell leukemia that can be transactivated by human T-cell lymphotropic virus type 1 Tax. 
L-selectin is an adhesion molecule of the selectin family that mediates the initial step of leukocyte adhesion to vascular endothelium. Upon cellular activation, expression of the L-selectin gene is downregulated at both the protein and mRNA levels. To understand the mechanism of leukemic cell infiltration into organs, we studied the expression and regulation of L-selectin mRNA in fresh leukemic cells of adult T-cell leukemia (ATL) patients and investigated the response of the L-selectin promoter to human T-cell lymphotropic virus type 1 (HTLV-1) Tax, which is a viral transcriptional transactivator. Flow cytometry showed that L-selectin was expressed on fresh ATL cells along with other activation antigens. Northern blot analysis showed that ATL cells overexpressed that L-selectin mRNA and that the level was aberrantly upregulated after PMA stimulation. Studies using in situ hybridization showed expression of the L-selectin mRNA in the infiltrating leukemic cells in the liver of two ATL patients. Intravenous injection of a rat T-cell line that overexpresses L-selectin showed increased organ infiltration. The induction of Tax expression in JPX9 cells resulted in about a twofold increase in the mRNA expression levels compared with the basal level. Chloramphenicol acetyltransferase (CAT) assay after transient cotransfection showed about a fivefold transactivation of the L-selectin promoter by Tax. The serum level of the shed form of L-selectin was significantly increased in ATL patients (mean +/- SD, 4,215.4 +/- 4,111 ng/mL) compared with those of asymptomatic carriers and healthy blood donors (mean +/- SD, 1,148.0 +/- 269.0 ng/mL and 991.9 +/- 224 ng/mL, respectively). These results indicated that ATL cells constitutively overexpress the L-selectin gene that can be transactivated by HTLV-1 Tax. The overexpression of L-selectin, as well as of inflammatory cytokines, by ATL cells may provide a basis for ATL cells to attach the vascular endothelium, leading to transmigration and organ infitration. 


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Nonopsonic phagocytosis of Pseudomonas aeruginosa by macrophages and polymorphonuclear leukocytes requires the presence of the bacterial flagellum. 
Whereas the mechanism of nonopsonic phagocytosis of Pseudomonas aeruginosa has been described, the bacterial ligands required are poorly understood. To identify the requisite bacterial ligands, studies with isogenic mutants of P. aeruginosa PAK lacking pili, flagella, and the RpoN sigma factor were undertaken. The RpoN mutant, lacking pili, flagella, and nonpilus adhesins, bound poorly and was resistant to ingestion by both macrophages and neutrophils. Pili were not absolutely required for binding or phagocytosis of P. aeruginosa. The presence of a flagellum was not required for binding of P. aeruginosa to macrophages but was critical for the subsequent internalization of the bacterium, suggesting that this factor or a surface ligand associated with its assembly was responsible for stimulation of nonopsonic phagocytosis. 


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Synergistic interactions between overlapping binding sites for the serum response factor and ELK-1 proteins mediate both basal enhancement and phorbol ester responsiveness of primate cytomegalovirus major immediate-early promoters in monocyte and T-lymphocyte cell types. 
Cytomegalovirus (CMV) infection is nonpermissive or persistent in many lymphoid and myeloid cell types but can be activated in differentiated macrophages. We have shown elsewhere that both the major immediate-early gene (MIE) and lytic cycle infectious progeny virus expression can be induced in otherwise nonpermissive monocyte-like U-937 cell cultures infected with either human CMV (HCMV) or simian CMV (SCMV) by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Two multicopy basal enhancer motifs within the SCMV MIE enhancer, namely, 11 copies of the 16-bp cyclic AMP response element (CRE) and 3 copies of novel 17-bp serum response factor (SRF) binding sites referred to as the SNE (SRF/NFkappaB-like element), as well as four classical NFkappaB sites within the HCMV version, contribute to TPA responsiveness in transient assays in monocyte and T-cell types. The SCMV SNE sites contain potential overlapping core recognition binding motifs for SRF, Rel/NFkappaB, ETS, and YY1 class transcription factors but fail to respond to either serum or tumor necrosis factor alpha. Therefore, to evaluate the mechanism of TPA responsiveness of the SNE motifs and of a related 16-bp SEE (SRF/ETS element) motif found in the HCMV and chimpanzee CMV MIE enhancers, we have examined the functional responses and protein binding properties of multimerized wild-type and mutant elements added upstream to the SCMV MIE or simian virus 40 minimal promoter regions in the U-937, K-562, HL-60, THP-1, and Jurkat cell lines. Unlike classical NFkappaB sites, neither the SNE nor the SEE motif responded to phosphatase inhibition by okadaic acid. However, the TPA responsiveness of both CMV elements proved to involve synergistic interactions between the core SRF binding site (CCATATATGG) and the adjacent inverted ETS binding motifs (TTCC), which correlated directly with formation of a bound tripartite complex containing both the cellular SRF and ELK-1 proteins. This protein complex was more abundant in U-937, K-562, and HeLa cell extracts than in Raji, HF, BALB/c 3T3, or HL-60 cells, but the binding activity was altered only twofold after TPA treatment. A 40-fold stimulation of chloramphenicol acetyltransferase activity mediated by four tandem repeats of the SNE could be induced within 2 h (and up to 250-fold within 6 h) after addition of TPA in DNA-transfected U-937 cells, indicating that the stimulation appeared likely to be a true protein kinase C-mediated signal transduction event rather than a differentiation response. Slight differences in the sequence of the core SRF binding site compared with that of the classical c-Fos promoter serum response element, together with differences in the spacing between the SRF and ETS motifs, appear to account for the inability of the SCMV SNEs to respond to serum induction. 


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Human T-cell leukemia virus type I Tax associates with and is negatively regulated by the NF-kappa B2 p100 gene product: implications for viral latency. 
Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of the adult T-cell leukemia, an aggressive and often fatal malignancy of activated human CD4 T cells. HTLV-I encodes an essential 40-kDa protein termed Tax that not only transactivates the long terminal repeat of this retrovirus but also induces an array of cellular genes. Tax-mediated transformation of T cells likely involves the deregulated expression of various cellular genes that normally regulate lymphocyte growth produced by altered activity of various endogenous host transcription factors. In particular, Tax is capable of modulating the expression or activity of various host transcription factors, including members of the NF-kappa B/Rel and CREB/ATF families, as well as the cellular factors HEB-1 and p67SRF. An additional distinguishing characteristic of HTLV-I infection is the profound state of viral latency that is present in circulating primary leukemic T cells. In this study, we demonstrate that HTLV-I Tax can physically associate with p100, the product of the Rel-related NF-kappa B2 gene, both in transfected cells and in HTLV-I-infected leukemic T-cell lines. Furthermore, the physical interaction of Tax with p100 leads to the inhibition of Tax-induced activation of the HTLV-I and human immunodeficiency virus type 1 long terminal repeats, reflecting p100-mediated cytoplasmic sequestration of the normally nuclearly expressed Tax protein. In contrast, a mutant of Tax that selectively fails to activate nuclear NF-kappa B expression does not associate with p100. (ABSTRACT TRUNCATED AT 250 WORDS) 


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A direct interaction between the adaptor protein Cbl-b and the kinase zap-70 induces a positive signal in T cells. 
Engagement of the T-cell receptor (TCR)-CD3 complex induces a rapid increase in the activities of Src-family and Syk/Zap-70-family kinases [1] [2]. These activated kinases then induce the tyrosine phosphorylation of multiple intracellular proteins, eventually leading to T-cell activation. One of the prominent substrates for these kinases is the adaptor protein Cbl [3] and recent studies suggest that Cbl negatively regulates upstream kinases such as Syk and Zap-70 [4] [5]. Cbl-b, a homologue of Cbl, is widely expressed in many tissues and cells including hematopoietic cells [6] [7]. Cbl-b undergoes rapid tyrosine phosphorylation upon stimulation of the TCR and cytokine receptors [8] [9]. The role of Cbl-b is unclear, however. Here, we show that overexpression of Cbl-b in T cells induced the constitutive activation of the transcription factor nuclear factor of activated T cells (NFAT). A loss-of-function mutation in Cbl-b disrupted the interaction between Cbl-b and Zap-70 and nearly completely abrogated the Cbl-b-mediated activation of NFAT. Unlike the proposed role of Cbl as a negative regulator, our results suggest that the Cbl homologue Cbl-b has a positive role in T-cell signaling, most likely via a direct interaction with the upstream kinase Zap-70. 


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Disruption of the human SCL locus by "illegitimate" V-(D)-J recombinase activity. 
A fusion complementary DNA in the T cell line HSB-2 elucidates a provocative mechanism for the disruption of the putative hematopoietic transcription factor SCL. The fusion cDNA results from an interstitial deletion between a previously unknown locus, SIL (SCL interrupting locus), and the 5' untranslated region of SCL. Similar to 1;14 translocations, this deletion disrupts the SCL 5' regulatory region. This event is probably mediated by V-(D)-J recombinase activity, although neither locus is an immunoglobulin or a T cell receptor. Two other T cell lines, CEM and RPMI 8402, have essentially identical deletions. Thus, in lymphocytes, growth-affecting genes other than immune receptors risk rearrangements. 


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Differential regulation of IL-6 gene transcription and expression by IL-4 and IL-10 in human monocytic cell lines. 
IL-4 and IL-10 inhibit the cytokine production and mRNA expression by monocytes/macrophages. To investigate the molecular mechanism of the inhibitory effect on transcriptional or post-transcriptional regulation of IL-6 gene expression by IL-4 and IL-10, we studied IL-6 production, expression level of IL-6 mRNA, IL-6 promoter activity, transcriptional activity of NF-kappaB and NF-IL-6, and IL-6 mRNA stability in human monocytic cell lines, THP-1 and U937, stimulated by PMA and LPS in the absence or the presence of IL-4 or IL-10. Both IL-4 and IL-10 were seen to inhibit IL-6 production and the expression of IL-6 mRNA in both monocytic cell lines studied. In chloramphenicol acetyltransferase assays, utilizing the transient transfection of a chloramphenicol acetyltransferase reporter plasmid containing the IL-6 gene promoter, IL-4, but not IL-10, suppressed the transcriptional activity of the IL-6 gene promoter stimulated by PMA and LPS. Electrophoretic mobility shift assays showed that IL-4, but not IL-10, inhibited nuclear NF-kappaB activity, and that IL-4 and IL-10 did not affect NF-IL-6 activity. On the other hand, IL-10 enhanced the degradation of IL-6 mRNA in a mRNA stability assay. These results suggest that IL-4 may inhibit the transcription of the IL-6 gene by affecting NF-kappaB binding activity, while IL-10 may inhibit the IL-6 mRNA levels post-transcriptionally, without suppressing promoter activity. Therefore, we conclude that IL-4 and IL-10 inhibit IL-6 production by different mechanisms in human monocytic cell lines. 


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Flutamide in the treatment of hirsutism: long-term clinical effects, endocrine changes, and androgen receptor behavior. 
OBJECTIVE: To investigate the long-term effects of treatment with low doses of flutamide on clinical and hormonal parameters, as well as on the androgen receptor status, in hirsute women. DESIGN: Eighteen hirsute patients with regular menses were studied basally and during treatment with 125 mg flutamide, three times per day for 12 months. Barrier or intrauterine contraception was used during the study in sexually active women. Safety parameters were assessed throughout the study. Hirsutism, graded by the modified Ferriman-Gallwey score, and hormonal parameters were evaluated basally and at 4-month intervals during treatment. Gonadotropin-releasing hormone and ACTH stimulation tests were performed before and after 3 to 4 months of therapy. In addition, the concentration of androgen receptors in mononuclear leukocytes was measured, in both the follicular and luteal phases of the menstrual cycle, basally and after 4 months of flutamide treatment. RESULTS: Flutamide was well tolerated in all women, with the noticeable exception of one patient who presented increased serum transaminase after 8 months of therapy. Hirsutism markedly improved in all women during the treatment (Ferriman-Gallwey score after 1 year: 4.1 +/- 0.5 versus 14.1 +/- 0.9). A reduction of serum androgens was found, whereas no change was observed in either basal or GnRH-stimulated gonadotropins or in the cortisol and 17 alpha-hydroxyprogesterone response to ACTH. Cycles remained ovulatory. Before treatment, the number of androgen receptors was higher in the luteal than in the follicular phase. This rhythmic differentiation disappeared after the patients had been given the antiandrogen drug. CONCLUSIONS: Flutamide is effective in the treatment of hirsutism but requires constant surveillance of liver function. Androgen receptor blockade might be potentiated by a reduction of serum androgens. Flutamide affects androgen receptor behavior during the menstrual cycle. The meaning of this finding remains to be elucidated . 


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E1A gene expression induces susceptibility to killing by NK cells following immortalization but not adenovirus infection of human cells. 
Adenovirus (Ad) infection and E1A transfection were used to model changes in susceptibility to NK cell killing caused by transient vs stable E1A expression in human cells. Only stably transfected target cells exhibited cytolytic susceptibility, despite expression of equivalent levels of E1A proteins in Ad-infected targets. The inability of E1A gene products to induce cytolytic susceptibility during infection was not explained by an inhibitory effect of viral infection on otherwise susceptible target cells or by viral gene effects on class I MHC antigen expression on target cells. This differential effect of E1A expression on the cytolytic phenotypes of infected and stably transfected human cells suggests that human NK cells provide an effective immunologic barrier against the in vivo survival and neoplastic progression of E1A-immortalized cells that may emerge from the reservoir of persistently infected cells in the human host. 


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Inhibition of the differentiation of human myeloid cell lines by redox changes induced through glutathione depletion. 
We have investigated the effect of redox changes in vivo on the differentiation of two human myeloid cell lines, HL-60 and KG-1. The glutathione-depleting agent diethyl maleate (DEM) prevented the development of differentiated features in response to phorbol esters, including adherence of the cells to plastic surfaces and repression of the myeloperoxidase and CD34 genes. Moreover, DEM abolished phorbol 12-myristate 13-acetate-induced activation of the transcription factors AP-1 and Egr-1, suggesting that inhibition of differentiation may be due, at least in part, to redox modifications of these proteins. 


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A new member of the leucine zipper class of proteins that binds to the HLA DR alpha promoter. 
Several mutants derived from transformed human B cell lines are defective in expressing major histocompatibility complex (MHC) class II genes. The failure to express a class II gene in at least one such mutant line has been mapped to the MHC class II X box, a conserved transcriptional element in the promoter region. A complementary DNA encoding a DNA-binding protein (human X box binding protein, hXBP-1) whose target is the human DR alpha X box and the 3' flanking region has now been cloned. This complementary DNA encoded a protein with structural similarities to the c-jun proto-oncogene product, and its target sequence was closely related to the palindromic target sequence of c-jun. Mutation of the hXBP-1 DNA target sequence decreased DR alpha promoter activity in vivo. These studies suggest that the hXBP-1 protein acts as a transcription factor in B cells. 


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Binding characteristics of the glucocorticoid receptor in peripheral blood lymphocytes in multiple sclerosis. 
Although the exact etiology of multiple sclerosis (MS) remains unresolved, immune reactions are believed to be the central pathogenic mechanisms. Endogenous and therapeutic steroid hormones affect the immune system, and inflammatory diseases are associated with activation of the hypothalamic-pituitary-adrenal axis, providing evidence of an immune-endocrine interplay. Function tests in MS have revealed dysregulation of the hypothalamic-pituitary-adrenal system in a substantial proportion of patients. We characterized glucocorticoid receptor (GR) binding in peripheral blood lymphocytes from 39 MS patients and 14 age- and sex-matched controls with respect to dissociation constant and binding capacity, using a whole-cell binding assay with [3H]dexamethasone as the ligand. GR binding parameters did not differ significantly between patients (Kd 8.98 +/- 1.07 nM, Bmax 183 +/- 29.8 fmol/mg) and controls (Kd 9.36 +/- 1.17 nM, Bmax 158 +/- 16 fmol/mg). No effect of age, sex, course, duration or severity of disease, or prior steroid treatments was detected. GR binding parameters were analyzed in relation to the results of the combined dexamethasone-CRH test, which reflects corticosteroid receptor function at the hypothalamus, in 30 patients and 9 controls. While controls showed a moderate correlation between binding affinity of the GR in lymphocytes and regulatory function at the hypothalamic level, the patients did not. These data suggest that the physiological relationship between binding and function of the glucocorticoid receptor is disturbed in MS. 


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Molecular mechanisms of steroid action: a novel type of cross-talk between glucocorticoids and NF-kappa B transcription factors. 
Despite the widespread use of glucocorticoids in the treatment of diseases characterized by inflammation, the molecular mechanism(s) by which these hormones exert this beneficial effect in patients with asthma remains to be elucidated. Therefore, we have studied the transcriptional regulation of intercellular adhesion molecule-1 (ICAM-1) as adhesion molecules are likely to play a causal role in inflammation in promoting cell-cell and cell-matrix interactions. We observed that in a monocytic (U937) and a bronchial epithelial (H292) cell-line dexamethasone strongly suppressed basal and induced ICAM-1 expression. Subsequent analysis of the human ICAM-1 promoter has revealed that both 12-O-tetradecanoylphorbol 13-acetate (TPA) and tumour necrosis factor-alpha (TNF-alpha) upregulate ICAM-1 expression through the presence of a nuclear factor (NF-kappa B) target sequence (TGGAAATTCC). No glucocorticoid recognition sequences are present in this promoter region and dexamethasone is still able to repress transcription when the multimerized NF-kappa B sequence is transactivated by TNF-alpha upon transfection in 293 cells. We propose that direct interaction between the glucocorticoid receptor and nuclear factor-kappa B factors is at least a partial explanation for the effects of this hormone in inflammatory diseases. 


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Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1. 
Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-1-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the -512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation. 


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Microtubules mediate cellular 25-hydroxyvitamin D3 trafficking and the genomic response to 1,25-dihydroxyvitamin D3 in normal human monocytes. 
The genomic actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the intracellular vitamin D receptor (VDR). Although immunocytochemistry has shown that disruption of microtubular assembly prevents nuclear access of the sterol-VDR complex, the role of microtubules in the response to 1,25(OH)2D3 has not been studied in viable cells. Our studies examined this interaction in normal human monocytes. Monocytes convert 25(OH)D3 to 1,25(OH)2D3 and to 24-hydroxylated metabolites more polar than 1,25(OH)2D3. Microtubule disruption totally abolished the ability of exogenous 1,25(OH)2D3 to suppress its own synthesis and to induce 24-hydroxylase mRNA and activity, without affecting either total 1,25(OH)2D3 uptake or maximal 1,25(OH)2D3-VDR binding. Thus, intact microtubules are essential for 1,25(OH)2D3-dependent modulation of gene transcription. Interestingly, microtubule disruption also decreased monocyte 1,25(OH)2D3 synthesis, not by decreasing the Vmax of monocyte mitochondrial 1 alpha-hydroxylase but through an increase in the Km for 25(OH)2D3. We examined 25(OH)D3 transport. Microtubule disruption did not affect total cellular 25(OH)D3 uptake but reduced its intracellular trafficking to the mitochondria. Thus, microtubules participate in intracellular 25(OH)D3 transport, and their integrity determines normal 1,25(OH)2D3 synthesis. 


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Regulation of interferon-gamma gene expression. 
Interferon-gamma ( IFN-gamma ), also known as type II interferon, is an important immunoregulatory gene that has multiple effects on the development, maturation, and function of the immune system. IFN-gamma mRNA and protein are expressed predominantly by T cells and large granular lymphocytes. The IFN-gamma mRNA is induced/inhibited in these cell types by a wide variety of extracellular signals, thus implicating a number of diverse, yet convergent signal transduction pathways in its transcriptional control. In this review, I describe how DNA methylation and specific DNA binding proteins may regulate transcription of the IFN-gamma gene in response to extracellular signals. 


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Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. 
Tumor necrosis factor receptor-1 (TNFR-1) and CD95 (also called Fas or APO-1) are cytokine receptors that engage the apoptosis pathway through a region of intracellular homology, designated the "death domain." Another death domain-containing member of the TNFR family, death receptor 3 (DR3), was identified and was shown to induce both apoptosis and activation of nuclear factor kappaB. Expression of DR3 appears to be restricted to tissues enriched in lymphocytes. DR3 signal transduction is mediated by a complex of intracellular signaling molecules including TRADD, TRAF2, FADD, and FLICE. Thus, DR3 likely plays a role in regulating lymphocyte homeostasis. 


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In vivo modulation of glucocorticoid receptor mRNA by inhaled fluticasone propionate in bronchial mucosa and blood lymphocytes in subjects with mild asthma. 
BACKGROUND: In vivo regulation of the glucocorticoid receptor (GR) by glucocorticoids provides a means of modulating sensitivity of targeted cells. OBJECTIVE: We sought to determine the in vivo modulation of GR mRNA expression by fluticasone propionate (FP) in subjects with mild asthma. METHODS: Ten atopic asthmatic subjects were treated with FP 250 microg twice daily for 4 weeks. Before and after treatment, the patients underwent fiberoptic bronchoscopy with endobronchial biopsy and sampling of venous blood for measurements of GR mRNA levels. A solution hybridization assay was used for quantitative analysis of GR mRNA. In addition, a 24-hour urinary cortisol excretion and an adrenocorticotropic hormone test before and after treatment with FP were performed. RESULTS: A high interindividual variation in GR mRNA expression was seen. However, we detected a significant reduction of the GR mRNA levels in the endobronchial biopsy specimens after FP treatment (36.6 +/- 23.1 and 25.0 +/- 10.9 amol GR mRNA/microg RNA, respectively; P <.01). In the peripheral blood lymphocytes an even more striking downregulation of the GR by its cognate ligand was documented (30.3 +/- 26.5 and 8.8 +/- 5 amol GR mRNA/microg RNA, respectively; P <.001), possibly reflecting differences in glucocorticoid sensitivity between tissues. A small but significant reduction of the 24-hour urinary cortisol excretion was observed (233 +/- 109 and 157 +/- 66 nmol/L, respectively; P <.01), whereas the feedback regulation of glucocorticoid synthesis by means of the hypothalamic-pituitary-adrenal axis as assessed by the adrenocorticotropic hormone test remained normal after treatment with FP. CONCLUSION: The results in this study confirm the potency of the inhaled corticosteroid FP and provide evidence for a considerable tissue-specific interindividual variation in the expression of the GR. 


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Multiple NF-ATc isoforms with individual transcriptional properties are synthesized in T lymphocytes. 
The transcription factor NF-ATc that controls gene expression in T lymphocytes and embryonic cardiac cells is expressed in three prominent isoforms. This is due to alternative splice/polyadenylation events that lead to the predominant synthesis of two long isoforms in naive T cells and a shorter NF-ATc isoform in effector T cells. Whereas the previously described isoform NF-ATc/A contains a relatively short C terminus, the longer isoforms, B and C, span extra C-terminal peptides of 128 and 246 aa, respectively. We show here that in addition to the strong N-terminal trans-activation domain, TAD-A, which is common to all three NF-ATc isoforms, NF-ATc/C contains a second trans-activation domain, TAD-B, in its C-terminal peptide. Various stimuli of T cells that induce the activity of TAD-A also enhance the activity of TAD-B, but, unlike TAD-A, TAD-B remains unphosphorylated by protein from 12-O-tetradecanoyl 12-phorbol 13-acetate-stimulated T cells. The shorter C-terminal peptide of isoform NF-ATc/B exerts a suppressive transcriptional effect. These properties of NF-ATc/B and -C might be of importance for gene regulation in naive T lymphocytes in which NF-ATc/B and -C are predominantly synthesized. 


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Latent membrane protein-1 induces cyclin D2 expression, pRb hyperphosphorylation, and loss of TGF-beta 1-mediated growth inhibition in EBV-positive B cells. 
The normal cell cycle is regulated by several molecules, such as the tumor-suppressor protein pRb, the G1 cyclins, the cyclin-dependent kinases, and their inhibitors. These regulators are targeted by negative growth regulatory signals, such as that provided by TGF-beta. Here, we show that the presence of either wild-type EBV or its transforming latent membrane protein-1 (LMP-1) results in the loss of TGF-beta 1-mediated growth inhibition in human B cells. Chemical cross-linking with 125I-labeled TGF-beta 1 showed an essentially normal TGF-beta receptor profile in EBV-positive and EBV-negative Burkitt's lymphoma cell lines, and these receptors were shown to be functional in transducing signals, as evidenced by the TGF-beta 1-mediated modulation of junB gene expression. However, TGF-beta 1 did not induce dephosphorylation of pRb in EBV (or LMP-1)-positive cells as opposed to EBV-negative cells, suggesting a dichotomy in the TGF-beta 1 signaling pathway leading to separable gene regulatory and growth inhibitory responses. Furthermore, LMP-1 was found to induce the expression of cyclin D2; normal B cells or EBV-negative Burkitt's lymphoma cells do not express D-type cyclins. Taken together, these data point to a potential mechanism underlying EBV-mediated B cell transformation whereby constitutive induction of key cell cycle regulators by LMP-1 can lead to pRb hyperphosphorylation and uncontrolled cell proliferation. 


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Cellular and molecular mechanisms of IFN-gamma production induced by IL-2 and IL-12 in a human NK cell line. 
Interferon-gamma (IFN-gamma) is an important immunoregulatory protein produced predominantly by T cells and large granular lymphocytes (LGL) in response to different extracellular signals. In particular, two interleukins (ILs), IL-2 and IL-12, have been shown to be potent inducers of IFN-gamma gene expression in both T cells and LGL. Although it has been reported that there are some T cell lines that produce IFN-gamma in response to IL-2 and IL-12 stimulation, there has as yet been no report of a natural killer (NK) cell line that responds in a similar manner. In this report we present evidence that the cell line NK3.3 derived from human NK cells, responds to both IL-2 and IL-12, as measured by increases in IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) cytoplasmic mRNA and protein expression. In addition, when used together IL-2 and IL-12 synergized in the induction of IFN-gamma and GM-CSF and this synergy was attributed to an increased accumulation and stability of the IFN-gamma and GM-CSF mRNAs. To investigate the signaling pathways involved in the gene induction, five inhibitors, cyclosporin A (CsA), transforming growth factor-beta, cycloheximide, genistein, and staurosporine A, were used in analyzing the effects of IL-2 and IL-12 on NK3.3 cells. The results suggest that activation of protein kinase C, but not new protein synthesis, is required for IL-2 induction of IFN-gamma and GM-CSF cytoplasmic mRNA. In contrast, IL-12 induction of IFN-gamma cytoplasmic mRNA appears to only partially depend on activation of protein kinase C. Furthermore, both transforming growth factor-beta and genistein, a tyrosine kinase inhibitor, could suppress IL-2 and IL-12 signaling but CsA was generally inactive. It also was observed that suppression of cytokine gene expression by these agents was independent of the inhibition of proliferation. In addition, IL-2 but not IL-12 induced nuclear factors NF-kappa B and AP1, and regulation of the nuclear levels of these two DNA binding protein complexes is correlated with IFN-gamma and GM-CSF gene expression. These data indicate that IL-2 and IL-12 may have distinct signaling pathways leading to the induction of IFN-gamma and GM-CSF gene expression, and that the NK3.3 cell line may serve as a novel model for dissecting the biochemical and molecular events involved in these pathways. 


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Jeg-3 human choriocarcinoma-induced immunosuppression: downregulation of interleukin-2, interleukin-2 receptor alpha-chain, and its Jak/Stat signaling pathway. 
PROBLEM: The mechanisms of the immunosuppressive and immunosuppression-inducing capacities of Jeg-3 human choriocarcinoma cell line supernatants (HCSs) are not yet completely understood. The influence on interleukin (IL)-2, IL-4 and interferon (IFN)-gamma production; IL-2 receptor (IL-2R) alpha-, beta-, and gamma-chain; and the signaling pathway molecules Janus kinase (Jak)1, Jak3, signal transducers and activators of transcription (Stat)1, Stat3, and Stat5 should be investigated. METHOD OF STUDY: For assessment of IL production, whole peripheral venous blood from healthy donors was stimulated with phorbol-myristate-acetate and ionomycine. Secretion of ILs was blocked with monensine. Intracellular ILs were analyzed by flow cytometry. For IL-2R and signaling pathway molecule analysis, peripheral blood lymphocytes were stimulated with phytohemagglutinin (PHA). IL-2R chains were measured by flow cytometry, and Jaks/Stats by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. RESULTS: Phorbol-myristate-acetate and ionomycine strongly increase the percent-age of IL-2+ cells; an additional 50% HCSs significantly suppresses the percentage to, or below the level of unstimulated cells. IFN-gamma production is strongly decreased by HCSs in some cases, but not in others. PHA stimulates IL-2R alpha-, beta-, and gamma-chain expression and their signaling pathway molecules Jak1, Jak3, Stat1, Stat3, and Stat5. 50% HCS downregulates the alpha-chain and slightly upregulates the beta-chain. Jak1, Jak3, Stat1, Stat3, and Stat5 expression is suppressed approximately to, or below the level of unstimulated cells. CONCLUSIONS: HCS forcefully blocks the production of IL-2; the IL-2R alpha-chain; and Jak1, Jak3, Stat1, Stat3, and Stat5 expression. The observed phenomena might be caused by downregulation of an IL-2R regulation gene, and might play a key role in the expansion of choriocarcinoma, and possibly in the survival of the fetal allograft. 


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Signal transduction through interferon-gamma receptor on human eosinophils. 
BACKGROUND: We reported on the constitutive interferon-gamma receptor (IFN-gammaR) expression on eosinophils. But signal transduction through IFN-gammaR on eosinophils remains to be elucidated. In this study, we examined the involvement of the Jak/Stat pathway in the signaling of eosinophils after IFN-gammaR conjugation by the ligand binding. METHODS: Purified peripheral eosinophils were stimulated with IFN-gamma at 37 degrees C for 1-60 min. Tyrosine phosphorylation of IFN-gammaR, Jak1, Jak2, and Stat1alpha was examined by immunoblotting. Gel-shift assay was also examined to show the formation of Stat1alpha-DNA complexes. RESULTS: We show that binding of IFN-gamma to human eosinophils initiated a series of events that resulted in the rapid tyrosine phosphorylation of not only the IFN-gammaRalpha chain but also Jak1, Jak2, and Stat1alpha. In addition, IFN-gamma enhanced the DNA-binding activity of Stat1alpha. CONCLUSION: These data indicate that IFN-gamma affects eosinophils through its specific receptor and utilizes the Jak/Stat pathway as its mode of signaling. 


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Ras oncogene transformation of human B lymphoblasts is associated with lymphocyte activation and with a block of differentiation. 
The p21ras small GTP binding proteins participate in signal transduction from cell surface receptors and affect neoplastic transformation and development in many different cell types. In the present study, we examined the relationship between ras transformation and differentiation of human B lymphocytes. We show that the constitutive expression of the T24 Ha-ras oncogene in EBV-immortalized B lymphoblasts was associated with the induction of the interleukin 2 receptor alpha subunit, with an impaired immunoglobulin gene expression, altered adhesion properties and increased survival in serum-free medium. Since induction of the IL-2 receptor alpha subunit is a hallmark of lymphocyte activation, we suggest that p21ras naturally triggers B cell activation. The ras-transformed lymphocytes displayed a fully functional IL-2r, as assessed by c-fos induction following treatment with IL-2; nevertheless, they were not growth stimulated by this lymphokine. The decreased expression of immunoglobulin genes indicates that the ras oncogene blocks terminal differentiation to plasma cells, possibly by inhibiting the activity of lymphocyte-specific transcription factors. Somewhat unexpectedly, the constitutive p21ras activity did not cause an increased DNA binding of transcription factors PEA1 (AP1), PEA3, Oct-2 or NF-kB. 


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Inhibition of dexamethasone binding to human glucocorticoid receptor by New World primate cell extracts. 
To determine if New World primates express an inhibitor that influences glucocorticoid receptor (GR) binding characteristics, we examined [3H]dexamethasone binding in cytosol prepared from B95-8 lymphoid cells, derived from the cotton top tamarin (Saguinus oedipus), in combination with cytosol prepared from human or rat tissues. B95-8 cytosol inhibited specific binding of [3H]dexamethasone (P < 0.01) when mixed with cytosol prepared from either a human lymphoid cell line (HL) or rat thymus. The inhibitory activity was heat labile and trypsin sensitive. Peak inhibitory activity was found in the 150-200 kd fractions after Sephacryl G-200 ultrafiltration. Scatchard analysis of [3H]dexamethasone binding using mixed cytosol showed a diminished GR apparent binding affinity when compared to HL cytosol. Kinetic studies using mixed cytosol indicated that B95-8 cytosol did not affect the apparent dissociation rate of [3H]dexamethasone. These data demonstrate that B95-8 cells contain a competitive inhibitor that prevents binding of dexamethasone to its cognate receptor. 


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Age-related decreases in IL-2 production by human T cells are associated with impaired activation of nuclear transcriptional factors AP-1 and NF-AT. 
Although transcriptional factors AP-1 and nuclear factor of activated T cells (NF-AT) are important for the normal induction of IL-2, it is unknown if the age-related decline in IL-2 production by activated human T cells may be associated with aberrancies in transcriptional regulatory proteins. In the current studies, IL-2 production by T cells from elderly (mean 78 years) and young (mean 37 years) humans was measured in cultures stimulated with PHA, PHA plus PMA, crosslinked anti-CD3 mAB OKT3 plus PMA, or PMA plus ionomycin. Substantial decreases of IL-2 production were observed for cell cultures from 7 of 12 elderly individuals in response to the different stimuli, whereas the levels of IL-2 produced by stimulated T cells from other elderly individuals were equivalent to those observed for stimulated T cells of young subjects. Analyses of nuclear extracts by electrophoretic DNA mobility shift assays showed that decreased IL-2 production by stimulated T cells of elderly individuals was closely associated with impairments in the activation of both AP-1 and NF-AT. By contrast, T cells from elderly subjects with normal levels of IL-2 production exhibited normal activation of AP-1 and NF-AT. In addition, the results of competition experiments analyzing the normal components of NF-AT showed that the age-related reductions in stimulus-dependent NF-AT complexes corresponded to the slow migrating complexes that were composed of c-Fos/c-Jun AP-1. The resting and stimulated levels of NF kappa B were reduced in T cells from certain elderly individuals; however, alterations of NF kappa B did not correlate with changes in IL-2 expression. Thus, these results show that age-related impairments in the activation of AP-1 and NF-AT are closely associated with decreased expression of IL-2 and further suggest that aberrancies in the signaling pathways important for the induction of transcriptionally active c-Fos/c-Jun AP-1 may contribute to the impaired activation of NF-AT. 


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Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3). 
Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Interferon-alpha induction of STATs1, -3 DNA binding and growth arrest is independent of Lck and active mitogen-activated kinase in T cells. 
Type I interferons (IFNs) are a family of cytokines that have antiviral and antiproliferative effects. Data regarding the processes by which these cytokines transduce signals from the cell membrane to the nucleus are becoming increasingly complex. The most characterized pathway is via JAK-STAT signaling. Previous studies established a potential role for the Src-family kinase Lck in JAK-STAT signaling. Therefore, this study was designed to analyze the role of Lck in IFN-alpha signaling by using the Jurkat, JCam (an Lck-defective cell line derived from Jurkat), and JCam/Lck (JCam cells with Lck restored). The results show that IFN-alpha can induce MAPK activity, but only in cells containing Lck. Furthermore, STATs1 and -3 are effectively phosphorylated and activated to bind DNA in the absence of Lck expression in IFN-alpha-treated cells. Finally, the results demonstrate that IFN-alpha exerts an antiproliferative effect in all three cell lines. These data indicate that Lck and active MAPK do not affect IFN-alpha-induced growth arrest or induction of STAT1s1 and -3 DNA binding ability. Copyright 1999 Academic Press. 


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Evidence for a polyclonal etiology of palmar fibromatosis. 
X chromosome inactivation patterns at the androgen receptor locus were evaluated to determine clonality in microdissected lesional tissue and in leukocytes from 2 women with Dupuytren's disease. The tissue from both patients generated a polyclonal pattern of X chromosome inactivation of the human androgen receptor gene. This finding supports a polyclonal reactive process as the underlying etiology for palmar fibromatosis. 


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Comparative analysis identifies conserved tumor necrosis factor receptor-associated factor 3 binding sites in the human and simian Epstein-Barr virus oncogene LMP1. 
Nonhuman primates are naturally infected with a B-lymphotropic herpesvirus closely related to Epstein-Barr virus (EBV). These simian EBV share considerable genetic, biologic, and epidemiologic features with human EBV, including virus-induced tumorigenesis. However, latent, transformation-associated viral genes demonstrate marked sequence divergence among species despite the conserved functions. We have cloned the latent membrane protein 1 (LMP1) homologs from the simian EBV naturally infecting baboons (cercopithicine herpesvirus 12, herpesvirus papio) and rhesus monkeys (cercopithicine herpesvirus 15) for a comparative study with the human EBV oncogene. The transmembrane domains are well conserved, but there is striking sequence divergence of the carboxy-terminal cytoplasmic domain essential for B-cell immortalization and interaction with the tumor necrosis factor receptor signaling pathway. Nevertheless, the simian EBV LMP1s retain most functions in common with EBV LMP1, including the ability to induce NF-(kappa)B activity in human cells, to bind the tumor necrosis factor-associated factor 3 (TRAF3) in vitro, and to induce expression of tumor necrosis factor-responsive genes, such as ICAM1, in human B lymphocytes. Multiple TRAF3 binding sites containing a PXQXT/S core sequence can be identified in the simian EBV LMP1s by an in vitro binding assay. A PXQXT/S-containing sequence is also present in the cytoplasmic domain of the Hodgkin's disease marker, CD30, and binds TRAF3 in vitro. The last 13 amino acids containing a PXQXT/S sequence are highly conserved in human and simian EBV LMP1 but do not bind TRAF3, suggesting a distinct role for this conserved region of LMP1. The conserved TRAF3 binding sites in LMP1 and the CD30 Hodgkin's disease marker provides further evidence that a TRAF3-mediated signal transduction pathway may be important in malignant transformation. 


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Overexpression of DR-nm23, a protein encoded by a member of the nm23 gene family, inhibits granulocyte differentiation and induces apoptosis in 32Dc13 myeloid cells. 
Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, DR-nm23, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-nm23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granulocyte colony-stimulating factor and causes apoptotic cell death. These results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia. 


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Induction of ICAM-1 and LFA-3 by Tax1 of human T-cell leukemia virus type 1 and mechanism of down-regulation of ICAM-1 or LFA-1 in adult-T-cell-leukemia cell lines. 
The present study was undertaken to determine the role of HTLV-I TaxI in the up-regulation of ICAM-I and LFA-3 in human T cells transformed with HTLV-I and the mechanism of down-regulation of ICAM-I and LFA-I in ATL-derived cell lines. Induction of TaxI in a human T-cell line Jurkat carrying the TaxI gene under the metallothionein promoter led to increases in mRNA and surface expression of ICAM-I. The response of LFA-3 to TaxI induction was, on the other hand, relatively slow and weak, and might be indirect. Transactivation of the ICAM-I promoter by TaxI was further shown by co-transfection of a CAT reporter construct with the ICAM-I promoter and a plasmid expressing TaxI. The mechanism of down-regulation of ICAM-I or LFA-I in 4 ATL cell lines was next examined. ICAM-I mRNA was quite low in MT-I, but no genomic changes were found. The CAT reporter with the ICAM-I promoter was inactive in MT-I. Finally, combined treatment of MT-I with 5-azacytidine and IFN-gamma induced re-expression of ICAM-I. Collectively, (a) transcriptional factor(s) necessary for expression of ICAM-I gene may be repressed in MT-I through DNA methylation. Three other ATL cell lines (TL-OmI, H582, HuT102) were found to have little mRNA for the LFA-I beta chain (CD18). H582 and HuT102 were also negative for the LFA-I alpha chain (CDIIa) mRNA. No genomic changes were found, and a CAT reporter gene with the CD18 promoter was inactive in the 3 of them, again suggesting lack of (a) transcriptional factor(s) necessary for CD18 expression. 


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SOCS-3 is tyrosine phosphorylated in response to interleukin-2 and suppresses STAT5 phosphorylation and lymphocyte proliferation. 
Members of the recently discovered SOCS/CIS/SSI family have been proposed as regulators of cytokine signaling, and while targets and mechanisms have been suggested for some family members, the precise role of these proteins remains to be defined. To date no SOCS proteins have been specifically implicated in interleukin-2 (IL-2) signaling in T cells. Here we report SOCS-3 expression in response to IL-2 in both T-cell lines and human peripheral blood lymphocytes. SOCS-3 protein was detectable as early as 30 min following IL-2 stimulation, while CIS was seen only at low levels after 2 h. Unlike CIS, SOCS-3 was rapidly tyrosine phosphorylated in response to IL-2. Tyrosine phosphorylation of SOCS-3 was observed upon coexpression with Jak1 and Jak2 but only weakly with Jak3. In these experiments, SOCS-3 associated with Jak1 and inhibited Jak1 phosphorylation, and this inhibition was markedly enhanced by the presence of IL-2 receptor beta chain (IL-2Rbeta). Moreover, following IL-2 stimulation of T cells, SOCS-3 was able to interact with the IL-2 receptor complex, and in particular tyrosine phosphorylated Jak1 and IL-2Rbeta. Additionally, in lymphocytes expressing SOCS-3 but not CIS, IL-2-induced tyrosine phosphorylation of STAT5b was markedly reduced, while there was only a weak effect on IL-3-mediated STAT5b tyrosine phosphorylation. Finally, proliferation induced by both IL-2- and IL-3 was significantly inhibited in the presence of SOCS-3. The findings suggest that when SOCS-3 is rapidly induced by IL-2 in T cells, it acts to inhibit IL-2 responses in a classical negative feedback loop. 


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HIV-1 LTR activity in human CD40-activated B lymphocytes is dependent on NF-kappaB. 
CD40-stimulated human B lymphocytes are highly permissive to a productive infection by the human immunodeficiency virus type 1. In these cells, nuclear factors involved in activation of the HIV-1 LTR, which contains the transcriptional control elements of the virus, are unknown. Transient expression assays with plasmids containing deleted parts of the LTR region linked to a reporter gene showed that the NF-kappaB binding site was essential for HIV-1 LTR activity in CD40-stimulated B lymphocytes. In addition, electrophoretic mobility shift and supershift assays revealed that important NF-kappaB binding activity composed of at least p50, p65, and c-Rel NF-kappaB subunits was present in nuclei of CD40-stimulated B cells. These results confirm at a molecular level the ability of HIV-1 to replicate in B cells and that this activity is strongly associated with NF-kappaB. 


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Oxidative stress triggers STAT3 tyrosine phosphorylation and nuclear translocation in human lymphocytes. 
Oxidizing agents are powerful activators of factors responsible for the transcriptional activation of cytokine-encoding genes involved in tissue injury. In this study we show evidence that STAT3 is a transcription factor whose activity is modulated by H2O2 in human lymphocytes, in which endogenous catalase had previously been inhibited. H2O2-induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by immunoblotting of nuclear fractions and electrophoretic mobility shift assays, and vanadate was found to strongly synergize with H2O2. Moreover, anti-STAT3 antibodies specifically precipitated a protein of 92 kDa that becomes phosphorylated on tyrosine upon lymphocyte treatment with H2O2. Phenylarsine oxide, a tyrosine phosphatase inhibitor, and genistein, a tyrosine kinase inhibitor, cooperated and cancelled, respectively, the H2O2-promoted STAT3 nuclear translocation. Evidence is also presented, using Fe2+/Cu2+ ions, that.OH generated from H2O2 through Fenton reactions could be a candidate oxygen reactive species to directly activate STAT3. Present data suggest that H2O2 and vanadate are likely to inhibit the activity of intracellular tyrosine phosphatase(s), leading to enhanced STAT3 tyrosine phosphorylation and hence its translocation to the nucleus. These results demonstrate that the DNA binding activity of STAT3 can be modulated by oxidizing agents and provide a framework to understand the effects of oxidative stress on the JAK-STAT signaling pathway. 


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20-Epi analogues of 1,25-dihydroxyvitamin D3 are highly potent inducers of DRIP coactivator complex binding to the vitamin D3 receptor. 
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in the stimulation of bone growth, mineralization, and intestinal calcium and phosphate absorption; it also acts as a general inhibitor of cellular proliferation. Several new, clinically relevant compounds dissociate antiproliferative and calcemic activities of 1,25(OH)2D3, but the molecular basis for this has not been clearly elucidated. Here, we tested whether the potency of one class of compounds, 20-epi analogues, to induce myeloid cell differentiation, is because of direct molecular effects on vitamin D receptor (VDR). We report that two 20-epi analogues, MC1627 and MC1288, induced differentiation and transcription of p21(Waf1,Cip1), a key VDR target gene involved in growth inhibition, at a concentration 100-fold lower than that of 1,25(OH)2D3. We compared this sensitivity to analogue effects on VDR interacting proteins: RXR, GRIP-1, and DRIP205, a subunit of the DRIP coactivator complex. Compared with the interaction of VDR with RXR or GRIP-1, the differentiation dose-response most closely correlated to the ligand-dependent recruitment of the DRIP coactivator complex to VDR and to the ability of the receptor to activate transcription in a cell-free system. These results provide compelling links between the efficiency of the 20-epi analogue in inducing VDR/DRIP interactions, transactivation in vitro, and its enhanced ability to induce cellular differentiation. 


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Expression of Th1 and Th2 type cytokines responding to HBsAg and HBxAg in chronic hepatitis B patients. 
The cytokine pattern on viral antigen recognition is believed to exert a profound influence on the resolution of viral infections and viral clearance. This study was initiated to investigate whether a cytokine imbalance oriented toward Th2 type response plays a role in chronic hepatitis B. Cytokine profiles of peripheral blood mononuclear cells associated with chronic hepatitis B were analysed by RT-PCR. Upon HBsAg stimulation, expression of IFN-gamma, IL-2, IL-4, and IL-10 was detected in 41%, 8%, 41%, and 50% of the patients, respectively. Among these cytokines, the expression of IFN-gamma was associated with high levels of serum AST/ALT. However, we could not prove that Th2 type cytokines had a protective effect on hepatocytes. Upon HBxAg stimulation, there was no recognizable association of cytokine patterns with AST/ALT levels. In conclusion, production of a Th1 cytokine, IFN-gamma, by HBsAg-reactive cells was associated with hepatocyte damage in chronic hepatitis B, while no counteracting effect of Th2 cytokines produced by those cells was observed. 


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Expression of the chemokine receptor BLR2/EBI1 is specifically transactivated by Epstein-Barr virus nuclear antigen 2. 
In our attempt to identify chemokine receptors that are related to Burkitt's lymphoma receptor 1 (BLR1) and are expressed in activated lymphocytes we used RT-PCR resulting in the isolation of a cDNA encoding a seven transmembrane receptor termed BLR2. The protein shows significant sequence similarities to the family of G-protein coupled chemokine receptors and turned out to be identical to the recently described receptor EBI1. Northern blot analysis revealed that BLR2 mRNA could be highly stimulated in mitogen- and anti-CD3-treated peripheral blood lymphocytes. BLR2-specific mRNA could be detected in all Epstein-Barr virus positive B cell lines. We show that transcription of the BLR2 gene could be specifically induced in Epstein-Barr virus negative BL 41 cells via estrogen-mediated activation of Epstein-Barr virus nuclear antigen 2, a key regulator of viral and cellular genes in immortalized B cells. Our data suggest an involvement of BLR2 in the regulation of migration in activated lymphocytes and in viral pathogenesis. 


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Functional roles of in vivo footprinted DNA motifs within an alpha-globin enhancer. Erythroid lineage and developmental stage specificities. 
Transcriptional regulation of the human alpha-like globin genes, embryonic zeta 2 and adult alpha, during erythroid development is mediated by a distal enhancer, HS-40. Previous protein-DNA binding studies have shown that HS-40 consists of multiple nuclear factor binding motifs that are occupied in vivo in an erythroid lineage- and developmental stage-specific manner. We have systematically analyzed the functional roles of these factor binding motifs of HS-40 by site-directed mutagenesis and transient expression assay in erythroid cell cultures. Three of these HS-40 enhancer motifs, 5'NF-E2/AP1, GT II, and GATA-1(c), positively regulate the zeta 2-globin promoter activity in embryonic/fetal erythroid K562 cells and the adult alpha-globin promoter activity in adult erythroid MEL cells. On the other hand, the 3'NF-E2/AP1 motif is able to exert both positive and negative regulatory effects on the zeta 2-globin promoter activity in K562 cells, and this dual function appears to be modulated through differential binding of the ubiquitous AP1 factors and the erythroid-enriched NF-E2 factor. Mutation in the GATA-1(d) motif, which exhibits an adult erythroid-specific genomic footprint, decreases the HS-40 enhancer function in dimethyl sulfoxide-induced MEL cells but not in K562 cells. These studies have defined the regulatory roles of the different HS-40 motifs. The remarkable correlation between genomic footprinting data and the mutagenesis results also suggests that the erythroid lineage- and developmental stage-specific regulation of human alpha-like globin promoters is indeed modulated by stable binding of specific nuclear factors in vivo. 


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Expression of c-fos, c-jun and jun B in peripheral blood lymphocytes from young and elderly adults. 
The expression of c-fos, c-jun and jun B proto-oncogenes was studied in phytohemagglutinin (PHA) activated peripheral blood lymphocytes (PBL) from young and aged humans. Specific mRNAs for c-fos and c-jun were detectable within 30 min after cell activation and reached maximal levels within 2 h. Both c-fos and jun B mRNAs decreased to pre-activation levels within 6 h, while c-jun mRNA remained elevated. In PHA-activated PBL, no age-related differences were observed in c-fos or jun B mRNA expression. However, c-jun mRNA levels decreased significantly (1.73 +/- 0.08 vs. 1.16 +/- 0.09 arbitrary units, P < 0.01, young vs. old) in PBL from elderly individuals activated with PHA. Because previous work has demonstrated that T cells from elderly individuals may display normal proliferative responses when activated via the anti-CD2 pathway, c-jun and jun B mRNA expression was also studied in anti-CD2-activated purified T cells. No age-related differences were found in the expression of either of these two proto-oncogenes by anti-CD2 activated T cells. These results suggest that the decreased IL-2 production and proliferative response displayed by PHA-activated PBL from elderly adults may be related to age-related changes in c-jun mRNA expression and in the ratio of c-fos to c-jun mRNA. 


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Lineage-specific activation of STAT3 by interferon-gamma in human neutrophils. 
Binding of interferon-gamma (IFN-gamma) to its heterodimeric receptor induces activation of the tyrosine kinases JAK1 and JAK2 followed by tyrosine phosphorylation of STAT1alpha. Selective activation of STAT1alpha at the IFN-gamma receptor is achieved by specific interaction between a cytosolic tyrosine motif including Y440 in the IFN-gamma receptor alpha-chain and the SH2 domain of STAT1alpha. We demonstrate that, in addition to STAT1alpha, STAT3 is also activated by IFN-gamma in human neutrophils. The activation of STAT3 was not found in human eosinophils, monocytes, and HL-60 cells, although the STAT3 protein was expressed in these cells. The cell type-specific activation of STAT3 by IFN-gamma was also observed in neutrophils that are differentiated in vitro from human CD34+ hematopoietic stem cells. These results indicate that a single cytokine receptor can activate different STAT family members in a cell-specific manner, which might result in cell-specific gene transcription. 


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Identification and purification of human Stat proteins activated in response to interleukin-2. 
A key cytokine induced during the immune response is IL-2. Following T cell activation, the genes encoding IL-2 and the various chains of its receptor are transcriptionally induced. In turn, secreted IL-2 serves to stimulate the proliferation and differentiation of T lymphocytes. Several recent studies have implicated Jak kinases in the signaling pathway induced by IL-2. Following this lead, we set out to identify transcription factors induced in response to IL-2. Human peripheral blood lymphocytes were observed to contain several IL-2-inducible DNA binding activities. Similar activities were also observed in a transformed human lymphocyte line, termed YT. We have purified these activities and found that the principal IL-2-inducible component bears significant relatedness to a prolactin-induced transcription factor first identified in sheep mammary gland tissue. We hypothesize that activation of this protein, designated hStat5, helps govern the biological effects of IL-2 during the immune response. 


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Retinoic acid activates interferon regulatory factor-1 gene expression in myeloid cells. 
All-trans-retinoic acid (ATRA) is the drug of choice in the treatment of acute promyelocytic leukemia (APL). ATRA induces both in vitro and in vivo differentiation of APL cells into mature granulocytes. However, the molecular mechanisms involved in ATRA-dependent growth inhibition and cellular differentiation are not presently understood. The NB4 cell line, which is derived from the bone marrow of a patient with APL during relapse, can be used as a model system to study the growth and differentiation of APL cells. Because interferon (IFN) regulatory factors (IRF-1 and IRF-2) and other IFN-inducible gene products regulate cell growth, we analyzed the effects of ATRA on the expression of these genes. We show that ATRA directly activates IRF-1 gene expression, followed by activation of IRF-2 and 2'-5' oligoadenylate synthetase (OAS) gene expression with slower kinetics. In addition to NB4 cells, ATRA also activated IRF-1 gene expression in HL-60, U937, and THP-1 cells, which all respond to ATRA by growth inhibition. A more than additive increase in IRF-1 gene expression was seen with ATRA and IFN-gamma in NB4 cells. ATRA did not activate nuclear factor kappa B or signal transducer and activator of transcription (STAT) activation pathways, suggesting that an alternate mechanism is involved in IRF-1 gene activation. The ATRA-induced expression of IRF-1, an activator of transcription and repressor of transformation, may be one of the molecular mechanisms of ATRA-induced growth inhibition, and the basis for the synergistic actions of ATRA and IFNs in myeloid leukemia cells. 


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Differential nuclear localization of p50, p52, and RelB proteins in human accessory cells of the immune response in situ. 
The Rel/NF-kappa B proteins, p50, p52, p65, c-Rel, and RelB, constitute a family of transcription factors involved in the positive regulation of a variety of genes during the immune response. Recently, it has been shown that RelB knockout mice have no dendritic cells (DC). An overexpression of p50 has been described in follicular dendritic cells (FDC). A constitutive NF-kappa B activity has been reported in mature macrophages. This led to the hypothesis that some of the Rel/NF-kappa B proteins were key nuclear factors in functions of accessory cells of the immune response. Therefore, we investigated in situ the nuclear localization of Rel/NF-kappa B proteins in accessory cells of the immune system by immunohistochemistry and double labeling by immunofluorescence from five normal human tonsils and five lymph nodes with follicular hyperplasia. Nuclear p65 and c-Rel proteins were found in all cell types including lymphocytes. In germinal centers GC, p50, p52, and RelB were found in the nuclei of FDC only and were not detected in the nuclei of CD68+ cells. In T cell areas, p50, p52, and RelB were found in the nuclei of HLA-DR+ cells with an antigen-presenting cell (APC) morphology. p52 and RelB were detected in the nuclei in both CD1a+ and CD68+ cells from the T cell area, whereas p50 was found only in CD68- and CD1a- cells. Cells with nuclear p50 were negative for the CD38, CD20 and CD2 markers. These results show that, physiologically, high levels of nuclear of p50, p52 and RelB are restricted to accessory cells of the immune system, which include FDC in GC, and DC and macrophages in the T cell zone, that specialized scavenger macrophages from GC do not have detectable levels of p52 and RelB, whereas macrophages from the T cell area, known to present the antigen to T cells, do have both nuclear p52 and RelB, and that in the T cell zone, p52 and RelB are located in nuclei of both CD1a+, CD68+ or both, cells APC, whereas p50 is restricted to CD1a- and CD68- APC. The different patterns of p50, p52 and RelB protein nuclear localization may provide insight into their different roles during the immune response in vivo. 


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Glucocorticoid-mediated inhibition of interleukin-2 receptor alpha and -beta subunit expression by human T cells. 
To determine the mechanism of glucocorticoid (GC)-mediated inhibition of T cell functions, the effect of dexamethasone (DM) on T cell proliferation and interleukin-2 receptor (IL-2R) generation were studied. Dexamethasone inhibited IL-2-induced T cell proliferation by 30%-88%, relative to its concentration within the cultures. The effect of DM on expression of IL-2R alpha (Tac, p55, CD25) and beta (p75) genes in activated T cells was examined next. In T cells stimulated with purified phytohemagglutinin (PHA-p) and 4 beta-phorbol 12-myristate 13-acetate (PMA) addition of DM to the cultures resulted in a 60% reduction in IL-2R alpha and a 30% reduction in IL-2R beta membrane expression compared to T cells cultured in the absence of DM (p < 0.01). Inhibition of membrane IL-2R alpha and IL-2R beta expression by 10(-6) M DM was partially reversible by recombinant human IL-2 (rhIL-2). By Northern blot analysis, DM caused a comparable decrease in IL-2R alpha and in IL-2R beta mRNA levels to membrane receptor expression in mitogen-stimulated T cells. By in vitro transcription assays, DM regulated IL-2R alpha gene expression at a transcriptional level while transcription of IL-2R beta gene was unaffected by DM. The mechanism of action of DM on IL-2R alpha transcription was examined by determining the mRNA levels of the p50 subunit of nuclear factor kappa B (NF-kappa B), a transcription factor that stimulates IL-2R alpha gene expression. The data indicate that 10(-6) M DM increased T cell p50 NF-kappa B mRNA levels by four-fold compared to the levels obtained in the absence of DM. Further, the level of nuclear proteins capable of binding to the NF-kappa B sites in activated T cells increased in response to DM. In sum, DM regulates T cell membrane expression of IL-2R by more than one molecular mechanism. 


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Glucocorticoid receptor in patients with lupus nephritis: relationship between receptor levels in mononuclear leukocytes and effect of glucocorticoid therapy. 
We investigated the clinical significance of glucocorticoid receptor determination in 20 patients with systemic lupus erythematosus (SLE) who afterwards developed nephrotic syndrome. Glucocorticoid receptor concentrations in mononuclear leukocytes (MNL) in these patients were comparable with those in both other patients with SLE and healthy persons. Improvement in urinary protein excretion and in disease activity, which was scored according to the SLE Disease Activity Index system of the University of Toronto, closely related to the glucocorticoid receptor concentrations in MNL isolated from the corresponding patients. In summary, glucocorticoid receptor determination in patients with lupus nephritis may be a predictive clue for assessing responsiveness to glucocorticoid therapy. 


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rel Is rapidly tyrosine-phosphorylated following granulocyte-colony stimulating factor treatment of human neutrophils. 
Stimulation of neutrophils with granulocyte-colony stimulating factor (G-CSF) results in an enhanced respiratory burst, prolonged survival, and increased tumor cell killing. The effects of G-CSF are mediated by binding to specific, high affinity receptors. G-CSF receptors lack intrinsic tyrosine kinase activity, but activation of the receptor results in the rapid induction of tyrosine kinase activity. Antiphosphotyrosine immunoblots of whole cell lysates prepared from neutrophils show that the G-CSF rapidly induces prominent tyrosine phosphorylation of a protein of a relative molecular mass of 80 kDa. Using monospecific antibodies, the 80-kDa tyrosine-phosphorylated protein has been shown to be p80c-rel, a proto-oncogene belonging to a family of transcriptional regulators which include NF-kB. The induction of tyrosine phosphorylation of p80c-rel was unique to G-CSF in that granulocyte-macrophage colony stimulating factor which also stimulates neutrophils and induces tyrosine phosphorylation does not result in tyrosine phosphorylation of p80c-rel. The consequences of p80c-rel tyrosine phosphorylation are not yet known; however, tyrosine-phosphorylated p80c-rel is capable of binding to DNA, and G-CSF stimulation results in an increase in the amount of p80c-rel which binds to DNA. These results demonstrate that one of the first biochemical events which occurs in neutrophils following G-CSF stimulation, activation of a tyrosine kinase, leads directly to the tyrosine phosphorylation of p80c-rel. Thus, the tyrosine kinase activated by G-CSF appears to directly transduce a signal to a protein which functions as a transcriptional regulator. 


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Activation of STAT5 by IL-4 relies on Janus kinase function but not on receptor tyrosine phosphorylation, and can contribute to both cell proliferation and gene regulation. 
We have investigated mechanisms and consequences of STAT5 activation through the human IL-4 receptor (IL-4R). By functionally expressing receptor mutants in the murine pro-B cell line Ba/F3, we could show that phosphorylated tyrosine residues within the IL-4R alpha chain are dispensable for IL-4-induced STAT5 activity. However, disruption of a membrane-proximal proline-rich sequence motif ('box1') in either subunit of the bipartite IL-4R abolished not only ligand-induced tyrosine phosphorylation of Janus kinases JAK1 and JAK3, but also IL-4-triggered activation of STAT5 and concomitant cell proliferation. A dominant-negative version of STAT5b, but not of STAT5a, interfered with IL-4-induced DNA synthesis in Ba/F3 cells, suggesting an involvement of STAT5b in the control of cell proliferation through IL-4R. Reporter gene experiments finally showed that transcription from promoters of STAT5 target genes can be specifically induced by challenging cells with IL-4, and that both STAT5a and STAT5b can contribute to IL-4-triggered transcriptional control. 


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HTLV-1 Tax induces expression of various immediate early serum responsive genes. 
Human T-cell leukemia virus type 1 (HTLV-1) is an etiological agent of adult T-cell leukemia (ATL). We showed here by mobility-shift assay that T-cell lines transformed with the virus contained high levels of AP-1 activities. Consistent with this result, these cell lines expressed increased levels of mRNAs encoding the AP-1 proteins, c-Fos, Fra-1, c-Jun, JunB, and JunD. Previously, transcription of the c-fos gene has been reported to be transactivated by the viral transcription factor, Tax1. By using the human T-cell line (JPX-9), in which expression of the Tax1 is inducible, we showed that expression of mRNAs for Fra-1, c-Jun, and JunD was also transactivated by Tax1. Moreover, Tax1 activated expression of two other transcription factors having zinc finger motifs, Egr-1 and Egr-2, in the same cells. The Tax1-inducible transcription factors identified here are encoded by the members of immediate early genes under the control of growth signals. Thus, Tax1 was suggested to replace growth signals, at least in part, by this mechanism. 


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Monoallelic expression of Pax5: a paradigm for the haploinsufficiency of mammalian Pax genes? 
It is generally assumed that most mammalian genes are transcribed from both alleles. Hence, the diploid state of the genome offers the advantage that a loss-of-function mutation in one allele can be compensated for by the remaining wild-type allele of the same gene. Indeed, the vast majority of human disease syndromes and engineered mutations in the mouse genome are recessive, indicating that recessiveness is the 'default' state. However, a minority of genes are semi-dominant, as heterozygous loss-of-function mutation in these genes leads to phenotypic abnormalities. This condition, known as haploinsufficiency, has been described for five of the nine mammalian Pax genes, which are associated with mouse developmental mutants and human disease syndromes. Recently we have reported that the Pax5 gene is subject to allele-specific regulation during B cell development. Pax5 is predominantly transcribed from only one of its two alleles in early B-lymphoid progenitors and mature B cells, while it transiently switches to a biallelic mode of transcription in pre-B and immature B cells. As a consequence, B-lymphoid tissues are mosaic with regard to the transcribed allele, and heterozygous mutation of Pax5 therefore results in deletion of B lymphocytes expressing only the mutant allele. The allele-specific regulation of Pax5 raises the intriguing possibility that monoallelic expression may also be the mechanism causing the haploinsufficiency of other Pax genes. In this review, we discuss different models accounting for the haploinsufficiency of mammalian Pax genes, provide further evidence in support of the allele-specific regulation of Pax5 and discuss the implication of these findings in the context of the recent literature describing the stochastic and monoallelic activation of other hematopoietic genes. 


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[The effect of 24,25-dihydroxyvitamin D3 (dioxyvit) on Ca metabolism and immune status during chronic kidney failure] 
Active metabolite of vitamin D3, 24R,25 (OH)2D3 (dioxyvit) was used at a daily dose of 100 micrograms in treatment of children affected with tubulointerstitial disease of kidney and with chronic glomerulonephritis under conditions of kidney insufficiency. The drug exhibited distinct normalizing effect on patterns of calcium metabolism: increase of total and ionized Ca2+ and of 25-OHD, decrease in concentration of parath hormone and osteocalcine in blood serum as well as on immunological parameters: restoration of decreased content of T- and 0-lymphocytes. Concentration of receptors of hormonal form of 1,25(OH)2D3 was found to be minimal in lymphocytes under conditions of chronic kidney insufficiency, while their expression, after the dioxyvit action, was detected only in patients with glomerulonephritis. Specific calcitropic effect of dioxyvit with simultaneous correction of vitamin D deficiency were apparently responsible for high efficacy of the drug in treatment of calcium metabolism and immunity impairments in children with renal deteriorations at the step of chronic kidney insufficiency. 


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Detection of intracellular phosphorylated STAT-1 by flow cytometry. 
We have applied flow cytometry to the investigation of interferon-gamma activation of human monocytes. This approach uses monoclonal antibodies that distinguish between the native and phosphorylated forms of STAT-1. It enables rapid and quantitative assessment of STAT-1 phosphorylation on a discrete cell basis and is both more sensitive and less time consuming than immunoblotting. Furthermore, it allows for discrimination between a mixture of cells that differ in their response to interferon-gamma. This approach should allow for the evaluation of different intracellular signaling pathways using a combination of monoclonal reagents that are specific for native and activation modified proteins. Application of this form of testing should prove valuable in screening for signaling defects in selected patients with recurrent infections. In addition, this technique should permit dissection of a full range of cellular signaling pathways at the protein level. 


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Two glucocorticoid binding sites on the human glucocorticoid receptor. 
Glucocorticoids are known to have a lytic effect in leukemic cells via interactions with the glucocorticoid receptor (GR). Cortisol and various synthetic glucocorticoids bind to the GR with one-site kinetics. Cortivazol (CVZ) is a unique, high potency synthetic glucocorticoid, which has a phenylpyrazol fused to the A-ring of the steroid nucleus and displays binding consistent with two or more sites in the cytosol from CEM C7 cells (a human acute lymphoblastic T-cell line). It has previously been shown that the lower affinity class of sites are similar in affinity and site molarity to those recognized by dexamethasone. The higher affinity sites bind CVZ with 20- to 50-fold greater affinity, consistent with CVZ's enhanced biological effects. In mutant leukemic cells resistant to the lytic effects of dexamethasone, CVZ both lyses the cells and recognizes a single class of sites similar to the high affinity site in CEM C7 cells. We have carried out experiments to define the nature of the higher affinity CVZ binding site. We now show that: 1) CVZ has more than one binding site in a second, independent, B-cell line, IM-9; 2) the antiglucocorticoid RU 38486 is able to block both CVZ's higher and lower affinity sites; 3) all of CVZ's binding sites are on a protein immunologically indistinguishable from the human GR; and 4) freshly isolated clones of CVZ-resistant cells have lost all binding sites for CVZ. These data indicate that CVZ is recognizing two glucocorticoid binding sites on the human GR or a protein very similar to it. 


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Transcriptional activation of the macrophage colony-stimulating factor gene by IL-2 is associated with secretion of bioactive macrophage colony-stimulating factor protein by monocytes and involves activation of the transcription factor NF-kappa B. 
Human peripheral blood monocytes (Mo) constitutively display the beta-chain of the receptor for IL-2, whereas expression of the IL-2R alpha-chain is not constitutive but inducible with IL-2. Here we report that binding of human IL-2 to its binding site leads to transcriptional activation of the macrophage CSF (M-CSF) gene in Mo resulting in accumulation of M-CSF mRNA and subsequent release of bioactive M-CSF protein as demonstrated by ELISA and inhibition of IL-2 induced release of an activity-stimulating growth of monocyte-type colonies by a neutralizing anti-M-CSF antibody. Transcriptional activation of the M-CSF gene by IL-2 is preceded by enhanced binding activity of the transcription factor NF-kappa B to its recognition sequence in the 5' regulatory enhancer region of the M-CSF gene. Moreover, using a heterologous promoter (herpes thymidine kinase) construct containing the NF-kappa B consensus sequence, it is shown that NF-kappa B binding by an IL-2-induced monocyte-derived nuclear protein confers reporter gene (human growth hormone) activity. Taken together, our findings indicate that IL-2 induces gene expression of M-CSF in human blood-derived Mo and provide evidence for involvement of NF-kappa B in transcriptional regulation of this gene. 


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Human T-cell leukemia virus type 1 Tax protein induces the expression of STAT1 and STAT5 genes in T-cells. 
Human T-cell leukemia virus type 1 (HTLV-1) Tax transforms normal T-cells in the presence of interleukin (IL)-2 in vitro. STAT is a family of transcription factors that play a pivotal role in cytokine-induced functions of a various type of cells. We investigated the involvement of STATs in the transformation of T-cells by HTLV-1. HTLV-1-transformed T-cell lines expressed higher amounts of STAT1, STAT3 and STAT5 RNA and proteins than virus-negative T cells. The expression of STAT1 and STAT5 in a human T-cell line was induced by Tax. IL-2 induced the DNA binding activity of STAT3 and STAT5 of a HTLV-1-transformed cell line and then stimulated its proliferation. In contrast, IL-2 did neither in a cell line lacking STAT3 and STAT5. The expression of STAT1, STAT3 and STAT5 mRNAs were also induced by a T-cell mitogen in normal human peripheral blood mononuclear cells. Our results suggest that the induction of STAT1 and STAT5 by Tax enhances cytokine-induced functions of virus-infected T-cells, hence the induction may play a role in IL-2-dependent transformation steps of T-cells by HTLV-1. 


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A microtitre assay system for glucocorticoid receptors: decreased receptor concentration in myocardial infarction. 
A major difficulty in determination of glucocorticoid receptor sites is the very complicated assay procedure. Therefore, we describe a microtitre assay system for glucocorticoid receptors which is a whole-cell competitive binding radioassay using [3H]-dexamethasone as radioligand. This modification of a previously described protocol simplifies and reduces laboratory work and allows assay reproducibility to be controlled more reliably. Thus enabled to perform the test on multiple blood samples in parallel, we investigated cardiac infarction patients over a 12-day period to test if glucocorticoid receptor binding is altered in this 'stressful' disease. On the first day of the disease, glucocorticoid receptor capacity was significantly decreased without alteration of the receptor-ligand affinity, whereas on days 4 and 12 the number of receptor sites was normal again. This result fits well into the general observation of stress-induced down-regulation of immune responses. 


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The suppression of T cell function and NF(kappa)B expression by serine protease inhibitors is blocked by N-acetylcysteine. 
Direct evidence that N-acetylcysteine (NAC) enhances the immune response of peripheral blood T cells at the level of NF(kappa)B is presented. In addition, NAC blocks the suppression of T cell mitogenesis and cytokine production by protease inhibitors such as N-tosylphenylalanine chloromethyl ketone (TPCK). The proliferative responses of purified CD4+ or CD8+ T cells are suppressed more strongly by TPCK when anti-CD28 rather than the phorbol ester PMA is used as the mitogenic coactivator. Cytokine (IL-2, IL-6, INF-gamma) production is inhibited 95-100% by concentrations of TPCK that totally suppress the mitogenesis of CD4+ or CD8+ cells. Using electrophoretic mobility shift assays, we find that TPCK virtually abolishes (to less than 1%) the levels of NF(kappa)B (but not Oct-1) found in nuclear and whole cell extracts of activated T cells. Strikingly, the immunosuppressive effects of TPCK are blocked when T cells are pretreated for 15 min with 5 mM NAC. NAC not only blocks the effect of TPCK but enhances mitogenesis and cytokine production (>2.5-fold in some cases) upon activation of unsuppressed T cells. Our data support the notion that NF(kappa)B and I(kappa)B proteases play obligate roles in T cell activation and mitogenesis, roles that are enhanced significantly by NAC. 


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Detection in non-erythroid cells of a factor with the binding characteristics of the erythroid cell transcription factor EF1. 
The erythroid transcription factor erythroid factor-1 (EF1) plays a critical role in the transcription of erythroid-specific genes. Here we report the presence of a factor with the mobility and sequence-specific DNA-binding characteristics of EF1 at low abundance in a wide variety of non-erythroid cell types. This is the first report of an EF1-like activity in non-erythroid cells and indicates that this factor may play a role in the regulation of genes expressed in such cells. 


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A newly established megakaryoblastic/erythroid cell line that differentiates to red cells in the presence of erythropoietin and produces platelet-like particles. 
In August, 1992, we established a leukemic cell line (NS-Meg) from a patient in megakaryoblastic transformation of Philadelphia chromosome-positive chronic myeloid leukemia. The NS-Meg cells were positive for alpha-naphthyl acetate esterase and periodic acid-Schiff (PAS) staining and for surface CD4, CD7, CD13, CD34, CD41a, and glycophorin A antigens. Ultrastructurally, the cells had alpha-granules, demarcation membranes, and platelet peroxidase activity. The NS-Meg cells spontaneously produced platelet-like particles which contained alpha-granules, mitochondria and dense bodies, strongly suggesting platelet production. Erythropoietin (Epo), granulocyte/macrophage colony stimulating factor(GM-CSF), and interleukin 3 (IL-3) promoted the growth of NS-Meg cells. Phorbol-12-myristate-13-acetate increased the expression of both CD41a and CD61 antigens. Ten-day exposure to Epo induced mature erythroblasts and red cells. These benzidine-positive cells were positive for hemoglobin F staining. Untreated NS-Meg cells expressed mRNA for the Epo receptor (EpoR), for GATA-1, and for alpha 1, alpha 2 and gamma globin genes. These results indicate that NS-Meg cells undergo terminal differentiation of both megakaryocytic and erythroid lineages. This cell line should be a very useful tool for the investigation of both megakaryocytic and erythroid maturation. 


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Cytomegalovirus modulates interleukin-6 gene expression. 
Complications after lung transplantation include the development of rejection and an increased incidence of infection, particularly with cytomegalovirus (CMV). Several recent studies have suggested that interleukin (IL)-6 may be used to detect both infection and rejection after lung transplantation. In addition, IL-6 may play a role in the development of bronchiolitis obliterans after transplantation. Because CMV is also associated with the development of bronchiolitis obliterans after transplantation, we determined whether CMV induces IL-6 gene expression. We demonstrated that CMV infection increased both IL-6 protein and mRNA in peripheral blood mononuclear cells. We also demonstrated that the CMV immediate early 1 gene product increased expression of the IL-6 promoter. This effect of the CMV immediate early 1 gene product was dependent upon the presence of specific transcription factor binding sites in the IL-6 promoter. These studies demonstrate that CMV may be an important cofactor in the development of rejection and infection after transplantation through its effects on IL-6. 


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Signal transduction pathways in normal human monocytes stimulated by cytokines and mediators: comparative study with normal human neutrophils or transformed cells and the putative roles in functionality and cell biology. 
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) -3 induced tyrosine phosphorylation of 92-kDa protein in normal human monocytes. We identified this 92-kDa protein as STAT5, but not as STATs1, 3, and 6 nor c-fes and vav protooncogene products, and demonstrated its translocation to the nucleus, enhancement of specific DNA binding capacity, and potentiation of trancriptional activity by GM-CSF. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA) induced tyrosine phosphorylation of 42- and 44-kDa proteins, which were identified as extracellular signal-regulated kinase (ERK), in human monocytes. In marked contrast to neutrophils and MO7e cells, GM-CSF did not induce tyrosine phosphorylation and activation of ERK in monocytes. Among upstream signaling molecules of ERK, Shc was constitutively associated with Grb2 and was not tyrosine-phosphorylated by GM-CSF and FMLP, and Sos1 and c-Raf-1 were not phosphorylated by GM-CSF, IL-3, TNF, and FMLP in monocytes, whereas all these signaling molecules were affected and/or utilized by GM-CSF in MO7e cells. In contrast to neutrophils, p38 was constitutively phosphorylated and agonist-dependent phosphorylation and activation was not detected in human monocytes. Superoxide release stimulated by FMLP was inhibited partially by PD98059 or SB203580, a specific inhibitor of ERK or p38 pathway, and was almost completely inhibited by the combination of both inhibitors, whereas PMA-induced superoxide release was resistant to these two inhibitors in monocytes. PD98059 inhibited GM-CSF-dependent proliferation of MO7e cells. Present results indicate trancriptional roles of STAT5 and functional roles of ERK and/or p38 in normal human monocytes stimulated by physiological receptor-mediated agonists GM-CSF and FMLP. Possible roles of ERK in proliferation of transformed cells were also suggested. 


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Elevated cyclic AMP inhibits NF-kappaB-mediated transcription in human monocytic cells and endothelial cells. 
The NF-kappaB/Rel family of transcription factors regulates the inducible expression of many genes in activated human monocytes and endothelial cells. In this study, we examined the molecular mechanism by which agents that elevate intracellular cAMP inhibit the expression of the tumor necrosis factor alpha (TNFalpha), tissue factor, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1 genes. Both forskolin and dibutyryl cAMP, which elevate intracellular cAMP by independent mechanisms, inhibited TNFalpha and tissue factor expression at the level of transcription. Induction of NF-kappaB-dependent gene expression in transiently transfected human monocytic THP-1 cells and human umbilical vein endothelial cells was inhibited by elevated cAMP and by overexpression of the catalytic subunit of protein kinase A (PKA). Elevated cAMP did not prevent nuclear translocation of p50/p65 and c-Rel/p65 heterodimers, decrease nuclear translocation of p65, or significantly modify TNFalpha-induced phosphorylation of p65. Functional studies demonstrated that transcriptional activation of a plasmid containing multimerized kappaB sites by p65 was inhibited by agents that elevate cAMP and by overexpression of the catalytic subunit of PKA. This study indicates that activation of PKA reduces the induction of a distinct set of genes in monocytes and endothelial cells by inhibiting NF-kappaB-mediated transcription. 


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The Epstein-Barr virus (EBV) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell-specific manner. 
The Epstein-Barr virus early antigen diffuse component (EA-D) is essential for Epstein-Barr virus DNA polymerase activity, and its activity is suppressed during latent infection. We investigated the regulation of the promoter (BMRF1) for this early gene by studying its responsiveness in vitro to two immediate-early viral transactivators, BZLF1 (Z) and BRLF1 (R), focusing on the differences in response in lymphoid cells and epithelial cells. In lymphoid cells, Z or R alone produced only small increases in EA-D promoter activity, whereas both transactivators together produced a large stimulatory effect. In epithelial cells, the Z transactivator alone produced maximal stimulation of the EA-D promoter; the effect of R and Z together was no greater than that of Z alone. Deletional analysis and site-directed mutagenesis of the EA-D promoter demonstrated that in epithelial cells the potential AP-1 binding site plays an essential role in Z responsiveness, although sequences further upstream are also important. In lymphoid cells, only the upstream sequences are required for transactivation by the Z/R combination, and the AP-1 site is dispensable. These data suggest that EA-D (BMRF1) promoter regulation by Z and R is cell type specific and appears to involve different mechanisms in each cell type. 


Document 003003589 ends.


Bcl-6 expression in reactive follicular hyperplasia, follicular lymphoma, and angioimmunoblastic T-cell lymphoma with hyperplastic germinal centers: heterogeneity of intrafollicular T-cells and their altered distribution in the pathogenesis of angioimmunoblastic T-cell lymphoma. 
BACKGROUND: The Bcl-6 gene product, a nuclear phosphoprotein, is expressed independently of Bcl-6 gene rearrangement. In lymph nodes, expression of Bcl-6 protein is restricted to germinal center (GC) B-cells and 10% to 15% of CD3/CD4+ intrafollicular T cells. Interfollicular cells are negative for Bcl-6 protein, except for rare CD3+/CD4+ T cells. Recently, we reported cases of angioimmunoblastic T-cell lymphoma (AITL) with hyperplastic GCs (AITL/GC), and observed that borders of enlarged GCs were ill defined, with features suggestive of an outward migration of GC cells to surrounding interfollicular zones. This prompted a study of follicular borders with Bcl-6 staining in reactive follicular hyperplasias and follicular lymphomas to compare with AITL/GC. MATERIALS AND METHODS: Formalin-fixed paraffin sections were used for immunostaining of Bcl-6. Six cases of AITL/GC, 12 nonspecific reactive follicular hyperplasia (FH), 7 HIV adenopathy, 10 follicular lymphoma (FL), and 8 typical AITL (ie, AITL without GC) were studied. Double staining for Bcl-6/CD20, Bcl-6/CD3, and Bcl-6/CD57 was performed in selected cases. RESULTS: In FH and HIV adenopathy, staining for Bcl-6 revealed densely populated GCs with well-defined and regular GC borders, whereas Bcl-6+ cells were rare in the interfollicular areas. An occasional GC with an ill-defined border was invariably surrounded by a broad mantle zone; those with indistinct mantle zones had well-defined, regular borders. In FL, follicles were densely populated, and their borders were irregular, with some Bcl-6+ cells in the interfollicular zones. In AITL/GC, GCs were less dense, GC borders were ill defined and irregular, and the number of interfollicular Bcl-6+ cells was markedly increased. Double staining revealed that these interfollicular Bcl-6+ cells in AITL/GC were Bcl6+/CD3+/CD20-/CD57- T cells. Moreover, CD3+ intrafollicular T cells were depleted in AITL/GC, whereas they were abundant in FH. Intrafollicular CD57+ cells did not stain for Bcl-6, and were also depleted in AITL/GC. In typical AITL, some neoplastic cells were positive for Bcl-6, showing variable degrees of staining. CONCLUSIONS: (1) GCs of AITL/GC differed from those of other reactive follicular hyperplasias and follicular lymphomas, and staining for Bcl-6 was useful to discern them. (2) Intrafollicular CD3+ T cells, many of which were also positive for Bcl-6, were markedly depleted in AITL/GC, with increased interfollicular Bcl-6+/CD3+ cells, suggesting an outward migration of intrafollicular T cells in this condition. (3) Interfollicular Bcl-6+/CD3+ cells in AITL/GC were too numerous to be accounted for by migration alone, suggesting local proliferation. (4) Intrafollicular CD57+ cells were negative for Bcl-6, indicating heterogeneity of the intrafollicular T-cell population. (5) Some neoplastic cells in AITL stained for Bcl-6, suggesting up-regulation of Bcl-6 expression in this tumor. 


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Staphylococcal enterotoxins modulate interleukin 2 receptor expression and ligand-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription (Stat proteins). 
Staphylococcal enterotoxins (SE) stimulate T cells expressing the appropriate variable region beta chain of (V beta) T-cell receptors and have been implicated in the pathogenesis of several autoimmune diseases. Depending on costimulatory signals, SE induce either proliferation or anergy in T cells. In addition, SE can induce an interleukin-2 (IL-2) nonresponsive state and apoptosis. Here, we show that SE induce dynamic changes in the expression of and signal transduction through the IL-2 receptor (IL-2R) beta and gamma chains (IL-2R beta and IL-2R gamma) in human antigen-specific CD4+ T-cell lines. Thus, after 4 hr of exposure to SEA and SEB, the expression of IL-2R beta was down-regulated, IL-2R gamma was slightly up-regulated, while IL-2R alpha remained largely unaffected. The changes in the composition of IL-2Rs were accompanied by inhibition of IL-2-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription called Stat3 and Stat5. In parallel experiments, IL-2-driven proliferation was inhibited significantly. After 16 hr of exposure to SE, the expression of IL-2R beta remained low, while that of IL2R alpha and IL2R gamma was further up-regulated, and ligand-induced tyrosine phosphorylation of Jak3 and Stat proteins was partly normalized. Yet, IL-2-driven proliferation remained profoundly inhibited, suggesting that signaling events other than Jak3/Stat activation had also been changed following SE stimulation. In conclusion, our data suggest that SE can modulate IL-2R expression and signal transduction involving the Jak/Stat pathway in CD4+ T-cell lines. 


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The 56-59-kilodalton protein identified in untransformed steroid receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins. 
It has previously been shown that 9S, untransformed progestin, estrogen, androgen, and glucocorticoid receptor complexes in rabbit uterine and liver cytosols contain a 59-kDa protein [Tai, P.K., Maeda, Y., Nakao, K., Wakim, N.G., Duhring, J.L., & Faber, L.E.(1986) Biochemistry 25, 5269-5275]. In this work we show that the monoclonal antibody KN 382/EC1 raised against the rabbit 59-kDa protein reacts with 9S, untransformed glucocorticoid receptor complexes in cytosol prepared from human IM-9 lymphocytes but not with 4S salt-transformed receptors. The human protein recognized by the EC1 antibody is a 56-kDa protein (p56) of moderate abundance located predominantly in the cytoplasm by indirect immunofluorescence. There are at least six isomorphs of p56 by two-dimensional gel analysis. N-Terminal sequencing (20 amino acids) shows that p56 is a unique human protein. When p56 is immunoadsorbed from IM-9 cell cytosol, both the 70- and 90-kDa heat shock proteins are coadsorbed in an immune-specific manner. Neither heat shock protein reacts directly with the EC1 antibody. We conclude that p56 exists in cytosol in a higher order complex containing hsp70 and hsp90, both of which in turn have been found to be associated with untransformed steroid receptors. 


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Oncogenicity of human papillomavirus- or adenovirus-transformed cells correlates with resistance to lysis by natural killer cells. 
The reasons for the dissimilar oncogenicities of human adenoviruses and human papillomaviruses (HPV) in humans are unknown but may relate to differences in the capacities of the E1A and E7 proteins to target cells for rejection by the host natural killer (NK) cell response. As one test of this hypothesis, we compared the abilities of E1A- and E7-expressing human fibroblastic or keratinocyte-derived human cells to be selectively killed by either unstimulated or interferon (IFN)-activated NK cells. Cells expressing the E1A oncoprotein were selectively killed by unstimulated NK cells, while the same parental cells but expressing the HPV type 16 (HPV-16) or HPV-18 E7 oncoprotein were resistant to NK cell lysis. The ability of IFN-activated NK cells to selectively kill virally transformed cells depends on IFN's ability to induce resistance to NK cell lysis in normal (i.e., non-viral oncogene-expressing) but not virally transformed cells. E1A blocked IFN's induction of cytolytic resistance, resulting in the selective lysis of adenovirus-transformed cells by IFN-activated NK cells. The extent of IFN-induced NK cell killing of E1A-expressing cells was proportional to the level of E1A expression and correlated with the ability of E1A to block IFN-stimulated gene expression in target cells. In contrast, E7 blocked neither IFN-stimulated gene expression nor IFN's induction of cytolytic resistance, thereby precluding the selective lysis of HPV-transformed cells by IFN-activated NK cells. In conclusion, E1A expression marks cells for destruction by the host NK cell response, whereas the E7 oncoprotein lacks this activity. 


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Cloning a cDNA from human NK/T cells which codes for a protein with high proline content. 
A cDNA clone, B4-2, was isolated from a natural killer (NK) minus T cell subtractive library. The B4-2 clone coded for an mRNA of 2061 bp in length. It encodes a deduced 327 aa protein with a calculated molecular mass of 35.2 kDa. Searching of B4-2 DNA and protein sequences against various databases revealed no high homology to other sequences. However, B4-2 has an unusually high proline content (13%), contains a putative nuclear targeting sequence, and has several SPXX motifs which are frequently found in gene regulatory proteins. One of the stretches of prolines in B4-2 closely resembles the ligand for proteins with SH3 domains. Northern hybridization data showed that B4-2 is not a lymphoid specific gene and is expressed in a hepatoma cell line and also weakly transcribed or absent in a variety of other cells. A polyclonal antiserum raised against recombinant B4-2 recognizes a 32-34 kDa protein in lymphocytes. 


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Visualization of the endogenous NF-kappa B p50 subunit in the nucleus of follicular dendritic cells in germinal centers. 
NF-kappa B, a 50 kDa/65 kDa (p50/p65) heterodimer, is a ubiquitous transcription factor involved in the positive regulation of various immune genes. The aim of this study was to determine whether NF-kappa B is related to a particular cell type and/or differentiation step during immunopoiesis. Using in situ hybridization on sections from non HIV hyperplastic lymph nodes, we found that the gene of the 105 kDa precursor of p50 was overexpressed in the light zone of germinal centers, with a network aspect, which suggested the involvement of follicular dendritic cells (FDC). By immunohistochemistry, p50 protein was detected in the cytoplasm and nucleus of FDC, confirming the involvement of FDC. Furthermore, p50 protein was detected in the cytoplasm of all lymphocytes. Thus, we focused our study on isolated FDC clusters from normal tonsils. As showed on tissue sections, we detected the p50 in both cytoplasm and nucleus of FDC. Nuclei of lymphocytes from FDC clusters were negative. We next studied p65 and c-Rel protein expression in FDC clusters. p65 was detected in the cytoplasm of FDC, whereas nuclei were negative. Furthermore, p65 was detected in the nuclei of some lymphocytes. c-Rel protein was detected only in the cytoplasm of lymphocytes and not in the nucleus and cytoplasm of FDC. Our results indicated that, in the context of T cell-dependent B cell immunopoiesis occurring in FDC clusters, p50 is mainly related to FDC with a massive overexpression in the nuclei, whereas p65 is expressed in a scattered manner in the nuclei of lymphocytes and c-Rel protein exclusively in the cytoplasm of lymphocytes from FDC clusters. These results suggested that the two subunits of NF-kappa B and the c-Rel protein have different roles in different cell types during B cell immunopoiesis. 


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Sp1 functions in a chromatin-dependent manner to augment human alpha-globin promoter activity. 
The 5' flanking region of the human alpha-globin gene is highly G + C rich and contains multiple copies of the consensus sequence for the Sp1 binding site. We investigated the role of this G + C-rich region in augmenting alpha-globin promoter activity in the presence of the far-upstream alpha-globin enhancer, HS-40. We show that in transiently transfected erythroid cells, deletion of the alpha-globin G + C-rich 5' flanking region has no effect on alpha-globin promoter activity. However, upon stable integration into chromatin, deletion of this region causes a nearly 90% decrease in promoter activity compared with expression from an alpha-globin promoter retaining this region. These results suggest that the alpha-globin G + C-rich 5' flanking region augments alpha-globin promoter activity in a chromatin-dependent manner. We further show that this G + C-rich region is required for the activation of alpha-globin gene expression during erythroid differentiation. Finally, we show by both footprint analysis and functional assays that the ability of the G + C-rich region to increase alpha-globin promoter activity from a stably integrated alpha-globin gene is mediated by its multiple binding sites for the transcription factor Sp1. 


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A central role for a single c-Myb binding site in a thymic locus control region. 
Locus control regions (LCRs) are powerful assemblies of cis elements that organize the actions of cell-type-specific trans-acting factors. A 2.3-kb LCR in the human adenosine deaminase (ADA) gene first intron, which controls expression in thymocytes, is composed of a 200-bp enhancer domain and extended flanking sequences that facilitate activation from within chromatin. Prior analyses have demonstrated that the enhancer contains a 28-bp core region and local adjacent augmentative cis elements. We now show that the core contains a single critical c-Myb binding site. In both transiently cotransfected human cells and stable chromatin-integrated yeast cells, c-Myb strongly transactivated reporter constructs that contained polymerized core sequences. c-Myb protein was strongly evident in T lymphoblasts in which the enhancer was active and was localized within discrete nuclear structures. Fetal murine thymus exhibited a striking concordance of endogenous c-myb expression with that of mouse ADA and human ADA LCR-directed transgene expression. Point mutation of the c-Myb site within the intact 2.3-kb LCR severely attenuated enhancer activity in transfections and LCR activity in transgenic thymocytes. Within the context of a complex enhancer and LCR, c-Myb can act as an organizer of thymocyte-specific gene expression via a single binding site. 


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[Hormonal metabolic status in breast cancer patients after conservative surgery: comparison with known prognostic criteria] 
Body weight, body mass index, body fat, lean body mass, blood-glucose, cholesterol, HDL-cholesterol, triglyceride, beta-lipoproteins, insulin, gonadotropin, estradiol, testosterone, SHBG, T3, T4 and TSH levels as well as estradiol and progesterone receptor levels in excised tumor were studied in 40 patients with breast cancer prior to conservative treatment. Said anthropometric, metabolic and hormonal parameters were compared with the index of lymphocytic infiltration of tumor selected as a prognostic factor. A significant correlation between high lymphocytic infiltration (2.5 points), low body mass and fat was identified. Also, smoking contributed to loss of body mass and fat; however, it caused lymphocytic infiltration to rise. Moderate body mass, relatively low fat level and positive receptor status are among factors of good prognosis in breast cancer of early stages. 


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FK506 and ciclosporin: molecular probes for studying intracellular signal transduction. 
The immunosuppressants ciclosporin and FK506 block the Ca(2+)-dependent signal-transduction pathway emanating from the T-cell receptor, thereby inhibiting the activation of helper T cells. Using these drugs as probes, chemists and biologists have uncovered several intracellular signalling molecules bridging the generation of second-messenger Ca2+ ions and the transcriptional activation of IL-2, among which are calmodulin, calcineurin and the nuclear factor of activated T cells (NF-AT). Hence, Ca2+ binds to calmodulin, leading to the binding of calmodulin to calcineurin; the activated calcineurin, in turn, may dephosphorylate the cytoplasmic subunit of NF-AT, resulting in its translocation from the cytoplasm into the nucleus to form a competent transcriptional activator. As described by Jun Liu, these drugs manifest their effects in an unprecedented fashion. They do not directly intercept intracellular signalling molecules. Instead, they form tight complexes with two different classes of abundant cytosolic receptors called immunophilins upon entering the cell, and consequently inhibit their peptidyl prolyl cis-trans isomerase activities. The two structurally distinct immunophilin-drug complexes bind to, and inhibit, the phosphatase activity of calcineurin. 


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Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins. 
Latent membrane protein 1 (LMP1) acts like a permanently activated receptor of the tumor necrosis factor (TNF)-receptor superfamily and is absolutely required for B cell immortalization by Epstein-Barr virus. Molecular and biochemical approaches demonstrated that LMP1 usurps cellular signaling pathways resulting in the induction of NF-kappaB and AP-1 via two C-terminal activating regions. We demonstrate here that a third region encompassing a proline rich sequence within the 33 bp repetitive stretch of LMP1's C-terminus is required for the activation of Janus kinase 3 (JAK3). The interaction of LMP1 and JAK3 leads to the enhanced tyrosine auto/transphosphorylation of JAK3 within minutes after crosslinking of a conditional NGF-R:LMP1 chimera and is a prerequisite for the activation of STAT transcription factors. These results reveal a novel activating region in the LMP1 C-terminus and identify the JAK/STAT pathway as a target of this viral integral membrane protein in B cells. 


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Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development. 
Males with X-linked severe combined immunodeficiency (XSCID) have defects in the common cytokine receptor gamma chain (gamma c) gene that encodes a shared, essential component of the receptors of interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. The Janus family tyrosine kinase Jak3 is the only signaling molecule known to be associated with gamma c, so it was hypothesized that defects in Jak3 might cause an XSCID-like phenotype. A girl with immunological features indistinguishable from those of XSCID was therefore selected for analysis. An Epstein-Barr virus (EBV)-transformed cell line derived from her lymphocytes had normal gamma c expression but lacked Jak3 protein and had greatly diminished Jak3 messenger RNA. Sequencing revealed a different mutation on each allele: a single nucleotide insertion resulting in a frame shift and premature termination in the Jak3 JH4 domain and a nonsense mutation in the Jak3 JH2 domain. The lack of Jak3 expression correlated with impaired B cell signaling, as demonstrated by the inability of IL-4 to activate Stat6 in the EBV-transformed cell line from the patient. These observations indicate that the functions of gamma c are dependent on Jak3 and that Jak3 is essential for lymphoid development and signaling. 


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Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate. 
We have previously shown that minimally oxidized LDL (MM-LDL) activated endothelial cells to increase their interaction with monocytes but not neutrophils, inducing monocyte but not neutrophil binding and synthesis of monocyte chemotactic protein-1 and monocyte colony-stimulating factor (M-CSF). In the present studies we have examined the signaling pathways by which this monocyte-specific response is induced. Both induction of monocyte binding and mRNA levels for M-CSF by MM-LDL were not inhibited in protein kinase C-depleted endothelial cells. A number of our studies indicate that cAMP is the second messenger for the effects of MM-LDL cited above. Incubation of endothelial cells with MM-LDL caused a 173% increase in intracellular cAMP levels. Agents which increased cAMP levels, including cholera toxin, pertussis toxin, dibutyryl cAMP, and isoproterenol mimicked the actions of MM-LDL. Agents which elevated cAMP were also shown to activate NF kappa B, suggesting a role for this transcription factor in activation of monocyte-endothelial interactions. Although endothelial leukocyte adhesion molecule (ELAM) mRNA synthesis can be regulated by NF kappa B, ELAM was not expressed and ELAM mRNA was only slightly elevated in response to MM-LDL. We present evidence that induction of neutrophil binding by LPS is actually suppressed by agents that elevated cAMP levels. 


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The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. 
To understand the molecular bases for cytokine redundancy and pleiotropy, we have compared the Stat proteins activated in peripheral blood lymphocytes (PBLs) by cytokines with shared and distinct actions. Interleukin-2 (IL-2) rapidly activated Stat5 in fresh PBL, and Stat3 and Stat5 in preactivated PBL. IL-7 and IL-15 induced the same complexes as IL-2, a feature explained by the existence of similar tyrosine-phosphorylated motifs in the cytoplasmic domains of IL-2R beta and IL-7R that can serve as docking sites for Stat proteins. IL-13 Induced the same complexes as IL-4, a finding explained by our studies implicating IL-4R as a shared component of the receptors. These studies demonstrate that a single cytokine can activate different combinations of Stat proteins under different physiological conditions, and also indicate two mechanisms by which distinct cytokines can activate the same Stat protein. 


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Identification of essential GATA and Ets binding motifs within the promoter of the platelet glycoprotein Ib alpha gene. 
Platelet glycoprotein (GP) Ib-IX-V is a multisubunit adhesion receptor that supports platelet attachment to thrombogenic surfaces at sites of vascular injury. The congenital absence of the receptor results in a bleeding disorder associated with "giant" platelets, a condition linking the expression of the complex to platelet morphogenesis. To understand better the expression of the GP Ib-IX-V complex, studies were undertaken to define the essential genetic elements supporting the expression of the alpha-subunit of the complex (GP Ib alpha). GP Ib alpha promoter activity was evaluated by transfection of human erythroleukemia cells with reporter plasmids coding for the enzyme, luciferase. Studies were initiated with a fragment extending 2,738 nucleotides 5' to the transcription start site and lead to the identification of 253 nucleotides retaining full promoter activity in human erythroleukemia cells. In cells of nonhematopoietic lineage, human endothelial and HeLa cells, the GP Ib alpha promoter activity was no greater than background levels obtained with promoterless constructs. Gel shift assays and site-directed mutagenesis studies defined essential GATA and Ets binding motifs 93 and 150 nucleotides upstream of the transcription start site, a finding which further substantiates these elements as important determinants of megakaryocytic gene expression. The results define essential cis-acting elements responsible for the expression of GP Ib alpha and provide insights into molecular events coinciding with the release of normal platelets into the bloodstream. 


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Protein-tyrosine kinase activation is required for lipopolysaccharide induction of interleukin 1beta and NFkappaB activation, but not NFkappaB nuclear translocation. 
In human monocytes, interleukin 1beta protein production and steady state mRNA levels are increased in response to lipopolysaccharide, predominantly as a result of increased transcription of the interleukin 1beta gene. Expression of interleukin 1beta and other cytokines, such as interleukin 6 and tumor necrosis factor alpha, has been shown to be dependent on the activation of the transcription factor, NFkappaB. Since recent studies have shown that lipopolysaccharide-induced tyrosine kinase activation is not required for NFkappaB nuclear translocation, we sought to determine whether NFkappaB translocated in the absence of tyrosine kinase activity was active in stimulating transcription. We have found that, in the human pro-monocytic cell line, THP-1, the lipopolysaccharide-induced expression of interleukin 1beta is dependent on tyrosine kinase activation. Tyrosine kinases are not required for lipopolysaccharide-mediated nuclear translocation of NFkappaB. However, in the absence of tyrosine kinase activity, the ability of NFkappaB to stimulate transcription is impaired. This inhibition of transcription is specific for NFkappaB; in the absence of tyrosine kinase activity, AP-1-dependent transcription is enhanced. These results suggest that, while lipopolysaccharide-induced expression of inflammatory mediators requires tyrosine kinase activity, tyrosine kinase activity is not obligatory for lipopolysaccharide signal transduction. 


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Cleavage of transcription factor SP1 by caspases during anti-IgM-induced B-cell apoptosis. 
Apoptosis is instrumental in the processes generating the diversity of the B-cell repertoire. Autoreactive B-cells are eliminated by anti-IgM crosslinking after encountering self-antigens, but precise mechanisms leading to B-cell apoptosis are still not well understood. We report here the cleavage of the transcription factor SP1 in the human Burkitt lymphoma cell line BL60 during anti-IgM-induced apoptosis. Western blot analysis revealed two cleavage products of approximately 68 kDa and 45 kDa after induction of apoptosis. Cleavage could be completely inhibited by zDEVD-fmk, an inhibitor specific for caspase 3-like proteases. In-vitro cleavage of recombinant SP1 by recombinant caspase 3 (CPP32) or caspase 7 (Mch 3) results in similar cleavage products as those observed in vivo. Recombinant caspase 6 (Mch 2) primarily generates a 68-kDa cleavage product, as observed after calcium ionophore (CaI) induced B-cell apoptosis. In contrast, caspase 1 (ICE) did not cleave SP1 in vitro. The time course of SP1 cleavage during anti-IgM-induced apoptosis is paralleled by an increase of caspase activity measured by DEVD-p-nitroanilide (DEVD-pNA) cleavage. DNA band-shift assays revealed a decrease in the intensity of the full length SP1/DNA complex and an increase in the intensity of a smaller complex due to the binding of one SP1 cleavage product. By Edman sequencing we could identify a caspase 3 cleavage site after Asp584 (D584AQPQAGR), generating a 22-kDa C-terminal SP1 protein fragment which still contains the DNA binding site. Our results show the cleavage of the human transcription factor SP1 in vivo and in vitro, underlining the central role of caspase 3-like proteases during the process of anti-IgM-induced apoptosis. 


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[Effect of the regimen of kidney-tonifying and qi-invigorating on aging changes of glucocorticoid receptor] 
The plasma cortisol concentration and the sites of glucocorticoid receptor (GCR) in the peripheral lymphocytes were measured in 32 healthy aged persons and 13 young adults. In animal experiment, GCR of spleen lymphocytic cell was also measured in 18 aged rats and 9 young rats. The results showed that GCR was significantly lower in the aged persons or rats than that in the young while the plasma cortisol level didn't change with aging. So we think that GCR is more sensitive than the plasma cortisol level to reflect the aging change of the adrenal cortex function. After the treatment with the regimen of Kidney-tonifying and Qi-invigorating, the GCR of the aged persons and rats was enhanced, and in this way, the function of the aged adrenal cortex was improved. 


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Immune functions, clinical parameters and hormone receptor status in breast cancer patients. 
We have carried out a detailed analysis of the cellular immune functions of breast cancer patients in comparison with healthy controls. A possible correlation between immune and clinical parameters was analysed in 50 breast cancer patients. Immune parameters, natural killer cell and T lymphocyte functions and the numbers of circulating T lymphocytes were analysed against the clinical parameters comprising the tumour burden, the stage of the disease and the expression of hormone receptors on the tumour. In order to analyse the immune function data effectively, low responders were identified with stringent cut-off values. Considerably higher proportions of low responders were found among the patient population. Elevated numbers of circulating T lymphocytes and CD3-directed cytolysis correlated with the expression of oestrogen receptors independently of the clinical/histological parameters. 


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Charybdotoxin-sensitive, Ca(2+)-dependent membrane potential changes are not involved in human T or B cell activation and proliferation. 
The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding. Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is presumably through opening of Ca(2+)-stimulated K+ channels. We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, without affecting changes in cytosolic Ca2+. However, blockade of the Ca(2+)-activated K+ channel is not associated with changes in cell-cycle gene activation, IL-2 production, IL-2R expression or B and T cell mitogenesis. These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca(2+)-activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis. 


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Epstein-Barr virus nuclear antigen 2 and latent membrane protein independently transactivate p53 through induction of NF-kappaB activity. 
B-cell immortalization by Epstein-Barr virus (EBV) is dependent on permanent control of the cellular processes which normally regulate cell division and apoptosis, functions possessed by p53 in a number of normal cell types. In studies initiated to evaluate relationships between EBV latent genes and p53, p53 levels were found to increase approximately 10-fold 4 to 5 days after EBV infection of purified resting human B cells; the induced p53 was transcriptionally active. Latent membrane protein 1 and, to a lesser extent, EBV nuclear antigen 2 mediated the increase in p53 levels via activation of the NF-kappaB transcription factor. 


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Grf40, A novel Grb2 family member, is involved in T cell signaling through interaction with SLP-76 and LAT. 
We molecularly cloned a new Grb2 family member, named Grf40, containing the common SH3-SH2-SH3 motif. Expression of Grf40 is predominant in hematopoietic cells, particularly T cells. Grf40 binds to the SH2 domain-containing leukocyte protein of 76 kD (SLP-76) via its SH3 domain more tightly than Grb2. Incidentally, Grf40 binds to linker for activation of T cells (LAT) possibly via its SH2 domain. Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76-dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity. Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant. Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT. 


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Regulation of the human interleukin-2 gene by the alpha and beta isoforms of the glucocorticoid receptor. 
The immunosuppressive effects of glucocorticoids are largely due to transcriptional inhibition of immunologically relevant genes, such as the interleukin-2 (IL-2) gene. These effects are mediated by the intracellular glucocorticoid receptor (GR). In humans, alternative splicing of the GR precursor mRNA gives rise to two receptor isoforms, termed GRalpha and GRbeta. We previously demonstrated that GRbeta could antagonize GRalpha-mediated transactivation of a glucocorticoid-responsive element (GRE)-driven reporter gene in COS-7 cells. The present study was designed to analyze the roles of the two GR isoforms on glucocorticoid-mediated transrepression of the IL-2 gene. Using a recently developed transfection technique, we demonstrate that in primary human lymphocytes, stimulation of a 548 bp IL-2 promoter-luciferase reporter construct by phorbol ester and calcium ionophore is reversed by dexamethasone to a similar extent as in Jurkat T lymphoma cells transfected with a GRalpha expression vector. Transfection of a GRbeta expression vector alone did not result in IL-2 promoter repression in response to glucocorticoids. Furthermore, GRbeta did not antagonize the repressive effects of GRalpha on IL-2 promoter activity. Surprisingly, overexpression of GRbeta in Jurkat cells did not cause significant inhibition of GRalpha-induced transactivation of a GRE-dependent luciferase reporter gene either. We conclude that the transrepressive effect of glucocorticoids on IL-2 gene transcription is exclusively mediated by GRalpha. GRbeta can neither antagonize GRalpha-mediated transactivation nor transrepression in Jurkat cells, indicating a cell type-specific pattern of GRbeta-mediated antiglucocorticoid activity. 


Document 003003662 ends.


HLA-DR-, CD33+, CD56+, CD16- myeloid/natural killer cell acute leukemia: a previously unrecognized form of acute leukemia potentially misdiagnosed as French-American-British acute myeloid leukemia-M3 [see comments] 
We have identified and characterized a previously unrecognized form of acute leukemia that shares features of both myeloid and natural killer (NK) cells. From a consecutive series of 350 cases of adult de novo acute myeloid leukemia (AML), we identified 20 cases (6%) with a unique immunophenotype: CD33+, CD56+, CD11a+, CD13lo, CD15lo, CD34+/-, HLA-DR-, CD16-. Multicolor flow cytometric assays confirmed the coexpression of myeloid (CD33, CD13, CD15) and NK cell-associated (CD56) antigens in each case, whereas reverse transcription polymerase chain reaction (RT-PCR) assays confirmed the identity of CD56 (neural cell adhesion molecule) in leukemic blasts. Although two cases expressed CD4, no case expressed CD2, CD3, or CD8 and no case showed clonal rearrangement of genes encoding the T-cell receptor (TCR beta, gamma, delta). Leukemic blasts in the majority of cases shared unique morphologic features (deeply invaginated nuclear membranes, scant cytoplasm with fine azurophilic granularity, and finely granular Sudan black B and myeloperoxidase cytochemical reactivity) that were remarkably similar to those of acute promyelocytic leukemia (APL); particularly the microgranular variant (FAB AML-M3v). However, all 20 cases lacked the t(15;17) and 17 cases tested lacked the promyelocytic/retinoic acid receptor alpha (RAR alpha) fusion transcript in RT-PCR assays; 12 cases had 46,XX or 46,XY karyotypes, whereas 2 cases had abnormalities of chromosome 17q: 1 with del(17)(q25) and the other with t(11;17)(q23;q21) and the promyelocytic leukemia zinc finger/RAR alpha fusion transcript. All cases tested (6/20), including the case with t(11;17), failed to differentiate in vitro in response to all-trans retinoic acid (ATRA), suggesting that these cases may account for some APLs that have not shown a clinical response to ATRA. Four of 6 cases tested showed functional NK cell-mediated cytotoxicity, suggesting a relationship between these unique CD33+, CD56+, CD16- acute leukemias and normal CD56+, CD16- NK precursor cells. Using a combination of panning and multiparameter flow cytometric sorting, we identified a normal CD56+, CD33+, CD16- counterpart cell at a frequency of 1% to 2% in the peripheral blood of healthy individuals. Our studies suggest that this form of acute leukemia may arise from transformation of a precursor cell common to both the myeloid and NK cell lineages; thus we propose the designation myeloid/NK acute leukemia. Recognition of this new leukemic entity will be important in distinguishing these ATRA-nonresponsive cases from ATRA-responsive true APL. 


Document 003003667 ends.


Single point estimation of glucocorticoid receptors in lymphocytes of normal subjects and of children under long term glucocorticoid treatment. 
A single point assay of glucocorticoid receptors (GR) in human lymphocytes based on the measurement of specific dexamethasone binding has been developed and compared with a common multi-point Scatchard analysis. The assay conditions-concentration of the ligand 20 nmol/l, incubation time 2 h and the cell count 2-6 mil. cells/tube in the assay volume 0.25 ml were found to be optimal. An attempt was also undertaken to use a cell harvester for the separation of cells from unbound ligand. Though specifically bound dexamethasone measured by whole-cell assay and that using cell harvester correlated well, almost by one order lower values obtained with the latter method render it non-applicable for receptor quantitation. The results from 9 healthy volunteers (average GR concentration 7131 +/- 1256 sites/cell) correlated excellently with those obtained by the Scatchard analysis. The single point assay has been also applied for determination of GH in 10 children treated with large doses of prednisone. The average values from healthy volunteers did not differ significantly from those found in these children, though much broader range was found in patients. 


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Interleukin 2 signaling involves the phosphorylation of Stat proteins. 
One of the most important cytokines involved in immune response regulation is interleukin 2 (IL-2), a potent activator of the proliferation and function of T lymphocytes and natural killer cells. The mechanisms by which the effects of IL-2 are propagated within cells are not understood. While the binding of IL-2 to its receptor was recently shown to lead to the activation of two kinases, Jak-1 and Jak-3, subsequent steps in the signaling pathway to the nucleus that lead to the activation of specific genes had not been characterized. Since many cytokines that activate Jak kinases also lead to the tyrosine phosphorylation and activation of members of the Stat family of transcription factors, the ability of IL-2 to trigger Stat phosphorylation was examined. Exposure of activated human T lymphocytes or of a natural killer cell line (NKL) to IL-2 leads to the phosphorylation of Stat1 alpha, Stat1 beta, and Stat3, as well as of two Stat-related proteins, p94 and p95. p94 and p95 share homology with Stat1 at the phosphorylation site and in the Src homology 2 (SH2) domain, but otherwise are immunologically distinct from Stat1. These Stat proteins were found to translocate to the nucleus and to bind to a specific DNA sequence. These findings suggest a mechanism by which IL-2 binding to its receptor may activate specific genes involved in immune cell function. 


Document 003003671 ends.


Nuclear factor-90 of activated T-cells: A double-stranded RNA-binding protein and substrate for the double-stranded RNA-dependent protein kinase, PKR. 
NFAT transcription factors play a central role in initiating T-cell activation through the induction of immediate-early T-cell specific genes including interleukin-2 (IL-2). NFAT transcription factors bind to a sequence in the IL-2 enhancer known as the antigen receptor response element 2 (ARRE-2). Multiple proteins exhibiting ARRE-2 binding activity have been isolated, including a heterodimer from stimulated T-cell nuclear extracts consisting of Mr = 90 000 (NF90) and Mr = 45 000 (NF45) subunits. The subunits of this heterodimer have been cloned, and NF90 was found to encode a protein containing two domains that are predicted to form motifs capable of binding to double-stranded RNA. Using in vitro translated polypeptides, we have demonstrated that NF90 specifically binds to double-stranded RNA. Furthermore, NF90 was phosphorylated in a double-stranded RNA-dependent manner likely by the interferon-induced, double-stranded RNA-dependent protein kinase, PKR. The NF90 protein was found to be expressed not only in T-cells, but also in nonimmune HeLa cells. In HeLa cells, the protein was almost exclusively localized to the ribosome salt wash fraction of cell lysates. 


Document 003003674 ends.


Prolactin and interleukin-2 receptors in T lymphocytes signal through a MGF-STAT5-like transcription factor. 
The cell surface receptors for PRL and interleukin-2 (IL-2) are structurally distinct, but share regulatory tasks in T lymphocytes. They can stimulate proliferation and activate transcription of over-lapping sets of genes of T cells. PRL and IL-2 receptor activation are both linked to the Jak/Stat (signal transducer and activator of transcription) pathway. We investigated the ability of PRL and IL-2 to activate Stat proteins in different T cell lines. The DNA binding specificities, the reactivities toward Stat-specific antisera, and the mol wt of IL-2- and PRL-induced DNA-binding proteins in Nb2 and C196 T cell lines were investigated. A comparison with the Stat proteins induced by interferon-gamma, PRL, and IL-6 in T47D mammary tumor cells was made. We found that these parameters were indistinguishable for one of the PRL- and IL-2-induced factors. A transcription factor closely related to mammary gland factor-Stat5 is rapidly activated upon interaction of IL-2 and PRL with their respective receptors. Activation of a second protein related to Stat1 was also observed. Our results emphasize the role of PRL as a regulator of the immune response and indicate that the Stat factors mammary gland factor-Stat5 and Stat1 play a role in the regulation of gene expression during T cell development. 


Document 003003682 ends.


Mutations in the Pit-1 gene in children with combined pituitary hormone deficiency. 
Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone and prolactin genes. In three unrelated Japanese children with combined pituitary hormone deficiency, we identified three point mutations in the Pit-1 gene, Pro24Leu, Arg143Gln, and Arg271Trp, located on the major transactivation region, POU-specific domain, and POU-homeodomain, respectively. 


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Activation of the HIV-1 enhancer by the LEF-1 HMG protein on nucleosome-assembled DNA in vitro. 
Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobility group (HMG) protein that activates the T cell receptor alpha (TCR alpha) enhancer in a context-restricted manner in T cells. In this paper we demonstrate that the distal region of the human immunodeficiency virus-1 (HIV-1) enhancer, which contains DNA-binding sites for LEF-1 and Ets-1, also provides a functional context for activation by LEF-1. First, we show that mutations in the LEF-1-binding site inhibit the activity of multimerized copies of the HIV-1 enhancer in Jurkat T cells, and that LEF-1/GAL4 can activate a GAL4-substituted HIV-1 enhancer 80- to 100-fold in vivo. Second, recombinant LEF-1 is shown to activate HIV-1 transcription on chromatin-assembled DNA in vitro. By using a nucleosome-assembly system derived from Drosophila embryos, we find that the packaging of DNA into chromatin in vitro strongly represses HIV-1 transcription and that repression can be counteracted efficiently by preincubation of the DNA with LEF-1 (or LEF-1 and Ets-1) supplemented with fractions containing the promoter-binding protein, Sp1. Addition of TFE-3, which binds to an E-box motif upstream of the LEF-1 and Ets-1 sites, further augments transcription in this system. Individually or collectively, none of the three enhancer-binding proteins (LEF-1, Ets-1, and TFE-3) could activate transcription in the absence of Sp1. A truncation mutant of LEF-1 (HMG-88), which contains the HMG box but lacks the trans-activation domain, did not activate transcription from nucleosomal DNA, indicating that bending of DNA by the HMG domain is not sufficient to activate transcription in vitro. We conclude that transcription activation by LEF-1 in vitro is a chromatin-dependent process that requires a functional trans-activation domain in addition to the HMG domain. 


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Lymphocyte cell lines from vitamin D-dependent rickets type II show functional defects in the 1 alpha,25-dihydroxyvitamin D3 receptor. 
Lymphocyte cell lines were established from five patients with vitamin D-dependent rickets, type II (VDDR-II). These lines were established by infection with human T-lymphotrophic virus type I (HTLV-I). Binding of [3H]1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) to its receptor in these cell lines was compared to binding studies using a T-lymphocyte cell line (S-LB1) from a normal individual. The 1,25(OH)2D3 receptor of S-LB1 was comparable to the well-characterized chick intestinal 1,25(OH)2D3 receptor in terms of its ligand binding affinity and capacity, its mobility on 5-20% sucrose gradients, and its adsorption to and elution properties from DNA-cellulose. Three cell lines established from patients with VDDR-II (Rh-VDR, Sh-VDR, and Ab-VDR) showed no specific binding of 1,25(OH)2D3 to a receptor and treatment of the cultured cells with 1,25(OH)2D3 did not stimulate production of 24,25-dihydroxy-vitamin D3 (24,25(OH)2D3), a response which is diagnostic of the presence of a functional 1,25(OH)2D3 receptor. In a fourth cell line, A1-VDR, the receptor for 1,25(OH)2D3 had a low binding capacity and 25(OH)D3-24-hydroxylase activity was not detectable. Induction of 24,25-(OH)2D3 synthesis by 1,25(OH)2D3 was observed in the fifth cell line, designated Ro-VDR, although the sensitivity to hormone treatment was lower than in the control cell line from a normal donor. The capacity of the receptor for 1,25(OH)2D3 was low in Ro-VDR. In all cell lines where 1,25(OH)2D3 binding to a receptor was detectable, the receptor had the typical sedimentation coefficient of 3.7 S on sucrose density gradient analysis. Binding and elution properties to DNA-cellulose, however, differed from normal in both Ro-VDR and A1-VDR cells where elution from DNA-cellulose occurred at a lower salt concentration than is typical of the 1,25(OH)2D3 receptor. While Ro-VDR cells showed typical nuclear localization of the unoccupied 1,25(OH)2D3 receptor, neither the unoccupied nor the occupied receptor from A1-VDR cells was completely localized in the nucleus. In a series of functional studies we found that modulation of the level of the mRNAs coding for both the c-myc oncogene and the growth factor known as granulocyte-monocyte colony stimulating activity by 1,25(OH)2D3 correlated with the 1,25(OH)2D3 receptor status of these cells. Use of these cell lines will facilitate further study of the molecular defect(s) in the receptor for 1,25(OH)2D3 in vitamin D-dependent rickets type II and will allow a correlation with impairment of cellular functions. 


Document 003003693 ends.


Activation of human monocytic cells by Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides proceeds via a pathway distinct from that of lipopolysaccharide but involves the transcriptional activator NF-kappa B. 
There is increasing evidence that lipoproteins of Treponema pallidum and Borrelia burgdorferi are key inflammatory mediators during syphilis and Lyme disease. A principal objective of the present study was to identify more precisely similarities and divergences among lipopolysaccharide (LPS)- and lipoprotein-lipopeptide-induced immune cell signaling events. Like LPS, purified native B. burgdorferi OspA and synthetic analogs of OspA, OspB, and two T. pallidum lipoproteins (Tpp47 and Tpp17) all induced NF-kappa B translocation in THP-1 human monocytoid cells. Acylation of OspA and the synthetic peptides was requisite for cell activation. Polymyxin B abrogated only the response to LPS. By using 70Z/3-derived pre-B-cell lines either lacking or expressing human CD14 (the LPS receptor), it was observed that expression of human CD14 imparted responsiveness to LPS but not to OspA or spirochetal lipopeptides (assessed by induction of NF-kappa B and expression of surface immunoglobulin M). Finally, the biological relevance of the observation that T. pallidum lipoproteins-lipopeptides induce both NF-kappa B and cytokine production in monocytes was supported by the ability of the synthetic analogs to promote human immunodeficiency virus replication in chronically infected U1 monocytoid cells; these observations also suggest a potential mechanism whereby a syphilitic chancre can serve as a cofactor for human immunodeficiency virus transmission. The combined data lend additional support to the proposal that spirochetal lipoproteins and LPS initiate monocyte activation via different cell surface events but that the signaling pathways ultimately converge to produce qualitatively similar cellular responses. 


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The Epstein-Barr virus latency BamHI-Q promoter is positively regulated by STATs and Zta interference with JAK/STAT activation leads to loss of BamHI-Q promoter activity. 
In Epstein-Barr virus (EBV)-associated tumors in nonimmunocompromised patients, EBV gene expression is highly restricted. EBV-encoded nuclear antigen (EBNA)-1 is expressed, whereas the immunogenic and proliferative EBNAs are not. This pattern of EBNA expression is generated by usage of the BamHI-Q promoter (Qp). We have determined that the JAK/STAT pathway positively regulates Qp activity. In transient-transfection assays, a Qp-CAT reporter was activated by cotransfected JAK-1 and by treatment of cells with the cytokine IL-6. The ability of Qp to bind signal transducer and activator of transcription (STAT) proteins was directly demonstrated by electrophoretic mobility-shift assay, and mutation of potential STAT-binding sites reduced Qp responsiveness to Janus kinase (JAK)-1. Consistent with a role for STATs in Qp function, Qp using Burkitt's lymphoma Rael cells and cultured nasopharyngeal carcinoma (NPC) cells contained nuclear STAT protein. We investigated whether the inability to maintain EBV-positive NPC cell lines in culture was related to Qp activity. Passaging of the NPC cell line HK666 led to activation of expression of BZLF1, which encodes Zta and loss of Qp function. Transient expression assays linked Zta expression to the down-regulation of Qp. Cotransfection of Zta reduced Qp activity in reporter assays. This negative regulation required Zta DNA-binding activity. We provide evidence that Zta up-regulation of p53 leads to p53-mediated interference with JAK/STAT activation of Qp. The data imply that JAK/STAT signaling has a role in EBV-associated malignancies. 


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Lipopolysaccharide induces phosphorylation of MAD3 and activation of c-Rel and related NF-kappa B proteins in human monocytic THP-1 cells. 
Many effects of lipopolysaccharide (LPS) on gene expression, including that of human immunodeficiency virus (HIV), in monocytic cells are mediated by activation of kappa B DNA-binding proteins. However, the specific members of the NF-kappa B/Rel transcription factor family involved in the LPS response, and the mechanisms through which LPS-generated signals are transduced remain unclear. Here we show that LPS induces nuclear expression of c-Rel/p50 heterodimers as well as p50/p65 (NF-kappa B) kappa B DNA-binding complexes in human monocytic THP-1 cells. Nuclear localization of these proteins occurred concomitantly with a rapid decrease in their cytosolic levels and was independent of phorbol ester-sensitive protein kinase C. Within 24 h following LPS stimulation there was a striking increase in the levels of c-Rel, p105, and p50 in the cytosol. The increased levels of these proteins correlated with increases in the amounts of their mRNAs during LPS activation of THP-1 cells. LPS activation of THP-1 cells resulted in phosphorylation of MAD3 (an I kappa B-like protein), a rapid increase in MAD3 mRNA, and an increase in MAD3 protein by 2 h. Thus, LPS activation of human monocytic cells results in nuclear expression of c-Rel/p50 and p50/p65 (NF-kappa B) and induces phosphorylation of MAD3. 


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The role of NFATp in cyclosporin A-sensitive tumor necrosis factor-alpha gene transcription. 
The tumor necrosis factor-alpha (TNF alpha) gene is an immediate early gene in activated T cells, in that it is rapidly induced without a requirement for protein synthesis. Maximal induction of TNF alpha mRNA can be induced by treatment of T cells with calcium ionophores alone, via a calcineurin-dependent process that is blocked by cyclosporin A. We have previously identified a promoter element, kappa 3, that is required for calcium-stimulated, cyclosporin A-sensitive induction of the TNF alpha gene in activated T cells. Here, we demonstrate that the kappa 3 binding factor contains NFATp, a cyclosporin-sensitive DNA-binding protein required for interleukin-2 gene transcription. NFATp binds to two sites within the kappa 3 element, and occupancy of both sites is required for TNF alpha gene induction. Thus, although the kappa 3 element has little sequence similarity to other NFATp-binding sites, it appears to function as a cyclosporin-sensitive promoter element in T cells by virtue of its ability to bind NFATp. The involvement of NFATp in transcriptional activation of both the interleukin-2 and TNF alpha genes suggests that this factor plays an important role in the coordinate induction of multiple cytokine genes, starting at the earliest stages of T cell activation. 


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Comparison of retinoic acid and phorbol myristate acetate as inducers of monocytic differentiation. 
Several human myeloid leukemia cell lines growing in vitro can be induced to differentiate to more mature monocyte/macrophage-like cells by treatment with protein kinase C-activating phorbol esters, such as PMA. In addition to PMA, cells of the THP-1 myeloid leukemia cell line acquire macrophage-like characteristics after treatment with all-trans retinoic acid (RA). To analyze the signal transduction mechanisms induced by RA, we first compared the effects of PMA and RA on the expression of genes which are known to be regulated during monocytic differentiation. Both RA and PMA effectively down-regulated c-myc expression, while c-myb expression decreased only after PMA treatment. Expression of the beta 2-integrin genes, CD11a and CD11b, was clearly increased after both of these treatments. Their effects on the src-family tyrosine kinase genes were different: hck expression was similarly induced by these agents but lyn expression was stronger and more rapid after RA treatment. RA also enhanced lyn mRNA production rapidly in HL-60, indicating that the activation of lyn gene expression is common in monocytic and granulocytic maturation of myeloid leukemia cells. To examine whether the AP-1 enhancer activity is involved in RA-induced monocytic differentiation, THP-1 cells were transiently transfected with a chloramphenicol acetyl transferase (CAT)-reporter gene containing 5 copies of the AP-1 binding sites. In contrast to PMA, RA did not induce any CAT activity in these cells, thus suggesting that the RA-induced changes in the expression of those genes described above were not dependent on the AP-1 enhancer activity. 


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Modulation of E2F complexes during G0 to S phase transition in human primary B-lymphocytes. 
The pocket protein-E2F complexes are convergence points for cell cycle signaling. In the present report, we identified and monitored the pocket protein-E2F complexes in human primary B-lymphocytes after activation by phorbol 12-myristate 13-acetate. Consistent with previous data from human and mouse fibroblasts and T-lymphocytes, E2F4 and DP1 form the predominant E2F heterodimers both in G0 and G1 phases of the human B-lymphocyte cell cycle, whereas E2F1 and -3 are first detected in late G1, and their expression levels increase towards S phase. Intriguingly, the major E2F complex that we detected in quiescent human B-lymphocytes is consisted of pRB, E2F4, and DP1. Though the levels of DP1 and -2 increase when cells progress from G0 to S, the proportion of DP1 to DP2 remains relatively constant during the cell cycle. We also observed an increase in electrophoretic mobility of the predominant E2F components, DP1 and E2F4, as B-lymphocytes progressed from G0 into early G1. This increase in mobility was attributable to dephosphorylation, as lambda phosphatase treatment could convert the slower migrating forms into the corresponding faster mobility forms. We further demonstrated that this change in phosphorylation status correlates with a decrease in DNA binding activity. This modulation of DNA binding activity mediated through the dephosphorylation of DP1 and E2F4 could help to explain the lack of in vivo DNA footprinting in late G1 and S phases of gene promoters negatively regulated through E2F sites and suggests a novel mechanism for controlling E2F transcriptional activity during the transition from quiescence to proliferation. 


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SCL and related hemopoietic helix-loop-helix transcription factors. 
The helix-loop-helix (HLH) proteins are a family of transcription factors that include proteins critical to differentiation and development in species ranging from plants to mammals. Five members of this family (MYC, SCL, TAL-2, LYL-1 and E2A) are implicated in oncogenic events in human lymphoid tumors because of their consistent involvement in chromosomal translocations. Although activated in T cell leukemias, expression of SCL and LYL-1 is low or undetectable in normal T cell populations. SCL is expressed in erythroid, megakaryocyte and mast cell populations (the same cell lineages as GATA-1, a zinc-finger transcription factor). In addition, both SCL and GATA-1 undergo coordinate modulation during chemically induced erythroid differentiation of mouse erythroleukemia cells and are down-modulated during myeloid differentiation of human K562 cells, thus implying a role for SCL in erythroid differentiation events. However, in contrast to GATA-1, SCL is expressed in the developing brain. Studies of the function of SCL suggest it is also important in proliferation and self-renewal events in erythroid cells. 


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Tumor necrosis factor-alpha mRNA accumulation in human myelomonocytic cell lines. Role of transcriptional regulation by DNA sequence motifs and mRNA stabilization. 
The cytokine TNF mediates many of the pathologic signs of cachexia, inflammation, and sepsis. The current work describes the regulation of TNF in human myelomonocytic cell lines after PMA stimulation. The cell lines exhibit a low level of constitutive TNF mRNA expression. Within 2 to 4 h of PMA exposure, steady state levels of TNF mRNA are markedly elevated in all myelomonocytic cell lines studied. This rise is due to increased mRNA stability, which increased by almost twofold, and to an overall increase in transcription, which rises by more than sixfold. At the level of the genomic TNF gene, a DNase I hypersensitive site is detected within the TNF promoter between -200 to -100 bp relative to the transcription initiation site. Although absent in nonexpressing erythroleukemia cell lines, the DNase I site is present in uninduced myelomonocytic cell lines and is not changed after PMA induction. The PMA induction of c-fos mRNA correlated well with TNF gene induction; expression of genes encoding other proteins in the AP-1 complex (junB and junD) were also induced by PMA. The nuclear extracts from resting and induced ML-1 cells contain proteins binding specifically to the AP-1, AP-2, and NF kappa B sequence located within the TNF promoter. PMA induction increases the level of a number of specific binding complexes relative to the resting cells. The regulatory mechanisms of the human and murine TNF genes are discussed. 


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Binding of c-Rel to STAT5 target sequences in HTLV-I-transformed T cells. 
The type I human T-cell leukemia virus (HTLV-I) induces abnormal growth and subsequent transformation of T cells, which is associated with the development of an acute T-cell malignancy termed adult T-cell leukemia. A characteristic of HTLV-I-transformed T cells is the constitutive nuclear expression of NF-kappaB/Rel family of transcription factors, which appears to be essential for the growth of these transformed cells. Although NF-kappaB/Rel factors are known to induce the expression of T-cell growth factor interleukin (IL)-2, it is unclear how they participate in the IL-2-independent growth of HTLV-I-transformed cells. In this study, we show that certain NF-kappaB/Rel members, predominantly c-Rel, interact with enhancer sequences for STAT5, a key transcription factor mediating IL-2-induced T-cell proliferation. Reporter gene assays reveal that the binding of c-Rel to the STAT5 site present in the Fc gammaR1 gene leads to potent transactivation of this enhancer. Binding of c-Rel to the Fc gammaR1 STAT site also occurs in human peripheral blood T cells immortalized with HTLV-I in vitro and is correlated with enhanced levels of proliferation of these cells. These results raise the possibility that NF-kappaB/Rel may participate in the growth control of HTLV-I-transformed T cells by regulating genes driven by both kappaB and certain STAT enhancers. 


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Erythropoietin stimulates transcription of the TAL1/SCL gene and phosphorylation of its protein products. 
Activation of the TAL1 (or SCL) gene, originally identified through its involvement by a recurrent chromosomal translocation, is the most frequent molecular lesion recognized in T-cell acute lymphoblastic leukemia. The protein products of this gene contain the basic-helix-loop-helix motif characteristic of a large family of transcription factors that bind to the canonical DNA sequence CANNTG as protein heterodimers. TAL1 expression by erythroid cells in vivo and in chemical-induced erythroleukemia cell lines in vivo suggested the gene might regulate aspects of erythroid differentiation. Since the terminal events of erythropoiesis are controlled by the glycoprotein hormone erythropoietin (Epo), we investigated whether the expression or activity of the TAL1 gene and its protein products were affected by Epo in splenic erythroblasts from mice infected with an anemia-inducing strain of Friend virus (FVA cells). Epo elicited a rapid, dose-related increase in TAL1 mRNA by increasing transcription of the gene and stabilizing one of its mRNAs. An Epo-inducible TAL1 DNA binding activity was identified in FVA cell nuclear extracts that subsequently decayed despite accumulating mRNA and protein. Induction of DNA binding activity was associated temporally with Epo-induced phosphorylation of nuclear TAL1 protein. These results indicate that Epo acts at both transcriptional and posttranscriptional levels on the TAL1 locus in Friend virus-induced erythroblasts and establish a link between Epo signaling mechanisms and a member of a family of transcription factors involved in the differentiation of diverse cell lineages. 


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C/EBP proteins activate transcription from the human immunodeficiency virus type 1 long terminal repeat in macrophages/monocytes. 
Three binding sites for C/EBP proteins are found in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) (V.M. Tesmer, A.Rajadhyaksha, J.Babin, and M.Bina, Proc.Natl.Acad.Sci. USA 90:7298-7302, 1993). We have determined the functional role of C/EBP proteins and C/EBP sites in regulating transcription from the HIV- 1 LTR in monocytes/macrophages. Inhibition of endogenous C/EBP proteins, using either an excess of C/EBP binding sites or a trans- dominant negative inhibitor, demonstrated that C/EBP proteins are required for basal and activated levels of HIV-1 LTR transcription in the promonocytic cell line U937. Northern (RNA) blots and binding assays showed that NF-IL6 is the only known C/EBP family member which is increased when U937 cells are activated. Mutational analyses of the HIV-1 LTR showed that one C/EBP site is required for normal LTR transcription both before and after cellular activation and that the two 3' C/EBP sites are functionally equivalent. However, transcription from crippled HIV-1 LTRs lacking C/EBP sites can still be induced following activation of U937 cells. Several models are suggested for how elevated NF-IL6 may participate in an autostimulatory loop involving HIV infection, macrophage activation, cytokine expression, and HIV replication. 


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Effects of diesel organic extracts on chemokine production by peripheral blood mononuclear cells. 
BACKGROUND: Polyaromatic hydrocarbons (PAHs) associated with diesel exhaust particles (DEPs) are found in the atmospheric urban pollution. Such compounds have been shown to favor IgE production, bronchial hyperresponsiveness, and airway inflammation. Chemokines are a group of chemotactic cytokines involved in the recruitment of inflammatory cells. OBJECTIVE: We investigated the effect of DEP-PAHs on the release and mRNA expression of IL-8, MCP-1, and RANTES by PBMCs obtained from healthy subjects. METHODS: Protein production in supernatants was assessed by ELISA, and mRNA expression was evaluated by semiquantitative RT-PCR. RESULTS: Secretion of IL-8 and RANTES increased in a dose-dependent manner with increasing concentrations of DEP-PAHs (range, 0.5 ng to 50 ng/mL). On the contrary, the release of MCP-1 was significantly inhibited, also in a dose-dependent manner. Messenger RNA production coding for IL-8, RANTES, and MCP-1 showed parallel variations to the production of the correspondent proteins. Effects of DEP-PAHs became significant at 7 hours and up to 48 hours time culture for MCP-1, and up to 24 hours time culture for IL-8 and RANTES. Moreover, supernatants from DEP-PAH-activated cells, compared with those of controls, exhibited a significantly enhanced chemotactic activity for neutrophils and eosinophils, which was significantly inhibited by pretreatment with anti-IL-8 and anti-RANTES neutralizing antibodies, respectively. CONCLUSION: These findings suggest that the chemokine pathways are modulated by DEP-PAHs at the transcriptional level, reinforcing the idea that the development of inflammatory reactions might be affected by diesel exhaust emission. 


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Acetylsalicylic acid and sodium salicylate inhibit LPS-induced NF-kappa B/c-Rel nuclear translocation, and synthesis of tissue factor (TF) and tumor necrosis factor alfa (TNF-alpha) in human monocytes. 
We have investigated the effects of acetylsalicylic acid and sodium salicylate on the LPS-induced synthesis of the pro-coagulant protein tissue factor (TF) and the pro-inflammatory protein tumor necrosis factor-alpha (TNF-alpha), as well as the prostaglandin PGE2 in human monocytes. Both drugs dose-dependently inhibited LPS-induced TF and TNF-alpha synthesis at the mRNA and the protein level, and reduced PGE2 production. As evidenced by electro mobility shift assay (EMSA) and the use of a NF-kappa B prototypic probe, these drugs probably exert their inhibitory effects by interference with the nuclear translocation of NF-kappa B/c-Rel proteins. These data may expand the understanding of the anti-thrombotic and anti-inflammatory effects of these drugs when activation of monocytes occurs. 


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HLA class I-mediated induction of cell proliferation involves cyclin E-mediated inactivation of Rb function and induction of E2F activity. 
Chronic rejection of transplanted organs is manifested as atherosclerosis of the blood vessels of the allograft. HLA class I Ags have been implicated to play a major role in this process, since signaling via HLA class I molecules can induce the proliferation of aortic endothelial as well as smooth muscle cells. In this study, we show that HLA class I-mediated induction of cell proliferation correlates with inactivation of the Rb protein in the T cell line Jurkat as well as human aortic endothelial cells. HLA class I-mediated inactivation of Rb can be inhibited specifically by neutralizing Abs to basic fibroblast growth factor (bFGF), suggesting a role for FGF receptors in the signaling process. Signaling through HLA class I molecules induced cyclin E-associated kinase activity within 4 h in quiescent endothelial cells, and appeared to mediate the inactivation of Rb. A cdk2 inhibitor, Olomoucine, as well as a dominant-negative cdk2 construct prevented HLA class I-mediated inactivation of Rb; in contrast, dominant-negative cdk4 and cdk6 constructs had no effect. Furthermore, there was no increase in cyclin D-associated kinase activity upon HLA class I ligation, suggesting that cyclin E-dependent kinase activity mediates Rb inactivation, leading to E2F activation and cell proliferation. 


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A conserved motif in the promoters of several cytokines expressed by human Th2-type lymphocytes. 
We have recently found a novel conserved motif in the promoters of several T-cell-expressed cytokines [human interleukin-2, -4, -5 and -13 and human and mouse granulocyte/macrophage-colony stimulating factor (GM-CSF)]. It contains a core sequence CTTGG ... CCAAG which is present as part of larger palindromic sequences in each gene. This suggest that they may interact with a new family of trans-acting factors. In transfection assays, the human GM-CSF element has a strong positive effect on the expression of a reporter gene by the human T cell line Jurkat J6 upon stimulation. In DNA mobility shift assays, this sequence can give either six different specific bands which are competed out by different parts of the sequence or one specific band which is competed out by each of the inverted repeats, depending on the reconstitution conditions. In different genes, the core sequences are separated by integer numbers of helical turns. Considering the strong positive regulatory effect of this element and its presence in several T-cell-expressed cytokine genes, it may be crucial to the coordinated expression of these cytokines in T helper cells. 


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Human immunodeficiency viruses containing heterologous enhancer/promoters are replication competent and exhibit different lymphocyte tropisms. 
The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) contains binding sites for nuclear factor kappa B (NF-kappa B) and the constitutively expressed transcription factor Sp1, both of which are highly conserved in HIV and simian immunodeficiency virus isolates. To delineate the effects of these motifs on the replicative capacity of HIV and to explore the possibility of extending the virus host range, known heterologous enhancer/promoters were inserted into the HIV-1 LTR in place of the NF-kappa B and Sp1 binding sites. The effects of these substitutions on viral replication in transfected HeLa cells and on HIV infection of human peripheral blood lymphocytes or continuous T-leukemia cell lines were evaluated. HIVs in which the NF-kappa B/Sp1 enhancer plus the downstream TATA element were replaced with heterologous enhancer/promoters were also constructed. Viruses containing the human cytomegalovirus immediate-early enhancer exhibited infectious kinetics similar to that of wild-type HIV in activated human peripheral blood lymphocytes and AA2 cells but replicated less efficiently in H9 and CEM cells. These studies indicate that heterologous enhancer elements are capable of restoring Tat responsiveness to the HIV LTR in the context of directing reporter gene expression as well as in the production of infectious progeny virions. 


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Naive (CD45RA+) T lymphocytes are more sensitive to oxidative stress-induced signals than memory (CD45RO+) cells. 
Formation of reactive oxygen intermediates (ROI) after oxidative stress has been shown to be an activation signal for T lymphocytes, e.g., expression of IL-2 and its receptor are induced. These ROI-induced effects can, to a large extent, be attributed to the activation of the transcription factor NF-kappaB. Now we have examined whether naive and memory T lymphocytes differ in their sensitivity to ROI-mediated signals. When CD45RA+ (naive) and CD45RO+ (memory) T lymphocytes were directly stimulated with H2O2, NF-kappaB nuclear translocation was stronger in naive cells than in memory cells and it could be induced with lower doses. The composition of the induced nuclear NF-kappaB (levels of p50 and RelA proteins) was similar in these cell types. The magnitude and kinetics of intracellular ROI were similar, suggesting that there were no differences in ROI-forming mechanisms or antioxidative capacities. The probable regulatory point was the cytoplasmic IkappaB inhibitor: in CD45RA+ cells, H2O2 caused a more profound depression in the levels of IkappaB alpha. These findings indicate that T cells representing different activation and/or differentiation stages can be differentially responsive to ROI-mediated signals. 


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Transcription factors Sp1 and AP-2 mediate induction of acid sphingomyelinase during monocytic differentiation. 
Cells from the human monocytic leukemia cell line THP-1 differentiate towards a macrophage-like phenotype when stimulated with phorbol 12-myristate -13- acetate (PMA), 1,25-dihydroxy-vitamin D3, and various other agents. We demonstrate here that the expression of the lysosomal enzyme acid sphingomyelinase (ASM; E.C.3.1.4.12) is induced during this process and is strongly elevated in differentiated THP-1 cells, as well as in differentiated human mononuclear phagocytes. Using Northern blotting, RNase protection assay, and nuclear run-on analyses, we show that the up-regulation of ASM expression is regulated mainly at the level of transcription and that new protein synthesis is required for enhanced ASM activity. This cell-type specific induction by PMA treatment was further investigated with respect to transcriptional control. A series of 5' deletion derivatives of the upstream regulatory region were used in transient transfection assays to identify promoter elements required for basal and inducible gene expression. A PMA responsive element was localized to a region between -319 and -219 bp upstream of the initiation codon and co-transfections with transcription factor expression plasmids for AP-2 and Sp1 resulted in augmented ASM promoter activity, which was abolished when the binding sites for these two factors were deleted. Using electrophoretic mobility shift assays and supershift assays we demonstrate that this region is specifically bound by Sp1 and AP-2. These factors are present in nuclear extracts prepared from both induced and uninduced THP-1 cells. However, the intensity of the complex formed appeared to increase when nuclear extracts from PMA-treated cells were used. From these studies, we conclude that a concerted action of the transcription factors AP-2 and Sp1 is essential for the up -regulation of ASM expression during the process of macrophage differentiation. 


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Glucocorticoid hormone suppression of human neutrophil-mediated tumor cell cytostasis. 
In the present study, we have investigated the effect of glucocorticoid hormones on neutrophil-mediated tumor cell cytostasis and found that hydrocortisone and a synthetic hormone, dexamethasone (Dex), inhibited cytostasis in the presence or absence of tumor necrosis factor-alpha. The effect of Dex was completely reversed by a glucocorticoid receptor antagonist, RU38486. To clarify the underlying mechanisms, we examined effects of Dex on the binding avidity of beta2 integrin on the neutrophil surface and how these might in turn affect neutrophil-to-tumor cell binding. Dex was found to inhibit these neutrophil properties, and RU38486 completely suppressed both forms of Dex inhibition. Taken together, our findings suggest that glucocorticoid hormone inhibition of neutrophil-mediated tumor cell cytostasis is at least partially due to a lowering of the ligand binding avidity of beta2 integrin on the neutrophil surface. 


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Induction of vascular cell adhesion molecule-1 by low-density lipoprotein. 
Low-density lipoprotein (LDL) is a well-established risk factor for atherosclerosis. When endothelial cells are incubated with this lipoprotein in pathophysiologic amounts, the cells are activated. Among the documented cellular responses to LDL is increased recruitment of monocytes, which are believed to play a major role in promoting intimal plaque formation. The findings presented here link an atheogenic lipoprotein, LDL, with the induction of an adhesion molecule important in atherogenesis Human LDL induces the vascular cell adhesion molecule-1 (VCAM-1) transcriptionally with an increase in mRNA levels through activation of the VCAM promoter. This effect is blocked by anti-VCAM antibodies. After a 2-day incubation in LDL, the binding of NF-kappa B, which is believed to be a key oxidative-stress sensor for VCAM regulation, remains at basal level. In contrast, the binding activities of AP-1 and GATA, on the other hand, are increased by LDL. Thus, a component of LDL-enhanced endothelial recruitment of monocytes is attributed to VCAM-1 expression, which appears to be mediated through AP-1 and GATA. These data identify LDL as a VCAM-inducer possibly distinct from cytokines and endotoxin. 


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p38 mitogen-activated protein kinase mediates signal integration of TCR/CD28 costimulation in primary murine T cells. 
Optimal T cell activation requires two signals, one generated by TCR and another by the CD28 costimulatory receptor. In this study, we investigated the regulation of costimulation-induced mitogen-activated protein kinase (MAPK) activation in primary mouse T cells. In contrast to that reported for human Jurkat T cells, we found that p38 MAPK, but not Jun NH2-terminal kinase (JNK), is weakly activated upon stimulation with either anti-CD3 or anti-CD28 in murine thymocytes and splenic T cells. However, p38 MAPK is activated strongly and synergistically by either CD3/CD28 coligation or PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3/CD28-mediated signaling. Activation of p38 MAPK correlates closely with the stimulation of T cell proliferation. In contrast, PMA-induced JNK activation is inhibited by Ca2+ ionophore. T cell proliferation and production of IL-2, IL-4, and IFN-gamma induced by both CD3 and CD3/CD28 ligation and the nuclear expression of the c-Jun and ATF-2 proteins are each blocked by the p38 MAPK inhibitor SB203580. Our findings demonstrate that p38 MAPK 1) plays an important role in signal integration during costimulation of primary mouse T cells, 2) may be involved in the induction of c-Jun activation and augmentation of AP-1 transcriptional activity, and 3) regulates whether T cells enter a state of functional unresponsiveness. 


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The p65 subunit of NF-kappa B regulates I kappa B by two distinct mechanisms. 
Transcription factor NF-kappa B (p50/p65) is generally localized to the cytoplasm by its inhibitor I kappa B. Overproduced I kappa B, free from NF-kappa B, is rapidly degraded. Overexpression of p65 increases endogenous I kappa B protein in both carcinoma and lymphoid cells by two mechanisms: protein stabilization and increased transcription of I kappa B mRNA. In contrast, p65 delta, a naturally occurring splice variant, fails to markedly augment I kappa B protein levels. Both overexpressed p65 and coexpressed p50 are cytoplasmic, whereas p65 delta is partly nuclear, indicating that the I kappa B induced by p65 can maintain NF-kappa B in the cytoplasm. Thus, p65 and I kappa B are linked in an autoregulatory loop, ensuring that NF-kappa B is held in the cytoplasm until cells are specifically induced to translocate it to the nucleus. 


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Mononuclear leukocyte glucocorticoid receptor binding characteristics and down-regulation in major depression. 
Some patients with major depressive disorder (MDD) have elevated plasma cortisol concentrations and show failure to suppress cortisol secretion upon administration of dexamethasone (DEX), yet they do not have Cushingoid features. To study whether this represents glucocorticoid (GC) resistance, [3H]-DEX-binding assays were used to measure, in vitro, the GC receptor affinity (1/Kd) and number (Bmax) in mononuclear leukocytes of 11 MDD patients and 15 control subjects. No receptor abnormalities were detected in the MDD group; thus any cellular defect leading to a lack of responsiveness to GC in the MDD patients, if present, probably lies beyond the initial receptor binding. DEX (1.0 mg orally) was administered to study in vivo GC receptor down-regulation. Compared to the control group, fewer depressed subjects down-regulated Bmax after DEX. By paired t-test, Bmax decreased significantly in the control group but not in the depressed group. Receptor number on the control day did not correlate significantly with the degree of receptor down-regulation, severity of depression or cortisol concentrations across all the subjects. These results do not lend support to previous reports suggesting that GC resistance in MDD results from a GC receptor-binding abnormality, and they emphasize the importance of considering receptor studies in the context of GC-mediated cell processes in order to identify the exact cellular defect(s) leading to GC resistance. 


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Cutting edge: expression of the NF of activated T cells in eosinophils: regulation by IL-4 and IL-5. 
We report that NF-AT1 and NF-AT4 are expressed cytoplasmically in resting eosinophils, whereas NF-AT2 and NF-AT3 have not been seen. Likewise, NF-AT1 mRNA and NF-AT4 mRNA have been detected in resting eosinophils, and their levels can be significantly up-regulated by the Th2-associated cytokines IL-4 and IL-5. There is no detectable NF-AT protein expression in the nuclei of resting eosinophils. However NF-ATs appear in the nuclei of IL-4-, IL-5-, or ionomycin-stimulated eosinophils. Only NF-AT1 and NF-AT4, but not NF-AT2 and NF-AT3, have translocated into the nuclei in IL-4- or IL-5-stimulated eosinophils. These findings delineate a novel pathway in the cytokine network in which Th2 lymphocytes "control" eosinophils via the release of IL-4 and IL-5, and activation of NF-AT in eosinophils. The findings also suggest that a later feedback "talking" may exist between eosinophils and Th2 lymphocytes. 


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Osteoclast markers accumulate on cells developing from human peripheral blood mononuclear precursors. 
Recent studies show that human osteoclasts develop in vitro from hematopoietic cells; however, special cultures conditions and/or cytokine mobilized peripheral blood are apparently required. Here, we report that cells expressing osteoclast markers differentiate from precursors present in nonmobilized peripheral blood mononuclear cells (PBMC), without the addition of stromal cells, growth factors, cytokines or steroids; and characterize their phenotype. Three days after establishing high-density PBMC cultures (1.5 x 10(6) cells/cm2), in serum-containing medium, small adherent colonies of tartrate resistant acid phosphatase positive (TRAP+) cells emerge, amidst massive monocyte cell death. These adherent cells have an eccentrically placed, round nucleus, and express low levels of TRAP and sodium fluoride-resistant- alpha-naphthyl-acetate-esterase (NaF-R-NSE). Over the next week, this cell population accumulates phenotypic markers of osteoclasts (vitronectin receptor [VR], calcitonin receptor, TRAP, cathepsin K protein, and mRNA) with increased nuclearity, covering the entire surface by 15 days. When cultured on bone, VR+, TRAP+ cells of low multinuclearity appear and cover up to 50% of the surface. Resorption lacunae can be observed by day 22. Although these pits are not nearly as numerous as the cells of preosteoclast phenotype, they do represent the activity of a subset of osteoclast-like cells that has achieved osteoclastic maturity under these culture conditions. Transcripts for osteoprotegerin ligand (OPGL), an osteoclast differentiation factor (also known as RANKL and TRANCE) are expressed, likely by adherent cells. Thus, an adherent population of cells, with preosteoclast/osteoclast phenotypic properties, arises selectively under simple culture conditions from normal PBMC. Further characterization of these cells should identify factors involved in the growth, terminal differentiation and activation of osteoclasts. 


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Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. 
The NK-kappa B transcription factor complex is sequestered in the cytoplasm by the inhibitory protein I kappa B-alpha (MAD-3). Various cellular stimuli relieve this inhibition by mechanisms largely unknown, leading to NF-kappa B nuclear localization and transactivation of its target genes. It is demonstrated here with human T lymphocytes and monocytes that different stimuli, including tumor necrosis factor alpha and phorbol 12-myristate 13-acetate, cause rapid degradation of I kappa B-alpha, with concomitant activation of NF-kappa B, followed by a dramatic increase in I kappa B-alpha mRNA and protein synthesis. Transfection studies reveal that the I kappa B-alpha mRNA and the encoded protein are potently induced by NF-kappa B and by homodimers of p65 and of c-Rel. We propose a model in which NF-kappa B and I kappa B-alpha mutually regulate each other in a cycle: saturating amounts of the inhibitory I kappa B-alpha protein are destroyed upon stimulation, allowing rapid activation of NF-kappa B. Subsequently, I kappa B-alpha mRNA and protein levels are quickly induced by the activated NF-kappa B. This resurgence of I kappa B-alpha protein acts to restore an equilibrium in which NF-kappa B is again inhibited. 


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Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals. 
The mRNA for the Duffy blood group antigen, the erythrocyte receptor for the Plasmodium vivax malaria parasite, has recently been cloned and shown to encode a widely expressed chemokine receptor. Here, we show that the Duffy antigen/chemokine receptor gene (DARC) is composed of a single exon and that most Duffy-negative blacks carry a silent FY*B allele with a single T to C substitution at nucleotide -46. This mutation impairs the promoter activity in erythroid cells by disrupting a binding site for the GATA1 erythroid transcription factor. With the recent characterization of the FY*A and FY*B alleles, these findings provide the molecular basis of the Duffy blood group system and an explanation for the erythroid-specific repression of the DARC gene in Duffy-negative individuals. 


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The c-rel protooncogene product represses NF-kappa B p65-mediated transcriptional activation of the long terminal repeat of type 1 human immunodeficiency virus. 
The long terminal repeat (LTR) of the type 1 human immunodeficiency virus (HIV-1) and the 5' regulatory region of the gene encoding the interleukin 2 receptor alpha subunit (IL-2R alpha) share functional kappa B enhancer elements involved in the regulation of these inducible transcription units during T-cell activation. These kappa B enhancer elements are recognized by a structurally related family of interactive proteins that includes p50, p65, and the product of the c-rel protooncogene (c-Rel). Recent biochemical studies have shown that p65 and p50 form the prototypical NF-kappa B complex, which is rapidly translocated from the cytoplasm to the nucleus during T-cell activation. This intracellular signaling complex potently stimulates kappa B-directed transcription from either the HIV-1 LTR or the IL-2R alpha promoter via the strong transactivation domain present in p65. We now demonstrate that nuclear expression of human c-Rel, which is induced by either phorbol ester or tumor necrosis factor alpha with delayed kinetics relative to p65, markedly represses p65-mediated activation of these transcription units. These inhibitory effects of c-Rel correlate with its DNA-binding activity but not with its ability to heterodimerize with p50, suggesting that c-Rel inhibition involves competition with p50/p65 for occupancy of the kappa B enhancer element. Together, these findings suggest that one function of c-Rel is as a physiologic repressor of the HIV-1 LTR and IL-2R alpha promoters, serving to efficiently counter the strong transcriptional activating effects of p65. 


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Activation of human immunodeficiency virus type 1 expression by Gardnerella vaginalis. 
Bacterial vaginosis (BV) is associated with an increased rate of sexual transmission of human immunodeficiency virus (HIV) type 1, and Gardnerella vaginalis is frequently isolated from the genital tracts of women with BV. G. vaginalis lysates were found to significantly stimulate HIV expression in monocytoid cells. Stimulation was significantly higher when lysates were heated at 100 degrees C for 5 min but was reduced by treatment with lysozyme or protease. G. vaginalis lysates also activated HIV expression in certain T cell lines. G. vaginalis lysates activated HIV long-terminal repeat transcription in HIV-infected cells and increased NF-kappaB binding activity, indicating an effect by G. vaginalis on HIV transcription. The activation of HIV production by G. vaginalis suggests that genital tract infection with G. vaginalis increases the risk of HIV transmission by increasing HIV expression in the genital tract. This may explain, at least in part, the increased rate of HIV transmission in women with BV. 


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Transcriptional targeting of retroviral vectors to the erythroblastic progeny of transduced hematopoietic stem cells. 
Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3' LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5' LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34(+) stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (triangle upLNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells. 


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Four P-like elements are required for optimal transcription of the mouse IL-4 gene: involvement of a distinct set of nuclear factor of activated T cells and activator protein-1 family proteins. 
We previously identified the P sequence as a critical regulatory element of the human IL-4 promoter. In the mouse IL-4 promoter, there are five elements homologous to the human P sequence designated conserved lymphokine element 0 (CLE0), P, P2, P3 and P4. To characterize the role of these P-like elements and their binding factors in the native promoter, we did transient transfection and electrophoretic mobility shift assays (EMSA). Transfection of EL-4 cells with the IL-4 promoter-reporter constructs carrying mutated P-like elements showed that four P-like elements, CLE0, P, P2 and P4, but not P3, were required for optimal activation of the IL-4 promoter. EMSA showed that both constitutive and inducible complexes bound to CLE0, P, P2 and P4, whereas only a constitutive complex bound to P3. In competition and antibody supershift assays in EMSA, complexes formed with P or P2 proved to contain nuclear factor of activated T cells (NFAT) family proteins as major components. Activator protein (AP)-1 family proteins interacted with CLE0, P, P2 and P4. NFAT/AP-1 complex formed only with P and P2. Cross-competition assays among the P-like elements revealed element-specific and common complexes. Six tandem repeats of the P element linked to the SV40 promoter responded to phorbol 12-myristate 13-acetate, while that of other elements did not. It would thus appear that components of each P-like element-binding complexes are not identical and may coordinately contribute to transcriptional activity. 


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HIV enhancer activity perpetuated by NF-kappa B induction on infection of monocytes [see comments] 
Permissiveness to replication of human immunodeficiency virus (HIV) differs in T lymphocytes and macrophages. In T cells, HIV transcription is poorly detected in vivo. Cloned, normal T lymphocytes show very little, if any, basal activity of the HIV enhancer and low nuclear expression of NF-kappa B, a potent transcriptional activator of the HIV enhancer. In contrast, fixed tissue macrophages express detectable HIV proteins, indicating permanent virus transcription. One explanation for the perpetuation of virus infection in macrophages could be sustained nuclear NF-kappa B expression. However, the U937 monocytic cell line, which is fully permissive to HIV replication, is known to express only low levels of nuclear NF-kappa B. We show here that chronic HIV infection results in both induction of a nuclear factor with antigenic properties indistinguishable from those of NF-kappa B and permanently increased HIV enhancer activity. This phenomenon, which is independent of tumour necrosis factor, is associated with HIV replication, and is thus likely to explain at least in part the perpetuation of HIV infection in monocytes. 


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Functional roles of the transcription factor Oct-2A and the high mobility group protein I/Y in HLA-DRA gene expression. 
The class II major histocompatibility complex gene HLA-DRA is expressed in B cells, activated T lymphocytes, and in antigen-presenting cells. In addition, HLA-DRA gene expression is inducible in a variety of cell types by interferon-gamma (IFN-gamma). Here we show that the lymphoid-specific transcription factor Oct-2A plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) I/Y binds to multiple sites within the DRA promoter, including the Oct-2A binding site. Coexpression of HMG I/Y and Oct-2 in cell lines lacking Oct-2 results in high levels of HLA-DRA gene expression, and in vitro DNA-binding studies reveal that HMG I/Y stimulates Oct-2A binding to the HLA-DRA promoter. Thus, Oct-2A and HMG I/Y may synergize to activate HLA-DRA expression in B cells. By contrast, Oct-2A is not involved in the IFN-gamma induction of the HLA-DRA gene in HeLa cells, but antisense HMG I/Y dramatically decreases the level of induction. We conclude that distinct sets of transcription factors are involved in the two modes of HLA-DRA expression, and that HMG I/Y may be important for B cell-specific expression, and is essential for IFN-gamma induction. 


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Functional characterization of the murine homolog of the B cell-specific coactivator BOB.1/OBF.1. 
B cell-specific transcriptional promoter activity mediated by the octamer motif requires the Oct1 or Oct2 protein and additional B cell-restricted cofactors. One such cofactor, BOB.1/OBF.1, was recently isolated from human B cells. Here, we describe the isolation and detailed characterization of the murine homolog. Full-length cDNAs and genomic clones were isolated, and the gene structure was determined. Comparison of the deduced amino acids shows 88% sequence identity between mouse and human BOB.1/OBF.1. The NH2-terminal 126 amino acids of BOB.1/OBF.1 are both essential and sufficient for interaction with the POU domains of either Oct1 or Oct2. This protein-protein interaction does not require the simultaneous binding of Oct proteins to DNA, and high resolution footprinting of the Oct-DNA interaction reveals that binding of BOB.1/OBF.1 to Oct1 or Oct2 does not alter the interaction with DNA. BOB.1/OBF.1 can efficiently activate octamer-dependent promoters in fibroblasts; however, it fails to stimulate octamer-dependent enhancer activity. Fusion of subdomains of BOB.1/OBF.1 with the GAL4 DNA binding domain reveals that both NH2- and COOH-terminal domains of BOB.1/OBF.1 contribute to full transactivation function, the COOH-terminal domain is more efficient in this transactivation assay. Consistent with the failure of full-length BOB.1/OBF.1 to stimulate octamer-dependent enhancer elements in non B cells, the GAL4 fusions likewise only stimulate from a promoter-proximal position. 


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Activation of E2F-mediated transcription by human T-cell leukemia virus type I Tax protein in a p16(INK4A)-negative T-cell line. 
The human T-cell leukemia virus type I (HTLV-I) is a causative agent of adult T-cell leukemia. Although the exact mechanism by which HTLV-I contributes to leukemogenesis is still unclear, the Tax protein is thought to play a major role in this process. This 40-kDa polypeptide is able to interact with the tumor suppressor p16(INK4A). Consequently, Tax can activate the signaling pathway that lead to the release of E2F that in turn induces expression of factors required for cell cycle progression. In this paper, we demonstrate that Tax can also activate E2F-mediated transcription independently of p16(INK4A). Indeed, when Tax is coexpressed with the E2F-1 transcription factor in CEM T-cells, which lack expression of p16(INK4A), it strongly potentiates the E2F-dependent activation of a reporter construct driven by a promoter containing E2F binding sites. This stimulation is abrogated by mutations affecting the E2F-binding sites. In addition, Tax also stimulates the transcription of the E2F-1 gene itself. Using Tax mutants that fail to activate either ATF- or NF-kappaB-dependent promoters and different 5' truncation mutants of the E2F-1 promoter, we show that the Tax-dependent transcriptional control of the E2F1 gene involves, at least in part, the ATF binding site located in the E2F-1 promoter. 


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Activation-dependent transcriptional regulation of the human Fas promoter requires NF-kappaB p50-p65 recruitment. 
Fas (CD95) and Fas ligand (CD95L) are an interacting receptor-ligand pair required for immune homeostasis. Lymphocyte activation results in the upregulation of Fas expression and the acquisition of sensitivity to FasL-mediated apoptosis. Although Fas upregulation is central to the preservation of immunologic tolerance, little is known about the molecular machinery underlying this process. To investigate the events involved in activation-induced Fas upregulation, we have examined mRNA accumulation, fas promoter activity, and protein expression in the Jurkat T-cell line treated with phorbol myristate acetate and ionomycin (P/I), pharmacological mimics of T-cell receptor activation. Although resting Jurkat cells express Fas, Fas mRNA was induced approximately 10-fold in 2 h upon P/I stimulation. Using sequential deletion mutants of the human fas promoter in transient transfection assays, we identified a 47-bp sequence (positions -306 to -260 relative to the ATG) required for activation-driven fas upregulation. Sequence analysis revealed the presence of a previously unrecognized composite binding site for both the Sp1 and NF-kappaB transcription factors at positions -295 to -286. Electrophoretic mobility shift assay (EMSA) and supershift analyses of this region documented constitutive binding of Sp1 in unactivated nuclear extracts and inducible binding of p50-p65 NF-kappaB heterodimers after P/I activation. Sp1 and NF-kappaB transcription factor binding was shown to be mutually exclusive by EMSA displacement studies with purified recombinant Sp1 and recombinant p50. The functional contribution of the kappaB-Sp1 composite site in P/I-inducible fas promoter activation was verified by using kappaB-Sp1 concatamers (-295 to -286) in a thymidine kinase promoter-driven reporter construct and native promoter constructs in Jurkat cells overexpressing IkappaB-alpha. Site-directed mutagenesis of the critical guanine nucleotides in the kappaB-Sp1 element documented the essential role of this site in activation-dependent fas promoter induction. 


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HIV type 1 protease activation of NF-kappa B within T lymphoid cells. 
NF-kappa B is a nuclear protein of the rel oncogene family capable of enhancing transcription of several cellular genes, including IL-2 and the IL-2 receptor, and viral genes transcribed from the HIV-1 LTR. It has been reported that HIV-1 protease may cleave the NF-kappa B precursor to its active form in vitro. In this study the effects of HIV protease on NF-kappa B precursor activation were examined in Jurkat T cells by introducing a protease expression vector into the cells. Increased NF-kappa B activity was observed and this increased activity was blocked by a specific inhibitor of the viral protease. Viral transcription, as measured using LTR-CAT assays, was only slightly enhanced in the HIV-protease expressing cells, while secretion of IL-2 and expression of the IL-2 receptor were not affected. The limited activation of NF-kappa B by HIV protease appears unlikely to have a significant effect on virus expression or T cell function. 


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Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. 
To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1). In a concentration-dependent manner, NO inhibited interleukin (IL)-1 alpha-stimulated VCAM-1 expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, IL-1 beta, IL-4, tumor necrosis factor (TNF alpha), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL-8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-1, but did not augment cytokine-induced VCAM-1 expression. Nuclear run-on assays, transfection studies using various VCAM-1 promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses VCAM-1 gene transcription, in part, by inhibiting NF-kappa B. We propose that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall. 


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Activated platelets induce monocyte chemotactic protein-1 secretion and surface expression of intercellular adhesion molecule-1 on endothelial cells [see comments] 
BACKGROUND: Platelet/endothelium interaction plays an important role in the pathophysiology of inflammation and atherosclerosis. The role of platelets for monocyte chemotactic protein-1 (MCP-1) secretion and surface expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells has been assessed. METHODS AND RESULTS: Monolayers of human umbilical vein endothelial cells were incubated with nonstimulated or ADP-activated platelets for 6 hours, and secretion of MCP-1 and surface expression of ICAM-1 were determined by ELISA and flow cytometry, respectively. In the presence of ADP-activated platelets, both MCP-1 secretion and ICAM-1 surface expression were significantly increased compared with nonstimulated platelets (P<0.02). Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) determined by electrophoretic mobility shift assay and kappaB-dependent transcriptional activity was enhanced in the presence of activated platelets. In addition, ADP-activated platelets induced MCP-1 and ICAM-1 promoter-dependent transcription. Liposomal transfection of a double-stranded kappaB phosphorothioate oligonucleotide, but not of the mutated form, inhibited MCP-1 secretion and surface expression of ICAM-1 on activated endothelium (P<0.05). CONCLUSIONS: The present study indicates that activated platelets modulate chemotactic (MCP-1) and adhesive (ICAM-1) properties of endothelial cells via an NF-kappaB-dependent mechanism. Platelet-induced activation of the NF-kappaB system might contribute to early inflammatory events in atherogenesis. 


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Decreased proteasome-mediated degradation in T cells from the elderly: A role in immune senescence. 
Induction of NFkappaB is a highly regulated process requiring phosphorylation, ubiquitination, and proteasome-mediated degradation of the cytosolic inhibitor IkappaBalpha. Analyses of the regulation of IkappaBalpha in TNF-alpha-treated T lymphocytes from young and elderly donors revealed severely compromised degradation of IkappaBalpha in T cells from the elderly. Examination of activation-induced phosphorylation and ubiquitination of IkappaBalpha did not demonstrate any significant age-related alterations. However, examination of proteasome activity in these T cells using fluorogenic peptide assays revealed a significant age-related decline in chymotryptic activity. These results suggest that a decline in proteasome activity results in a failure to fully degrade IkappaBalpha in the elderly. This failure to degrade IkappaBalpha may underlie both the observed decrease in NFkappaB induction and the IL-2 receptor expression in TNF-treated T cells during aging. Thus, decreased proteasome-mediated degradation may be central to immune dysfunction that accompanies aging. Copyright 1999 Academic Press. 


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The regulation of HIV by retinoic acid correlates with cellular expression of the retinoic acid receptors. 
OBJECTIVES: To analyze the effect of retinoic acids (RA) on HIV-1 expression and correlate this effect with expression levels of RA receptors (RARs) in T-lymphoid and monocytoid cell lines. DESIGN AND METHODS: The effect of all-trans and 9-cis RA on HIV-1 production in T-lymphoid (H9, CEM) and monocytoid (U937,THP-1) cell lines was measured during acute and chronic infection. The expression levels of human RAR alpha (hRAR alpha, receptor for all-trans RA) and the human retinoid-X receptor alpha (hRXR alpha receptor for 9-cis RA) were determined by Northern blot analysis. RESULTS: Both all-trans and 9-cis RA inhibited virus replication in HIV-1 IIIB-infected monocytoid cells, in the presence and absence of the co-stimulatory agent phorbol myristate acetate (PMA). The retinoids had weak or no stimulatory effects on HIV production by T-cell lines. HIV production by PMA-stimulated T-cell lines was inhibited by these retinoids. The 9-cis RA was generally more effective than all-trans RA in inhibiting HIV production and in combination generally more effective than the single agents alone. Human RAR alpha was expressed in H9, U937 and THP-1 cells, but almost undetectable in CEM cells. Human RXR alpha was significantly expressed in U937 and THP-1 cells, weakly expressed in H9 cells and not detectable in CEM cells. After stimulation by PMA, RXR alpha expression increased in H9 and U937 cells but not in CEM cells. Human RAR alpha expression was unchanged in H9 and CEM cells, and elevated in U937 cells, after PMA stimulation. CONCLUSION: The effect of RA on HIV-1 expression was cell-type-dependent and partially correlated with cellular expression of RARs. Endogenous or exogenously administered RA may have a significant role in HIV regulation. 


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The state of maturation of monocytes into macrophages determines the effects of IL-4 and IL-13 on HIV replication. 
The molecular mechanisms of the effects of IL-4 and IL-13 on HIV infection in human monocytes as they matured into monocyte-derived macrophages over 7 days were investigated using HIV-1(BaL), and low passage clinical strains. IL-4 and IL-13 up-regulated the expression of both genomic and spliced HIV mRNA in monocytes cultured on Teflon, as determined by Northern analysis and p24 Ag assay. Using a nuclear run-on assay, IL-4 stimulation was shown to enhance transcription by two- to threefold. IL-4 stimulated nuclear factor-kappaB nuclear translocation and binding before enhancement of HIV RNA expression. Conversely, IL-4 and IL-13 markedly and significantly inhibited HIV replication at the transcriptional level in monocyte-derived macrophages, and this occurred whether these cytokines were added before or after HIV infection. The reversal from stimulation to inhibition occurred after 3 to 5 days of adherence to plastic. IL-4 had no significant effect on HIV reverse transcription. The effect of both cytokines on the monocyte maturation/differentiation (CD11b, CD13, and CD26) and other macrophage markers (CD14 and CD68) was examined. IL-4 enhanced CD11b, but inhibited CD26 expression and delayed CD13 loss. IL-13 had similar effects on CD11b and CD13, but no effect on CD26. Hence, these cytokines do not simply enhance monocyte differentiation, but have complex and slightly divergent effects that impact on HIV replication probably through cell signaling pathways and nuclear factor-kappaB translocation. 


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Transcriptional activity and constitutive nuclear localization of the ETS protein Elf-1. 
Elf-1 is a lymphoid-specific transcription factor that belongs to the ETS protein family. It can bind to DNA target sequences within a variety of cytokine genes. We demonstrate that Elf-1 is constitutively localized in the nucleus which is dependent on the presence of amino acids 86-265. Analysis of Gal4-Elf-1 fusion proteins revealed that the N-terminal 86 amino acids of Elf-1 contain a transcriptional activation domain, the activity of which is attenuated by an internal repression domain. Furthermore, Elf-1 interacts specifically with the E74 target sequence and can stimulate transcription driven by the E74 site independent of mitogenic signaling. Thus, Elf-1 is able to stimulate gene transcription which may be required for the development and activity of lymphocytes. 


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Reactive oxygen intermediate-release of fibre-exposed monocytes increases inflammatory cytokine-mRNA level, protein tyrosine kinase and NF-kappaB activity in co-cultured bronchial epithelial cells (BEAS-2B). 
Some pulmonary diseases like bronchitis or asthma bronchiale are mediated by inflammatory mechanisms in bronchial epithelial cells. Alveolar macrophages are located directly in the surrounding of these cells, so that we suppose an interaction between epithelial cells and macrophages regarding to the release of inflammatory mediators. For measuring the contribution of macrophages to the release of inflammatory mediators by bronchial epithelial cells, we established an in vitro model of co-cultured blood monocytes (BM) and BEAS-2B cells in a transwell system (Costar). BM were exposed to Chrysotile B and soot particle FR 101 in a concentration of 100 microg/10(6) cells. After up to 90 min exposure time ELISA, EMSA (electromobility shift assay) and RT-PCR were used to measure protein tyrosine kinase activity, protein activity of NF-kappaB and cytokine (IL-1beta, IL-6, TNF-alpha) specific mRNA levels in BEAS-2B cells. We observed an increase in protein tyrosine kinase activity (up to 1.8 +/- 0.5-fold) and NF-kappaB protein activity in BEAS-2B cells after particle or fibre exposure of co-cultured BM. Consecutive IL-1beta-, IL-6- and TNF-alpha-mRNA were elevated (up to 1.9 +/- 0.58-fold). Protein tyrosine kinase activity, NF-kappaB activity, and the synthesis of cytokine-specific mRNA were inhibited by antioxidants. These data suggest a ROI-dependent NF-kappaB mediated transcription of inflammatory cytokines in bronchial epithelial cells. 


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Characterization of the human myeloid cell nuclear differentiation antigen gene promoter. 
MNDA (myeloid cell nuclear differentiation antigen) is an interferon alpha regulated nuclear protein expressed only in cells of the human myelomonocytic lineage. To identify mechanisms responsible for this lineage-specific and interferon-regulated expression, the 5' flanking sequence of the gene has been characterized. Two interferon-stimulated response elements (ISRE) flank a multiple transcription start site region identifying MNDA as a TATA-less interferon-regulated gene. Other DNA elements present include a cluster of Myb sites, several Ets, an Ets related PU.1 site and an Sp1 site located within 600 bp of the transcription start sites. In addition, DNA methylation was revealed as one of the possible factors in establishing MNDA expression. The 5' flanking sequence has promoter activity which is elevated by interferon alpha. The findings indicate that MNDA expression is regulated by mechanisms similar to other myelomonocytic cell specific genes and genes up-regulated by interferon alpha. 


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Mycobacterium tuberculosis mannose-capped lipoarabinomannan can induce NF-kappaB-dependent activation of human immunodeficiency virus type 1 long terminal repeat in T cells. 
Tuberculosis has emerged as an epidemic, extended by the large number of individuals infected with human immunodeficiency virus type 1 (HIV-1). The major goal of this study was to determine whether the mycobacterial cell wall component mannose-capped lipoarabinomannan (ManLAM) of Mycobacterium tuberculosis (M. tuberculosis) could activate transcription of HIV-1 in T cells with the use of an in vitro cell culture system. These experiments are of prime importance considering that CD4-expressing T lymphocytes represent the major virus reservoir in the peripheral blood of infected individuals. Using the 1G5 cell line harbouring the luciferase reporter gene under the control of the HIV-1 LTR, it was first found that culture protein filtrates (CFP) from M. tuberculosis or purified ManLAM could activate HIV-1 LTR-dependent gene expression unlike similarly prepared CFP extracts devoid of ManLAM. The implication of protein tyrosine kinase(s), protein kinase A and/or protein kinase C was highlighted by the abrogation of the ManLAM-mediated activation of HIV-1 LTR-driven gene expression using herbimycin A and H7. It was also determined, using electrophoresis mobility shift assays, that M. tuberculosis ManLAM led to the nuclear translocation of the transcription factor NF-kappaB. M. tuberculosis ManLAM resulted in clear induction of the luciferase gene placed under the control of the wild-type, but not the kappaB-mutated, HIV-1 LTR region. Finally, the ManLAM-mediated activation of HIV-1 LTR transcription was found to be independent of the autocrine or paracrine action of endogenous TNF-alpha. The results suggest that M. tuberculosis can upregulate HIV-1 expression in T cells and could thus have the potential to influence the pathogenesis of HIV-1 infection. 


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[Corticoids and allergy] 
Inflammation is constantly observed in allergic reactions. Corticosteroids are most effective in preventing the late phase of allergic reaction. The action of glucocorticosteroids is mediated through glucocorticoid receptors present in the cellular cytoplasm. When activated, glucocorticoid receptors form a dimer and bind to DNA after migration into the nucleus. Interaction to DNA induces changes in the transcription rate, leading to either gene induction or gene repression. Glucocorticoid receptors are also able to interact with transcriptional factors such as AP-1 (activator protein-1) of NF-kappa B (nuclear factor-kappa B). Through these actions glucocorticosteroids are susceptible to modify functions of cells involved in the allergic inflammatory response. They are in particular able to inhibit most of the pro-inflammatory functions of the eosinophils. 


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Alpha-lipoic acid is a potent inhibitor of NF-kappa B activation in human T cells. 
Acquired immunodeficiency syndrome (AIDS) results from infection with a human immunodeficiency virus (HIV). The long terminal repeat (LTR) region of HIV proviral DNA contains binding sites for nuclear factor kappa B (NF-kappa B), and this transcriptional activator appears to regulate HIV activation. Recent findings suggest an involvement of reactive oxygen species (ROS) in signal transduction pathways leading to NF-kappa B activation. The present study was based on reports that antioxidants which eliminate ROS should block the activation of NF-kappa B and subsequently HIV transcription, and thus antioxidants can be used as therapeutic agents for AIDS. Incubation of Jurkat T cells (1 x 10(6) cells/ml) with a natural thiol antioxidant, alpha-lipoic acid, prior to the stimulation of cells was found to inhibit NF-kappa B activation induced by tumor necrosis factor-alpha (25 ng/ml) or by phorbol 12-myristate 13-acetate (50 ng/ml). The inhibitory action of alpha-lipoic acid was found to be very potent as only 4 mM was needed for a complete inhibition, whereas 20 mM was required for N-acetylcysteine. These results indicate that alpha-lipoic acid may be effective in AIDS therapeutics. 


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Effects of glucocorticoids on transcription factor activation in human peripheral blood mononuclear cells. 
Glucocorticoids have an inhibitory effect on inflammatory and immune responses, and this may be through the modulation of transcription factor binding to DNA. The interaction of the transcription factors, activator protein-1 (AP-1), nuclear factor kappa B (NF kappa B), and cAMP-responsive element binding protein (CREB) with DNA and glucocorticoid receptors (GR) was analyzed in human peripheral blood mononuclear cells by gel mobility shift assays. TNF-alpha, IL-1 beta and phorbol myristate acetate (PMA) treatment increased AP-1 and NF kappa B DNA binding by up to 200% but decreased CREB binding (38%) over a 60-min time course. Dexamethasone produced a rapid and sustained increase in glucocorticoid response element binding and a concomitant 40-50% decrease in AP-1, NF kappa B, and CREB DNA binding that was blocked by combined dexamethasone and cytokine or PMA treatment. These latter effects were due to increases in the nuclear localization of GR, not to reduced amounts of the other transcription factors. This suggests that in these cells GR within the nucleus interacts with cytokine-stimulated transcription factors by the process of cross coupling. This may be an important molecular site of steroid action. 


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Helenalin, an anti-inflammatory sesquiterpene lactone from Arnica, selectively inhibits transcription factor NF-kappaB [see comments] 
Alcoholic extracts prepared form Arnicae flos, the collective name for flowerheads from Arnica montana and A. chamissonis ssp. foliosa, are used therapeutically as anti-inflammatory remedies. The active ingredients mediating the pharmacological effect are mainly sesquiterpene lactones, such as helenalin, 11alpha,13-dihydrohelenalin, chamissonolid and their ester derivatives. While these compounds affect various cellular processes, current data do not fully explain how sesquiterpene lactones exert their anti-inflammatory effect. We show here that helenalin, and, to a much lesser degree, 11alpha,13-dihydrohelenalin and chamissonolid, inhibit activation of transcription factor NF-kappaB. This difference in efficacy, which correlates with the compounds' anti-inflammatory potency in vivo, may be explained by differences in structure and conformation. NF-kappaB, which resides in an inactive, cytoplasmic complex in unstimulated cells, is activated by phosphorylation and degradation of its inhibitory subunit, IkappaB. Helenalin inhibits NF-kappaB activation in response to four different stimuli in T-cells, B-cells and epithelial cells and abrogates kappaB-driven gene expression. This inhibition is selective, as the activity of four other transcription factors, Oct-1, TBP, Sp1 and STAT 5 was not affected. We show that inhibition is not due to a direct modification of the active NF-kappaB heterodimer. Rather, helenalin modifies the NF-kappaB/IkappaB complex, preventing the release of IkappaB. These data suggest a molecular mechanism for the anti-inflammatory effect of sesquiterpene lactones, which differs from that of other nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and acetyl salicylic acid. 


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Competent transcription initiation by RNA polymerase II in cell-free extracts from xeroderma pigmentosum groups B and D in an optimized RNA transcription assay. 
The human autosomal recessive disease, xeroderma pigmentosum (XP), can result from mutations in any one of seven genes, designated XPA through XPG. Of these, the XPB and XPD genes encode proteins that are subunits of a general transcription factor, TFIIH, involved in both nucleotide excision repair (NER) and initiation of mRNA transcription by RNA polymerase II. In humans, mutation of the XPB or XPD gene impairs NER, resulting in hyper-sensitivity to sunlight and greatly increased skin tumor formation. However, no transcription deficiency has been demonstrated in either XP-B or XP-D. We have employed an optimized cell-free RNA transcription assay to analyze transcription activity of XP-B and XP-D. Although the growth rate was normal, the XP-B and XP-D cells contained reduced amounts of TFIIH. Extracts prepared from XP-B and XP-D lymphoblastoid cells exhibited similar transcription activity from the adenovirus major late promoter when compared to that in extracts from normal cells. Thus, we conclude that the XP-B and XP-D lymphoblastoid cells do not have impaired RNA transcription activity. We consider the possible consequences of the reduced cellular content of TFIIH for the clinical symptoms in XP-B or XP-D patients, and discuss a 'conditional phenotype' that may involve an impairment of cellular function only under certain growth conditions. 


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Targeted remodeling of human beta-globin promoter chromatin structure produces increased expression and decreased silencing. 
The chromatin structure of the human beta-globin gene locus assumes a transcriptionally-active conformation in erythroid cells. One feature of this chromatin reorganization is the formation of DNase 1 hypersensitive sites in the regions of active globin gene promoters. This reorganization requires the globin locus control region and is associated with normal expression of the beta-like globin genes. To determine whether it is possible to artificially enhance the opening of the chromatin structure of a minimal beta-globin promoter, we placed a 101bp, erythroid-specific DNase 1 hypersensitive site-forming element (HSFE) immediately upstream of the beta-globin promoter and gene. This element includes binding sites for NF-E2, AP-1, GATA-1 and Sp-1. Constructs were stably transfected into murine erythroleukemia cells and promoter chromatin structure and gene expression were analyzed. The HSFE induced an area of enhanced DNase 1 hypersensitivity extending from the transcriptional start site to -300bp of the artificial promoter and significantly increased the proportion of beta-globin promoters in an open chromatin configuration. This remodeling of promoter chromatin structure resulted in 3-fold increases in beta-globin gene transcription and induction, and inhibited long-term beta-globin gene silencing. These results indicate that a relatively small cis-acting element is able to enhance remodeling of promoter chromatin structure resulting in increased beta-globin gene expression. 


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Transcriptional control of steroid-regulated apoptosis in murine thymoma cells. 
Early studies in murine T cell lines indicated that transcriptional transactivation functions encoded in the glucocorticoid receptor (GR) N-terminal domain are required for glucocorticoid-mediated apoptosis. However, more recent studies in human T cell lines have suggested that the N-terminal domain is not necessary for steroid-regulated apoptosis and that GR-mediated transrepression may be the more critical mechanism. To better understand the contribution of the GR N-terminal transactivation domain in mediating murine thymocyte apoptosis, we stably transfected GR, GR variants, and the androgen receptor (AR) into receptor-negative S49 murine thymoma cells. GR expression levels were shown to be rate-limiting for initiating the apoptotic pathway, and a positive correlation between steroid sensitivity and GR-mediated induction of an integrated mouse mammary tumor virus (MMTV) LTR reporter gene was observed. Analysis of GR chimeric receptors containing the potent VP16 and E1A viral transactivation domains in place of the GR N terminus revealed that even low level expression of these receptors resulted in both enhanced steroid sensitivity and MMTV induction, thus supporting a role for transactivation in apoptosis. In contrast, we found that AR can initiate apoptosis in S49 cells after treatment with 5 alpha-dihydrotestosterone, despite its relative inability to induce high level expression of MMTV. To investigate this further, we examined the steroid-regulated expression of an endogenous thymocyte-specific gene called GIG18. We found that GIG18 was rapidly induced to comparable levels by both AR and GR, demonstrating that AR can indeed function as a transcriptional activator in S49 cells and, moreover, that GIG18 induction may be a marker of early apoptotic events in steroid-treated cells. Taken together, these results support our conclusion that transcriptional transactivation is a necessary signaling component of S49 cell apoptosis, although an additional role for GR-mediated transrepression cannot be excluded. 


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RFLAT-1: a new zinc finger transcription factor that activates RANTES gene expression in T lymphocytes. 
RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) is a chemoattractant cytokine (chemokine) important in the generation of inflammatory infiltrate and human immunodeficiency virus entry into immune cells. RANTES is expressed late (3-5 days) after activation in T lymphocytes. Using expression cloning, we identified the first "late" T lymphocyte associated transcription factor and named it "RANTES Factor of Late Activated T Lymphocytes-1" (RFLAT-1). RFLAT-1 is a novel, phosphorylated, zinc finger transcription factor that is expressed in T cells 3 days after activation, coincident with RANTES expression. While Rel proteins play the dominant role in RANTES gene expression in fibroblasts, RFLAT-1 is a strong transactivator for RANTES in T cells. 


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Infection and replication of Tat- human immunodeficiency viruses: genetic analyses of LTR and tat mutations in primary and long-term human lymphoid cells. 
Tat is an essential regulatory protein for the replication of human immunodeficiency virus (HIV). Mutations in the tat gene have been shown to block HIV replication in human T cells. Several studies have established that Tat releases an elongation block to the transcription of HIV long terminal repeat (LTR); however, it is not known whether this mechanism alone is sufficient to explain the block to HIV replication in human T cells when Tat is absent. It is possible that Tat is also needed for other functions during HIV replication. To test these hypotheses, we studied several tat mutants, including two stop codon mutants and one deletion mutant using replication-competent HIV-1 constructs carrying wild-type or mutant LTRs with modifications in the NF-kappa B and/or Sp1 binding sites. In this study, we show that Tat- HIV-1 with wild-type LTRs can replicate in HeLa cells, and the virus produced from HeLa cells can infect primary peripheral blood lymphocytes and macrophages. It was found that the propagation of the Tat mutants containing wild-type LTRs was less efficient than that of the LTR-modified Tat mutants. Large amounts of viral RNA and particles were synthesized in infections established using the tat mutants that contain modified LTRs. However, this efficient propagation of the LTR-modified tat mutants was restricted to some lymphoid cell lines that have been transformed with other viruses. Thus, despite its essential role for releasing an elongation block, Tat is not otherwise absolutely required for synthesis of full-length HIV transcripts and assembly of virus particles. Direct sequencing of the viral genomes and reinfection kinetics showed no evidence of wild-type reversion even after prolonged infection with the Tat- virus. The implications for in vivo HIV-1 replication and potential application of this system to the study of alternative Tat function are discussed. 


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The human prointerleukin 1 beta gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction. 
In these studies, we have identified DNA sequences and specific protein interactions necessary for transcriptional regulation of the human prointerleukin 1 beta (proIL-1 beta) gene. A cell-type-independent lipopolysaccharide (LPS)-responsive enhancer element located between -3757 and -2729 bp upstream from the transcription start site (cap site) consisted of at least six discrete subregions which were essential to the maximal induction by LPS in transfected monocytes. The enhancer also appeared to mediate phorbol myristate acetate induction in monocytes and IL-1 responsiveness in fibroblasts. Deletion and base substitution mutations along with DNA binding studies demonstrated that the enhancer contained a minimum of three functional protein binding sequences, two of which appeared to be important for gene induction. One of the essential proteins which bound to the enhancer was similar or identical to members of the C/EBP family of transcription factors required for both IL-1- and LPS-specific induction of the IL-6 gene (i.e., the NF-IL6 proteins). When ligated to the proIL-1 beta cap site-proximal region (located between -131 to +12), both the proIL-1 beta and the simian virus 40 enhancer elements functioned more efficiently in monocytes than in HeLa cells, which are not normally competent for IL-1 beta expression. When ligated to the murine c-fos promoter, however, the proIL-1 beta enhancer was inducible in phorbol myristate acetate-stimulated HeLa cells, suggesting the existence of a proIL-1 beta promoter-proximal requirement for tissue specificity. 


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Interaction of HTLV-I Tax with the human proteasome: implications for NF-kappa B induction. 
The human T-cell leukemia virus type I (HTLV-I) has been etiologically associated with the development of the adult T-cell leukemia (ATL) as well as degenerative neurologic syndrome termed tropical spastic paraparesis (TSP). HTLV-I encodes a potent transactivator protein termed Tax that appears to play an important role in the process of T-cell immortalization. Even though the mechanisms by which Tax induces transformation are still unknown, it seems likely that the ability of Tax to alter the expression of many cellular genes plays an important part in this process. Tax does not bind directly to DNA but rather deregulates the activity of cellular transcription factors. One family of host transcription factors whose activity is altered by Tax includes NF-kappa B/Rel. These transcription factors are post-transcriptionally regulated by their assembly with a second family of inhibitory proteins termed I kappa B that serve to sequester the NF-kappa B/Rel complexes in the cytoplasm. Upon cellular activation, I kappa B alpha is phosphorylated, polyubiquitinated, and degraded in the proteasome. This proteolytic event liberates NF-kappa B, permitting its rapid translocation into the nucleus where it binds to its cognate enhancer elements. Similarly, the p105 precursor of the NF-kappa B p50 subunit is also post-translationally processed in the proteasome. The mechanisms by which Tax activates NF-kappa B remain unclear, and findings presented in the literature are often controversial. We identified a physical interaction between Tax and the HsN3 subunit of the human proteasome. This raises the intriguing possibility that physical association of the HsN3 proteasome subunit with HTLV-I Tax coupled with the independent interaction of Tax with either p100 or p65-I kappa B alpha targets these cytoplasmic NF-kappa B/Rel complexes to the proteasome for processing. 


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Angiotensin II activates the proinflammatory transcription factor nuclear factor-kappaB in human monocytes. 
The renin-angiotensin system may contribute to the pathogenesis of atherosclerosis. A common feature of all stages of atherosclerosis is inflammation of the vessel wall. The transcription factor nuclear factor-kappaB (NF-kappaB) participates in most signaling pathways involved in inflammation. This study therefore examined the effect of angiotensin (ANG) II on NF-kappaB activation in monocytic cells, a major cellular component of human atheroma, by electrophoretic mobility shift assay. ANG II, like TNFalpha, caused rapid activation of NF-kappaB in human mononuclear cells isolated from peripheral blood by Ficoll density gradient. This ANG II effect was blocked by the angiotensin AT1 receptor antagonist losartan. Specificity of ANG II-induced NF-kappaB activation was ascertained by supershift and competition experiments. Moreover, ANG II stimulated NF-kappaB activation in human monocytes, but not in lymphocytes from the same preparation. Together, the data demonstrate the ability of the vasoactive peptide ANG II to activate inflammatory pathways in human monocytes. Copyright 1999 Academic Press. 


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X-rays-induced secretion of cellular factor(s) that enhance(s) HIV-1 promoter transcription in various non-irradiated transfected cell lines. 
Various cellular stress agents like ionizing radiation exposure could activate human immunodeficiency virus type 1 (HIV- 1) replication or reporter gene expression. In addition, extracellular factor(s) released by X-ray-treated human colonic carcinoma cell line (HT29) might activate the long terminal repeat (LTR) of HIV-1 in non-irradiated HT29 cells. In the present report we show that in various transiently or stably transfected cell lines, X-ray irradiation up-regulates HIV-1 LTR transcription through the kappaB regulatory elements. A factor(s), which is processed by and acts upon a variety of cell types, was detected by addition to non-irradiated cells of either X-ray-treated cells or a conditioned medium taken from irradiated cultures. The magnitude of responsiveness is cell type dependent. In addition, X-ray activation of HIV-1 LTR in transiently or stably transfected cell lines is inhibited by a potent antioxidant drug, pyrrolidine dithiocarbamate and by another drug, known for its role in the trapping of growth factors, suramin. The importance of these observations in the pathophysiology of patients with AIDS-related cancers treated by radiotherapy remains to be established. 


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Novel therapies for inflammatory bowel disease. 
Looking back at successes and failures in newer approaches to treating IBD, it is tempting--although still difficult--to draw conclusions about pathogenesis. When a therapy proves effective, do clinicians truly know how it works? Even with a therapy as specific as anti-TNF antibody, it is not clear if the benefit is attributable to simple binding and clearance of TNF-alpha or to binding on the cell surface and subsequent deletion of the activated macrophage. When a drug appears to be less effective than preclinical models suggest, can failures in effectiveness from delivery or dosing be differentiated? The disappointing results of clinical trials with IL-10--so at odds with the prediction of benefit from animal models--bring into question the validity of those models as well as the soundness of design of the clinical trials on which efficacy of IL-10 is judged. The variability of response even to the most narrowly targeted agents suggests that these diseases are far more heterogeneous in humans than in their murine counterparts. Clinicians are only just beginning to recognize subclinical markers of response, and it may soon be possible to predict response on the basis of genetic composition. For the moment, however, the field of pharmacogenetics is embryonic. Challenges in developing new therapeutic strategies include not only identifying novel agents, but also improving the definitions of clinical endpoints and defining efficacy at the biologic level. Only through considered evaluation of clinical evidence may clinicians determine which therapies should remain novelties and which should become an accepted part of the armamentarium. 


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TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas(Apo-1/CD95). 
A novel member of the tumor necrosis factor (TNF) receptor family, designated TRAMP, has been identified. The structural organization of the 393 amino acid long human TRAMP is most homologous to TNF receptor 1. TRAMP is abundantly expressed on thymocytes and lymphocytes. Its extracellular domain is composed of four cysteine-rich domains, and the cytoplasmic region contains a death domain known to signal apoptosis. Overexpression of TRAMP leads to two major responses, NF-kappaB activation and apoptosis. TRAMP-induced cell death is inhibited by an inhibitor of ICE-like proteases, but not by Bcl-2. In addition, TRAMP does not appear to interact with any of the known apoptosis-inducing ligands of the TNF family. 


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Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils. 
Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis. 


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Thrombin and thrombin receptor agonist peptide induce early events of T cell activation and synergize with TCR cross-linking for CD69 expression and interleukin 2 production. 
Thrombin stimulation of the T leukemic cell line Jurkat induced a transient increase in [Ca2+]i. Proteolytic activity of the enzyme was required for this effect since diisopropyl fluorophosphate-thrombin failed to increase [Ca2+]i. Furthermore, hirudin and anti-thrombin III inhibited the thrombin-induced [Ca2+]i rise in Jurkat T cells. A synthetic thrombin receptor agonist peptide (TRP) of 7 residues (SFLLRNP) was found to be as effective as thrombin for [Ca2+]i mobilization, and both agonists induced Ca2+ release exclusively from internal stores. Thrombin stimulated tyrosine phosphorylation of several proteins of molecular mass 40, 42, 70, 120, and 130 kDa. There was a good correlation between thrombin-induced tyrosine phosphorylation of the latter three proteins and Ca2+ mobilization. Thrombin and TRP also caused translocation of protein kinase C from the cytosol to the plasma membrane. As a likely consequence of these events, thrombin activated the nuclear factor NF-kB. Several cell lines of hematopoietic origin including the leukemic T cell line HPB.ALL and the erythroleukemic cell line K562 were responsive to thrombin, whereas others such as THP1, a myelomonocytic cell line, and BL2, a Burkitt lymphoma were refractory to thrombin or TRP stimulation. The magnitude of the thrombin response in the different cell types paralleled the expression of the thrombin receptor mRNA. We found that activation of Jurkat T cells by a combination of phytohemagglutinin and phorbol 12-myristate 13-acetate led to a dramatic inhibition of thrombin receptor mRNA expression and to a concomitant loss of the thrombin response. Finally, we demonstrate that thrombin and TRP enhanced CD69 expression and interleukin 2 production induced by T cell receptor cross-linking in both Jurkat T cells and peripheral blood lymphocytes. These findings highlight the role of thrombin as a potential regulator of T lymphocyte activation. 


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The Oct-2 transcription factor. 
The Oct-2 transcription factor is a member of the POU (Pit-Oct-Unc) family of transcription factors and is expressed only in B lymphocytes and in neuronal cells but not in other cell types. The primary RNA transcript of the gene is subject to alternative splicing to yield different variants which can either activate or repress gene expression. The forms produced in B lymphocytes have a predominantly activating effect on gene expression whereas those produced in neuronal cells have a predominantly inhibitory effect and can repress the expression of both the herpes simplex virus immediate-early genes and the cellular tyrosine hydroxylase gene. Thus Oct-2 plays an important role in the regulation of cellular gene expression in both B cells and neuronal cells as well as in the control of viral latency. 


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Interactions between the class II transactivator and CREB binding protein increase transcription of major histocompatibility complex class II genes. 
Class II major histocompatibility (class II) genes are regulated in a B-cell-specific and gamma interferon-inducible fashion. The master switch for the expression of these genes is the class II transactivator (CIITA). In this report, we demonstrate that one of the functions of CIITA is to recruit the CREB binding protein (CBP) to class II promoters. Not only functional but also specific binding interactions between CIITA and CBP were demonstrated. Moreover, a dominant negative form of CBP decreased the activity of class II promoters and levels of class II determinants on the surface of cells. Finally, the inhibition of class II gene expression by the glucocorticoid hormone could be attributed to the squelching of CBP by the glucocorticoid receptor. We conclude that CBP, a histone acetyltransferase, plays an important role in the transcription of class II genes. 


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The myeloid zinc finger gene, MZF-1, regulates the CD34 promoter in vitro. 
MZF-1 is a C2H2 zinc finger gene encoding a putative transcriptional regulator of myeloid differentiation. The MZF-1 protein contains 13 C2H2 zinc fingers arranged in bipartite DNA binding domains containing zinc fingers through 4 and, in the carboxy-terminus, 5 through 13. We previously identified the DNA consensus binding site recognized by the two DNA binding domains. To assess the transcription regulatory function of MZF-1, the full-length MZF-1 coding region was fused to the DNA binding domain of the yeast transactivator GAL4. The expression vector was cotransfected with the chloramphenicol acetyl transferase (CAT) reporter gene regulated by the thymidine kinase promoter containing GAL4 DNA binding sites into NIH 3T3, 293, K562, and Jurkat cell lines. MZF-1 represses CAT reporter gene expression via GAL4 binding sites in the nonhematopoietic cell lines NIH 3T3 and 293. In contrast, MZF-1 activates CAT reporter gene expression in the hematopoietic cell lines K562 and Jurkat. The MZF-1 binding sites are present in the promoters of several genes expressed during myeloid differentiation, including the CD34 promoter. MZF-1 transcriptional regulation of this physiologically relevant promoter was assessed in both hematopoietic and nonhematopoietic cell lines. Recombinant MZF-1 protein specifically binds to the consensus binding sites in the CD34 promoter in mobility shift assays. MZF-1 expression vectors were cotransfected with the luciferase reporter plasmids regulated by the CD34 promoter into both nonhematopoietic and hematopoietic cell lines. As with the heterologous DNA binding domain, MZF-1 represses reporter gene expression in nonhematopoietic cell lines and activates expression in hematopoietic cell lines. Activation of CD34 expression in hematopoietic cell lines is dependent on the presence of intact MZF-1 binding sites. The cell type-specific regulation of the CD34 promoter by MZF-1 suggests the presence of tissue-specific regulators/adapters or differential MZF-1 modifications that determine MZF-1 transcriptional regulatory function. 


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A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p [see comments] 
Trapoxin is a microbially derived cyclotetrapeptide that inhibits histone deacetylation in vivo and causes mammalian cells to arrest in the cell cycle. A trapoxin affinity matrix was used to isolate two nuclear proteins that copurified with histone deacetylase activity. Both proteins were identified by peptide microsequencing, and a complementary DNA encoding the histone deacetylase catalytic subunit (HD1) was cloned from a human Jurkat T cell library. As the predicted protein is very similar to the yeast transcriptional regulator Rpd3p, these results support a role for histone deacetylase as a key regulator of eukaryotic transcription. 


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BCL-6 expression during B-cell activation. 
Translocations involving the BCL-6 gene are common in the diffuse large cell subtype of non-Hodgkin's lymphoma. Invariably, the BCL-6 coding region is intact, but its 5' untranslated region is replaced with sequences from the translocation partner. The present study shows that BCL-6 expression is regulated in lymphocytes during mitogenic stimulation. Resting B and T lymphocytes contain high levels of BCL-6 mRNA. Stimulation of mouse B cells with anti-IgM or IgD antibodies, bacterial lipopolysaccharide, phorbol 12-myristate 13-acetate plus ionomycin, or CD40 ligand led to a five-fold to 35-fold decrease in BCL-6 mRNA levels. Similar downregulation of BCL-6 mRNA was seen in human B cells stimulated with Staphylococcus aureus plus interleukin-2 or anti-IgM antibodies and in human T lymphocytes stimulated with phytohemagglutinin. BCL-6 mRNA levels began to decrease 8 to 16 hours after stimulation, before cells entered S phase. Although polyclonal activation of B cells in vitro invariably decreased BCL-6 MRNA expression, activated B cells from human germinal centers expressed BCL-6 mRNA at levels comparable to the levels in resting B cells. Despite these similar mRNA levels, BCL-6 protein expression was threefold to 34-fold higher in germinal center B cells than in resting B cells, suggesting that BCL-6 protein levels are controlled by translational or posttranslational mechanisms. These observations suggest that the germinal center reaction provides unique activation signals to B cells that allow for continued, high-level BCL-6 expression. 


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Involvement of an SAF-like transcription factor in the activation of serum amyloid A gene in monocyte/macrophage cells by lipopolysaccharide. 
Serum amyloid A (SAA) has been linked to atherosclerosis because of its ability to remodel high-density lipoprotein by the depletion of apolipoprotein A1, its ability to bind cholesterol, and its presence in the atherosclerotic plaques of coronary and carotid arteries. In the present study, we investigated the induction mechanism of SAA gene in THP-1 monocyte/macrophage cells which play a critical role in the development of atherosclerotic fatty streak and plaque formation. We and others have shown that SAA gene is induced in monocyte/macrophage cells by lipopolysaccharide (LPS). By promoter function analysis, we show that the SAA promoter sequence between -280 and -226 can confer LPS responsiveness. Gel electrophoretic mobility shift assay detected an induced DNA-binding activity in these cells in response to LPS. Characterization of the DNA-binding protein by UV cross-linking, Southwestern blot, and antibody ablation/supershift assays revealed that it is similar to a recently reported nuclear factor designated SAF. These results demonstrated that LPS-mediated SAA gene induction in monocyte/macrophage cells is primarily due to the induction of SAF activity. 


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Human T-cell leukemia virus type I Tax activation of NF-kappa B/Rel involves phosphorylation and degradation of I kappa B alpha and RelA (p65)-mediated induction of the c-rel gene. 
The tax gene product of human T-cell leukemia virus type I (HTLV-I) is a potent transcriptional activator that both stimulates viral gene expression and activates an array of cellular genes involved in T-cell growth. Tax acts indirectly by inducing or modifying the action of various host transcription factors, including members of the NF-kappa B/Rel family of enhancer-binding proteins. In resting T cells, many of these NF-kappa B/Rel factors are sequestered in the cytoplasm by various ankyrin-rich inhibitory proteins, including I kappa B alpha. HTLV-I Tax expression leads to the constitutive nuclear expression of biologically active NF-kappa B and c-Rel complexes; however, the biochemical mechanism(s) underlying this response remains poorly understood. In this study, we demonstrate that Tax-stimulated nuclear expression of NF-kappa B in both HTLV-I-infected and Tax-transfected human T cells is associated with the phosphorylation and rapid proteolytic degradation of I kappa B alpha. In contrast to prior in vitro studies, at least a fraction of the phosphorylated form of I kappa B alpha remains physically associated with the NF-kappa B complex in vivo but is subject to rapid degradation, thereby promoting the nuclear translocation of the active NF-kappa B complex. We further demonstrate that Tax induction of nuclear c-Rel expression is activated by the RelA (p65) subunit of NF-kappa B, which activates transcription of the c-rel gene through an intrinsic kappa B enhancer element. In normal cells, the subsequent accumulation of nuclear c-Rel acts to inhibit its own continued production, indicating the presence of an autoregulatory loop. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Nitric oxide-stimulated guanine nucleotide exchange on p21ras. 
The protooncogene p21ras, a monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical events. Here, we report that nitric oxide (NO) activates p21ras in human T cells as evidenced by an increase in GTP-bound p21ras. In vitro studies using pure recombinant p21ras demonstrate that the activation is direct and reversible. Circular dichroism analysis reveals that NO induces a profound conformational change in p21ras in association with GDP/GTP exchange. The mechanism of activation is due to S-nitrosylation of a critical cysteine residue which stimulates guanine nucleotide exchange. Furthermore, we demonstrate that p21ras is essential for NO-induced downstream signaling, such as NF-kappa B activation, and that endogenous NO can activate p21ras in the same cell. These studies identify p21ras as a target of the same cell. These studies identify p21ras as a target of NO in T cells and suggest that NO activates p21ras by an action which mimics that of guanine nucleotide exchange factors. 


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The spatial distribution of human immunoglobulin genes within the nucleus: evidence for gene topography independent of cell type and transcriptional activity. 
The three-dimensional positioning of immunoglobulin (Ig) genes within the nucleus of human cells was investigated using in situ hybridization and confocal microscopy. The visualization of heavy and light chain genes in B-lymphoid cells showed that the three Ig genes are differentially and nonrandomly distributed in different nuclear subvolumes: the kappa genes were found to be preferentially confined to an outer nuclear volume, whereas the gamma and lambda genes consistently occupied more central positions within the nucleus, the lambda genes being more interior when compared with the gamma genes. The data further show that these overall topographical distributions are independent of gene transcriptional activity and are conserved in different cell types. Although subtle gene movements within those defined topographical regions cannot be excluded by this study, the results indicate that tissue specificity of gene expression is not accompanied by drastic changes in gene nuclear topography, rather suggesting that gene organization within the nucleus may be primarily dependent on structural constraints imposed on the respective chromosomes. 


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V3 loop of human immunodeficiency virus type 1 suppresses interleukin 2-induced T cell growth [published erratum appears in AIDS Res Hum Retroviruses 1997 May 1;13(7):633] 
We tested the effect of three linear or two loop peptides derived from the V3 region of the HTLV-III BH10 clone or the SF2 strain of human immunodeficiency virus type 1 on IL-2-driven T cell proliferation. V3-BH10, which consists of 42 amino acids and has a loop structure, suppressed IL-2-driven proliferation of all IL-2-dependent cells [Kit225, ED-40515(+), KT-3, 7-day PHA-blasts, and fresh peripheral blood mononuclear cells] tested, whereas it did not suppress the cell growth of IL-2-independent cell lines (Hut102, Molt-4, and Jurkat). This suppressive effect was also seen in IL-2-driven cell growth of CD8-positive lymphocytes purified from 7-day PHA-blasts, indicating that CD4 molecules were not required for the suppression. The treatment with anti-V3 loop monoclonal antibody (902 antibody) completely abolished the suppressive effect of V3-BH10. In addition, V3-BH10 generated the arrest of Kit225 cells and also purified CD8-positive lymphocytes in G1 phase in the presence of IL-2. Neither chromatin condensation nor DNA fragmentation was detected in Kit225 cells cultured with V3-BH10 and IL-2. V3-BH10 neither blocked radiolabeled IL-2 binding to IL-2 receptors nor affected tyrosyl phosphorylation of several cellular proteins (p120, p98, p96, p54, and p38), which is immediately induced by IL-2 stimulation. However, V3-BH10 enhanced IL-2-induced mRNA expression of c-fos but not c-myc or junB. Thus, the binding of V3 loop of gp120 to the cell surface molecule(s) appears to affect intracellular IL-2 signaling, which leads to the suppression of IL-2-induced T cell growth. 


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T cell-specific negative regulation of transcription of the human cytokine IL-4. 
IL-4 secreted by activated T cells is a pleiotropic cytokine affecting growth and differentiation of diverse cell types such as T cells, B cells, and mast cells. We investigated the upstream regulatory elements of the human IL-4 promoter. A novel T cell-specific negative regulatory element (NRE) composed of two protein-binding sites were mapped in the 5' flanking region of the IL-4 gene: -311CTCCCTTCT-303 (NRE-I) and -288CTTTTTGCTT-TGC-300 (NRE-II). A T cell-specific protein Neg-1 and a ubiquitous protein Neg-2 binding to NRE-I and NRE-II, respectively, were identified. Furthermore, a positive regulatory element was found 45 bp downstream of the NRE. The enhancer activity of the PRE was completely suppressed when the NRE was present. These data suggest that IL-4 promoter activity is normally down-regulated by an NRE via repression of the enhancer positive regulatory element. These data may have implications for the stringent control of IL-4 expression in T cells. 


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A CD28-associated signaling pathway leading to cytokine gene transcription and T cell proliferation without TCR engagement. 
Stimulation of resting human T cells with the CD28-specific mAb BW 828 induces proliferation and cytokine synthesis without further requirement for TCR coengagement. This observation prompted us to postulate that signal 2 (costimulatory signal) alone without signal 1 (TCR signal) can activate T cells. To test whether this putative function of CD28 is mediated via a particular signaling pathway, we compared early signaling events initiated in resting T cells by the stimulatory mAb BW 828 with signals triggered by the nonstimulating CD28 mAb 9.3. Stimulation of T cells with BW 828 induced an increase in intracellular Ca2+, but did not lead to detectable activation of the protein kinases p56(lck) and c-Raf-1. This pathway resulted in the induction of the transcription factors NF-kappa B, NF-AT, and proteins binding to the CD28 response element of the IL-2 promoter. On the other hand, stimulation of T cells with mAb 9.3 increased the level of intracellular Ca2+ and triggered the activation of p56(lck) and c-Raf-1, but was unable to induce the binding of transcription factors to the IL-2 promoter. In contrast to the differential signaling of BW 828 and 9.3 in resting T cells, the two mAbs exhibited a similar pattern of early signaling events in activated T cells and Jurkat cells (p56(lck) activation, association of phosphatidylinositol 3-kinase with CD28), indicating that the signaling capacity of CD28 changes with activation. These data support the view that stimulation through CD28 can induce some effector functions in T cells and suggest that this capacity is associated with a particular pattern of early signaling events. 


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Thapsigargin induces IL-2 receptor alpha-chain in human peripheral and Jurkat T cells via a protein kinase C-independent mechanism. 
Thapsigargin (TG), an inhibitor of Ca(2+)-ATPase, depletes intracellular Ca2+ stores and induces a sustained Ca2+ influx without altering phosphatidyl inositol levels. TG plus phorbol myristate acetate (PMA) but not TG alone induced IL-2 in Jurkat T cells, suggesting that TG had no effect on protein kinase C (PKC). However, TG induced increases in IL-2R alpha protein as well as IL-2R alpha mRNA in Jurkat T cells in a dose-dependent manner. A similar increase in IL-2R alpha by TG was also observed in human peripheral T cells. Further, like PMA, TG markedly induced NF kappa B in Jurkat T cells. However, TG and PMA exhibited a synergistic action on IL-2R alpha expression, suggesting that TG and PMA induce IL-2R alpha through distinct pathways. PMA- but not TG-induced IL-2R alpha is inhibited by the PKC inhibitor H7, whereas TG- but not PMA-induced IL-2R alpha was inhibited by cholera toxin, forskolin and 1,9-dideoxy forskolin. In toto, these results suggest that TG induces IL-2R alpha in human T cells through a PKC-independent pathway. 


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Functions of glutathione and glutathione disulfide in immunology and immunopathology. 
Even a moderate increase in the cellular cysteine supply elevates the intracellular glutathione (GSH) and glutathione disulfide (GSSG) levels and potentiates immunological functions of lymphocytes in vitro. At low GSSG levels, T cells cannot optimally activate the immunologically important transcription factor NF kappa B, whereas high GSSG levels inhibit the DNA binding activity of NF kappa B. The effects of GSSG are antagonized by reduced thioredoxin (TRX). As the protein tyrosine kinase activities p56lck and p59fyn are activated in intact cells by hydrogen peroxide, they are likely targets for GSSG action. These redox-regulated enzymes trigger signal cascades for NF kappa B activation and transduce signals from the T cell antigen receptor, from CD4 and CD8 molecules, and from the IL-2 receptor beta-chain. The effector phase of cytotoxic T cell responses and IL-2-dependent functions are inhibited even by a partial depletion of the intracellular GSH pool. As signal transduction is facilitated by prooxidant conditions, we propose that the well-known immunological consequences of GSH depletion ultimately may be results of the accompanying GSSG deficiency. As HIV-infected patients and SIV-infected rhesus macaques have, on the average, significantly decreased plasma cyst(e)ine and intracellular GSH levels, we also hypothesize that AIDS may be the consequence of a GSSG deficiency as well. 


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Interleukin 6-induced differentiation of a human B cell line into IgM-secreting plasma cells is mediated by c-fos. 
The role of the protooncogene c-fos in interleukin (IL) 6-induced B cell differentiation was assessed. Treatment of SKW 6.4 cells with IL 6 induced a transient and early stimulation of c-fos sense mRNA expression. The effect appeared within 30 min and returned to basal levels after 2 h. The addition of antisense oligonucleotides to c-fos significantly inhibited IL 6-induced IgM production by SKW 6.4 cells (p less than 0.001), whereas control oligonucleotides had no inhibitory effect. These results indicate that activation of c-fos is involved in IL 6-induced differentiation of SKW 6.4 cells into IgM-secreting cells. 


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Dopamine stimulates expression of the human immunodeficiency virus type 1 via NF-kappaB in cells of the immune system. 
Recent studies have reported that lymphocytes produce, transport and bind dopamine present in plasma. However, the action of dopamine on HIV-1 gene expression in cells of the immune system has not yet been examined. Here, we have investigated the regulation of HIV-1 expression by dopamine in Jurkat T cells and in primary blood mononuclear cells (PBMC). HIV-1 replication was increased by dopamine, which correlated with the increased levels of HIV-1 transactivation. Our transient expression data revealed that dopamine stimulated transcription through the NF-kappaB element present in the long terminal repeat. The importance of NF-kappaB sites was confirmed by using vectors containing wild-type or mutant kappaB sites in a heterologous promoter. Consistent with the role of NF-kappaB in mediating dopamine responsiveness, the proteasome inhibitor MG132 abolished dopamine-induced transcriptional activation. We further explored the effect of dopamine in the presence of phorbol esters or tumor necrosis factor-alpha (TNF-alpha) known to activate NF-kappaB. The combination of dopamine and TNF-alpha led to a stimulation of HIV-1 transcription and replication. However, in contrast with TNF-alpha, dopamine treatment did not affect NF-kappaB DNA binding activity nor the concentrations of p50, p65 and IkappaB-alpha proteins, which suggests a distinct NF-kappaB activation mechanism. These results reveal a new link between the dopamine system, cytokine signaling pathway and regulation of gene expression via the involvement of NF-kappaB in T cells and PBMC. 


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Association of alterations in NF-kappa B moieties with HIV type 1 proviral latency in certain monocytic cells. 
Human immunodeficiency virus type 1 (HIV-1) replication is controlled by a complex array of virally encoded and cellular proteins. A wide spectrum of levels of HIV-1 expression have been demonstrated in various cells, both in cell culture and in vivo. Molecular mechanisms leading to restricted HIV-1 replication may differ between certain cell types. It is now demonstrated that HIV-1 proviral latency in the monocytic cell line U1, in which only extremely low levels of HIV-1 expression are detected in the baseline unstimulated state, is associated with alterations in nuclear factor-kappa B (NF-kappa B) moieties demonstrated in these cells by electrophoretic mobility shift assays (EMSAs) and in situ UV cross-linking studies. A predominance of p50 NF-kappa B moieties and possibly p50 homodimers or closely related species, rather than the p50-p56 heterodimer of NF-kappa B that is the predominant NF-kappa B species in most T lymphocytic and monocytic cells, is demonstrated in the nuclei of U1 cells. This pattern of NF-kappa B-related moieties differs from the latently infected T lymphocytic cell line ACH-2, and from the U937 monocytic line, the parental cell line of the U1 cellular clone. As such, these data suggest that different proximal mechanisms may lead to restricted HIV-1 replication in various cell types. 


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Differential induction of DNA-binding activities following CD19 cross-linking in human B lineage cells. 
The B cell-specific cell surface molecule CD19 is expressed at all stages of B cell development, including normal plasma cells, and mediates signal transduction via interaction with cytoplasmic effector proteins. Cross-linking CD19 on early human B lineage cells induces the formation of a CD19/Vav/phosphatidylinositol-3 kinase complex, tyrosine phosphorylation of CD19 and Vav, and activation of the Ras pathway. To further explore the ramifications of CD19 signaling, the current study examined whether phosphorylation of Elk-1, activation of activator protein-1 (AP-1), or activation of nuclear factor-kappaB (NF-kappaB) transcription factors occurred following CD19 cross-linking. The cells used were the BLIN-1 pre-B cell line expressing low levels of cell surface mu heavy chain associated with surrogate light chain and the 1E8 immature B cell line expressing cell surface mu/kappa. Lysates from CD19 cross-linked 1E8 cells induced robust phosphorylation of an Elk-1 fusion protein in vitro, whereas no phosphorylation of Elk-1 fusion protein occurred using lysates from CD19 cross-linked BLIN-1 cells. An electrophoretic mobility shift assay employing AP-1 and NF-kappaB consensus oligonucleotides was used to demonstrate that AP-1 -binding activity increased, while constitutive NF-kappaB-binding activity was not enhanced, following 2 h of CD19 cross-linking in 1E8 cells. Supershift experiments revealed that JunD and c-Fos proteins mediated anti-CD19 induced AP-1-binding activity in 1E8 cells. In contrast, CD19 cross-linking in BLIN-1 cells resulted in the induction of NF-kappaB, but had no apparent effect on AP-1-binding activity. These data suggest that CD19-mediated signal transduction activates different transcription factors at juxtaposed stages of B cell development that may culminate in the activation or suppression of distinct sets of genes. 


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[Regulation of transcription of the interleukin-2 gene in B-lymphocytes] 
Since most B cell clones immortalized with EBV virus can be induced to produce interleukin-2, a typical T cell cytokine, we studied the role of different elements of the IL-2 promoter in such clones by transfection. It was found, in particular, that the element TCEd, which binds the transcription factor NF-kB, is very active in all three B clones tested. This element has no activity in T cells of the Jurkat line. The NFATd element, which binds the transcription factor NFAT-1 and is very active in T cells, is only weakly active in one B clone and not at all in another. Different elements thus contribute to IL-2 promoter activity in different cells. 


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Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the beta-globin locus control region in vivo. 
The locus control region of the beta-globin cluster contains five DNase I hypersensitive sites (5'HS1-5) required for locus activation. 5'HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5'HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5'HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5'HS3. 


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Stimulation of the human immunodeficiency virus type 2 (HIV-2) gene expression by the cytomegalovirus and HIV-2 transactivator gene. 
Human immunodeficiency virus (HIV) often causes latent infection. Transactivation by some DNA viruses has been implicated in inducing HIV-1 replication and pathogenesis. The transactivator (IE-2) gene of the human cytomegalovirus (CMV) can enhance HIV-2 as well as HIV-1 gene expression in vitro. This inducer can act in concert with the HIV-2 tat gene and T-cell activation in enhancing gene expression in human CD4+ lymphocytes. While the HIV-2 and HIV-1 tat genes and T-cell activators apparently employ independent modes of action, the CMV transactivator in combination with the HIV-2 tat or T-cell activators may employ a gene activation pathway with some common and some distinct components. Both HIV-2 and CMV transactivators enhance HIV-2 gene expression by transcriptional activation involving transcript initiation as well as elongation, with CMV transactivator affecting elongation more than the initiation. A significant proportion of transcripts appear to terminate prematurely in the absence of transactivators. Deletion mutation analysis of the HIV-2 long terminal repeat (LTR) suggests that the element that responds to CMV transactivation in human CD4+ lymphocytes is either a diffuse one or located downstream of the HIV-2 enhancer element. 


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Tissue-specific expression of the platelet GPIIb gene. 
One of the major objectives in the study of thrombogenesis is to determine the mechanisms by which a hematopoietic progenitor is activated and committed to the megakaryocytic lineage. Recent development of primary cultures of human megakaryocytes and the molecular cloning of genes that are specific to this lineage offer the possibility of getting some insights into the genetic mechanisms that control megakaryocytopoiesis. One gene of interest is the glycoprotein IIb (GPIIb) gene; GPIIb, the alpha subunit of the platelet cytoadhesin GPIIb-IIIa, is produced in megakaryocytes at an early stage of the differentiation, whereas the other subunit of this complex, GPIIIa, is expressed in other cells. For these reasons, the 5'-flanking region of the GPIIb gene was used to identify the regions that interact with DNA-binding nuclear factors. A fragment extending from -643 to +33 is capable of controlling the tissue-specific expression of the CAT gene in transfection experiments. Within this region, we have identified several sequences that are implicated in DNA protein interactions as shown in DNAse I footprints and gel mobility shift assays. One region, centered at -54, is similar to a nuclear factor E1-binding site, and a region located at position -233 contains a CCAAT motif. Two domains centered at positions -345 and -540, respectively, bind proteins that are present in megakaryocytic cells and nonrelated cells as well. Finally, two other domains, located at positions -460 and -510, interact with proteins that are only present in megakaryocytic cells. In addition, deletion of the region containing these two domains results in a significant decrease of the promoter activity. It is very likely that these domains bind megakaryocyte-specific nuclear proteins acting as positive transcription factors. 


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HIV-1 Nef protein inhibits the recruitment of AP-1 DNA-binding activity in human T-cells. 
The human immunodeficiency virus type 1 long terminal repeat, HIV-1-LTR, contains binding sites for several cellular transcription factors which contribute to HIV-1 gene expression. Our previous studies on the function of the HIV-1-encoded Nef protein suggested that Nef may be an inhibitor HIV-1 transcription. To determine whether Nef affects the binding of cellular factors implicated in HIV-1 regulation, 32P-labeled oligonucleotides corresponding to the binding sites were incubated with nuclear extracts prepared from Nef-expressing T-cell lines that were not stimulated or were stimulated with T-cell mitogens. We found that Nef inhibited the recruitment of AP-1 DNA-binding activity in mitogen-stimulated human T-cells. Additionally, Nef expressing cells were transiently transfected with a plasmid in which HIV-1 AP-1 DNA recognition sequences were cloned downstream of the chloramphenicol acetyltransferase (CAT) gene. Mitogen-mediated transcriptional activation of the CAT gene in this construct was inhibited in Nef-expressing cells but not in control cells. These studies suggest that, by inhibiting AP-1 activation, Nef may play a role in regulating HIV-1 gene expression in infected T-cells. 


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Cyclosporin A inhibits early mRNA expression of G0/G1 switch gene 2 (G0S2) in cultured human blood mononuclear cells. 
Cyclosporin A (CsA) may achieve its immunosuppressive effects by inhibiting the calcium- and calmodulin-dependent phosphatase calcineurin which is required for activation of target genes by members of the NFAT (nuclear factor of activated T cells) transcription factor family. Among these target genes is the gene encoding interleukin-2 (IL2), a cytokine facilitating progression through the G1 phase of the cell cycle. However, IL2 does not reverse CsA inhibition, suggesting that at least one other NFAT-sensitive gene may be involved. The human G0/G1 switch gene, G0S2, has potential NFAT-binding sites in the 5' flank and encodes a small basic potential phosphoprotein of unknown function. Using a sensitive, reverse transcription-polymerase chain reaction (RT-PCR) assay, G0S2 mRNA levels were assayed in cultured blood mononuclear cells. Freshly isolated cells contain high levels of G0S2 mRNA which rapidly decline. This "spontaneous stimulation" is also noted with some other G0S genes and has been attributed to some aspect of the isolation procedure. In cells that have been preincubated to lower mRNA levels, there is a transient increase in G0S2 mRNA, peaking between 1-2 h, in response to Concanavalin-A (ConA), or to the combination of phorbol ester (TPA), and the calcium ionophore, ionomycin. Both these responses are inhibited by CsA. Our results suggest that G0S2 expression is required to commit cells to enter the G1 phase of the cell cycle, and that, while not excluding other possible targets, early inhibition of G0S2 expression by CsA may be important in achieving immunosuppression. G0S2 may be of value as a reporter gene for analyzing the mechanism of action of CsA and its influence on the positive and negative selection of lymphocytes in response to self and not-self antigens. 


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ETS1, NFkappaB and AP1 synergistically transactivate the human GM-CSF promoter. 
Activation of helper T cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony stimulating factor (GM-CSF) is one such cytokine, whose increased expression results mostly from increases in transcription. Cis-acting elements with NFkappaB, AP1 and ETS-like binding motifs have been identified in the promoter region of the GM-CSF gene, and are important or essential for transcriptional activity following T cell activation. ETS1 is a transcription factor of the ETS family that is expressed in T cells. We have previously shown that ETS1 can transactivate GM-CSF in Jurkat T cells, but only after the cells have been stimulated by treatment with PMA and ionomycin, agents that mimic T cell activation. Thus we proposed that ETS1, which is expressed constitutively in Jurkat cells, may act in concert with PMA/ionomycin inducible factors. Here we show that ETS1 can transactivate a GM-CSF reporter construct in unstimulated Jurkat cells, providing that either NFkappaB or AP1 transcription factors are supplied by co-transfection. We confirm that binding of endogenous NFkappaB and AP1 is induced following PMA/ionomycin treatment of T cells. Transactivation by ETS1, NFkappaB and AP1 is synergistic, and mutation of the individual binding sites reveals that the transcriptional activities of these factors are interdependent. Our results suggest that constitutive ETS1, and inducible NFkappaB and AP1, cooperate as part of a higher order transcriptional complex in activated T cells. 


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Synergism between two distinct elements of the HTLV-I enhancer during activation by the trans-activator of HTLV-I. 
We have conducted functional studies of the enhancer elements of human T-cell leukemia virus type I (HTLV-I) using the human T-cell lines Jurkat and MOLT 4, which are negative for HTLV-I, and MT-2 and TL-Mor, which carry the proviral genome of HTLV-I. Two distinct elements have been implicated in function of the HTLV-I enhancer. One is the 21-base-pair (bp) core element that is responsible for trans-activation by the HTLV-I trans-activator p40tax and that has the ability to bind to cyclic-AMP responsive element binding factor (CREB)-like factor(s). The other is a region interposed between the 21-bp elements. In this study we demonstrate that a subfragment (C26) in the region between the 21-bp elements is involved in trans-activation by p40tax, possibly through binding to an NF-kappa B-like nuclear factor or factors. Formation of the protein-DNA complex with the C26 subfragment was positively affected by p40tax. The C26 element conferred partial responsiveness to p40tax when linked to one copy of the 21-bp element that, by itself, showed little activation in response to p40tax. However, the C26 element alone, even when repeated, could not be activated by p40tax, unlike other NF-kappa B-binding elements. In contrast, the C26 element itself was profoundly activated upon stimulation with 12-O-tetradecanoylphorbol-13-acetate. These findings therefore suggest that the HTLV-I enhancer contains multiple functional elements, including binding sites for at least CREB- and NF-kappa B-like factors, which synergistically cooperate in activation of the HTLV-I enhancer in response to p40tax. Our results also demonstrate that TPA-dependent activation of the HTLV-I enhancer may be mediated through the C26 element. 


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Regulation of sialoadhesin expression on rat macrophages. Induction by glucocorticoids and enhancement by IFN-beta, IFN-gamma, IL-4, and lipopolysaccharide. 
Sialoadhesin is a macrophage-restricted member of the Ig superfamily that mediates adhesion with lymphoid and myeloid cells. It is expressed on a subpopulation of macrophages in lymphoid tissues and in chronic inflammation (e.g., during autoimmune diseases). We have studied the regulation of sialoadhesin expression in vitro and show that glucocorticoids (GC) induce sialoadhesin expression on freshly isolated rat macrophages and the rat macrophage cell line R2. The cytokines IFN-beta, IFN-gamma, IL-4, and LPS, although unable to induce sialoadhesin expression by themselves, were able to enhance GC-mediated induction of sialoadhesin. Sialoadhesin expression was functional as shown by cell adhesion assays with human RBCs. Northern blotting experiments indicated that regulation predominantly occurred at the mRNA level. Comparison of the different combinations of GC and cytokines/LPS revealed differences in the level of GC-dependent enhancement of sialoadhesin expression, with IFN-beta and IL-4 being more potent than IFN-gamma and LPS. Moreover, the effects of IFN-gamma and LPS could be reproduced by priming, whereas IFN-beta and IL-4 were required simultaneously with GC. The regulation of sialoadhesin expression was mediated by the GC receptor, and not by mineralocorticoid receptor, as shown by inhibition experiments with specific antagonists. Finally, it is demonstrated that macrophages in the adrenal gland, the major site of endogenous GC production, express sialoadhesin. This study demonstrates that GC act as a primary inducer of sialoadhesin expression on rat macrophages, and that the response can be enhanced by IFN-beta, T cell-derived cytokines, or LPS. 


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Transcription factor activation in lymphokine activated killer cells and lymphocytes from patients receiving IL-2 immunotherapy. 
Administration of the cytokine interleukin-2 (IL-2) can result in therapeutic benefits for individuals with renal cell carcinoma and melanoma. Here we report an analysis of the transcription factor families AP-1, Sp1, NF-kappaB, and signal transducers and activators of transcription (STAT) in cancer patients' lymphocytes before and after IL-2 immunotherapy, as assessed by a gel-shift assay. An in vitro surrogate of IL-2 immunotherapy is the incubation of fresh peripheral blood mononuclear cells (PBMC) from healthy individuals in IL-2 for several days, resulting in the production of lymphokine-activated killer (LAK) activity in these cultures. One purpose of this study was to describe the profile of transcription factor activation in these different populations, and assess whether the patterns observed correlated with functional differences in these cells. Prior to in vivo IL-2 administration, the typical binding pattern of transcription factors in PBMC from patients resembled that seen in fresh PBMC from healthy individuals. Over a 3-week course of IL-2 therapy, in most patients the binding patterns of AP-1 , Sp1, and NF-kappaB proteins changed to resemble those seen in PBMC activated by IL-2 in vitro. However, the cells obtained from IL-2-treated patients did not have low-level constitutive expression of STAT binding factors as did LAK cells. When these patient cells were further stimulated by IL-2 in vitro, additional differences in STAT induction patterns were noted. These data provide further information on the molecular events occurring in immune cells generated through in vivo and in vitro administration of IL-2, and further document that there is not a precise congruence between PBMC activated in vivo and in vitro by IL-2. 


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Positive and negative regulation of immunoglobulin gene expression by a novel B-cell-specific enhancer element. 
A new B-cell-specific enhancer element has been identified 3' of E4 and the octamerlike motifs in the human immunoglobulin heavy-chain gene enhancer. Tandem copies of this 67-bp MnlI-AluI fragment, when fused to the chloramphenicol acetyltransferase gene driven by the conalbumin promoter, stimulated transcription in B cells but not in Jurkat T cells or HeLa cells. Footprinting analysis revealed that the identical sequence CCGAAACTGAAAAGG, designated E6, was protected by nuclear extracts from B cells, T cells, or HeLa cells. Gel mobility shift assays using a synthetic E6 motif detected a B-cell-specific complex in addition to a ubiquitous band found also in T cells and HeLa cells. In agreement with the results of gel retardation assays, tandem copies of the E6 motif stimulated transcription in ARH77 and Raji cells but not in Jurkat or HeLa cells. Furthermore, a mutant E6 motif lost both in vitro binding activity and in vivo enhancer activity. In striking contrast to the mouse Ig heavy-chain enhancer, in which the octamer motif acts as a B-cell-specific enhancer element, the human enhancer contains an octamerlike sequence with one base substitution which bound octamer-binding proteins with only very low affinity and showed no enhancer activity of its own. Interestingly, the MnlI-AluI fragment could suppress the basal-level activity of the conalbumin promoter in both Jurkat and HeLa cells. Moreover, simian virus 40 enhancer activity was blocked by the MnlI-AluI fragment in HeLa cells but not in B cells. Thus, the novel enhancer element identified in this study is probably a target site for both positive and negative factors. 


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A family of serine proteases expressed exclusively in myelo-monocytic cells specifically processes the nuclear factor-kappa B subunit p65 in vitro and may impair human immunodeficiency virus replication in these cells. 
Two groups of U937 promonocytic cells were obtained by limiting dilution cloning which differed strikingly in their ability to support human immunodeficiency virus 1 (HIV-1) replication. "Plus" clones replicated the virus efficiently, whereas "minus" clones did not. We examined these clones for differences in nuclear factor (NF)-kappa B activity which might account for the observed phenomenon. Stimulation of plus clones liberated the classical p50-p65 complex from cytoplasmic pools, whereas minus clones produced an apparently novel, faster-migrating complex, as judged by electrophoretic mobility shift assays. It is surprising that the faster-migrating complex was composed also of p50 and p65. However, the p65 subunit was COOH-terminally truncated, as shown by immunoprecipitation. The truncation resulted from limited proteolysis of p65 during cellular extraction which released particular lysosomal serine proteases, such as elastase, cathepsin G, and proteinase 3. These specific proteases are coordinately expressed and were present exclusively in the minus U937 clones, but not in the plus clones, as demonstrated in the case of cathepsin G. In addition, these proteases were detected in certain subclones of THP-1 and HL-60 cells and in primary monocytes, in each case correlating with the truncated from of p65. We demonstrate in vitro cleavage of p65 by purified elastase and cathepsin G. It is possible that particular serine proteases may have inhibiting effects on the replication of HIV-1 in myelo-monocytic cells. The data also demonstrate that special precautions must be taken when making extracts from myelo-monocytic cells. 


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Nuclear factor-kappaB activation in human monocytes stimulated with lipopolysaccharide is inhibited by fibroblast conditioned medium and exogenous PGE2. 
The nuclear factor kappaB (NF-kappaB) is thought to be crucially involved in the gene activation of several cytokines, including tumor necrosis factor alpha (TNF). Previously, we showed that fibroblast conditioned medium (FCM) is able to inhibit both TNF mRNA accumulation and protein release in peripheral blood-derived human monocytes (PBM) stimulated with lipopolysaccharide (LPS). In this study we have investigated the effect of FCM on the LPS-induced DNA-binding activity of NF-kappaB, by means of electrophoretic shift assay (EMSA). We provide evidence that FCM strongly inhibits the LPS-induced NF-kappaB activation in PBM. Furthermore, we show that exogenous PGE2 mimics the NF-kappaB inhibitory effect of FCM. On the other hand, FCM produced in the presence of indomethacin does not inhibit NF-kappaB activation by LPS. Our results lend further support to the hypothesis that inflammatory and immune responses of monocytes/macrophages may be modulated at the molecular level by signals originating from tissue structural cells such as fibroblasts. 


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Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels, AP-1 binding and AP-1 transcriptional activity. 
We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 (AP-1) binding and enhancer activity in Jurkat T-cells. Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation. Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C (PKC), the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested. PMA, which directly activates PKC, mimicked the effect of the lectins on c-Fos and c-Jun, but elevation of either intracellular Ca2+ or cAMP levels had little or no effect. The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7, a kinase inhibitor with relatively high specificity for PKC, and less efficiently by H-8, a structurally related kinase inhibitor less active on PKC, but more active on cyclic nucleotide-dependent kinases. Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence, TRE, and AP-1 enhancer activity, in Jurkat cells. PMA was also found to increase the AP-1 enhancer activity, whereas elevation of Ca2+ or cAMP had only minor effects. We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels, AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC. 


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The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression: marked enhancement of lipopolysaccharide-stimulated interleukin-1 beta messenger RNA levels by 1,25-dihydroxyvitamin D3. 
The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a potent regulator of human monocyte/macrophage function in vitro. To establish a model for 1,25-(OH)2D3 regulation of human monocyte monokine synthesis, three human cell lines (U-937, THP-1, and HL-60) were examined for: 1) the presence of functional 1,25-(OH)2D3 receptors; 2) the accumulation of interleukin-1 beta (IL-1 beta) mRNA and IL-1 beta protein in response to lipopolysaccharide (LPS); and 3) the regulation of this response by 1,25-(OH)2D3. All three cell lines expressed vitamin D receptor and had increased levels of IL-1 beta mRNA in response to LPS. Preincubation of cells with 1,25-(OH)2D3 augmented IL-1 beta mRNA levels only in U-937 and HL-60 cells. From these data, and taking into consideration their state of differentiation and relative ease of culture, U-937 was chosen over HL-60 and THP-1 as the cell line we further characterized. In U-937 cells, optimum time and dose of pretreatment with 1,25-(OH)2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25-(OH)2D3 (10 nM). Preincubation of cells with 1,25-(OH)2D3 had no effect on the time course of IL-1 beta mRNA appearance in response to LPS. However, exposure of U-937 cells to 1,25-(OH)2D3 increased by 200% the level of IL-1 beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone.2+o 


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Interferon-gamma potentiates the antiviral activity and the expression of interferon-stimulated genes induced by interferon-alpha in U937 cells. 
Binding of type I interferon (IFN-alpha/beta) to specific receptors results in the rapid transcriptional activation, independent of protein synthesis, of IFN-alpha-stimulated genes (ISGs) in human fibroblasts and HeLa and Daudi cell lines. The binding of ISGF3 (IFN-stimulated gene factor 3) to the conserved IFN-stimulated response element (ISRE) results in transcriptional activation. This factor is composed of a DNA-binding protein (ISGF3 gamma), which normally is present in the cytoplasm, and other IFN-alpha-activated proteins which preexist as latent cytoplasmic precursors (ISGF3 alpha). We have found that ISG expression in the monocytic U937 cell line differs from most cell lines previously examined. U937 cells express both type I and type II IFN receptors, but only IFN-alpha is capable of inducing antiviral protection in these cells. Pretreatment with IFN-gamma potentiates the IFN-alpha-induced protection, but IFN-gamma alone does not have any antiviral activity. ISG15 mRNA accumulation in U937 cells is not detectable before 6 h of IFN-alpha treatment, peaks at 24 h, and requires protein synthesis. Although IFN-gamma alone does not induce ISG expression, IFN-gamma pretreatment markedly increases and hastens ISG expression and transcriptional induction. Nuclear extracts assayed for the presence of ISRE binding factors by electrophoretic mobility shift assays show that ISGF3 is induced by IFN-alpha within 6 h from undetectable basal levels in untreated U937 cells. Activation of ISGF3 alpha, the latent component of ISGF3, occurs rapidly. However, the increase in ISGF3 activity ultimately correlates with the accumulation of ISGF3 gamma induced by IFN-alpha or IFN-gamma. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Clone pAT 133 identifies a gene that encodes another human member of a class of growth factor-induced genes with almost identical zinc-finger domains. 
We report the structure and regulation of a gene represented by clone pAT 133, which is induced upon transition from a resting state (G0) through the early phase of the cell cycle (G1). The pAT 133 gene is immediately induced, with FOS-like kinetics, in human T cells and in fibroblasts. Primary structure analysis showed that the encoded protein contains three tandem zinc-finger sequences of the type Cys2-Xaa12-His2. This zinc-finger region, which is thought to bind DNA in a sequence-specific manner, is similar (greater than 80% on the amino acid level) to two previously described transcription factors pAT 225/EGR1 and pAT 591/EGR2. Except for the conserved zinc-finger domains, the amino acid sequences of the three proteins are distinct. This structural similarity suggests that the pAT 133 gene encodes a transcription factor with a specific biological function. Comparing the regulation of these related zinc-finger-encoding genes showed coordinate induction upon mitogenic stimulation of resting T lymphocytes and of resting fibroblasts. However, upon transition from a proliferating (G1) to a resting state of the cell cycle the three genes were differently regulated. In human histiocytic U937 cells mRNA of clone pAT 133 was constitutively expressed, whereas mRNA of pAT 225/EGR1 was induced upon induction of terminal differentiation. In contrast mRNA representing pAT 591/EGR2 was not expressed in these cells. This difference in gene regulation suggests distinct biological roles in the control of cell proliferation for the respective proteins. 


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Interferon-alpha activates multiple STAT proteins and upregulates proliferation-associated IL-2Ralpha, c-myc, and pim-1 genes in human T cells. 
Interferon-alpha (IFN-alpha) is a pleiotropic cytokine that has antiviral, antiproliferative, and immunoregulatory functions. There is increasing evidence that IFN-alpha has an important role in T-cell biology. We have analyzed the expression of IL-2Ralpha, c-myc, and pim-1 genes in anti-CD3-activated human T lymphocytes. The induction of these genes is associated with interleukin-2 (IL-2)-induced T-cell proliferation. Treatment of T lymphocytes with IFN-alpha, IL-2, IL-12, and IL-15 upregulated IL-2Ralpha, c-myc, and pim-1 gene expression. IFN-alpha also sensitized T cells to IL-2-induced proliferation, further suggesting that IFN-alpha may be involved in the regulation of T-cell mitogenesis. When we analyzed the nature of STAT proteins capable of binding to IL-2Ralpha, pim-1, and IRF-1 GAS elements after cytokine stimulation, we observed IFN-alpha-induced binding of STAT1, STAT3, and STAT4, but not STAT5 to all of these elements. Yet, IFN-alpha was able to activate binding of STAT5 to the high-affinity IFP53 GAS site. IFN-alpha enhanced tyrosine phosphorylation of STAT1, STAT3, STAT4, STAT5a, and STAT5b. IL-12 induced STAT4 and IL-2 and IL-15 induced STAT5 binding to the GAS elements. Taken together, our results suggest that IFN-alpha, IL-2, IL-12, and IL-15 have overlapping activities on human T cells. These findings thus emphasize the importance of IFN-alpha as a T-cell regulatory cytokine. 


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An alternatively spliced isoform of the Spi-B transcription factor. 
Spi-B is an Ets transcription factor related to the oncoprotein Spi-1/PU.1 and highly expressed in B lymphoid cells. The Ets proteins share a conserved Ets domain that mediates specific DNA binding. Spi-B binds DNA sequences containing a core 5'-GGAA-3' and activates transcription through this motif. Up to date, the biological function of Spi-B remains unknown. Here, we describe the characterization of an alternatively spliced variant of Spi-B, named deltaSpi-B, which has lost the Ets domain. In B lymphoid cells, deltaspi-B and spi-B mRNAs were present simultaneously in a ratio of around 10%. DeltaSpi-B product was not able to bind DNA and was recovered in cytoplasmic cellular extracts. We raise the hypothesis that delta Spi-B might affect Spi-B function by recruiting factors involved in Spi-B activity. 


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ZAP-70 tyrosine kinase, CD45, and T cell receptor involvement in UV- and H2O2-induced T cell signal transduction. 
Several mammalian responses to UV irradiation, including the activation of NF-kappa B, are believed to involve tyrosine phosphorylation. UV irradiation and H2O2 treatment of T lymphocytes induce protein tyrosine phosphorylation and Ca2+ signals similar to those observed following biological stimulation. We have examined the role of cell surface molecules in these responses. Normal T lymphocytes whose surface expression of CD3 was depleted showed impaired UV-induced tyrosine phosphorylation and Ca2+ signals. Similarly, Jurkat T cell lines deficient in CD3 or CD45 expression also gave impaired UV responses. However, all these cell types still gave strong Ca2+ and tyrosine phosphorylation responses to H2O2. The T cell tyrosine kinase ZAP-70 was found to be highly responsive to UV and H2O2 treatment. ZAP-70 responsiveness to UV required expression of both CD3 and CD45, whereas only CD3 was required for the response to H2O2. UV-induced activation of NF-kappa B was blocked by CD3 depletion, indicating the importance of such cell surface molecules in biological responses to UV. In nonlymphoid cells, the epidermal growth factor receptor displayed increased tyrosine phosphorylation within seconds of UV irradiation. These results suggest that UV-induced signal transduction is mediated via cell surface receptors that normally respond to biological stimulation, whereas H2O2 is able to partially bypass this requirement. 


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Bcl-2 protein inhibits bufalin-induced apoptosis through inhibition of mitogen-activated protein kinase activation in human leukemia U937 cells. 
In a previous study, we demonstrated that bufalin, which is an active principle of Chinese medicine, chan'su, caused apoptosis in human leukemia U937 cells by anomalous activation of mitogen-activated protein kinase (MAPK) via the signaling pathway of Ras, Raf-1, and MAPK kinase-1. Here, we report the effect of overexpression of bcl-2 in U937 cells on the signaling pathway of apoptosis that is induced by bufalin. The results indicated that the apoptosis induced by bufalin in U937 cells was significantly inhibited by overexpression of the Bcl-2 protein. No significant difference was detected in the activation of MAPK kinase-1 that is induced by bufalin in wild-type or Bcl-2-overexpressed U937 cells; however, the activation of MAPK by bufalin was significantly attenuated in the cells overexpressing Bcl-2. Bufalin treatment activated activator protein-1 transcriptional activity; however, this activation was decreased to 40% in bcl-2-overexpressed U937 cells. These results indicate that Bcl-2 acts downstream of MAPK kinase-1 but upstream of MAPK and suggest that, in the signaling pathway of the apoptotic process induced by bufalin, the transcriptional activity of activator protein-1 may be down-regulated through the inhibition of MAPK activity by Bcl-2. 


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Activation of NF-kappaB in Mycobacterium tuberculosis- induced interleukin-2 receptor expression in mononuclear phagocytes. 
Soluble interleukin-2 receptor-alpha (IL-2Ralpha) has been reported to be increased in the sera of patients with advanced tuberculosis, and levels decline after therapy in accordance with improvement of radiologic findings. We investigated expression of the IL-2Ralpha in bronchoalveolar lavage (BAL) cells in active pulmonary tuberculosis, and evaluated the mechanism Mycobacterium tuberculosis induces in the IL-2Ralpha using the THP-1 mononuclear phagocyte cell line. We found IL-2Ralpha expression to be increased in BAL cells from involved sites of active pulmonary tuberculosis. Expression of the alpha-chain of IL-2Ralpha on peripheral blood monocytes (PBM) was induced by M. tuberculosis by flow cytometry evaluation. Northern analysis demonstrated increased IL-2Ralpha gene expression after stimulation with M. tuberculosis which was further induced by interferon-gamma (IFN-gamma). The IL-2Ralpha promoter containing the nuclear factor kappa B (NF-kappaB) site was transcriptionally induced by M. tuberculosis and this NF-kappaB site could confer inducibility to a heterologous herpes thymidine kinase (TK) promoter by M. tuberculosis. Electrophoretic mobility shift assays (EMSAs) revealed specific binding of nuclear protein to the NF-kappaB site upon induction with M. tuberculosis. Using antibodies against the p50 and p65 subunits of NF-kappaB in EMSAs, the involvement of both p50 and p65 proteins was further demonstrated. Functional expression of the IL-2Ralpha on mononuclear phagocytes in M. tuberculosis infection may play an important immunomodulatory role in the host response. 


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Regulation of the beta-globin locus. 
Transcription of the human beta-globin gene cluster depends upon upstream regulatory sequences, which are collectively termed the locus control region. Recent studies have provided new insights into how the individual genes of the cluster are regulated through development. The crux of transcriptional activation is how the locus control region communicates with the gene-proximal regulatory elements. 


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Induction of NF-KB during monocyte differentiation by HIV type 1 infection. 
The production of human immunodeficiency virus type 1 (HIV-1) progeny was followed in the U937 promonocytic cell line after stimulation either with retinoic acid or PMA, and in purified human monocytes and macrophages. Electrophoretic mobility shift assays and Southwestern blotting experiments were used to detect the binding of cellular transactivation factor NF-KB to the double repeat-KB enhancer sequence located in the long terminal repeat. PMA treatment, and not retinoic acid treatment of the U937 cells acts in inducing NF-KB expression in the nuclei. In nuclear extracts from monocytes or macrophages, induction of NF-KB occurred only if the cells were previously infected with HIV-1. When U937 cells were infected with HIV-1, no induction of NF-KB factor was detected, whereas high level of progeny virions was produced, suggesting that this factor was not required for viral replication. These results indicate that in monocytic cell lineage, HIV-1 could mimic some differentiation/activation stimuli allowing nuclear NF-KB expression. 


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STAT1 activation during monocyte to macrophage maturation: role of adhesion molecules. 
Human monocytes isolated from peripheral blood of healthy donors show a time-dependent differentiation into macrophages upon in vitro cultivation, closely mimicking their in vivo migration and maturation into extravascular tissues. The mediator(s) of this maturation process has not been yet defined. We investigated the involvement of signal transducers and activators of transcription (STAT) factors in this phenomenon and reported the specific, time-dependent, activation of STAT1 protein starting at day 0/1 of cultivation and maximally expressed at day 5. STAT1 activity was evident on the STAT binding sequences (SBE) present in the promoters of genes which are up-regulated during monocyte to macrophage maturation such as FcgammaRI and ICAM-1, and in the promoter of the transcription factor IFN regulatory factor-1. Moreover, the effect of cell adhesion to fibronectin or laminin was studied to investigate mechanisms involved in STAT1 activation. Compared with monocytes adherent on plastic surfaces, freshly isolated cells allowed to adhere either to fibronectin- or laminin-coated flasks exhibited an increased STAT1 binding activity both in control and in IFN-gamma-treated cells. The molecular events leading to enhanced STAT1 activation and cytokine responsiveness concerned both Y701 and S727 STAT1 phosphorylation. Exogenous addition of transforming growth factor-beta, which exerts an inhibitory effect on some monocytic differentiation markers, inhibited macrophage maturation, integrin expression and STAT1 binding activity. Taken together these results indicate that STAT1 plays a pivotal role in the differentiation/maturation process of monocytes as an early transcription factor initially activated by adherence and then able to modulate the expression of functional genes, such as ICAM-1 and FcgammaRI. 


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Quantification of vitamin D receptor mRNA by competitive polymerase chain reaction in PBMC: lack of correspondence with common allelic variants. 
It has been recently claimed that polymorphism for the vitamin D receptor (VDR) influences several aspects of calcium and bone metabolism. To evaluate the physiologic plausibility of these claims, we compared the abundance of the VDR mRNA in peripheral blood mononuclear cells (PBMCs) between different VDR genotypes using a quantitative reverse transcribed polymerase chain reaction-based method. The method is based on the coamplification of VDR cDNA and an internal standard consisting of known concentrations of a human VDR CDNA mutated at a BglII restriction site; the interassay coefficient of variation is 11%. To validate the method, we made use of earlier receptor binding studies indicating that normal human monocytes and activated, but not resting, lymphocytes expressed the VDR. The concentration of the VDR mRNA was 10(-8) to 10(-7) g/g of total RNA in cell-sorted monocytes and in in vitro activated lymphocytes, but only 10(-12) g/g of total mRNA in resting lymphocytes, establishing that the VDR mRNA determined by our method in PBMCs is due to constitutive expression in monocytes. Following an initial genotype screening of 85 normal volunteers by polymerase chain reaction or restriction fragment length polymorphism analysis, 14 individuals with the Bb genotype, 12 with the bb genotype, and 12 with the BB genotype were selected. The concentration of the VDR mRNA, corrected for the number of monocytes, was similar among the three genotype groups, as were the other variables examined: serum calcitriol, serum osteocalcin, and vertebral and hip bone density. We conclude that VDR polymorphism does not affect the abundance of the VDR mRNA. 


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Nuclear localization and formation of beta-catenin-lymphoid enhancer factor 1 complexes are not sufficient for activation of gene expression. 
In response to activation of the Wnt signaling pathway, beta-catenin accumulates in the nucleus, where it cooperates with LEF/TCF (for lymphoid enhancer factor and T-cell factor) transcription factors to activate gene expression. The mechanisms by which beta-catenin undergoes this shift in location and participates in activation of gene transcription are unknown. We demonstrate here that beta-catenin can be imported into the nucleus independently of LEF/TCF binding, and it may also be exported from nuclei. We have introduced a small deletion within beta-catenin (Delta19) that disrupts binding to LEF-1, E-cadherin, and APC but not axin. This Delta19 beta-catenin mutant localizes to the nucleus because it may not be efficiently sequestered in the cytoplasm. The nuclear localization of Delta19 definitively demonstrates that the mechanisms by which beta-catenin localizes in the nucleus are completely independent of LEF/TCF factors. beta-Catenin and LEF-1 complexes can activate reporter gene expression in a transformed T-lymphocyte cell line (Jurkat) but not in normal T lymphocytes, even though both factors are nuclear. Thus, localization of both factors to the nucleus is not sufficient for activation of gene expression. Excess beta-catenin can squelch reporter gene activation by LEF-1-beta-catenin complexes but not activation by the transcription factor VP16. Taken together, these data suggest that a third component is necessary for gene activation and that this third component may vary with cell type. 


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Every enhancer works with every promoter for all the combinations tested: could new regulatory pathways evolve by enhancer shuffling? 
The promoters and enhancers of cell type-specific genes are often conserved in evolution, and hence one might expect that a given enhancer has evolved to work best with its own promoter. While this expectation may be realized in some cases, we have not found evidence for it. A total of 27 combinations of different promoters and enhancers were tested by transfection into cultured cells. We found that the relative efficiency of the enhancers is approximately the same, irrespective of the type of promoter used, i.e., there was no strong preference for any given enhancer/promoter combination. Notably, we do not see particularly strong transcription when the immunoglobulin kappa enhancer (or the immunoglobulin heavy chain enhancer) is used to activate a kappa gene promoter. We propose that a generally permissive enhancer/promoter interaction is of evolutionary benefit for higher eukaryotes: by enhancer shuffling, genes could be easily brought under a new type of inducibility/cell type specificity. 


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Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. 
Stimulation of T lymphocytes through their antigen receptor leads to the appearance of several transcription factors, including NF-AT and NF-kappa B, which are involved in regulating genes required for immunologic activation. To investigate the activity of a single transcription factor in individual viable cells, we have applied an assay that uses the fluorescence-activated cell sorter to quantitate beta-galactosidase (beta-gal). We have analyzed the distribution of NF-AT transcriptional activity among T cells undergoing activation by using a construct in which three tandem copies of the NF-AT-binding site directs transcription of the lacZ gene. Unexpectedly, stimulation of cloned stably transfected Jurkat T cells leads to a bimodal pattern of beta-gal expression in which some cells express no beta-gal and others express high levels. This expression pattern cannot be accounted for by cell-cycle position or heritable variation. Further results, in which beta-gal activity is correlated with NF-AT-binding activity, indicate that the concentration of NF-AT must exceed a critical threshold before transcription initiates. This threshold likely reflects the NF-AT concentration-dependent assembly of transcription complexes at the promoter. Similar constructs controlled by NF-kappa B or the entire interleukin-2 enhancer show bimodal expression patterns during induction, suggesting that thresholds set by the concentration of transcription factors may be a common property of inducible genes. 


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One gene, two transcripts: isolation of an alternative transcript encoding for the autoantigen La/SS-B from a cDNA library of a patient with primary Sjogrens' syndrome. 
A cDNA library was prepared from peripheral blood lymphocytes of an autoimmune patient with primary Sjogrens' syndrome. The cDNA library was screened with the patients own autoimmune serum being monospecific for the nuclear autoantigen La/SS-B. Thereby an alternative type of La mRNA was identified that differed from the known La mRNA due to an exchange of the exon 1. Sequencing of the genomic region between the exons 1 and 2 showed that the alternative 5'-end is a part of the intron. In addition, the presence of an alternative promoter site, which exists within the intron downstream of the exon 1, became evident. In consequence, the alternative La mRNA is the result of a promoter switching combined with an alternative splicing mechanism. In the intron, further transcription factor binding sites, including a NF-kappa B element, were identified leading to the suggestion that the expression of the gene encoding for the nuclear autoantigen La/SS-B alters in dependence on disease conditions. 


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The promoter and 5' flanking sequences controlling human B29 gene expression. 
The product of the B-cell-specific B29 gene (B29, Ig beta, CD79b) is essential for Ig-mediated B-cell activation via the B-cell antigen receptor complex (BCR) on human and murine B lymphocytes. To better understand the regulation of this pivotal gene, we have analyzed the human genomic DNA sequence upstream of the B29 ATG start codon for transcriptional control activity. The human B29 gene lacks either a TATA or a CAAT box and transcription is initiated at multiple sites. The minimal promoter of the human B29 gene is contained within a 193-bp region 5' of these multiple start sites. This minimal promoter exhibits B-cell-specific activity and contains SP1, ETS, OCT, and IKAROS/LYF-1 transcription factor motifs. All these motifs are strikingly conserved in sequence and placement relative to the previously characterized murine B29 promoter. Additional upstream gene segments dramatically affected B29 minimal promoter activity. A newly identified motif called the B29 conserved sequence (BCS), found upstream of both human and murine B29 promoters, appears to stimulate B29 transcription through a novel mechanism. A single BCS had little effect either on the minimal B29 promoter or on a heterologous promoter. Instead, the BCS stimulated transcription by counteracting 5' negative regulatory DNA sequences that block the activity of the B29 minimal promoter in its absence. These findings indicate that B29 gene expression is controlled by the complex interplay of positive and negative regulatory elements. 


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Regulation of the tissue factor gene in human monocytic cells. Role of AP-1, NF-kappa B/Rel, and Sp1 proteins in uninduced and lipopolysaccharide-induced expression. 
Tissue factor (TF) expression by peripheral blood monocytes during sepsis initiates intravascular thrombosis. Bacterial lipopolysaccharide (LPS) rapidly induces TF gene transcription in monocytes. The human TF promoter contains binding sites for the transcription factors AP-1, c-Rel/p65, Egr-1, and Sp1. NF-kappa B/Rel proteins have been shown to physically interact with both AP-1 and Sp1 proteins. In this study, we investigated the role of these transcription factors in uninduced and LPS-induced TF gene expression in human monocytic THP-1 cells. Deletional analysis indicated that five Sp1 sites mediated basal expression in uninduced cells. The two AP-1 sites bound c-Fos/c-Jun heterodimers in both unstimulated and LPS-stimulated cells. Maximal LPS induction of the TF promoter required the two AP-1 sites and the kappa B site within the LPS response element. Disruption of the conserved spacing between the proximal AP-1 site and the kappa B site abolished LPS induction. Replacement of the two AP-1 sites with intrinsically bent DNA partially restored LPS induction, suggesting an additional structural role for the AP-1 sites. Synergistic transactivation of the LPS response element in Drosophila Schneider cells by coexpression of c-Fos, c-Jun, c-Rel, and p65 or c-Jun and p65 required the transactivation domains of c-Jun and p65. These data indicated that c-Fos/c-Jun, c-Rel/p65, and Sp1 regulate TF gene expression in human monocytic cells. 


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Nuclear factor-IL6 activates the human IL-4 promoter in T cells. 
Positive regulatory element I (PRE-I) is a strong enhancer element essential for expression of the human IL-4 gene. To identify transcription factors binding to PRE-I, we screened a cDNA expression library from Jurkat T cells and isolated a cDNA encoding nuclear factor (NF)-IL6 (also known as C/EBP beta). NF-IL6 mRNA was found in human Jurkat T cells and in the mouse Th2 clone D10, but not in Th1 clone 29. rNF-IL6 expressed in bacteria was shown to specifically bind to PRE-I. PRE-I forms multiple DNA-protein complexes with nuclear extracts from Jurkat cells. Some of these complexes were demonstrated to contain NF-IL6 by using anti-C/EBP beta Abs. Overexpression of NF-IL6 enhanced expression of the chloramphenicol acetyl transferase reporter gene linked to the PRE-I-thymidine kinase or the human IL-4 promoter more than 10-fold in Jurkat cells. Promoter deletion studies revealed two additional NF-IL6 binding sites located at positions -44 to -36 (C/EBP proximal) and -87 to -79 (C/EBP medial), respectively. Our results demonstrate that NF-IL6 is involved in transcriptional activation of the human IL-4 promoter in T cells. 


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An 11-base-pair DNA sequence motif apparently unique to the human interleukin 4 gene confers responsiveness to T-cell activation signals. 
We have identified a DNA segment that confers responsiveness to antigen stimulation signals on the human interleukin (IL) 4 gene in Jurkat cells. The human IL-4 gene, of 10 kilobases, is composed of four exons and three introns. A cis-acting element (P sequence) resides in the 5' upstream region; no additional DNA segments with enhancer activity were identified in the human IL-4 gene. For further mapping purposes, a fusion promoter was constructed with the granulocyte/macrophage colony-stimulating factor basic promoter containing 60 base pairs of sequence upstream from the cap site of the mouse granulocyte/macrophage colony-stimulating factor gene and various lengths of the 5' upstream sequence of the IL-4 gene. The P sequence was located between positions -79 and -69 relative to the transcription start site of the human IL-4 gene, and this location was confirmed by base-substitution mutations. The plasmids carrying multiple copies of the P sequence showed higher responsiveness to the stimulation. The binding protein(s) that recognize the P sequence of the IL-4 gene were identified by DNA-mobility-shift assays. The binding of NF(P) (a DNA binding protein that specifically recognizes the P sequence) to the P sequence was abolished when oligonucleotides carrying base substitutions were used, indicating that the NF(P) interaction is sequence-specific and that binding specificity of the protein paralleled the sequence requirements for IL-4 expression in vivo. The P sequence does not share homology with the 5' upstream sequence of the IL-2 gene, even though surrounding sequences of the IL-4 gene share high homology with the IL-2 gene. We conclude that a different set of proteins recognize IL-2 and IL-4 genes. 


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PPARalpha activators inhibit cytokine-induced vascular cell adhesion molecule-1 expression in human endothelial cells. 
BACKGROUND: Adhesion molecule expression on the endothelial cell (EC) surface is critical for leukocyte recruitment to atherosclerotic lesions. Better understanding of transcriptional regulation of adhesion molecules in ECs may provide important insight into plaque formation. Peroxisome proliferator-activated receptor-alpha (PPARalpha), a member of the nuclear receptor family, regulates gene expression in response to certain fatty acids and fibric acid derivatives. The present study investigated PPARalpha expression in human ECs and their regulation of vascular cell adhesion molecule-1 (VCAM-1). METHODS AND RESULTS: Immunohistochemistry revealed that human carotid artery ECs express PPARalpha. Pretreatment of cultured human ECs with the PPARalpha activators fenofibrate or WY14643 inhibited TNF-alpha-induced VCAM-1 in a time- and concentration-dependent manner, an effect not seen with PPARgamma activators. Both PPARalpha activators decreased cytokine-induced VCAM-1 mRNA expression without altering its mRNA half-life. Transient transfection of deletional VCAM-1 promoter constructs and electrophoretic mobility shift assays suggest that fenofibrate inhibits VCAM-1 transcription in part by inhibiting NF-kappaB. Finally, PPARalpha activators significantly reduced adhesion of U937 cells to cultured human ECs. CONCLUSIONS: Human ECs express PPARalpha, a potentially important regulator of atherogenesis through its transcriptional control of VCAM-1 gene expression. Such findings also have implications regarding the clinical use of lipid-lowering agents, like fibric acids, which can activate PPARalpha. 


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S-allyl cysteine inhibits activation of nuclear factor kappa B in human T cells. 
Reactive oxygen species are involved in signal transduction pathways leading to nuclear factor kappa B (NF-kappa B) activation which has been implicated in the regulation of gene transcription. We recently reported that a garlic compound, S-allyl cysteine (SAC), protects bovine pulmonary artery endothelial cells from oxidant injury induced by hydrogen peroxide (H2O2). In this study we determined the effects of SAC on NF-kappa B activation in human T lymphocytes (Jurkat cells) induced by tumor necrosis factor alpha (TNF- alpha) and H2O2. Activated NF-kappa B in nuclear extracts was measured by an electrophoretic mobility shift assay using 32P-labeled probe. SAC consistently exhibited a dose-dependent inhibition of NF-kappa B activation induced by both TNF-alpha and H2O2. Supershift with specific antibodies to NF-kappa B subunits confirmed that the inducible retarded bands observed in the EMSA and p65-p50 heterodimer of the NF-kappa B/Rel protein. Our data suggest that SAC may act via antioxidant mechanisms to block NF-kappa B activation in Jurkat cells. 


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Inducible expression and phosphorylation of coactivator BOB.1/OBF.1 in T cells [see comments] 
BOB.1/OBF.1 is a transcriptional coactivator that is constitutively expressed in B cells and interacts with the Oct1 and Oct2 transcription factors. Upon activation of Jurkat T cells and primary murine thymocytes with phorbol esters and ionomycin, BOB.1/OBF.1 expression and transactivation function were induced. BOB.1/OBF.1 was phosphorylated at Ser184 both in vivo and in vitro, and this modification was required for inducible activation. Mutation of Ser184 also diminished transactivation function in B cells, suggesting that the activating phosphorylation that is inducible in T cells is constitutively present in B cells. Thus, BOB.1/OBF.1 is a transcriptional coactivator that is critically regulated by posttranslational modifications to mediate cell type-specific gene expression. 


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Molecular basis of a multiple lymphokine deficiency in a patient with severe combined immunodeficiency. 
We have previously reported that the T lymphocytes of a child with severe combined immunodeficiency are defective in the transcription of several lymphokine genes that include IL2, IL3, IL4, and IL5, which encode interleukins 2, 3, 4, and 5 (IL-2, -3, -4, and -5). To determine whether the defect in the patient's T lymphocytes involved a trans-acting factor common to the affected lymphokine genes, we examined the ability of nuclear factors from the patient's T lymphocytes to bind response elements present in the regulatory region of IL2. Nuclear factor NF-kB, activation protein 1 (AP-1), OCT-1, and NF-IL-2B binding activity were normal. In contrast, the binding of the nuclear factor of activated T cells (NF-AT) to its response element in the IL2 enhancer and to an NF-AT-like response element present in the IL4 enhancer was abnormal. To ascertain whether the abnormal NF-AT binding activity was related to an impaired function, we transfected patient and control T lymphocytes with constructs containing the reporter gene encoding chloramphenicol acetyl transferase (CAT) under the control of the entire IL2 regulatory region or of multimers of individual enhancer sequences. CAT expression directed by the IL2 regulatory region or by a multimer of the NF-AT-binding site was markedly lower in the patient relative to controls. In contrast, CAT gene expression directed by a multimer of the OCT-1 proximal (OCT-1p)-binding site was equivalent in patient and controls. These results indicate that an abnormality of/or influencing NF-AT may underlie the multiple lymphokine deficiency in this patient. 


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Oncogenic forms of NOTCH1 lacking either the primary binding site for RBP-Jkappa or nuclear localization sequences retain the ability to associate with RBP-Jkappa and activate transcription. 
Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (DeltaE) or consisting only of the intracellular domain (ICN) were found to activate transcription in cultured cells, presumably through RBP-Jkappa response elements within DNA. Both truncated forms also bound to the transcription factor RBP-Jkappa in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation required the presence of a weak RBP-Jkappa-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-Jkappa-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-Jkappa, was not needed for oncogenesis or for transcriptional activation. While ICN appeared primarily in the nucleus, DeltaE localized to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreased nuclear localization and increased transcriptional activation by membrane-bound DeltaE. Transcriptional activation by this mutant form of membrane-bound DeltaE was approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-Jkappa at promoter sites. 


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Impaired induction of c-fos/c-jun genes and of transcriptional regulatory proteins binding distinct c-fos/c-jun promoter elements in activated human T cells during aging. 
The activation of transcriptional factor c-Fos/c-Jun AP-1 is essential for normal T cell responsiveness and is often impaired in T cells during aging. In the present study, we investigated whether aberrancies in the regulation of c-fos/c-jun at the mRNA or protein level might underlie the age-associated impairments of AP-1 in human T cells. Whereas T cells from young subjects stimulated with cross-linked anti-CD3epsilon mAb OKT3 plus PMA or with the lectin PHA plus PMA demonstrated considerable increases in c-Fos protein expression, the expression of c-Fos but not c-Jun was markedly reduced in stimulated T cells from certain elderly subjects. In addition, RNase protection assays revealed that anti-CD3/PMA-stimulated T cells from a substantial proportion of elderly subjects exhibited decreased levels of c-fos and/or c-jun mRNA compared to T cells from young subjects. Using electrophoretic mobility shift assays, the levels of nuclear regulatory proteins recognizing the AP-1 consensus TRE motif, the proximal c-jun TRE-like promoter element, and the c-fos serum response element (SRE) were determined in resting and stimulated T cells. Although the stimulation of T cells from young subjects resulted in coordinated increases of nuclear protein complexes binding the AP-1 TRE, c-jun TRE, and c-fos SRE DNA sequence motifs, age-related reductions in the activation of AP-1 were accompanied by decreased levels of c-jun TRE and c-fos SRE binding complexes. Furthermore, the nuclear protein complexes binding the SRE motif induced in activated T cells of young and elderly subjects contained serum response factor and Elk-1 pointing toward age-related defects in the activation of transcriptional regulatory proteins distinct from c-jun/AP-1. These results suggest that underlying aberrancies in the induction of c-fos/c-jun as well as their nuclear regulatory proteins may contribute to the age-related impairments of AP-1 activation in human T cells. 


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Effects of alpha-lipoic acid and dihydrolipoic acid on expression of proto-oncogene c-fos. 
The transcription factor AP-1 is an important human mediator of the cellular response to serum, growth factors, and phorbol esters such as 12-O-tetradecanoyl-phorbol-13 acetate (TPA). The AP-1 complex consists of distinct protein heterodimers encoded by the proto-oncogene c-fos and c-jun mRNA whose gene expression can be induced by TPA, cyclic AMP and growth factors. Recent findings suggest an involvement of reactive oxygen species in the pathway of TPA and protein kinase C leading to expression of c-fos and c-jun mRNA. To investigate the role of reactive oxygen species we studied the effects of alpha-lipoic acid and dihydrolipoic acid (natural thiol antioxidants) on the expression of c-fos mRNA in human Jurkat T cells. When cells were preincubated with dihydrolipoic acid (0.2 mM) the expression of c-fos mRNA was suppressed at 30 min after stimulation of TPA (0.5 microM) whereas in the case of preincubation of alpha-lipoic acid (0.2 microM), the expression was enhanced at 30 min. These studies support the idea that superoxide anion radical plays a role in the expression of c-fos mRNA. 


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SLP-76 and Vav function in separate, but overlapping pathways to augment interleukin-2 promoter activity. 
SLP-76 and Vav, two hematopoietic cell specific molecules, are critical for T cell development and activation. Following T cell antigen receptor stimulation, SLP-76 and Vav both undergo tyrosine phosphorylation and associate with each other via the SH2 domain of Vav and phosphorylated tyrosines of SLP-76. Furthermore, SLP-76 and Vav have a synergistic effect on interleukin (IL)-2 promoter activity in T cells. In this report, we show that two tyrosines, Tyr-113 and Tyr-128, of SLP-76 are required for its binding to Vav, both in vitro and in intact cells. Surprisingly, we find also that the interaction between SLP-76 and Vav is not required for their cooperation in augmenting IL-2 promoter activity, as the two molecules appear to function in different signaling pathways upstream of IL-2 gene expression. Overexpression of SLP-76 in the Jurkat T cell line potentiates the activities of both nuclear factor of activated T cells and AP-1 transcription factors. In contrast, overexpression of Vav leads to enhanced nuclear factor of activated T cells activity without affecting AP-1. Additionally, overexpression of Vav, but not SLP-76, augments CD28-induced IL-2 promoter activity. These findings suggest that the synergy between SLP-76 and Vav in regulating IL-2 gene expression reflects the cooperation between different signaling pathways. 


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Human immunodeficiency virus type-2 gene expression: two enhancers and their activation by T-cell activators. 
The human immunodeficiency viruses (HIVs) may include a spectrum of retroviruses with varying potential to infect their host, undergo long periods of latent infection, and induce pathology. Since expression of the viruses is in large part regulated by the sequence elements in their long terminal repeats (LTRs), this study was directed to an analysis of the regulatory elements in the HIV-2 LTR. The HIV-2 LTR was found to contain two enhancers. One of these enhancers is, in part, identical to the HIV-1 enhancer. This enhancer in HIV-1 is the T-cell activation response element; in HIV-2, however, it is the second enhancer that is mainly responsible for activation in response to T-cell activators. The second enhancer interacts with two nuclear binding proteins (85 kD and 27 kD mobility) that appear to be required for optimal enhancer function and activation. Observations such as these encourage the speculation that there may be subtle differences in the regulation of HIV-1 and HIV-2 expression that may be relevant to the possible longer latency and reduced pathogenicity of HIV-2. 


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Engagement of the Lewis X antigen (CD15) results in monocyte activation. 
We previously reported that monocyte adhesion to tumor necrosis factor-alpha (TNF-alpha)-treated endothelial cells increased expression of tissue factor and CD36 on monocytes. Using immunological cross-linking to mimic receptor engagement by natural ligands, we now show that CD15 (Lewis X), a monocyte counter-receptor for endothelial selectins may participate in this response. We used cytokine production as a readout for monocyte activation and found that CD15 cross-linking induced TNF-alpha release from peripheral blood monocytes and cells from the monocytic cell line MM6. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed an increase in steady-state TNF-alpha mRNA after 3 to 4 hours of cross-linking. CD15 cross-linking also concomitantly increased interleukin-1 beta (IL-1 beta) mRNA, while no apparent change was observed in the levels of beta-actin mRNA, indicating specificity. To examine transcriptional regulation of cytokine genes by CD15 engagement, a CAT plasmid reporter construct containing IL-1 beta promoter/enhancer sequences was introduced into MM6. Subsequent cross-linking of CD15 increased CAT activity. CD15 engagement by monoclonal antibody also attenuated IL-1 beta transcript degradation, demonstrating that signaling via CD15 also had posttranscriptional effects. Nuclear extracts of anti-CD15 cross-linked cells demonstrated enhanced levels of the transcriptional factor activator protein-1, minimally changed nuclear factor-kappa B, and did not affect SV40 promoter specific protein-1. We conclude that engagement of CD15 on monocytes results in monocyte activation. In addition to its well-recognized adhesive role, CD15 may function as an important signaling molecule capable of initiating proinflammatory events in monocytes that come into contact with activated endothelium. 


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Vitamin E therapy of acute CCl4-induced hepatic injury in mice is associated with inhibition of nuclear factor kappa B binding. 
Oxidative stress, with reactive oxygen intermediate formation, may represent a common mechanism by which liver injury is induced by diverse etiologies. Oxidative stress enhances nuclear factor kappa B (NF-kappa B) activity, and NF-kappa B activity has been shown to enhance the expression of cytotoxic cytokines. Acute hepatic injury caused by reactive oxygen intermediate production was induced by an intraperitoneal injection of CCl4 in mice. This injury was significantly inhibited by intravenous pretreatment of the mice with a water-soluble emulsion of alpha-tocopherol. Alpha-tocopherol treatment of the mice given the CCl4 also reduced the NF-kappa B binding to levels approaching those found in normal mice. In vitro treatment of a monocyte/macrophage cell line with CCl4 led to enhanced NF-kappa B binding and an increase in tumor necrosis factor-alpha (TNF-alpha) messenger RNA levels. Liver specimens taken from patients with acute fulminant hepatitis had markedly increased NF-kappa B binding activity in comparison with the binding of normal livers. These data demonstrate that abolishing acute hepatic injury with alpha-tocopherol, a free radical scavenger, also eliminated increased NF-kappa B binding. It is tempting to speculate that enhanced NF-kappa B expression caused by free radical production/oxidative stress may modulate liver injury, perhaps through an effect on cytotoxic cytokine synthesis. 


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Fludarabine-induced immunosuppression is associated with inhibition of STAT1 signaling. 
Fludarabine is a nucleoside analog used in the treatment of hematologic malignancies that can induce severe and prolonged immunosuppression. Although it can be incorporated into the DNA of dividing cells, fludarabine is also a potent inhibitor of cells with a low growth fraction, thus it must have other mechanisms of action. STAT1, which is activated in response to many lymphocyte-activating cytokines including the interferons, is essential for cell-mediated immunity, as the absence of this protein is associated with prominent defects in the ability to control viral infections. Here we show that fludarabine, but not the immunosuppressant cyclosporine A, inhibits the cytokine-induced activation of STAT1 and STAT1-dependent gene transcription in normal resting or activated lymphocytes. Fludarabine caused a specific depletion of STAT1 protein (and mRNA) but not of other STATs. This loss of STAT1 was also seen in cells from patients treated with fludarabine in vivo. Brief exposure to fludarabine led to a sustained loss of STAT1, analogous to the prolonged period of immunosuppression induced by exposure to the drug in vivo. Thus, STAT1 may be a useful target in the development of new immunosuppressive and antineoplastic agents. 


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Nuclear factor-kappa B activity in T cells from patients with rheumatic diseases: a preliminary report. 
OBJECTIVE: The NF-kappa B/Rel family of transcription factors regulates the expression of many genes involved in the immune or inflammatory response at the transcriptional level. The aim of this study was to determine whether distinctive patterns of NF-kappa B activation are seen in different forms of joint disease. METHODS: The DNA binding activity of these nucleoproteins was examined in purified synovial and peripheral T cells from patients with various chronic rheumatic diseases (12: four with rheumatoid arthritis; five with spondyloarthropathies; and three with osteoarthritis). RESULTS: Electrophoretic mobility shift assays disclosed two specific complexes bound to a NF-kappa B specific 32P-labelled oligonucleotide in nucleoproteins extracted from purified T cells isolated from synovial fluid and peripheral blood of patients with rheumatoid arthritis. The complexes consisted of p50/p50 homodimers and p50/p65 heterodimers. Increased NF-kappa B binding to DNA in synovial T cells was observed relative to peripheral T cells. In non-rheumatoid arthritis, binding of NF-kappa B in synovial T cells was exclusively mediated by p50/p50 homodimers. CONCLUSION: Overall, the results suggest that NF-kappa B may play a central part in the activation of infiltrating T cells in chronic rheumatoid arthritis. The activation of this nuclear factor is qualitatively different in rheumatoid synovial T cells to that in other forms of non-rheumatoid arthritis (for example, osteoarthritis, spondyloarthropathies). 


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Interleukin 4 activates a signal transducer and activator of transcription (Stat) protein which interacts with an interferon-gamma activation site-like sequence upstream of the I epsilon exon in a human B cell line. Evidence for the involvement of Janus kinase 3 and interleukin-4 Stat. 
Germ line C transcripts can be induced by IL-4 in the human B cell line, BL-2. Utilizing a IFN-gamma activation site-like DNA sequence element located upstream of the I epsilon exon, we demonstrated by gel mobility shift assays that IL-4 induced a binding activity in the cytosol and nucleus of BL-2 cells. This factor was designated IL-4 NAF (IL-4-induced nuclear-activating factors) and was identified as a tyrosine phosphoprotein, which translocates from the cytosol to the nucleus upon IL-4 treatment. Because these are the characteristics of a signal transducer and activator of transcription (Stat) protein, we determined whether antibodies to Stat proteins will interfere with gel mobility shift and found that antibodies to IL-4 Stat, also known as Stat6, but not antibodies to other Stat proteins, interfere with the formation of the IL-4 NAF complex. Congruous with the involvement of a Stat protein, IL-4 induced robust Janus kinase 3 (JAK3) activity in BL-2 cells. Cotransfection of JAK3 with IL-4 Stat into COS-7 cells produced an intracellular activity which bound the same IFN-gamma activation site-like sequence and comigrated with IL-4 NAF in electrophoretic mobility shift assay. These results show that IL-4 NAF is IL-4 Stat, which is activated by JAK3 in response to IL-4 receptor engagement. 


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Limited proteolysis for assaying ligand binding affinities of nuclear receptors. 
The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand. 


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Isolation of a candidate repressor/activator, NF-E1 (YY-1, delta), that binds to the immunoglobulin kappa 3' enhancer and the immunoglobulin heavy-chain mu E1 site. 
We have determined that the developmental control of immunoglobulin kappa 3' enhancer (kappa E3') activity is the result of the combined influence of positive- and negative-acting elements. We show that a central core in the kappa E3' enhancer is active at the pre-B-cell stage but is repressed by flanking negative-acting elements. The negative-acting sequences repress enhancer activity in a position- and orientation-independent manner at the pre-B-cell stage. We have isolated a human cDNA clone encoding a zinc finger protein (NF-E1) that binds to the negative-acting segment of the kappa E3' enhancer. This protein also binds to the immunoglobulin heavy-chain enhancer mu E1 site. NF-E1 is encoded by the same gene as the YY-1 protein, which binds to the adeno-associated virus P5 promoter. NF-E1 is also the human homologue of the mouse delta protein, which binds to ribosomal protein gene promoters. The predicted amino acid sequence of this protein contains features characteristic of transcriptional activators as well as transcriptional repressors. Cotransfection studies with this cDNA indicate that it can repress basal promoter activity. The apparent dual function of this protein is discussed. 


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Cyclosporin A interferes with the inducible degradation of NF-kappa B inhibitors, but not with the processing of p105/NF-kappa B1 in T cells. 
The transcription factor NF-kappa B controls the induction of numerous cytokine promoters during the activation of T lymphocytes. Inhibition of T cell activation by the immunosuppressants cyclosporin A (CsA) and FK506 exerts a suppressive effect on the induction of these NF-kappa B-controlled cytokine promoters. We show for human Jurkat T leukemia cells, as well as human and mouse primary T lymphocytes, that this inhibitory effect is accompanied by an impaired nuclear translocation of the Rel proteins c-Rel, RelA/p65 and NF-kappa B1/p50, whereas the nuclear appearance of RelB remains unaffected. CsA does not interfere with the synthesis of Rel proteins, but prevents the inducible degradation of cytosolic NF-kappa B inhibitors I kappa B alpha and I kappa B beta upon T cell activation. CsA neither inhibits the processing of the NF-kappa B1 precursor p105 to p50, nor does it "stabilize" the C-terminal portion of p105, I kappa B gamma, which is degraded during p105 processing to mature p50. These results indicate that CsA interferes with a specific event in the signal-induced degradation of I kappa B alpha and I kappa B beta, but does not affect the processing of NF-kappa B1/p105 to p50. 


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Suppression of TNFalpha-mediated NFkappaB activity by myricetin and other flavonoids through downregulating the activity of IKK in ECV304 cells. 
Flavonoids are a group of naturally-occurring phenolic compounds in the plant kingdom, and many flavonoids are found with vascular protective properties. Nevertheless how the protective response is exerted by flavonoids is not well characterized. In view of the nuclear factor-kappaB (NFkappaB) may play a central role in the initiation of atherosclerosis, prevention of the activation of NFkappaB represents an important role in protecting vascular injury. In this study, the effects of flavonoids on NFkappaB/inhibitor-kappaB (IkappaB) system in ECV304 cells activated with tumor necrosis factor-alpha (TNFalpha) were examined. We investigated the inhibitory action of six flavonoids on IkappaB kinase (IKK) activity, an enzyme recently found to phosphorylate critical serine residues of IkappaB for degradation. Of six flavonoids tested, myricetin was found to strongly inhibit IKK kinase activity, and prevent the degradation of IkappaBalpha and IkappaBbeta in activated endothelial cells. Furthermore, myricetin was also found to inhibit NFkappaB activity correlated with suppression of monocyte adhesion to ECV304 cells. Therefore we conclude that flavonoids may be of therapeutic value for vascular disease through down regulation of NFkappaB/IkappaB system. Copyright 1999 Wiley-Liss, Inc. 


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Structural and functional characterization of the human CD36 gene promoter: identification of a proximal PEBP2/CBF site. 
CD36 is a cell surface glycoprotein composed of a single polypeptide chain, which interacts with thrombospondin, collagens type I and IV, oxidized low density lipoprotein, fatty acids, anionic phospholipids, and erythrocytes parasitized with Plasmodium falciparum. Its expression is restricted to a few cell types, including monocyte/macrophages. In these cells, CD36 is involved in phagocytosis of apoptotic cells, and foam cell formation by uptake of oxidized low density lipoprotein. To study the molecular mechanisms that control the transcription of the CD36 gene in monocytic cells we have isolated and analyzed the CD36 promoter. Transient expression experiments of 5'-deletion fragments of the CD36 promoter coupled to luciferase demonstrated that as few as 158 base pairs upstream from the transcription initiation site were sufficient to direct the monocyte-specific transcription of the reporter gene. Within the above region, the fragment spanning nucleotides -158 to -90 was required for optimal transcription in monocytic cells. Biochemical analysis of the region -158/-90 revealed a binding site for transcription factors of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family at position -103. Disruption of the PEBP2/CBF site markedly diminished the role of the PEBP2/CBF factors in the constitutive transcription of the CD36 gene. The involvement of members of the PEBP2/CBF family in chromosome translocations associated with acute myeloid leukemia, and in the transcriptional regulation of the myeloid-specific genes encoding for myeloperoxidase, elastase, and the colony-stimulating factor receptor, highlights the relevance of the regulation of the CD36 gene promoter in monocytic cells by members of the PEBP2/CBF family. 


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Transcription factor activation and functional stimulation of human monocytes. 
Activation of expression of genes encoding transcription factors: c-fos and c-jun and formation of AP1 transcriptional complex in human monocytes was investigated. It was found that lipopolysaccharide induced strongly both c-fos and c-jun expression as well as AP1 formation. Interferon gamma activated strongly c-fos and weakly c-jun and AP1. Tumor necrosis factor induced slightly c-fos and had almost no effect on c-jun and AP1. The data suggest that differences in functional responses elicited in monocytes by all three factors may be dependent on different routes on nuclear signalling employed by the factors. 


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Interferon-gamma modulates the lipopolysaccharide-induced expression of AP-1 and NF-kappa B at the mRNA and protein level in human monocytes. 
Interferon-gamma (IFN-gamma) modulates the expression of several cytokines by human monocytes at the transcriptional level. In view of these findings, we analyzed the effects of IFN-gamma on the expression of different transcription factors in activated human monocytes. Priming of human monocytes with IFN-gamma resulted in the down regulation of c-fos and c-jun mRNA in response to stimulation with lipopolysaccharide (LPS) compared to the effects of LPS alone. Not only was this effect observed at the mRNA level, but activator protein-1 (AP-1) DNA binding capacity was affected as well, A strong reduction was observed in the LPS-induced DNA-binding activity of AP-1 in the presence of IFN-gamma. LPS-stimulated monocytes showed an increased expression of p105 mRNA, the precursor of the p50 subunit of the transcription factor nuclear factor-kappa B (NF-kappa B), while no effect was noticed on the expression of p65 mRNA. In contrast, IFN-gamma priming did not affect the expression of p105 transcripts but enhanced the expression of p65 mRNA (two-fold). Priming with IFN-gamma followed by LPS stimulation resulted in a further increase in the expression of p65 mRNA. This was due to an increase in the half-life of p65 mRNA (75 vs 150 minutes). Electrophoretic mobility shift assays (EMSAs) demonstrated that unstimulated monocytes predominantly expressed p50 NF-kappa B. Stimulation with LPS or IFN-gamma resulted in the expression of p50 and p65 subunits, while the combination of IFN-gamma plus LPS caused a further increase in the expression of NF-kappa B. With Western blotting, it was shown that nuclear extracts from monocytes contained p50 and p65 protein in response to LPS and IFN-gamma stimulation. However, the combined stimulation did not result in enhanced p50 and p65 protein expression. The effects of IFN-gamma on the transcription factors were specific, since no change was observed in the expression of NF-IL-6 or I kappa B alpha, the inhibitor of NF-kappa B. We conclude that the effects of IFN-gamma on the expression of the transcription factors AP-1 and NF-kappa B may be important for the modulatory effects of IFN-gamma on the cytokine expression in activated human monocytes. 


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Potent and stable attenuation of live-HIV-1 by gain of a proteolysis-resistant inhibitor of NF-kappaB (IkappaB-alphaS32/36A) and the implications for vaccine development. 
Live-attenuated human immunodeficiency viruses (HIVs) are candidates for Acquired Immunodeficiency Syndrome (AIDS) vaccine. Based on the simian immunodeficiency virus (SIV) model for AIDS, loss-of-function (e.g. deletion of accessory genes such as nef) has been forwarded as a primary approach for creating enfeebled, but replication-competent, HIV-1/SIV. Regrettably, recent evidence suggests that loss-of-function alone is not always sufficient to prevent the emergence of virulent mutants. New strategies that attenuate via mechanisms distinct from loss-of-function are needed for enhancing the safety phenotype of viral genome. Here, we propose gain-of-function to be used simultaneously with loss-of-function as a novel approach for attenuating HIV-1. We have constructed an HIV-1 genome carrying the cDNA of a proteolysis-resistant nuclear factor-kappaB inhibitor (IkappaB-alphaS32/36A) in the nef region. HIV-1 expressing IkappaB-alphaS32/36A down-regulates viral expression and is highly attenuated in both Jurkat and peripheral blood mononuclear cells. We provide formal proof that the phenotypic and attenuating characteristics of IkappaB-alphaS32/36A permit its stable maintenance in a live, replicating HIV-1 despite 180 days of forced ex vivo passaging in tissue culture. As compared with other open-reading frames embedded into HIV/SIV genome, this degree of stability is unprecedented. Thus, IkappaB-alphaS32/36A offers proof-of-principle that artifactually gained functions, when used to attenuate the replication of live HIV-1, can be stable. These findings illustrate gain-of-function as a feasible strategy for developing safer live-attenuated HIVs to be tested as candidates for AIDS vaccine. 


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Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. 
I kappa B-alpha inhibits transcription factor NF-kappa B by retaining it in the cytoplasm. Various stimuli, typically those associated with stress or pathogens, rapidly inactivate I kappa B-alpha. This liberates NF-kappa B to translocate to the nucleus and initiate transcription of genes important for the defense of the organism. Activation of NF-kappa B correlates with phosphorylation of I kappa B-alpha and requires the proteolysis of this inhibitor. When either serine-32 or serine-36 of I kappa B-alpha was mutated, the protein did not undergo signal-induced phosphorylation or degradation, and NF-kappa B could not be activated. These results suggest that phosphorylation at one or both of these residues is critical for activation of NF-kappa B. 


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TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation. 
The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for productive infection and is a potential target for antiviral therapy. Tat, a potent activator of HIV-1 gene expression, serves to greatly increase the rate of transcription directed by the viral promoter. This induction, which seems to be an important component in the progression of acquired immune deficiency syndrome (AIDS), may be due to increased transcriptional initiation, increased transcriptional elongation, or a combination of these processes. Much attention has been focused on the interaction of Tat with a specific RNA target termed TAR (transactivation responsive) which is present in the leader sequence of all HIV-1 mRNAs. This interaction is believed to be an important component of the mechanism of transactivation. In this report we demonstrate that in certain CNS-derived cells Tat is capable of activating HIV-1 through a TAR-independent pathway. A Tat-responsive element is found upstream within the viral promoter that in glial-derived cell lines allows transactivation in the absence of TAR. Deletion mapping and hybrid promoter constructs demonstrate that the newly identified Tat-responsive element corresponds to a sequence within the viral long terminal repeat (LTR) previously identified as the HIV-1 enhancer, or NF-kappa B domain. DNA band-shift analysis reveals NF-kappa B binding activity in glial cells that differs from that present in T lymphoid cells. Further, we observe that TAR-deleted mutants of HIV-1 demonstrate normal late gene expression in glial cells as evidenced by syncytia formation and production of viral p24 antigen. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Biphasic control of nuclear factor-kappa B activation by the T cell receptor complex: role of tumor necrosis factor alpha. 
The regulation of nuclear factor (NF)-kappa B activation by the T cell receptor (TcR)/CD3 complex in primary human T cells has been studied at various times after activation. Only p50 NF-kappa B protein bound the kappa B element of interleukin-2 receptor (IL-2R) alpha chain promoter on resting T cells. However, immediately after TcR/CD3 cross-linking (after approximately 1 h; immediate) binding of p50.p65 heterodimers was observed. p50.c-rel heterodimers were also detected bound to this sequence at early time points (7-16 h; early), and both remained active at later time points (40 h; late) after activation. This regulation takes place mainly at the level of nuclear translocation of p65 and c-rel, at immediate and early time points. Activation also induced c-rel and p105/p50 mRNA synthesis, but not p65 mRNA whose expression was constitutive. Interestingly, all those early and late events, but not the immediate ones, were inhibited by a neutralizing anti-tumor necrosis factor alpha (TNF-alpha) monoclonal antibody. Similarly, cycloheximide prevented the p65 and c-rel translocation and consequent formation of active binding heterodimers, at early and late times. Cyclosporin A impaired not only early and late, but also immediate events; however, addition of TNF-alpha prevented all inhibition. These results indicate that the regulation of NF-kappa B activation during T cell activation by TcR/CD3 signals is biphasic: TcR/CD3 triggers its immediate translocation, which is transient if no TNF-alpha is present. TNF-alpha, therefore, emerges as the main factor responsible for a second phase of NF-kappa B regulation, controlling both translocation of p65 and c-rel, and new mRNA synthesis for c-rel and p105/p50. 


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ATF1 and CREB trans-activate a cell cycle regulated histone H4 gene at a distal nuclear matrix associated promoter element. 
Proteins of the ATF/CREB class of transcription factors stimulate gene expression of several cell growth-related genes through protein kinase A-related cAMP response elements. The promoter activity of cell cycle regulated histone H4 genes is regulated by at least four principal cis-acting elements which mediate G1/S phase control and/or enhancement of transcription during the cell cycle. Using protein-DNA interaction assays we show that the H4 promoter contains two ATF/CREB recognition motifs which interact with CREB, ATF1, and ATF2 but not with ATF4/CREB2. One ATF/CRE motif is located in the distal promoter at the nuclear matrix-associated Site IV, and the second motif is present in the proximal promoter at Site I. Both ATF/CRE motifs overlap binding sequences for the multifunctional YY1 transcription factor, which has previously been shown to be nuclear matrix associated. Subnuclear fractionation reveals that there are two ATF1 isoforms which appear to differ with respect to DNA binding activity and partition selectively between nuclear matrix and nonmatrix compartments, consistent with the role of the nuclear matrix in regulating gene expression. Site-directed mutational studies demonstrate that Site I and Site IV together support ATF1- and CREB-induced trans-activation of the H4 promoter. Thus, our data establish that ATF/CREB factors functionally modulate histone H4 gene transcription at distal and proximal promoter elements. 


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Transcriptional and post-transcriptional regulation of c-jun expression during monocytic differentiation of human myeloid leukemic cells. 
AP-1, the polypeptide product of c-jun, recognizes and binds to specific DNA sequences and stimulates transcription of genes responsive to certain growth factors and phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). We studied the effects of TPA on the regulation of c-jun gene expression in HL-60 cells during monocytic differentiation. Low levels of c-jun transcripts were detectable in untreated HL-60 leukemic cells, increased significantly by 6 h, and reached near maximal levels by 24 h of exposure to 32 nM TPA. Similar kinetics of c-jun induction by TPA were observed in human U-937 and THP-1 monocytic leukemia cells. Similar findings were obtained with bryostatin 1 (10 nM), another activator of protein kinase C and inducer of monocytic differentiation. Furthermore, 1,25-dihydroxyvitamin D3 (0.5 microM), a structurally distinct agent which also induces HL-60 monocytic differentiation, increased c-jun expression. TPA treatment of HL-60 cells in the presence of cycloheximide was associated with superinduction of c-jun transcripts. Run-on analysis demonstrated detectable levels of c-jun gene transcription in untreated HL-60 cells, and that exposure to TPA increases this rate 3.3-fold. Treatment of HL-60 cells with both TPA and cycloheximide had no effect on the rates of c-jun transcription. The half-life of c-jun RNA as determined by treating HL-60 cells with TPA and actinomycin D was 30 min. In contrast, the half-life of c-jun RNA in TPA-treated HL-60 cells exposed to cycloheximide and actinomycin D was greater than 2 h. These findings suggested that the increase in c-jun RNA observed during TPA-induced monocytic differentiation is mediated by both transcriptional and post-transcriptional mechanisms. 


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Transcriptional induction of collagenase-1 in differentiated monocyte-like (U937) cells is regulated by AP-1 and an upstream C/EBP-beta site. 
In this report, we demonstrate that the AP-1 site and a distal promoter element regulate transcriptional induction of collagenase-1 during monocytic differentiation. Chloramphenicol acetyltransferase expression constructs containing regions of the human collagenase-1 promoter were stably or transiently transfected into U937 cells, and reporter activity was assessed at various times after the onset of phorbol 12-myristate 13-acetate (PMA)-mediated differentiation. Rapid and strong induction of promoter activity was lost in constructs with a mutant AP-1 element; however, at 16-96 h post-PMA, the mutant collagenase-1 promoter displayed AP-1 independent PMA-mediated transactivation. The AP-1 mutant constructs also showed delayed transcriptional activation in PMA-treated fibroblasts. Western and supershift analyses indicated that functional Jun and Fos proteins were present in nuclear extracts of PMA-differentiated U937 cells. Promoter deletion constructs demonstrated the potential role of distal promoter sequences in regulating collagenase-1 transcription. In particular, Western, supershift, and promoter deletion analyses suggested a role for CCAAT/enhancer-binding protein-beta (C/EBP-beta) binding site between -2010 and -1954 in regulating transcription of collagenase-1 in monocytic cells. Our findings suggest that distinct regulatory elements, acting somewhat independently of each other, control expression of collagenase-1. In addition, our data suggests that the rapid PMA-mediated induction of collagenase-1 transcription is controlled by a mechanism distinct from that regulating the sustained expression of this proteinase in activated macrophages. 


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Cloning of a mitogen-inducible gene encoding a kappa B DNA-binding protein with homology to the rel oncogene and to cell-cycle motifs. 
We have cloned and characterized a mitogen-inducible gene isolated from human T cells that predicts a protein of 968 amino acids. The amino-terminal domain has regions homologous to the oncogene rel and to the developmentally important gene dorsal of Drosophila. The carboxy-terminal domain contains repeat structures found in a variety of proteins that are involved in cell-cycle control of yeast and in tissue differentiation in Drosophila and Ceanorhabditis elegans, as well as in the putative human oncogene bcl-3 and in the ankyrin protein. A truncated form of the product of this gene translated in vitro is a DNA-binding protein which interacts specifically with the kappa B binding site found in many inducible genes, including the enhancer in human immunodeficiency virus. This gene is yet another in a growing list of important regulatory molecules whose expression is transcriptionally induced upon cellular activation. 


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NF-kappa B/Rel family members regulating the ICAM-1 promoter in monocytic THP-1 cells. 
A kappa B-site was identified in the promoter of the intercellular adhesion molecule-1 (ICAM-1) gene, which is involved in regulation of ICAM-1 expression by tumor necrosis factor alpha (TNF-alpha) and glucocorticoids. We now report on the transcription factors which bind and transactivate this enhancer sequence. In vitro, the ICAM-1 kappa B site appeared to bind RelA and c-Rel homodimers as well as heterodimers with NF-kappa B1, but weakly NF-kappa B1 homodimers. In addition, both RelA and c-Rel, but not NF-kappa B1, were shown to transactivate an ICAM-1 kappa B-reporter construct. In monocytic THP-1 cells TNF-alpha induced two nuclear complexes which in vitro bound to the ICAM-1 kappa B site. Using antibodies in an electrophoretic mobility supershift assay, one of these complexes was shown to contain NF-kappa B1 and RelA, and to bind with higher affinity to the consensus kappa B site in the HIV long terminal repeat. The second complex contained RelA, and exhibited higher affinity towards the ICAM-1 kappa B than to the HIV kappa B site. The glucocorticoid receptor was shown to repress activity of both the RelA homodimer and the NF-kappa B1/RelA heterodimer. We argue that in vivo RelA homodimers are likely to play a dominant role in TNF-alpha-induced ICAM-1 transcription in monocytic cells. 


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Activity of the kappa B enhancer of the interleukin-2 receptor alpha chain in somatic cell hybrids is accompanied by the nuclear localization of NF-kappa B. 
The two nuclear proteins NF-kappa B (consisting of subunits p50 and p65) and the DNA-binding subunit of NF-kappa B (p50) by itself, also called KBF1, are constitutively expressed and localized in the nucleus of the human T-cell line IARC 301.5. In order to define the roles of these two factors, which bind to the same kappa B enhancers, in transcription activation we have prepared somatic cell hybrids between IARC 301.5 and a murine myeloma. Most hybrids express both KBF1 and NF-kappa B in their nuclei, but one hybrid expresses only KBF1. The kappa B enhancer of the gene encoding the interleukin-2 (IL-2) receptor alpha chain (IL-2R alpha) is functional only in the hybrids expressing nuclear NF-kappa B. These findings show that nuclear NF-kappa B is necessary to activate the kappa B enhancer, while KBF1 by itself is not sufficient. We propose that KBF1 is a competitive inhibitor of NF-kappa B and discuss how these factors may be involved in the transient expression of IL-2 and IL-2R alpha genes during the immune response. 


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Differential regulation of proto-oncogenes c-jun and c-fos in T lymphocytes activated through CD28. 
The T cell surface molecule CD28 binds to ligands on accessory cells and APCs, playing an important costimulatory role in the response of T cells to Ags. Our knowledge of the intracellular signaling pathways coupled to this receptor is incomplete. In addition to activation of phospholipase C gamma 1, ligation of this receptor also seems to activate a calcium-independent, CD28-specific pathway. In this paper, we report that cross-linking of CD28 (but not CD2, CD5, LFA-1, or CD7) leads to an elevation of c-jun mRNA, with only minimal activation of c-fos expression. CD28-dependent induction of c-jun expression requires protein tyrosine kinase activity, but does not depend on activation of a phorbol ester-responsive protein kinase C or elevation of cytosolic calcium. Furthermore, CD28-dependent elevation of c-jun mRNA does not appear to be mediated at the level of mRNA stability. A mechanism is suggested whereby expression of c-jun and junB, in the absence of members of the fos family, can prevent inappropriate activation of T cells caused by ligation of CD28 in the absence of a specific antigenic stimulus. 


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Antioxidants inhibit monocyte adhesion by suppressing nuclear factor-kappa B mobilization and induction of vascular cell adhesion molecule-1 in endothelial cells stimulated to generate radicals. 
Cell adhesion to endothelial cells stimulated by tumor necrosis factor-alpha (TNF) is due to induction of surface receptors, such as vascular cell adhesion molecule-1 (VCAM-1). The antioxidant pyrrolidine dithiocarbamate (PDTC) specifically inhibits activation of nuclear factor-kappa B (NF-kappa B). Since kappa B motifs are present in VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) promoters, we used PDTC to study the regulatory mechanisms of VCAM-1 and ICAM-1 induction and subsequent monocyte adhesion in TNF-treated human umbilical vein endothelial cells (HUVECs). PDTC or N-acetylcysteine dose dependently reduced TNF-induced VCAM-1 but not ICAM-1 surface protein (also in human umbilical arterial endothelial cells) and mRNA expression (by 70% at 100 mumol/L PDTC) in HUVECs as assessed by flow cytometry and polymerase chain reaction. Gel-shift analysis in HUVECs demonstrated that PDTC prevented NF-kappa B mobilization by TNF, suggesting that only VCAM-1 induction was controlled by NF-kappa B. Since HUVECs released superoxide anions in response to TNF, and H2O2 induces VCAM-1, PDTC may act as a radical scavenger. Although ICAM-1 induction was unaffected, inhibitors of NADPH oxidase (apocynin) or cytochrome P-450 (SKF525a) suppressed VCAM-1 induction by TNF, revealing that several radical-generating systems are involved in its regulation. PDTC, apocynin, or SKF525a decreased adhesion of monocytic U937 cells to TNF-treated HUVECs (by 75% at 100 mumol/L PDTC). Inhibition by anti-VCAM-1 monoclonal antibody 1G11 indicated that U937 adhesion was VCAM-1 dependent and suppression by antioxidants was due to reduced VCAM-1 induction. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Reduction of tumour necrosis factor alpha expression and signalling in peripheral blood mononuclear cells from patients with thalassaemia or sickle cell anaemia upon treatment with desferrioxamine. 
Recent evidence indicates that the rate of progression of the HIV-1 disease is significantly reduced in thalassaemia major patients upon treatment with high doses of desferrioxamine (DFX). The authors have previously demonstrated that in vitro exposure of mononuclear cells to DFX decreases the bioavailability of tumour necrosis factor alpha (TNF-alpha) which has a stimulatory effect on HIV-1 replication. In this study, therefore, TNF-alpha bioavailability from mononuclear cells isolated from 10 patients with thalassaemia or sickle cell anaemia given DFX as compared to 10 untreated subjects has been evaluated. Evidence is presented showing that DFX treatment reduces TNF-alpha bioavailability (P<0.05) by inhibiting its steady state (P<0.05) and by enhancing its inactivation through binding to soluble TNF-alpha receptor type II (P<0.05). We also show that DFX treatment limits the in vivo activation of NF-kappaB, a transcription factor involved in both TNF-alpha gene transcription and TNF-alpha signalling (P<0.005). We conclude that TNF-alpha bioavailability and signalling are impaired in patients upon DFX treatment. This mechanism may contribute to delayed progression of the HIV-1 infection in vivo. Copyright 1999 Academic Press. 


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Heterodimerization and transcriptional activation in vitro by NF-kappa B proteins. 
The NF-kappa B family of transcription proteins represents multiple DNA binding, rel related polypeptides that contribute to regulation of genes involved in immune responsiveness and inflammation, as well as activation of the HIV long terminal repeat. In this study multiple NF-kappa B related polypeptides ranging from 85 to 45 kDa were examined for their capacity to interact with the PRDII regulatory element of interferon beta and were shown to possess distinct intrinsic DNA binding affinities for this NF-kappa B site and form multiple DNA binding homo- and heterodimer complexes in co-renaturation experiments. Furthermore, using DNA templates containing two copies of the PRDII domain linked to the rabbit beta globin gene, the purified polypeptides specifically stimulated NF-kappa B dependent transcription in an in vitro reconstitution assay as heterodimers but not as p50 homodimers. These experiments emphasize the role of NF-kappa B dimerization as a distinct level of transcriptional control that may permit functional diversification of a limited number of regulatory proteins. 


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A nuclear factor NF-GM2 that interacts with a regulatory region of the GM-CSF gene essential for its induction in responses to T-cell activation: purification from human T-cell leukemia line Jurkat cells and similarity to NF-kappa B. 
Activation of T cells by antigen, lectin, or a combination of phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) leads to the induction of genes for a set of lymphokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF). We demonstrated in earlier studies that the upstream region of the mouse GM-CSF promoter at positions between -95 and -73 is essential for transcriptional activation in response to PMA/A23187. This region contains two DNA-binding motifs, GM2 and GC-box. The GM2 sequence (GGTAGTTCCC) is recognized by an inducible factor NF-GM2; the other (CCGCCC) by constitutive factors A1, A2, and B. To elucidate the mechanism of GM-CSF gene activation, we have purified the inducible factor NF-GM2 from the nuclear extract of stimulated Jurkat cells on the basis of specific DNA-binding activity. The purified NF-GM2 consists of 50 (p50) and 65 kDa (p65) polypeptides and has a binding activity specific for both the GM-CSF and immunoglobulin kappa (GGAAAGTCCC) enhancers. Electrophoretically purified p50 alone can form a protein-DNA complex, but in the mixture, p50 associates preferentially with p65 to form the NF-GM2 complex. In addition, p65 gave per se, with low affinity, a protein-DNA complex that migrated more slowly than native NF-GM2 complex. Furthermore, an antiserum against KBF1 (identical to 50 kDa NF-kappa B protein) reacted with the p50 of NF-GM2, indicating that the NF-GM2 polypeptide cannot be immunologically differentiated from the 50 kDa subunit of NF-kappa B. The purified NF-GM2 activated in vitro transcription from the kappa B enhancer, while it failed to stimulate transcription from the GM-CSF promoter harboring the GM2 sequence. This suggests that the activation mechanism of the GM-CSF gene through the GM2/GC-box sequence is different from that of genes carrying the kappa B enhancer alone. 


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Retinoic acid-induced modulation of IL-2 mRNA production and IL-2 receptor expression on T cells. 
BACKGROUND: Retinoic acid (RA) has important immune-modulating effects on both T and B cell function. Our laboratory has shown that RA can enhance in vitro polyclonal B cell immunoglobulin (Ig) response. Investigating cytokines known to affect B cell differentiation, we have recently shown that IL-6 production is augmented by RA. In the present study we have examined the immune modulating effects of RA on IL-2 mRNA, another important cytokine for B cell immunoglobulin production, the expression of IL-2 receptors on T cells, and the RA nuclear receptors. METHODS: Purified T cells were obtained from adenoidal tissues, and incubated with RA (10(-7) M) or DMSO solvent/media control for 0, 6-8, and 24 h. Total mRNA was extracted from T cells, and using RT-PCR, changes in the production of IL-2 and RA receptors (RAR)-alpha,beta,gamma mRNA were determined. The effects of RA on IL-2-alpha receptor expression was determined by flow cytometry on T cells. CONCLUSION: These studies suggest that RA can augment IL-2 mRNA production by T cells with a possible paracrine effect on IL-2R-alpha expression. These changes appear to be mediated by RAR-alpha. Thus, IL-2 may be another important cytokine modulated by RA in the immune response. 


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Interleukin-3 expression by activated T cells involves an inducible, T-cell-specific factor and an octamer binding protein. 
Interleukin-3 (IL-3) is exclusively expressed by activated T and natural killer cells, a function that is tightly controlled both in a lineage-specific and in a stimulation-dependent manner. We have investigated the protein binding characteristics and functional importance of the ACT-1-activating region of the IL-3 promoter. This region binds an inducible, T-cell-specific factor over its 5' end, a site that is necessary for the expression of IL-3 in the absence of other upstream elements. Over its 3' end, it binds a factor that is ubiquitously and constitutively expressed. This factor is Oct-1 or an immunologically related octamer-binding protein, and it plays a role in coordinating the activity of several regulatory elements. These characteristics make the ACT-1 site analogous to the activating ARRE-1 site in the IL-2 promoter. Furthermore, and despite a lack of sequence homology, the promoters of IL-3 and IL-2 share an organizational pattern of regulatory elements that is likely to be important for the T-cell-specific expression of these genes. 


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IL-1 receptor and TCR signals synergize to activate NF-kappa B-mediated gene transcription. 
Previous studies have demonstrated that IL-1 receptor (IL-1R)- and TCR-initiated signals can interact synergistically to increase the rate of transcription of several lymphokine and lymphokine receptor genes during the competence phase of the activation program in T helper lymphocytes. In this report we describe how signals initiated through the type I IL-1R interact with signals from the antigen receptor to synergistically augment the transactivating properties of NF-kappa B. The synergistic antigen receptor initiated signals are mediated through protein kinase C because they can be mimicked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, but not with calcium ionophores; and are staurosporine sensitive but cyclosporine resistant. Gel shift analyses demonstrate that NF-kappa B nuclear translocation is stimulated primarily by IL-1 rather than by antigen receptor signals. Western blot and phosphorylation analyses demonstrate that the synergistic effect on NF-kappa B functional activity is independent of I kappa B alpha (MAD3)-NF-kappa B dissociation in the cytosol and is not associated with I kappa B nuclear translocation. The IL-1-induced NF-kappa B DNA nuclear localization is transient and can be prolonged either by an antigen receptor-initiated signal or by inhibiting protein synthesis. These results suggest that IL-1 induces both NF-kappa B nuclear translocation and the synthesis of a protein(s) responsible for terminating NF-kappa B-DNA interaction in the nucleus. Antigen receptor signals prolong NF-kappa B-DNA interaction, probably by functionally antagonizing the IL-1-induced synthesis of a protein(s) responsible for the transient NF-kappa B-DNA interaction and consequently synergistically enhance IL-1-induced NF-kappa B-dependent gene transcription. 


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The DNA binding domain of the A-MYB transcription factor is responsible for its B cell-specific activity and binds to a B cell 110-kDa nuclear protein. 
Expression studies as well as the use of transgenic animals have demonstrated that the A-MYB transcription factor plays central and specific role in the regulation of mature B cell proliferation and/or differentiation. Furthermore, it is highly expressed in Burkitt's lymphoma cells and may participate in the pathogenesis of this disease. We have therefore investigated the transcriptional activity of A-MYB and its regulation in several human lymphoid cell lines using co-transfection assays and show that A-MYB is transcriptionally active in all the B cell lines studied, but not in T cells. In particular the best responder cell line was the Burkitt's cell line Namalwa. The activity of A-MYB in B and not T cells was observed when either an artificial construct or the c-MYC promoter was used as a reporter. Furthermore, the functional domains responsible for DNA binding, transactivation, and negative regulation, previously characterized in a fibroblast context, were found to have similar activity in B cells. The region of A-MYB responsible for the B cell specific activity was defined to be the N-terminal 218 amino acids containing the DNA binding domain. Finally, a 110-kDa protein has been identified in the nuclei of all the B, but not T, cell lines that specifically binds to this A-MYB N-terminal domain. We hypothesize that this 110-kDa protein may be a functionally important B cell-specific co-activator of A-MYB. 


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The normal cell cycle activation program is exploited during the infection of quiescent B lymphocytes by Epstein-Barr virus. 
B lymphocytes in the peripheral circulation are maintained in a non-proliferative state. Antigen recognition stimulates limited proliferation, whereas infection with Epstein-Barr virus (EBV) results in continual proliferation and the outgrowth of immortal cell lines. Because it is not clear at which point in cell cycle the peripheral B lymphocytes are arrested, we characterized the expression of several cell cycle-associated genes in quiescent and stimulated cells. We show that the expression of four cell genes, cdc-2, cyclin E, CD23, and cyclin D2, are up-regulated approximately 100-fold as a result of EBV-mediated immortalization. Because these genes play a positive role in cell proliferation, we suggest that this regulatory switch contributes to controlling entry into the cell cycle. Transient stimulation of quiescent B lymphocytes with either a cocktail of anti-CD40, anti-IgM, and IL4, or EBV results in the rapid expression of the same four genes, suggesting that, after infection, EBV exploits the normal program of B-lymphocyte cell cycle activation. 


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Anti-Ehrlichia chaffeensis antibody complexed with E. chaffeensis induces potent proinflammatory cytokine mRNA expression in human monocytes through sustained reduction of IkappaB-alpha and activation of NF-kappaB. 
Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects monocytes and macrophages and is the etiologic agent of human ehrlichiosis in the United States. Our previous studies showed that the exposure of human monocytes to E. chaffeensis induces the expression of interleukin-1beta (IL-1beta), IL-8, and IL-10 genes in vitro but not the expression of tumor necrosis factor alpha (TNF-alpha) and IL-6 mRNAs. In this study, the effect of anti-E. chaffeensis antibody complexed with E. chaffeensis on the expression of major proinflammatory cytokines in human monocytes was examined. Human monocytic cell line THP-1 was treated with E. chaffeensis which had been preincubated with human anti-E. chaffeensis serum for 2 h, and the levels of cytokine mRNAs were evaluated by competitive reverse transcription-PCR. Anti-E. chaffeensis antibody complexed with E. chaffeensis significantly enhanced mRNA expression of IL-1beta in THP-1 cells. The expression of TNF-alpha and IL-6 mRNAs was also induced. The levels of secreted IL-1beta, TNF-alpha, and IL-6 during 24 h of stimulation were comparable to those induced by Escherichia coli lipopolysaccharide at 1 microg/ml. Fab fragment of anti-E. chaffeensis immunoglobulin G complexed with E. chaffeensis did not induce any of these three cytokines, indicating that ehrlichial binding is required for IL-1beta mRNA expression and that binding of the immune complex to the Fc gamma receptor is required for TNF-alpha and IL-6 mRNA expression and enhanced IL-1beta mRNA expression. Furthermore, prolonged degradation of IkappaB-alpha and activation of NF-kappaB were demonstrated in THP-1 cells exposed to anti-E. chaffeensis serum and E. chaffeensis. This result implies that development of anti-E. chaffeensis antibody in patients can result in the production of major proinflammatory cytokines, which may play an important role in the pathophysiology of ehrlichiosis and immune responses to it. 


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Inhibition of NF-AT-dependent transcription by NF-kappa B: implications for differential gene expression in T helper cell subsets. 
Activation of individual CD4+ T cells results in differential lymphokine expression: interleukin 2 (IL-2) is preferentially produced by T helper type 1 (TH1) cells, which are involved in cell-mediated immune responses, whereas IL-4 is synthesized by TH2 cells, which are essential for humoral immunity. The Ca(2+)-dependent factor NF-ATp plays a key role in the inducible transcription of both these lymphokine genes. However, while IL2 expression requires the contribution of Ca(2+)- and protein kinase C-dependent signals, we report that activation of human IL4 transcription through the Ca(2+)-dependent pathway is diminished by protein kinase C stimulation in Jurkat T cells. This phenomenon is due to mutually exclusive binding of NF-ATp and NF-kappa B to the P sequence, an element located 69 bp upstream of the IL4 transcription initiation site. Human IL4 promoter-mediated transcription is downregulated in Jurkat cells stimulated with the NF-kappa B-activating cytokine tumor necrosis factor alpha and suppressed in RelA-overexpressing cells. In contrast, protein kinase C stimulation or RelA overexpression does not affect the activity of a human IL4 promoter containing a mouse P sequence, which is a higher-affinity site for NF-ATp and a lower-affinity site for RelA. Thus, competition between two general transcriptional activators, RelA and NF-ATp, mediates the inhibitory effect of protein kinase C stimulation on IL4 expression and may contribute to differential gene expression in TH cells. 


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Epstein-Barr virus-transforming protein latent infection membrane protein 1 activates transcription factor NF-kappaB through a pathway that includes the NF-kappaB-inducing kinase and the IkappaB kinases IKKalpha and IKKbeta. 
The Epstein-Barr virus oncoprotein latent infection membrane protein 1 (LMP1) is a constitutively aggregated pseudo-tumor necrosis factor receptor (TNFR) that activates transcription factor NF-kappaB through two sites in its C-terminal cytoplasmic domain. One site is similar to activated TNFRII in associating with TNFR-associated factors TRAF1 and TRAF2, and the second site is similar to TNFRI in associating with the TNFRI death domain interacting protein TRADD. TNFRI has been recently shown to activate NF-kappaB through association with TRADD, RIP, and TRAF2; activation of the NF-kappaB-inducing kinase (NIK); activation of the IkappaB alpha kinases (IKKalpha and IKKbeta); and phosphorylation of IkappaB alpha. IkappaB alpha phosphorylation on Ser-32 and Ser-36 is followed by its degradation and NF-kappaB activation. In this report, we show that NF-kappaB activation by LMP1 or by each of its effector sites is mediated by a pathway that includes NIK, IKKalpha, and IKKbeta. Dominant negative mutants of NIK, IKKalpha, or IKKbeta substantially inhibited NF-kappaB activation by LMP1 or by each of its effector sites. 


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Inhibition of NF-kappa B activation in vitro and in vivo: role of 26S proteasome. 
It is becoming increasingly apparent that NF-kappa B plays a critical role in regulating the inflammatory response. Data obtained from studies in our laboratories demonstrate that the proteasome plays an important role in the inflammatory cascade by regulating the activation of NF-kappa B. Indeed, the availability of selective and orally active proteasome inhibitors should prove useful in delineating the roles of the proteasome and NF-kappa B in other pathophysiological conditions such as cancer and heart disease. 


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Transactivation by CIITA, the type II bare lymphocyte syndrome-associated factor, requires participation of multiple regions of the TATA box binding protein. 
CIITA is a positive regulator of class II major histocompatibility complex gene transcription that has been found to be defective in one of the five complementation groups of class II major histocompatibility complex-negative cell lines. Its N-terminal region is capable of activating transcription from a reporter gene when fused to a DNA binding domain. We have investigated the mechanism of transactivation mediated by the CIITA activation domain by studying its role in the process of transcription initiation and elongation. Specifically the altered specificity TBP (TATA box binding protein) assay has been used to analyze the response of the CIITA activation domain to mutations in TBP known to disrupt its interaction with its associated general factors. Transactivation by CIITA was extremely sensitive to a mutation in TBP that in yeast is known to abolish VP16-mediated transcription but leaves basal transcription unaffected. A TBP mutant defective in interaction with TBP-associated factor TAFII250 also failed to mediate transactivation through the CIITA activation domain. Certain interactions between TBP and general factors that are specifically required for acidic activation domains were also required for CIITA-mediated transactivation to reach its full potential. Finally, like VP16, CIITA was able to stimulate elongation of transcription. Overall the mechanism of transactivation by the human B-cell-specific CIITA is very similar to that mediated by the herpes virus transactivator VP16 in the ways that have been tested. 


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Transactivation of the human immunodeficiency virus promoter by human herpesvirus 6 (HHV-6) strains GS and Z-29 in primary human T lymphocytes and identification of transactivating HHV-6(GS) gene fragments. 
Human herpesvirus 6 (HHV-6) can activate the human immunodeficiency virus (HIV) promoter and accelerate cytopathic effects in HIV-infected human T cells. This study examines the regions of the HIV promoter required for HHV-6 transactivation in a heterogeneous population of primary human T lymphocytes with or without antigenic stimulation. Two different strains of HHV-6, GS and Z29, transactivated the HIV promoter. The GS strain transactivated the promoter in both stimulated and resting T cells, while the Z29 strain increased HIV promoter activity only in stimulated T cells. Three DNA clones containing HHV-6(GS) genomic fragments transactivated the HIV promoter in cotransfected T cells. A 21.4-kb DNA clone, pZVB70, showed the highest transactivating ability, while two other DNA fragments, pZVB10 (6.2 kb) and pZVH14 (8.7 kb), showed lower activity. One of these clones, pZVH14, activated the HIV promoter construct containing a mutation in the NF kappa B site. However, this mutated NF kappa B promoter was not transactivated during HHV-6(GS) infection or after cotransfection with pZVB70 or pZVB10. These data indicate that the NF kappa B sites of the HIV promoter are essential for its transactivation during HHV-6(GS) infection. By increasing HIV promoter activity in primary T lymphocytes, HHV-6 may consequently increase HIV replication, leading to an increase in the cytopathic effect on coinfected human T cells. 


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HIV1 infection of human monocytes and macrophages promotes induction or translocation of NF-KB-related factors. 
In 1991, we demonstrated, using electrophoretic mobility shift assays, that 3 different factors (termed B1, B2 and B3) with affinity for the KB-enhancer target sequence were specifically detected in nuclear extracts from HIV1-infected monocytes and macrophages. The B2 factor was induced in the nuclei of these cells only upon HIV1 infection. The B3 factor was only slightly evident in nuclei of uninfected cells but was readily detectable in nuclei of infected monocytes. Its expression remained very low in nuclei of HIV1-infected macrophages. In this paper, we demonstrate that the B2 factor is expressed in the cytosol of monocytes and macrophages as a DNA-binding protein, indicating that it is not associated with an inhibitor (IKB). This factor remained clustered in the cytosol and was translocated to the nuclei only after HIV1 infection. The B3 factor is detected in the cytosol only when cells are HIV1-infected. The role of HIV1 infection in the expression and the translocation of these factors is discussed. 


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The intracellular parasite Theileria parva protects infected T cells from apoptosis. 
Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasite Theileria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-kappaB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-kappaB activation in T. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-kappaB activation plays a crucial role in the survival of T. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Consequently, inhibition of NF-kappaB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-kappaB activation pathway, such as IkappaBalpha or p65, prompt rapid apoptosis of T. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-kappaB activation is an essential step in maintaining the transformed phenotype of the infected cells. 


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TAL1 and LIM-only proteins synergistically induce retinaldehyde dehydrogenase 2 expression in T-cell acute lymphoblastic leukemia by acting as cofactors for GATA3. 
Previously, we have shown that TAL1 and the LIM-only protein gene (LMO) are regularly coactivated in T-cell acute lymphoblastic leukemia (T-ALL). This observation is likely to relate to the findings that TAL1 and LMO are highly synergistic in T-cell tumorigenesis in double-transgenic mice. To understand the molecular mechanisms of functional synergy between TAL1 and LMO in tumorigenesis and transcriptional regulation, we tried to identify downstream target genes regulated by TAL1 and LMO by a subtractive PCR method. One of the isolated genes, that for retinaldehyde dehydrogenase 2 (RALDH2), was regularly expressed in most of the T-ALL cell lines that coexpressed TAL1 and LMO. Exogenously transfected TAL1 and LMO, but not either alone, induced RALDH2 expression in a T-ALL cell line, HPB-ALL, not expressing endogeneous TAL1 or LMO. The RALDH2 transcripts in T-ALL were, however, mostly initiated within the second intron. Promoter analysis revealed that a GATA site in a cryptic promoter in the second intron was essential and sufficient for the TAL1- and LMO-dependent transcriptional activation, and GATA3 binds to this site. In addition, forced expression of GATA3 potentiated the induction of RALDH2 by TAL1 and LMO, and these three factors formed a complex in vivo. Furthermore, a TAL1 mutant not binding to DNA also activated the transcription of RALDH2 in the presence of LMO and GATA3. Collectively, we have identified the RALDH2 gene as a first example of direct transcriptional target genes regulated by TAL1 and LMO in T-ALL. In this case, TAL1 and LMO act as cofactors for GATA3 to activate the transcription of RALDH2. 


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Posttranscriptional regulation of macrophage tissue factor expression by antioxidants. 
Tissue factor (TF) expression by cells of monocyte/macrophage lineage represents an important mechanism underlying the initiation of fibrin deposition at sites of extravascular inflammation. Recent evidence suggests a role for oxidant stress in the signalling pathway of various cell types by virtue of its ability to induce DNA binding of various transcription factors, including nuclear factor kappa B and AP-1. The effect of antioxidant treatment on lipopolysaccharide (LPS)-induced TF expression was examined in murine peritoneal macrophages and human monocytes. Both pyrrolidine dithiocarbamate, an oxidant scavenger, and N-acetyl-cysteine, a precursor of the endogenous antioxidant glutathione, inhibited stimulation of macrophage procoagulant activity by LPS. Northern blot analysis showed that neither of these agents reduced LPS-stimulated TF mRNA accumulation, thereby suggesting a posttranscriptional mechanism for the effect. Immunofluorescence studies of human monocytes using polyclonal anti-TF antibody showed that N-acetyl-cysteine treatment prevented the characteristic plasmalemmal localization of TF antigen that occurs in response to LPS. Western blot analysis showed that N-acetyl-cysteine reduced the accumulation of the 47-kD mature glycoprotein in LPS-treated cells, a finding consistent with the results of the immunofluorescence studies. Furthermore, these conditions did not result in an accumulation of the less mature forms of TF. When considered together, these data suggest that antioxidants exert their effects by impairing translation and/or by causing degradation of newly translated protein. The effect of antioxidants on tumor necrosis factor appeared to be species specific, with no effect on LPS-induced tumor necrosis factor in murine cells, but with inhibition in human monocytes. The posttranscriptional effect of antioxidants on TF expression data suggests a novel mechanism whereby these agents might modulate monocyte/macrophage activation. 


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Natural variants of the HIV-1 long terminal repeat: analysis of promoters with duplicated DNA regulatory motifs. 
Sequence variation in the long terminal repeat (LTR) region of HIV-1 was analyzed in viral isolates of 17 infected individuals. Two classes of LTR size variants were found. One HIV-1 variant was detected containing an additional binding site for the transcription factor Sp1. Another LTR size variation was observed in four patients in a region just upstream of the NF-kappa B enhancer. This variation was the result of a duplication of a short DNA sequence (CTG-motif). Cell culture experiments demonstrated that the natural variant with four Sp1 sites had a slightly higher promoter activity and viral replication rate than the isogenic control LTR with three Sp1 sites. No positive effect of the duplicated CTG-motif could be detected. In order to measure small differences in virus production more accurately, equal amounts of a size variant and the wild-type plasmid were cotransfected into T-cells. The virus with four Sp1 sites did outgrow the three Sp1 virus in 35 days of culture and CTG-monomer virus outcompeted the CTG-dimer virus in 42 days. Based on these results we estimate a 5-10% difference in virus production of the LTR variants when compared to that of wild-type. 


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Human immunodeficiency virus type 1 Nef protein down-regulates transcription factors NF-kappa B and AP-1 in human T cells in vitro after T-cell receptor stimulation. 
Human immunodeficiency virus type 1 (HIV-1) negative factor (Nef) has been shown to down-regulate the transcription factors NF-kappa B and AP-1 in vitro. To define the mechanism of action of the Nef protein, the signal transduction pathways which may be affected in T cells by constitutive expression of the nef gene were examined. Stimulation of T cells with tumor necrosis factor, interleukin-1, or lipopolysaccharide resulted in the recruitment of transcriptional factors to a similar level whether or not the cells expressed the nef gene. On the other hand, stimulation of T cells by mitogens or antibodies to the T-cell receptor (TCR)-CD3 complex resulted in the down-regulation of transcriptional factors NF-kappa B and AP-1 in cells expressing the nef gene compared with cells not expressing the nef gene. Because the Nef protein does not affect the surface expression of the CD3-TCR complex, we conclude that the Nef protein down-regulates the transcriptional factors NF-kappa B and AP-1 in T cells in vitro through an effect on the TCR-dependent signal transduction pathway. 


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Involvement of cyclic AMP-dependent protein kinases in the signal transduction pathway for interleukin-1. 
Expression of a highly specific protein inhibitor for cyclic AMP-dependent protein kinases in interleukin-1 (IL-1)-responsive cells blocked IL-1-induced gene transcription that was driven by the kappa immunoglobulin enhancer or the human immunodeficiency virus long terminal repeat. This inhibitor did not affect protein kinase C-mediated gene transcription, suggesting that cyclic AMP-dependent protein kinases are involved in the signal transduction pathway for IL-1 in a number of responsive cell types. 


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Interleukin-10 inhibits expression of both interferon alpha- and interferon gamma- induced genes by suppressing tyrosine phosphorylation of STAT1. 
Interleukin-10 (IL-10) helps maintain polarized T-helper cells in a T-helper lymphocyte 2 (Th2) phenotype. Part of this process involves the prevention of the development of Th1 cells, which are a primary source of interferon gamma (IFNgamma), a potent activator of monocytes and an inhibitor of Th2 proliferation. Because monocytes and macrophages are important mediators of Th1-type responses, such as delayed-type hypersensitivity, we sought to determine if IL-10 could directly mediate inhibition of IFNgamma- and IFNalpha-induced gene expression in these cells. Highly purified monocytes were incubated with IL-10 for 60 to 90 minutes before the addition of IFNgamma or IFNalpha. IL-10 preincubation resulted in the inhibition of gene expression for several IFN-induced genes, such as IP-10, ISG54, and intercellular adhesion molecule-1. The reduction in gene expression resulted from the ability of IL-10 to suppress IFN-induced assembly of signal transducer and activator of transcription (STAT) factors to specific promoter motifs on IFNalpha- and IFNgamma-inducible genes. This was accomplished by preventing the IFN-induced tyrosine phosphorylation of STAT1, a component of both IFNalpha- and IFNgamma-induced DNA binding complexes. Therefore, IL-10 can directly inhibit STAT-dependent early response gene expression induced by both IFNalpha and IFNgamma in monocytes by suppressing the tyrosine phosphorylation of STAT1. This may occur through the ability of IL-10 to induce expression of the gene, suppressor of cytokine signaling 3 (SOCS3). 


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Okadaic acid is a potent inducer of AP-1, NF-kappa B, and tumor necrosis factor-alpha in human B lymphocytes. 
Treatment of human B lymphocytes with an optimal concentration of okadaic acid, an inhibitor of phosphatases 1 and 2A, resulted in the induction of the transcription factor, AP-1 and a marked increase in NF-kappa B levels. In contrast, no effect on the levels of the octamer binding proteins, Oct-1 or Oct-2, were found. Since both AP-1 and NF-kappa B have been reported to be important in the induction of the tumor necrosis factor-alpha (TNF-alpha) gene we examined the effects of okadaic acid on TNF-alpha mRNA levels. Treatment with okadaic acid resulted in a striking increase in TNF-alpha mRNA transcripts within 1 h of stimulation and large amounts of TNF-alpha were released into the culture media. Although okadaic acid provides a potent inductive signal for AP-1 and NF-kappa B it did not induce either B cell proliferation or immunoglobulin secretion. 


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Long-term inositol phosphate release, but not tyrosine kinase activity, correlates with IL-2 secretion and NF-AT induction in anti-CD3-activated peripheral human T lymphocytes. 
The cascade of events within the first few minutes of T cell stimulation has been well characterized. Although many second messengers have been shown to be necessary and sufficient for T cell activation in a number of model systems, the rate-limiting step in peripheral T cells has not been demonstrated. To model effective versus ineffective CD3-mediated stimulation in peripheral T cells, we used two anti-CD3 mAbs that differ in their ability to stimulate purified T cells: OKT3, which causes early second messenger generation but is unable to activate T cells without a second signal, and 64.1, which stimulates T cell proliferation on its own. We found that tyrosine kinase activity was similar for both mAbs over a period of hours. However, the inositol phosphate response was stronger for 64.1 than for OKT3. To tie these events to gene activation, we measured NF-kappa B and NF-AT activity in the nucleus after anti-CD3 stimulation. Both stimuli induced the appearance of the NF-kappa B components (c-Rel, p65 (RelA), and p50 (NF-kappa B1)) and NF-kappa B DNA binding activity in the nucleus. However, only 64.1 induced NF-AT in the nucleus, correlating with its ability to activate T cells. Thus, NF-AT induction and IL-2 secretion were correlated with the levels of inositol phosphate release but not with gross levels of tyrosine kinase activity induced late following the response. On the other hand, NF-kappa B induction and IL-2 receptor expression occurred even with the smaller second messenger response generated by OKT3. 


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Differentiation of T-helper lymphocytes: selective regulation by members of the STAT family of transcription factors. 
Interleukin-4 (IL-4) and interleukin-12 (IL-12) control the differentiation of T-helper cells. Here we summarize studies which investigate the mechanism by which these cytokines selectively reprogramme gene expression in T-lymphocytes. Cytokine stimulation leads to the phosphorylation of specific tyrosine residues within the intracellular domain of the corresponding cytokine receptor. These phosphotyrosines serve as docking sites for latent, cytoplasmic transcription factors known as signal transducers and activators of transcription (Stat) proteins. Receptor/Stat interaction is mediated by the src homology 2 (SH2) domain of the corresponding Stat protein. Although Stat binding to the intracellular domain of the cytokine receptor strongly depends on the phosphotyrosine residue, the recruitment of a specific Stat protein is dictated by amino acid residues C-terminal to the phosphotyrosine. Specific docking sites within individual cytokine receptors have been identified for almost all Stat proteins. The direct coupling between cytokine receptor and transcription factor helps to explain how different cytokines elicit distinct patterns of gene expression. 


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Function of NF-kappa B/Rel binding sites in the major histocompatibility complex class II invariant chain promoter is dependent on cell-specific binding of different NF-kappa B/Rel subunits. 
The promoter of the human major histocompatibility complex class II-associated invariant-chain gene (Ii) contains two NF-kappa B/Rel binding sites located at -109 to -118 (Ii kappa B-1) and -163 to -172 (Ii kappa B-2) from the transcription start site. We report here that the differential function of each of these NF-kappa B/Rel sites in several distinct cell types depends on cell-specific binding of NF-kappa B/Rel transcription factors. Ii kappa B-1 is a positive regulatory element in B-cell lines and in the Ii-expressing T-cell line, H9, but acts as a negative regulatory element in myelomonocytic and glia cell lines. In vivo protein-DNA contacts are detectable at Ii kappa B-1 in cell lines in which this site is functional as either a positive or negative regulator. Electrophoretic mobility supershift assays determine that members of the NF-kappa B/Rel family of transcription factors can bind to this site in vitro and that DNA-binding complexes that contain p50, p52, p65, and cRel correlate with positive regulation whereas the presence of p50 correlates with negative regulation. Ii kappa B-2 is a site of positive regulation in B-cell lines and a site of negative regulation in H9 T cells, myelomonocytic, and glial cell lines. In vivo occupancy of this site is observed only in the H9 T-cell line. Again, in vitro supershift studies indicate that the presence of p50, p52, p65, and cRel correlates with positive function whereas the presence of only p50 and p52 correlates with negative function. This differential binding of specific NF-kappa B/Rel subunits is likely to mediate the disparate functions of these two NF-kappa B/Rel binding sites. 


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Induction of relA(p65) and I kappa B alpha subunit expression during differentiation of human peripheral blood monocytes to macrophages. 
We evaluated the expression and DNA binding activity of nuclear factor (NF)-kappa B subunits in human peripheral blood monocytes and in monocyte-derived macrophages (MDMs). Constitutive DNA binding activity consisting of p50 homodimers was detected in nuclear extracts from both cell types. An additional complex composed of p50/RelA(p65) heterodimers appeared only in nuclear extracts from 7-day MDMs. Immunoblot analysis showed that the p50 subunit was constitutively expressed in monocytes and MDMs. In contrast, the RelA(p65) subunit was barely detectable in monocytes, but its level increased markedly in MDMs. Analysis of RelA(p65) mRNA revealed that the stability of RelA(p65) mRNA was significantly higher in MDMs, compared with monocytes. In MDMs, an upregulation of I kappa B alpha synthesis as well as the appearance of a novel M(r) 40,000 form of I kappa B alpha were also observed. These results suggest that macrophage differentiation results in the expression of active p50/RelA(p65) heterodimers with the capacity to activate target gene expression. The parallel induction of I kappa B alpha synthesis may allow for the continuous presence of a cytoplasmic reservoir of p50/RelA(p65) complexes that are readily available for inducer-mediated stimulation. 


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CholecystokininB receptor from human Jurkat lymphoblastic T cells is involved in activator protein-1-responsive gene activation. 
The aim of this study was to analyze the role of cholecystokinin (CCK(B)) receptor in human lymphoblastic Jurkat T cells. We investigated the trophic effect resulting from activation of such a receptor by using the reporter gene strategy. For this purpose, we transiently transfected Jurkat T cells with the reporter plasmid p[(TRE)3-tk-Luc] and found that CCK-8 was able to dose-dependently induce luciferase expression related to activator protein-1 (AP-1) activation with a maximal response identical to that obtained with compounds known to activate AP-1 complex (quantitatively, the same level of induction was obtained with 1 nM 12-O-tetradecanoylphorbol-13-acetate, 100 microM diacylglycerol, or 4 nM epidermal growth factor). The involvement of the CCK(B) receptor in such a stimulation was demonstrated by the inhibiting effect of the selective CCK(B) receptor antagonist PD-135,158. This effect was confirmed in COS-7 cells transfected with the cDNA of CCK(B) receptor cloned from Jurkat T cells. To better understand the AP-1-dependent luciferase expression in Jurkat T cells, we tested two specific inhibitors of serine/threonine phosphatases-1 and -2A: okadaic acid and calyculin A. These compounds strongly increased the phorbol-12-myristate-13-acetate response, whereas we have not observed a contribution of phosphatase inhibitors on a CCK-8-induced luciferase activity. To confirm that CCK(B) receptors are involved in AP-1 response, we investigated the CCK-8 effect on interleukin-2 expression, a natural endogenous gene regulated by several factors, including AP-1. In Jurkat T cells activated by phorbol-12-myristate-13-acetate and phytohemagglutinin, CCK-8 induced IL-2 expression. This induction was abolished by PD-135,158. Our results indicate that CCK-8 exerts a trophic effect in Jurkat T cells through stimulation of CCK(B) receptors by modulation of expression of AP-1-regulated genes. 


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Phosphatidylinositides bind to plasma membrane CD14 and can prevent monocyte activation by bacterial lipopolysaccharide. 
Although bacterial lipopolysaccharides (LPS) and several other microbial agonists can bind to mCD14 (membrane CD14), a cell-surface receptor found principally on monocytes and neutrophils, host-derived mCD14 ligands are poorly defined. We report here that phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate, and other phosphatidylinositides can bind to mCD14. Phosphatidylserine (PS), another anionic glycerophospholipid, binds to mCD14 with lower apparent affinity than does PtdIns. LPS-binding protein, a lipid transfer protein found in serum, facilitates both PS- and PtdIns-mCD14 binding. PtdIns binding to mCD14 can be blocked by anti-CD14 monoclonal antibodies that inhibit LPS-mCD14 binding, and PtdIns can inhibit both LPS-mCD14 binding and LPS-induced responses in monocytes. Serum-equilibrated PtdIns also binds to mCD14-expressing cells, raising the possibility that endogenous PtdIns may modulate cellular responses to LPS and other mCD14 ligands in vivo. 


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Transcriptional regulation of the pyruvate kinase erythroid-specific promoter. 
Mammal pyruvate kinases are encoded by two genes. The L gene produces the erythroid (R-PK) or the hepatic (L-PK) isozymes by the alternative use of two promoters. We report the characterization of the cis- and trans-acting elements involved in the tissue-specific activity of the L gene erythroid promoter. A R-PK DNA fragment extending from -870 to +54 relative to the cap site confers erythroid specificity to a reporter gene. Within this region, we define a minimal promoter (-62 to +54) that displays erythroid-specific activity and contains two DNA binding sites. One, located at -50, binds members of the CCACC/Sp1 family and the other, located at -20, binds the erythroid factor GATA-1. Although the -20 GATA binding site (AGATAA) is also a potential TFIID binding site, it does not bind TFIID. Furthermore, the substitution of this GATA binding site by a canonical TFIID binding site suppresses the promoter activity. Mutations and deletions of both sites indicate that only the association of CCACC/Sp1 and GATA binding sites can drive efficient and tissue-specific expression of this R-PK minimal promoter. Finally, by co-transfection experiments, we study the elements involved in the hGATA-1 transactivation of the R-PK promoter in HeLa cells. 


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The peri-kappa B site mediates human immunodeficiency virus type 2 enhancer activation in monocytes but not in T cells. 
Human immunodeficiency virus type 2 (HIV-2), like HIV-1, causes AIDS and is associated with AIDS cases primarily in West Africa. HIV-1 and HIV-2 display significant differences in nucleic acid sequence and in the natural history of clinical disease. Consistent with these differences, we have previously demonstrated that the enhancer/promoter region of HIV-2 functions quite differently from that of HIV-1. Whereas activation of the HIV-1 enhancer following T-cell stimulation is mediated largely through binding of the transcription factor NF-kappa B to two adjacent kappa B sites in the HIV-1 long terminal repeat, activation of the HIV-2 enhancer in monocytes and T cells is dependent on four cis-acting elements: a single kappa B site, two purine-rich binding sites, PuB1 and PuB2, and a pets site. We have now identified a novel cis-acting element within the HIV-2 enhancer, immediately upstream of the kappa B site, designated peri-kappa B. This site is conserved among isolates of HIV-2 and the closely related simian immunodeficiency virus, and transfection assays show this site to mediate HIV-2 enhancer activation following stimulation of monocytic but not T-cell lines. This is the first description of an HIV-2 enhancer element which displays such monocyte specificity, and no comparable enhancer element has been clearly defined for HIV-1. While a nuclear factor(s) from both peripheral blood monocytes and T cells binds the peri-kappa B site, electrophoretic mobility shift assays suggest that either a different protein binds to this site in monocytes versus T cells or that the protein recognizing this enhancer element undergoes differential modification in monocytes and T cells, thus supporting the transfection data. Further, while specific constitutive binding to the peri-kappa B site is seen in monocytes, stimulation with phorbol esters induces additional, specific binding. Understanding the monocyte-specific function of the peri-kappa B factor may ultimately provide insight into the different role monocytes and T cells play in HIV pathogenesis. 


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Suppressive effects of anti-inflammatory agents on human endothelial cell activation and induction of heat shock proteins. 
BACKGROUND: Studies from our laboratory have shown that the earliest stages of atherosclerosis may be mediated by an autoimmune reaction against heat shock protein 60 (Hsp60). The interactions of Hsp60-specific T cells with arterial endothelial cells (EC) require expression of both Hsp60 and certain adhesion molecules shown to be induced simultaneously in EC by mechanical and other types of stress. Recently, it was shown that suppression of T cell-mediated immune responses by cyclosporin A (CyA) enhanced atherosclerotic lesion formation in mice. In contrast, aspirin was found to lower the risk of myocardial infarction in men. These conflicting observations may be due to different effects of anti-inflammatory agents on adhesion molecule and Hsp expression in EC, respectively. MATERIAL AND METHODS: In the present study, we analyzed the effects of CyA, aspirin, and indomethacin on T cell proliferation using a proliferation assay. To explore the expression of adhesion molecules, monocyte chemoattractant protein-1 (MCP-1), and Hsp60 in human umbilical vein endothelial cells (HUVECs), Northern blot analyses were used. To examine the activation status of the transcription factors nuclear factor kappaB (NF-kappaB) and heat shock factor-1 (HSF-1), electrophoretic mobility shift assays were performed. RESULTS: With the exception of indomethacin, the used immunosuppressive and anti-inflammatory agents significantly inhibited T cell proliferation in response to influenza virus antigen in a dose-dependent manner. Interestingly, CyA and indomethacin did not suppress tumor necrosis factor-alpha (TNF-alpha)-induced adhesion molecule expression on HUVECs, whereas aspirin had an inhibitory effect. These observations correlated with the modulation of NF-kappaB activity in EC. All agents tested induced expression of Hsp60 6 hr after application. In addition, aspirin and indomethacin, but not CyA, induced Hsp70 expression in HUVECs that correlated with induction of HSF-1 activity. CONCLUSION: Our results show that the tested agents (except indomethacin) are inhibitors of the T cell-mediated immune response, as expected, that aspirin is an effective suppressor of adhesion molecule expression, and that all three agents can induce Hsp60 in HUVECs. These data provide the molecular basis for the notion that (1) part of the anti-atherogenic effect of aspirin may be due to the prevention of the adhesion of sensitized T cells to stressed EC; (2) that part of the atherosclerosis-promoting effect of CyA may be due to its potential as an inducer of Hsp60 expression and its inability to down-regulate adhesion molecule expression on EC; and (3) that down-regulation of MCP-1 expression by aspirin may result in decreased recruitment of monocytes into the arterial intima beneath stressed EC. 


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Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains. 
The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90. 


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New immunosuppressive drug PNU156804 blocks IL-2-dependent proliferation and NF-kappa B and AP-1 activation. 
We had previously shown that the drug undecylprodigiosin (UP) blocks human lymphocyte proliferation in vitro. We have now investigated the mechanism of action of a new analogue of UP, PNU156804, which shows a more favorable activity profile than UP in mice. We demonstrate here that the biological effect of PNU156804 in vitro is indistinguishable from UP: PNU156804 blocks human T cell proliferation in mid-late G1, as determined by cell cycle analysis, expression of cyclins, and cyclin-dependent kinases and retinoblastoma phosphorylation. In addition, we show that PNU156804 does not block significantly the induction of either IL-2 or IL-2R alpha- and gamma-chains but inhibits IL-2-dependent T cell proliferation. We have investigated several molecular pathways that are known to be activated by IL-2 in T cells. We show that PNU156804 does not inhibit c-myc and bcl-2 mRNA induction. On the other hand, PNU156804 efficiently inhibits the activation of the NF-kappa B and AP-1 transcription factors. PNU156804 inhibition of NF-kappa B activation is due to the inhibition of the degradation of I kappa B-alpha and I kappa B-beta. PNU156804 action is restricted to some signaling pathways; it does not affect NF-kappa B activation by PMA in T cells but blocks that induced by CD40 cross-linking in B lymphocytes. We conclude that the prodigiosin family of immunosuppressants is a new family of molecules that show a novel target specificity clearly distinct from that of other immunosuppressive drugs such as cyclosporin A, FK506, and rapamycin. 


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Interaction of transcription factors RFX1 and MIBP1 with the gamma motif of the negative regulatory element of the hepatitis B virus core promoter. 
The negative regulatory element (NRE) of the hepatitis B virus (HBV) core promoter contains three subregions which act synergistically to suppress core promoter activity. One of these subregions, NRE gamma, is active in both HeLa cervical carcinoma cells and Huh7 hepatoma cells and was found to be bound by a protein factor present in both cell types. Here we show that the transcription factor RFX1 can bind to NRE gamma and transactivate the core promoter through this site. Mutations which abrogated the gene-suppressive activity of NRE gamma prevented RFX1 from binding to NRE gamma. In addition, RFX1 can bind simultaneously, most likely as a heterodimer, with the transcription factor MIBP1 to NRE gamma. In the absence of a cloned MIBP1 gene for further studies, we hypothesize that RFX1 acts with MIBP1 to negatively regulate the core promoter activity through the NRE gamma site. The ability of RFX1 to transactivate the core promoter raises the possibility that RFX1 may play a dual role in regulating HBV gene expression. 


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Danazol decreases transcription of estrogen receptor gene in human monocytes. 
1. Administration of danazol for over one month reduced the levels of estrogen receptor (ER) and its mRNA to approximately 50 and 20%, respectively in monocytes. 2. Danazol did not alter the degradation rate of ER mRNA in monocytes. 3. Danazol decreased the transcription rate of ER gene to approximately 50% in monocytes in a run-on assay. 4. Danazol may release estrogen predominance via the reduction of transcription for ER gene, which leads to the reduction of ER mRNA and ER expressions in monocytes. 


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Cross-linking of CD30 induces HIV expression in chronically infected T cells. 
CD30, a member of the tumor necrosis factor (TNF) receptor family, is expressed constitutively on the surface of the human T cell line ACH-2, which is chronically infected with human immunodeficiency virus type-1 (HIV)-1. We demonstrate that cross-linking CD30 with an anti-CD30-specific monoclonal antibody, which mimics the described biological activities of the CD30 ligand (CD30L), results in HIV expression. CD30 cross-linking does not alter proliferation of ACH-2 cells and the induction of HIV expression is not mediated by endogenous TNF alpha/beta. Furthermore, cross-linking of CD30 leads to NF-kappa B activation and enhanced HIV transcription. Thus, CD30-CD30L interactions mediate the induction of HIV expression by a kappa B-dependent pathway that is independent of TNF. This mechanism may be important in the activation of HIV expression from latently infected CD4+ T cells, especially in lymphoid organs where cell to cell contact is conducive to receptor-ligand interactions. 


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An intricate arrangement of binding sites for the Ets family of transcription factors regulates activity of the alpha 4 integrin gene promoter. 
alpha 4 integrins mediate cell-cell and cell-extracellular matrix interactions that are critical for maturation and function of the immune system as well as differentiation of skeletal muscle. Here we examine molecular mechanisms controlling the pattern of alpha 4 expression. The activity of constructs containing 5' deletion mutants of the alpha 4 gene promoter was compared in transfection assays into cell lines that express alpha 4 and cell lines that do not. The sequence between position -42 and -76 base pairs (bp) was required for efficient transcription in cells that express alpha 4, but it showed no activity in HeLa cells, which do not express alpha 4. Three binding sites for the Ets family of transcription factors are found in this region: two adjacent sites at positions -50 and -54 bp and a more 5' site at position -67 bp. Using a series of constructs containing deletions and mutations in this region, we found that the 3'-most site alone was sufficient for binding GA-binding protein alpha (GABP alpha)/GABP beta and for a low level of transcriptional activation. When all three sites were present, a second complex "a" was detected, which contains an unknown member of the Ets family. Formation of complex a was cell-type specific and correlated with a high level of transcription. Deletion of the 5'-most Ets site had no effect on binding to GABP alpha/GABP beta, but it eliminated a. Concomitant with this loss of a, a new Ets-1-containing complex "c" appeared. Complex c substituted efficiently for complex a in transcriptional activation. We conclude that although neither of the two 5'-most Ets sites alone binds nuclear protein, they appear to act as modulators which control the pattern of Ets proteins that bind the alpha 4 gene promoter. This arrangement of Ets sites, coupled with the tissue- and developmental-specific expression of Ets members, likely play a key role in defining the pattern of alpha 4 integrin. 


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Kappa B-specific DNA binding proteins are differentially inhibited by enhancer mutations and biological oxidation. 
Kappa B (kappa B) enhancer binding proteins isolated from the nuclei of activated human T cells produce two distinct nucleoprotein complexes when incubated with the kappa B element from the interleukin-2 receptor-alpha (IL-2R alpha) gene. These two DNA-protein complexes are composed of at least four host proteins (p50, p55, p75, p85), each of which shares structural similarity with the v-rel oncogene product. Nuclear expression of these proteins is induced with distinctly biphasic kinetics following phorbol ester activation of T cells (p55/p75 early and p50/p85 late). DNA-protein crosslinking studies have revealed that the more rapidly migrating B2 complex contains both p50 and p55 while the more slowly migrating B1 complex is composed of p50, p55, p75, and p85. Site-directed mutagenesis of the wild-type IL-2R alpha kappa B enhancer (GGGGAATCTCCC) has revealed that the binding of p50 and p55 (B2 complex) is particularly sensitive to alteration of the 5' triplet of deoxyguanosine residues. In contrast, formation of the B1 complex, reflecting the binding of p75 and p85, critically depends upon the more 3' sequences of this enhancer element. DNA binding by all four of these Rel-related factors is blocked by selective chemical modification of lysine and arginine residues, suggesting that both of these basic amino acids are required for binding to the kappa B element. Similarly, covalent modification of free sulfhydryl groups with diamide (reversible) or N-ethylmaleimide (irreversible) results in a complete loss of DNA binding activity. In contrast, mild oxidation with glucose oxidase selectively inhibits p75 and p85 binding while not blocking p50 and p55 interactions. These findings suggest that reduced cysteine thiols play an important role in the DNA binding activity of this family of Rel-related transcription factors. 


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Involvement of tyrosine phosphorylation in endothelial adhesion molecule induction. 
Induction of endothelial adhesion molecules by the cytokine tumor necrosis factor-alpha (TNF) can occur independently of protein kinase C and activation of a protein tyrosine kinase (PTK) has recently been implicated in the upregulation of vascular cell adhesion molecule 1 (VCAM-1) by interleukin-4 (IL-4) on endothelial cells. We demonstrate that the PTK inhibitors herbimycin A or genistein suppress induction of endothelial VCAM-1 and E-selectin, as well as subsequent monocytic cell adhesion to endothelial cells stimulated by TNF. Inhibition studies indicate that specific tyrosine phosphorylation following PTK activation is involved in the mobilization of the transcription factor, nuclear factor kappa B, and VCAM-1 mRNA expression. This may have implications for pathophysiological conditions that involve the upregulation of these molecules (e.g. inflammation and atherosclerosis). 


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Nonpituitary human prolactin gene transcription is independent of Pit-1 and differentially controlled in lymphocytes and in endometrial stroma. 
Expression of the human PRL (hPRL) gene in extrapituitary sites such as the uterus (decidualized endometrial stroma and myometrium) and cells of the hematopoietic lineage is directed by an alternative promoter which is located approximately 6 kilobases (kb) upstream of the pituitary-specific start site. In order to delineate the tissue-specific mechanisms governing the control of nonpituitary PRL gene expression, we have cloned and sequenced 3 kb 5'-flanking DNA of the upstream decidual/lymphoid (dPRL) promoter. Based on sequence homology we identified two binding motifs for Pit-1 and seven half-sites for glucocorticoid receptor/progesterone receptor (PR) binding. We focused our studies on the role of Pit-1 and of PR as potential transcriptional regulators, since the POU domain protein Pit-1 is essential in the control of pituitary PRL expression, and progesterone induces decidual transformation of the endometrial stroma, a differentiation process during which the decidual PRL gene is activated. We demonstrate in a variety of cell types, including lymphocytes and endometrial stroma, that Pit-1 is not involved in the regulation of dPRL promoter/reporter gene constructs carrying 3 kb 5'-flanking DNA. Our experiments also show that activated PR does not confer direct transcriptional control on the dPRL promoter. When we compared the activity of the transfected dPRL promoter in PRL-secreting and nonsecreting lymphoid cells, we found that the 3 kb 5'-flanking region of the dPRL promoter did not contain elements restricting expression to only those lymphocytes that produce PRL but allowed expression of fusion reporter genes irrespective of the status of the endogenous PRL gene. This was in sharp contrast to endometrial cells where 3 kb 5'-flanking DNA conferred strong transcriptional activation on the dPRL promoter in decidualized endometrial stromal cells actively secreting PRL, but did not allow transcription in undifferentiated non-PRL-secreting endometrial stromal cells. Activation of the dPRL promoter construct in these undifferentiated cells could however be induced by the addition of cAMP, in the absence of progesterone, suggesting that a signal transduced through the cAMP signaling pathway is a primary inducer of decidual PRL gene expression. 


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HIV-1 reactivation in resting peripheral blood mononuclear cells of infected adults upon in vitro CD4 cross-linking by ligands of the CDR2-loop in extracellular domain 1. 
HIV-1 infects resting peripheral blood mononuclear cells (PBMCs) but remains inactive state until subsequent cell activation. We have demonstrated that the cross-linking of cell surface CD4 by gp120-anti-gp120 immune complexes or heat-inactivated HIV-1 (iHIV-1) is sufficient to trigger activation signals leading to virus reactivation (9). In this study, we demonstrate that NF-kappaB nuclear translocation and stimulation of virus production by iHIV-1 were strictly linked to the concentrations of viral proteins used as exogenous stimuli. Moreover, we further investigated the physiologic relevance of these observations. When submitted to an in vitro CD4 cross-linking by iHIV-1, PBMCs from HIV-1-infected patients were found to produce virus. This viral reactivation was associated with increased NF-kappaB nuclear translocation in patients' PBMCs. Additionally, virus reactivation in resting PBMCs infected in vitro with HIV-1 was found to be specifically induced by ligands of the CDR2-loop in domain 1 (D1) of CD4 (virus envelope and anti-CD4 monoclonal antibodies). In contrast, virus reactivation was not observed following CD4 oligomerization by antibodies that bind other epitopes in D1, including the D1/CDR3-loop. Finally, soluble CD4 (sCD4) prevented virus reactivation by D1/CDR2-loop ligands. Our results indicate that the signaling events initiated in PBMCs by oligomerization of CD4 at the D1/CDR2-loop can trigger HIV-1 upregulation in infected individuals. 


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Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells. 
Prolonged poor glycemic control in non-insulin-dependent diabetes mellitus patients often leads to a decline in insulin secretion from pancreatic beta cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the so-called glucose toxicity to pancreatic beta cells, we induced glycation in HIT-T15 cells using a sugar with strong deoxidizing activity, D-ribose, and examined the effects on insulin gene transcription. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control beta-actin gene promoter; approximately 50 and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 d in the presence of 40 and 60 mM D-ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycation-induced cells. Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF1/STF-1. These effects of D-ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediators of the phenomena. These observations suggest that protein glycation in pancreatic beta cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the beta cell glucose toxicity. 


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Lactobacilli and vaginal host defense: activation of the human immunodeficiency virus type 1 long terminal repeat, cytokine production, and NF-kappaB. 
Lactobacilli, a component of the normal vaginal flora, can activate the human immunodeficiency virus (HIV)-1 long terminal repeat (LTR) in the Jurkat T lymphocyte and THP-1 macrophage cell lines. Activation of the LTR in Jurkat cells was strongly enhanced by vanadate and inhibited by catalase, implicating H2O2. In contrast, activation in THP-1 cells occurred in the absence of vanadate and was unaffected by catalase. The active material partitioned into the phenol layer on hot aqueous phenol extraction. Lactobacilli also increased tumor necrosis factor-alphaand interleukin-1betaproduction and activated NF-kappaB in THP-1 cells and increased tumor necrosis factor-alphaproduction by human monocytes. Human vaginal fluid specimens had comparable properties, which correlated with their bacterial content. These findings suggest the presence in vaginal fluid of agent(s) derived from indigenous bacteria that can activate the HIV-1 LTR, cytokine production, and NF-kappaB in cells of macrophage lineage, with possible influence on vaginal physiology and host defense. 


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Human T-cell leukemia virus type 1 Tax induction of NF-kappaB involves activation of the IkappaB kinase alpha (IKKalpha) and IKKbeta cellular kinases. 
Tax corresponds to a 40-kDa transforming protein from the pathogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) that activates nuclear expression of the NF-kappaB/Rel family of transcription factors by an unknown mechanism. Tax expression promotes N-terminal phosphorylation and degradation of IkappaB alpha, a principal cytoplasmic inhibitor of NF-kappaB. Our studies now demonstrate that HTLV-1 Tax activates the recently identified cellular kinases IkappaB kinase alpha (IKKalpha) and IKKbeta, which normally phosphorylate IkappaB alpha on both of its N-terminal regulatory serines in response to tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) stimulation. In contrast, a mutant of Tax termed M22, which does not induce NF-kappaB, fails to activate either IKKalpha or IKKbeta. Furthermore, endogenous IKK enzymatic activity was significantly elevated in HTLV-1-infected and Tax-expressing T-cell lines. Transfection of kinase-deficient mutants of IKKalpha and IKKbeta into either human Jurkat T or 293 cells also inhibits NF-kappaB-dependent reporter gene expression induced by Tax. Similarly, a kinase-deficient mutant of NIK (NF-kappaB-inducing kinase), which represents an upstream kinase in the TNF-alpha and IL-1 signaling pathways leading to IKKalpha and IKKbeta activation, blocks Tax induction of NF-kappaB. However, plasma membrane-proximal elements in these proinflammatory cytokine pathways are apparently not involved since dominant negative mutants of the TRAF2 and TRAF6 adaptors, which effectively block signaling through the cytoplasmic tails of the TNF-alpha and IL-1 receptors, respectively, do not inhibit Tax induction of NF-kappaB. Together, these studies demonstrate that HTLV-1 Tax exploits a distal part of the proinflammatory cytokine signaling cascade leading to induction of NF-kappaB. The pathological alteration of this cytokine pathway leading to NF-kappaB activation by Tax may play a central role in HTLV-1-mediated transformation of human T cells, clinically manifested as the adult T-cell leukemia. 


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A cell type-specific enhancer in the human B7.1 gene regulated by NF-kappaB. 
The costimulatory molecule B7.1 provides a second signal critical for T cell activation. The distribution of this integral membrane protein is restricted to certain tissues where its level of expression is modulated by multiple exogenous stimuli. To identify the molecular basis for specificity and inducibility, the chromatin configuration of the human B7.1 gene was examined in intact nuclei from various cell types. The identification of a tissue-specific deoxyribonuclease I hypersensitive site approximately 3kb upstream of the transcription start site led to the characterization of a cell type-specific enhancer region. This 183-bp region was both cell type specific and responsive to two distinct stimuli, lipopolysaccharide and dibutyryl cAMP, known to regulate B7.1 expression. Deletional and site-directed mutagenesis revealed the presence of multiple functionally critical cis elements within this region, one of which was a nuclear factor (NF)-kappaB consensus sequence. In B7.1-positive B cells, this element bound several members of the NF-kappaB family, transcription factors already implicated in signal transduction pathways relevant to B7.1 expression. This is the first description, to our knowledge, of regulatory elements that control expression of a gene encoding a B7 costimulatory molecule. 


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Transcriptional analysis of Epstein-Barr virus gene expression in EBV-positive gastric carcinoma: unique viral latency in the tumour cells. 
Although case-oriented evidence for an association of Epstein-Barr virus (EBV) with gastric carcinoma has been accumulating recently, the interaction(s) between EBV and gastric epithelial cells is/are largely unknown. In this study, we examined seven EBV-positive gastric carcinoma tissues for viral gene expression at the mRNA level, from which studies on the EBV oncogenicity in human epithelial cells will benefit. Reverse transcription-PCR analysis showed that all seven EBV-positive tumour tissues constitutively expressed EBV nuclear antigen (EBNA) 1 mRNA, but not EBNA2 mRNA. The EBNA transcription was initiated from one of three EBNA promoters, Qp: by contrast, both Cp and Wp were silent, thus resulting in the lack of EBNA2 mRNA. Latent membrane protein (LMP) 2A mRNA was detected in three of seven cases; however, neither LMP1 nor LMP2B mRNA was detected in any of the tumours tested. Transcripts from the BamHI-A region of the viral genome were detectable in all cases. BZLF1 mRNA and the product, an immediate-early gene for EBV replication, was not expressed in any of them, thereby suggesting that the tumour cells carried EBV genomes in a tightly latent form. These findings further extended our previous data regarding EBV latency in gastric carcinoma cells at the protein level, and have affirmed that the programme of viral gene expression in the tumour more closely resembles 'latency I' represented by Burkitt's lymphoma than 'latency II' represented by the majority of nasopharyngeal carcinomas. 


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Differential interaction of nuclear factors with the PRE-I enhancer element of the human IL-4 promoter in different T cell subsets. 
The immunomodulatory cytokine IL-4 affects cells of most hemopoietic lineages. IL-4 is secreted by activated Th2 but not Th1 cells and plays a major role in the immune response by modulating the differentiation of naive Th cells toward the Th2 phenotype. We have previously identified an enhancer element, PRE-I, that is essential for the function of the human IL-4 promoter. To investigate the mechanisms responsible for tissue-specific expression of the IL-4 gene, we analyzed nuclear factors binding to the PRE-I site and compared the binding activities of these factors to the IL-4 promoter of Th1 and Th2 cells. We show that PRE-I interacts with PMA- and PMA/ionomycin-inducible, cyclosporin A-sensitive nuclear factors. Using anti-C/EBPbeta (NF-IL6), anti-C/EBPdelta (NF-IL6beta), anti-NF-ATc, anti-NF-ATp, anti-Fos, and anti-Jun Abs we demonstrate that the previously identified PRE-I binding factor POS-1 is composed of different transcription factors in different Th cell subsets. In the IL-4-producing Th0-like human Jurkat and mouse EL-4 cells, POS-1 (designated POS-1a) contains NF-IL6beta and Jun. In the mouse Th2 D10 cells and in the human Th2 clones, POS-1 (designated POS-1b) contains NF-IL6beta, Jun, and NF-ATc/p. In contrast, POS-1 was not found in nuclear extracts of human Th1 clones. These findings suggest that PRE-I may play a role in the differential regulation of IL-4 gene expression levels. 


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Inhibition of protein phosphatases by okadaic acid induces AP1 in human T cells. 
To examine the role of protein phosphatases in T cell activation, Jurkat cells were treated with okadaic acid, an inhibitor of type 1 and 2A phosphatases, and nuclear extracts were examined for the presence of AP1 as a measure of early T cell activation. Okadaic acid was found to be a potent inducer of AP1. In contrast to phorbol esters such as phorbol myristate acetate (PMA), the induction of AP1 by okadaic acid occurs predominantly by transcriptional activation of the jun and fos family of proto-oncogenes. Surprisingly, while the addition of phytohemagglutinin further enhanced the induction of AP1, the addition of PMA inhibited it. Okadaic acid treatment was found to dramatically increase mRNA transcripts of the jun family of proto-oncogenes including c-jun, junD, and junB and to a lesser extent the fos family including c-fos and fra-1. By comparison, PMA is a very inefficient inducer of the jun gene family in Jurkat cells. Similar to its effect on the induction of AP1 by okadaic acid, PMA inhibits the induction of c-jun mRNA by okadaic acid. Transfection of c-jun promoter constructs confirmed the marked difference between PMA and okadaic acid in inducing c-jun transcription. The induction of AP1 by okadaic acid suggests that protein phosphatases 1 and 2A (PP1 and PP2A) may be involved in T cell activation as important negative regulators of the transcription factor AP1. 


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Molecular mechanisms of anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells. 
The objectives of this study were to (1) determine the time course of neutrophil adhesion to monolayers of human umbilical vein endothelial cells (HUVECs) that were exposed to 60 minutes of anoxia followed by 30 to 600 minutes of reoxygenation and (2) define the mechanisms responsible for both the early (minutes) and late (hours) hyperadhesivity of postanoxic HUVECs to human neutrophils. The results clearly demonstrate that anoxia/reoxygenation (A/R) leads to a biphasic increase in neutrophil adhesion to HUVECs, with peak responses occurring at 30 minutes (phase 1) and 240 minutes (phase 2) after reoxygenation. Oxypurinol and catalase inhibited phase-1 adhesion, suggesting a role for xanthine oxidase and H2O2. In comparison, platelet activating factor (PAF) contributed to both phases of neutrophil adhesion. Anti-intercellular adhesion molecule-1 (ICAM-1) and anti-P-selectin antibodies (monoclonal antibodies [mAbs]) attenuated phase-1 neutrophil adhesion, consistent with roles for constitutively expressed ICAM-1 and enhanced surface expression of preformed P-selectin. Phase-2 neutrophil adhesion was attenuated by an anti-E-selectin mAb, indicating a dominant role of this adhesion molecule in the late phase response. Pretreatment with actinomycin D and cycloheximide or with competing ds-oligonucleotides containing the nuclear factor-kappa B or activator protein-1 cognate DNA sequences significantly attenuated phase-2 response, suggesting a role for de novo macromolecule synthesis. Surface expression of ICAM-1, P-selectin, and E-selectin on HUVECs correlated with the phase-1 and -2 neutrophil adhesion responses. Collectively, these findings indicate that A/R elicits a two-phase neutrophil-endothelial cell adhesion response that involves transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules. 


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Costimulation of human CD4+ T cells with LFA-3 and B7 induce distinct effects on AP-1 and NF-kappa B transcription factors. 
We have earlier shown that stimulation of human CD4+ T cells with SEA presented on Chinese hamster ovary (CHO)-DR transfectants coexpressing either B7 or LFA-3 resulted in distinct cytokine profiles. We now demonstrate that B7, but not LFA-3, strongly costimulated IL-2 transcription and mRNA expression in CD4+ T cells. Maximal increase in IL-2 transcription was recorded with CHO-DR/B7/LFA-3, suggesting a cooperative effect of B7 and LFA-3 at the transcriptional level. Gel-shift analysis demonstrated that stimulation of CD4+ T cells with CHO-DR and staphylococcal enterotoxin A was sufficient to induce significant amounts of NF-kappa B binding proteins, whereas induction of AP-1 binding proteins required costimulation. LFA-3 induced moderate levels of AP-1, but did not influence the levels of NF-kappa B, while B7 costimulation strongly induced both AP-1 and substantially enhanced NF-kappa B binding proteins. The CHO-DR/B7/LFA-3 triple transfectant induced a further increase in AP-1 and NF-kappa B binding proteins compared with the double transfectants. The level of Oct-1 binding proteins remained similar in all samples. Super-shift analysis revealed that the NF-kappa B complex of costimulated CD4+ T cells contained large amounts of p50, substantial amounts of p65, and marginal levels of c-Rel proteins. The AP-1 binding proteins contained c-Jun, Jun-D, and Fra-1, but marginal amounts of Jun-B and c-Fos. Our results indicate distinct effects of B7 and LFA-3 costimulation on the activity of AP-1 and NF-kappa B. These may partly account for the differential effects of B7 and LFA-3 costimulation on IL-2 expression. 


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The expression of c-fos, c-jun, and c-myc genes is regulated by heat shock in human lymphoid cells. 
The effect of heat shock on the expression of the nuclear protooncogenes c-fos, c-jun, and c-myc was studied in human lymphoid cells. Heat shock caused an increase in c-fos and c-jun mRNA levels and a decrease in c-myc mRNA levels in pre-B (Hyon) and T (DND-41) cell lines as well as in freshly isolated normal human thymocytes. The changes in the mRNA levels of these protooncogenes in Hyon cells were most pronounced at 42 and 43 degrees C; kinetic analysis demonstrated that the changes could be detected within 30 min of heat shock. Altered transcription of c-fos and c-myc genes was the primary effect of heat shock. Secondarily, heat shock of Hyon cells stabilized the c-myc mRNA level by increasing its half-life from 24 to 45 min. The overall effect of heat shock on c-myc mRNA level, however, was a marked inhibition of its transcription. These results demonstrate that the transcription of nuclear protooncogenes is regulated by heat shock indicating a role for nuclear protooncogenes in the stress response of lymphoid cells. 


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Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells. 
Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants. 


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Activation of human macrophages by mechanical ventilation in vitro. 
Positive-pressure mechanical ventilation supports gas exchange in patients with respiratory failure but is also responsible for significant lung injury. In this study, we have developed an in vitro model in which isolated lung cells can be submitted to a prolonged cyclic pressure-stretching strain resembling that of conventional mechanical ventilation. In this model, cells cultured on a Silastic membrane were elongated up to 7% of their initial diameter, corresponding to a 12% increase in cell surface. The lung macrophage was identified as the main cellular source for critical inflammatory mediators such as tumor necrosis factor-alpha, the chemokines interleukin (IL)-8 and -6, and matrix metalloproteinase-9 in this model system of mechanical ventilation. These mediators were measured in supernatants from ventilated alveolar macrophages, monocyte-derived macrophages, and promonocytic THP-1 cells. Nuclear factor-kappaB was found to be activated in ventilated macrophages. Synergistic proinflammatory effects of mechanical stress and molecules such as bacterial endotoxin were observed, suggesting that mechanical ventilation might be particularly deleterious in preinjured or infected lungs. Dexamethasone prevented IL-8 and tumor necrosis factor-alpha secretion in ventilated macrophages. Mechanical ventilation induced low levels of IL-8 secretion by alveolar type II-like cells. Other lung cell types such as endothelial cells, bronchial cells, and fibroblasts failed to produce IL-8 in response to a prolonged cyclic pressure-stretching load. This model is of particular value for exploring physical stress-induced signaling pathways, as well as for testing the effects of novel ventilatory strategies or adjunctive substances aimed at modulating cell activation induced by mechanical ventilation. 


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Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer. 
A family of transcriptional activators has recently been identified in chickens; these transcriptional activators recognize a common consensus motif (WGATAR) through a conserved C4 zinc finger DNA-binding domain. One of the members of this multigene family, cGATA-3, is most abundantly expressed in the T-lymphocyte cell lineage. Analysis of human and murine GATA-3 factors shows a striking degree of amino acid sequence identity and similar patterns of tissue specificity of expression in these three organisms. The murine and human factors are abundantly expressed in a variety of human and murine T-cell lines and can activate transcription through a tissue-specific GATA-binding site identified within the human T-cell receptor delta gene enhancer. We infer that the murine and human GATA-3 proteins play a central and highly conserved role in vertebrate T-cell-specific transcriptional regulation. 


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Transforming growth factor-beta suppresses human B lymphocyte Ig production by inhibiting synthesis and the switch from the membrane form to the secreted form of Ig mRNA. 
Transforming growth factor-beta (TGF-beta) inhibits B cell Ig secretion and reduces B cell membrane Ig expression. The addition of TGF-beta to human B lymphocyte cultures stimulated with Staphylococcus aureus Cowan strain I and IL-2 completely inhibited B cell Ig secretion (greater than 90%) and decreased B cell surface IgM, IgD, kappa L chain, and lambda L chain expression. In contrast, TGF-beta had only minimal effects on two other B cell membrane proteins, HLA-DR and CD20. Internal labeling with [35S]methionine and immunoprecipitation with anti-IgM, anti-kappa, and anti-lambda antibodies revealed a striking reduction in kappa L chain in the presence of TGF-beta. A less pronounced reduction in lambda L chain and microH chain was also noted. Northern blot analysis of RNA purified from B cells treated with TGF-beta for varying time intervals revealed a significant decrease in steady state kappa and lambda L chain mRNA levels. Furthermore, a significant decrease in the switch from the membrane forms of mu and gamma to their respective secreted forms was noted in the presence of TGF-beta. Nuclear run-on experiments demonstrated decreased transcription of kappa L chain. The effects of TGF-beta on two transcriptional regulatory factors, Oct-2 and nuclear factor (NF) kappa B, known to be important in Ig gene transcription were examined. Oct-2 mRNA levels and both Oct-2 and NF-kappa B proteins in nuclear extracts were not altered by treatment with TGF-beta. In contrast, levels of the transcriptional factor AP-1, which is not known to be important in B cell Ig production, were reduced by TGF-beta. These findings demonstrate that TGF-beta decreases B lymphocyte Ig secretion by inhibiting the synthesis of Ig mRNA and inhibiting the switch from the membrane form to the secreted forms of mu and gamma mRNA. The mechanism by which TGF-beta inhibits Ig chain synthesis is unclear although it does not involve inhibition of the binding of NF-kappa B or Oct-2 to their respective target sequences. 


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Temporal control of IgH gene expression in developing B cells by the 3' locus control region. 
The suggested roles of the downstream 3' regions acting as a Locus Control Region (LCR), have allowed comparisons to be made between the regulation of the IgH locus with other model systems whose gene expression is governed by LCR activity. Here we summarize the importance of the IgH 3'LCR and its putative functional role in IgH gene expression and compare it with the 5'LCR regulatory region of the human beta-globin locus. 


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ERP, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B-lymphocyte development. 
The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation. 


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Inhibitory effect of growth hormone on TNF-alpha secretion and nuclear factor-kappaB translocation in lipopolysaccharide-stimulated human monocytes. 
Several studies have pointed to a link between immune and endocrine systems, including a regulatory function of GH on monocyte activation. The present study demonstrates that human THP-1 promonocytic cells, engineered by gene transfer to constitutively produce human growth hormone (hGH), secreted depressed amounts of TNF-alpha in response to challenge by LPS. The effect of GH appears to occur in an autocrine fashion, since the inhibitory effect on TNF-alpha secretion by constitutive GH production could be abolished in the presence of anti-hGH mAb. The GH-induced inhibitory effect was also observed using normal human monocytes and monocyte-derived macrophages. Inhibition of TNF-alpha production by THP-1-hGH-transfected cells cultured in the presence of LPS is dependent on a selective pathway, since no inhibition of TNF-alpha production was observed when cells were cultured in the presence of PMA. Inhibition of TNF-alpha secretion by LPS-stimulated THP-1-hGH cells was associated with a decrease in nuclear translocation of nuclear factor-kappaB. The capacity of GH to inhibit LPS-induced TNF-alpha production by monocytes without altering other pathways leading to TNF-alpha production may be of potential relevance in septic shock, since GH is available for clinical use. 


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Transcriptional regulation of interleukin 3 (IL3) in primary human T lymphocytes. Role of AP-1- and octamer-binding proteins in control of IL3 gene expression. 
We have investigated the molecular and biochemical basis for activation of interleukin 3 (IL3) gene expression in primary human T lymphocytes following CD3 and CD2 receptor stimulation or activation by phytohemagglutinin plus phorbol 12-myristate 13-acetate. Using transfection and reporter gene assays specifically designed for primary T lymphocytes in conjunction with gel retardation assays, Western blot analyses and UV cross-linking studies, we found that c-Jun, c-Fos, and octamer-binding proteins play a major role in transcriptional activation of the IL3 gene via their interaction with two specific regions contained within the IL3 5'-flanking sequence. Additionally, the region between bases -107 and -59 of the IL3 promoter containing putative AP-2 and Sp1 binding motifs appears necessary for basal level expression of the IL3 gene. The data also indicate that CD2 receptor activation and phytohemagglutinin plus phorbol 12-myristate 13-acetate stimulation augment T cell IL3 gene expression through the same cis- and trans-activating signals. These results should contribute to a better understanding of the regulation of IL3 gene expression in human T lymphocytes. 


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Tcf-1-mediated transcription in T lymphocytes: differential role for glycogen synthase kinase-3 in fibroblasts and T cells. 
Beta-catenin is the vertebrate homolog of the Drosophila segment polarity gene Armadillo and plays roles in both cell-cell adhesion and transduction of the Wnt signaling cascade. Recently, members of the Lef/Tcf transcription factor family have been identified as protein partners of beta-catenin, explaining how beta-catenin alters gene expression. Here we report that in T cells, Tcf-1 also becomes transcriptionally active through interaction with beta-catenin, suggesting that the Wnt signal transduction pathway is operational in T lymphocytes as well. However, although Wnt signals are known to inhibit the activity of the negative regulatory protein kinase glycogen synthase kinase-3beta (GSK-3beta), resulting in increased levels of beta-catenin, we find no evidence for involvement of GSK-3beta in Tcf-mediated transcription in T cells. That is, a dominant negative GSK-3beta does not specifically activate Tcf transcription and stimuli (lithium or phytohemagglutinin) that inhibit GSK-3beta activity also do not activate Tcf reporter genes. Thus, inhibition of GSK-3beta is insufficient to activate Tcf-dependent transcription in T lymphocytes. In contrast, in C57MG fibroblast cells, lithium inactivates GSK-3beta and induces Tcf-controlled transcription. This is the first demonstration that lithium can alter gene expression of Tcf-responsive genes, and points to a difference in regulation of Wnt signaling between fibroblasts and lymphocytes. 


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Identification of a novel factor that interacts with an immunoglobulin heavy-chain promoter and stimulates transcription in conjunction with the lymphoid cell-specific factor OTF2. 
The tissue-specific expression of the MOPC 141 immunoglobulin heavy-chain gene was studied by using in vitro transcription. B-cell-specific transcription of this gene was dependent on the octamer element 5'-ATGCAAAG-3', located in the upstream region of this promoter and in the promoters of all other immunoglobulin heavy- and light-chain genes. The interaction of purified octamer transcription factors 1 and 2 (OTF1 and OTF2) with the MOPC 141 promoter was studied by using electrophoretic mobility shift assays and DNase I footprinting. Purified OTF1 from HeLa cells and OTF1 and OTF2 from B cells bound to identical sequences within the heavy-chain promoter. The OTF interactions we observed extended over the heptamer element 5'-CTCAGGA-3', and it seems likely that the binding of the purified factors involves cooperation between octamer and heptamer sites in this promoter. In addition to these elements, we identified a second regulatory element, the N element with the sequence 5'-GGAACCTCCCCC-3'. The N element could independently mediate low levels of transcription in both B-cell and HeLa-cell extracts, and, in conjunction with the octamer element, it can promote high levels of transcription in B-cell extracts. The N element bound a transcription factor, NTF, that is ubiquitous in cell-type distribution, and NTF was distinct from any of the previously described proteins that bind to similar sequences. Based on these results, we propose that NTF and OTF2 interactions (both with their cognate DNA elements and possibly at the protein-protein level) may be critical to B-cell-specific expression and that these interactions provide additional pathways for regulating gene expression. 


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Fibroblast growth factor-1 (FGF-1) enhances IL-2 production and nuclear translocation of NF-kappaB in FGF receptor-bearing Jurkat T cells. 
Fibroblast growth factors (FGFs) are heparin-binding proteins crucial to embryogenesis, angiogenesis, and wound healing. FGF-1 is abundantly expressed in the synovium in rheumatoid arthritis and in rejecting allografts, sites of chronic immune-mediated inflammation. The frequency of FGF-1-responsive T cells is increased in the peripheral blood of these disorders, and a high percentage of infiltrating T cells in rheumatoid arthritis synovium express receptors for FGF-1. To understand the action of FGF-1 in T cells, studies were initiated in Jurkat T cells that express the signaling isoform of FGF receptor-1. These experiments show that FGF-1 stimulation of Jurkat T cells provides a second signal that augments TCR-mediated IL-2 production. Analogous to costimulation via CD28, this activity is mediated through activation of Rel/kappaB, a family of transcription factors known to regulate IL-2 and other activation-inducible proteins. FGF-1 alone induces modest nuclear translocation of kappaB-binding proteins, and this translocation is enhanced by the combination of anti-CD3 and FGF-1. This NF-kappaB binding complex is composed of transcriptionally active p65(RelA)/p50 heterodimers and results primarily from the targeted degradation of IkappaB-alpha, an inhibitor that sequesters Rel/kappaB in the cytoplasm. These data are the first to show a connection between FGF-1 signaling and NF-kappaB activation outside of embryonic development. The signaling events that link FGF receptor-1 engagement and NF-kappaB activation in Jurkat are probably distinct from the CD28 costimulation pathway, since FGF-1-induced Rel/kappaB binding proteins do not contain significant levels of c-Rel and are not identical with the CD28 response complex. 


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Activation of NF-kappa B by interleukin 2 in human blood monocytes. 
We report here that interleukin 2 (IL-2) acts on human blood monocytes by enhancing binding activity of the transcription factor NF-kappa B to its consensus sequence in the 5' regulatory enhancer region of the IL-2 receptor alpha chain (p55). Similarly, IL-2 activates NF-kappa B in the human monocytic cell line U 937, but not in resting human T-cells. This effect is detectable within 15 min and peaks 1 h after exposure to IL-2. Enhanced NF-kappa B binding activity is followed by functional activation in that inducibility of the IL-2 receptor alpha chain is mediated by enhanced NF-kappa B binding and that a heterologous promoter containing the NF-kappa B consensus sequence (-291 to -245) of the IL-2 receptor alpha chain gene is activated. In addition, IL-2 is capable of increasing transcript levels of the p50 gene coding for the p50 subunit of the NF-kappa B transcription factor, whereas mRNA levels of the p65 NF-kappa B gene remained unchanged. 


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A transcriptional regulatory element is associated with a nuclease-hypersensitive site in the pol gene of human immunodeficiency virus type 1. 
Analysis of the chromatin organization of the integrated human immunodeficiency virus type 1 (HIV-1) genome has previously revealed a major constitutive DNase I-hypersensitive site associated with the pol gene (E. Verdin, J. Virol. 65:6790-6799, 1991). In the present report, high-resolution mapping of this site with DNase I and micrococcal nuclease identified a nucleosome-free region centered around nucleotides (nt) 4490 to 4766. A 500-bp fragment encompassing this hypersensitive site (nt 4481 to 4982) exhibited transcription-enhancing activity (two- to threefold) when it was cloned in its natural position with respect to the HIV-1 promoter after transient transfection in U937 and CEM cells. Using in vitro footprinting and gel shift assays, we have identified four distinct binding sites for nuclear proteins within this positive regulatory element. Site B (nt 4519 to 4545) specifically bound four distinct nuclear protein complexes: a ubiquitous factor, a T-cell-specific factor, a B-cell-specific factor, and the monocyte/macrophage- and B-cell-specific transcription factor PU.1/Spi-1. In most HIV-1 isolates in which this PU box was not conserved, it was replaced by a binding site for the related factor Ets1. Factors binding to site C (nt 4681 to 4701) had a DNA-binding specificity similar to that of factors binding to site B, except for PU.1/Spi-1. A GC box containing a binding site for Sp1 was identified (nt 4623 to 4631). Site D (nt 4816 to 4851) specifically bound a ubiquitously expressed factor. These results identify a transcriptional regulatory element associated with a nuclease-hypersensitive site in the pol gene of HIV-1 and suggest that its activity may be controlled by a complex interplay of cis-regulatory elements. 


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Induction of endothelial cell surface adhesion molecules by tumor necrosis factor is blocked by protein tyrosine phosphatase inhibitors: role of the nuclear transcription factor NF-kappa B. 
Recent studies from our laboratory have indicated that protein tyrosine phosphatase (PTPase) inhibitors can down-modulate the tumor necrosis factor (TNF)-mediated activation of the nuclear transcription factor NF-kappa B in ML-1a, a monocytic cell line (Singh and Aggarwal, J. Biol. Chem. 1995: 270: 10631). Since TNF is one of the major inducers of various adhesion molecules in human endothelial cells and their expression is known to require the activation of NF-kappa B, we examined the effect of PTPase inhibitors on the TNF-mediated induction of intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and endothelial leukocyte adhesion molecule (ELAM)-1. Like ML-1a, human dermal microvessel endothelial cells (MVEC) treated with TNF rapidly activated (within 30 min) NF-kappa B; this effect was completely abolished by co-treatment with phenylarsine oxide (PAO), a specific inhibitor of PTPase. The induction of ICAM-1, VCAM-1, and ELAM-1 by TNF in MVEC occurred within 6 h and was also completely down-regulated by PAO in a dose-dependent manner. PAO was found to be effective even when added 3 h after TNF, suggesting a rapid mode of action of this inhibitor. Besides PAO, other inhibitors of PTPase, including pervanadate and diamide, also blocked TNF-dependent NF-kappa B activation and induction of all the three adhesion proteins. Consistent with these results, the attachment of monocytes to MVEC was also blocked by the PTPase inhibitors. Thus, overall, our results demonstrate that a PTPase is involved either directly or indirectly in the pathway leading to the induction of endothelial cell adhesion molecules by TNF. Because of their role in cell adhesion, PTPase may provide a novel target of drug development for treatment of inflammation, atherogenesis, and tumor metastasis. 


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Inhibition of activation of transcription factor AP-1 by CD28 signalling in human T-cells. 
Co-stimulation of T-lymphocytes by T-cell receptor (TcR) occupancy and activation of the CD28 surface molecule results in enhanced proliferation and interleukin 2 (IL-2) production. The increase in IL-2 gene expression triggered by CD28 involves a kappa B-like sequence in the 5'-regulatory region of the IL-2 promoter, called CD28-responsive element. Stimulation of T-cells by agonistic anti-CD28 antibodies in conjunction with phorbol 12-myristate 13-acetate (PMA)- or TcR-derived signals induces the enhanced activation of the transcription factor NF-kappa B. Here we report that CD28 engagement, however, exerts opposite effects on the transcription factor AP-1. Whereas anti-CD28 together with PMA increased the DNA binding and trans-activation activity of NF-kappa B, PMA-induced activation of AP-1 was significantly suppressed. The inhibitory effect exerted by anti-CD28 was observed at the level of DNA binding as well as in functional reporter-gene assays. These results suggest that the two transcription factors are independently regulated and may perform different functions during T-cell activation. 


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Integrin-mediated tyrosine phosphorylation and cytokine message induction in monocytic cells. A possible signaling role for the Syk tyrosine kinase. 
Activation of cytoplasmic tyrosine kinases is an important aspect of signal transduction mediated by integrins. In the human monocytic cell line THP-1, either integrin-dependent cell adhesion to fibronectin or ligation of beta 1 integrins with antibodies causes a rapid and intense tyrosine phosphorylation of two sets of proteins of about 65-75 and 120-125 kDa. In addition, integrin ligation leads to nuclear translocation of the p50 and p65 subunits of the NF-kappa B transcription factor, to activation of a reporter gene driven by a promoter containing NF-kappa B sites, and to increased levels of mRNAs for immediate-early genes, including the cytokine interleukin (IL)-1 beta. The tyrosine kinase inhibitors genistein and herbimycin A block both integrin-mediated tyrosine phosphorylation and increases in IL-1 beta message levels, indicating a causal relationship between the two events. The components tyrosine phosphorylated subsequent to cell adhesion include paxillin, pp125FAK, and the SH2 domain containing tyrosine kinase Syk. In contrast, integrin ligation with antibodies induces tyrosine phosphorylation of Syk but not of FAK or paxillin. In adhering cells, pre-treatment with cytochalasin D suppresses tyrosine phosphorylation of FAK and paxillin but not of Syk, while IL-1 beta message induction is unaffected. These observations indicate that the Syk tyrosine kinase may be an important component of an integrin signaling pathway in monocytic cells, leading to activation of NF-kappa B and to increased levels of cytokine messages. 


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Induction of human immunodeficiency virus type 1 expression in monocytic cells by Cryptococcus neoformans and Candida albicans. 
Because candidiasis and cryptococcosis are common in human immunodeficiency virus (HIV)-infected persons, the effect of Cryptococcus neoformans and Candida albicans on HIV expression in monocytic cells was examined. Stimulation of the latently HIV-infected myelomonocytic cell line OM-10.1 with C. neoformans and C. albicans in the presence of pooled human serum caused a ratio-dependent increase in HIV production. Induction of HIV by C. neoformans was enhanced by anti-capsular antibody, while induction by both organisms was inhibited by anti-TNF-alpha antibody. In THP-1 cells transfected with HIV plasmid constructs, both organisms induced transcription from the HIV long terminal repeat that was dependent on intact NF-kappaB binding sequences. Thus, C. neoformans and C. albicans enhance HIV expression in monocytic cells through a TNF-alpha- and NF-kappaB-dependent mechanism. In HIV-infected patients, such enhancement may further impair host immunity and could accelerate the course of HIV disease. 


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Sterol dependent LDL-receptor gene transcription in lymphocytes from normal and CML patients. 
Sterol regulatory element (SRE) has been recognized to regulate various key genes coding for especially low density lipoprotein (LDL)-receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase known to play a crucial role in the cholesterol feedback mechanism. The deranged cholesterol feedback mechanism has been widely recognised in initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Consequently, the present study was addressed to understand this phenomenon and revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when the CML patients achieved complete haematological remission (CHR) through alpha-interferon therapy. Further, an inverse relationship was also observed between sterol modulated LDL-receptor gene transcription and the binding affinity of this 47 kDa factor to the SRE sequence. Based upon these results we propose that alpha-interferon through its receptor initiates phosphatidic acid dependent signalling which in turn regulates the affinity of 47 kDa sterol regulatory element binding factor as well as LDL-receptor gene transcription in lymphocytes from CML patients. 


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Regulation of IL-6 synthesis in human peripheral blood mononuclear cells by C3a and C3a(desArg). 
The anaphylatoxin C3a has been reported to have immunomodulatory effects on a number of different cell types. In this study we investigated the effects of C3a and C3a(desArg) on gene expression and protein secretion of IL-6 in human PBMCs, either alone or in combination with LPS or IL-1beta. C3a or C3a(desArg) alone exhibited no effect on the expression or secretion of IL-6. However, when PBMC were stimulated with LPS or IL-1beta, both C3a and C3a(desArg) were found to enhance IL-6 release by PBMC in a dose-dependent manner. Since C3a has been shown to induce PGE2 production by monocytes, and PGE2 has been shown to influence cytokine production, we investigated the potential role of PGE2 in C3a-mediated enhancement of LPS- and IL-1beta-induced IL-6 production. Indomethacin blocked PGE2 release, but had no influence on the observed effects of C3a, suggesting that the effects of C3a on IL-6 production are independent of PGE2 formation by monocytes. Northern blot analysis showed that C3a as well as C3a(desArg) enhanced LPS-induced mRNA levels for IL-6. Pretreatment of PBMCs with pertussis toxin blocked the functions of C3a and C3a(desArg), indicating that the actions of these two molecules are mediated by a G protein-coupled pathway. Furthermore, we investigated the effects of C3a and C3a(desArg) on induction of NF-kappaB and activating protein-1 binding. Both molecules enhanced LPS-induced NF-kappaB and activating protein-1 binding activity. These results demonstrate the capacity of intact C3a and its circulating des-Arg form to exert immunmodulatory effects in vitro. 


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Towards a molecular understanding of T-cell differentiation. 
Lymphoid differentiation is one of the best studied examples of mammalian development. Here Hans Clevers and Michael Owen describe how the cloning of the genes that encode T-cell-specific membrane proteins allows the identification of transcription factors that control the expression of these T-cell genes. Such transcription factors play a key role in the development of the mature T-cell phenotype by functioning as 'master regulators of T-cell differentiation'. 


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Identification of an ionomycin/cyclosporin A-responsive element within the human T cell receptor gamma enhancer. 
Activation through the Ca2+/calcineurin pathway is essential to the transcription of many cytokine genes. The conserved cis-acting sequence, GGAAAA, and transcription factors binding to this sequence are involved in the response to increased intracellular Ca2+ concentrations. Here we report the identification and importance of the same sequence in a non-cytokine gene, the human T cell receptor gamma (TCRG) enhancer. Results from site-directed mutations and electrophoretic mobility shift assays strongly suggest that this sequence mediates the ionomycin-induced activation of the TCRG enhancer. Our studies provide an explanation for a previous observation that TCRG mRNA levels, but not mRNA levels for T cell receptor alpha and -beta, are increased by ionomycin treatment. 


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Ex vivo activation of tumor-draining lymph node T cells reverses defects in signal transduction molecules. 
The adoptive transfer of tumor-draining lymph node (LN) T cells activated ex vivo with anti-CD3 and interleukin 2 (IL-2) mediates the regression of the poorly immunogenic murine melanoma D5. The efficacy of the activated LN cells is augmented when the sensitizing tumor is a genetically modified variant (designated D5G6) that secretes granulocyte/macrophage-colony-stimulating factor. In contrast to anti-CD3/IL-2-activated LN cells, adoptive transfer of freshly isolated tumor-draining LN T cells has no therapeutic activity. To determine whether the acquisition of antitumor function during ex vivo activation is associated with modifications in signal transduction capacity, the protein tyrosine kinases p56lck and p59fyn and proteins of the NF-kappaB family were analyzed in tumor-draining LN T cells. The levels of p56lck and p59fyn were lower in tumor-draining than in normal LN T cells and production of tyrosine-phosphorylated substrates was markedly depressed following anti-CD3 stimulation. After 5-day anti-CD3/IL-2 activation, levels of p56lck and p59fyn and protein tyrosine kinase activity increased. Interestingly, the levels of p56lck, p59fyn, and tyrosine kinase activity were higher in activated T cells derived from LN that drained D5G6 than they were in those from D5 tumors. In contrast, the cytoplasmic levels of c-Rel and Rel A were normal in freshly isolated tumor-draining LN, as was nuclear kappaB DNA-binding activity induced by anti-CD3 mAb or phorbol myristate acetate. Stimulation of activated LN cells with D5 tumor cells induced the nuclear translocation of NF-kappaB. These findings indicate that the recovery of proteins mediating signal transduction through the T cell receptor/CD3 complex in LN T cells activated ex vivo was associated with the acquisition of antitumor function. 


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NF-kappa B activation by tumor necrosis factor alpha in the Jurkat T cell line is independent of protein kinase A, protein kinase C, and Ca(2+)-regulated kinases. 
NF-kappa B is a DNA-binding regulatory factor able to control transcription of a number of genes, including human immunodeficiency virus (HIV) genes. In T cells, NF-kappa B is activated upon cellular treatment by phorbol esters and the cytokine tumor necrosis factor alpha (TNF alpha). In the present work, we investigated the molecular events leading to NF-kappa B activation by TNF alpha in a human T cell line (Jurkat) and its subclone JCT6, which presents a deficiency in the PKA transduction pathway. We found that in both cell lines, both phorbol ester and TNF alpha were able to activate NF-kappa B. Phorbol activation was positively modulated by Ca2+ influx while TNF alpha activation was not. Furthermore, while PMA activation was inhibited by the PKC inhibitor staurosporin, the TNF alpha effect was unchanged. TNF alpha did not activate cAMP production and its signal was not modulated by cAMP activators. Moreover, cAMP activators did not activate NF-kappa B in Jurkat cells. Thus, TNF alpha-induced NF-kappa B activation was found to be mediated by none of the major signal-mediating kinases such as protein kinase C (PKC), protein kinase A, or Ca(2+)-regulated kinases. Furthermore, we found that cytoplasmic acidification facilitated NF-kappa B activation by both TNF alpha and PKC, by a mechanism that increases NF-kappa B/I kappa B dissociation without affecting the NF-kappa B translocation step. 


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Abnormal apoptosis and cell cycle progression in humans exposed to methyl tertiary-butyl ether and benzene contaminating water. 
1. In this study we hypothesized that in individuals with certain genetic makeup, MTBE, benzene or their metabolites act as adducts and may induce programmed cell death. 2. Our study involved a group of 60 male and female subjects who were exposed to MTBE and benzene-contaminated water concentrations up to 76 PPB for MTBE and 14 PPB for benzene, for a period of 5 to 8 years. For comparison, we recruited a control group consisting of 32 healthy males and females with similar age distribution and without a history of exposure to MTBE or benzene. 3. Peripheral blood lymphocytes (PBL) of both groups were tested for the percentage of apoptotic cells and cell cycle progression using flow cytometry. 4. When apoptotic lymphocytes from exposed individuals were compared to apoptotic lymphocytes from the control group, statistically-significant differences between each mean group were detected (26.4 +/- 1.8 and 12.1 +/- 1.3, respectively), indicating an increased rate of apoptosis in 80.5% of exposed individuals (P < 0.0001, Mann-Whitney U-Test). MTBE and benzene-induced apoptosis is attributed to a discrete block within the cell cycle progression. Because cell cycle analysis showed that in PBL from chemically-exposed individuals, between 20-50% of cells were accumulated at the S-G2/M boundaries. 5. One of the signaling molecules which mediates programmed cell death is nuclear factor Kappa-B (NF-kappa B). NF-kappa B was examined as one of the many molecular mechanisms for mediating cell death by MTBE and benzene. Indeed, addition of inhibitors of NF-kappa B activation pyrrolidine dithiocarbamate (PDTC), to the lymphocytes of the chemically-exposed group was capable of inhibiting programmed cell death by 40%. This reversal of apoptosis almost to the control level by inhibitor of NF-kappa B activation may indicate involvement of this signaling molecule in MTBE and benzene induction of programmed cell death. 


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Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. 
The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B-cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes. Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells. BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but not in nonexpressing subclones of the B-cell line WEHI-231. Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone. A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter. Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector. The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line. These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44. The relevance of these molecular events to BCR signal transduction and antigen-stimulated B-cell-mediated immune responses is discussed. 


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Differential monocyte adhesion and adhesion molecule expression in venous and arterial endothelial cells. 
We compared U-937 cell adhesion and adhesion molecule expression in human umbilical venous (HUVECs) and arterial (HUAECs) endothelial cells exposed to tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide (LPS). TNF and LPS stimulated vascular cell adhesion molecule (VCAM)-1 surface expression and adhesion of U-937 monocyte-like cells to HUVECs but not to HUAECs. Antibody studies demonstrated that in HUVECs at least 75% of the adhesion response is VCAM-1 mediated. Interleukin-1 stimulated U-937 cell adhesion to and VCAM-1 surface expression in both HUVECs and HUAECs. Pyrrolidinedithiocarbamate and the proteasome inhibitor MG-132 blocked TNF- and LPS-stimulated U-937 cell adhesion to HUVECs. These agents also significantly decreased TNF- and LPS-stimulated increases in HUVEC surface VCAM-1. TNF increased VCAM-1 protein and mRNA in HUVECs that was blocked by pyrrolidinedithiocarbamate. However, neither TNF or LPS stimulated VCAM-1 expression in HUAECs. TNF stimulated expression of both intercellular adhesion molecule-1 and E-selectin in HUVECs, but in HUAECs, only intercellular adhesion molecule-1 was increased. Electrophoretic mobility shift assays demonstrated no difference in the pattern of TNF-stimulated nuclear factor-kappaB activation between HUVECs and HUAECs. These studies demonstrate a novel and striking insensitivity of arterial endothelium to the effects of TNF and LPS and indicate a dissociation between the ability of HUAECs to upregulate nuclear factor-kappaB and VCAM-1. 


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Surfactant suppresses NF-kappa B activation in human monocytic cells. 
In addition to biophysical properties, pulmonary surfactant has immunomodulatory activity. We previously demonstrated that both synthetic (Exosurf) and modified natural surfactant (Survanta) downregulated endotoxin-stimulated inflammatory cytokine mRNA levels and protein products (tumor necrosis factor-alpha [TNF], interleukin-1-beta [IL-1], interleukin-6 [IL-6]) in human alveolar macrophages. In this study, we report that both Exosurf and Survanta suppress TNF mRNA and secretion (85 +/- 4% mean percent inhibition +/- SEM by Exosurf; 71 +/- 6% by Survanta) by endotoxin-stimulated THP-1, a human monocytic cell line. Because surfactant downregulated inflammatory cytokine production similarly in both normal human alveolar macrophages and the THP-1 cell line, we used this cell line to investigate whether surfactant affected transcriptional mechanisms. Specifically, we examined nuclear factor-kappa B (NF-kappa B) activation because it is crucial in transcriptional regulation of many inflammatory cytokine genes including TNF, IL-1, and IL-6. Electrophoretic mobility shift assays showed that both surfactants decreased activation of NF-kappa B. The presence of both p65 and p50 NF-kappa B components in LPS-activated THP-1 cells was confirmed by specific antibody induction of supershifts in mobility assays. These results are the first to suggest that surfactant's suppressive effects on inflammatory cytokine production may involve transcriptional regulation through inhibition of NF-kappa B activation. 


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The regulation of the human tumor necrosis factor alpha promoter region in macrophage, T cell, and B cell lines. 
The 1311-base pair human tumor necrosis factor (TNF) alpha promoter region was fused to the luciferase (Luc) reporter gene and studied in a transient transfection system in three TNF producing cell lines, the U937 macrophage cell line, the MLA 144 T cell line, and the 729-6 B cell line. This full length promoter construct can be induced by phorbol 13-myristate acetate (PMA) in each of these cell types. Analysis of a series of 5'-truncations showed several peaks of basal and PMA induced activity suggesting the presence of several positive and negative regulatory elements. A PMA responsive element was localized to a region between -95 and -36 bp relative to the transcription start site. Within this region, single AP-2- and AP-1- like consensus sequences were noted. These AP-2 and AP-1 sites were each modified with a double point mutation. A modest (20-50%) reduction in TNF promoter activity was observed with the AP-2 site mutation. However, mutation of the AP-1 site markedly diminished both the basal and PMA-activated promoter activity. Also co-transfections of the wild-type promoter construct with an AP-1/c-jun expression vector resulted in augmented basal and PMA-induced promoter activity. 


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Specific complex formation between the type II bare lymphocyte syndrome-associated transactivators CIITA and RFX5. 
Two of the genes defective in the five complementation groups identified in the class II-negative bare lymphocyte syndrome or corresponding laboratory mutants have been cloned. One gene encodes a protein, RFX5, that is a member of the RFX family of DNA binding proteins. The other, CIITA, encodes a large protein with a defined acidic transcriptional activation domain; this protein does not interact with DNA. Expression plasmids encoding regions of RFX5 fused to the GAL4 DNA binding domain activated transcription from a reporter construct containing GAL4 sites in a cotransfection assay in the Raji human B cell line. However, these plasmids produced transcriptional activity in HeLa cells only in conjunction with interferon gamma stimulation, a condition in which expression of both CIITA and class II major histocompatibility complex surface proteins are induced. Furthermore, these plasmids were not active in RJ2.2.5, an in vitro mutagenized derivative of Raji in which both copies of CIITA are defective. Transcriptional activation by the RFX5 fusion protein could be restored in RJ2.2.5 by cotransfection with a CIITA expression plasmid. Finally, a direct interaction between RFX5 and CIITA was detected with the yeast two-hybrid and far-Western blot assays. Thus, RFX5 can activate transcription only in cooperation with CIITA. RFX5 and CIITA associate to form a complex capable of activating transcription from class II major histocompatibility complex promoters. In this complex, promoter specificity is determined by the DNA binding domain of RFX5 and the general transcription apparatus is recruited by the acidic activation domain of CIITA. 


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Redox status of cells influences constitutive or induced NF-kappa B translocation and HIV long terminal repeat activity in human T and monocytic cell lines. 
We have tested the hypothesis that cellular activation events occurring in T lymphocytes and monocytes and mediated through translocation of the transcription factor NF-kappa B are dependent upon the constitutive redox status of these cells. We used phenolic, lipid-soluble, chain-breaking antioxidants (butylated hydroxyanisole (BHA), nordihydroquairetic acid, or alpha-tocopherol (vitamin E) to show that peroxyl radical scavenging in unstimulated and PMA- or TNF-stimulated cells blocks the functions depending on NF-kappa B activation. BHA was found to suppress not only PMA- or TNF-induced, but also constitutive, HIV-enhancer activity concomitant to an inhibition of NF-kappa B binding activity in both lymphoblastoid T (J.Jhan) and monocytic (U937) cell lines. This was also true for KBF (p50 homodimer) binding activity in U937 cells. Secretion of TNF, the product of another NF-kappa B-dependent gene, was abolished by BHA in PMA-stimulated U937 cells. The anti-oxidative effect of BHA was accompanied by an increase in thiol, but not glutathione, content in stimulated and unstimulated T cell, whereas TNF stimulation itself barely modified the cellular thiol level. Oxidative stress obtained by the addition of H2O2 to the culture medium of J.Jhan or U937 cells could not by itself induce NF-kappa B activation. These observations suggest that TNF and PMA do not lead to NF-kappa B activation through induction of changes in the cell redox status. Rather, TNF and PMA can exert their effect only if cells are in an appropriate redox status, because prior modification toward reduction with BHA treatment prevents this activation. It appears that a basal redox equilibrium tending toward oxidation is a prerequisite for full activation of transduction pathways regulating the activity of NF-kappa B-dependent genes. 


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Constitutive expression of p50 homodimer in freshly isolated human monocytes decreases with in vitro and in vivo differentiation: a possible mechanism influencing human immunodeficiency virus replication in monocytes and mature macrophages. 
Human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in vitro in differentiated macrophages than in freshly isolated monocytes. We investigated whether this may be partly explained by changes in expression of NF-kappaB with monocyte differentiation. We demonstrated that constitutive expression of NF-kappaB in primary human monocytes changed significantly with differentiation in vitro to monocyte-derived macrophages (MDMs) and differentiation in vivo to alveolar macrophages (AMs). Freshly isolated monocytes constitutively expressed high levels of transcriptionally inactive p50 homodimer which decreased with time in culture in favor of the transcriptionally active p50/p65 and p50/RelB heterodimers. As in MDMs, AMs constitutively expressed p50/p65 and p50/RelB although at lower levels. HIV infection of fresh monocytes failed to induce p50/p65 as seen in MDMs. The replacement of p50 homodimers with transcriptionally active heterodimers following time in culture may partially explain the progressive increase in susceptibility of monocytes to HIV infection during in vitro culture. The change in NF-kappaB components with monocyte differentiation in vivo may also explain the different transcriptional activities of these cell populations in HIV-infected individuals. 


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A regulatory element in the human interleukin 2 gene promoter is a binding site for the zinc finger proteins Sp1 and EGR-1. 
Activation of the interleukin 2 (IL-2) gene after antigen recognition is a critical event for T cell proliferation and effector function. Prior studies have identified several transcription factors that contribute to the activity of the IL-2 promoter in stimulated T lymphocytes. Here we describe a novel regulatory element within the IL-2 promoter located immediately upstream of the nuclear factor of activated T cell (NFAT) domain. This region (termed the zinc finger protein binding region (ZIP)) serves as binding site for two differently regulated zinc finger proteins: the constitutively expressed transcription factor Sp1 and the inducible early growth response protein EGR-1. In unstimulated cells which do not secrete IL-2, only Sp1 binds to this region, while in stimulated IL-2 secreting cells the inducible EGR-1 protein recognizes this element. In Jurkat T cells, the ZIP site serves as an activator for IL-2 gene expression, and a combination of ZIP and NFAT binding sites is required for maximal IL-2 promoter activity. These results suggest a critical role of the ZIP site for IL-2 promoter activity. 


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Involvement of Alu sequences in the cell-specific regulation of transcription of the gamma chain of Fc and T cell receptors. 
The Fc epsilon RI-gamma chains are expressed in a variety of hematopoietic cells where they play a critical role in signal transduction. They are part of the high affinity IgE receptor in mast cells, basophils, Langerhans cells, and possibly other cells; a component of the low affinity receptor for IgG (Fc gamma RIIIA or CD16) in natural killer cells and macrophages; and part of the T cell antigen receptor in subsets of T cells. Here we have investigated the transcriptional regulation of the gamma chain gene by analyzing the 2.5-kilobase sequence upstream of the transcription start site. This sequence contains a promoter specific to cells of hematopoietic lineage. However, the tissue specificity of this promoter is only partial because it is active in all of the hematopoietic cells tested here, regardless of whether they constitutively express Fc epsilon RI- gamma chain transcripts. We have identified two adjacent cis-acting regulatory elements, both of which are part of an Alu repeat. The first (-445/-366) is a positive element active in both basophils and T cells. The second (-365/-264) binds to nuclear factors, which appear to be different in basophils and T cells, and acts as a negative element in basophils and as a positive one in T cells. Thus, this Alu repeat (90% identical to Alu consensus sequences) has evolved to become both a positive and negative regulator. 


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Differential autoregulation of glucocorticoid receptor expression in human T- and B-cell lines. 
Regulation of glucocorticoid receptor (GR) expression by its cognate ligand was examined in the glucocorticoid-sensitive human leukemic T-cell line 6TG1.1 and in the human B-cell line IM-9. In contrast to the decrease in GR mRNA seen in IM-9 cells after treatment with 1 microM dexamethasone for 16-18 h, treatment of 6TG1.1 cells resulted in an 8-fold increase in GR mRNA, as determined by Northern blot and RNase protection analysis, with a corresponding 3- to 4-fold increase in GR protein. Half-maximal induction of GR mRNA and protein in 6TG1.1 cells was observed between 10-100 nM dexamethasone, and inclusion of 1 microM RU 38486 completely blocked the effects of 100 nM dexamethasone, demonstrating that positive autoregulation of GR expression in 6TG1.1 cells is a receptor-mediated response. Positive autoregulation of GR expression was also observed in glucocorticoid-resistant CEM-C1 cells, which contain functional GR, but whose growth is unaffected by glucocorticoids. Thus, positive autoregulation is neither a consequence nor the sole cause of growth arrest. The degree of negative autoregulation in IM-9 cells and positive autoregulation in 6TG1.1 cells was unaffected by inhibition of protein synthesis with cycloheximide. Measurement of GR mRNA turnover in 6TG1.1 cells treated with actinomycin-D revealed a half-life of 2.5 h, which was unaffected by dexamethasone treatment. A similar half-life was determined in IM-9 cells and was also unaffected by steroid treatment. These results are consistent with the interpretation that glucocorticoid-mediated autoregulation of GR expression is a tissue-specific primary transcriptional response. 


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Fcgamma receptor-mediated mitogen-activated protein kinase activation in monocytes is independent of Ras. 
Receptors for the Fc portion of immunoglobulin molecules (FcR) present on leukocyte cell membranes mediate a large number of cellular responses that are very important in host defense, including phagocytosis, cell cytotoxicity, production and secretion of inflammatory mediators, and modulation of the immune response. Cross-linking of FcR with immune complexes leads, first to activation of protein-tyrosine kinases. The molecular events that follow and that transduce signals from these receptors to the nucleus are still poorly defined. We have investigated the signal transduction pathway from Fc receptors that leads to gene activation and production of cytokines in monocytes. Cross-linking of FcR, on the THP-1 monocytic cell line, by immune complexes resulted in both activation of the transcription factor NF-kappaB and interleukin 1 production. These responses were completely blocked by tyrosine kinase inhibitors. In contrast, expression of dominant negative mutants of Ras and Raf-1, in these cells, did not have any effect on FcR-mediated nuclear factor activation, suggesting that the mitogen-activated protein kinase (MAPK) signaling pathway was not used by these receptors. However, MAPK activation was easily detected by in vitro kinase assays, after FcR cross-linking with immune complexes. Using the specific MAPK/extracellular signal-regulated kinase kinase (MAPK kinase) inhibitor PD98059, we found that MAPK activation is necessary for FcR-dependent activation of the nuclear factor NF-kappaB. These results strongly suggest that the signaling pathway from Fc receptors leading to expression of different genes important to leukocyte biology, initiates with tyrosine kinases and requires MAPK activation; but in contrast to other tyrosine kinase receptors, FcR-mediated MAPK activation does not involve Ras and Raf. 


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In vivo inhibition of NF-kappa B in T-lineage cells leads to a dramatic decrease in cell proliferation and cytokine production and to increased cell apoptosis in response to mitogenic stimuli, but not to abnormal thymopoiesis. 
To understand the role of NF-kappa B complexes in T cell development and activation, we have generated transgenic mice in which RelA and c-Rel complexes were selectively inhibited in the T-lineage cells by specific expression of a trans-dominant form of I kappa B alpha. Transgene expression did not affect the thymic development, but led to lowered numbers of splenic T cells and to a dramatic decrease in the ex vivo proliferative response of splenic T lymphocytes. Analysis of IL-2 and IL-2R alpha expression demonstrated that the perturbation of the proliferation response was not attributable to an abnormal expression of these genes. In contrast, expression of IL-4, IL-10, and IFN-gamma was strongly inhibited in the transgenic T cells. The proliferative deficiency of the transgenic T cells was associated with an increased apoptosis. These results point out the involvement of NF-kappa B/Rel family proteins in growth signaling pathways by either regulating proteins involved in the IL-2 signaling or by functionally interfering with the cell cycle progression. 


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Pentoxifylline for the treatment of infection with human immunodeficiency virus. 
Cytokine dysregulation in human immunodeficiency virus type 1 (HIV-1) infection has been documented in numerous studies and has been cited as an important component in the pathogenesis of this retroviral infection. Pharmacological modification of cytokine dysregulation, therefore, has been suggested as a therapeutic modality for HIV-1 infection. Dr. Dezube of Beth Israel Hospital (Boston) concisely reviews the state of our knowledge regarding the effects of pentoxifylline on expression of tumor necrosis factor-alpha, a cytokine known to influence HIV-1 replication and to play a possible role in the clinical manifestations of advanced infection with this virus. Pentoxifylline, a trisubstituted xanthine derivative, has been used to decrease blood viscosity and is reasonably well tolerated by most recipients of the drug. Results of preliminary studies, many of which were conducted by Dr. Dezube, suggest that use of this agent in combination with antiretroviral compounds may prove useful in the treatment of patients with HIV-1 infection. 


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DNA-binding studies of the Epstein-Barr virus nuclear antigen 2 (EBNA-2): evidence for complex formation by latent membrane protein gene promoter-binding proteins in EBNA-2-positive cell lines. 
The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) protein is essential for the immortalization of human primary B cells by EBV. EBNA-2 trans-activates cellular and viral genes like CD23, c-fgr, latent membrane protein 1 (LMP1) and terminal protein 1 (TP1). Trans-activation of the TP1 promoter and of the BamHI C promoter has already been investigated in detail and appears to be mediated via protein-protein interactions and not by direct binding of EBNA-2 type A (of EBV type 1) to the DNA. EBNA-2 is able to trans-activate the expression of the LMP gene in several cell lines. Various reports have delineated the cis-acting elements of the LMP promoter through which EBNA-2 mediates trans-activation. To determine whether EBNA-2 also trans-activates the LMP promoter by protein-protein interactions, we performed a series of gel retardation assays and competition experiments with LMP promoter fragments of different sizes. We determined that the protein-binding region on the LMP promoter was within a 42 bp fragment encompassing nucleotides -135 to -176 relative to the LMP transcriptional start site. None of the DNA fragments investigated indicated interaction of EBNA-2 with the DNA via protein-protein interactions. No significant differences between EBNA-2-positive and EBNA-2-negative nuclear extracts could be seen in the gel retardation assay under conditions that clearly showed binding of EBNA-2A to the TP1 promoter. However, analysis of sucrose gradient fractions in the gel retardation assay provided evidence that the LMP promoter-binding proteins form a complex of higher M(r) in EBNA-2-positive cell extracts. These complexes were destroyed by detergent. We deduce from these results that EBNA-2-positive cells might indeed contain specific complexes bound to the LMP promoter which are, however, too labile to be detected in a standard gel retardation assay. 


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Immunosuppressant PG490 (triptolide) inhibits T-cell interleukin-2 expression at the level of purine-box/nuclear factor of activated T-cells and NF-kappaB transcriptional activation. 
PG490 (triptolide) is a diterpene triepoxide with potent immunosuppressive and antiinflammatory properties. PG490 inhibits interleukin(IL)-2 expression by normal human peripheral blood lymphocytes stimulated with phorbol 12-myristate 13-acetate (PMA) and antibody to CD3 (IC50 of 10 ng/ml), and with PMA and ionomycin (Iono, IC50 of 40 ng/ml). In Jurkat T-cells, PG490 inhibits PMA/Iono-stimulated IL-2 transcription. PG490 inhibits the induction of DNA binding activity at the purine-box/antigen receptor response element (ARRE)/nuclear factor of activated T-cells (NF-AT) target sequence but not at the NF-kappaB site. PG490 can completely inhibit transcriptional activation at the purine-box/ARRE/NF-AT and NF-kappaB target DNA sequences triggered by all stimuli examined (PMA, PMA/Iono, tumor necrosis factor-alpha). PG490 also inhibits PMA-stimulated activation of a chimeric transcription factor in which the C-terminal TA1 transactivation domain of NF-kappaB p65 is fused to the DNA binding domain of GAL4. In 16HBE human bronchial epithelial cells, IL-8 expression is regulated predominantly by NF-kappaB, and PG490 but not cyclosporin A can completely inhibit expression of IL-8. The mechanism of PG490 inhibition of cytokine gene expression differs from cyclosporin A and involves nuclear inhibition of transcriptional activation of NF-kappaB and the purine-box regulator operating at the ARRE/NF-AT site at a step after specific DNA binding. 


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NF-kappaB functions as both a proapoptotic and antiapoptotic regulatory factor within a single cell type. 
Recently NF-kappaB has been shown to have both proapoptotic and antiapoptotic functions. In T cell hybridomas, both T cell activators and glucocorticoids induce apoptosis. Here we show that blockade of NF-kappaB activity, using a dominant negative IkappaBalpha, has opposite effects on these two apoptotic signals. Treatment with PMA plus ionomycin (P/I) results in the upregulation of Fas Ligand (FasL) and induction of apoptosis. Inhibition of NF-kappaB activity inhibits the P/I mediated induction of FasL mRNA and decreases the level of apoptosis in these cultures, thus establishing NF-kappaB as a proapoptotic factor in this context. Conversely, inhibition of NF-kappaB confers a tenfold increase in glucocorticoid mediated apoptosis, establishing that NF-kappaB also functions as an antiapoptotic factor. We conclude that NF-kappaB is a context-dependent apoptosis regulator. Our data suggests that NF-kappaB may function as an antiapoptotic factor in thymocytes while functioning as a proapoptotic factor in mature peripheral T cells. 


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Immune hyperactivation of HIV-1-infected T cells mediated by Tat and the CD28 pathway. 
Human immunodeficiency virus-type 1 (HIV-1) infection is characterized by a chronic state of immune hyperactivation in patients. Infection of human peripheral blood lymphocytes with HIV-1 in vitro resulted in increased interleukin-2 (IL-2) secretion in response to T cell activation via the CD3 and CD28 receptors. Expression of the HIV-1 transactivator Tat recapitulated this phenotype and was associated with increased IL-2 secretion in response to costimulation with CD3 plus CD28. IL-2 superinduction by Tat occurred at the transcriptional level, was mediated by the CD28-responsive element in the IL-2 promoter, and was exclusively dependent on the 29 amino acids encoded by the second exon of Tat. 


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Inhibition of nuclear factor kappaB activation attenuates apoptosis resistance in lymphoid cells. 
Death-inducing ligands (DILs) such as tumor necrosis factor alpha (TNFalpha) or the cytotoxic drug doxorubicin have been shown to activate a nuclear factor kappaB (NFkappaB)-dependent program that may rescue cells from apoptosis induction. We demonstrate here that TRAIL (TNF-related apoptosis-inducing ligand), a recently identified DIL, also activates NFkappaB in lymphoid cell lines in a kinetic similar to TNFalpha. NFkappaB activity is independent from FADD, caspases, and apoptosis induction. To study the influence of NFkappaB activity on apoptosis mediated by TRAIL, CD95, TNFalpha, or doxorubicin, NFkappaB activation was inhibited using the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal or transient overexpression of mutant IkappaBalpha. Sensitivity for induction of apoptosis was markedly increased by these treatments in apoptosis sensitive cell lines. Moreover, both in cell lines and in primary leukemia cells that are resistant towards induction of apoptosis by DILs and doxorubicin, antagonization of NFkappaB activity partially restored apoptosis sensitivity. These data suggest that inhibition of NFkappaB activation may provide a molecular approach to increase apoptosis sensitivity in anticancer treatment. 


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Regulation of CD14 expression during monocytic differentiation induced with 1 alpha,25-dihydroxyvitamin D3. 
CD14, a monocyte/macrophage receptor for the complex of LPS and LPS binding protein, is a differentiation marker for the monocyte/macrophage lineage. We have analyzed the regulation of CD14 expression during 1 alpha,25-dihydroxyvitamin D3 (VitD3)-induced monocytic differentiation. Using FACS, Northern blotting, and nuclear run-on analyses, we demonstrate that the up-regulation of CD14 expression during monocytic cell maturation is regulated mainly at the level of gene transcription, and that new protein synthesis is required for CD14 induction. We have recently cloned the CD14 5' upstream sequence and demonstrated its tissue-specific promoter activity. Using stable transfection of the monocytoid U937 cell line with a series of deletion mutants of the CD14 5' upstream sequence coupled to a reporter gene construct, we show that bp -128 to -70 is the critical region for the induction of CD14 expression. This region contains two binding sites for the Sp1 transcription factor. A 3-bp mutation at the distal Sp1-binding site not only eliminates Sp1 interaction, but also abolishes most of the VitD3 induction of CD14 expression. Electrophoretic mobility shift analysis does not detect a direct interaction of the CD14 distal Sp1-binding site with the vitamin D3 receptor and its partner, the retinoid X receptor. These data demonstrate that VitD3 induces CD14 indirectly through some intermediary factor, and suggest a critical role for Sp1 in this process. 


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Activation of the transcription factor NF-kappaB in lipopolysaccharide-stimulated U937 cells. 
During the course of serious bacterial infections, lipopolysaccharide (LPS) interacts with monocyte/macrophage receptors, resulting in the generation of inflammatory cytokines. Transcription factor NF-kappaB is crucial in activating the transcription of genes encoding proinflammatory cytokines. In this paper, we demonstrate that the activation of NF-kappaB by LPS in a promonocytic cell line (U937) followed a rather slow kinetics, depending on the rate of IkappaB-alpha inhibitor hydrolysis. No degradation of p105 and p100 inhibitors was observed under these conditions. The transduction pathway leading to NF-kappaB activation in U937 cells involved the intracellular generation of reactive oxygen species (ROS), as demonstrated by the concomitant inhibitory effects of antioxidants on NF-kappaB activation and the emission of a fluorescent probe reacting intracellularly with hydrogen peroxide. This ROS pathway was also characterized by the use of other inhibitors. This finding indicates that phospholipase A2 and 5-lipoxygenase are also involved. However, the NF-kappaB activation pathway involving the acidic sphingomyelinase of the endolysosomial membrane did not seem to participate in the LPS-induced NF-kappaB activation in U937 cells. 


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Overexpression of HSF2-beta inhibits hemin-induced heat shock gene expression and erythroid differentiation in K562 cells. 
Acquisition of heat shock factor 2 (HSF2) DNA binding activity is accompanied by induced transcription of heat shock genes in hemin-treated K562 cells undergoing erythroid differentiation. Previous studies revealed that HSF2 consists of two alternatively spliced isoforms, HSF2-alpha and HSF2-beta, whose relative abundance is developmentally regulated and varies between different tissues. To investigate whether the molar ratio of HSF2-alpha and HSF2-beta isoforms is crucial for the activation of HSF2 and whether the HSF2 isoforms play functionally distinct roles during the hemin-mediated erythroid differentiation, we generated cell clones expressing different levels of HSF2-alpha and HSF2-beta. We show that in parental K562 cells, the HSF2-alpha isoform is predominantly expressed and HSF2 can be activated upon hemin treatment. In contrast, when HSF2-beta is expressed at levels exceeding those of endogenous HSF2-alpha, the hemin-induced DNA binding activity and transcription of heat shock genes are repressed, whereas overexpression of HSF2-alpha results in an enhanced hemin response. Furthermore, the hemin-induced accumulation of globin, known as a marker of erythroid differentiation, is decreased in cells overexpressing HSF2-beta. We suggest that HSF2-beta acts as a negative regulator of HSF2 activity during hemin-mediated erythroid differentiation of K562 cells. 


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Activation of the human immunodeficiency virus type 1 enhancer is not dependent on NFAT-1. 
The function of a putative NFAT-1 site in the human immunodeficiency virus type 1 enhancer has been analyzed. Activation by the T-cell antigen receptor is minimal in Jurkat cells and is mediated by the kappa B sites. The putative NFAT-1 region is not required for the response to anti-CD3 or to mitogens in T-cell, B-cell, or monocyte/macrophage leukemia lines, nor is it a cis-acting negative regulatory element. 


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Involvement of intracellular Ca2+ in oxidant-induced NF-kappa B activation. 
In human Jurkat T cells and its subclone Wurzburg cells oxidant challenge elevated [Ca2+]i by mobilizing Ca2+ from intracellular stores. In Jurkat cells this effect was rapid and transient, but in Wurzburg cells the response was slow and sustained. H2O2-induced NF-kappaB activation in Wurzburg cells was not influenced by the presence of extracellular EGTA but was totally inhibited in cells that were loaded with esterified EGTA. In Jurkat cells that are not sensitive to H2O2-induced NF-kappaB activation, H2O2 potentiated NF-kappaB activation in the presence of sustained high [Ca2+]i following thapsigargin treatment. NF-kappaB regulatory effect of alpha-lipoate and N-acetylcysteine appeared to be, at least in part, due to their ability to stabilize elevation of [Ca2+]i following oxidant challenge. Results of this study indicate that a sustained elevated [Ca2+]i is a significant factor in oxidant-induced NF-kappaB activation. 


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Pancreatic islet expression studies and polymorphic DNA markers in the genes encoding hepatocyte nuclear factor-3alpha, -3beta, -3gamma, -4gamma, and -6. 
The genes encoding the functionally related hepatocyte nuclear factors HNF-1alpha and HNF-4alpha play a critical role in normal pancreatic beta-cell function. Mutations in these liver-enriched transcription factors result in two forms of early-onset type 2 diabetes (maturity-onset diabetes of the young [MODY]), MODY3 and MODY1, which are characterized by impaired glucose-stimulated insulin secretion, early disease onset, and autosomal dominant inheritance. The transcriptional hierarchy of HNFs suggests that other proteins of the regulatory cascade might be responsible for other forms of MODY and/or late-onset type 2 diabetes. In this study, we show that HNF-3alpha, -3beta, -3gamma, -4gamma, and -6 are expressed in pancreatic beta-cells. We report the identification and characterization of simple tandem repeat DNA polymorphisms in the genes encoding HNF-3alpha, -3beta, -3gamma, -4gamma, and -6 and the mapping of HNF-6 to chromosome bands 15q21.1-21.2 by fluorescence in situ hybridization. These markers will be useful to study the role of genetic variation in these genes in the pathogenesis of type 2 diabetes. 


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Steel factor affects SCL expression during normal erythroid differentiation. 
Steel factor is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and SCL, also known as Tcl-5 or Tal-1, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of Steel factor on SCL expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. SCL protein levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of SCL with erythroid proliferation. In contrast, SCL mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in SCL protein observed. The role of SCL in Steel factor-induced erythroid proliferation was then examined. In BFU-E-derived colonies cultured with Steel factor, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with Steel factor, SCL protein was increased significantly compared to control. The increase in SCL protein levels in early erythroid precursors stimulated with Steel factor suggests one mechanism through which Steel factor may enhance normal erythroid proliferation. SCL mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to Steel factor stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in SCL protein observed in response to Steel. 


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AM580, a stable benzoic derivative of retinoic acid, has powerful and selective cyto-differentiating effects on acute promyelocytic leukemia cells. 
All-trans retinoic acid (ATRA) is successfully used in the cyto-differentiating treatment of acute promyelocytic leukemia (APL). Paradoxically, APL cells express PML-RAR, an aberrant form of the retinoic acid receptor type alpha (RAR alpha) derived from the leukemia-specific t(15;17) chromosomal translocation. We show here that AM580, a stable retinobenzoic derivative originally synthesized as a RAR alpha agonist, is a powerful inducer of granulocytic maturation in NB4, an APL-derived cell line, and in freshly isolated APL blasts. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF), the compound induces granulocytic maturation, as assessed by determination of the levels of leukocyte alkaline phosphatase, CD11b, CD33, and G-CSF receptor mRNA, at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. By contrast, AM580 is not effective as ATRA in modulating the expression of these differentiation markers in the HL-60 cell line and in freshly isolated granulocytes obtained from the peripheral blood of chronic myelogenous leukemia patients during the stable phase of the disease. In NB4 cells, two other synthetic nonselective RAR ligands are capable of inducing LAP as much as AM580, whereas RAR beta- or RAR gamma-specific ligands are totally ineffective. These results show that AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. Binding experiments, using COS-7 cells transiently transfected with PML-RAR and the normal RAR alpha, show that AM580 has a lower affinity than ATRA for both receptors. However, in the presence of PML-RAR, the synthetic retinoid is a much better transactivator of retinoic acid-responsive element-containing promoters than the natural retinoid, whereas, in the presence of RAR alpha, AM580 and ATRA have similar activity. This may explain the strong cyto-differentiating potential of AM580 in PML-RAR-containing leukemic cells. 


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Suppression of nuclear factor kappa B and CD18-mediated leukocyte adhesion to the corneal endothelium by dexamethasone. 
PURPOSE: To demonstrate that leukocyte adhesion to cultured corneal endothelial cells is mediated by the CD18 antigen, and to determine whether dexamethasone directly suppresses adhesion by inhibiting activation of nuclear factor kappa B (NFkappaB). METHODS: Cultured bovine corneal endothelium was stimulated for 6 hours by 40 micron/ml tumor necrosis factor alpha (TNFalpha). Dexamethasone was added 1 hour before TNFalpha stimulation in the dexamethasone group. After stimulation, neutrophils separated from a healthy human volunteer were added with or without anti-CD18 antibody. The culture plate was settled for 15 minutes at 37 degrees C, and then neutrophils were activated by N-formyl-methionyl-leucyl-phenylalanine for 5 minutes. Nonadherent neutrophils were removed by sealing and inverting the culture well. The intracellular localization of NFkappaB after TNFalpha simulation was determined by confocal immunocytochemistry using an anti-p65 antibody. RESULTS: Neutrophil adhesion to cultured corneal endothelial cells increased significantly on exposure to TNFalpha (451.4+/-45.4 cells/mm2, n = 16) compared to control (156.7+/-27.3 cells/mm2, n = 16, P < 0.01). This increased adhesion was suppressed by the addition of anti-CD18 antibody (157.6+/-25.1 cells/mm2, n = 8, P < 0.01) and by pretreatment with 10(-7) M dexamethasone (207.9+/-31.5 cells/mm2, n = 10, P < 0.01). Immunocytochemistry 60 minutes after stimulation revealed that NFkappaB was located in the cytoplasm in unstimulated cells; however, the addition of TNFalpha caused NFkappaB to translocate into the nucleus. Pretreatment with dexamethasone tapered NFkappaB translocation into the nucleus. CONCLUSIONS: Leukocyte adhesion to the corneal endothelium was shown to be mediated by CD18 expressed on activated leukocytes. Pretreatment of the endothelium with dexamethasone inhibited leukocyte adhesion; this may be due in part to the suppression of NFkappaB entry into the nucleus. 


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The B cell-specific nuclear factor OTF-2 positively regulates transcription of the human class II transplantation gene, DRA. 
The promoter of the major histocompatibility class II gene DRA contains an octamer element (ATTTGCAT) that is required for efficient DRA expression in B cells. Several DNA-binding proteins are known to bind this sequence. The best characterized are the B cell-specific OTF-2 and the ubiquitous OTF-1. This report directly demonstrates that OTF-2 but not OTF-1 regulates the DRA gene. In vitro transcription analysis using protein fractions enriched for the octamer-binding protein OTF-2 demonstrate a positive functional role for OTF-2 in DRA gene transcription. In contrast, OTF-1-enriched protein fractions did not affect DRA gene transcription although it functionally enhanced the transcription of another gene. Recombinant OTF-2 protein produced by in vitro transcription/translation could also enhance DRA gene transcription in vitro. In vivo transient transfection studies utilizing an OTF-2 expression vector resulted in similar findings: that OTF-2 protein enhanced DRA gene transcription, and that this effect requires an intact octamer element. Together these results constitute the first direct evidence of a positive role for the lymphoid-specific octamer-binding factor in DRA gene transcription. 


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Modulation of the expression of the IFN-gamma receptor beta-chain controls responsiveness to IFN-gamma in human peripheral blood T cells. 
IFN-gamma has potent antiproliferative and apoptotic effects in T cells that are important in determining T cell development and polarized differentiation. Therefore, any event that enables T cells to become less responsive to IFN- gamma may potentially alter immune responsiveness to Ag. In this work, we show that human peripheral blood T cells that are stimulated through the TCR and expanded with IL-2 are unresponsive to IFN-gamma, as determined by a lack of activation of jak kinases and the transcription factor, STAT1(alpha), a signal transducer and activator of transcription. This nonresponsiveness occurs because of a lack of expression of the beta- chain (accessory factor) of the IFN-gamma receptor, while at the same time maintaining IFN-gamma receptor alpha-chain expression. Expression of the beta-chain can be restored by secondary TCR ligation or PMA treatment. T cell blasts treated with PMA are now responsive to IFN-gamma. When freshly isolated, highly enriched (>98%) T cells are examined for IFN-gamma responsiveness; these cells can respond to IFN-gamma and express beta-chain. Therefore, as T cells progress from primary TCR activation through IL-2-dependent proliferation, followed by secondary TCR stimulation, their responsiveness to IFN-gamma varies, and this may affect their ability to participate in an ongoing immune response. 


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Regulation of gene expression with double-stranded phosphorothioate oligonucleotides. 
Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins is necessary for determining how these factors participate in cellular differentiation. The functions of these proteins can be antagonized by several methods, each with specific limitations. Inhibition of sequence-specific DNA-binding proteins was achieved with double-stranded (ds) phosphorothioate oligonucleotides that contained octamer or kappa B consensus sequences. The phosphorothioate oligonucleotides specifically bound either octamer transcription factor or nuclear factor (NF)-kappa B. The modified oligonucleotides accumulated in cells more effectively than standard ds oligonucleotides and modulated gene expression in a specific manner. Octamer-dependent activation of a reporter plasmid or NF-kappa B-dependent activation of the human immunodeficiency virus (HIV) enhancer was inhibited when the appropriate phosphorothioate oligonucleotide was added to a transiently transfected B cell line. Addition of phosphorothioate oligonucleotides that contained the octamer consensus to Jurkat T leukemia cells inhibited interleukin-2 (IL-2) secretion to a degree similar to that observed with a mutated octamer site in the IL-2 enhancer. The ds phosphorothioate oligonucleotides probably compete for binding of specific transcription factors and may provide anti-viral, immunosuppressive, or other therapeutic effects. 


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Signals and nuclear factors that regulate the expression of interleukin-4 and interleukin-5 genes in helper T cells. 
Mouse thymoma line EL-4 cells produce cytokines such as interleukin (IL)-2, IL-3, IL-4, IL-10, and granulocyte-macrophage colony-stimulating factor in response to phorbol 12-myristate 13-acetate (PMA). EL-4 cells also produce low levels of IL-5 when stimulated by PMA alone; however, cAMP greatly augments PMA-dependent IL-5 production. A transient transfection assay revealed that two signals, PMA and cAMP, are required for optimal activation of the IL-5 promoter. In contrast, cAMP almost completely inhibited the PMA-dependent activation of the endogenous IL-2 gene, as well as the transfected IL-2 promoter. These results indicate that the IL-5 gene is positively regulated by cAMP in a manner opposite to that for the IL-2 gene. One of the nuclear factors (NFs) that regulates the response of the IL-5 promoter to cAMP and PMA has properties similar to NF for activated t cell. The P sequence of the IL-4 gene, defined as a responsive element for PMA and calcium ionophore (A23187), shares sequence similarity with the NF kappa B and the NF-activated T cell binding sites. We attempted to determine whether NF(P), a nuclear factor specific for the P sequence, is related to NF-kappa B and nuclear factor for activated T cell (NF-AT). In electromobility shift assays both NF-kappa B (P65 or P65/P50 heterodimer) and NF-AT bound to the P sequence. However, sequence specificity of NF-AT was more similar to that of NF(P), and only a small amount of P65 was detected in NF(P). These results indicate that a component or components of NF-AT have the potential to reconstitute NF(P), whereas NF-kappa B alone does not account for NF(P) in Jurkat crude extract. Taken together, these results suggest that NF-AT-like factors are involved in the regulation of IL-4 and IL-5 genes. 


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Octamer transcription factors and the cell type-specificity of immunoglobulin gene expression. 
Antibodies are produced exclusively in B lymphocytes. The expression of the antibody-encoding genes, the immunoglobulin (Ig) genes, is also restricted to B cells. The octamer sequence ATGCAAAT is present in the promoter and the enhancer of Ig genes, and plays an important role in its tissue-specific expression. This sequence motif is a binding site for nuclear proteins, the so-called octamer transcription factors (Oct or OTF factors). The Oct-1 protein is present in all cell types analyzed so far, whereas Oct-2A and Oct-2B are found mainly in B lymphocytes. All three proteins show the same sequence specificity and binding affinity. It appears that the B cell-specific expression of Ig genes is mediated at least in part by cell type-specific Oct factors, and that there are both quantitative and qualitative differences between Oct-1 and Oct-2 factors. Recently, a number of other octamer factor variants were identified. Many of these may be created by alternative splicing of a primary transcript of one Oct factor gene and may serve a specific function in the fine tuning of gene expression. 


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Retinoblastoma protein expression leads to reduced Oct-1 DNA binding activity and enhances interleukin-8 expression. 
Tumor cell lines with a defective retinoblastoma gene are unable to transcribe the HLA class II genes in response to IFN-gamma treatment, and reconstitution of functional Rb rescues IFN-gamma-induced class II gene expression. However, the molecular mechanism of Rb rescue of the class II genes is unknown. We have examined the effect of Rb expression on the activation of the promoter for HLA-DRA, the prototype class II gene. Oct-1, a POU domain transcription factor, was identified as a repressor of HLA-DRA promoter activity in the Rb-defective cells. Rb expression led to phosphorylation of Oct-1, thus relieving its repressive effect. Oct-1 has also been shown to repress interleukin 8 promoter activity. Consistent with reduced levels of Oct-1 DNA binding activity in the Rb-transformed cell lines, interleukin 8 expression is higher in these cell lines. 


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Inhibitor (IK) of IFN-gamma induced HLA class II antigens expression also inhibits HLA class II constitutive expression in the human Raji B cell line. 
The expression of major histocompatibility complex (MHC) class II antigens is constitutive in professional antigen presenting cells (APCs) but can also be induced by interferon-gamma (IFN-gamma) on the majority of the non professional APCs (e.g. fibroblasts). We have recently characterised a new factor called IK which is an efficient inhibitor of IFN-gamma induction of MHC class II antigens expression. Here, we demonstrate a novel role for IK in MHC class II expression since over-expression of this protein by stable transfection into human B cells led to a total disappearance of constitutive MHC class II mRNA expression. The class II transactivator (CIITA) is necessary for both constitutive and IFN-gamma induced MHC class II expressions. Examination of CIITA mRNA in IK stably transfected clones revealed a marked reduction of CIITA mRNA transcription. Taken together these results demonstrate that the IK protein plays a key role in the constitutive expression of MHC class II antigens and that inhibition induced by IK is upstream of CIITA in this regulatory pathway. 


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Epstein-Barr virus binding to CD21 activates the initial viral promoter via NF-kappaB induction. 
Epstein-Barr virus (EBV), an oncogenic human herpesvirus, binds to and infects normal human B lymphocytes via CD21, the CR2 complement receptor. Studies of the mechanisms that enable EBV to infect nonactivated, noncycling B cells provide compelling evidence for a sequence of events in which EBV binding to CD21 on purified resting human B cells rapidly activates the NF-kappaB transcription factor, which, in turn, binds to and mediates transcriptional activation of Wp, the initial viral latent gene promoter. Thus, EBV binding to its cellular receptor on resting B cells triggers an NF-kappaB-dependent intracellular signaling pathway which is required for infection. 


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PPARgamma activation induces the expression of the adipocyte fatty acid binding protein gene in human monocytes. 
The peroxisome-proliferator activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily of ligand activated transcription factors, plays a key role in the anti-diabetic actions of the thiazolidinediones (TZDs). PPARgamma induces the expression of many genes involved in lipid anabolism, including the adipocyte fatty acid binding protein (aP2), and is a key regulator of adipocyte differentiation. PPARgamma is also expressed in hematopoietic cells and is up-regulated in activated monocytes/macrophages. Activation of PPARgamma may play a role in the induction of differentiation of macrophages to foam cells that are associated with atherosclerotic lesions. We report that both natural and synthetic PPARgamma agonists induce time- and dose-dependent increases in aP2 mRNA in both primary human monocytes and the monocytic cell line, THP-1. These data suggest that PPARgamma activation may play a role in monocyte differentiation and function analogous to its well-characterized role in adipocytes. 


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An enhancer-blocking element between alpha and delta gene segments within the human T cell receptor alpha/delta locus. 
T cell receptor (TCR) alpha and delta gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3' of TCR delta gene segments and 5' of TCR alpha joining gene segments within this locus. BEAD-1 blocked the ability of the TCR delta enhancer (Edelta) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR alpha/delta locus into distinct regulatory domains controlled by Edelta and the TCR alpha enhancer, and that it prevents Edelta from opening the chromatin of the TCR alpha joining gene segments for VDJ recombination at an early stage of T cell development. 


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Nuclear factor-kappaB induction in CD45RO+ and CD45RA+ T cell subsets during aging. 
An increase in the ratio of memory to naive T cells has been postulated to underlie immune hyporesponsiveness accompanying aging. Our analyses of the induction of nuclear factor-kappaB (NFkappaB) in activated memory (CD45RO+) and naive (CD45RA+) T cell subsets from young and elderly donors has demonstrated that, regardless of donor age, memory T cells are not significantly altered in their responsiveness to TNF-alpha-mediated induction of NFkappaB. Although treatment with TNF-alpha induced nuclear localization of NFkappaB in both memory and naive T cell subsets, irrespective of the age of the donor, the levels of induced NFkappaB were significantly lower in both subsets of T cells obtained from the elderly, when compared to those in young. Examination of IkappaB alpha regulation revealed that TNF-alpha-mediated degradation of IkappaB alpha in both memory and naive T cells from the elderly was severely impaired, thus contributing to the lowered induction of the observed NFkappaB. In addition, this age-related decrease in induction of nuclear NFkappaB correlated with decrease in intracellular IL-2 receptor expression and anti-CD3-induced proliferation of both memory and naive T cells subsets. Taken together, our results suggest that the age-related hyporesponsiveness cannot be attributed to a skewing of the T cell population towards a memory phenotype in the elderly. 


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Patterns of Pan expression and role of Pan proteins in endocrine cell type-specific complex formation. 
The Pan gene encodes at least two distinct transcripts, Pan-1 and Pan-2 (also known as E47 and E12, respectively), by the mechanism of alternative RNA splicing. Northern blot analyses performed on rat and mouse tissues have detected ubiquitously expressed Pan transcripts, but the abundance, distribution, and form of Pan proteins have not been clearly defined. Studies of cell lines representing endocrine, fibroblast, and lymphoid lineages using polyclonal antisera to detect E2A proteins have suggested that significant E2A protein expression is restricted to B-lymphocytes. We have developed a monoclonal antibody, Yae, which is specific for Pan/E2A proteins, and have used the Yae antibody to examine a variety of endocrine and nonendocrine cell lineages for differences in Pan/E2A protein expression, subcellular localization, and heteromeric complex formation. In contrast to previous results obtained using polyclonal antiseras to detect Pan/E2A proteins, we report comparable levels of Pan proteins in GH/PRL- and insulin-producing, B- and T-lymphocyte cells. IEF-1, a pancreatic beta-cell type-specific complex believed to regulate insulin expression, is demonstrated to consist of at least two distinct species, one of which does not contain Pan molecules. Although it has been postulated that pituitary endocrine cells and pancreatic endocrine beta-cells share identical Pan/E2A complexes, native-Western analyses of pituitary and endocrine beta-cells detect Pan proteins in distinct cell type-specific complexes. 


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Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation. 
The eukaryotic transcription factor NF-kappa B plays a central role in the induced expression of human immunodeficiency virus type 1 and in many aspects of the genetic program mediating normal T-cell activation and growth. The nuclear activity of NF-kappa B is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called I kappa B alpha. This cytoplasmic inhibitor is rapidly phosphorylated and degraded in response to a diverse set of NF-kappa B-inducing agents, including T-cell mitogens, proinflammatory cytokines, and viral transactivators such as the Tax protein of human T-cell leukemia virus type 1. To explore these I kappa B alpha-dependent mechanisms for NF-kappa B induction, we identified novel mutants of I kappa B alpha that uncouple its inhibitory and signal-transducing functions in human T lymphocytes. Specifically, removal of the N-terminal 36 amino acids of I kappa B alpha failed to disrupt its ability to form latent complexes with NF-kappa B in the cytoplasm. However, this deletion mutation prevented the induced phosphorylation, degradative loss, and functional release of I kappa B alpha from NF-kappa B in Tax-expressing cells. Alanine substitutions introduced at two serine residues positioned within this N-terminal regulatory region of I kappa B alpha also yielded constitutive repressors that escaped from Tax-induced turnover and that potently inhibited immune activation pathways for NF-kappa B induction, including those initiated from antigen and cytokine receptors. In contrast, introduction of a phosphoserine mimetic at these sites rectified this functional defect, a finding consistent with a causal linkage between the phosphorylation status and proteolytic stability of this cytoplasmic inhibitor. Together, these in vivo studies define a critical signal response domain in I kappa B alpha that coordinately controls the biologic activities of I kappa B alpha and NF-kappa B in response to viral and immune stimuli. 


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Molecular cloning and functional characterization of murine cDNA encoding transcription factor NFATc. 
Transcription factors of the NFAT (nuclear factor of activated T cells) family play important roles in immune and inflammatory responses by regulating the expression of genes encoding cytokines and immunoregulatory proteins. Here we describe cloning and characterization of full-length cDNA encoding murine (m) NFATc which predicts that the protein has all the conserved structural motifs of NFAT family members, including the rel homology domain, the NFAT homology domain and the nuclear translocation signals. mNFATc complexed with AP-1 bound specifically to the murine IL-2 NFAT recognition sequence and activated transcription from the co-transfected IL-2 promoter in COS-7 cells. Northern blot analysis showed that the cDNA probe hybridized with a 4.5 kb transcript which is highly inducible in murine T cells. By Northern and in situ hybridization, mNFATc transcript was detected from the early stage of development. In the mouse embryo, mNFATc transcript was strongly expressed in thymus, lung and submandibular gland and weakly in skeletal muscle and heart suggesting that mNFATc may have a role both in embryogenesis and in mature T cells. 


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Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin. 
Proliferation and immunologic function of T lymphocytes are initiated by signals from the antigen receptor that are inhibited by the immunosuppressant FK506 but not by its structural analog, rapamycin. On the other hand, interleukin 2 (IL-2)-induced signals are blocked by rapamycin but not by FK506. Remarkably, these two drugs inhibit each other's actions, raising the possibility that both act by means of a common immunophilin (immunosuppressant binding protein). We find that the dissociation constant of rapamycin to the FK506 binding protein FKBP (Kd = 0.2 nM) is close to the dissociation constant of FK506 to FKBP (Kd = 0.4 nM) and to their effective biologic inhibitory concentrations. However, an excess of rapamycin is needed to revert FK506-mediated inhibition of IL-2 production, apoptosis, and transcriptional activation of NF-AT, a T-cell-specific transcription factor necessary for IL-2 gene activation. Similarly, an excess of FK506 is needed to revert rapamycin-mediated inhibition of IL-2-induced proliferation. The drug concentrations required for antagonism may be explained by the relative affinity of the drugs to, and by the abundance of, the immunophilin FKBP. FKBP has been shown to catalyze the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptide substrates; here we show that rapamycin, like FK506, is a potent inhibitor of the rotamase activity of FKBP (Ki = 0.2 nM). Neither FKBP binding nor inhibition of rotamase activity of FKBP alone is sufficient to explain the biologic actions of these drugs. Rather, these findings suggest that immunophilin bound to FK506 interferes with antigen receptor-induced signals, while rapamycin bound to the immunophilin interferes with IL-2-induced signals. 


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Signals transduced through the CD4 molecule on T lymphocytes activate NF-kappa B. 
We have demonstrated that native envelope glycoproteins of HIV-1, gp160 can induce activation of the transcription factor, NF-kappa B. The stimulatory effects of gp160 are mediated through the CD4 molecule, since pretreatment with soluble CD4 abrogates its activity. The gp160-induced NF-kappa B complex consists of p65, p50 and c-rel proteins. The stimulatory effect of gp160 on NF-kappa B activation is protein synthesis independent, is dependent upon protein tyrosine phosphorylation, and abrogated by inhibitors of protein kinase C. The gp160-mediated activation of NF-kappa B in CD4 positive T cells may be involved in biological effects, e.g., enhanced HIV replication, hypergammaglobulinemia, increased cytokine secretion, hypercellularity in bone marrow and apoptosis. 


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Constitutive activation of different Jak tyrosine kinases in human T cell leukemia virus type 1 (HTLV-1) tax protein or virus-transformed cells. 
HTLV-1 infection causes an adult T cell leukemia in humans. The viral encoded protein tax, is thought to play an important role in oncogenesis. Our previous data obtained from a tax transgenic mouse model revealed that tax transforms mouse fibroblasts but not thymocytes, despite comparable levels of tax expression in both tissues. Constitutive tyrosine phosphorylation of a 130-kD protein(s) was observed in the tax transformed fibroblast B line and in HTLV-1 transformed human lymphoid lines, but not in thymocytes from Thy-tax transgenic mice. Phosphotyrosine immunoprecipitation followed by Western blot analysis with a set of Jak kinase specific antibodies, identified p130 as Jak2 in the tax transformed mouse fibroblastic cell line and Jak3 in HTLV-1 transformed human T cell lines. Phosphorylation of Jak2 in tax transformed cells resulted from high expression of IL-6. Tyrosine phosphorylation of this protein could also be induced in Balb/c3T3 cells using a supernatant from the B line, which was associated with induction of cell proliferation. Both phosphorylation and proliferation were inhibited by IL-6 neutralizing antibodies. Constitutive phosphorylation of Jak kinases may facilitate tumor growth in both HTLV-1 infected human T cells and the transgenic mouse model. 


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Regulation of interleukin-1 beta production by glucocorticoids in human monocytes: the mechanism of action depends on the activation signal. 
Glucocorticoids are known to downregulate interleukin-1 beta production in monocytic cells by two different mechanims: direct inhibition of the gene transcription and destabilization of the preformed interleukin-1 beta mRNA. Now we have examined the effect of the nature of the monocyte activating signal on these two inhibitory mechanims. When human monocytes were preincubated with dexamethasone for 1 hour and then stimulated either with bacterial lipopolysaccharide or phorbol myristate, it was found that dexamethasone inhibited the lipopolysaccharide-induced interleukin-1 beta protein production, but the phorbol myristate-induced production was increased 3-10 fold. This difference was also seen at the mRNA level. When dexamethasone was added to the cultures 3 hours after the stimulators, it clearly decreased the interleukin-1 beta mRNA levels regardless of the stimulator used (although the effect was clearly weaker on the PMA-induced mRNA). Thus these data suggest that the phorbol myristate-induced signal (prolonged protein kinase C activation?) cannot be inhibited by prior incubation with dexamethasone and it also protects the induced mRNA for the degradative action of dexamethasone. 


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Heat shock induces HIV-1 replication in chronically infected promyelocyte cell line OM10.1. 
A long period of clinical latency before development of symptoms is characteristic of human immunodeficiency virus type 1 (HIV-1) infection. OM10.1, a promyelocyte cell line latently infected with HIV-1, has been developed as a model for studying the mechanism of viral latency and the activation of virus expression. We found that this latently infected cell line with heat shock at 42 degrees C for 2 h resulted in a high level of HIV-1 production without addition of any cytokines. The mechanism of activation was analyzed by using anti-TNF-alpha antibody and various inhibitors. Although the TNF-alpha level in culture supernatants was below the sensitivity of an ELISA assay system, addition of anti-TNF-alpha antibody in culture medium could partially suppress the heat shock induced HIV-1 production. Staurosporine (PKC inhibitor), pentoxifylline (NF-kappa B inhibitor), and Ro5-3335 (HIV-1 Tat inhibitor) also inhibited significantly the heat shock induced virus activation. In particular, staurosporine achieved approximately 90% inhibition of the HIV-1 antigen expression in heat shock-treated OM10.1 at a non-toxic concentration. Although the mechanism of HIV-1 activation with heat shock has not been fully elucidated yet, it is presumed PKC plays an important role in HIV-1 activation. Thus, the present observations will provide a further insight into the pathogenesis of HIV-1 infections. 


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SHP2-interacting transmembrane adaptor protein (SIT), a novel disulfide-linked dimer regulating human T cell activation. 
T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain-containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes. After tyrosine phosphorylation by src and possibly syk protein tyrosine kinases SIT recruits the SH2 domain-containing tyrosine phosphatase SHP2 via an immunoreceptor tyrosine-based inhibition motif. Overexpression of SIT in Jurkat cells downmodulates T cell receptor- and phytohemagglutinin-mediated activation of the nuclear factor of activated T cells (NF-AT) by interfering with signaling processes that are probably located upstream of activation of phospholipase C. However, binding of SHP2 to SIT is not required for inhibition of NF-AT induction, suggesting that SIT not only regulates NF-AT activity but also controls NF-AT unrelated pathways of T cell activation involving SHP2. 


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Thiol modulation inhibits the interleukin (IL)-1-mediated activation of an IL-1 receptor-associated protein kinase and NF-kappa B. 
The interleukin-1 receptor type I (IL-1RI) is associated with other proteins thus forming a complex system by which IL-1 exerts its various signals. The initiating event is still uncertain, but activation of a recently described receptor-associated protein kinase is one of the earliest events detectable (Martin et al., Eur.J.Immunol.1994.24: 1566). IL-1 signaling is commonly accompanied by oxidative processes and is thought to be subject to redox regulation. We therefore investigated whether the activation of the IL-1RI-associated protein kinase could be a target for redox regulation and whether an altered activity of the kinase could influence IL-1-mediated NF-kappa B activation. A murine T cell line, EL4, was stimulated with IL-1 with and without pretreatment with different compounds known to influence the cellular redox status. Thiol modifying agents like diamide, menadione, pyrrolidine dithiocarbamate (PDTC), diethyl dithiocarbamate or phenylarsine oxide inhibited the IL-1-induced activation of the IL-1RI-associated protein kinase. N-Acetylcysteine, alpha,alpha'-dipyridyl, aminotriazole or nitrofurantoin did not show any effect. The inhibition by PDTC was reversible unless glutathione synthesis was blocked by buthionine sulfoximine. The described conditions which inhibited or prevented the activation of the IL-1RI-associated kinase similarly impaired the activation of NF-kappa B in EL4 cells. From these observations we conclude that free thiols in the IL-1RI complex are essential for the activation of the IL-1RI-associated protein kinase and that this process is mandatory for IL-1 signaling leading to NF-kappa B activation. 


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Cellular and molecular mechanisms of IL-5 synthesis in atopic diseases: a study with allergen-specific human helper T cells. 
BACKGROUND: Cytokines produced by helper T cells are intimately involved in chronic allergic diseases associated with eosinophilic inflammation. OBJECTIVE: We investigated the production of IL-5, a potent growth factor and chemotactic factor for eosinophils, by CD4+ T lymphocytes in patients with asthma. METHODS: Allergen-specific T cell clones and T cell hybridomas were established from the peripheral blood lymphocytes of patients with asthma, and the responses to various stimuli were determined. RESULTS: After nonspecific stimulation, IL-5 production by CD4+ T cells from both atopic and nonatopic subjects with asthma was significantly enhanced compared with that by cells from healthy controls. Peripheral blood mononuclear cells from atopic asthma patients both proliferated and produced IL-5 after incubation with mite allergen, suggesting that mite-specific helper T cells were involved in the eosinophilic inflammation of atopic asthma. A human IL-5 promoter/enhancer luciferase gene construct transfected into IL-5-producing T cell clones was clearly transcribed after stimulation, indicating that the 515 base pair IL-5 gene segment upstream of the coding region was sufficient to respond to activating signals in human helper T cells. The same gene segment was not transcribed in IL-5-nonproducing T cell clones, suggesting that human T cell IL-5 synthesis is regulated at the transcriptional level. Experiments with T cell hybridomas confirmed these findings and suggested that a unique transcription factor may be essential for human IL-5 gene transcription. CONCLUSION: Enhanced IL-5 production by helper T cells seems to cause the eosinophilic inflammation of both atopic and nonatopic asthma. Elucidation of IL-5-specific regulatory mechanisms may facilitate the development of novel treatments for allergic diseases associated with eosinophilic inflammation. 


Document 003001990 ends.


Renal cell carcinoma-derived gangliosides suppress nuclear factor-kappaB activation in T cells. 
Activation of the transcription factor nuclear factor-kappaB (NFkappaB) is impaired in T cells from patients with renal cell carcinomas (RCCs). In circulating T cells from a subset of patients with RCCs, the suppression of NFkappaB binding activity is downstream from the stimulus-induced degradation of the cytoplasmic factor IkappaBalpha. Tumor-derived soluble products from cultured RCC explants inhibit NFkappaB activity in T cells from healthy volunteers, despite a normal level of stimulus-induced IkappaBalpha degradation in these cells. The inhibitory agent has several features characteristic of a ganglioside, including sensitivity to neuraminidase but not protease treatment; hydrophobicity; and molecular weight less than 3 kDa. Indeed, we detected gangliosides in supernatants from RCC explants and not from adjacent normal kidney tissue. Gangliosides prepared from RCC supernatants, as well as the purified bovine gangliosides G(m1) and G(d1a), suppressed NFkappaB binding activity in T cells and reduced expression of the cytokines IL-2 and IFN-gamma. Taken together, our findings suggest that tumor-derived gangliosides may blunt antitumor immune responses in patients with RCCs. 


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3-deazaadenosine, a S-adenosylhomocysteine hydrolase inhibitor, has dual effects on NF-kappaB regulation. Inhibition of NF-kappaB transcriptional activity and promotion of IkappaBalpha degradation. 
Previously we reported that 3-deazaadenosine (DZA), a potent inhibitor and substrate for S-adenosylhomocysteine hydrolase inhibits bacterial lipopolysaccharide-induced transcription of tumor necrosis factor-alpha and interleukin-1beta in mouse macrophage RAW 264.7 cells. In this study, we demonstrate the effects of DZA on nuclear factor-kappaB (NF-kappaB) regulation. DZA inhibits the transcriptional activity of NF-kappaB through the hindrance of p65 (Rel-A) phosphorylation without reduction of its nuclear translocation and DNA binding activity. The inhibitory effect of DZA on NF-kappaB transcriptional activity is potentiated by the addition of homocysteine. Taken together, DZA promotes the proteolytic degradation of IkappaBalpha, but not IkappaBbeta, resulting in an increase of DNA binding activity of NF-kappaB in the nucleus in the absence of its transcriptional activity in RAW 264.7 cells. The reduction of IkappaBalpha by DZA is neither involved in IkappaB kinase complex activation nor modulated by the addition of homocysteine. This study strongly suggests that DZA may be a potent drug for the treatment of diseases in which NF-kappaB plays a central pathogenic role, as well as a useful tool for studying the regulation and physiological functions of NF-kappaB. 


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Inducible binding to the c-fos serum response element during T cell activation is regulated by a phosphotyrosine-containing protein. 
The proto-oncogene c-fos is an immediate-early gene, and one of the first genes transcribed after stimulation of most cells with a variety of ligands. Fos expression may be a pivotal event in converting ligand-receptor interactions at the membrane into functional modulation of cell phenotype. The serum response element (SRE) in the c-fos regulatory region participates in induction of transcription by various growth factors and by phorbol esters and subsequent squelching of transcription. We show that an inducible protein complex (Band A) binds to SRE DNA within 10 min after mitogenic stimulation of human PBL-T, and becomes nondetectable by 60 min. Band A contains the serum response factor plus additional factor(s). A protein that is phosphorylated on a tyrosine residue in resting PBL-T suppresses binding of a component of Band A to the SRE motif. Upon stimulation of the cells, this protein no longer prevents binding of DNA by Band A, and suppression of binding is restored within 30 min. The phosphorylated tyrosine residue itself is important for the protein-protein interaction. 


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Rel/NF-kappaB can trigger the Notch signaling pathway by inducing the expression of Jagged1, a ligand for Notch receptors. 
Jagged1 belongs to the DSL family of ligands for Notch receptors that control the proliferation and differentiation of various cell lineages. However, little is known about the transcription factors that regulate its expression. Here, we show that Jagged1 is a Rel/NF-kappaB-responsive gene. Both c-Rel and RelA induced jagged1 gene expression, whereas a mutant defective for transactivation did not. Importantly, jagged1 transcripts were also upregulated by endogenous NF-kappaB activation and this effect was inhibited by a dominant mutant of IkappaBalpha, a physiological inhibitor of NF-kappaB. Cell surface expression of Jagged1 in c-Rel-expressing cell monolayers led to a functional interaction with lymphocytes expressing the Notch1/TAN-1 receptor. This correlated with the initiation of signaling downstream of Notch, as evidenced by increased levels of HES-1 transcripts in co-cultivated T cells and of CD23 transcripts in co-cultivated B cells. Consistent with its Rel/NF-kappaB-dependent induction, Jagged1 was found to be highly expressed in splenic B cells where c-Rel is expressed constitutively. These results demonstrate that c-Rel can trigger the Notch signaling pathway in neighboring cells by inducing jagged1 gene expression, and suggest a role for Jagged1 in B-cell activation, differentiation or function. These findings also highlight the potential for an interplay between the Notch and NF-kappaB signaling pathways in the immune system. 


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Transcription factor AP-2 activates gene expression of HTLV-I. 
The HTLV-I LTR contains three conserved regulatory elements known as 21 base pair repeats which are required for stimulation of gene expression by the transactivator protein tax. Mutagenesis indicates that the 21 bp repeats can be subdivided into three motifs, A, B and C, each of which influences the level of tax activation. The A site in the 21 bp repeat has strong homology with previously described binding sites for the transcription factor AP-2. We demonstrated that AP-2 mRNA was present in T-lymphocytes and that cellular factors from both non-transformed and transformed T-lymphocytes specifically bound to the consensus motif for AP-2 in each 21 bp. To determine the role of AP-2 in the regulation of the HTLV-I LTR gene expression, we used an AP-2 cDNA in DNA binding and transient expression assays. Gel retardation and methylation interference studies revealed that bacterially produced AP-2 bound specifically and with high affinity to all three 21 bp repeats, and that it required the core sequence AGGC for specific binding. Binding of AP-2 prevented the subsequent binding of members of the CREB/ATF family to an adjacent regulatory motif in the 21 bp repeat. Transfection of an AP-2 expression construct into T-lymphocytes activated gene expression from the HTLV-I LTR. At least two 21 bp repeats were required for high levels of AP-2 activation and mutagenesis of the AP-2 consensus binding sequences in the 21 bp repeats eliminate this activation. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Two distinct transcription factors that bind the immunoglobulin enhancer microE5/kappa 2 motif. 
Activity of the immunoglobulin heavy and kappa light chain gene enhancers depends on a complex interplay of ubiquitous and developmentally regulated proteins. Two complementary DNAs were isolated that encode proteins, denoted ITF-1 and ITF-2, that are expressed in a variety of cell types and bind the microE5/kappa 2 motif found in both heavy and kappa light chain enhancers. The complementary DNAs are the products of distinct genes, yet both ITF-1 and ITF-2 are structurally and functionally similar. The two proteins interact with one another through their putative helix-loop-helix motifs and each possesses a distinct domain that dictates transcription activation. 


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Interaction of sickle erythrocytes with endothelial cells in the presence of endothelial cell conditioned medium induces oxidant stress leading to transendothelial migration of monocytes. 
The abnormal adherence of sickle red blood cells (SS RBC) to endothelial cells has been thought to contribute to vascular occlusion, a major cause of morbidity in sickle cell disease (SCD). We determined whether the interaction of SS RBC with cultured endothelial cells induced cellular oxidant stress that would culminate in expression of cell adhesion molecules (CAMs) involved in the adhesion and diapedesis of monocytes and the adherence of SS reticulocytes. We showed that the interaction of SS RBC at 2% concentration in the presence of multimers of von Willebrand factor (vWf), derived from endothelial cell-derived conditioned medium (E-CM) with cultured human umbilical vein endothelial cells (HUVEC), resulted in a fivefold increased formation of thiobarbituric acid-reactive substances (TBARS) and activation of the transcription factor NF-kB, both indicators of cellular oxidant stress. Normal RBC show none of these phenomena. The oxidant stress-induced signaling resulted in an increased surface expression of a subset of CAMs, ICAM-1, E-selectin, and VCAM-1 in HUVEC. The addition of oxygen radical scavenger enzymes (catalase, superoxide dismutase) and antioxidant (probucol) inhibited these events. Additionally, preincubation of HUVEC with a synthetic peptide Arg-Gly-Asp (RGD) that prevents vWf-mediated adhesion of SS RBC reduced the surface expression of VCAM-1 and NF-kB activation. Furthermore, SS RBC-induced oxidant stress resulted in a twofold increase in the transendothelial migration of both monocyte-like HL-60 cells and human peripheral blood monocytes, and approximately a sixfold increase in platelet-endothelial cell adhesion molecule-1 (PECAM-1) phosphorylation, each of which was blocked by protein kinase C inhibitor and antioxidants. These results suggest that the adherence/contact of SS RBC to endothelial cells in large vessel can generate enhanced oxidant stress leading to increased adhesion and diapedesis of monocytes, as well as heightened adherence of SS reticulocytes, indicating that injury/activation of endothelium can contribute to vaso-occlusion in SCD. 


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Two distinct signalling pathways are involved in the control of the biphasic junB transcription induced by interleukin-6 in the B cell hybridoma 7TD1. 
We have measured the level of junB mRNA in the B hybridoma cell line 7TD1, under interleukin-6 (IL-6) stimulation. IL-6 increases junB mRNA in a biphasic fashion. The first early-induced peak was transient and likely corresponds to the well documented typical junB mRNA, stimulated in response to numerous growth factors, including IL-6. At variance, the second peak which has never been reported previously, lasted several hours. As a consequence of its effect on junB mRNA, IL-6 stimulated, in a biphasic fashion, the nuclear accumulation of the JunB protein. In this study, we demonstrated that IL-6 regulation occurred exclusively at the transcriptional level and that the bimodal increase of junB mRNA and JunB protein can be accounted for by a biphasic stimulation of junB transcription. Furthermore, our data point to two major differences between the mechanism of control of the early and the late IL-6-induced junB transcription waves. First, cycloheximide strongly potentiated the transcription of the second wave, whereas it failed to affect the early-induced burst. Second, tyrphostin, a tyrosine kinase inhibitor, impaired the expression of the first but not the second junB mRNA peak. Conversely, genistein, another tyrosine kinase inhibitor, totally abolished the expression of the second peak of junB mRNA whereas it did not affect the expression of the first peak. Altogether these data indicate that, in 7TD1 cells, IL-6 controls junB transcription in a biphasic fashion by means of two separate transduction pathways. 


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IFN-gamma priming of monocytes enhances LPS-induced TNF production by augmenting both transcription and MRNA stability. 
The induction of cytokine expression in monocytes/macrophages by bacterial endotoxin or lipopolysaccharide is a critical, highly regulated host defence response. The augmentation of LPS responses by interferon gamma (IFN-gamma), referred to as priming, is well established. However, the mechanism(s) by which priming occurs is poorly defined. Using tumour necrosis factor (TNF) induction as a model, experiments were designed to analyse in detail the priming effect on the LPS response in human monocytes. Priming by IFN-gamma was primarily manifested at the level of TNF mRNA accumulation. IFN-gamma pre-treatment affected the magnitude rather than the sensitivity of the LPS response. Priming occurred after several hours of treatment, and the primed state was induced by either IFN-gamma or GM-CSF, but not M-CSF. Primed monocytes transcribed TNF mRNA at a higher rate than freshly isolated monocytes upon activation with LPS. The increased transcriptional rate correlated with a marked increase in nuclear factor-kappa B activity in these cells as determined by electrophoretic mobility shift assay using a consensus NF-kappa B oligonucleotide. An additional significant finding was than TNF mRNA induced in primed cells was much more stable than in unprimed cells (T1/2 increased 6-8-fold). Consistent with the increased mRNA stability, the duration of mRNA accumulation was longer following LPS stimulation in primed monocytes, in addition to being of greater magnitude. Finally, primed and unprimed cells possessed a differential sensitivity to the kinase inhibitor H-89. H-89 substantially suppressed LPS-induced TNF mRNA accumulation in unprimed cells, but had no effect on primed monocytes following LPS stimulation. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Platelet-activating factor (PAF) positively auto-regulates the expression of human PAF receptor transcript 1 (leukocyte-type) through NF-kappa B. 
The human platelet-activating factor receptor (PAFR) gene is transcribed by two distinct promoters (promoter 1 and promoter 2) to generate two transcripts (designated as PAFR transcript 1 and PAFR transcript 2), though their open reading frames are identical. By primer extension analysis to discriminate two transcripts, we found that the levels of PAFR transcript 1 (leukocyte-type), but not PAFR transcript 2 (tissue-type), are upregulated by PAF as well as by 12-O-tetradecanoylphorbol-13-acetate (TPA) in the human stomach cancer cell line (JR-St cells) which expresses both functional PAFR transcript 1 and PAFR transcript 2 endogenously. Functional analysis of the promoter 1 with a transient expression assay using chloramphenicol acetyltransferase (CAT) gene as a reporter showed that both PAF and TPA activated the promoter 1 but not the deleted promoter lacking the three consensus binding sites for NF-kappa B located from -571 bp to -459 bp. These findings suggest a molecular mechanism of positive regulation of PAFR gene expression by PAF through NF-kappa B, possibly by a phosphorylation reaction involving protein kinase C by PAF. 


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Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis [see comments] 
Glucocorticoids are among the most potent anti-inflammatory and immunosuppressive agents. They inhibit synthesis of almost all known cytokines and of several cell surface molecules required for immune function, but the mechanism underlying this activity has been unclear. Here it is shown that glucocorticoids are potent inhibitors of nuclear factor kappa B (NF-kappa B) activation in mice and cultured cells. This inhibition is mediated by induction of the I kappa B alpha inhibitory protein, which traps activated NF-kappa B in inactive cytoplasmic complexes. Because NF-kappa B activates many immunoregulatory genes in response to pro-inflammatory stimuli, the inhibition of its activity can be a major component of the anti-inflammatory activity of glucocorticoids. 


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Calcium signalling in T cells stimulated by a cyclophilin B-binding protein. 
The immunosuppressant drug cyclosporin A blocks a calcium-dependent signal from the T-cell receptor (TCR) that normally leads to T-cell activation. When bound to cyclophilin, cyclosporin A binds and inactivates the key signalling intermediate calcineurin. To identify potential cellular homologues of cyclosporin A that might regulate calcium signalling, we have cloned human genes encoding cyclophilin B-binding-proteins using the yeast two-hybrid system. One gene product, when overexpressed in Jurkat T cells, specifically induced transcription from the interleukin-2 enhancer, by activating the T-cell-specific transcription factors NF-AT and NF-IL2A. This protein, termed calcium-signal modulating cyclophilin ligand (CAML), acts downstream of the TCR and upstream of calcineurin by causing an influx of calcium. CAML appears to be a new participant in the calcium-signal transduction pathway, implicating cyclophilin B in calcium signalling, even in the absence of cyclosporin. 


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Characterization of defensin resistance phenotypes associated with mutations in the phoP virulence regulon of Salmonella typhimurium. 
The defensin sensitivities of Salmonella typhimurium strains with mutations in the phoP/phoQ two-component virulence regulon were tested by using purified defensins NP-1 and NP-2. Strains with mutations in either gene of the regulatory pair (phoP [transcriptional activator] or phoQ [membrane sensor kinase]) had increased sensitivities to defensin. The predicted periplasmic domain of the PhoQ protein contained a markedly anionic domain that could interact with cationic proteins and that could be responsible for resistance to defensin. Because insertion mutations in phoP are polar on phoQ, we constructed strains that expressed the PhoQ protein in the absence of PhoP to test whether resistance to defensin requires only the phoQ gene product. We found that resistance to defensin requires the function of both components of this regulatory system, because strains expressing PhoQ without PhoP were still markedly sensitive to defensins. This implied that a pag (phoP-activated gene) product is responsible for defensin resistance. We also tested for the ability of defensins NP-1, NP-5, and HNP-1 to activate pag expression and found that these peptides have no effect. Defensin resistance is not the only virulence characteristic controlled by the PhoP-PhoQ regulon because mutations in pagC, as well as ones in the phoP locus that resulted in constitutive pag activation (phenotype PhoPc), had no effect on defensin resistance, even though they rendered the organism avirulent and deficient in survival within macrophages. The virulence defect conferred by mutations in the phoP-phoQ two-component regulatory system is not completely explained by alterations in resistance to cationic proteins and involves the control of other proteins necessary for S. typhimurium survival within macrophages. 


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AP-1 derived from mature monocytes and astrocytes preferentially interacts with the HTLV-I promoter central 21 bp repeat. 
Characterization of the cellular transcription factors interacting with the human T cell lymphotropic virus type I (HTLV-I) long terminal repeat (LTR) is essential to dissecting the mechanisms involved in viral transcription that may be pertinent to the oncogenic and neuropathogenic processes associated with HTLV-I infection in both the immune and nervous systems. Electrophoretic mobility shift (EMS) analyses utilizing oligonucleotides homologous to each of the 21 bp repeat elements reacted with nuclear extracts derived from cell lines of lymphocytic, monocytic, neuronal, and glial cell origin have demonstrated differential binding of cellular factors to the three 21 bp repeats (1-4). ATF/CREB and Sp family members interacted with the 21 bp repeats to form DNA-protein complexes common to all cell types examined. However, a unique DNA-protein complex was detected when the promoter central 21 bp repeat was reacted with nuclear extracts derived from either the U-373 MG glioblastoma cell line or the THP-1 mature monocytic cell line. Based on nucleotide sequence requirements and immunoreactivity, we demonstrate that this DNA-protein complex is comprised of the AP-1 components, Fos and Jun. 


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Histamine modulates the expression of c-fos through cyclic AMP production via the H2 receptor in the human promonocytic cell line U937. 
We examined the effects of histamine and its agonists on the expression of the c-fos and c-myc proto-oncogenes at the transcriptional and translational levels in the human promonocytic U937 cell line. Histamine transiently increased cAMP and c-fos expression through H2 receptors. Dibutyryl cAMP also increased c-fos mRNA and protein, and levels remained elevated even after 12 hr of treatment. Dose-dependence studies using histamine and dimaprit showed that the EC50 values for cAMP production and c-fos increase were similar, suggesting that cAMP might be involved in c-fos induction via H2 receptors. Furthermore, studies carried out using H7, a protein kinase A/protein kinase C inhibitor, blocked c-fos induction, whereas no effect was observed with bisindolylmaleimide, a specific protein kinase C inhibitor. No modification of c-myc expression could be detected on treatment with histamine or its analogues. Nevertheless, dibutyryl cAMP induced a down-regulation of the levels of this proto-oncogene. In addition, dibutyryl cAMP inhibited cell growth in a dose-dependent manner, whereas histamine failed to affect proliferation and differentiation of U937 cells. Cells pretreated with dimaprit showed a decrease in the cAMP response to subsequent addition of H2 agonists, whereas the cAMP response to prostaglandin E2 remained unaltered. This homologous mechanism of H2 receptor desensitization was time dependent. These results indicate that histamine activates several mechanisms involved in the induction of differentiation, such as cAMP and c-fos production, but fails to promote differentiation of U937 cells, apparently due to the rapid desensitization of H2 receptors. 


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Transcription mediated by NFAT is highly inducible in effector CD4+ T helper 2 (Th2) cells but not in Th1 cells. 
Transcriptional factors of the NFAT family play an important role in regulating the expression of several cytokine genes during the immune response, such as the genes for interleukin 2 (IL-2) and IL-4, among others. Upon antigen stimulation, precursor CD4+ T helper (pTh) cells proliferate and differentiate into two populations of effector cells (eTh1 and eTh2), each one expressing a specific pattern of cytokines that distinguishes them from their precursors. eTh2 cells are the major source of IL-4, while gamma interferon is produced by eTh1 cells. Here we have used reporter transgenic mice to show that DNA binding and transcriptional activities of NFAT are transiently induced during the differentiation of pTh cells into either eTh1 or eTh2 cells to mediate the expression of IL-2 as a common growth factor in both pathways. However, although NFAT DNA binding is similarly induced in both eTh1 and eTh2 cells upon antigen stimulation, only the NFAT complexes present in eTh2 cells are able to mediate high-level transcription, and relatively little NFAT transcriptional activity was induced in eTh1 cells. In contrast to activated pTh cells, neither eTh1 nor eTh2 cells produced significant IL-2 upon stimulation, but the high levels of NFAT transcriptional activities directly correlate with the IL-4 production induced in response to antigen stimulation in eTh2 cells. These data suggest that activated NFAT is involved in the effector function of eTh2 cells and that the failure of eTh1 cells to produce IL-4 in response to an antigen is due, at least partially, to a failure to induce high-level transcription of the IL-4 gene by NFAT. Regulation of NFAT could be therefore a critical element in the polarization to eTh1 or eTh2. 


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Protein kinase C and calcineurin synergize to activate IkappaB kinase and NF-kappaB in T lymphocytes. 
The nuclear factor of kappaB (NF-kappaB) is a ubiquitous transcription factor that is key in the regulation of the immune response and inflammation. T cell receptor (TCR) cross-linking is in part required for activation of NF-kappaB, which is dependent on the phosphorylation and degradation of IkappaBalpha. By using Jurkat and primary human T lymphocytes, we demonstrate that the simultaneous activation of two second messengers of the TCR-initiated signal transduction, protein kinase C (PKC) and calcineurin, results in the synergistic activation of the IkappaBalpha kinase (IKK) complex but not of another putative IkappaBalpha kinase, p90(rsk). We also demonstrate that the IKK complex, but not p90(rsk), is responsible for the in vivo phosphorylation of IkappaBalpha mediated by the co-activation of PKC and calcineurin. Each second messenger is necessary, as inhibition of either one reverses the activation of the IKK complex and IkappaBalpha phosphorylation in vivo. Overexpression of dominant negative forms of IKKalpha and -beta demonstrates that only IKKbeta is the target for PKC and calcineurin. These results indicate that within the TCR/CD3 signal transduction pathway both PKC and calcineurin are required for the effective activation of the IKK complex and NF-kappaB in T lymphocytes. 


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Attenuated function of a variant form of the helix-loop-helix protein, Id-3, generated by an alternative splicing mechanism. 
The Id family of helix-loop-helix proteins function as negative regulators of DNA binding, basic helix-loop-helix proteins in the regulation of cell growth and differentiation. We report here on the identification of a 17 kDa variant of the 14 kDa Id-3 protein termed Id-3L (long version) which possesses a unique 60 amino acid carboxy-terminus generated by read through of a 'coding intron' and alternative splicing. Northern analysis revealed expression of a minor 1.1 kb Id-3L transcript together with the predominant 0.95 kb Id-3 transcript in the majority of adult human tissues analysed. The variant Id-3L protein is functionally distinguishable from conventional Id-3 since in in vitro DNA mobility shift assays, it was greatly impaired in its ability to abrogate binding of the basic helix-loop-helix protein, E47, to an E box recognition sequence. 


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Lipopolysaccharide is a potent monocyte/macrophage-specific stimulator of human immunodeficiency virus type 1 expression. 
Lipopolysaccharide (LPS) potently stimulates human immunodeficiency virus type 1-long terminal repeat (HIV-1-LTR) CAT constructs transfected into monocyte/macrophage-like cell lines but not a T cell line. This effect appears to be mediated through the induction of nuclear factor kappa B (NF-kappa B). Electrophoretic mobility shift assays demonstrate that LPS induces a DNA binding activity indistinguishable from NF-kappa B in U937 and THP-1 cells. LPS is also shown to dramatically increase HIV-1 production from a chronically infected monocyte/macrophage-like cloned cell line, U1, which produces very low levels of HIV-1 at baseline. The stimulation of viral production from this cell line occurs only if these cells are treated with granulocyte/macrophage colony-stimulating factor (GM-CSF) before treatment with LPS. This stimulation of HIV-1 production is correlated with an increase in the level of HIV-1 RNA and and activation of NF-kappa B. LPS is not able to induce HIV-1 production in a cloned T cell line. The effect of LPS on HIV-1 replication occurs at picogram per milliliter concentrations and may be clinically significant in understanding the variability of the natural history of HIV-1 infection. 


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Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. 
MyD88, originally isolated as a myeloid differentiation primary response gene, is shown to act as an adaptor in interleukin-1 (IL-1) signaling by interacting with both the IL-1 receptor complex and IL-1 receptor-associated kinase (IRAK). Mice generated by gene targeting to lack MyD88 have defects in T cell proliferation as well as induction of acute phase proteins and cytokines in response to IL-1. Increases in interferon-gamma production and natural killer cell activity in response to IL-18 are abrogated. In vivo Th1 response is also impaired. Furthermore, IL-18-induced activation of NF-kappaB and c-Jun N-terminal kinase (JNK) is blocked in MyD88-/- Th1-developing cells. Taken together, these results demonstrate that MyD88 is a critical component in the signaling cascade that is mediated by IL-1 receptor as well as IL-18 receptor. 


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Glucocorticoid-induced apoptosis and regulation of NF-kappaB activity in human leukemic T cells. 
Glucocorticoid-induced apoptosis was investigated in glucocorticoid-sensitive 6TG1.1 and resistant ICR27TK.3 human leukemic T cells. Following glucocorticoid treatment of 6TG1.1 cells, chromatin fragmentation was observed after a delay of 24 h. Fragmentation was not observed in ICR27TK.3 cells containing mutant glucocorticoid receptors (L753F) that are activation-deficient but retain the ability to repress AP-1 activity. Nor was fragmentation observed after treatment with RU38486, indicating that repression of AP-1 activity is not involved. As described in other systems, fragmentation required ongoing protein synthesis. However, inhibition of protein synthesis with cycloheximide anytime during the first 18 h of steroid treatment was as effective in blocking chromatin fragmentation as inhibition for the entire period, suggesting that synthesis of a component with a rapid turnover rate is required. Dexamethasone treatment completely blocked 12-O-tetradecanoylphorbol 13-acetate induction of nuclear factor-kappaB (NF-kappaB) activity and elicited an increase in the amount of immunoreactive IkappaB alpha in sensitive 6TG1.1 cells but not in resistant ICR27TK.3 cells. In addition, mild detergent treatment of cell extracts indicated that a substantial amount of cytoplasmic NF-kappaB is complexed with IkappaB alpha or some other inhibitory factor. These results suggest that induction of a labile inhibitory factor such as IkappaB alpha may contribute to glucocorticoid-induced apoptosis. 


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Transcriptional regulation of the beta-casein gene by cytokines: cross-talk between STAT5 and other signaling molecules. 
The beta-casein promoter has been widely used to monitor the activation of STAT (signal transducer and activator of transcription)5 since STAT5 was originally found as a mediator of PRL-inducible beta-casein expression. However, not only is expression of the beta-casein gene regulated by STAT5 but it is also affected by other molecules such as glucocorticoid and Ras. In this report, we describe the transcriptional regulation of the beta-casein gene by cytokines in T cells. We have found that the beta-casein gene is expressed in a cytotoxic T cell line, CTLL-2, in response to interleukin-2 (IL-2), which activates STAT5. While IL-4 does not activate STAT5, it induces expression of STAT5-regulated genes in CTLL-2, i.e. beta-casein, a cytokine-inducible SH2-containing protein (CIS), and oncostatin M (OSM), suggesting that STAT6 activated by IL-4 substitutes for the function of STAT5 in T cells. IL-2-induced beta-casein expression was enhanced by dexamethasone, and this synergistic effect of Dexamethasone requires the sequence between -155 and -193 in the beta-casein promoter. Coincidentally, a deletion of this region enhanced the IL-2-induced expression of beta-casein. Expression of an active form of Ras, Ras(G12V), suppressed the IL-2-induced beta-casein and OSM gene expression, and the negative effect of Ras is mediated by the region between -105 and -193 in the beta-casein promoter. In apparent contradiction, expression of a dominant negative form of Ras, RasN17, also inhibited IL-2-induced activation of the promoter containing the minimal beta-casein STAT5 element as well as the promoters of CIS and OSM. In addition, Ras(G12V) complemented signaling by an erythropoietin receptor mutant defective in Ras activation and augmented the activation of the beta-casein promoter by the mutant erythropoietin receptor signaling, suggesting a possible role of Ras in Stat5-mediated gene expression. These results collectively reveal a complex interaction of STAT5 with other signaling pathways and illustrate that regulation of gene expression requires integration of opposing signals. 


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Induction of transcription factors in human T lymphocytes by aspirin-like drugs. 
Aspirin-like drugs (ALD) induce calcium mobilization, an essential component of T cell activation, but do not induce the biosynthesis of IL-2. To understand the extent to which ALD may mimic mitogenic stimulation, we studied cytoplasmic and nuclear signaling steps in ALD-treated T cells. We found that ALD induce a transient activation of protein kinase (PKC) but have no effect (in comparison to anti-CD3 antibodies) on protein tyrosine phosphorylation nor on PCL gamma 1 tyrosine phosphorylation. ALD-induced calcium mobilization and PKC activation are independent of tyrosine protein kinase activity as shown by the lack of effect of herbimycin, a tyrosine-protein kinase-specific inhibitor. Although we detected no IL-2 mRNA in ALD-treated cells, the nuclei of these cells contain proteins capable of binding to three regulatory sequences in the IL-2 promoter region: NFAT, NF kappa B, and AP-1. These binding activities are expressed only in activated T cells. The expression of AP-1 depended on calcium mobilization and PKC activation. These data suggest that ALD cause transient but significant changes in T cell transmembrane signaling, although some events induced by stimulation with anti-CD3 antibodies are not induced by ALD. The signal is transmitted to the nucleus and induces DNA-binding activity by several transcription factors. However, the ALD stimulus is not capable of causing complete T cell activation. 


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Comparison of the transactivation domains of Stat5 and Stat6 in lymphoid cells and mammary epithelial cells. 
Stat (signal transducers and activators of transcription) and Jak (Janus kinases) proteins are central components in the signal transduction events in hematopoietic and epithelial cells. They are rapidly activated by various cytokines, hormones, and growth factors. Upon ligand binding and cytokine receptor dimerization, Stat proteins are phosphorylated on tyrosine residues by Jak kinases. Activated Stat proteins form homo- or heterodimers, translocate to the nucleus, and induce transcription from responsive genes. Stat5 and Stat6 are transcription factors active in mammary epithelial cells and immune cells. Prolactin activates Stat5, and interleukin-4 (IL-4) activates Stat6. Both cytokines are able to stimulate cell proliferation, differentiation, and survival. We investigated the transactivation potential of Stat6 and found that it is not restricted to lymphocytes. IL-4-dependent activation of Stat6 was also observed in HC11 mammary epithelial cells. In these cells, Stat6 activation led to the induction of the beta-casein gene promoter. The induction of this promoter was confirmed in COS7 cells. The glucocorticoid receptor was able to further enhance IL-4-induced gene transcription through the action of Stat6. Deletion analysis of the carboxyl-terminal region of Stat6 and recombination of this region with a heterologous DNA binding domain allowed the delimitation and characterization of the transactivation domain of Stat6. The potencies of the transactivation domains of Stat5, Stat6, and viral protein VP16 were compared. Stat6 had a transactivation domain which was about 10-fold stronger than that of Stat5. In pre-B cells (Ba/F3), the transactivation domain of Stat6 was IL-4 regulated, independently from its DNA binding function. 


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Induction of activator protein (AP)-1 and nuclear factor-kappaB by CD28 stimulation involves both phosphatidylinositol 3-kinase and acidic sphingomyelinase signals. 
A major obstacle in understanding the signaling events that follow CD28 receptor ligation arises from the fact that CD28 acts as a costimulus to TCR engagement, making it difficult to assess the relative contribution of CD28 signals as distinct from those of the TCR. To overcome this problem, we have exploited the observation that activated human T cell blasts can be stimulated via the CD28 surface molecule in the absence of antigenic challenge; thus, we have been able to observe the response of normal T cells to CD28 activation in isolation. Using this system, we observed that CD28 stimulation by B7-transfected CHO cells induced a proliferative response in T cells that was not accompanied by measurable IL-2 production. However, subsequent analysis of transcription factor generation revealed that B7 stimulation induced both activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) complexes, but not NF-AT. In contrast, engagement of the TCR by class II MHC/superantigen, either with or without CD28 ligation, resulted in the induction of NF-AT, AP-1, and NF-kappaB as well as IL-2 production. Using selective inhibitors, we investigated the signaling pathways involved in the CD28-mediated induction of AP-1 and NF-kappaB. This revealed that NF-kappaB generation was sensitive to chloroquine, an inhibitor of acidic sphingomyelinase, but not to the phosphatidylinositol 3-kinase inhibitor, wortmannin. In contrast, AP-1 generation was inhibited by wortmannin and was also variably sensitive to chloroquine. These data suggest that in activated normal T cells, CD28-derived signals can stimulate proliferation at least in part via NF-kappaB and AP-1 generation, and that this response uses both acidic sphingomyelinase and phosphatidylinositol 3-kinase-linked pathways. 


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Different sequence requirements for expression in erythroid and megakaryocytic cells within a regulatory element upstream of the GATA-1 gene. 
The lineage-restricted transcription factor GATA-1 is required for differentiation of erythroid and megakaryocytic cells. We have localized a 317 base pair cis-acting regulatory element, HS I, associated with a hematopoietic-specific DNase I hypersensitive site, which lies approx. 3.7 kilobases upstream of the murine hematopoietic-specific GATA-1 IE promoter. HS I directs high-level expression of reporter GATA-1/lacZ genes to primitive and definitive erythroid cells and megakaryocytes in transgenic mice. Comparative sequence analysis of HS I between human and mouse shows approx. 63% nucleotide identity with a more conserved core of 169 base pairs (86% identity). This core contains a GATA site separated by 10 base pairs from an E-box motif. The composite motif binds a multi-protein hematopoietic-specific transcription factor complex which includes GATA-1, SCL/tal-1, E2A, Lmo2 and Ldb-1. Point mutations of the GATA site abolishes HS I function, whereas mutation of the E-box motif still allows reporter gene expression in both lineages. Strict dependence of HS I activity on a GATA site implies that assembly of a protein complex containing a GATA-factor, presumably GATA-1 or GATA-2, is critical to activating or maintaining its function. Further dissection of the 317 base pair region demonstrates that, whereas all 317 base pairs are required for expression in megakaryocytes, only the 5' 62 base pairs are needed for erythroid-specific reporter expression. These findings demonstrate differential lineage requirements for expression within the HS I element. 


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Rescue by cytokines of apoptotic cell death induced by IL-2 deprivation of human antigen-specific T cell clones. 
The control of cell survival and cell death is of central importance in tissues with high cell turnover such as the lymphoid system. We have examined the effect of cytokines on IL-2 deprivation-induced apoptosis of human antigen-specific T helper clones with different cytokine production profiles. We found that IL-2, interferon-alpha (IFN-alpha), and IFN-beta inhibited IL-2 deprivation apoptosis in Th0, Th1, and Th2 clones. We also found that IL-2 protects T cell clones from IL-2 deprivation apoptosis accompanying active proliferation and enhanced expression of P53, Rb and Bcl-xL proteins. In contrast, IFN-alpha/beta rescued T cell clones from apoptosis without active proliferation, and expression of apoptosis-associated proteins tested so far was unaffected. This may be due to the fact that T cells treated with IL-2 contained those located in S + G2/M phases of the cell cycle, whereas the vast majority of T cells treated with IFN-alpha/beta were located in G0/G1 phase. IFN-alpha/beta specifically induced tyrosine phosphorylation and translocation into nucleus of signal transducers and activators of transcription (STAT) 2 protein in the T cell clones. In addition, over-expression of STAT2 by transfection of the cDNA prevented apoptosis of the T cell clones. Our present study shows that IFN-alpha and -beta mediate anti-apoptotic effect through other pathways than that of IL-2 in growth factor deprivation apoptosis. 


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Anti-rheumatic compound aurothioglucose inhibits tumor necrosis factor-alpha-induced HIV-1 replication in latently infected OM10.1 and Ach2 cells. 
NF-kappaB is a potent cellular activator of HIV-1 gene expression. Down-regulation of NF-kappaB activation is known to inhibit HIV replication from the latently infected cells. Gold compounds have been effectively used for many decades in the treatment of rheumatoid arthritis. We previously reported that gold compounds, especially aurothioglucose (AuTG) containing monovalent gold ion, inhibited the DNA-binding of NF-kappaB in vitro. In this report we have examined the efficacy of the gold compound AuTG as an inhibitor of HIV replication in latently infected OM10.1 and Ach2 cells. Tumor necrosis factor (TNF)-alpha-induced HIV-1 replication in OM10.1 or Ach2 cells was significantly inhibited by non-cytotoxic doses of AuTG (>10 microM in OM10.1 cells and >25 F.M in Ach2 cells), while 25 microM of the counter-anion thioglucose (TG) or gold compound containing divalent gold ion, HAuCl3, had no effect. The effect of AuTG on NF-kappaB-dependent gene expression was confirmed by a transient CAT assay. Specific staining as well as electron microscopic examinations revealed the accumulation of metal gold in the cells, supporting our previous hypothesis that gold ions could block NF-kappaB-DNA binding by a redox mechanism. These observations indicate that the monovalent gold compound AuTG is a potentially useful drug for the treatment of patients infected with HIV. 


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T-lymphocytes from individuals with filarial inflammatory disease have increased transendothelial migration in vitro. 
The in vitro transendothelial migration of circulating filarial antigen-specific T-cells was examined in Wuchereria banerofti infection. Circulating T-cells from individuals with filaria-induced lymphatic pathology (LP) had significantly greater migration through unstimulated HUVEC monolayers than did T-cells from asymptomatic infected (MF) individuals (P = 0.04). In contrast to the MF individuals where no effect was seen, transendothelial migration of 48-hr filarial antigen stimulated T-cells from LP individuals was significantly (P = 0.01) greater than migration of 48-hr media-stimulated T-cells. In six of seven patients examined, inhibition of the VLA-4/VCAM-1 pathway resulted in greater than 50% inhibition of transendothelial migration of T-cells. 


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A low NM23.H1 gene expression identifying high malignancy human melanomas. 
The NM23 gene has been proposed as a metastasis-suppressor gene, and its use has been suggested as prognostic factor. NM23 was identified in a system of murine melanoma cell lines, in which an inverse relationship was found between NM23 expression and metastatic ability. In a human malignant melanoma study NM23 expression was found to be significantly lower in metastases that developed less than 24 months after diagnosis of the primary tumours. The present paper studies the expression of the NM23.H1 gene in cell lines which derive from primary or metastatic human malignant melanomas in relation to staging, infiltration degree, lymphocytic infiltration, cell morphology, cell pigmentation, karyotype, and disease-free survival. The level of mRNA expression of the NM23 gene is significantly lower in cell lines that derive from more infiltrating primary melanomas than in cell lines obtained from less infiltrating tumours. Moreover, cell lines derived from tumours of patients with a disease-free survival of more than 24 months (24-58 months) express the NM23 gene at higher levels than cell lines obtained from melanomas of patients with a disease-free survival of less than 24 months (6-15 months). 


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Tissue factor expression of human monocytes is suppressed by lysophosphatidylcholine. 
The expression of tissue factor (TF), the principal initiator of coagulation, is increased during inflammation and atherosclerosis. Both conditions are promoted by lysophosphatidylcholine (lysoPC). We observed in the present study that lysoPC (1 to 10 micromol/L) dose-dependently reduced TF activity in human monocytes, as elicited by lipopolysaccharide (LPS). Lysophosphatidylethanolamine (lysoPE) and other lysophospholipids did not affect LPS-induced TF activity of human monocytes. TF antigen expression as elicited by LPS was also lowered by lysoPC. Phospholipid analyses indicated a selective increase in the lysoPC content of the monocytes after preincubation with the lysophospholipid. LysoPC inhibited the TF activity of Mono Mac-6 cells to a similar extent as in the monocytes. LPS binding to plasma membrane receptors and internalization of LPS into monocytes were not affected by lysoPC. In contrast, LPS-mediated nuclear binding of nuclear factor-kappaB/Rel to a TF-specific kappaB site was inhibited by lysoPC. Induction of TF mRNA expression by LPS tended to be partially reduced by the lysophospholipid. Preincubation with lysoPC increased monocytic cAMP levels. Inhibition of adenylyl cyclase by pretreatment with 2'-deoxy-3'-adenosine monophosphate partially reversed the inhibition of TF activity promoted by lysoPC. In conclusion, lysoPC markedly decreases LPS-mediated TF expression of human monocytes, the effect probably being mediated by both transcriptional and posttranscriptional mechanisms. LysoPC may thus attenuate activation of coagulation during inflammation and atherosclerosis. 


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IL-2-independent activation and proliferation in human T cells induced by CD28. 
Although the role of CD28 in T cell costimulation is firmly established, the mechanisms by which it exerts its costimulatory actions are less clear. In many circumstances it is difficult to distinguish the effects of CD28 from subsequent actions of cytokines, such as IL-2, on T cell proliferation. Here, we report a model of CD28 costimulation using PMA plus the natural ligand CD80 that resulted in very limited stimulation of IL-2, as evidenced by both cytokine production and IL-2 promoter stimulation. Promoter assays revealed CD28-dependent effects on both NF-kappaB and AP-1, but not on NF-AT or the intact IL-2 promoter. In addition, T cell proliferation was completely resistant to the actions of the immunosuppressant cyclosporin A (CsA). Moreover T cell proliferation was unaffected by the addition of blocking Abs to both IL-2 and the IL-2 receptor, demonstrating that this form of costimulation by CD28 was independent of IL-2. We also investigated the effects of stimulating T cell blasts with CD80 alone and found that there was a limited requirement for IL-2 in this system. We conclude that CD28 costimulation can cause substantial T cell proliferation in the absence of IL-2, which is driven by a soluble factor independent of NF-AT transactivation. 


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Characterization of the nuclear and cytoplasmic components of the lymphoid-specific nuclear factor of activated T cells (NF-AT) complex. 
The lymphoid-specific transcription complex, NF-AT, is involved in early gene activation in T cells and is assembled from a pre-existing, T cell restricted cytoplasmic factor and an inducible ubiquitous nuclear component within 30 min after activation through the antigen receptor. Recent studies have implicated the family of AP1 factors as components of the murine NF-AT complex. Evidence is provided here that the nuclear component of human NF-AT contains the phorbol ester-inducible transcription factor AP1 (Jun/Fos). We further characterize which AP1 family members can assume this role. Antisera to Fos inhibits NF-AT DNA binding as does an oligonucleotide containing a binding site for AP1. Constitutive expression in vivo of Fos, and to a lesser extent Fra-1, eliminates the requirement for phorbol 12-myristate 13-acetate (PMA) stimulation, leaving NF-AT-directed transcription responsive to calcium ionophore alone. Overexpression of cJun or JunD, but not JunB, also eliminates the requirement for PMA, indicating that many but not all Jun- and Fos-related proteins functionally activate NF-AT-dependent transcription in the presence of the cytoplasmic component. NF-AT DNA binding can be reconstituted in vitro using semi-purified AP1 proteins mixed with cytosol from T lymphocytes. Fos proteins are not needed for this reconstitution, and although JunB is not functional, it can participate in the NF-AT DNA binding complex. Finally, we have partially purified the cytoplasmic component of NF-AT and show by elution and renaturation from SDS-polyacrylamide gel electrophoresis gels that it has a molecular mass between 94 and 116 kDa and may have multiple differentially modified forms. 


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Amelioration of rat cerulein pancreatitis by guamerin-derived peptide, a novel elastase inhibitor. 
Increased activity of various proteases is observed in both human and experimental pancreatitis; however, the information on the effects of specific protease inhibitors on the disease is limited. In this study we show that a novel elastase inhibitor, guamerin-derived synthetic peptide (GDSP), improves the parameters of cerulein-induced acute pancreatitis in the rat. The effects of GDSP on pancreatic weight, serum amylase and lipase, morphologic changes in the pancreas, neutrophil infiltration, and nuclear factor KB (NF-KB) activation were measured in rats infused with supramaximal dose of cerulein (5 (g/kg/h) for 6 h. The effects of GDSP were also measured on superoxide formation by activated human neutrophils. The effects of GDSP were compared with those of another elastase inhibitor, elastatinal. GDSP significantly inhibited edema formation, neutrophil infiltration, acinar cell damage, and plasma lipase and amylase increases caused by cerulein. GDSP also completely inhibited superoxide formation in the human neutrophils stimulated by N-formyl-methionine-leucine-phenyl-alanine (fMLP) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Elastatinal had some of the same effects as GDSP but was less potent and effective. These results demonstrate a beneficial effect of GDSP, a novel specific elastase inhibitor, on the development of rat cerulein pancreatitis. 


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Human neutrophils express GH-N gene transcripts and the pituitary transcription factor Pit-1b. 
Since GH stimulates the development and function of granulocytes, we investigated the expression of GH in granulocyte subsets. By immunocytochemistry, 25 +/- 7% of the human neutrophils were shown to express immunoreactive GH, whereas eosinophils were negative. Reversed transcription (RT)-PCR analysis demonstrated GH mRNA in neutrophils. Restriction analysis revealed that neutrophils express the GH-N gene but not the GH-V gene. Furthermore, we demonstrated by western blot analysis that neutrophils express an alternatively spliced variant of the pituitary transcription factor Pit-1, designated Pit-1b. 


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N- and C-terminal sequences control degradation of MAD3/I kappa B alpha in response to inducers of NF-kappa B activity. 
The proteolytic degradation of the inhibitory protein MAD3/I kappa B alpha in response to extracellular stimulation is a prerequisite step in the activation of the transcription factor NF-kappa B. Analysis of the expression of human I kappa B alpha protein in stable transfectants of mouse 70Z/3 cells shows that, as for the endogenous murine protein, exogenous I kappa B alpha is degraded in response to inducers of NF-kappa B activity, such as phorbol myristate acetate or lipopolysaccharide. In addition, pretreatment of the cells with the proteasome inhibitor N-Ac-Leu-Leu-norleucinal inhibits this ligand-induced degradation and, in agreement with previous studies, stabilizes a hyperphosphorylated form of the human I kappa B alpha protein. By expressing mutant forms of the human protein in this cell line, we have been able to delineate the sequences responsible for both the ligand-induced phosphorylation and the degradation of I kappa B alpha. Our results show that deletion of the C terminus of the I kappa B alpha molecule up to amino acid 279 abolishes constitutive but not ligand-inducible phosphorylation and inhibits ligand-inducible degradation. Further analysis reveals that the inducible phosphorylation of I kappa B alpha maps to two serines in the N terminus of the protein (residues 32 and 36) and that the mutation of either residue is sufficient to abolish ligand-induced degradation, whereas both residues must be mutated to abolish inducible phosphorylation of the protein. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F. 
Initiation of DNA replication requires the function of MCM gene products, which participate in ensuring that DNA replication occurs only once in the cell cycle. Expression of all mammalian genes of the MCM family is induced by growth stimulation, unlike yeast, and the mRNA levels peak at G1/S boundary. In this study, we examined the transcriptional activities of isolated human MCM gene promoters. Human MCM5 and MCM6 promoters with mutation in the E2F sites failed in promoter regulation following serum stimulation and exogenous E2F expression. In addition, we identified a novel E2F-like sequence in human MCM6 promoter which cooperates with the authentic E2F sites in E2F-dependent regulation. Forced expression of E2F1 could induce expression of all members of the endogenous MCM genes in rat embryonal fibroblast REF52 cells. Our results demonstrated that the growth-regulated expression of mammalian MCM5 and MCM6 genes, and presumably other MCM members, is primarily regulated by E2F through binding to multiple E2F sites in the promoters. 


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Cyclosporin A inhibits monocyte tissue factor activation in cardiac transplant recipients. 
BACKGROUND: Fibrin deposition and thrombosis have been implicated in both allograft rejection and vasculopathy after cardiac transplantation. Because monocytes play a pivotal role in the pathophysiology of intravascular coagulation activation through their ability to synthesize tissue factor (TF), we asked (1) whether monocyte TF activation occurs in cardiac transplant recipients and (2) whether monocyte TF expression is affected by treatment with cyclosporin A (CsA). METHODS AND RESULTS: We measured levels of TF activity in peripheral blood mononuclear cells and highly purified monocytes/macrophages from 10 consecutive cardiac transplant recipients and 10 healthy control subjects. TF activity generated by both unstimulated and endotoxin-stimulated cells was significantly higher in transplant recipients than in control subjects (P<.05). Increased monocyte TF expression in transplant recipients was shown to be adversely affected by treatment with CsA: TF induction was markedly reduced by CsA serum concentrations reaching peak CsA drug levels. Inhibition of TF induction in the presence of high CsA blood concentrations was also observed when stimulation of cells was performed with interferon-gamma or interleukin-1beta. As shown by reverse transcription-polymerase chain reaction and electrophoretic mobility shift assay, respectively, treatment with CsA leads to decreased TF mRNA expression and reduced activation of the NF-kappaB transcription factor, which is known to contribute to the induction of the TF promotor in human monocytes. CONCLUSIONS: This study demonstrates that TF activation, occurring in mononuclear cells of cardiac transplant recipients, is inhibited by treatment with CsA. Inhibition of monocyte TF induction by CsA may contribute to its successful use in cardiac transplant medicine and might be useful in managing further settings of vascular pathology also known to involve TF expression and NF-kappaB activation. 


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c-Maf induces monocytic differentiation and apoptosis in bipotent myeloid progenitors. 
The transcriptional mechanisms that drive colony-forming unit granulocyte-macrophage (CFU-GM) myeloid progenitors to differentiate into cells of either the granulocytic or monocytic lineage are not fully understood. We have shown that the c-Maf and c-Myb transcription factors physically interact in myeloid cells to form inhibitory complexes that hinder transactivation of c-Myb target genes through direct binding to Myb consensus sites. These complexes arise in a developmentally regulated pattern, peaking at the promyelocyte stage, or in cell model systems, appearing soon after the induction of monocytic differentiation. We wished to determine if this developmentally related interaction is a consequence of myeloid differentiation or an intrinsic differentiating stimulus. Because the elevated Myb:Maf status seen in differentiating cells can be recapitulated by overexpression of c-Maf in myeloid cell lines, we inducibly expressed the c-Maf cDNA in 2 bipotent human myeloid progenitor cells. Elevated levels of c-Maf protein led to marked increases in Myb:Maf complexes and the accumulation of monocyte/macrophage cells, followed by eventual programmed cell death. Analysis of targets that could mediate these phenotypic changes indicated that c-Maf likely plays a key role in myeloid cell development through dual mechanisms; inhibition of a select set of c-Myb regulated targets, such as Bcl-2 and CD13/APN, coupled with the activation of as yet undefined differentiation-promoting genes. 


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Activation of transcription factor NF-kappa B by phagocytic stimuli in human neutrophils. 
Phagocytosis represents an important physiological trigger for the inducible expression of several genes in human neutrophils. Here, we report that a DNA-binding activity primarily consisting of the classical NF-kappa B heterodimer, p50/RelA, is induced in phagocytosing neutrophils. Under these conditions, NF-kappa B activation was found to be a rapid and transient response, reaching a maximum by 10-15 min, and returning to near-basal levels by 30 min. In neutrophils undergoing the phagocytosis of opsonized yeasts, the onset of NF-kappa B activation was paralleled by a decline in immunoreactive I kappa B-alpha protein levels, and the cellular I kappa B-alpha pool was replenished by 30 min, in agreement with our gel shift data. We conclude that NF-kappa B activation could constitute one of the mechanisms whereby the expression of kappa B-responsive genes is enhanced in phagocytosing neutrophils. To our knowledge, this represents the first demonstration that phagocytic stimuli can induce NF-kappa B activation in human neutrophils. 


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Binding of erythroid and non-erythroid nuclear proteins to the silencer of the human epsilon-globin-encoding gene. 
To clarify the molecular mechanisms involved in the developmental control of hemoglobin-encoding genes we have been studying the expression of these genes in human cells in continuous culture. We have previously reported the presence of a transcriptional control element with the properties of a silencer extending from -392 to -177 bp relative to the cap site of the human epsilon-globin-encoding gene [Cao et al., Proc.Natl.Acad.Sci.USA 86 (1989) 5306-5309]. We also showed that this silencer has stronger inhibitory activity in HeLa cells, as compared to K562 human erythroleukemia cells. Using deletion mutants and cis-cloned synthetic oligodeoxyribonucleotides in transient expression assays, nucleotide sequences responsible for this effect have now been further delimited to 44 bp located from -294 to -251 bp. Gel electrophoresis mobility shift assays and DNaseI footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells. Two binding proteins are detected in K562 nuclear extracts, while only one is found in extracts from HeLa cells. Possible mechanisms by which these proteins may regulate transcription of the epsilon-globin-encoding gene in erythroid and non-erythroid cells are discussed. 


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cDNA cloning of a NGFI-B/nur77-related transcription factor from an apoptotic human T cell line. 
A human T lymphoid cell line, PEER, dies by apoptosis in the presence of PMA and calcium ionophore. A new gene, TINUR, was cloned from apoptotic PEER cells. The expression of the TINUR gene is induced within 1 h after the cross-linking of the T cell Ag receptor complex. TINUR belongs to the NGFI-B/nur77 family of the steroid receptor superfamily and is an orphan receptor. TINUR binds to the same DNA sequence as NGFI-B/nur77. We also propose that the NGFI-B/nur77 family can be classified into two subtypes. 


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Constitutive nuclear NF-kappa B in cells of the monocyte lineage. 
In monocytes, the nuclear factor NF-kappa B has been invoked as an important transcription factor in the expression of cytokine genes, of cell-surface receptors and in the expression of human immunodeficiency virus. In such cells, DNA binding activity of NF-kappa B can be detected without intentional stimulation. In our studies, cells of the human monocytic line Mono Mac 6, cultured in medium containing fetal-calf serum and low levels of lipopolysaccharide (LPS), also exhibit such 'constitutive' NF-kappa B, as demonstrated by mobility-shift analysis of nuclear extracts. This nuclear NF-kappa B was still present when contaminant LPS was removed by ultrafiltration and when serum was omitted. Protein-DNA complexes of constitutive NF-kappa B are similar in mobility to the LPS-induced NF-kappa B and both are recognized by an antibody specific to the p50 subunit of NF-kappa B. By contrast, treatment of cells with pyrrolidine dithiocarbamate (PDTC) will only block LPS-induced NF-kappa B, but not the constitutive binding protein. Using LPS-free and serum-free conditions, constitutive NF-kappa B can be detected in different cell lines of the monocytic lineage (HL60, U937, THP-1, Mono Mac 1 and Mono Mac 6), but not in Molt 4 T cells or K562 stem cells. When ordered according to stage of maturation, the amount of constitutive NF-kappa B was not increased in more mature cell lines. Furthermore, when inducing differentiation in Mono Mac 6 cells, with vitamin D3, no change in constitutive or inducible NF-kappa B can be detected. Analysis of primary cells revealed substantial constitutive NF-kappa B-binding activity in blood monocytes, pleural macrophages and alveolar macrophages. The constitutive NF-kappa B appears to be functionally active, since a low level of tumour necrosis factor (TNF) transcript is detectable in monocytes, and this level can be increased by blocking transcript degradation using cycloheximide. The level of constitutive NF-kappa B in these cells is variable and is frequently found to be lower in the more mature macrophages. Constitutive NF-kappa B was not maintained by autocrine action of cytokines TNF, interleukin 6, interleukin 10, granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor, since neutralizing antibodies did not reduce constitutive DNA-binding activity. Furthermore, blockade of prostaglandin or leukotriene biosynthesis did not affect constitutive NF-kappa B. (ABSTRACT TRUNCATED AT 400 WORDS) 


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In vivo anergized CD4+ T cells express perturbed AP-1 and NF-kappa B transcription factors. 
Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell receptor (TCR) variable region beta-chain families and induces strong and rapid production of interleukin 2 (IL-2). In contrast, repeated injections of SEA cause CD4+ T-cell deletion and anergy in the remaining CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. We analyzed expression of AP-1, NF-kappa B, NF-AT, and octamer binding transcription factors, which are known to be involved in the regulation of IL-2 gene promoter activity. Large amounts of AP-1 and NF-kappa B and significant quantities of NF-AT were induced in SEA-activated CD4+ spleen T cells, whereas Oct-1 and Oct-2 DNA binding activity was similar in both resting and activated T cells. In contrast, anergic CD4+ T cells contained severely reduced levels of AP-1 and Fos/Jun-containing NF-AT complexes but expressed significant amounts of NF-kappa B and Oct binding proteins after SEA stimulation. Resolution of the NF-kappa B complex demonstrated predominant expression of p50-p65 heterodimers in activated CD4+ T cells, while anergic cells mainly expressed the transcriptionally inactive p50 homodimer. These alterations of transcription factors are likely to be responsible for repression of IL-2 in anergic T cells. 


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A novel SP-1 site in the human interleukin-1 beta promoter confers preferential transcriptional activity in keratinocytes. 
To investigate the mechanisms of transcriptional activation of interleukin-1beta (IL-1beta) in non-monocytic cells, we constructed a series of reporter plasmids with the bacterial chloramphenicol acetyltransferase gene linked to various parts of the human IL-1beta promoter and performed transient transfection experiments. We identified a promoter segment that activates transcription most efficiently in keratinocytes. Electrophoretic mobility shift assays (EMSA) with a 43-mer oligonucleotide derived from the functionally identified cis-acting element revealed specific complexes. By competition analysis with transcription factor consensus sequence oligonucleotides and by immunosupershift, transcription factor SP-1 or a closely related protein was shown to bind to this regulatory element. The closest match to the known SP-1 consensus sequence within the respective region is a TCCCCTCCCCT motif. Mutation of this motif almost completely, and specifically, abolished the binding of two low-mobility complexes and led to a 95% decrease of constitutive transcriptional activation of a reporter construct IL-1beta (-170/+108). Likewise, activation of this reporter construct by tumor necrosis factor-alpha depended on the SP-1 site. These observations suggest that a so-far-unrecognized SP-1 site in the human IL-1beta promoter may participate in the transcriptional regulation of this gene in keratinocytes. 


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Multifactor cis-dominant negative regulation of IL-2 gene expression in anergized T cells. 
The molecular mechanism underlying IL-2 transcriptional blockade in anergic T cell clones is not fully understood. To examine whether an active negative regulatory process occurs, we created a reporter construct containing as an enhancer four copies of the NF-AT site and one copy of the octamer site (4X NF-AT-Oct). This construct was only slightly reduced (1.3-fold) in its expression when stimulated under anergic conditions, while a whole mouse IL-2 enhancer construct showed a reduction of 4.3-fold. Addition of the -176 to -96 sequence to the 4X NF-AT-Oct construct did not impart the ability to be affected by anergy, but addition of the -236 to -96 sequence did, demonstrating that anergy is an active inhibitory process and that more than the presence of the -150 AP-1 binding site (-152 to -147) is required to mediate the effect. Mutational studies of the -236 to -96 sequence indicated that the presence of both the -130 AP-1-like site (-187 to -181) and the -150 proximal AP-1 site were necessary to observe anergy. Because the -180 site is not required for trans-activation, it was possible to confirm by mutation in the normal mouse IL-2 enhancer that this site is absolutely essential for anergy induction. The simplest model to explain these results is that anergy is mediated by a complex of multiple transcription factors that exert a cis-acting dominant negative regulatory effect on the trans-activation of the IL-2 gene. 


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Cytokine modulation of HIV expression. 
Cytokines, the peptide hormones which control the homeostasis of the immune system and also play a fundamental role in inflammatory and immune mediated reactions, have been involved at multiple levels in the pathogenesis of the acquired immune deficiency syndrome (AIDS). Infection with the human immunodeficiency virus (HIV) has been shown to induce production of several cytokines both in vitro and in vivo. Conversely, several cytokines modulate the levels of HIV expression in infected cells of both T lymphocytic and mononuclear phagocytic lineage. Activated mononuclear cells, particularly B cells which are in a state of chronic activation in HIV infected individuals, release HIV-inductive cytokines and thus play a potentially important role in the pathogenesis of HIV infection. 


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An erythroid specific enhancer upstream to the gene encoding the cell-type specific transcription factor GATA-1. 
The transcription factor GATA-1 is expressed in a subset of hemopoietic cells, where it mediates the cell-type specific expression of several genes. We have cloned the mouse and human GATA-1 genes. A region upstream to the first exon, and highly conserved between mouse and man, acts as an erythroid specific enhancer in transient assays, if linked to the GATA-1 or to the SV40 promoter. The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site. 


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Constitutive expression of specific interferon isotypes in peripheral blood leukocytes from normal individuals and in promonocytic U937 cells. 
Constitutive expression of IFN-alpha5 and IFN-beta was detected in different lymphoid cells including peripheral blood mononuclear cells from normal individuals following amplification of IFN mRNA by reverse transcriptase-polymerase chain reaction and direct sequencing of the amplified product. The activated form of the interferon-induced transcription factor complex ISGF3 was also detected in nuclear extracts from uninduced cells. Culture supernatants from uninduced U937 cells were also found to activate an ISRE cloned upstream of the luciferase reporter gene, indicating the presence of endogenous IFN activity equivalent to approximately 0.3 to 0.5 IU/mL. This endogenous IFN was also shown to play a role in maintaining the basal level of expression of the major histocompatibility class I genes in lymphoid cells. These results suggest that IFN-alpha5 and IFN-beta are produced at low levels in normal tissues and play an important role in the regulation of cell function and in the maintenance of homeostasis. 


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Constitutive activation of NF-kB in human thymocytes. 
NF-kB is a eukaryotic transcription regulatory factor. In T cells and T cell lines, NF-kB is bound to a cytoplasmic proteic inhibitor, the IkB. Treatment of T cells with mitogens (phorbol esters) or cytokines (TNF alpha) induces NF-kB nuclear translocation and the subsequent expression of NF-kB dependent T cell genes. Here we examined the activation of NF-kB in human T cell thymic progenitors. We report differences in (Ca2+)i requirement for NF-kB activation in thymocytes as compared to mature T cells. Furthermore, our results indicated that thymocytes have a constitutively active form of NF-kB, suggesting that they are activated in vivo. 


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Requirements for induction of vitamin D-mediated gene regulation in normal human B lymphocytes. 
Mature human lymphocytes are unique targets of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in that vitamin D receptors (VDR) are not constitutively expressed, and specific cellular activation signals are required for both the up-regulation of VDR and establishment of reactivity to the lipophilic ligand. Treatment of B lymphocytes with the cytokine IL-4 (IL-4), in the absence of prior activation, induces a weak up-regulation of VDR expression but fails to generate vitamin D-responsive element (VDRE)-reactive nuclear protein complexes or to initiate the genomic transcription of 25-hydroxyvitamin D3 24-hydroxylase. Stimulation of B lymphocytes by either ligation of CD40 Ag or cross-linking the Ig receptor is also insufficient to render B lymphocytes responsive to 1 alpha,25(OH)2D3. However, this apparent lack of response to the secosterol can be overcome by stimulation of B lymphocytes with a combination of these cellular activation signals, which are sufficient to lead to G1 cell cycle progression. In the presence of 1 alpha,25(OH)2D3, cellular activation associated with stimulation of such a progression appears to be sufficient for the up-regulation of VDR message and protein and necessary for the establishment of VDRE binding complexes and the induction of 24-hydroxylase message. Furthermore, biologic functions are modulated, in that the hormone inhibits proliferation in a subset of the activated B cells. These observations suggest that reactivity to 1 alpha,25(OH)2D3 is tightly regulated in B lymphocytes, requiring specific signals for its initiation. 


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Functional analysis of cis-linked regulatory sequences in the HLA DRA promoter by transcription in vitro. 
Two consensus sequences, called X and Y boxes, capable of binding nuclear proteins and regulating expression in B cells have been defined within the immediate upstream region of major histocompatibility complex (MHC) class II promoters. Unlike other class II promoters, the HLA-DR alpha (DRA) promoter also contains one element identical to the "octamer" motif of immunoglobulin variable region promoters that is responsible for B cell-specific transcription. This "octamer" in the context of DRA appears capable of binding both the ubiquitous (OTF-1) and lymphoid-specific (OTF-2) "octamer" binding proteins, but at least one other distinct "octamer" complex was found. In order to characterize the function of cis-acting elements, we have developed an in vitro system in which a DRA promoter construct is transcribed more efficiently in extracts from B cells than in extracts from class II-negative HeLa cells. 5' deletion constructs which lacked the Y box, but retained the "octamer" motif and TATA box were completely inactive, and internal deletion of the Y box reduced transcription by 95%. Using supercoiled, but not linear templates, we observed differences in transcription efficiencies from templates lacking or disrupting the X consensus element that reflect effects of random replacement of X box sequences in transient expression assays. Demonstration of the complete dependence on the Y box in this system suggests that, despite its demonstrated importance in the DRA promoter, the DRA "octamer" does not utilize OTF-2 in a manner analogous to immunoglobulin promoters in B cells. 


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Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors. 
Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint. 


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Physical interactions between Ets and NF-kappaB/NFAT proteins play an important role in their cooperative activation of the human immunodeficiency virus enhancer in T cells. 
The transcriptional regulatory elements of many inducible T-cell genes contain adjacent or overlapping binding sites for the Ets and NF-kappaB/NFAT families of transcription factors. Similar arrays of functionally important NF-kappaB/NFAT and Ets binding sites are present in the transcriptional enhancers of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2), suggesting that this pattern of nuclear protein binding sites reflects an evolutionarily conserved mechanism for regulating inducible T-cell gene expression that has been co-opted during HIV evolution. Despite these findings, the molecular mechanisms by which Ets and NF-kappaB/NFAT proteins cooperatively regulate inducible T-cell gene expression remained unknown. In the studies described in this report, we demonstrated a physical interaction between multiple Ets and NF-kappaB/NFAT proteins both in vitro and in activated normal human T cells. This interaction is mediated by the Ets domain of Ets proteins and the C-terminal region of the Rel homology domains of NF-kappaB/NFAT proteins. In addition, the Ets-NF-kappaB/NFAT interaction requires the presence of DNA binding sites for both proteins, as it is abolished by the DNA intercalating agents propidium iodide and ethidium bromide and enhanced by the presence of synthetic oligonucleotides containing binding sites for Ets and NF-kappaB proteins. A dominant-negative mutant of NF-kappaB p50 that binds DNA but fails to interact with Ets proteins inhibits the synergistic activation of the HIV-1 and HIV-2 enhancers by NF-kappaB (p50 + p65) and Ets-1, suggesting that physical interaction between Ets and NF-kappaB proteins is required for the transcriptional activity of the HIV-1 and HIV-2 enhancers. Taken together, these findings suggest that evolutionarily conserved physical interactions between Ets and NF-kappaB/NFAT proteins are important in regulating the inducible expression of T-cell genes and viruses. These interactions represent a potential target for the development of novel immunosuppressive and antiviral therapies. 


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Human CD3-CD16+ natural killer cells express the hGATA-3 T cell transcription factor and an unrearranged 2.3-kb TcR delta transcript. 
In this study we analyzed the T cell receptor(TcR) delta transcripts expressed by CD3-CD16+ cells and we investigated whether these cells expressed the hGATA-3 T cell transcription factor and the recombination-activating gene (RAG)-1. Multiple TcR delta transcripts deriving from an unrearranged TcR delta gene were detected in both polyclonal and clonal CD3-CD16+ natural killer(NK) cell lines. Two unrearranged TcR delta transcripts had a size similar to that of the functional TcR delta mRNA (2.3 and 1.3 kb) found in TcR gamma/delta+ T lymphocytes. Sequence analysis of nine different 2.3-kb cDNA clones obtained from NK-derived polyA+ RNA confirmed that they corresponded to an unrearranged TcR delta gene. These cDNA were 2343 bp long and their transcription initiation site was located 814 bp upstream from the J delta 1 segment. The sequence located upstream of the J delta 1 segment corresponded to the previously reported germ-line sequence. The J delta 1 segment was correctly spliced to C delta; in addition the four C delta exons were found to be already assembled. Two polyadenylation sites were present in the fourth C delta exon. However, only that located at the 3' end appeared to be utilized in the 2.3-kb cDNA. The expression of hGATA-3, a T cell-specific factor known to be involved in the regulation of the transcription of TcR delta locus, was analyzed by Northern blot, in cultured NK cell population and clones (but not in freshly derived cell populations). All NK clones and cell lines studied were found to express hGATA-3-specific mRNA, suggesting that hGATA-3 may be involved in the regulation of the unrearranged TcR delta gene expression in NK cells. Finally, no transcription of the RAG-1 gene could be detected in all NK cell lines or clones analyzed. 


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NF-AT activation induced by a CAML-interacting member of the tumor necrosis factor receptor superfamily. 
Activation of the nuclear factor of activated T cells transcription factor (NF-AT) is a key event underlying lymphocyte action. The CAML (calcium-modulator and cyclophilin ligand) protein is a coinducer of NF-AT activation when overexpressed in Jurkat T cells. A member of the tumor necrosis factor receptor superfamily was isolated by virtue of its affinity for CAML. Cross-linking of this lymphocyte-specific protein, designated TACI (transmembrane activator and CAML-interactor), on the surface of transfected Jurkat cells with TACI-specific antibodies led to activation of the transcription factors NF-AT, AP-1, and NFkappaB. TACI-induced activation of NF-AT was specifically blocked by a dominant-negative CAML mutant, thus implicating CAML as a signaling intermediate. 


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Distinct mechanisms for N-acetylcysteine inhibition of cytokine-induced E-selectin and VCAM-1 expression. 
We have examined the effects of N-acetyl-L-cysteine (NAC), a well-characterized, thiol-containing antioxidant, on agonist-induced monocytic cell adhesion to endothelial cells (EC). NAC inhibited interleukin-1 (IL-1 beta)-induced, but not basal, adhesion with 50% inhibition at approximately 20 mM. Monocytic cell adhesion to EC in response to tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA) was similarly inhibited by NAC. Unlike published studies with pyrrolidinedithiocarbamate, which specifically inhibited vascular cell adhesion molecule 1 (VCAM-1), NAC inhibited IL-1 beta-induced mRNA and cell surface expression of both E-selectin and VCAM-1. NAC had no effect on the half-life of E-selectin or VCAM-1 mRNA. Although NAC reduced nuclear factor-kappa B (NF-kappa B) activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding to the consensus NF-kappa B binding sites of the VCAM-1 gene (VCAM-NF-kappa B), the antioxidant had no appreciable effect when an oligomer corresponding to the consensus NF-kappa B binding site of the E-selectin gene (E-selectin-NF-kappa B) was used. Because NF-kappa B has been reported to be redox sensitive, we studied the effects of NAC on the EC redox environment. NAC caused an expected dramatic increase in the reduced glutathione (GSH) levels in EC. In vitro studies demonstrated that whereas the binding affinity of NF-kappa B to the VCAM-NF-kappa B oligomer peaked at a GSH-to-oxidized glutathione (GSSG) ratio of approximately 200 and decreased at higher ratios, the binding to the E-selectin-NF-kappa B oligomer appeared relatively unaffected even at ratios > 400, i.e., those achieved in EC treated with 40 mM NAC. These results suggest that NF-kappa B binding to its consensus sequences in the VCAM-1 and E-selectin gene exhibits marked differences in redox sensitivity, allowing for differential gene expression regulated by the same transcription factor. Our data also demonstrate that NAC increases the GSH-to-GSSG ratio within the EC suggesting one possible mechanism through which this antioxidant inhibits agonist-induced monocyte adhesion to EC. 


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Dual effects of LPS antibodies on cellular uptake of LPS and LPS-induced proinflammatory functions. 
Human phagocytes recognize bacterial LPS (endotoxin) through membrane CD14 (mCD14), a proinflammatory LPS receptor. This study tested the hypothesis that anti-LPS Abs neutralize endotoxin by blocking cellular uptake through mCD14. Ab-associated changes in the uptake and cellular distribution of FITC-LPS were assessed by flow cytometry and laser scanning confocal microscopy in human CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and human peripheral blood monocytes. LPS core- and O-side chain-specific mAbs inhibited mCD14-mediated LPS uptake by both cell types in the presence of serum. O-side chain-specific mAb concurrently enhanced complement-dependent LPS uptake by monocytes through complement receptor-1 (CR1) and uptake by CHO-CD14 cells involving another heat-labile serum factor(s) and cell-associated recognition molecule(s). Core-specific mAb inhibited mCD14-mediated uptake of homologous and heterologous LPS, while producing less concurrent enhancement of non-mCD14-mediated LPS uptake. The modulation by anti-LPS mAbs of mCD14-mediated LPS uptake was associated with inhibition of LPS-induced nuclear factor-kappaB (NF-kappaB) translocation and TNF-alpha secretion in CHO-CD14 cells and monocytes, respectively, while mAb enhancement of non-mCD14-mediated LPS uptake stimulated these activities. LPS-specific Abs thus mediate anti-inflammatory and proinflammatory functions, respectively, by preventing target cell uptake of LPS through mCD14 and augmenting uptake through CR1 or other cell receptors. 


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Increased IkappaB expression and diminished nuclear NF-kappaB in human mononuclear cells following hydrocortisone injection. 
We have recently demonstrated that hydrocortisone and other glucocorticoids inhibit reactive oxygen species (ROS) generation by mononuclear (MNC) and polymorphonuclear leucocytes (PMNL). Since NF-kappaB/IkappaB system regulates the transcription of proinflammatory genes, including those responsible for ROS generation, we tested the hypothesis that hydrocortisone may stimulate IkappaB production thus inhibiting NF-kappaB translocation from the cytosol into the nucleus in MNC, in vivo. One hundred milligram of hydrocortisone was injected intravenously into 4 normal subjects. Blood samples were obtained prior to the injection and at 1, 2, 4, 8 and 24 hr after the injection. Nuclear extracts and total cell lysates were prepared from MNC by standard techniques. IkappaB levels in MNC homogenates increased at 1 hr, peaked at 2-4 hr, started to decrease at 8 hr, and returned to baseline levels at 24 hr. NF-kappaB in MNC nuclear extracts decreased at 1 hr, reached a nadir at 4 hr, gradually increased at 8 hr and returned back to baseline levels at 24 hr. The total protein content of NF-kappaB subunit (P65) in MNC lysates also showed a decrease following hydrocortisone injection. This decrease was observed at 2 hr, reached a nadir at 4 hr, and returned to baseline levels at 24 hr. ROS generation inhibition paralleled NF-kappaB levels in the nucleus. It was inhibited at 1 hr, reached a nadir at 2-4 hr, started to increase at 8 hr, and returned to basal levels at 24 hr. Our data demonstrate that hydrocortisone induces IkappaB and suppresses NF-kappaB expression in MNC in parallel. IkappaB further reduces the translocation of NF-kappaB into the nucleus thus preventing the expression of proinflammatory genes. 


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Anaphylatoxins C5a and C3a induce nuclear factor kappaB activation in human peripheral blood monocytes. 
The anaphylatoxins C5a and C3a are involved in the regulation of cytokine production. In this study the capability of C5a and C3a to induce transcription factor activation was examined. C5a and C3a stimulation of human peripheral blood monocytes resulted in nuclear expression of a DNA binding activity with specificity to the kappaB sequence. The p50 and p65 proteins, constituents of the prototypic nuclear factor kappaB, were identified as components of the DNA-protein complexes by anti-peptide antibodies in gel supershift assays. C5a induced kappaB binding activity was detected 15 min after agonist stimulation, peaked at 30-40 min, and remained detectable at 2 h. Binding to kappaB sequence was accompanied by an initial decrease and subsequent increase in the cytoplasmic IkappaBalpha levels, as detected by Western blotting using an anti-IkappaBalpha antibody. Pertussis toxin treatment markedly decreased kappaB binding activities induced by both C5a and C3a, whereas cholera toxin displayed no inhibitory effect. Neither of the two toxins affected kappaB binding activity induced by TNFalpha in the same cells. These results imply a potential role of the anaphylatoxins C5a and C3a in regulating leukocytes gene expression through G protein-coupled transcription factor activation. 


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Platelet-activating factor stimulates transcription of the heparin-binding epidermal growth factor-like growth factor in monocytes. Correlation with an increased kappa B binding activity. 
Human peripheral blood monocytes responded to stimulation of platelet-activating factor (PAF) with up-regulation of the transcript for heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells. This function of PAF was observed at nanomolar concentrations of the ligand, starting at 30 min after stimulation. The PAF-induced up-regulation of HB-EGF mRNA was accompanied by an increase in kappa B binding activity. These functions of PAF appeared to be mediated through the cell surface PAF receptors, as two PAF receptor antagonists, WEB 2086 and L-659,989, blocked both the up-regulation of HB-EGF mRNA and kappa B binding activity induced by PAF. The antagonists, however, had no effect on phorbol ester-induced up-regulation of HB-EGF mRNA and kappa B binding activity. Pretreatment of monocytes with pertussis toxin inhibited these functions of PAF, whereas cholera toxin had no inhibitory effect. Pyrrolidine dithiocarbamate, an inhibitor for NF-kappa B activation, markedly reduced PAF-stimulated kappa B binding activity as well as up-regulation of HB-EGF mRNA. These results suggest a potential role of PAF in HB-EGF expression and provide evidence that this stimulation may occur through increased kappa B binding activity. 


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Transcriptional down-regulation of c-myc expression by protein synthesis-dependent and -independent pathways in a human T lymphoblastic tumor cell line. 
We show that in the human T lymphoblastic tumor cell line Molt4 c-myc mRNA and protein expression is down-regulated after exposure to dimethyl sulfoxide, to phorbol myristate acetate, or to the calcium ionophore A23187, which raises the intracellular calcium concentration. A block to RNA elongation is largely responsible for decreased c-myc transcription. Although negative regulation by dimethyl sulfoxide takes place even when protein synthesis is inhibited by cycloheximide, the phorbol myristate acetate effect is blocked to some extent only by cycloheximide. The calcium ionophore-induced c-myc suppression, however, strictly requires de novo protein synthesis. Therefore, two different negative regulatory pathways are involved in c-myc regulation: one which is independent and one which depends on de novo protein synthesis. The latter one appears to be mediated by a rapidly calcium-dependent induced gene product. 


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Expression of the Runt domain-encoding PEBP2 alpha genes in T cells during thymic development. 
The PEBP2 alpha A and PEBP2 alpha B genes encode the DNA-binding subunit of a murine transcription factor, PEBP2, which is implicated as a T-cell-specific transcriptional regulator. These two related genes share the evolutionarily conserved region encoding the Runt domain. PEBP2 alpha B is the murine counterpart of human AML1, which is located at the breakpoints of the 8;21 and 3;21 chromosome translocations associated with acute myeloid leukemia. Northern (RNA) blots of various adult mouse tissues revealed that the levels of expression of both genes were most prominent in the thymus. Furthermore, transcripts of PEBP2 alpha A and mouse AML1/PEBP2 alpha B were detected in T lymphocytes in the thymuses from day 16 embryos and newborns, as well as 4-week-old adult mice, by in situ hybridization. The expression of the genes persisted in peripheral lymph nodes of adult mice. The transcripts were detected in all the CD4- CD8-, CD4+ CD8+, CD4+ CD8-, and CD4- CD8+ cell populations. The results indicated that both genes are expressed in T cells throughout their development, supporting the notion that PEBP2 is a T-cell-specific transcription factor. Transcripts of mouse AML1/PEBP2 alpha B were also detected in day 12 fetal hematopoietic liver and in the bone marrow cells of newborn mice. The implication of mouse AML1/PEBP2 alpha B expression in hematopoietic cells other than those of T-cell lineage is discussed in relation to myeloid leukemogenesis. 


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Regulation of granulocyte-macrophage colony-stimulating factor and E-selectin expression in endothelial cells by cyclosporin A and the T-cell transcription factor NFAT. 
Nuclear factor of activated T cells (NFAT) was originally described as a T-cell-specific transcription factor athat supported the activation of cytokine gene expression and mediated the immunoregulatory effects of cyclosporin A (CsA). As we observed that activated endothelial cells also expressed NFAT, we tested the antiinflammatory properties of CsA in endothelial cells. Significantly, CsA completely suppressed the induction of NFAT in endothelial cells and inhibited the activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene regulatory elements that use NFAT by 60%. CsA similarly mediated a reduction of up to 65% in GM-CSF mRNA and protein expression in activated endothelial cells. CsA also suppressed E-selectin, but not vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells, even though the E-selectin promoter is activated by NF-kappa B rather than NFAT. Hence, induction of cell surface expression of this leukocyte adhesion molecule by tumor necrosis factor (TNF)-alpha was reduced by 40% in the presence of CsA, and this was reflected by a 29% decrease in neutrophil adhesion. The effects of CsA on endothelial cells were also detected at the chromatin structure level, as DNasel hypersensitive sites within both the GM-CSF enhancer and the E-selectin promoter were suppressed by CsA. This represents the first report of NFAT in endothelial cells and suggests mechanisms by which CsA could function as an antiinflammatory agent. 


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Cross-linking of CD44 on rheumatoid synovial cells up-regulates VCAM-1. 
CD44 is a ubiquitous molecule also known as hyaluronic acid or homing receptor. However, the cellular functions and its role in inflammation, for example, rheumatoid synovitis, are currently unknown. In this study, we propose a novel function for CD44. Using synovial cells from rheumatoid arthritis (RA) patients, we demonstrated that CD44 cross-linking and binding to hyaluronan augmented VCAM-1 expression and subsequently VCAM-1-mediated cell adhesion. Briefly, we found that 1) rheumatoid synovial cells highly expressed CD44; 2) cross-linking of CD44 markedly but transiently augmented VCAM-1 expression and its mRNA transcription much more than did IL-1beta and TNF-alpha; 3) hyaluronan, especially when fragmented, also up-regulated VCAM-1; 4) CD44 activated the transcription factor AP-1; and 5) the integrin-dependent adhesive function of RA synovial cells to T cells was also amplified by CD44 cross-linking. These results indicate that the adhesion of RA synovial cells to matrices such as hyaluronic acid through CD44 could up-regulate VCAM-1 expression and VCAM-1-mediated adhesion to T cells, which might in turn cause activation of T cells and synovial cells in RA synovitis. We therefore propose that such cross-talking among distinct adhesion molecules may be involved in the pathogenesis of inflammation, including RA synovitis. 


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Suppression of a cellular differentiation program by phorbol esters coincides with inhibition of binding of a cell-specific transcription factor (NF-E2) to an enhancer element required for expression of an erythroid-specific gene. 
Induction by hemin increases, while induction with 12-O-tetradecanoylphorbol-13-acetate (TPA) represses, erythroid-specific gene expression in the human cell line K562. We analyzed the effects of hemin or TPA induction on the binding and activity of transcription factors at a regulatory element found within the transcriptional regulatory sequences of many erythroid-specific genes. TPA induction increases the binding of ubiquitous AP-1 factors to this element. TPA induction inhibits the binding of the lineage limited transcription factor NF-E2 to this transcriptional control element. Hemin induction of K562 cells does not facilitate the binding of NF-E2 to its recognition site. Hemin induction appears to nonspecifically increase the expression of transiently transfected genes in K562 cells. Beyond this nonspecific increase in gene expression, hemin induction acts to increase the activity of the lineage limited transcription factor NF-E2. The divergent effects of hemin and TPA on gene expression in K562 cells are mediated, in part, by their contrasting effects on the transcription factor NF-E2. 


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Identification of a human LIM-Hox gene, hLH-2, aberrantly expressed in chronic myelogenous leukaemia and located on 9q33-34.1. 
We describe the isolation of human LH-2, a putative transcription factor containing two cysteine-rich regions (LIM domains) and a homeobox (Hox) DNA-binding domain. High levels of hLH-2 expression were observed in all cases of chronic myelogenous leukaemia (CML) tested, regardless of disease status. hLH-2 was mapped to chromosome 9Q33-34.1, in the same region as the reciprocal translocation that creates the BCR-ABL chimera of the Philadelphia chromosome (Ph'), the hallmark of CML; hLH-2 was retained on the derivative 9 chromosome and is therefore centromeric of c-ABL. The proximity of hLH-2 to the breakpoint on chromosome 9 raises the possibility of cis-activation by the t(9;22)(q34;q11) translocation. In addition to finding hLH-2 expression in all cases of CML, expression was observed in lymphoid malignancies and myeloid cell lines, but not in primary cases of acute myelogenous leukaemia. The role of hLH-2 in the development or progression of leukaemia is not known. However, hLH-2 may prove useful as a marker of CML for monitoring residual disease. 


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Evidence for lowered induction of nuclear factor kappa B in activated human T lymphocytes during aging. 
Transcription factor NF kappa B (nuclear factor kappa B) is induced in T lymphocytes from young individuals following activation with a variety of stimuli including anti-CD3, phorbol myristate acetate (PMA), and tumor necrosis factor-alpha (TNF-alpha). In contrast, activated T lymphocytes from older individuals show a significant reduction in the induction of NF kappa B in response to the same stimuli. The age-related decline in induction of NF kappa B could not be attributed to alteration in the composition of subunits, p50 and p65 were found to be the predominant subunits of induced NF kappa B in T cells from young as well as elderly donors. Furthermore, similar levels of NF kappa B were found in the cytosols of unactivated T cells from both young and elderly donors suggesting that precursor levels of NF kappa B remain unaltered during aging. These results suggest that an age-associated decline in the induction of NF kappa B in activated T cells from elderly individuals may be attributable to altered regulation of the inhibitor, I kappa B, and may play an important role in immune dysregulation accompanying aging. 


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Expression of Ah receptor (TCDD receptor) during human monocytic differentiation. 
We have previously found a high expression of human Ah receptor (TCDD receptor) mRNA in peripheral blood cells of individuals. In this paper, the expression of this gene in blood cells was first investigated in fractions of nucleated cells, revealing predominant expression of the Ah receptor gene in the monocyte fraction. Then the expression levels of AhR mRNA in various hematopoietic cell lines were examined together with those of Arnt and P450IA1. AhR was expressed at high levels in monocytoid U937, THP1, and HEL/S cells, and at moderate levels in promyelocytic HL60 cells and erythroblastic HEL cells. However, it was not detected in lymphoid cells MOLT4 (T cell) and BALL1 (B cell), nor in K562 erythroblasts. Furthermore, a specific induction of AhR during monocytic differentiation was investigated in HL60 and HEL cells. HL60 cells were induced to differentiate toward monocytes-macrophages by incubation with phorbol ester, showing a 5- to 2-fold increase of AhR mRNA. The incubation with transforming growth factor beta 1 and 1 alpha,25-dihydroxyvitamin D3 resulted in a 5- to 7-fold increase of AhR mRNA. The HEL cells also exhibited a similar elevation of AhR mRNA level, when they had differentiated toward monocyte-macrophage cells by these combined inducers, but little change in the mRNA level was observed when the cells were induced to differentiate into other cell types. Treatment of the differentiated HL60 cells with 3-methylcholanthrene, a ligand of AhR, induced the expression of the P450IA1 gene. These results indicated that expression of AhR mRNA was significantly induced during monocytic differentiation and that the differentiated cells were responsive to xenobiotics. Our results suggest that AhR may play an important role in the function of monocytes and also in the eventual activation of environmental carcinogens. 


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Induction of IL-8 expression in T cells uses the CD28 costimulatory pathway. 
IL-8, a potent chemotactic factor for neutrophil granulocytes and lymphocytes, is a proinflammatory cytokine secreted by a variety of cell types, including T cells. Stimulation of the CD28 cell surface molecule delivers costimulatory signals essential for lymphokine production in activated T cells via a conserved sequence element found in the promoter of several lymphokine genes. Anti-CD28-stimulated T cells produced significant amounts of IL-8; additionally, costimulation with anti-CD3 and anti-CD28 Abs resulted in a synergistic induction of IL-8 secretion. Sequence homology, single nucleotide mutations, and anti-CD28 Ab stimulation studies established that the NF-kappa B-like sequence in the promoter of the IL-8 gene functioned as a CD28 response element. Furthermore, cyclosporin A, but not rapamycin, blocked the synergistic induction of IL-8 expression achieved with anti-CD3 and anti-CD28 costimulation. The involvement of a CD28 response element in the induction of IL-8 expression in activated T cells may provide new insights into the pathogenesis and persistence of immune disorders characterized by increased levels of IL-8, such as psoriasis and rheumatoid arthritis. 


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Genes encoding general initiation factors for RNA polymerase II transcription are dispersed in the human genome. 
General transcription factors are required for accurate initiation of transcription by RNA polymerase II. Human cDNAs encoding subunits of these factors have been cloned and sequenced. Using fluorescence in situ hybridization (FISH), we show here that the genes encoding the TATA-box binding protein (TBP), TFIIB, TFIIE alpha, TFIIE beta, RAP30, RAP74 and the 62 kDa subunit, of TFIIH are located at the human chromosomal bands 6q26-27, 1p21-22, 3q21-24, 8p12, 13q14, 19p13.3 and 11p14-15.1, respectively. This dispersed localization of a group of functionally related gene provides insights into the molecular mechanism of human genome evolution and their possible involvement in human diseases. 


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Interferon-gamma and the sexual dimorphism of autoimmunity. 
The sexual difference in the incidence of autoimmune diseases has remained an enigma for many years. In the examination of the induction of autoimmunity in transgenic mice, evidence has been obtained further implicating the lymphokine interferon-gamma in the etiology of autoimmunity. Sex steroid regulation of the production of this molecule, as well as other cytokines, may help explain the gender-specific differences in the immune system, including autoimmunity. 


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Alternative splicing of RNA transcripts encoded by the murine p105 NF-kappa B gene generates I kappa B gamma isoforms with different inhibitory activities. 
The gene encoding the 105-kDa protein (p105) precursor of the p50 subunit of transcription factor NF-kappa B also encodes a p70 I kappa B protein, I kappa B gamma, which is identical to the C-terminal 607 amino acids of p105. Here we show that alternative RNA splicing generates I kappa B gamma isoforms with properties different from those of p70. One 63-kDa isoform, termed I kappa B gamma-1, which lacks 59 amino acids C-terminal to ankyrin repeat 7, has a novel 35-amino acid C terminus encoded by an alternative reading frame of the p105 gene. A 55-kDa isoform, I kappa B gamma-2, lacks the 190 C-terminal amino acids of p70I kappa B gamma. In contrast to p70I kappa B gamma, which is a cytoplasmic protein, I kappa B gamma-1 is found in both the cytoplasm and nucleus, whereas I kappa B gamma-2 is predominantly nuclear. The I kappa B gamma isoforms also display differences in specificity and affinity for Rel/NF-kappa B proteins. While p70I kappa B gamma inhibits p50-, p65-, and c-Rel-mediated transactivation and/or DNA binding, both I kappa B gamma-1 and I kappa B gamma-2 are specific for p50 and have different affinities for this subunit. The absence in I kappa B gamma-1 and I kappa B gamma-2 of a protein kinase A site whose phosphorylation modulates p70I kappa B gamma inhibitory activity suggests that alternative RNA splicing may be used to generate I kappa B gamma isoforms that respond differently to intracellular signals. 


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Stimulation of interleukin-1 alpha and interleukin-1 beta production in human monocytes by protein phosphatase 1 and 2A inhibitors. 
Protein phosphatases 1 and 2A are important in regulating cellular functions by controlling the phosphorylation state of their substrates. In human monocytes, the inhibitors of these phosphatases, okadaic acid and calyculin A, were found to increase the mRNA accumulation and cytokine production of interleukin-1 beta and interleukin-1 alpha. The increased mRNA accumulation was found to be primarily because of the increase in the transcription rate of the interleukin-1 genes. Stimulation of interleukin-1 gene transcription may be caused by the stimulation of transcription factor activities, including those of AP-1, by these protein phosphatase inhibitors. Okadaic acid increased the synthesis of the interleukin-1 beta precursor and mature forms and their secretion. This increased processing and secretion correlated with the stimulation of IL-1 beta convertase mRNA accumulation. The stimulation of interleukin-1 alpha production by okadaic acid was more modest than that of interleukin-1 beta. However, the phosphorylation of the precursor interleukin-1 alpha cytokine was increased. These results show that protein phosphatase 1 and 2A inhibitors exert multiple effects on cytokine production in human monocytes and suggest that these two phosphatases play important roles in regulating interleukin-1 production. 


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Structure and function analysis of the human myeloid cell nuclear differentiation antigen promoter: evidence for the role of Sp1 and not of c-Myb or PU.1 in myelomonocytic lineage-specific expression. 
The human myeloid nuclear differentiation antigen (MNDA) is expressed specifically in maturing cells of the myelomonocytic lineage and in monocytes and granulocytes. Epitope enhancement was used to confirm the strict lineage- and stage-specific expression of MNDA in bone marrow as well as in other paraffin-embedded fixed tissues. A 1-kb region of the gene that includes 5' flanking sequence was reported earlier to contain functional promoter activity and was specifically demethylated in expressing cells in contrast to null cells. Further analysis has revealed that this 1-kb fragment promotes higher reporter gene activity in MNDA-expressing cells than non-expressing cells, indicating cell-specific differences in transactivation. This sequence contains consensus elements consistent with myeloid-specific gene expression, including a PU.1 consensus site near the major transcription start site and a cluster of c-Myb sites located several hundred bases upstream of this region. However, analysis of deletion mutants localized nearly all of the promoter activity to a short region (-73 to -16) that did not include the cluster of c-Myb sites. A 4-bp mutation of the core Sp1 consensus element (GC box) (-20) reduced overall promoter activity of the 1-kb fragment. Mutation of the PU.1 site did not significantly affect promoter activity. Only a small region (-35 to +22) including the Sp1 element and transcription start site, but not the PU.1 site was footprinted. The 4-bp mutation of the core Sp1 consensus element abolished footprinting at the site and an antibody super-shift reaction showed that Sp1 is one of the factors binding the consensus site. The Sp1 site also co-localizes with a DNase I hypersensitive site. The results indicate that DNA methylation, chromatin structure, and transactivation at an Sp1 site contribute to the highly restricted expression of this myelomonocytic lineage specific gene. 


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Molecular cloning of FKHRL1P2, a member of the developmentally regulated fork head domain transcription factor family. 
Here we report the expression of a fork head domain protein in human T helper cells. We cloned and characterized a fork head cDNA from human T helper cell mRNA using differential display RT-PCR. The cDNA contains a 546-nucleotide (nt) open reading frame (ORF) that codes for the carboxyl-terminal 180 amino acids (aa) of the recently identified fkhrl1 gene. This ORF does not contain the characteristic DNA-binding domain found in members of the forkhead protein family. In-vitro transcription/translation of this cDNA expressed a protein of approximately 20 kDa. We have generated antibodies that specifically immunoprecipitated the in-vitro-translated 20-kDa protein. This antibody also recognizes in human T lymphocytes a 70-kDa protein corresponding in size to that predicted for the fkhrl1 gene product. The mRNA levels for fkhrl1 is elevated in T helper-induced lymphocytes in comparison to PHA-stimulated T lymphocytes. Further characterization of FKHRL1 and its related family members should shed light on the transcriptional mechanisms of this fork head gene subfamily and their role in T helper cell differentiation and regulation of cell growth. 


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The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP. 
The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene. 


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Simple derivation of TFIID-dependent RNA polymerase II transcription systems from Schizosaccharomyces pombe and other organisms, and factors required for transcriptional activation. 
Resolution of whole cell extract through two chromatographic steps yields a single protein fraction requiring only the addition of TFIID for the initiation of transcription at RNA polymerase II promoters. This approach allows the convenient generation of RNA polymerase II transcription systems from Saccharomyces cerevisiae, human lymphocytes, and Schizosaccharomyces pombe. TFIIDs from all three organisms are interchangeable among all three systems. The S. cerevisiae and Sch. pombe systems support effects of acidic activator proteins, provided a further protein fraction from S. cerevisiae is supplied. This further fraction is distinct from the mediator of transcriptional activation described previously and represents a second component in addition to general initiation factors that may facilitate a response to acidic activators. 


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Hypoxia enhances induction of endothelial ICAM-1: role for metabolic acidosis and proteasomes. 
Intercellular adhesion molecule 1 (ICAM-1) is an important molecule in promotion of polymorphonuclear neutrophil transendothelial migration during inflammation. Coincident with many inflammatory diseases is tissue hypoxia. Thus we hypothesized that combinations of hypoxia and inflammatory stimuli may differentially regulate expression of endothelial ICAM-1. Human endothelial cells were exposed to hypoxia in the presence or absence of added lipopolysaccharide (LPS) and examined for expression of functional ICAM-1. Although hypoxia alone did not induce ICAM-1, the combination of LPS and hypoxia enhanced (3 +/- 0.4-fold over normoxia) ICAM-1 expression. Combinations of hypoxia and LPS significantly increased lymphocyte binding, and such increases were inhibited by addition of anti-ICAM-1 antibodies or antisense oligonucleotides. Hypoxic endothelia showed a > 10-fold increase in sensitivity to inhibitors of proteasome activation, and combinations of hypoxia and LPS enhanced proteasome-dependent cytoplasmic-to-nuclear localization of the nuclear transcription factor-kappa B p65 (Rel A) subunit. Such proteasome activation correlated with hypoxia-evoked decreases in both extracellular and intracellular pH. We conclude from these studies that endothelial hypoxia provides a novel, proteasome-dependent stimulus for ICAM-1 induction. 


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Expression of c-fos correlates with IFN-alpha responsiveness in Philadelphia chromosome positive chronic myelogenous leukemia. 
This study evaluates (i) constitutive levels of oncogene and p53 transcripts in chronic phase CML patients and (ii) their modulations subsequent to in vivo therapy with rIFN-alpha 2c. Peripheral blood mononuclear cells (pbmc) and bone marrow cells of 26 patients were examined for c-fos, c-myc, p53 and the hybrid bcr/abl mRNA levels. Results indicated that (i) constitutive c-fos transcript levels are significantly higher in patients subsequently responding to IFN-alpha therapy (p < 0.01) and positively correlated with the proportion of lymphocytes (r = 0.6895, p < 0.01) and negatively with the proportion of immature cells (r = -0.568, p < 0.01) contained in the pbmc preparations tested, (ii) constitutive mRNA levels of the hybrid bcr/abl, c-myc and p53 are positively correlated with each other, but failed to relate to disease parameters, and (iii) acute and chronic in vivo exposure to IFN-alpha is accompanied by upregulation of c-fos and downregulation of c-myc mRNA levels in responder patients. 


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IL-4 down-regulates IL-2-, IL-3-, and GM-CSF-induced cytokine gene expression in peripheral blood monocytes. 
IL-4, a product of the T-helper 0 (Th0) and 2 (Th2) subset, was originally described as a B-cell stimulatory factor and has subsequently been found to suppress IL-1 alpha, IL-1 beta, IL-6, IL-8, and TNF-alpha gene expression in monocytes stimulated with LPS, and to upregulate IL-1 receptor antagonist (IL1-RA) gene expression. In this study we investigated the effect of IL-4 on the expression of cytokine genes in monocytes evoked by other T-helper cell cytokines: IL-2, IL-3, and GM-CSF. IL-4 down-regulated mRNA accumulation of the proinflammatory cytokines IL-1 beta, IL-8, and TNF-alpha in monocytes stimulated with IL-2, IL-3, and GM-CSF. IL-4 also suppressed the IL-2-induced IL-6 mRNA expression. Temporal analysis of the IL-4 down-regulatory effect on the IL-2-, IL-3-, or GM-CSF-induced proinflammatory cytokine gene expression in monocytes provided evidence that IL-4 acts predominantly on the post-transcriptional level. This was supported by the observation that the down-regulatory capacity of IL-4 appeared to be dependent on de novo protein synthesis. IL-4 did not exert significant influence on the induction of expression of IL-1-RA or various CSFs by IL-2, IL-3, and GM-CSF. (ABSTRACT TRUNCATED AT 250 WORDS) 


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I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding. 
The active nuclear form of the NF-kappa B transcription factor complex is composed of two DNA binding subunits, NF-kappa B p65 and NF-kappa B p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. The NF-kappa B p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-kappa B, termed I kappa B. In contrast, NF-kappa B p50 alone fails to stimulate kappa B-directed transcription, and based on prior in vitro studies, is not directly regulated by I kappa B. To investigate the molecular basis for the critical regulatory interaction between NF-kappa B and I kappa B/MAD-3, a series of human NF-kappa B p65 mutants was identified that functionally segregated DNA binding, I kappa B-mediated inhibition, and I kappa B-induced nuclear exclusion of this transcription factor. Results from in vivo expression studies performed with these NF-kappa B p65 mutants revealed the following: 1) I kappa B/MAD-3 completely inhibits NF-kappa B p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 kappa B enhancer in human T lymphocytes, 2) the binding of I kappa B/MAD-3 to NF-kappa B p65 is sufficient to retarget NF-kappa B p65 from the nucleus to the cytoplasm, 3) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-kappa B p65 disrupts its ability to engage I kappa B/MAD-3, and 4) the unique C-terminus of NF-kappa B p65 attenuates its own nuclear localization and contains sequences that are required for I kappa B-mediated inhibition of NF-kappa B p65 DNA binding activity. Together, these findings suggest that the nuclear localization signal and transactivation domain of NF-kappa B p65 constitute a bipartite system that is critically involved in the inhibitory function of I kappa B/MAD-3. Unexpectedly, our in vivo studies also demonstrate that I kappa B/MAD-3 binds directly to NF-kappa B p50. This interaction is functional as it leads to retargeting of NF-kappa B p50 from the nucleus to the cytoplasm. However, no loss of DNA binding activity is observed, presumably reflecting the unique C-terminal domain that is distinct from that present in NF-kappa B p65. 


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Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized human glucocorticoid receptor. 
We have used a DNA-binding/immunoprecipitation assay to analyze the capacity of human glucocorticoid receptor (hGR), generated in rabbit reticulocyte lysates, to bind DNA. In vitro translated hGR was indistinguishable from native hGR, as determined by migration on sodium dodecyl sulfate-polyacrylamide gels, sedimentation on sucrose density gradients, and reactivity with antipeptide antibodies generated against hGR. In addition, cell-free synthesized hGR was capable of specific binding to glucocorticoid response element (GRE)-containing DNA fragments. Using this assay system, we have evaluated the contributions of ligand binding and heat activation to DNA binding by these glucocorticoid receptors. In vitro translated hGR was capable of selective DNA binding even in the absence of glucocorticoid. Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C (no activation). In contrast, addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors. Agonist (dexamethasone) was slightly more effective in supporting specific DNA binding than antagonist (RU486). DNA binding by in vitro synthesized GR was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation. Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes. The specificity of DNA binding by cell-free synthesized hGR was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat. Oligonucleotides containing the consensus GRE were the most efficient competitors, and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive, whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding, except when competitor was present at extremely high concentrations. Together these studies indicate that hGR synthesized in rabbit reticulocyte lysates displays many of the same properties, including GRE-specific DNA binding, observed for glucocorticoid receptor present in cytosolic extracts of mammalian cells and tissues. Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding. 


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Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion. 
We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation. 


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Constitutive expression c-fos, c-jun, and NF kappa B mRNA is in nucleated fetal blood cells and up-regulation of c-fos and c-jun with anti-CD3 stimulation. 
Fetal and neonatal lymphocytes are relatively resistant to activation and cytokine production when stimulated either via their T-cell antigen receptors or lectins. The molecular mechanism(s) responsible for this phenomenon have not been clearly elucidated. We have hypothesized that such defects in fetal/neonatal T-cell activation may be due to lack of expression of the transcriptional regulatory elements required for T-cell activation. We used reverse transcriptase-polymerase chain reaction to examine both fetal and term neonatal cord bloods for mRNA expression of three transcription factors implicated in T-cell activation: c-jun, c-fos, and NF kappa B (p50 subunit). We demonstrate that mRNAs for all three of these regulatory factors are expressed in fetal blood cells by the 27th week of gestation and in term cord bloods. Activation of term infant cord blood mononuclear cells with anti-CD3 monoclonal antibodies resulted in up-regulation of both c-jun and c-fos mRNAs within 15 min of stimulation. However, secretion of IL-2 by anti-CD3-stimulated cord blood mononuclear cells was still blunted compared with control cells from adults. We conclude that fetal nucleated blood cells constitutively express important genes for cytokine regulation and are able to increase intracellular accumulation of the mRNAs for these factors in response to anti-CD3 stimulation. Thus, qualitative differences in the capacity to regulate these factors could not be shown in fetal blood cells. Quantitative experiments comparing binding of these transcription factors to the IL-2 promoter are currently under investigation. 


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Triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) induces nuclear translocation of NF-kappa B (p50/p65) in human monocytes and enhances viral replication in HIV-infected monocytic cells. 
Monocyte/macrophages may harbor HIV in a nonproductive fashion for prolonged periods of time. Viral gene expression may be reactivated by stimulation of the cells with LPS or cytokines such as TNF-alpha in vitro. The effect of LPS and TNF-alpha is mediated by their ability to induce nuclear translocation of the DNA-binding heterodimer NF-kappa B (p50/p65), which binds to a specific sequence in the HIV-long terminal repeat. The present study demonstrates that triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) enhances viral replication in HIV-infected human monocytic cells. Monocytic cell lines and normal peripheral blood monocytes were infected with HIV-1 in vitro and cultured in the presence or absence of F(ab')2 fragments of monoclonal anti-CR1 or anti-CR3 Abs or with C3 fragments. Stimulation of CR1 or CR3 induces a two- to fourfold increase in the amount of cell-associated and released p24 Ag in cell cultures that was equivalent to that observed in control cultures triggered with LPS. We further observed that stimulation of CR1 or CR3 induces the nuclear translocation of NF-kappa B p50/p65 in infected cells. Translocation of NF-kappa B p50/p65 was also observed following stimulation of CR1 or CR3 of uninfected peripheral blood monocytes from HIV-seronegative donors. The amount of protein translocated was similar to that observed when cells were stimulated with rhTNF-alpha. TNF-alpha did not mediate the translocation of NF-kappa B p50/p65 induced by triggering of complement receptors. Taken together, these observations suggest that HIV gene expression may be activated in infected monocytes through interaction of the cells with complement-opsonized particles and that enhanced viral replication is associated with C3 receptor-mediated nuclear translocation of the NF-kappa B complex. 


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The carboxyl-terminal cytoplasmic domain of CD36 is required for oxidized low-density lipoprotein modulation of NF-kappaB activity by tumor necrosis factor-alpha. 
The binding of oxidized low-density lipoprotein (Ox LDL) by monocyte-macrophages causes pleiotropic effects, including changes in gene expression, and is thought to represent an early event in atherogenesis. The integral membrane glycoprotein CD36 appears to play a physiological role in binding and uptake of Ox LDL by monocyte-macrophages, although the molecular events associated with CD36-Ox LDL interaction are unknown. To approach this issue, we used CD36 transfected Chinese hampster ovary (CHO) cells, exposed them to Ox LDL, and determined changes in the activity of the transcription factor NF-kappaB. We report here that Ox LDL enhanced DNA binding activity of nuclear extracts to an NF-kappaB sequence following activation of CD36-producing CHO cells with the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). This enhanced DNA binding activity was inhibited by coincubation of CD36 transfected cells with the human CD36-specific antibody OKM5. We also determined that activation of NF-kappaB DNA binding activity required an intact carboxyl-terminal cytoplasmic segment on CD36. Our results support the idea that human CD36 mediates signal transduction events in response to Ox LDL. 


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One base pair change abolishes the T cell-restricted activity of a kB-like proto-enhancer element from the interleukin 2 promoter. 
The inducible, T cell-specific enhancers of murine and human Interleukin 2 (Il-2) genes contain the kB-like sequence GGGATTTCACC as an essential cis-acting enhancer motif. When cloned in multiple copies this so-called TCEd (distal T cell element) acts as an inducible proto-enhancer element in E14 T lymphoma cells, but not in HeLa cells. In extracts of induced, Il-2 secreting El4 cells three individual protein factors bind to TCEd DNA. The binding of the most prominent factor, named TCF-1 (T cell factor 1), is correlated with the proto-enhancer activity of TCEd. TCF-1 consists of two polypeptides of about 50 kD and 105 kD; the former seems to be related to the 50 kD polypeptide of NF-kB. Purified NF-kB is also able to bind to the TCEd, but TCF-1 binds stronger than NF-kB to TCEd DNA. The conversion of the TCEd to a 'perfect' NF-kB binding site leads to a tighter binding of NF-kB to TCEd DNA and, as a functional consequence, to the activity of the 'converted' TCEd motifs in HeLa cells. Thus, the substitution of the underlined A residue to a C within the GGGATTTCACC motif abolishes its T cell-restricted activity and leads to its functioning in both El4 cells and HeLa cells. These results indicate that lymphocyte-specific factors binding to the TCEd are involved in the control of T cell specific-transcription of the Il-2 gene. 


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IL-2 gene expression and NF-kappa B activation through CD28 requires reactive oxygen production by 5-lipoxygenase. 
Activation of the CD28 surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of interleukin-2 (IL-2) and cell proliferation. In primary T lymphocytes we show that CD28 ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for CD28-mediated activation of the NF-kappa B/CD28-responsive complex and IL-2 expression. Delineation of the CD28 signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of phospholipase A2 and 5-lipoxygenase. Our data suggest that lipoxygenase metabolites activate ROI formation which then induce IL-2 expression via NF-kappa B activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the CD28 costimulatory pathway. 


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CD2 signalling induces phosphorylation of CREB in primary lymphocytes. 
Promoter sequences responsive to cyclic AMP (cAMP) are found in a number of cellular genes, and bind transcription factors of the cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1) family. We have used a human T-lymphotropic virus type 1 (HTLV-1) model of cAMP response element (CRE) transcription to investigate the influence of lymphocyte activation on transcription from homologous regions in the viral promoter. We previously demonstrated increased HTLV-1 transcription following CD2 but not CD3 receptor cross-linking. We hypothesized that this increased viral transcription was mediated, in part, through the phosphorylation of CREB. Therefore, we investigated CD2 and CD3 receptor-mediated signalling in primary human peripheral blood mononuclear cells (PBMC). CD2, but not CD3, cross-linking increased cAMP detected by competitive enzyme-linked immunosorbent assay (ELISA) approximately fourfold. CD2 cross-linking concurrently increased phosphorylation of CREB detected by immunoblot assay eightfold. Consistent with post-translational regulation, no change in total level of CREB protein was observed. Phosphorylation of CREB occurred through a herbimycin A and Rp-cAMP- sensitive pathway, suggesting phosphorylation required antecedent activation of both protein tyrosine kinases (PTK) and protein kinase A (PKA). Both CD2 and CD3 cross-linking increased binding of nuclear proteins to a radiolabelled CRE oligonucleotide probe in electrophoretic mobility shift assays suggesting that lymphocyte activation enhances binding independently of phosphorylation of CREB at serine 133. These data indicate specific modulation of the CREB/ATF-1 family of transcription factors by the CD2 signalling pathway and suggest CD2 receptor modulation of CRE-mediated transcription following ligand engagement (e.g. cell-to-cell contact). 


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Human immunodeficiency virus type-1 transcription: role of the 5'-untranslated leader region (review). 
Human immunodeficiency virus type-1 (HIV-1) transcription is dependent on the interaction of host-cell transcription factors with cis-regulatory DNA elements within the viral long terminal repeat (LTR). Much attention has focused on the series of sequence elements upstream of the transcriptional initiation site in the U3 region of the LTR including the Sp1 and NF-kappaB binding sites. Recent studies, however, demonstrate that the transcribed 5'-untranslated leader region (5'-UTR) also contains important transcriptional elements. These regulatory elements situated downstream of transcription interact with constitutive and inducible transcription factors, mediate transmission of cellular activation signals, and are important for efficient HIV-1 transcription and replication. The 5'-UTR contains binding sites for the transcription factors AP-1, NF-kappaB, NF-AT, IRF, and Sp1. Mutations in these binding sites can interfere with the viral response to cell activation signals, decrease LTR transcription, and inhibit viral replication. The 5'-UTR also interacts with a specific nucleosome that is rapidly displaced during transcriptional activation of the latent provirus. We propose that the inducible transcription factor binding sites in the 5'-UTR comprise a downstream enhancer domain that can function independent of, or in concert with, the LTR promoter to rapidly increase latent proviral transcription in response to cell activation signals. In this review, we describe the host-cell transcription factors that interact with the 5'-UTR and discuss their role in the transcriptional regulation of HIV-1 gene expression. 


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Regulation of M-CSF expression by M-CSF: role of protein kinase C and transcription factor NF kappa B. 
Macrophage-colony-stimulating factor (M-CSF), also referred to as CSF-1, regulates the survival, growth, differentiation and functional activity of monocytes by binding to a single class of high-affinity cell surface receptors, known to be the product of the c-fms protooncogene. The detection of both M-CSF and c-fms expression by cells of the monocyte lineage has suggested that M-CSF may act by an autocrine mechanism. Interestingly, it has been shown that M-CSF can induce the expression of its own gene. Although sensitivity to M-CSF can be modulated by regulation of receptor expression and function, M-CSF responsiveness is largely determined at a postreceptor level. To date, little is known about the intracellular pathway of M-CSF signal transduction. We have therefore investigated the changes in protein kinase C (PKC) activity upon exposure of monocytes to M-CSF. We show that M-CSF activates and translocates PKC. Inhibition of PKC by the isoquinoline derivative H7 abolishes induction of M-CSF by M-CSF. Furthermore, activation of PKC was pertussis-toxin-sensitive and was associated with the detection of an NF kappa B protein in nuclear extracts of M-CSF-induced blood monocytes but not in monocytes exposed to medium treatment only. The results suggest that M-CSF induction of M-CSF involves G proteins, PKC and NF kappa B. 


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T-cell expression of the human GATA-3 gene is regulated by a non-lineage-specific silencer. 
The GATA-3 transcription factor is required for development of the T-cell lineage and Th2 cytokine gene expression in CD4 T-cells. We have mapped the DNase-I-hypersensitive (HS) regions of the human GATA-3 gene in T-cells and non-T-cells and studied their transcriptional activities. HS I-III, located 5' from the transcriptional initiation site, were found in hematopoietic and non-hematopoietic cells, whereas HS IV-VII, located 3' from the transcriptional start site, were exclusively observed in T-cells. Among these hypersensitive sites, two transcriptional control elements were found, one in the first intron of the GATA-3 gene and the other between 8.3 and 5.9 kilobases 5' from the GATA-3 transcriptional initiation site. The first intron acted as a strong transcriptional activator in a position-dependent manner and with no cell-type specificity. The upstream regulatory element could confer T-cell specificity to the GATA-3 promoter activity, and analysis of this region revealed a 707-base pair silencer that drastically inhibited GATA-3 promoter activity in non-T-cells. Two CAGGTG E-boxes, located at the 5'- and 3'-ends of the silencer, were necessary for this silencer activity. The 3'-CAGGTG E-box could bind USF proteins, the ubiquitous repressor ZEB, or the basic helix-loop-helix proteins E2A and HEB, and we showed that a competition between ZEB and E2A/HEB proteins is involved in the silencer activity. 


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Negative transcriptional regulation of human interleukin 2 (IL-2) gene by glucocorticoids through interference with nuclear transcription factors AP-1 and NF-AT. 
IL-2 gene transcription is affected by several nuclear proteins. We asked whether dexamethasone (Dex) and cyclosporin A (CsA) inhibit IL-2 gene transcription by interfering with the activity of nuclear proteins that bind to the IL-2 promoter. Nuclear extracts from primary human T lymphocytes were analyzed by electrophoretic DNA mobility shift assays. Both Dex and CsA inhibited the binding of transcription factors AP-1 and NF-AT, but not of NF-kB and OCT-1/OAF, to their corresponding sites on the IL-2 gene promoter. To correlate changes in nuclear factor binding in vitro with transcriptional activity in vivo and define the structural requirements for IL-2 promoter repression, we used transient DNA transfections. Jurkat cells were transfected with plasmids containing either the intact IL-2 promoter or its AP-1, NF-AT, and NF-kB motifs. Dex inhibited the IL-2 promoter and the AP-1, but not the NF-AT and NF-kB plasmids. In contrast, CsA inhibited the IL-2 promoter and the NF-AT, but not the AP-1 and NF-kB plasmids. These results suggest that in human T lymphocytes both Dex and CsA inhibited IL-2 gene transcription through interference with transcription factors AP-1 and NF-AT. We propose that, while maximum inhibition may involve interaction with both transcription factors, AP-1 is the primary target of Dex. 


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Analysis of the ligand-binding domain of human retinoic acid receptor alpha by site-directed mutagenesis. 
Three subtypes of retinoic acid receptors (RAR), termed RAR alpha, RAR beta, and RAR gamma, have been described. They are composed of different structural domains, including distinct domains for DNA and ligand binding. RARs specifically bind all-trans-retinoic acid (RA), 9-cis-RA, and retinoid analogs. In this study, we examined the functional role of cysteine and arginine residues in the ligand-binding domain of hRAR alpha (hRAR alpha-LBD, amino acids 154 to 462). All conserved cysteine and arginine residues in this domain were mutated by site-directed mutagenesis, and the mutant proteins were characterized by blocking reactions, ligand-binding experiments, transactivation assays, and protease mapping. Changes of any cysteine residue of the hRAR alpha-LBD had no significant influence on the binding of all-trans RA or 9-cis RA. Interestingly, residue C-235 is specifically important in antagonist binding. With respect to arginine residues, only the two single mutations of R-276 and R-394 to alanine showed a dramatic decrease of agonist and antagonist binding whereas the R272A mutation showed only a slight effect. For all other arginine mutations, no differences in affinity were detectable. The two mutations R217A and R294A caused an increased binding efficiency for antagonists but no change in agonist binding. From these results, we can conclude that electrostatic interactions of retinoids with the RAR alpha-LBD play a significant role in ligand binding. In addition, antagonists show distinctly different requirements for efficient binding, which may contribute to their interference in the ligand-inducible transactivation function of RAR alpha. 


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Nuclear Rel-A and c-Rel protein complexes are differentially distributed within human thymocytes. 
Nuclear factor-kappa B (NF-kappa B)/Rel proteins are inducible transcriptional regulators of numerous cellular genes. They are particularly abundant in lymphoid tissues and are thought to be critical for the transcription of genes involved in immune and inflammatory responses. We have reported previously that a nuclear NF-kappa B activity was present in freshly extracted human thymocytes in the absence of in vitro treatment of these cells. In the present report, we identified NF-kappa B proteins extracted from human thymocyte nuclei as being p50/p65 and p50/c-Rel complexes. Immunochemical and immunofluorescent staining of thymus sections using specific Abs allowed visualization of nuclear NF-kappa B proteins in both thymocytes and nonthymocyte cells. This detection suggested a preferential activation of p50/c-Rel in medullary thymocytes, whereas p50/p65 was present in both cortical and medullary regions of human thymus lobules. However, the intensity of p65 labeling was much higher in several thymocytes from the medulla. p65, p50, and c-Rel activities were found in both CD4- and CD8-positive thymocytes. These observations suggest that p65 and c-Rel complexes play distinct roles in gene expression and that both forms of NF-kappa B play critical roles during late stages of the intrathymic maturation of T cells. 


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Abundant expression of erythroid transcription factor P45 NF-E2 mRNA in human peripheral granurocytes. 
Transcription factor NF-E2 is crucial for regulation of erythroid-specific gene expression. p45 subunit of NF-E2 contains a basic-leucine zipper domain and dimerizes with the small Maf family protein to form functional NF-E2 complex. While p45 expression was shown to be restricted to erythroid cells, megakaryocytes and mast cells in hematopoietic lineage, we found in this study that p45 mRNA is abundantly transcribed in the granulocyte fraction of human peripheral blood cells. As neutrophils occupy approximately 92% of the cells in granulocyte fraction of human peripheral blood cells. As neutrophils occupy approximately 92% of the cells in this fraction, the cells expressing p45 is most likely to be neutrophils. p45 mRNA is also expressed in HL-60 promyelocytes, albeit the expression level is much lower than that of the granulocyte fraction. HL-60 cells were found to express mafK mRNA, indicating the presence of genuine NF-E2 complex in the cells. Although p45 mRNA is transcribed from two different promoters, aNF-E2 promoter and fNF-E2 promoter, in erythroid and megakaryocytic lineage cells, p45 mRNA is transcribed only from aNF-E2 promoter. The expression of p45 megakaryocytic lineage cells, p45 mRNA is transcribed only from aNF-E2 promoter. The expression of p45 mRNA in the neutrophils declined rapidly after transfer of the cells to in vitro culture and G-CSF could not sustain the expression from the down-regulation, suggesting the E2 may also participate in the regulation of neutrophil-specific gene expression. 


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Jak3 is associated with CD40 and is critical for CD40 induction of gene expression in B cells. 
CD40 is a receptor that is critical for the survival, growth, differentiation, and isotype switching of B lymphocytes. Although CD40 lacks intrinsic tyrosine kinase activity, its ligation induces protein tyrosine phosphorylation, which is necessary for several CD40-mediated events. We show that engagement of CD40 induces tyrosine phosphorylation and activation of Jak3 as well as of STAT3. Jak3 is constitutively associated with CD40, and this interaction requires a proline-rich sequence in the membrane-proximal region of CD40. Deletion of this sequence abolishes the capacity of CD40 to induce expression of CD23, ICAM-1, and lymphotoxin-alpha genes in B cells. These results indicate that signaling through Jak3 is activated by CD40 and plays an important role in CD40-mediated functions. 


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Thymocyte-thymic epithelial cell interaction leads to high-level replication of human immunodeficiency virus exclusively in mature CD4(+) CD8(-) CD3(+) thymocytes: a critical role for tumor necrosis factor and interleukin-7. 
This work aims at identifying the thymocyte subpopulation able to support human immunodeficiency virus (HIV) replication under the biological stimuli of the thymic microenvironment. In this report we demonstrate that interaction with thymic epithelial cells (TEC) induces a high-level replication of the T-tropic primary isolate HIV-1(B-LAIp) exclusively in the mature CD4(+) CD8(-) CD3(+) thymocytes. Tumor necrosis factor (TNF) and interleukin-7 (IL-7), secreted during this interaction, are critical cytokines for HIV long terminal repeat transactivation through NF-kappaB-dependent activation. TNF is the major inducer of NF-kappaB and particularly of the p50-p65 complex, whereas IL-7 acts as a cofactor by sustaining the expression of the p75 TNF receptor. The requirement for TNF is further confirmed by the observation that the inability of the intermediate CD4(+) CD8(-) CD3(-) thymocytes to replicate the virus is associated with a defect in TNF production during their interaction with TEC and correlates with the absence of nuclear NF-kappaB activity in these freshly isolated thymocytes. Addition of exogenous TNF to the intermediate thymocyte cultures induces NF-kappaB activity and is sufficient to promote HIV replication in the cocultures with TEC. The other major subpopulation expressing the CD4 receptor, namely, the double-positive (DP) CD4(+) CD8(+) CD3(+/-) thymocytes, despite the entry of the virus, do not produce a significant level of virus, presumably because they are unresponsive to TNF and IL-7. Together, these data suggest that in vivo, despite an efficient entry of the virus in all the CD4(+) subpopulations, a high viral load may be generated exclusively within the mature CD4(+) CD8(-) CD3(+) subset of thymocytes. However, under conditions of inflammatory response after infection, TNF might also be present in the intermediate thymocyte compartment, leading to efficient HIV replication in these cells. 


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Regulation of CD95 (Fas) ligand expression by TCR-mediated signaling events. 
Stimulation of mature peripheral T cells by TCR engagement results in activation of signals that drive induction of cytokine gene expression and clonal expansion. However, under some conditions, engagement of the TCR leads instead to apoptosis. Recent studies demonstrate that TCR-stimulated apoptosis requires expression of CD95 ligand on activated T cells followed by an interaction between CD95 ligand and the CD95 receptor also expressed on this population. The experiments reported in this study were designed to address the signaling events triggered by TCR engagement that are important for regulating CD95 ligand gene expression. To approach this, we generated a luciferase reporter construct containing elements of the CD95 ligand promoter. Using a previously described mutant of the Jurkat T cell line, we show that proximal signaling events dependent on the presence of the CD45 tyrosine phosphatase are required for TCR-stimulated CD95 ligand expression. Transient transfection studies demonstrate further that TCR-stimulated activation of the Ras signaling pathway is required for optimal activation of CD95 ligand. Next, in an effort to determine critical transcription factors that regulate CD95 ligand expression, we demonstrate a cyclosporin A-sensitive nuclear factor-AT response element in the promoter region of this gene that is critical for optimal CD95 ligand reporter activity in stimulated T cells. Together, these studies begin a dissection of the biochemical events that lead to expression of CD95 ligand, a required step for TCR-induced apoptosis. 


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Involvement of Egr-1/RelA synergy in distinguishing T cell activation from tumor necrosis factor-alpha-induced NF-kappa B1 transcription. 
NF-kappa B is an important transcription factor required for T cell proliferation and other immunological functions. The NF-kappa B1 gene encodes a 105-kD protein that is the precursor of the p50 component of NF-kappa B. Previously, we and others have demonstrated that NF-kappa B regulates the NF-kappa B1 gene. In this manuscript we have investigated the molecular mechanisms by which T cell lines stimulated with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutin (PHA) display significantly higher levels of NF-kappa B1 encoding transcripts than cells stimulated with tumor necrosis factor-alpha, despite the fact that both stimuli activate NF-kappa B. Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1. Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription. Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression. 


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Regulation of NF-kappa B, AP-1, NFAT, and STAT1 nuclear import in T lymphocytes by noninvasive delivery of peptide carrying the nuclear localization sequence of NF-kappa B p50. 
Activation of T lymphocytes by Ags or cytokines results in translocation of the transcription factors NF-kappa B, AP-1, NFAT, and STAT from the cytoplasm into the nucleus. The first step in the nuclear import process is recognition of a nuclear localization sequence (NLS) within the karyophilic protein by a cytoplasmic receptor such as the importin (karyopherin)-alpha subunit. The NLSs of NF-kappa B, AP-1, and NFAT differ and the NLS of STAT1 has not yet been identified. Herein we demonstrate that the inducible nuclear import of NF-kappa B, AP-1, NFAT, and STAT1 in Jurkat T lymphocytes is significantly inhibited by a cell-permeable peptide carrying the NLS of the NF-kappa B p50 subunit. NLS peptide-mediated disruption of the nuclear import of these transcription factors results in inhibition of I kappa B alpha and IL-2 gene expression, processes dependent on NF-kappa B or the combination of NF-kappa B, AP-1, and NFAT. Further, we show that inhibitory NLS peptide interacts in vitro with a cytoplasmic NLS receptor complex comprised of the Rch1/importin (karyopherin)-beta heterodimer expressed in Jurkat T cells. Taken together, these data indicate that the inducible nuclear import of NF-kappa B, AP-1, NFAT, and STAT1 in Jurkat T cells can be regulated by NLS peptide delivered noninvasively to the cytoplasm of Jurkat T cells to target members of the importin (karyopherin)-alpha beta NLS receptor complex. 


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Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene. 
Interleukin-5 (IL-5), which is produced by CD4(+) T helper 2 (Th2) cells, but not by Th1 cells, plays a key role in the development of eosinophilia in asthma. Despite increasing evidence that the outcome of many diseases is determined by the ratio of the two subsets of CD4(+) T helper cells, Th1 and Th2, the molecular basis for Th1- and Th2- specific gene expression remains to be elucidated. We previously established a critical role for the transcription factor GATA-3 in IL-5 promoter activation in EL-4 cells, which express both Th1- and Th2-type cytokines. Our studies reported here demonstrate that GATA-3 is critical for expression of the IL-5 gene in bona fide Th2 cells. Whereas mutations in the GATA-3 site abolished antigen- or cAMP- stimulated IL-5 promoter activation in Th2 cells, ectopic expression of GATA-3 in Th1 cells or in a non-lymphoid, non-IL-5-producing cell line activated the IL-5 promoter. During the differentiation of naive CD4(+) T cells isolated from T cell receptor transgenic mice, GATA-3 gene expression was up-regulated in developing Th2 cells, but was down-regulated in Th1 cells, and antigen- or cAMP-activated Th2 cells (but not Th1 cells) expressed the GATA-3 protein. Thus, GATA-3 may play an important role in the balance between Th1 and Th2 subsets in immune responses. Inhibition of GATA-3 activity has therapeutic potential in the treatment of asthma and other hypereosinophilic diseases. 


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Inhibition of human immunodeficiency virus type 1 replication in vitro by a novel combination of anti-Tat single-chain intrabodies and NF-kappa B antagonists. 
Human immunodeficiency virus type 1 (HIV-1) Tat, an early regulatory protein that is critical for viral gene expression and replication, transactivates the HIV-1 long terminal repeat (LTR) via its binding to the transactivation response element (TAR) and, along with other cellular factors, increases viral transcription initiation and elongation. Tat also superactivates the HIV-1 promoter through a TAR-independent mechanism, including tumor necrosis factor alpha-induced and protein kinase C (PKC)-dependent activation of NF-kappa B, and inhibitors of Tat and NF-kappa B cooperatively down-regulate this Tat-mediated LTR superactivation. In this study, a combined pharmacologic and genetic strategy using two PKC (NF-kappa B) inhibitors, pentoxifylline (PTX) and Go-6976, and a stably expressed anti-Tat single-chain intracellular antibody (sFv intrabody) was employed to obtain cooperative inhibition of both HIV-1 LTR-driven gene expression and HIV-1 replication. Treatment of cells with PTX and Go-6976 resulted in cooperative inhibition of both HIV-1 LTR-driven gene expression and HIV-1 replication. In addition, the combined use of anti-Tat sFv intrabodies and the two NF-kappa B inhibitors retained the virus in the latent state for as long as 45 days. The combined treatment resulted in more durable inhibition of HIV-1 replication than was seen with the NF-kappa B inhibitors alone or the anti-Tat sFv intrabodies alone. Together, these results suggest that in future clinical gene therapy trials, a combined pharmacologic and genetic strategy like the one reported here may improve the survival of transduced cells and prolong clinical benefit. 


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N-acetyl-L-cysteine inhibits primary human T cell responses at the dendritic cell level: association with NF-kappaB inhibition. 
N-acetyl-L-cysteine (NAC) is an antioxidant molecule endowed with immunomodulatory properties. To investigate the effect of NAC on the induction phase of T cell responses, we analyzed its action on human dendritic cells (DC) derived from adherent PBMC cultured with IL-4 and granulocyte-macrophage CSF. We first found that NAC inhibited the constitutive as well as the LPS-induced activity of the transcription factor NF-kappaB. In parallel, NAC was shown to down-regulate the production of cytokines by DC as well as their surface expression of HLA-DR, CD86 (B7-2), and CD40 molecules both at the basal state and upon LPS activation. NAC also inhibited DC responses induced by CD40 engagement. The inhibitory effects of NAC were not due to nonspecific toxicity as neither the viability of DC nor their mannose receptor-mediated endocytosis were modified by NAC. Finally, we found that the addition of NAC to MLR between naive T cells and allogeneic DC resulted in a profound inhibition of alloreactive responses, which could be attributed to a defect of DC as APC-independent T cell responses were not inhibited by NAC. Altogether, our results suggest that NAC might impair the generation of primary immune responses in humans through its inhibitory action on DC. 


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Extracellular-regulated kinase 1/2, Jun N-terminal kinase, and c-Jun are involved in NF-kappa B-dependent IL-6 expression in human monocytes. 
In the present study we investigated the possible involvement of the mitogen-activated protein kinase family members extracellular-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in mediating IL-6 gene expression in human monocytes, in particular their role in enhancing NF-kappa B activity. Freshly isolated monocytes treated with the protein phosphatase inhibitor okadaic acid secreted high levels of IL-6 protein, which coincided with enhanced binding activity of NF-kappa B as well as with phosphorylation and activation of the ERK1/2 and JNK proteins. The ERK pathway-specific inhibitor PD98059 inhibited IL-6 secretion from monocytes. Transient overexpression of inactive mutants of either Raf-1 or JNK1 showed that both pathways were involved in kappa B-dependent IL-6 promoter activity. By using PD98059, we demonstrated that the Raf1/MEK1/ERK1/2 pathway did not affect the DNA binding of NF-kappa B but, rather, acted at the level of transcriptional activity of NF-kappa B. Interestingly, it was shown that NF-kappa B-mediated gene transcription, both in the context of the IL-6 promoter as well as on its own, was dependent on both serine kinase activity and interaction with c-Jun protein. We conclude that okadaic acid-induced IL-6 gene expression is at least partly mediated through the ERK1/2 and JNK pathway-dependent activation of NF-kappa B transcriptional capacity. Our results suggest that the JNK pathway may regulate NF-kappa B-mediated gene transcription through its phosphorylation and activation of c-Jun. 


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Differential effects of protein kinase C inhibitors on fibronectin-induced interleukin-beta gene transcription, protein synthesis and secretion in human monocytic cells. 
Human monocytic cells express interleukin-1beta (IL-1beta) when stimulated with the extracellular matrix glycoprotein, fibronectin (FN). Protein kinase C (PKC) activation is considered important for this process; however, the metabolic steps at which PKC acts upon to mediate the FN-induced IL-1beta response remain unclear. We performed an analysis of the mechanisms by which two PKC inhibitors, Calphostin C and Staurosporine, prevent the FN-induced IL-1beta response. Both inhibitors blocked the secretion of IL-1beta protein into the media of peripheral blood mononuclear cells exposed to FN. Immunoprecipitation analysis revealed that under these circumstances, Calphostin C inhibited the production of IL-1beta protein, whereas Staurosporine allowed protein production, but inhibited its secretion. To determine the mechanisms responsible for these differences, we turned to human U937 promonocytic cells. U937 cells transfected with the human full-length IL-1beta promoter connected to a luciferase reporter gene were submitted to transcription assays, Northern blotting, and DNA electrophoresis mobility gel shift assays. These studies revealed that Calphostin C inhibited the nuclear translocation of the transcription factor activator protein-1 (AP-1) which is considered necessary for FN induction of IL-1beta gene transcription, and prevented the transcription of the IL-1beta gene. In contrast, Staurosporine alone induced AP-1 translocation and stimulation of the gene. Overall, our data indicate that Calphostin C prevents the transcription of the IL-1beta gene thereby inhibiting protein synthesis. Based on the high specificity of this compound for PKC, we conclude that PKC is necessary for FN-induced IL-1beta protein production. In contrast, Staurosporine prevented secretion of IL-1beta by unknown mechanisms. 


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Dependence for the proliferative response to erythropoietin on an established erythroid differentiation program in a human hematopoietic cell line, UT-7. 
Erythroid differentiation involves the activation of a number of erythroid-specific genes, most of which, including the globin genes and the erythropoietin receptor (Epo-R) gene, are, at least in part, regulated by the transcription factor GATA-1. In order to understand the relationship, if any, between expression of GATA-1, response to Epo and erythroid differentiation, we analyzed the expression of GATA-1, Epo-R and globin genes in an Epo-dependent human cell line, UT-7 Epo. The results were compared to those obtained with the parental granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line, UT-7, which has a predominantly megakaryoblastic phenotype and is unable to proliferate continuously in the presence of Epo. UT-7 Epo and UT-7 expressed similar levels of GATA-1 mRNA and binding activity. The two lines also expressed comparable levels of Epo-R mRNA while the number of Epo-binding sites on UT-7 Epo cells was one-sixth the number of UT-7 cells (2400 +/- 3 vs. 13,800 +/- 300). This difference in the number of binding sites could be due to differences in cell surface (UT-7 cells are 20% smaller than the parental UT-7 cells) or in receptor turnover. By Northern analysis, UT-7 cells expressed detectable levels of beta- and gamma-globin but not alpha-globin. In comparison, UT-7 Epo cells expressed alpha-globin and higher levels of gamma-globin (5-fold) and beta-globin (from barely to clearly detectable). Globin chains (alpha, beta and gamma) were clearly detectable by affinity chromatography in UT-7 Epo but not in UT-7 cells. The frequency of the cells which expressed beta- and gamma- globin genes in the two cell populations was measured by immunofluorescence with beta- and gamma-specific antibodies. The number of gamma-positive cells and their fluorescence intensity were higher in UT-7 Epo than in UT-7 cells (0 to 17% barely positive cells and 23 to 40% clearly positive cells, respectively), indicating that the increase in globin mRNA observed in UT-7 Epo is due to both an increase of gene expression per cell and an increase in numbers of cells containing gamma-globin. The levels of GATA-1, Epo-R and globin mRNA expressed were not affected by a 24-hour incubation of either cell line with Epo, GM-CSF or interleukin-3 (IL-3). (ABSTRACT TRUNCATED AT 400 WORDS) 


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Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism. 
Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a human herpesvirus associated with epithelial cell malignancies (nasopharyngeal carcinoma) as well as B-cell malignancies. Understanding how viral latency is disrupted is a central issue in herpesvirus biology. Epithelial cells are the major site of lytic EBV replication within the human host, and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas. It is known that expression of a single viral immediate-early protein, BZLF1, is sufficient to initiate the switch from latent to lytic infection in B cells. Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency. Here we show that, unexpectedly, expression of another viral immediate-early protein, BRLF1, can disrupt viral latency in an epithelial cell-specific fashion. Therefore, the mechanisms leading to disruption of EBV latency appear to be cell-type specific. 


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Regulation of c-jun mRNA expression by hydroxyurea in human K562 cells during erythroid differentiation [published erratum appears in Biochim Biophys Acta 1995 Dec 27;1264(3):409] 
Hydroxyurea (HU) is an antitumor agent which also induces hemoglobinization during erythroid differentiation. In addition, HU stimulates the synthesis of fetal hemoglobin in sickle cell anemia patients. To further understand its mechanism of action, we investigated the effects of HU on regulation of c-jun expression prior to the onset of erythroid differentiation of K562 cells. HU induced a dose-dependent stimulation of c-jun synthesis. The levels of c-jun mRNA was elevated 4 to 7.5-fold by HU within 2 h. This was followed by a gradual decline to the basal level by 24 h. Both nuclear run-on and actinomycin D pulse experiments strongly indicate that HU regulates c-jun mRNA expression by increasing the rate of synthesis as well as stabilizing the c-jun mRNA. In addition, the level of jun protein was elevated by 2 to 5-fold within 4 h in HU treated cells. Furthermore, concentrations of HU below 250 microM slightly increased the 5X AP-1/CAT activity. These results strongly suggest that HU induces both transcriptional and post-transcription regulation of c-jun during erythroid differentiation. 


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Lymphocytes from CML patients lack a 47 kDa factor having affinity for a genomic sterol regulatory sequence. 
Deranged cellular cholesterol homeostasis has been widely recognized in the initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Since the human genomic sterol regulatory element (SRE) has been shown to regulate various key genes involved in this phenomenon, the present study revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects, as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when these CML patients achieved complete haematological remission (CHR) through alpha-interferon therapy. Furthermore, an inverse relationship was also observed between the LDL receptor gene expression at the transcriptional level and the binding affinity of this 47 kDa protein factor to the SRE sequence. Based upon these results we propose that this factor may have a role in pathophysiology of chronic myeloid leukaemia. 


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Positive regulators of the lineage-specific transcription factor GATA-1 in differentiating erythroid cells. 
The zinc finger transcription factor GATA-1 is a major regulator of gene expression in erythroid, megakaryocyte, and mast cell lineages. GATA-1 binds to WGATAR consensus motifs in the regulatory regions of virtually all erythroid cell-specific genes. Analyses with cultured cells and cell-free systems have provided strong evidence that GATA-1 is involved in control of globin gene expression during erythroid differentiation. Targeted mutagenesis of the GATA-1 gene in embryonic stem cells has demonstrated its requirement in normal erythroid development. Efficient rescue of the defect requires an intact GATA element in the distal promoter, suggesting autoregulatory control of GATA-1 transcription. To examine whether GATA-1 expression involves additional regulatory factors or is maintained entirely by an autoregulatory loop, we have used a transient heterokaryon system to test the ability of erythroid factors to activate the GATA-1 gene in nonerythroid nuclei. We show here that proerythroblasts and mature erythroid cells contain a diffusible activity (TAG) capable of transcriptional activation of GATA-1 and that this activity decreases during the terminal differentiation of erythroid cells. Nuclei from GATA-1- mutant embryonic stem cells can still be reprogrammed to express their globin genes in erythroid heterokaryons, indicating that de novo induction of GATA-1 is not required for globin gene activation following cell fusion. 


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Possible role of nuclear factor-kappa B activity in germline C epsilon transcription in a human Burkitt lymphoma B cell line. 
Nuclear factor-kappa B (NF-kappa B) plays a broad role in gene regulation, but it is not evident whether NF-kappa B acts as a messenger system for germline C epsilon transcription. We report here that the signaling cascade triggered by interleukin-4 (IL-4) or anti-CD40 monoclonal antibody (mAb) participates in NF-kappa B activation responsible for germline C epsilon transcription in a human Burkitt lymphoma B cell line, DND39. Both IL-4 and anti-CD40 mAb induced activation of phosphatidylinositol 3-kinase (PI3-kinase), translocation of a zeta isoform of protein kinase C, and nuclear expression of NF-kappa B. All such events were abrogated by treatment with LY294002, a specific inhibitor of PI3-kinase. In addition, N-acetyl-L-cysteine (NAC), a potent antioxidant, decreased NF-kappa B activation caused by IL-4, anti-CD40 mAb, or their combination. NAC was also effective in diminishing germline C epsilon transcription, and its potency was higher in cultures costimulated with IL-4 and anti-CD40 mAb than in those stimulated with IL-4 alone. These results indicate that IL-4 and ligation of CD40 induce NF-kappa B expression via at least a mechanism dependent on the PI3-kinase pathway and suggest that NF-kappa B sensitive to NAC may play a role in regulating germline C epsilon transcription. 


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Cross-linking of Fc gamma receptors activates HIV-1 long terminal repeat-driven transcription in human monocytes. 
Elevation of the levels of circulating immune complexes frequently accompanies HIV-1 infection and is a prognostic indicator of clinical progression from asymptomatic infection to AIDS. Here we report that cross-linking of Fc gamma RI or Fc gamma RII by adherent human IgG or by specific anti-Fc gamma R mAb activates HIV-1 gene expression in the human monocytic cell line BF24 and increased HIV RNA expression in monocytes from HIV infected patients as assayed by reverse transcription-PCR. In THP-1 cells, Fc gamma R cross-linking induced NF-kappa B, which is known to bind to the regulatory region of the long terminal repeat (LTR) of HIV-1 and to activate HIV-1 transcription. Anti-TNF-alpha antibody but not anti-IL-1 beta antibody strongly inhibited both the induction of HIV-1-LTR-driven transcription and the induction of NF-kappa B by Fc gamma R cross-linking. These results indicate that Fc gamma R can mediate a TNF-alpha-dependent induction of HIV-1 gene transcription and suggest that immune complexes may contribute to the pathophysiology of HIV-1 infection by augmenting viral replication in monocytes. 


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Tobacco smoke induces coordinate activation of HSF and inhibition of NFkappaB in human monocytes: effects on TNFalpha release. 
Tobacco smoke (TS) exposure is a major risk factor for human disease, and macrophages of healthy smokers have a depressed capacity to release cytokines, including tumor necrosis factor (TNF)alpha. TS induces the synthesis of heat shock (HS)/stress proteins (HSP), and, in particular, of Hsp70. We determined whether Hsp70 induction by TS was mediated by the activation of the HS transcription factor, HSF. HSF activation has been shown to inhibit NFkappaB. Thus, we also determined the effects of TS on NFkappaB. U937 cells and human peripheral blood monocytes were exposed to TS, binding activities of the respective transcription factors were analyzed, and Hsp70 expression and TNFalpha release were determined in parallel. TS activated HSF, which was associated with Hsp70 overexpression and inhibition of NFkappaB binding activity and TNFalpha release. The altered cytokine profile observed in smokers may relate to an HSF/Hsp70-mediated inhibition of NFkappaB activity. Copyright 1998 Academic Press. 


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Adherence-dependent increase in human monocyte PDGF(B) mRNA is associated with increases in c-fos, c-jun, and EGR2 mRNA. 
Adherence is an important initial step in the transition of a circulating monocyte to a tissue macrophage. This differentiation is accompanied by an augmented capacity to generate growth factors. We hypothesized that adherence itself might be an important trigger for a sequence of gene activation culminating in cells with increased mRNA encoding profibrotic growth factors such as platelet-derived growth factor B subunit (PDGF[B]) and transforming growth factor-beta (TGF-beta). After in vitro adherence, human monocytes had a biphasic increase in PDGF(B) mRNA with peaks at 6 h and 13 d. No increase in TGF-beta mRNA was observed. The 6-h increase in PDGF(B) mRNA was adherence dependent, and in addition, was abrogated when the cytoskeletal integrity was compromised by cytochalasin D. The 6-h increase in PDGF(B) mRNA was unaltered by adherence in the presence of the monocyte stimulus lipopolysaccharide. Adherence to either fibronectin or collagen-coated plastic had little consistent effect on PDGF(B) mRNA accumulation. The increased PDGF(B) mRNA observed in adherent monocytes was accompanied by increases in mRNAs of the early growth response genes c-fos (maximal at 20 min), c-jun, and EGR2 (maximal at 6-24 h). The increase in c-jun and EGR2, but not c-fos, mRNA was also abrogated by cytochalasin D. These observations suggest that adherence results in increases of c-fos, c-jun, EGR2, and PDGF(B) mRNA. In addition, the increases in c-jun, EGR2, and PDGF(B) may depend on cytoskeletal rearrangement. Modulation of these events at the time of adherence offers a mechanism by which differential priming of the cells may be accomplished. 


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NF-kappaB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. 
Activation of CD40 is essential for thymus-dependent humoral immune responses and rescuing B cells from apoptosis. Many of the effects of CD40 are believed to be achieved through altered gene expression. In addition to Bcl-x, a known CD40-regulated antiapoptotic molecule, we identified a related antiapoptotic molecule, A1/Bfl-1, as a CD40-inducible gene. Inhibition of the NF-kappaB pathway by overexpression of a dominant-active inhibitor of NF-kappaB abolished CD40-induced up-regulation of both the Bfl-1 and Bcl-x genes and also eliminated the ability of CD40 to rescue Fas-induced cell death. Within the upstream promoter region of Bcl-x, a potential NF-kappaB-binding sequence was found to support NF-kappaB-dependent transcriptional activation. Furthermore, expression of physiological levels of Bcl-x protected B cells from Fas-mediated apoptosis in the absence of NF-kappaB signaling. Thus, our results suggest that CD40-mediated cell survival proceeds through NF-kappaB-dependent up-regulation of Bcl-2 family members. 


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Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion. 
We recently reported that prolonged exposure of human aortic endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the transcriptional regulator nuclear factor-kappaB (NF-kappaB). Here we investigate the hypothesis that low shear-induced activation of NF-kappaB is responsible for enhanced expression of vascular cell adhesion molecule (VCAM-1) resulting in augmented endothelial cell-monocyte (EC-Mn) adhesion and that this activation is dependent on intracellular oxidant activity. Before exposure to low shear (2 dyn/cm2) for 6 h, HAEC were preincubated with or without the antioxidants pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC). PDTC strongly inhibited low shear-induced activation of NF-kappaB, expression of VCAM-1, and EC-Mn adhesion. Paradoxically, NAC exerted a positive effect on low shear-induced VCAM-1 expression and EC-Mn adhesion and only slightly downregulated NF-kappaB activation. However, cytokine-induced NF-kappaB activation and VCAM-1 expression are blocked by both PDTC and NAC. These data suggest that NF-kappaB plays a key role in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated. 


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The small GTP-binding protein Rho potentiates AP-1 transcription in T cells. 
The Rho family of small GTP-binding proteins is involved in the regulation of cytoskeletal structure, gene transcription, specific cell fate development, and transformation. We demonstrate in this report that overexpression of an activated form of Rho enhances AP-1 activity in Jurkat T cells in the presence of phorbol myristate acetate (PMA), but activated Rho (V14Rho) has little or no effect on NFAT, Oct-1, and NF-kappaB enhancer element activities under similar conditions. Overexpression of a V14Rho construct incapable of membrane localization (CAAX deleted) abolishes PMA-induced AP-1 transcriptional activation. The effect of Rho on AP-1 is independent of the mitogen-activated protein kinase pathway, as a dominant-negative MEK and a MEK inhibitor (PD98059) did not affect Rho-induced AP-1 activity. V14Rho binds strongly to protein kinase Calpha (PKCalpha) in vivo; however, deletion of the CAAX site on V14Rho severely diminished this association. Evidence for a role for PKCalpha as an effector of Rho was obtained by the observation that coexpression of the N-terminal domain of PKCalpha blocked the effects of activated Rho plus PMA on AP-1 transcriptional activity. These data suggest that Rho potentiates AP-1 transcription during T-cell activation. 


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Fibrinogen activates NF-kappa B transcription factors in mononuclear phagocytes. 
Adhesion to extracellular matrices is known to modulate leukocyte activation, although the mechanisms are not fully understood. Mononuclear phagocytes are exposed to fibrinous provisional matrix throughout migration into inflammatory foci, so this study was undertaken to determine whether fibrinogen triggers activation of NF-kappa B transcription factors. U937 cells differentiated with PMA in nonadherent culture were shown to express two fibrinogen-binding integrins, predominately CD11b/CD18, and to a lesser extent, CD11c/CD18. Cells stimulated with fibrinogen (10-100 microg/ml)/Mn2+ (50 microM) for 2 h were examined by electrophoretic mobility shift assay. NF-kappa B activation, minimal in unstimulated cells, was substantially up-regulated by fibrinogen. Fibrinogen also caused activation of AP-1, but not SP1 or cAMP response element-binding protein (CREB) factors. Blocking mAbs against CD18 and CD11b abrogated fibrinogen-induced NF-kappa B activation. To determine the effects on transcriptional regulation, U937 cells were transfected with a plasmid containing the HIV-1 enhancer (bearing two NF-kappa B sites) coupled to a chloramphenicol acetyltransferase (CAT) reporter. Cells were subsequently stimulated with 1) PMA for 24 h, inducing CAT activity by 2.6-fold, 2) fibrinogen/Mn2+ for 2 h, inducing CAT activity by 3.2-fold, or 3) costimulation with fibrinogen and PMA, inducing 5.7-fold the CAT activity induced by PMA alone. We conclude that contact with fibrinogen-derived proteins may contribute to mononuclear phagocyte activation by signaling through CD11b/CD18, resulting in selective activation of transcriptional regulatory factors, including NF-kappa B. 


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Alteration of a single serine in the basic domain of the Epstein-Barr virus ZEBRA protein separates its functions of transcriptional activation and disruption of latency. 
The ZEBRA protein from Epstein-Barr virus (EBV) activates a switch from the latent to the lytic expression program of the virus. ZEBRA, a member of the bZIP family of DNA-binding proteins, is a transcriptional activator capable of inducing expression from viral lytic cycle promoters. It had previously been thought that ZEBRA's capacity to disrupt EBV latency resided primarily in its ability to activate transcription of genes that encode products required for lytic replication. We generated a point mutant of ZEBRA, Z(S186A), that was not impaired in its ability to activate transcription; however, this mutation abolished its ability to initiate the viral lytic cascade. The mutant, containing a serine-to-alanine substitution in the DNA-binding domain of the protein, bound to several known ZEBRA-binding sites and activated transcription from reporters bearing known ZEBRA-responsive promoters but did not disrupt latency in EBV-infected cell lines. Therefore, initiation of the EBV lytic cycle by the ZEBRA protein requires a function in addition to transcriptional activation; a change of serine 186 to alanine in the DNA-binding domain of ZEBRA abolished this additional function and uncovered a new role for the ZEBRA protein in disruption of EBV latency. The additional function that is required for initiation of the lytic viral life cycle is likely to require phosphorylation of serine 186 of the ZEBRA protein, which may influence either DNA recognition or transcriptional activation of lytic viral promoters in a chromatinized viral episome. 


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[An overexpression of retinoic acid receptor alpha blocks myeloid cell differentiation at the promyelocyte stage] 
Retinoic acid (RA), a vitamin A derivative, exerts a wide range of biological effects related to cell proliferation and differentiation. The pleiotropic effects of RA are thought to be mediated through specific nuclear RA receptors (RARs). RARs are members of the steroid/thyroid hormone receptor superfamily and exhibit a molecular structure that possess discrete DNA-binding and RA (ligand)-binding domains. In hematopoietic system, RA and RARs, predominantly RAR alpha may play key roles for the proliferation and differentiation of hematopoietic progenitors. However, it is currently unknown how RA and RARs are involved in regulating normal hematopoietic differentiation. To make clear the roles of RA and RAR alpha in the normal hematopoiesis, I have introduced the construct of human RAR alpha (hRAR alpha) into murine bone marrow cells with retroviral vector, and selected infected cells with drug resistant marker (Neo(r)) cultured on the stroma cell line (PA6-neo), and analyzed the behavior of infected cells. All of procedure were done in vitro. Most cells infected with hRAR alpha exhibited promyelocytic morphology and were thought to be blocked at the promyelocytic stage in their myeloid differentiation. Furthermore, these immature cells differentiated terminally into mature granulocytes by adding with RA (10(-6) M). RAR alpha infected cells were also able to differentiate into mature macrophages in the both of long term culture and IL3 colony. These observations suggest that an overexpression of RAR alpha alone is effective to suppress myeloid cell differentiation and RAR alpha plays a crucial role in the terminal differentiation of myeloid precursors. The system described here may serve as a model for studying the the essential genes for differentiation of normal bone marrow cells. 


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Phosphatidylinositol 3-kinase couples the interleukin-2 receptor to the cell cycle regulator E2F. 
Cell cycle progression initiated by interleukin-2 (IL-2) in T cells is critical for lymphoproliferation and an immune response. Phosphatidyl inositol 3-kinase (PI3K) is activated by IL-2. However, nuclear targets for PI3K are not known. Here we identify the cell cycle regulator E2F as an IL-2 target in T lymphocytes and PI3K as the critical signaling pathway. We eliminate both Stat5 and Raf/MEK pathways from E2F regulation. Protein kinase B (PKB) is activated by IL-2 via PI3K. The expression of an active PKB is sufficient to induce E2F activity. Inhibition of PI3K inhibits phosphorylation of Rb, induction of cyclin D3, and degradation of p27kip1. These results establish a crucial PI3K/PKB-mediated link between the IL-2 teceptor and the cell cycle machinery. 


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Erythropoietin-dependent induction of hemoglobin synthesis in a cytokine-dependent cell line M-TAT. 
M-TAT is a cytokine-dependent cell line with the potential to differentiate along the erythroid and megakaryocytic lineages. We cultured M-TAT cells long term (> 1 year) in the continuous presence of erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), or stem cell factor (SCF). These long term cultures are referred to as M-TAT/EPO, M-TAT/GM-CSF, and M-TAT/SCF cells, respectively. Hemoglobin concentration and gamma-globin and erythroid delta-aminolevulinate synthase mRNA levels were significantly higher in M-TAT/EPO cells than in M-TAT/GM-CSF cells. When the supplemented cytokine was switched from GM-CSF to EPO, hemoglobin synthesis in M-TAT/GM-CSF cells increased rapidly (within 5 h), and the level of GATA-1 mRNA increased. In contrast, the addition of GM-CSF to the M-TAT/EPO cell culture decreased the amount of hemoglobin, even in the presence of EPO, indicating that the EPO signal for erythroid differentiation is suppressed by GM-CSF. Thus, erythroid development of M-TAT cells is promoted by EPO and suppressed by GM-CSF. These results support the hypothesis that EPO actively influences the programming of gene expression required for erythroid progenitor cell differentiation. 


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Transcription factor NF-kappaB regulation of renal fibrosis during ureteral obstruction. 
Irrespective of the etiology, many kidney diseases result in inflammation and fibrosis of the tubulointerstitium, with the subsequent loss of renal function. To initiate any disease process or for any disease process to progress, there must be changes in the transcription of genes within the affected tissue. The nuclear factor-kappa B (NF-kappaB) family of transcription factors regulates genes involved in inflammation, cell proliferation, and cell differentiation. This review discusses the NF-kappaB transcription factor family in general and the association of NF-kappaB activation with cellular/molecular events of renal inflammation and fibrosis. 


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A negative role for phosphoinositide 3-kinase in T-cell antigen receptor function. 
BACKGROUND: A delicate balance between positive and negative regulatory mechanisms during T-cell activation determines the specificity and magnitude of an immune response. Phosphoinositide 3-kinase (PI 3-kinase) is activated by a diverse set of receptors that determine T-cell function, including the T-cell antigen receptor (TCR), the costimulatory receptor CD28, and negative regulators of T-cell activation such as CTLA-4. PI 3-kinase is also regulated by the haematopoietic cytokines that determine T-cell differentiation and lymphocyte proliferation. PI 3-kinase can thus dynamically influence the outcome of the immune reactions at various stages. In this study, we investigated the importance of PI 3-kinase in TCR-directed T-cell activation using activated or inhibitory versions of PI 3-kinase. RESULTS: Certain aspects of TCR responses such as the induction of transcriptional activity of AP1 and serum response factor were not affected by expression of the mutant forms of PI 3-kinase. We found, however, that PI 3-kinase profoundly influenced the transactivation capacity of 'nuclear factor of activated T cells' (NF-AT) elicited by the TCR: expression of an activated form of PI 3-kinase inhibited TCR-mediated NF-AT responses, whereas expression of a dominant negative mutant of PI 3-kinase potently enhanced TCR-controlled NF-AT induction. These effects of PI 3-kinase were not mediated by previously identified PI 3-kinase effectors, such as protein kinase B, a positive regulator of PI 3-kinase, or the GTPase Rac, and are therefore likely to involve a novel, as yet unknown, effector molecule. CONCLUSIONS: Our results establish that PI 3-kinase can both positively and negatively regulate T-cell function, and uncover a previously unrecognized function for PI 3-kinase in T cells as a selective negative regulator of TCR-signalling events and therefore as a determinant of T-cell homeostasis. 


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Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-1 and Sp1 motifs. 
Erythropoietin (Epo), the primary regulator of the production of erythroid cells, acts by binding to a cell surface receptor (EpoR) on erythroid progenitors. We used deletion analysis and transfection assays with reporter gene constructs to examine the transcription control elements in the 5' flanking region of the human EpoR gene. In erythroid cells most of the transcription activity was contained in a 150 bp promoter fragment with binding sites for transcription factors AP2, Sp1 and the erythroid-specific GATA-1. The 150 bp hEpoR promoter exhibited high and low activity in erythroid OCIM1 and K562 cells, respectively, reflecting the high and low levels of constitutive hEpoR expression. The GATA-1 and Sp1 binding sites in this promoter lacking a TATA sequence were necessary for a high level of transcription activation. Protein-DNA binding studies suggested that Sp1 and two other CCGCCC binding proteins from erythroid and non-erythroid cells could bind to the Sp1 binding motif. By increasing GATA-1 levels via co-transfection, we were able to transactivate the hEpoR promoter in K562 cells and non-erythroid cells, but not in the highly active OCIM1 cells, although GATA-1 mRNA levels were comparable in OCIM1 and K562. Interestingly, when we mutated the Sp1 site, resulting in a marked decrease in hEpoR promoter activity, we could restore transactivation by increasing GATA-1 levels in OCIM1 cells. These data suggest that while GATA-1 can transactivate the EpoR promoter, the level of hEpoR gene expression does not depend on GATA-1 alone. Rather, hEpoR transcription activity depends on coordination between Sp1 and GATA-1 with other cell-specific factors, including possibly other Sp1-like binding proteins, to provide high level, tissue-specific expression. 


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Alcohol-induced regulation of nuclear regulatory factor-kappa beta in human monocytes. 
Acute ethanol exposure has the capacity to modulate immune functions, particularly, to down regulate monocyte production of inflammatory cytokines. However, the intracellular mechanisms for these effects of ethanol are yet to be understood. Considering that nuclear regulatory factor-kappa beta (NF-kappa B)/Rel is a common regulatory element of the promoter region of the inflammatory cytokine genes, herein, we tested the hypothesis that acute ethanol affects NF-kappa B activation in human monocytes. Adherence-isolated monocytes showed constitutive DNA binding activity of NF-kappa B. A clinically relevant dose (25 mM) of acute ethanol treatment in vitro increased NF-kappa B binding activity in monocytes with a preferential induction of the inhibitory, p50/p50, NF-kappa B/Rel homodimer, and resulted in no induction of the p65/p50 heterodimer. In contrast, lipopolysaccharide stimulation primarily induced the p65/p50 heterodimer that has been shown to result in gene activation. Thus, such unique activation of the inhibitory p50/p50 homodimer by acute ethanol treatment may result in inhibition rather than activation of NF-kappa B-regulated inflammatory cytokine genes. Consequently, these results suggest that physiologically relevant concentrations of ethanol may affect production of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 by disrupting NF-kappa B signaling in monocytes. 


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Cloning and functional characterization of early B-cell factor, a regulator of lymphocyte-specific gene expression. 
Early B-cell factor (EBF) was identified previously as a tissue-specific and differentiation stage-specific DNA-binding protein that participates in the regulation of the pre-B and B lymphocyte-specific mb-1 gene. Partial amino acid sequences obtained from purified EBF were used to isolate cDNA clones, which by multiple criteria encode EBF. The recombinant polypeptide formed sequence-specific complexes with the EBF-binding site in the mb-1 promoter. The cDNA hybridized to multiple transcripts in pre-B and B-cell lines, but transcripts were not detected at significant levels in plasmacytoma, T-cell, and nonlymphoid cell lines. Expression of recombinant EBF in transfected nonlymphoid cells strongly activated transcription from reporter plasmids containing functional EBF-binding sites. Analysis of DNA binding by deletion mutants of EBF identified an amino-terminal cysteine-rich DNA-binding domain lacking obvious sequence similarity to known transcription factors. DNA-binding assays with cotranslated wild-type and truncated forms of EBF indicated that the protein interacts with its site as a homodimer. Deletions delineated a carboxy-terminal dimerization region containing two repeats of 15 amino acids that show similarity with the dimerization domains of basic-helix-loop-helix proteins. Together, these data suggest that EBF represents a novel regulator of B lymphocyte-specific gene expression. 


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Signal transduction pathways triggered by the FcepsilonRIIb receptor (CD23) in human monocytes lead to nuclear factor-kappaB activation. 
BACKGROUND: Alveolar macrophages play a key role in the initiation of the inflammatory reaction of allergic asthma. Alveolar macrophages and peripheral blood monocytes are activated when IgE/allergen immune complexes bind to the CD23 receptor, which leads to the production of inflammatory cytokines. OBJECTIVE: We sought to investigate the molecular mechanisms regulating this early inflammatory response. We have focused on the study of the signal transduction pathways triggered by CD23 in human monocytes and the promonocytic cell line U937. METHODS: CD23 was cross-linked in human monocytes and U937 cells with IgE immune complexes. Surface expression of CD23 was determined by FACS analysis. Transcription factor activation and gene transcription were studied by gel-shift assays and Northern blot analysis, respectively. IkappaBalpha phosphorylation and degradation was analyzed by Western blot. RESULTS: Nuclear factor (NF)-kappaB is the main transcription factor involved in the gene activation that follows CD23 cross-linking in monocytes. CD23-induced NF-kappaB is a heterodimer composed of p65/p50 subunits. NF-kappaB nuclear translocation is secondary to the phosphorylation and subsequent degradation of the NF-kappaB inhibitory molecule IkappaBalpha. Tyrosine kinase-dependent, and not protein kinase C-dependent, pathways mediate CD23-triggered NF-kappaB activation but do not participate in the direct phosphorylation of IkappaBalpha. IkappaBalpha degradation and NF-kappaB nuclear translocation correlate with transcriptional activation of the inflammatory cytokines TNF-alpha and IL-1beta. CONCLUSIONS: NF-kappaB is the main transcription factor involved in the signal transduction pathway of CD23 in monocytes. 


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Role of Egr-2 in up-regulation of Fas ligand in normal T cells and aberrant double-negative lpr and gld T cells. 
We previously identified a Fas ligand regulatory element (FLRE) in the Fas ligand (fasL) promoter that binds Egr family proteins and demonstrated that Egr-3 (PILOT) but not Egr-1 (NGFI-A, Krox-24, Tis-8, and Zif-268) induces transcription of fasL. The aberrant CD4(-)CD8(-) T cells from lpr/lpr and gld/gld mice, which have mutations in the genes encoding Fas and FasL, respectively, have an activated phenotype and constitutively express high levels of fasL mRNA, prompting us to ask what role if any the FLRE and Egr family proteins have in this aberrant expression of fasL. Unstimulated MRL-lpr/lpr and C3H-gld/gld CD4(-)CD8(-) T cells constitutively contained high levels of two proteins that bound to the FLRE. Supershift analysis revealed these proteins to be Egr-1 and Egr-2 (Krox-20); Egr-3 was not detected. Activation of normal lymph node cells resulted in increased expression of Egr-1, -2, and -3. As with egr-3, expression of egr-2 was blocked by cyclosporin A. Although overexpressed Egr-1 was ineffective, overexpressed Egr-2 was as potent as Egr-3 in inducing fasL promoter-dependent reporter constructs in T cell hybridomas and HeLa cells, and both up-regulated endogenous fasL mRNA in HeLa cells. FasL-dependent reporter constructs in MRL-lpr/lpr and C3H-gld/gld CD4(-)CD8(-) T cells were constitutively active, and this activity was largely prevented by mutation of the critical Egr family binding element. Thus, Egr-2, in addition to Egr-3, regulates FasL expression in activated normal T cells, and Egr-2 is likely to play a direct role in aberrant fasL up-regulation in lpr/lpr and gld/gld CD4(-)CD8(-) T cells. 


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Human TAFII 105 is a cell type-specific TFIID subunit related to hTAFII130. 
We previously characterized Drosophila and human TAF subunits that make up the core TFIID complex found in all cells. Here, we report that differentiated B cells contain a novel substoichiometric TAF of 105 kDa not found associated with TFIID isolated from other cell types. The cDNA encoding hTAFII105 reveals a highly conserved C-terminal domain shared by hTAFII130 and oTAFII110, while the N-terminal coactivator domain has diverged significantly. All cells tested express TAFII105 mRNA, but only B cells contain significant levels of protein associated with TFIID. Transient overexpression of hTAFII105 selectively squelches the transcription of some genes in B cells. These properties suggest that TAFII105 is a cell type-specific subunit of TFIID that may be responsible for mediating transcription by a subset of activators in B cells. 


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CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency. 
Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G.A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K.R., Tortolani, A.J., Cerami, A.& Tracey, K.J.(1995) Mol.Med.1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A.& Tracey, J.(1996) J.Exp.Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI-1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI-1493. Identification of the molecular target through which CNI- 1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation. 


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Cortivazol mediated induction of glucocorticoid receptor messenger ribonucleic acid in wild-type and dexamethasone-resistant human leukemic (CEM) cells. 
Cortivazol is a phenylpyrazolo glucocorticoid of high potency and unusual structure. In both wild-type and highly dexamethasone(dex)-resistant clones of the human leukemic cell line CEM, exposure to cortivazol leads to cell death. It has been shown recently that in wild-type CEM cells but not in a dex-resistant, glucocorticoid receptor(GR)-defective clone ICR-27 TK-3, dex induces GR mRNA. To test the hypothesis that cortivazol acts in dex-resistant cells by making use of the residual GR found there, wild-type and dex-resistant clones were treated with various concentrations of cortivazol and induction of GR mRNA was studied. Cortivazol significantly induced GR mRNA in the normal CEM-C7 as well as in two classes of dex-resistant clones, although the dex-resistant clones needed at least 10 times more cortivazol than the normal cells for significant GR mRNA induction. Increased levels of GR mRNA were noticed as early as 3 h after treatment. A general correlation between induction of GR mRNA and lysis of the normal and dex-resistant cells was found. Positive induction of GR mRNA might be one of the earliest crucial steps in the lysis of normal and dex-resistant CEM cells, or might serve as a marker for the process. However, the lysis pathway in the dex-resistant cells is defective in that dex-resistant clones needed significantly more cortivazol than the normal cells for lysis of the cells. 


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Expression of PILOT, a putative transcription factor, requires two signals and is cyclosporin A sensitive in T cells. 
Few known genes (IL-2, members of the IL-8 family, interferon-gamma) are induced in T cells only through the combined effect of phorbol myristic acetate (PMA) and a Ca(2+)-ionophore, and expression of only these genes can be fully suppressed by Cyclosporin A (CyA). We have identified a putative transcription factor, designated PILOT, with an identical dual signal requirement for expression. Induction of the PILOT gene is detectable in human T cells 20 min following activation in the presence of cycloheximide and is fully suppressed by CyA. The PILOT protein has a calculated M(r) of 42.6 kDa and contains three zinc fingers of the C2H2-type at the carboxyl-terminus which are highly homologous to the zinc finger regions of the transcription factors EGR1, EGR2, and pAT 133. In contrast to T cells, in fibroblasts PILOT gene expression requires only one signal (PMA) and is not affected by CyA. This observation directly demonstrates the existence of a Ca2+ signal-dependent regulatory element obligatory for expression of some genes in T cells but not in fibroblasts. This differential expression model will be valuable in the dissection of the dual signal pathway in T cells and the effects of CyA upon it. 


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Expression and genomic configuration of GM-CSF, IL-3, M-CSF receptor (C-FMS), early growth response gene-1 (EGR-1) and M-CSF genes in primary myelodysplastic syndromes. 
Peripheral blood mononuclear cells from seventeen patients with primary myelodysplastic syndromes (MDS) in advanced stage were enriched for blasts and tested for (1) karyotype, (2) genomic configuration and (3) expression of IL-3, GM-CSF, FMS and EGR-1 genes which are all located on the long arm of chromosome 5. The expression of the M-CSF gene, that has been recently reassigned to the short arm of chromosome 1 (lp), was also investigated. Aims of the study were to (1) assess the potential role of the expression of these genes in the maintenance and expansion of the neoplastic clones and (2) search for constitutional losses or rearrangements of one allele followed by a deletion of the second allele of the same genes in the leukemic cells. The latter issue was investigated by comparing, in 8 cases, constitutive DNA from skin fibroblasts with leukemic DNA. Eleven of the 17 patients had abnormal karyotypes. The M-CSF gene was expressed in 6 cases and the FMS and the EGR-1 genes were expressed in 2 of the latter cases. An autocrine mechanism of growth could be hypothesized only for the 2 patients whose cells expressed both the M-CSF and FMS genes. No germline changes or rearrangements were observed in any of the genes studied. Thus, deregulation of genes encoding for certain hemopoietic growth factors or receptors does not seem to represent a major mechanism of MDS progression. 


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Tax-independent binding of multiple cellular factors to Tax-response element DNA of HTLV-I. 
The human T-cell leukemia virus type I (HTLV-I) promoter contains three copies of imperfect repeats of a 21-base pair sequence designated here as TRE (Tax-response element) that is responsive to the virally encoded transactivator protein Tax. We have identified and separated four nuclear proteins from C81-66-45 cells, an HTLV-I immortalized Tax-expressing human T-lymphocyte line (Salahuddin et al., 1983), that interact with the TRE-DNA, none of which are identical with the Tax-protein. The proteins identified have molecular weights of about 32, 36 to 42, 50 and 110 kD. Four different methods were used to identify the proteins. First, from different cell lines three or all four of the nuclear proteins were specifically cross-linked by UV irradiation to the radioactively labeled TRE-DNA fragment. Second, TRE-DNA binding proteins sedimented through a glycerol density gradient at rates corresponding to proteins of native molecular weights of 35 to 50 kD and 110 kD. Third, only the 50 kD protein was retained on a biotinylated DNA-streptavidin matrix when the DNA fragment contained the TRE-DNA. Fourth, extensive purification by several cycles of TRE-DNA affinity chromatography resulted in the 32, 36 to 42 and 110 kD proteins and to less extent the 50 kD factor. Two abundant proteins of 75 and 80 kD were competed out by poly[d(I-C)] in all reactions. The cAMP-response element CRE, TGACGTCA, present in the 21 base-pair sequence, appears to be essential for specific protein-TRE-DNA interactions because mutation of the two G's destroys this complex. This result suggests that the cAMP response element binding protein, CREB, is involved in the protein-TRE-DNA complex and in mediating the Tax response. 


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ras protein activity is essential for T-cell antigen receptor signal transduction. 
In a Jurkat cell model of T-cell activation an interleukin-2 promoter/reporter gene construct was activated by antigen receptor agonism in combination with the lymphokine interleukin-1. Antigen receptor signals could be mimicked by suboptimal activation of protein kinase C (PKC) with phorbol esters in combination with calcium mobilization by an ionophore. In cotransfection experiments, oncogenic rats obviated the need for PKC stimulation but did not replace either the calcium signal or interleukin-1. Activated ras expression also replaced the requirement for PKC stimulation in activation of the T-cell transcription factor NF-AT. A dominant inhibitory ras mutant specifically blocked antigen receptor agonism, indicating that ras activity is required for antigen receptor signaling. In addition, an inhibitor of PKC blocked both activated ras and phorbol ester stimulation, suggesting a role for ras upstream of PKC. 


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Enhanced responsiveness to nuclear factor kappa B contributes to the unique phenotype of simian immunodeficiency virus variant SIVsmmPBj14. 
Infection with a variant of simian immunodeficiency virus, SIVsmmPBj14, leads to severe acute disease in macaques. This study was designed to investigate the functional significance of previously described mutations in the viral long terminal repeat (LTR) and to elucidate their contribution to the unique phenotype of SIVsmmPBj14. LTR-directed transcription was measured by using luciferase reporter constructs that were transiently transfected into cultured cells. In a wide range of cell types, the basal transcriptional activity of the LTR from SIVsmmPBj14 was found to be 2- to 4.5-fold higher than that of an LTR from a non-acutely pathogenic strain. These LTRs differ by five point mutations and a 22-bp duplication in SIVsmmPBj14, which includes a nuclear factor kappa B (NF kappa B) site. Transcriptional differences between these LTRs were further enhanced by two- to threefold upon treatment of cells with phorbol ester or tumor necrosis factor alpha or by cotransfection with plasmids expressing NF kappa B subunits. Mutagenesis studies, and the use of a reporter construct containing an enhancerless promoter, indicate that these transcriptional effects are due principally to the 22-bp sequence duplication and the NF kappa B site contained within it. Finally, infectious virus stocks that were isogenic except for the LTR were generated. The LTR from SIVsmmPBj14 was found to confer an increase in the kinetics of virus replication in cultured cells. Inclusion of this LTR in recombinant SIVs also resulted in a two- to threefold rise in the extent of cellular proliferation that was induced in quiescent simian peripheral blood mononuclear cells. These studies are consistent with the hypothesis that LTR mutations assist SIVsmmPBj14 in responding efficiently to cellular stimulation and allow it to replicate to high titers during the acute phase of viral infection. 


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[Molecular-biologic aspects of interaction between nervous and immune systems] 
The problem of the neuro-immuno interactions on the level of the protein trans-factors, stimulating interleukin-2 (IL-2) gene expression was discussed. The physico-chemical and functional parameters of the low molecular nuclear proteins (SP and BP- 14, 18, 19 kDs) isolated from splenic and brain cells of immunized rats were studied. The binding of these proteins to the regulatory region of IL-2 gene in vitro and stimulation of the IL-2mRNA synthesis in splenic T-lymphocytes culture in normal conditions were shown. The protective effect of SP and BP on the IL-2mRNA synthesis in stressful conditions and by the T-cells treatment with the CsA was demonstrated. 


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Interleukin-7 upregulates the interleukin-2-gene expression in activated human T lymphocytes at the transcriptional level by enhancing the DNA binding activities of both nuclear factor of activated T cells and activator protein-1. 
In the present report, we studied the role of the stromal-derived cytokine interleukin-7 (IL-7) in the IL-2-gene regulation in activated T lymphocytes. Production of IL-2 requires the formation of transcription factors involved in the IL-2-gene regulation. T-cell receptor (TCR)/CD3 engagement results in the activation of nuclear factor of activated T cells (NFAT), activator protein-1 (AP-1), and nuclear factor kappaB (NFkappaB), whereas the CD28 responsive complex (CD28RC) is activated in response to the CD28 signal. Costimulation of phytohemagglutinin/anti-CD28 activated T lymphocytes with IL-7 induces a fivefold enhanced IL-2-mRNA accumulation and a 2.5-fold enhanced protein secretion. The IL-2-gene transcription rate is increased 3.4-fold, indicating that the effect of IL-7 is in part mediated at the transcriptional level. The molecular mechanisms underlying the IL-7 effect involve the upregulation of the DNA binding activity of NFAT (60%) and AP-1 (120%), without affecting the activities of NFkappaB and CD28RC, which was confirmed by transfection assays. We also show that the IL-7-induced enhancement of the AP-1-DNA binding activity is not cyclosporin A-sensitive. Since AP-1 is part of the NFAT complex, we conclude that the IL-7-signaling pathway is involved in the activation of the fos and jun proteins of which AP-1 consists. 


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Differential induction of interferon (IFN)-inducible protein 10 following differentiation of a monocyte, macrophage cell lineage is related to the changes of nuclear proteins bound to IFN stimulus response element and kappaB sites. 
We examined chemokine gene expression following the differentiation of a monocyte, macrophage cell lineage. The human monoblastic cell line, U937 was differentiated to macrophages by the treatment with either phorbol 12-myristate 13-acetate (PMA), retinoic acid (RA), or vitamin D3 (VitD3). The gene expression of interferon (IFN)-inducible protein 10 (IP-10) (a CXC chemokine) was markedly augmented by the IFNgamma treatment in PMA- or RA-differentiated U937 cells, but only marginally in undifferentiated or VitD3-treated cells. In contrast, another inducible gene expression of monocyte chemotactic protein-1 (a CC chemokine) and the activation of the transcriptional factor (FcRFgamma) bound to the gamma response region were similarly or less abundantly induced by IFNgamma treatment in PMA- or RA-differentiated U937 cells, indicating that increased IP-10 mRNA induction was not due to the augmented ability of the cells to respond to the presence of IFNgamma. Increased expression of IFNgamma-induced IP-10 mRNA following the differentiation of U937 cells was mediated largely by augmented transcriptional activity of the gene and was related to differentiation-dependent changes of the proteins bound to IFN stimulus response element (ISRE) and kB sites, suggesting that these nuclear proteins may determine the IP-10 mRNA inducibility by IFNgamma. 


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CD28 costimulation augments IL-2 secretion of activated lamina propria T cells by increasing mRNA stability without enhancing IL-2 gene transactivation. 
The pathways leading to activation in lamina propria (LP) T cells are different from peripheral T cells. LP T cells exhibit enhanced IL-2 secretion when activated through the CD2 pathway. Coligation of CD28 leads to synergistic enhancement of IL-2 secretion. Previous studies have characterized the CD28 augmentation of TCR-mediated signaling in peripheral blood T cells through transcriptional activation of an IL-2 promoter CD28 response element (CD28RE), along with enhanced mRNA stability. This study characterized molecular events involved in CD28 costimulation of IL-2 production in LP mononuclear cells (LPMC). LPMC exhibited increased IL-2 production in response to CD28 costimulation, compared with cells activated through CD2 alone. IL-2 secretion was paralleled by increased expression of IL-2 mRNA, resulting from enhanced IL-2 mRNA stability. In contrast to transcriptional activation in PBMC, EMSA revealed that CD28 coligation of CD2-activated LPMC does not result in increased binding of trans-factors to the CD28RE, nor did Western blots detect changes in I-kappaBalpha or I-kappaBbeta levels following CD28 coligation. Furthermore, CD28 coligation fails to enhance IL-2 promoter-reporter or RE/AP construct expression in CD2-activated LPMC. The results reported herein indicate that the molecular mechanisms involved in CD28 cosignaling and regulation of IL-2 secretion in LP T cells are unique to that compartment and differ from those seen in peripheral blood T cells. These observations suggest a biological significance for different mechanisms of IL-2 activation in initiation and maintenance of the cytokine repertoire found in the mucosa. 


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Transcription-independent turnover of I kappa B alpha during monocyte adherence: implications for a translational component regulating I kappa B alpha/MAD-3 mRNA levels. 
We identified I kappa B alpha/MAD-3 as an immediate-early gene in human monocytes that is expressed in response to a variety of signals, including adhesion, lipopolysaccharide, and phorbol myristate acetate. Within 5 min of monocyte adhesion, the level of the I kappa B alpha protein is markedly diminished but is rapidly replaced in a cycloheximide-sensitive manner within 20 min. Accompanying the rapid turnover of the I kappa B alpha protein is simultaneous translocation of NF-kappa B-related transcription factors to nuclei of adhered monocytes. The demonstration that NF-kappa B can regulate I kappa B alpha/MAD-3 gene transcription in other cell types suggested that the rapid increase in steady-state I kappa B alpha/MAD-3 mRNA levels we observed within 30 min of monocyte adherence would result from NF-kappa B-dependent transcriptional stimulation of the I kappa B alpha/MAD-3 gene. Nuclear run-on analyses indicated that, instead, while several immediate-early cytokine genes, such as the interleukin 1 beta (IL-1 beta) gene, were transcriptionally activated during monocyte adhesion, the rate of I kappa B alpha/MAD-3 gene transcription remained constant. The adherence-dependent increase in I kappa B alpha/MAD-3 mRNA levels was also not a consequence of mRNA stabilization events. Interestingly, while increases in both IL-1 beta and I kappa B alpha/MAD-3 mRNA levels were detected in nuclei of adherent monocytes, cytoplasmic levels of IL-1 beta mRNA increased during adherence whereas those of I kappa B alpha/MAD-3 mRNA did not. Taken together, our data suggest that two interactive mechanisms regulate monocytic I kappa B alpha/MAD-3 mRNA levels. We propose that adherent monocytes regulate nuclear processing (or decay) of I kappa B alpha/MAD-3 mRNA, thereby increasing mRNA levels without stimulating I kappa B alpha/MAD-3 gene transcription. Moreover, since inhibition of protein synthesis leads to accumulation of I kappa B alpha/MAD-3 mRNA without stimulating I kappa B alpha/MAD-3 gene transcription, we suggest that low cytoplasmic levels of I kappa B alpha/MAD-3 mRNA are maintained by a translation-dependent degradation mechanism. 


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Cloning and characterization of the beta subunit of human proximal sequence element-binding transcription factor and its involvement in transcription of small nuclear RNA genes by RNA polymerases II and III. 
The proximal sequence element (PSE)-binding transcription factor (PTF), which binds the PSE of both RNA polymerase II- and RNA polymerase III-transcribed mammalian small nuclear RNA (snRNA) genes, is essential for their transcription. We previously reported the purification of human PTF, a complex of four subunits, and the molecular cloning and characterization of PTF gamma and delta subunits. Here we describe the isolation and expression of a cDNA encoding PTF beta, as well as functional studies using anti-PTF beta antibodies. Native PTF beta, in either protein fractions or a PTF-Oct-1-DNA complex, can be recognized by polyclonal antibodies raised against recombinant PTF beta. Immunodepletion studies show that PTF beta is required for transcription of both classes of snRNA genes in vitro. In addition, immunoprecipitation analyses demonstrate that substantial and similar molar amounts of TATA-binding protein (TBP) and TFIIIB90 can weakly associate with PTF at low salt conditions, but this association is dramatically reduced at high salt concentrations. Along with our previous demonstration of both physical interactions between PTF gamma/PTF delta and TBP and the involvement of TFIIIB90 in the transcription of class III snRNA genes, these results are consistent with the notion that a TBP-containing complex related to TFIIIB is required for the transcription of class III snRNA genes, and acts through weak interaction with the four-subunit PTF. 


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Functional block for 1 alpha,25-dihydroxyvitamin D3-mediated gene regulation in human B lymphocytes. 
Elements necessary for the steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) to induce a biological response include the presence of specific intracellular receptors (vitamin D3 receptors (VDR)) and modulation of gene expression via hormone-activated receptor binding to regulatory regions of target genes. These parameters were examined in normal and Epstein-Barr virus-immortalized human B cells and compared with 1 alpha,25-(OH)2D3-responsive cells of the T and monocytic lineages. Although resting tonsillar B cells did not express VDR mRNA, activation of these cells with interleukin-4 induced VDR in the absence of exogenously supplemented 1 alpha,25-(OH)2D3. As indicators of hormone-mediated gene regulation we analyzed modulation of CD23, a common B cell/monocyte surface antigen, and 24-hydroxylase. 1 alpha,25-(OH)2D3 inhibited CD23 expression in U937 cells, yet failed to modulate CD23 expression in B cells. Furthermore, 1 alpha,25-(OH)2D3 induced 24-hydroxylase mRNA expression and metabolic activity in both U937 cells and lectin-activated T cells, yet failed to induce 24-hydroxylase mRNA or its metabolic activity in B cells. These findings suggest that although human B lymphocytes can express VDR mRNA and protein, they exhibit a functional block for vitamin D-dependent gene regulation. 


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Tandem AP-1-binding sites within the human beta-globin dominant control region function as an inducible enhancer in erythroid cells. 
A powerful enhancer has been mapped to an 18-bp DNA segment located 11 kb 5' to the human epsilon-globin gene within the dominant control or locus-activating region. This enhancer is inducible in K562 human erythroleukemia cells, increasing linked gamma-globin promoter/luciferase gene expression to 170-fold over an enhancerless construct. The enhancer consists of tandem AP-1-binding sites, phased 10 bp apart, which are both required for full activity. DNA-protein binding assays with nuclear extracts from induced cells demonstrate a high molecular weight complex on the enhancer. The formation of this complex also requires both AP-1 sites and correlates with maximal enhancer activity. Induction of the enhancer may have a role in the increase in globin gene transcription that characterizes erythroid maturation. Enhancer activity appears to be mediated by the binding of a complex of proteins from the jun and fos families to tandem AP-1 consensus sequences. 


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Human immunodeficiency virus type 1 Nef protein inhibits NF-kappa B induction in human T cells. 
Human immunodeficiency virus type 1 (HIV-1) can establish a persistent and latent infection in CD4+ T lymphocytes (W.C.Greene, N.Engl.J. Med.324:308-317, 1991; S.M.Schnittman, M.C.Psallidopoulos, H.C. Lane, L.Thompson, M.Baseler, F.Massari, C.H.Fox, N.P.Salzman, and A.S.Fauci, Science 245:305-308, 1989). Production of HIV-1 from latently infected cells requires host cell activation by T-cell mitogens (T.Folks, D.M.Powell, M.M.Lightfoote, S.Benn, M.A. Martin, and A.S.Fauci, Science 231:600-602, 1986; D.Zagury, J. Bernard, R.Leonard, R.Cheynier, M.Feldman, P.S.Sarin, and R.C. Gallo, Science 231:850-853, 1986). This activation is mediated by the host transcription factor NF-kappa B [G.Nabel and D.Baltimore, Nature (London) 326:711-717, 1987]. We report here that the HIV-1-encoded Nef protein inhibits the induction of NF-kappa B DNA-binding activity by T- cell mitogens. However, Nef does not affect the DNA-binding activity of other transcription factors implicated in HIV-1 regulation, including SP-1, USF, URS, and NF-AT. Additionally, Nef inhibits the induction of HIV-1- and interleukin 2-directed gene expression, and the effect on HIV-1 transcription depends on an intact NF-kappa B-binding site. These results indicate that defective recruitment of NF-kappa B may underlie Nef's negative transcriptional effects on the HIV-1 and interleukin 2 promoters. Further evidence suggests that Nef inhibits NF-kappa B induction by interfering with a signal derived from the T-cell receptor complex. 


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Activation of NF-kappa B by phosphatase inhibitors involves the phosphorylation of I kappa B alpha at phosphatase 2A-sensitive sites. 
Activation of NF-kappa B by various cellular stimuli involves the phosphorylation and subsequent degradation of its inhibitor, I kappa B alpha, although the underlying mechanism remains unclear. In the present study, the role of serine/threonine phosphatases in the regulation of I kappa B alpha phosphorylation was investigated. Our studies demonstrate that incubation of human T cells with low concentrations (approximately 1-5 nM) of calyculin A or okadaic acid, potent inhibitors of protein phosphatase type 1 (PP-1) and type 2A (PP-2A), induces the phosphorylation of I kappa B alpha even in the absence of any cellular stimulus. This action of the phosphatase inhibitors, which is associated with the activation of the RelA.p50 NF-kappa B heterodimer, is not affected by agents that block the induction of I kappa B alpha phosphorylation by tumor necrosis factor alpha (TNF-alpha). Furthermore, the phosphorylated I kappa B alpha from calyculin A-treated cells, but not that from TNF-alpha-stimulated cells, is sensitive to PP-2A in vitro, suggesting the existence of fundamental differences in the phosphorylation of I kappa B alpha induced by the two different NF-kappa B inducers. However, induction of I kappa B alpha phosphorylation by both TNF-alpha and the phosphatase inhibitors is associated with the subsequent degradation of I kappa B alpha. We further demonstrate that TNF-alpha- and calyculin A-induced I kappa B alpha degradation exhibits similar but not identical sensitivities to a proteasome inhibitor. Together, these results suggest that phosphorylation of I kappa B alpha, mediated through both the TNF-alpha-inducible and the PP-2A-opposing kinases, may serve to target I kappa B alpha for proteasome-mediated degradation. 


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LPS-Induced NF-kappaB activation and TNF-alpha release in human monocytes are protein tyrosine kinase dependent and protein kinase C independent. 
BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is an important mediator of septic shock. Endotoxin (LPS) signal transduction in human monocytes leads to activation of nuclear factor-kappa B (NF-kappaB) and TNF-alpha release. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-kappaB activation and TNF-alpha production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-kappaB DNA binding and TNF-alpha release in human monocytes. MATERIALS AND METHODS: Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ml) with and without a nonspecific serine/threonine protein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibitor bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-alpha release in culture supernatants was measured by an ELISA. NF-kappaB DNA binding was evaluated by electrophoretic mobility shift assay. RESULTS: LPS increased NF-kappaB DNA binding and TNF-alpha release in human monocytes. Nonspecific protein kinase inhibition inhibited NF-kappaB activation and TNF-alpha release, while specific PKC inhibition with Bis had no effect on LPS-induced NF-kappaB DNA binding or TNF-alpha release. PTK inhibition with Gen attenuated both LPS-induced NF-kappaB DNA binding and TNF-alpha production in human monocytes. Direct activation of PKC with PMA induced both NF-kappaB activation and TNF-alpha production by human monocytes. CONCLUSIONS: These results suggest that LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes are independent of PKC activity. Furthermore, our results provide evidence that PTK plays a role in LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes and thus could be a potential therapeutic target in inflammatory states. Copyright 1999 Academic Press. 


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Functional characterization of novel IL-2 transcriptional inhibitors. 
IL-2-mediated T cell proliferation is a critical early event in the inflammatory process. Formation of the NFAT-1 transcriptional complex on the IL-2 promoter is essential for IL-2 transcription. Using a cell line that is stably transfected with a trimer of the NFAT-1 regulatory element linked to a lac-Z reporter gene, we screened for inhibitors of NFAT-1-mediated beta-galactosidase activity. WIN 61058 and WIN 53071 were identified as microM inhibitors. These compounds also inhibited beta-galactosidase mRNA levels. Similar inhibition of NFAT-1-mediated gene expression was observed in a second cell line, which is stably transfected with NFAT-1 regulatory elements linked to the reporter gene for sCD8. At 10 microM, both compounds inhibited IL-2 mRNA and protein levels in the NFAT-1-linked lac-Z transfectants, and in human lymphocytes. Both compounds inhibited the mixed lymphocyte reaction, and this inhibition was reversed by exogenous IL-2. WIN 53071 inhibited IL-2 production induced in the calcium-dependent PMA and ionomycin pathway. Conversely, calcium-independent anti-CD28 Ab and PMA-induced IL-2 production was resistant. Both compounds altered the NFAT-1 transcriptional complex, causing its retarded mobility on gels. By these functional criteria, we believe we have identified two structurally distinct, novel inhibitors of NFAT-1-mediated transcription. 


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Inhibition of HIV-1 replication by combination of a novel inhibitor of TNF-alpha with AZT. 
The small molecule S9a was derived from an established tumor necrosis factor-alpha (TNF-alpha) inhibitor (Canventol) by replacement of the isopropylidine group with a phenyl ring. S9a at 10 to 100 nM inhibited HIV production as potently as 3'-azido-3'-deoxythymidine (AZT), an inhibitor of viral reverse transcriptase. Furthermore, S9a and AZT in combination, at noncytoxic concentrations strongly inhibited HIV-1 replication that was more than additive and substantially prolonged the appearance of virus both in acutely infected CD4+ lymphocytes (SupT) in culture and in peripheral blood mononuclear cells (PBMCs) infected with a primary HIV-1 isolate. S9a inhibited TNF-alpha promoter-driven reporter gene activity. It was proposed that the mechanism of antiviral action of S9a was on the host cell, by blocking TNF-alpha transcription via a Tat-induced tar-independent loop, which decreases downstream NF-kappaB activation of HIV-1 long terminal repeat (LTR). S9a was superior to the first generation compound Canventol, which was superior to the natural compound sarcophytol A, demonstrating that further structure-based enhancement of potency of these compounds is feasible. This study suggests a therapeutic approach against AIDS by application of two drugs, one against a cellular and the other a viral target, which may provide an approach to the problem of frequent emergence of resistant variants to combinations of drugs that target only HIV genes. 


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Differentiation of U-937 promonocytic cells by etoposide and ICRF-193, two antitumour DNA topoisomerase II inhibitors with different mechanisms of action. 
We have compared the action on U-937 human promonocytic leukemia cells of two DNA topoisomerase II inhibitors, namely the epipodophyllotoxin etoposide and the bisdioxopiperazine ICRF-193. One hour pulse-treatment with 3 microM etoposide caused topoisomerase associated, primary DNA breakage, which was rapidly followed by apoptosis. By contrast, these effects were not observed upon pulse-treatment with 6 microM ICRF-193. However, continuous treatments with subcytotoxic concentrations of etoposide (0.15 microM) and ICRF-193 (0.3 microM) produced several similar effects, namely decreased cell proliferation, accumulation of cells at G2, increase in cell mass, and induction of differentiation. Under these conditions, etoposide produced a biphasic activation of protein kinase C, which consisted in an early transient activation (from hours 1 to 6) of the membrane-bound enzyme followed by a later activation (hour 48) of the total, membrane-bound and cytosolic enzyme. By contrast, ICRF-193 only provoked a late activation (from hours 72 to 96) of the total enzyme. When used at differentiation-inducing concentrations, both topoisomerase inhibitors caused a great stimulation of AP-1 binding activity, with maximum value at hour 12 in etoposide-treated cells and at hour 48 in ICRF-193-treated cells. By contrast, the binding activity of the NF-kappa(B) and EGR-1 transcription factors was little affected. It is concluded that topoisomerase II inhibitors may induce the differentiation of promonocytic cells, independently of their capacity to cause DNA strand breaks. However, there are other effects, such as the early activation of protein kinase C, which are probably derived from the production of primary DNA breakage by some anti-topoisomerase drugs. 


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Oct2 transactivation from a remote enhancer position requires a B-cell-restricted activity. 
Previous cotransfection experiments had demonstrated that ectopic expression of the lymphocyte-specific transcription factor Oct2 could efficiently activate a promoter containing an octamer motif. Oct2 expression was unable to stimulate a multimerized octamer enhancer element in HeLa cells, however. We have tested a variety of Oct2 isoforms generated by alternative splicing for the capability to activate an octamer enhancer in nonlymphoid cells and a B-cell line. Our analyses show that several Oct2 isoforms can stimulate from a remote position but that this stimulation is restricted to B cells. This result indicates the involvement of either a B-cell-specific cofactor or a specific modification of a cofactor or the Oct2 protein in Oct2-mediated enhancer activation. Mutational analyses indicate that the carboxy-terminal domain of Oct2 is critical for enhancer activation. Moreover, this domain conferred enhancing activity when fused to the Oct1 protein, which by itself was unable to stimulate from a remote position. The glutamine-rich activation domain present in the amino-terminal portion of Oct2 and the POU domain contribute only marginally to the transactivation function from a distal position. 


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Effect of adenovirus 2 on cellular gene activation in blood-derived monocytes and macrophages. 
We have investigated the effect of adenovirus 2 (Ad2) infection on human monocytes and monocyte-derived macrophages with regard to expression of TNF-alpha and IL-1 beta. In monocytes, the virus was bound to the surface without being internalized. On the other hand, Ad2 was internalized by macrophages. No virus replication and no transcription of the Ad2 early genes was observed in either of the cells. Ad2 infection induced transient increase in the mRNA levels for TNF-alpha and IL-1 beta in both monocytes and in macrophages, although the kinetics of the transcription was slightly different. The production of both cytokines, measured by ELISA tests, was enhanced in monocytes. In macrophages, a slight enhancement of TNF-alpha production was seen, whereas IL-1 beta was not detected. The data indicate that cellular genes might be activated by Ad2 virus infection in nonpermissive cells where no viral gene products could be detected. 


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Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity. 
We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (MAD-3) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the cdc10/ankyrin motif, which is 60% similar (46% identical) to the ankyrin repeat region of the precursor of NF-kappa B/KBF1 p50. The C-terminus has a putative protein kinase C phosphorylation site. In vitro translated MAD-3 protein was found to specifically inhibit the DNA-binding activity of the p50/p65 NF-kappa B complex but not that of the p50/p50 KBF1 factor or of other DNA-binding proteins. The MAD-3 cDNA encodes an I kappa B-like protein that is likely to be involved in regulation of transcriptional responses to NF-kappa B, including adhesion-dependent pathways of monocyte activation. 


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Inhibition of NF-kappa B activation in human T-cell lines by anetholdithiolthione. 
Nuclear factor (NF)-kappa B is a redox sensitive cytosolic transcription factor. Redox regulation of NF-kappa B has been implicated in the activation of the human immuno-deficiency virus (HIV). Therefore, inhibition of NF-kappa B activation may be an effective strategy for acquired immunodeficiency syndrome therapy. Anetholdithiolthione (ADT, 5-[p-methoxyphenyl]-3H-1,2-dithiol-3-thione) is an antioxidant which has been used to protect against acetaminophen- and CCl4-induced hepatotoxicity, lipid peroxidation, radiation injury, and also has been used clinically as an anti-choleretic agent. The present study examined the effect of ADT pretreatment on NF-kappa B activation in response to a variety of stimuli such as H2O2, phorbol myristate acetate (PMA) or tumor necrosis factor alpha (TNF alpha). PMA and TNF alpha induced activation of (NF)-kappa B in human Jurkat T-cells was partially inhibited by ADT (0.1 mM) pretreatment. ADT (0.1 mM) also inhibited H2O2 induced activation of the transcription factor in the peroxide sensitive human Wurzburg T-cells. Furthermore, ADT treated Wurzburg cells had significantly higher glutathione levels as compared with untreated cells. H2O2 induced lipid peroxidation in Wurzburg cells was remarkably inhibited by ADT pretreatment. ADT, a pro-glutathione antioxidant, was observed to be capable of modulating NF-kappa B activation. 


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Thrombopoietin supports in vitro erythroid differentiation via its specific receptor c-Mpl in a human leukemia cell line. 
Thrombopoietin (TPO) acts on megakaryopoiesis and erythropoiesis in vitro and in vivo. We isolated a novel subline, UT-7/GMT, from the human leukemia cell line UT-7/GM (N. Komatsu, et al., Blood, 89: 4021-4033, 1997). A small population of UT-7/GM cells positively stained for hemoglobin (Hb) after a 7-day exposure to TPO. More than 50% of TPO-treated UT-7/GMT cells positively stained for Hb. Using UT-7/GMT cells, we examined how TPO promotes hemoglobinization. TPO induced tyrosine phosphorylation of the TPO receptor but not the erythropoietin (EPO) receptor. There was no competition between TPO and EPO for binding to EPO receptor. These findings suggest that TPO has a direct effect on hemoglobinization via a specific receptor on UT-7/GMT cells. Isoelectric focusing demonstrated that TPO induced fetal and adult Hb synthesis, whereas EPO induced embryonic, fetal, and adult Hb synthesis. Thus, our data suggest that TPO has a distinct action on erythropoiesis. 


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Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for virus infectivity. 
When transcriptionally active, the human immunodeficiency virus (HIV) promoter contains a nucleosome-free region encompassing both the promoter/enhancer region and a large region (255 nucleotides [nt]) downstream of the transcription start site. We have previously identified new binding sites for transcription factors downstream of the transcription start site (nt 465 to 720): three AP-1 sites (I, II, and III), an AP3-like motif (AP3-L), a downstream binding factor (DBF) site, and juxtaposed Sp1 sites. Here, we show that the DBF site is an interferon-responsive factor (IRF) binding site and that the AP3-L motif binds the T-cell-specific factor NF-AT. Mutations that abolish the binding of each factor to its cognate site are introduced in an infectious HIV-1 molecular clone to study their effect on HIV-1 transcription and replication. Individual mutation of the DBF or AP3-L site as well as the double mutation AP-1(III)/AP3-L did not affect HIV-1 replication compared to that of the wild-type virus. In contrast, proviruses carrying mutations in the Sp1 sites were totally defective in terms of replication. Virus production occurred with slightly delayed kinetics for viruses containing combined mutations in the AP-1(III), AP3-L, and DBF sites and in the AP3-L and DBF-sites, whereas viruses mutated in the AP-1(I,II,III) and AP3-L sites and in the AP-1(I,II,III), AP3-L, and DBF sites exhibited a severely defective replicative phenotype. No RNA-packaging defect could be measured for any of the mutant viruses as determined by quantification of their HIV genomic RNA. Measurement of the transcriptional activity of the HIV-1 promoter after transient transfection of the HIV-1 provirus DNA or of long terminal repeat-luciferase constructs showed a positive correlation between the transcriptional and the replication defects for most mutants. 


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High-level replication of human immunodeficiency virus in thymocytes requires NF-kappaB activation through interaction with thymic epithelial cells. 
We have previously demonstrated that interaction of infected thymocytes with autologous thymic epithelial cells (TEC) is a prerequisite for a high level of human immunodeficiency virus type 1 (HIV-1) replication in thymocytes (M.Rothe, L.Chene, M.Nugeyre, F.Barre-Sinoussi, and N.Israel, J.Virol.72:5852-5861, 1998). We report here that this activation of HIV replication takes place at the transcriptional level through activation of the Rel/NF-kappaB transcription factors. We first demonstrate that an HIV-1 provirus (SF-2 strain) very effectively replicates in thymocytes cocultured with TEC whereas this provirus, with kappaB sites deleted, fails to replicate. We provide evidence that several NF-kappaB complexes are constitutively found in the nuclei of thymocytes either freshly isolated from the thymus or maintained in coculture with autologous or heterologous TEC. The prevalent complex is the heterodimer p50-p65. NF-kappaB activity is tightly correlated with the transcriptional activity of a long terminal repeat (LTR) of HIV-1 transfected in thymocytes. The cotransfection of this LTR with a mutated IkappaBalpha molecule formally demonstrates that LTR transactivation is regulated by members of the Rel/NF-kappaB family in thymocytes. We also showed that tumor necrosis factor (TNF) and to a lesser extent interleukin-1 (IL-1), secreted within the coculture, induce NF-kappaB activity and a correlative LTR transactivation. However IL-7, a crucial factor for thymopoiesis that is secreted mainly by TEC, is a necessary cofactor for NF-kappaB activation elicited by TNF or IL-1. Together, these data indicate that NF-kappaB activation, required for a high level of HIV replication in thymocytes, is regulated in a specific manner in the thymic microenvironment which provides the necessary cytokines: TNF, IL-1, and IL-7. 


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Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. 
Apoptosis is implicated in the generation and resolution of inflammation in response to bacterial pathogens. All bacterial pathogens produce lipoproteins (BLPs), which trigger the innate immune response. BLPs were found to induce apoptosis in THP-1 monocytic cells through human Toll-like receptor-2 (hTLR2). BLPs also initiated apoptosis in an epithelial cell line transfected with hTLR2. In addition, BLPs stimulated nuclear factor-kappaB, a transcriptional activator of multiple host defense genes, and activated the respiratory burst through hTLR2. Thus, hTLR2 is a molecular link between microbial products, apoptosis, and host defense mechanisms. 


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A lymphoid cell-specific nuclear factor containing c-Rel-like proteins preferentially interacts with interleukin-6 kappa B-related motifs whose activities are repressed in lymphoid cells. 
The proto-oncoprotein c-Rel is a member of the nuclear factor kappa B transcription factor family, which includes the p50 and p65 subunits of nuclear factor kappa B. We show here that c-Rel binds to kappa B sites as homodimers as well as heterodimers with p50. These homodimers and heterodimers show distinct DNA-binding specificities and affinities for various kappa B motifs. In particular, the c-Rel homodimer has a high affinity for interleukin-6 (IL-6) and beta interferon kappa B sites. In spite of its association with p50 in vitro, however, we found a lymphoid cell-specific nuclear factor in vivo that contains c-Rel but not p50 epitopes; this factor, termed IL-6 kappa B binding factor II, appears to contain the c-Rel homodimer and preferentially recognizes several IL-6 kappa B-related kappa B motifs. Although it has been previously shown that the IL-6 kappa B motif functions as a potent IL-1/tumor necrosis factor-responsive element in nonlymphoid cells, its activity was found to be repressed in lymphoid cells such as a Jurkat T-cell line. We also present evidence that IL-6 kappa B binding factor II functions as a repressor specific for IL-6 kappa B-related kappa B motifs in lymphoid cells. 


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Monocyte arrest and transmigration on inflamed endothelium in shear flow is inhibited by adenovirus-mediated gene transfer of IkappaB-alpha. 
Mobilization of nuclear factor-kappaB (NF-kappaB) activates transcription of genes encoding endothelial adhesion molecules and chemokines that contribute to monocyte infiltration critical in atherogenesis. Inhibition of NF-kappaB has been achieved by pharmacological and genetic approaches; however, monocyte interactions with activated endothelium in shear flow following gene transfer of the NF-kappaB inhibitor IkappaB-alpha have not been studied. We found that overexpression of IkappaB-alpha in endothelial cells using a recombinant adenovirus prevented tumor necrosis factor-alpha (TNF-alpha)-induced degradation of IkappaB-alpha and suppressed the upregulation of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin mRNA and surface protein expression and the upregulation of transcripts for the chemokines monocyte chemoattractant protein 1 (MCP-1) and growth-related activity-alpha (GRO-alpha) by TNF-alpha. This was associated with a reduction in endothelial MCP-1 secretion and GRO-alpha immobilization. Adhesion assays under physiological shear flow conditions showed that firm arrest, spreading, and transmigration of monocytes on TNF-alpha-activated endothelium was markedly inhibited by IkappaB-alpha overexpression. Inhibition with monoclonal antibodies and peptide antagonists inferred that this was due to reduced expression of Ig integrin ligand as well as of chemokines specifically involved in these events. In contrast, rolling of monocytes was increased by IkappaB-alpha transfer and was partly mediated by P-selectin; however, it appeared to be unaffected by the inhibition of E-selectin induction. Thus, our data provide novel evidence that selective modulation of NF-kappaB by adenoviral transfer of IkappaB-alpha impairs the expression of multiple endothelial gene products required for subsequent monocyte arrest and emigration in shear flow and thus for monocyte infiltration in atherosclerotic plaques. 


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The hematopoietic transcription factor PU.1 is downregulated in human multiple myeloma cell lines. 
PU.1 is a hematopoietic transcription factor belonging to the Ets-family. It is identical to the Spi-1 oncogene, which is implicated in spleen focus-forming virus-induced murine erythroleukemias. PU.1 seems to be required for early development of multiple hematopoietic lineages, but its expression in mature cells is preferentially observed in cells of the B-cell-and monocyte/macrophage-differentiation lineage. It binds the so-called Pu box, an important tissue-specific regulatory DNA element present in a number of genes expressed in these cell lineages. We have analyzed the expression and activity of PU.1 during human B-cell development using a panel of B-cell lines representing different stages of maturation, from early precursors to differentiated plasma cells. PU.1 mRNA expression and PU.1 DNA binding activity, as measured by Northern blot analysis and electrophoretic mobility shift assay, respectively, were evident in cell lines representing pro-B, pre-B, and mature B cells. We could also show Pu box-dependent transactivation of a reporter gene in transient transfections in these cell lines. In contrast, in a number of multiple myeloma cell lines, representing differentiated, plasma cell-like B cells, PU.1 DNA binding activity, mRNA expression, and Pu box-dependent transactivation were absent or detectable at a very low level. In lymphoblastoid cell lines, which exemplify an intermediate stage of B-cell differentiation, a reduced expression and activity were observed. The findings in the human multiple myeloma cell lines represent the first examples of B cells with downregulated PU.1 expression and apparently contradict observations in the murine system in which PU.1 is expressed and active in plasmacytoma cell lines. At present, it is unclear whether the lack of PU.1 expression and activity in human multiple myeloma cell lines represents a malignancy-associated defect in these cells or exemplifies a normal developmental regulation in terminally differentiated B cells. 


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Expression of v-src in T cells correlates with nuclear expression of NF-kappa B. 
NF-kappa B is a rapidly inducible transcriptional activator that responds to a variety of signals and influences the expression of many genes involved in the immune response. Protein tyrosine kinases transmit signals from cytokine and immune receptors. Very little information exists linking these two important classes of signaling molecules. We now demonstrate that v-src expression correlates with nuclear expression of a kappa B binding complex similar to that induced by phorbol ester and ionomycin, as detected by electrophoretic mobility shift assay using a variety of kappa B sites. This complex was blocked by the tyrosine kinase inhibitor, herbimycin A. The v-src-induced complex comprised the p50 and p65 components of NF-kappa B, as determined by supershift and immunoblot analysis. As a functional correlate of this finding, transient co-transfection of HIV-1 LTR reporter constructs in a different T cell line demonstrated that v-src activated this promoter in a kappa B-dependent manner. We found that transactivation of the HIV-1 LTR by v-src was more sensitive to mutations of the proximal, rather than the distal, kappa B element. The implications for T cell receptor signaling and HIV-1 gene expression are considered. 


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Comparison of constitutive and inducible transcriptional enhancement mediated by kappa B-related sequences: modulation of activity in B cells by human T-cell leukemia virus type I tax gene. 
The kappa B sequence (GGGACTTTCC) binds a factor, NF-kappa B, that is constitutively found in its functional, DNA binding form only in B lymphocytes. A factor with apparently indistinguishable sequence specificity can be induced in many other cell types, where it is used to regulate inducible gene expression. For example, kappa B-related sequences have been shown to be important for the transcription of a few inducible genes, such as the interleukin 2 receptor alpha-chain gene and the beta-interferon gene. However, these genes are not constitutively active in B lymphocytes, suggesting that other regulatory mechanisms must play a role in determining the patterns of expression. We have investigated the constitutive and inducible transcriptional activity mediated by five kappa B-related sequence elements in two different cell types. We show that in S194 plasma cells the activity of each element correlates well with the relative affinity of B-cell-derived NF-kappa B for that element. This leads to significantly lower transcription enhancement by sites derived from the interleukin 2 receptor or T-cell receptor genes in S194 cells. However, in either EL-4 (T) cells or S194 cells, both lower-affinity sites can be significantly induced by the tax gene product of human T-cell leukemia virus type I, showing that NF-kappa B activity can be modulated even in a B-cell line that constitutively expresses this factor. 


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Cell-specific differences in activation of NF-kappa B regulatory elements of human immunodeficiency virus and beta interferon promoters by tumor necrosis factor. 
Three aspects of the involvement of tumor necrosis factor in human immunodeficiency virus (HIV) pathogenesis were examined. Tumor necrosis factor alpha (TNF-alpha) mRNA production was analyzed by polymerase chain reaction amplification in monocytic U937 cells and in a chronically HIV infected U937 cell line (U9-IIIB). TNF-alpha RNA was undetectable in U937 cells, whereas a low constitutive level was detected in U9-IIIB cells. Paramyxovirus infection induced a 5- to 10-fold increase in the steady-state level of TNF-alpha RNA in U9-IIIB cells compared with U937 cells, suggesting that HIV-infected monocytic cells produced higher levels of TNF-alpha than did normal cells after a secondary virus infection. The effects of TNF-alpha on gene expression were examined by transient expression assays using reporter chloramphenicol acetyltransferase plasmids linked to regulatory elements from the HIV long terminal repeat (LTR) and the beta interferon promoter. In U937 and Jurkat T lymphoid cells, the inducibility of the different hybrid promoters by TNF-alpha or phorbol ester varied in a cell type- and promoter context-specific manner; the levels of gene activity of NF-kappa B-containing plasmids correlated directly with induction of NF-kappa B DNA-binding activity. Although the intact beta interferon promoter was only weakly stimulated by phorbol ester or TNF-alpha, multimers of the PRDII NF-kappa B-binding domain were inducible by both agents. TNF-alpha was able to increase expression of the HIV LTR in T cells, but in monocytic cells, TNF-alpha did not induce the HIV LTR above a constitutive level of activity. This level of NF-kappa B-independent activity appears to be sufficient for virus multiplication, since TNF-alpha treatment had no effect on the kinetics of de novo HIV type 1 (HIV-1) infection and viral RNA production in U937 cells. However, in Jurkat cells, TNF-alpha dramatically enhanced the spread of HIV-1 through the cell population and increased viral RNA synthesis, indicating that in T cells HIV-1 multiplication was stimulated by TNF-alpha treatment. 


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Polymorphic nucleotides within the human IL-4 promoter that mediate overexpression of the gene. 
Atopy, which predisposes individuals to develop asthma, severe systemic anaphylaxis, and atopic dermatitis, is usually associated with dramatically elevated total serum IgE levels and is thought to be controlled by a major susceptibility gene and multiple minor susceptibility genes. A recent sib-pair analysis revealed a tight linkage between markers on 5q31.1 and a major susceptibility gene controlling total serum IgE levels. Due to its location within this cluster and its biologic role in Ig class switching and Th2 cell differentiation, the IL-4 gene has emerged as one major candidate for the atopy gene. In one model, polymorphisms within IL-4 regulatory elements might result in overexpression of the gene, amplifying Th2 cell differentiation and class switching to IgE. In support of this model, we report that the human IL-4 promoter exists in multiple allelic forms that exhibit distinct transcriptional activities in IL-4-positive T cells. A particular allele has an unusually high transcriptional activity. A nucleotide substitution within a recently described OAP40 element located just upstream of an NF-AT site (P sequence) appears to be largely responsible for the increased promotor strength of this particular allelic form of the IL-4 promoter. In EMSAs, this substitution results in a markedly enhanced affinity for sequence-specific complexes exhibiting an AP-1 specificity. The identification of allelic nucleotides, which results in overexpression of the IL-4 gene, provides specific targets for a comprehensive screening of atopic and nonatopic individuals and may provide a clue for genetic predisposition for atopy. 


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Comparative analysis of NFAT (nuclear factor of activated T cells) complex in human T and B lymphocytes. 
Nuclear factor of activated T cells (NFAT) is a transcriptional activator that binds to sequences in the interleukin-2 (IL-2) promoter and is thought to be largely responsible for the T cell-specific inducibility of IL-2 expression. Electrophoretic mobility shift assays (EMSA) showed that specific NFAT binding activity could also be induced in human B cells. The B cell NFAT complex, however, was not functional, since it failed to activate transcription from an NFAT-driven chloramphenicol acetyltransferase (CAT) construct. Competition with an AP-1 motif or with anti-Jun and anti-Fos antibodies abolished binding to the NFAT motif in both T and B cells, indicating that Jun and Fos are critical for NFAT complex formation in both cell types. Purified recombinant Jun and Fos proteins failed to bind directly to the NFAT motif. However, when combined with unstimulated B or T cell extracts, full-length, but not truncated, Jun/Fos heterodimers were able to form an NFAT complex, indicating the presence of a constitutively expressed nuclear factor(s) in B and T cells necessary for the formation of the NFAT complex in both cell types. An NFAT oligonucleotide carrying mutations in the 5' purine-rich part of the NFAT sequence failed to form a complex and to compete with the wild type motif for NFAT complex formation in both T and B cells. We therefore propose a model whereby a core NFAT complex consisting of Jun, Fos, and a constitutive nuclear factor is formed in both T and B cells, but an additional factor and/or post-translational modification of a factor, missing in B cells, might be required for transactivation by NFAT. 


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Dimethyldithiocarbamate inhibits in vitro activation of primary human CD4+ T lymphocytes. 
Dithiocarbamates (DTC), a diverse group of industrial and therapeutic chemicals, have been reported to inhibit, enhance or have no effect on the immune system. These apparent inconsistencies reflect the complexity of the DTCs biological activities and are probably due in part to differences in dose, route of exposure, animal species used and/or specific compound tested. The studies described herein were undertaken to investigate the immunotoxicity of one member of this family, dimethyldithiocarbamate (DMDTC). We demonstrate that 0.1-0.5 microM DMDTC inhibits TNF-alpha-induced activation of NF-kappaB in primary human CD4+ T cells. This inhibition is not accompanied by a loss in viability, and DMDTC-treated T cells retain other active signaling pathways throughout the exposure duration. The inhibition of NF-kappaB is apparently permanent as DMDTC-treated T cells did not regain normal TNF-alpha activation, even after 72 h in culture. DMDTC does not appear to alter NF-kappaB directly as pre-incubation of nuclear extracts with DMDTC does not diminish binding activity of this protein. We further demonstrate that 0.1-0.5 microM DMDTC inhibits intracellular IL-2 production and decreases surface expression of CD25 (the alpha subunit of the IL-2 receptor) in T cells stimulated with phorbol ester. These data demonstrate that DMDTC is a potent immunosuppressive compound in vitro. 


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Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. 
cDNA microarrays and a clustering algorithm were used to identify patterns of gene expression in human mammary epithelial cells growing in culture and in primary human breast tumors. Clusters of coexpressed genes identified through manipulations of mammary epithelial cells in vitro also showed consistent patterns of variation in expression among breast tumor samples. By using immunohistochemistry with antibodies against proteins encoded by a particular gene in a cluster, the identity of the cell type within the tumor specimen that contributed the observed gene expression pattern could be determined. Clusters of genes with coherent expression patterns in cultured cells and in the breast tumors samples could be related to specific features of biological variation among the samples. Two such clusters were found to have patterns that correlated with variation in cell proliferation rates and with activation of the IFN-regulated signal transduction pathway, respectively. Clusters of genes expressed by stromal cells and lymphocytes in the breast tumors also were identified in this analysis. These results support the feasibility and usefulness of this systematic approach to studying variation in gene expression patterns in human cancers as a means to dissect and classify solid tumors. 


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Nuclear levels of NF-kappaB correlate with syncytium-forming capacity of 8e51 cells, expressing a defective HIV virus. 
The double NF-kappaB site identified in the LTR of the human immunodeficiency virus-1 (HIV-1) has been demonstrated to be necessary for efficient viral transcription. In this report we present the characterisation of NF-kappaB subunits engaged in complexes binding to the HIV-1 NF-kappaB site in human 8e51 T-cells, that harbour a defective HIV-1. At least four different specific NF-kappaB complexes are present in the nucleus of these cells. With the use of specific antibodies we have determined the composition of each complex using electrophoretic mobility shift assays. The results show the presence of several NF-kappaB family members, with the transactivating RelA being engaged in multiple complexes. The importance of NF-kappaB complexes in viral functions has been established comparing the level of NF-kappaB DNA-binding complexes with syncytia-forming activity of 8e51 cells. In fact, 8e51 cells that had almost lost their syncytia-forming capacity were found to contain at least 10 times less active NF-kappaB DNA-binding complex than the actively fusing cells. The correlation is specific as the level of at least three other transcription factors did not change. 


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DNA triplex formation selectively inhibits granulocyte-macrophage colony-stimulating factor gene expression in human T cells. 
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hemopoietic growth factor that is expressed in activated T cells, fibroblasts, macrophages, and endothelial cells. Although GM-CSF does not appear to be essential for normal hemopoiesis, overexpression of GM-CSF has been implicated in the pathogenesis of some diseases such as myeloid leukemia and chronic inflammation. An NF-kappaB/Rel binding site within the GM-CSF promoter, termed the kappaB element appears to be important for controlling expression in reporter gene assays in response to a number of stimuli in T cells. We investigated oligonucleotide-directed triple helix formation across this regulatory sequence as a potential tool to inhibit GM-CSF gene transcription. A 15-base oligonucleotide, GM3, was targeted to a purine-rich region in the GM-CSF proximal promoter, which overlaps the kappaB element. Gel mobility shift assays and DNase I footprinting demonstrated that GM3 formed a sequence-specific collinear triplex with its double-stranded DNA target. Triplex formation by GM3 blocked recombinant and nuclear NF-kappaB proteins binding to the GM-CSF element. GM3 also caused selective inhibition of the human T-cell lymphotrophic virus-1 Tax transactivator-induced luciferase activity from a reporter construct driven by the GM-CSF promoter in Jurkat T cells. Finally, GM3 greatly reduced the concentration of endogenous GM-CSF mRNA induced by different stimuli in Jurkat T cells but did not affect interleukin 3 mRNA levels in the same cells. We conclude that the kappaB element in the GM-CSF promoter plays a central role in the transcriptional activation of the endogenous GM-CSF gene. Colinear triplex formation acts as a selective transcriptional repressor of the GM-CSF gene and may have potential therapeutic application in cases of undesirable overexpression of this protein. 


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Association between expression of intercellular adhesion molecule-1 and integration of human T-cell-leukemia virus type 1 in adult T-cell leukemia cells. 
It is known that the expression levels of intercellular adhesion molecule-1 (ICAM-1) in adult T cell leukemia(ATL) cells are high, whereas those in T-lymphoid cells are not. In order to investigate the factors that influence the induction of ICAM-1 molecules, Northern blot analysis to measure the expression level of ICAM-1 mRNAs and Southern blot hybridization to analyze the integration of human T-cell-leukemia virus type 1 (HTLV-1) provirus were done. The levels of ICAM-1 mRNA expression of ATL cells were generally higher than those of T-lymphoid cells. However, ILT-mat cells and ATL16T(-) cells, although they were ATL cells, showed rather low surface ICAM-1 expression and ICAM-1 mRNA expression. Southern blot hybridization showed that only two and four bands were found in ILT-mat and ATL16T(-) cells, respectively, whereas > 10 bands were detected in other ATL cells. These results suggest that monoclonal integration of HTLV-1 provirus to the genome of T cell, especially the number of integration sites, is one of the factors for induction of ICAM-1 molecules. 


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Induction of nuclear factor kappa B/Rel nuclear activity in human peripheral blood T lymphocytes by anti-HLA class I monoclonal antibodies. 
Monoclonal antibodies against either monomorphic or polymorphic determinants of class I antigen induced in PBMC and highly purified T lymphocytes the nuclear activity of NF-kappa B/Rel complexes. These included both p50/p50 and p50/p65 dimers, recognized by specific antibodies in EMSA. The induced complexes were detectable in extracts of cells incubated with anti-class I monoclonal antibody (mAb) for 1.5 h; the induction was maximal at 5 h, persistent at 16 h and no longer observed at 40 h. The mAb failed to induce NF-kappa B/Rel nuclear activity in cells incubated in the presence of 3,4-dichloroisocoumarin, an inhibitor of I kappa B-alpha degradation. Together, these results suggest that class I triggering can induce the activity of NF-kappa B/Rel nuclear activity in peripheral blood T lymphocytes, thereby modulating the expression of genes regulated by these transcription factors. 


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IL-12-induced activation of NK and T cells occurs in the absence of immediate-early activation gene expression. 
The responses of lymphocytes to IL-2 and IL-12, involving proliferation, differentiation, and cytokine production, are only partially overlapping, and may depend on induced differential expression of specific sets of genes. Using reverse-transcription PCR differential display, we isolated an mRNA species expressed in IL-2- but not IL-12-stimulated NK cells. This was identified as the mRNA encoding the transcription factor egr-1, which is expressed with fast kinetics in T and NK cells upon IL-2, but not IL-12, stimulation. Analysis of the accumulation of mRNA-encoding members of the AP-1 transcription factor family demonstrated that c-fos and junB are also expressed upon stimulation of NK and T cells with IL-2, but not IL-12, whereas expression of c-jun and junD is not modified by either cytokine. Accordingly, increased AP-1 DNA-binding activity and AP-1-dependent transcriptional activity were detected exclusively in IL-2-stimulated cells. Analysis of the expression of genes reported to regulate cytokine-induced proliferation demonstrated that both IL-2 and IL-12 induce c-myc mRNA accumulation in NK and T cells, whereas only IL-2 induces bcl-2 expression. Our data provide the first demonstration that IL-12-mediated activation of T and NK cells does not involve expression of members of the immediate-early activation genes family (egr-1, c-fos, and junB), AP-1 transcriptional activity, or bcl-2 expression. This indicates that functional differences observed in IL-2- and IL-12-stimulated cells may depend, at least in part, on differential gene regulation. 


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Regulation of gene expression at early stages of B-cell and T-cell differentiation. 
The expression of distinct sets of genes at different stages of B-lymphocyte and T-lymphocyte differentiation is controlled at the level of transcription. A number of recent studies have described interactions between transcription factors in lymphocytes that provide new insights into mechanisms regulating gene expression. These mechanisms include the assembly of higher order nucleoprotein complexes and other protein-protein interactions that enhance the functional specificity of transcriptional regulators in lymphocytes. 


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Oleic acid inhibits endothelial activation : A direct vascular antiatherogenic mechanism of a nutritional component in the mediterranean diet. 
Because oleic acid is implicated in the antiatherogenic effects attributed to the Mediterranean diet, we investigated whether this fatty acid can modulate endothelial activation, ie, the concerted expression of gene products involved in leukocyte recruitment and early atherogenesis. We incubated sodium oleate with human umbilical vein endothelial cells for 0 to 72 hours, followed by coincubation of oleate with human recombinant tumor necrosis factor, interleukin (IL)-1alpha, IL-1beta, IL-4, Escherichia coli lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate for a further 6 to 24 hours. The endothelial expression of vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and intercellular adhesion molecule-1 was monitored by cell surface enzyme immunoassays or flow cytometry, and steady-state levels of VCAM-1 mRNA were assessed by Northern blot analysis. At 10 to 100 micromol/L for >24 hours, oleate inhibited the expression of all adhesion molecules tested. After a 72-hour incubation with oleate and a further 16-hour incubation with oleate plus 1 microg/mL LPS, VCAM-1 expression was reduced by >40% compared with control. Adhesion of monocytoid U937 cells to LPS-treated endothelial cells was reduced concomitantly. Oleate also produced a quantitatively similar reduction of VCAM-1 mRNA levels on Northern blot analysis and inhibited nuclear factor-kappaB activation on electrophoretic mobility shift assays. Incubation of endothelial cells with oleate for 72 hours decreased the relative proportions of saturated (palmitic and stearic) acids in total cell lipids and increased the proportions of oleate in total cell lipids without significantly changing the relative proportions of polyunsaturated fatty acids. Although less potent than polyunsaturated fatty acids in inhibiting endothelial activation, oleic acid may contribute to the prevention of atherogenesis through selective displacement of saturated fatty acids in cell membrane phospholipids and a consequent modulation of gene expression for molecules involved in monocyte recruitment. 


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Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes. 
We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters. 


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Neuronal (type I) nitric oxide synthase regulates nuclear factor kappaB activity and immunologic (type II) nitric oxide synthase expression. 
Nitric oxide subserves diverse physiologic roles in the nervous system. NO is produced from at least three different NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS, and immunologic NOS (iNOS). We show that nNOS is the predominant isoform constitutively expressed in glia. NO derived from nNOS in glia inhibits the transcription factor nuclear factor kappaB (NF kappaB) as NOS inhibitors enhance basal NF kappaB activation. Pyrrolidine dithiocarbamate (PDTC) is an inhibitor of NF kappaB in most cells; however, we show that PDTC is also a potent scavenger of NO through formation of mononitrosyl iron complexes with PDTC. In Jurkat cells, a human T-cell lymphoma cell line, tumor necrosis factor-alpha (TNF-alpha) induces NF kappaB activation that is inhibited by PDTC. Contrary to the results in Jurkat cells, PDTC did not inhibit tumor necrosis factor-alpha-induced NF kappaB activation in astrocytes; instead PDTC itself induces NF kappaB activation in astrocytes, and this may be related to scavenging of endogenously produced NO by the PDTC iron complex. In astrocytes PDTC also dramatically induces the NF kappaB-dependent enzyme, iNOS, supporting the physiologic relevance of endogenous NO regulation of NF kappaB. NF kappaB activation in glia from mice lacking nNOS responds more rapidly to PDTC compared with astrocytes from wild-type mice. Our data suggest that nNOS in astrocytes regulates NF kappaB activity and iNOS expression, and indicate a novel regulatory role for nNOS in tonically suppressing central nervous system, NF kappaB-regulated genes. 


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Inhibition of HIV-1 latency reactivation by dehydroepiandrosterone (DHEA) and an analog of DHEA. 
The initial infection with human immunodeficiency virus type 1 (HIV-1) in most individuals usually results in the establishment of a latent or chronic infection before eventual progression toward acquired immunodeficiency syndrome. HIV-1 can also establish a latent or persistent infection in some T cell lines that show minimal constitutive virus expression. However, activation of the T cell lines leading to enhanced HIV-1 replication can be induced by antigens, mitogens, and cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 1, and interleukin-2). Various gene products from other viruses (HTLV-1, HSV, EBV, CMV, HBV, and HHV-6) can also enhance HIV-1 long terminal repeat (LTR)-driven reporter gene activity. On the basis of these observations, it has been proposed that reactivation of latent HIV-1 harbored in chronically infected T lymphocytes, monocytes, or macrophages plays an important role in the pathogenesis of AIDS. So far, there are no drugs or therapy available that can provide protection against HIV-1 latency reactivation. ACH-2, derived from a human T cell line (CEM), is chronically infected with HIV-1, with low levels of constitutive virus expression. ACH-2 can be converted to productive infection by stimulation of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), mitogen or cytokines (TNF-alpha), or infection with HSV. Therefore the ACH-2 cell line is a good candidate for studying the effects of drugs on HIV-1 activation. Previously, we have reported that DHEA and synthetic analogs of DHEA can be modest inhibitors of HIV-1 IIIB replication in phytohemagglutinin-stimulated peripheral blood lymphocyte cultures. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Activation of a novel gene in 3q21 and identification of intergenic fusion transcripts with ecotropic viral insertion site I in leukemia. 
We have identified a novel gene, GR6, located within the leukemia breakpoint region of 3q21, that is normally expressed in early fetal development but not in adult peripheral blood. GR6 is activated in the UCSD-AML1 cell line and in a leukemic sample, both of which carry a t(3;3)(q21;q26). In UCSD-AML1, we have also identified fusion transcripts between the ecotropic viral insertion site I (EVI1) gene in 3q26 and GR6 and between EVI1 and Ribophorin I that maps 30 kb telomeric to GR6 in 3q21. All fusions splice the 5' ends of the 3q21 genes into exon 2 of the EVI1 gene, an event that is similar to the normal intergenic splicing of MDS1-EVI1 and to those previously documented in leukemias with t(3;21) and t(3;12), in which acute myelogenous leukemia 1-EVI1 fusions and ETV6-EVI1 fusions, respectively, occur. The Ribophorin I-EVI1 fusion in particular may be a common occurrence in t(3;3). 


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Immortalization of CD4(+) and CD8(+) T lymphocytes by human T-cell leukemia virus type 1 Tax mutants expressed in a functional molecular clone. 
The human T-cell leukemia virus type 1 (HTLV-1) transcriptional trans-activator Tax has been demonstrated to have transforming activity in multiple cell culture and transgenic-mouse models. In addition to activating transcription from the viral long terminal repeat (LTR) through the cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors, Tax activates the expression of multiple cellular promoters through the NF-kappaB pathway of transcriptional activation. The Tax mutants M22 and M47 have previously been demonstrated to selectively abrogate the ability of Tax to activate transcription through the NF-kappaB or CREB/ATF pathway, respectively. These mutations were introduced in the tax gene of the ACH functional molecular clone of HTLV-1, and virus produced from the mutant ACH clones was examined for the ability to replicate and immortalize primary human lymphocytes. While virus derived from the clone containing the M47 mutation retained the ability to immortalize T lymphocytes, the M22 mutant lost the ability to immortalize infected cells. These results indicate that activation of the CREB/ATF pathway by Tax is dispensable for the immortalization of T cells by HTLV-1, whereas activation of the NF-kappaB pathway may be critical. 


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Human immunodeficiency virus type 1 long terminal repeat quasispecies differ in basal transcription and nuclear factor recruitment in human glial cells and lymphocytes. 
The generation of genomic diversity during the course of infection has the potential to affect all aspects of HIV-1 replication, including expression of the proviral genome. To gain a better understanding of the impact of long terminal repeat (LTR) sequence diversity on LTR-directed gene expression in cells of the central nervous system (CNS) and immune system, we amplified and cloned LTRs from proviral DNA in HIV-1-infected peripheral blood. Sequence analysis of nineteen LTRs cloned from 2 adult and 3 pediatric patients revealed an average of 33 nucleotide changes (with respect to the sequence of the LAI LTR) within the 455-bp U3 region. Transient expression analyses in cells of neuroglial and lymphocytic origin demonstrated that some of these LTRs had activities which varied significantly from the LAI LTR in U-373 MG cells (an astrocytoma cell line) as well as in Jurkat cells (a CD4-positive lymphocyte cell line). While LTRs which demonstrated the highest activities in U-373 MG cells also yielded high activities in Jurkat cells, the LTRs were generally more active in Jurkat cells when compared to the LAI LTR. Differences in LTR sequence also resulted in differences in transcription factor recruitment to cis-acting sites within the U3 region of the LTR, as demonstrated by electrophoretic mobility shift assays. In particular, naturally occurring sequence variation impacted transcription factor binding to an activating transcription factor/cAMP response element binding (ATF/CREB) binding site (located between the LEF-1 and distal NF-kappaB transcription factor binding sites) that we identified in previous studies of the HIV-1 LTR. These findings suggest that LTR sequence changes can significantly affect basal LTR function and transcription factor recruitment, which may, in turn, alter the course of viral replication in cells of CNS and immune system origin. 


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GATA transcription factors associate with a novel class of nuclear bodies in erythroblasts and megakaryocytes. 
The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co-localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c-Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and -3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain. 


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Inhibition of NF-kappa B by sodium salicylate and aspirin [see comments] 
The transcription factor nuclear factor-kappa B (NF-kappa B) is critical for the inducible expression of multiple cellular and viral genes involved in inflammation and infection including interleukin-1 (IL-1), IL-6, and adhesion molecules. The anti-inflammatory drugs sodium salicylate and aspirin inhibited the activation of NF-kappa B, which further explains the mechanism of action of these drugs. This inhibition prevented the degradation of the NF-kappa B inhibitor, I kappa B, and therefore NF-kappa B was retained in the cytosol. Sodium salicylate and aspirin also inhibited NF-kappa B-dependent transcription from the Ig kappa enhancer and the human immunodeficiency virus (HIV) long terminal repeat (LTR) in transfected T cells. 


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Costimulation of peripheral blood T cell activation by human endothelial cells. Enhanced IL-2 transcription correlates with increased c-fos synthesis and increased Fos content of AP-1. 
Endothelial cells (EC) act as APC for resting PBL in vitro, and may have important roles in vivo in the pathogenesis of allograft rejection and delayed hypersensitivity. We previously reported that human umbilical vein EC provide costimulatory signals to PHA-stimulated PBL via CD2:lymphocyte function-associated Ag-3 and an unidentified ligand pair, resulting in a three- to eight-fold enhancement of IL-2 production. The physiologic relevance of this increase was demonstrated by the proliferative advantage provided by EC to PBL suboptimally stimulated with mAb OKT3. We now report that EC costimulation causes increased levels of IL-2 mRNA as a result of increased IL-2 transcription in PBL. We therefore examined the effects of EC on T cell nuclear factors known to regulate IL-2 transcription, including c-jun and c-fos-two components of the transcription factor AP-1, NFAT, and others. PBL constitutively express c-jun transcripts, and the level of c-jun mRNA is not altered by PHA activation in the absence or presence of EC. In contrast, c-fos mRNA is absent from resting T cells and is induced on PHA activation. EC alone do not induce c-fos mRNA but augment the level of c-fos mRNA in PHA-activated T cells by 3- to 10-fold. This effect is largely independent of the CD2:lymphocyte function-associated Ag-3 pathway. Gel-shift analysis reveals the constitutive presence of nuclear factors in resting PBL that bind to the proximal AP-1 site of the IL-2 promoter and that contain immunoreactive c-Jun but not c-Fos protein. In contrast, AP-1 from PHA-activated cells contains c-Jun and low levels of c-Fos. Strikingly, costimulation with EC results in a dramatic increase (up to 15-fold) in the c-Fos content of AP-1. Levels of other nuclear factors involved in IL-2 regulation were not altered by EC, although NFAT-DNA complexes migrated at a slightly different mobility. In summary, our data suggest that changes in the composition of transcription factor AP-1 is a key molecular mechanism for increasing IL-2 transcription and may underlie the phenomenon of costimulation by EC. 


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c-Rel is a target of pentoxifylline-mediated inhibition of T lymphocyte activation. 
The possible clinical use of the methyl xanthine derivative, pentoxifylline (PF), for the treatment of T cell-dependent diseases is being noted with increasing interest. In this paper, we studied the molecular consequences of PF treatment during lymphocyte activation. We found that in T cells, anti-CD3-induced c-Rel expression was blocked by PF, whereas the induction of other NF-kappaB family members was not significantly affected. However, induction of NF-AT, which has the same signaling requirements as c-Rel induction, was not inhibited by PF. Among genes that respond to these transcription factors, IL-2 mRNA induction was suppressed by PF, whereas IL-2R(alpha) chain mRNA induction was not affected. These observations implicated c-Rel as an IL-2 promoter factor, for which experimental support was obtained from transient transfection experiments. In contrast with the observation in T cells, c-Rel induction was not blocked by PF in B cells. The greater selectivity of PF, compared with FK506, at both the molecular and cellular levels may prove advantageous in manipulating T cell responses in vivo. 


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Calcineurin potentiates activation of the granulocyte-macrophage colony-stimulating factor gene in T cells: involvement of the conserved lymphokine element 0. 
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) are produced by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jurkat cells. The expression of GM-CSF and IL-2 is inhibited by immunosuppressive drugs such as cyclosporin A (CsA) and FK506. Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, can stimulate transcription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for CN A (catalytic) and CN B (regulatory) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lacks the auto-inhibitory and calmodulin-binding domains, acts in synergy with PMA to activate transcription from the GM-CSF promoter. We also found that the active CN partially replaces calcium ionophore in synergy with PMA to induce expression of endogenous GM-CSF and IL-2. By multimerizing the regulatory elements of the GM-CSF promoter, we found that one of the target sites for the CN action is the conserved lymphokine element 0 (CLE0), located at positions between -54 and -40. Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-CLE0 gamma binding is induced by PMA/A23187 and is inhibited by treatment with CsA. These results suggest that CN is involved in the coordinated induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter, which mediates signals downstream of T cell activation. 


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Distinct signaling properties identify functionally different CD4 epitopes. 
The CD4 coreceptor interacts with non-polymorphic regions of major histocompatibility complex class II molecules on antigen-presenting cells and contributes to T cell activation. We have investigated the effect of CD4 triggering on T cell activating signals in a lymphoma model using monoclonal antibodies (mAb) which recognize different CD4 epitopes. We demonstrate that CD4 triggering delivers signals capable of activating the NF-AT transcription factor which is required for interleukin-2 gene expression. Whereas different anti-CD4 mAb or HIV-1 gp120 could all trigger activation of the protein tyrosine kinases p56lck and p59fyn and phosphorylation of the Shc adaptor protein, which mediates signals to Ras, they differed significantly in their ability to activate NF-AT. Lack of full activation of NF-AT could be correlated to a dramatically reduced capacity to induce calcium flux and could be complemented with a calcium ionophore. The results identify functionally distinct epitopes on the CD4 coreceptor involved in activation of the Ras/protein kinase C and calcium pathways. 


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Inhibition of human immunodeficiency virus type 1 replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells. 
We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1-infected cells, HIV-1 LTR was modified. Deletion of the NF-kappa B elements of the HIV-1 LTR significantly inhibited Tat-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the NF-kappa B elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored Tat-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat-expressing cells, while this promoter was not responsive to tumor necrosis factor alpha-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of IFN A (IFNA) constitutively, and their abilities to express interleukin-2 and interleukin-2 receptor upon stimulation with phytohemagglutinin and phorbol myristate acetate were retained. Enhancement of IFNA synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV-1 replication for a period of at least 30 days. Virus isolated from IFNA-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the IFNA gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication. 


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Expression levels of the thyrotropin receptor gene in autoimmune thyroid disease: coregulation with parameters of thyroid function and inverse relation to major histocompatibility complex classes I and II. 
Using a human TSH receptor (TSH-R) cDNA probe, we investigated TSH-R transcript levels in 13 human thyroid fragments by Northern blot analysis; 7 Graves' disease, 2 Hashimoto's disease, 3 endemic goiter, and 1 healthy thyroid gland were studied. TSH-R expression levels were variable, but displayed a close correlation to the expression of thyroid peroxidase (r = 0.703; P < 0.05), thyroglobulin (r = 0.817; P < 0.01), and the nuclear oncogene c-fos (r = 0.935; P < 0.001), but not c-myc. Overall, TSH-R transcript levels were low or absent in those thyroids in which expression of the major histocompatibility complex class I or II (MHC I or II) was high, thus establishing an inverse relation (MHC I, r = -0.791; P < 0.01; MHC II, r = -0.784; P < 0.01). In situ hybridization showed that apart from lymphocytes, thyroid cells themselves were the source of MHC II transcripts. gamma-Interferon expression was only detectable in 1 Hashimoto's goiter. Our findings suggest that next to lymphocyte infiltration, active regulatory events in the thyrocyte are responsible for the inverse relation between functional parameters (TSH-R, thyroid peroxidase, thyroglobulin, and c-fos) and immunological markers (MHC I and II). 


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An in vitro globin gene switching model based on differentiated embryonic stem cells. 
We used mouse embryonic stem (ES) cells to study globin gene expression and switching in vitro. We show that ES-derived embryoid bodies express the full complement of mouse embryonic globin genes in the correct temporal order and that on further differentiation, a switch occurs to the fetal/adult genes. In addition, the erythroid-specific transcription factor NF-E1 was shown to be expressed coordinately with that of globin in embryoid bodies. We conclude from these experiments that the ES cell system provides a good model to study hematopoietic development. When the human epsilon- or beta-globin genes driven by the dominant control region (DCR) are introduced into this system, the human epsilon-globin gene, in contrast to the beta-globin gene, is not deregulated by the presence of the DCR and is expressed strictly as an embryonic gene. We conclude from this that the epsilon-globin gene is not regulated by competition with other genes in the human beta-globin locus. 


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Expression of LAZ3/BCL6 in follicular center (FC) B cells of reactive lymph nodes and FC-derived non-Hodgkin lymphomas. 
Chromosomal translocation resulting in abnormal expression of the LAZ3/BCL6 gene in B cells has been implicated in the tumorigenesis of non-Hodgkin lymphoma (NHL). Therefore we studied the expression pattern of LAZ3/BCL6 by in situ hybridization with synthetic oligonucleotide probes in frozen tissue sections from five reactive lymph nodes and 38 B cell and non-B NHL. In addition, we investigated the expression of LAZ3/BCL6 by Northern blot analysis on multiple human tissues. The LAZ3/BCL6 transcript was found in a variety of tissues, including skeletal muscle, peripheral blood leukocytes, and weakly in normal lymph nodes. In the tumor samples, expression of LAZ3/BCL6 was observed in 68% of all B cell NHL and none of the non-B lymphomas. All cases of follicular, mixed small and large cell lymphomas showed LAZ3/BCL6 expression confined to the neoplastic follicles. A follicular expression pattern was also found in all non-malignant reactive lymph nodes. Hence, the expression of LAZ3/BCL6 does not correlate to malignancy, but reflects the origin of B cells from the germinal centers. 


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Protein kinase C-zeta mediates NF-kappa B activation in human immunodeficiency virus-infected monocytes. 
The molecular mechanisms regulating human immunodeficiency virus (HIV) persistence in a major cell reservoir such as the macrophage remain unknown. NF-kappa B is a transcription factor involved in the regulation of the HIV long terminal repeat and is selectively activated following HIV infection of human macrophages. Although little information as to what signal transduction pathways mediate NF-kappa B activation in monocytes-macrophages is available, our previous work indicated that classical protein kinase C (PKC) isoenzymes were not involved in the HIV-mediated NF-kappa B activation. In this study, we have focused on atypical PKC isoenzymes. PKC-zeta belongs to this family and is known to be an important step in NF-kappa B activation in other cell systems. Immunoblotting experiments with U937 cells demonstrate that PKC-zeta is present in these cells, and its expression can be downmodulated by antisense oligonucleotides (AO). The HIV-mediated NF-kappa B activation is selectively reduced by AO to PKC-zeta. In addition, cotransfection of a negative dominant molecule of PKC-zeta (PKC-zeta mut) with NF-kappa B-dependent reporter genes selectively inhibits the HIV- but not phorbol myristate acetate- or lipopolysaccharide-mediated activation of NF-kappa B. That PKC-zeta is specific in regulating NF-kappa B is concluded from the inability of PKC-zeta(mut) to interfere with the basal or phorbol myristate acetate-inducible CREB- or AP1-dependent transcriptional activity. Lastly, we demonstrate a selective inhibition of p24 production by HIV-infected human macrophages when treated with AO to PKC-zeta. Altogether, these results suggest that atypical PKC isoenzymes, including PKC-zeta, participate in the signal transduction pathways by which HIV infection results in the activation of NF-kappa B in human monocytic cells and macrophages. 


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Regulation of I kappa B alpha and p105 in monocytes and macrophages persistently infected with human immunodeficiency virus. 
The mechanisms regulating human immunodeficiency virus (HIV) persistence in human monocytes/macrophages are partially understood. Persistent HIV infection of U937 monocytic cells results in NF-kappa B activation. Whether virus-induced NF-kappa B activation is a mechanism that favors continuous viral replication in macrophages remains unknown. To further delineate the molecular mechanisms involved in the activation of NF-kappa B in HIV-infected monocytes and macrophages, we have focused on the regulation of the I kappa B molecules. First, we show that persistent HIV infection results in the activation of NF-kappa B not only in monocytic cells but also in macrophages. In HIV-infected cells, I kappa B alpha protein levels are decreased secondary to enhanced protein degradation. This parallels the increased I kappa B alpha synthesis secondary to increased I kappa B alpha gene transcription, i.e., increased RNA and transcriptional activity of its promoter-enhancer. Another protein with I kappa B function, p105, is also modified in HIV-infected cells: p105 and p50 steady-state protein levels are increased as a result of increased synthesis and proteolytic processing of p105. Transcriptional activity of p105 is also increased in infected cells and is also mediated by NF-kappa B through a specific kappa B motif. These results demonstrate the existence of a triple autoregulatory loop in monocytes and macrophages involving HIV, p105 and p50, and MAD3, with the end result of persistent NF-kappa B activation and viral persistence. Furthermore, persistent HIV infection of monocytes and macrophages provides a useful model with which to study concomitant modifications of different I kappa B molecules. 


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Characterization of 5' end of human thromboxane receptor gene. Organizational analysis and mapping of protein kinase C--responsive elements regulating expression in platelets. 
Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells. 


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The effect of Toremifene on the expression of some genes in human mononuclear cells. 
Toremifene exerts multiple and varied effects on the gene expression of human peripheral mononuclear cells. After short-term, in vitro exposure to therapeutical levels, distinct changes in P-glycoprotein, steroid receptors, p53 and Bcl-2 expression take place. In view of the increasing use of antiestrogens in cancer therapy and prevention, there is obvious merit in long-term in vivo studies to be conducted. 


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Role of HIV-1 Nef expression in activation pathways in CD4+ T cells. 
The role of the human immunodeficiency virus (HIV-1) Nef protein in T cell activation pathways was investigated using a Jurkat CD4+ cell line stably transfected with a Nef expression vector. Secretion of IL-2 and TNF-alpha, surface expression of IL-2R, and DNA-binding activity of NF-kappa B and AP-1 (Fos/Jun) complex in response to phorbol myristate acetate, TNF-alpha, or immobilized antibodies to CD3 were monitored. These parameters were not modified by Nef expression in Jurkat cells, whereas stimulation with the same stimuli resulted in partial inhibition of LTR activation in Nef+ Jurkat cells. This inhibition was not mediated through Nef phosphorylation on Thr-15 or GTP-binding activity because mutations in critical sites did not alter this inhibition. Analysis of truncated LTRs confirmed that inhibition of LTR activation was not mediated through NF-kappa B-binding activity but through the region containing the negative responding elements (NREs). These results suggest that Nef downmodulates LTR activation without significantly inhibiting the capacity of T cells to respond to immunological activations. 


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Apoptosis mediated by HIV protease is preceded by cleavage of Bcl-2. 
Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor alpha. We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NF kappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site. 


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Differentiation-dependent expression of a human carboxylesterase in monocytic cells and transcription factor binding to the promoter. 
Carboxylesterases play an important role in defense and clearance mechanisms of the monocyte/macrophage system. During the differentiation process of cells from the monocytic cell line THP-1 we observed a transient transcriptional upregulation of a human carboxylesterase analyzed by means of Northern blots. In PMA-treated THP-1 cells we could detect three major transcription initiation sites as revealed by Nuclease Protection Assay carried out with two overlapping antisense RNA probes. We have recently cloned the carboxylesterase upstream sequence and showed its basal promoter activity in CHO cells. Using electrophoretic mobility shift analysis we demonstrated that the promoter region spanning base pairs -1 to -275, which contains several putative binding sites for transcription factors, is bound by nuclear factors Sp1 and IRBP but not by C/EBPs. Taken together these data indicate that carboxylesterase gene transcription in THP-1 cells starts at multiple initiation sites and that Sp1 and IRBP may be critical factors for modulating the differentiation-dependent transcription of this human carboxylesterase gene. 


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Engagement of natural cytotoxicity programs regulates AP-1 expression in the NKL human NK cell line. 
NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1. 


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Octamer independent activation of transcription from the kappa immunoglobulin germline promoter. 
Previous analyses of immunoglobulin V region promoters has led to the discovery of a common octamer motif which is functionally important in the tissue-specific and developmentally regulated transcriptional activation of immunoglobulin genes. The germline promoters (Ko) located upstream of the J region gene segments of the kappa locus also contain an octamer motif (containing a single base pair mutation and referred to as the variant octamer) which has been shown previously to bind Oct-1 and Oct-2 transcription factors in vitro. To further elucidate the role of this variant octamer motif in the regulation of germline transcription from the unrearranged kappa locus, we have quantitated the relative binding affinity of Oct-1 and Oct-2 for the variant octamer motif and determined the functional role of this octamer motif in transcriptional activation. We find that, although the variant octamer motif binds Oct-1 and Oct-2 in vitro with 5-fold lower affinity than the consensus octamer motif, mutation of the variant octamer motif to either a consensus octamer or non-octamer motif has no effect on transcriptional activation from the germline promoter. We also find significant differences in activation of germline and V region promoters by kappa enhancers. Our results suggest that the germline promoters and V region promoters differ in their dependence on octamer for activation and respond differently to enhancer activation. These findings have important implications in regulation of germline transcription as well as concomitant activation of the V-J recombination of the kappa light chain locus. 


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Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes. 
Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. We have identified two new isoforms of the transcription factor NFAT1 (previously termed NFATp) that are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive agent cyclosporin A (CsA). Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol 12-myristate 13-acetate; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol 12-myristate 13-acetate. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response. 


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A novel immunosuppressive factor in bovine colostrum blocks activation of the interleukin 2 gene enhancer at the NFAT site. 
A factor in bovine colostrum (colostrum inhibitory factor, CIF) inhibits interleukin 2 (IL2) production in activated T helper cells by blocking the accumulation of IL2 mRNA. To determine whether CIF blocks at the level of IL2 transcription, we introduced reporter plasmids into the human T leukemia cell line Jurkat by transient transfection. These contained the luciferase gene under the control of either the human IL2 upstream enhancer region (segments -326 to +45) or three repeats of the NFAT element contained within it (segments -255 to -285). Expression of luciferase in these cells was induced by phorbol myristate acetate plus a calcium ionophore. CIF inhibited induction of either construct as did cyclosporine, which is known to block activation of the NFAT element. CIF failed to inhibit several other enhancer elements. The NFAT-controlled luciferase gene system distinguishes CIF from other T cell inhibitory activities present in colostrum, in particular, TGF beta 1 and TGF beta 2 and the glucocorticoids. Stably transfected Jurkat cells behaved similarly to the transiently transfected ones with respect to inhibition by CIF and cyclosporine. The NFAT-luc assay is a useful technique for the rapid, sensitive measurement of CIF or other immunosuppressants with a similar mode of action. 


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Phenylarsine oxide inhibits ex vivo HIV-1 expression. 
Phenylarsine oxide (PAO), which is described as an inhibitor of tyrosine phosphatase activity, inhibits H2O2 release from human peripheral blood mononuclear cells (PBMCs) as measured by electrochemistry. Since human immunodeficiency virus type 1 (HIV-1) replication is known to be favored under oxidative stress conditions, ex vivo experiments using uninfected PBMCs, primary monocytes or a latently infected promonocytic U1 cell line show that HIV-1 replication and reactivation, monitored by p24 antigen measurement, are inhibited by PAO in a time- and concentration-dependent manner. These observations can be linked with the inhibition of NF-kappa B activation when uninfected monocytes are induced by either tumor necrosis factor alpha (TNF-alpha) phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). 


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Tissue-specific activity of the gammac chain gene promoter depends upon an Ets binding site and is regulated by GA-binding protein. 
The gammac chain is a subunit of multiple cytokine receptors (interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15), the expression of which is restricted to hematopoietic lineages. A defect in gammac leads to the X-linked severe combined immunodeficiency characterized by a block in T cell differentiation. In order to better characterize the human gammac promoter and define the minimal tissue-specific promoter region, progressive 5'-deletion constructs of a segment extending 1053 base pairs upstream of the major transcription start site were generated and tested for promoter activity in various hematopoietic and nonhematopoietic cell types. The -1053/+34 construct allowed promoter activity only in cells of hematopoietic origin, and tissue specificity was conserved in all other constructs tested. The region downstream of -90 appeared critical for basal promoter activity. It contains two potential Ets binding sites conserved in the murine gammac promoter gene, one of which was found essential for functional promoter activity as determined by mutational analysis. The functional Ets binding site was found to bind Ets family proteins, principally GA-binding protein and Elf-1 and could be transactivated by GABPalpha and -beta synergistically. These results indicate that, as already reported for the IL2Rbeta promoter, GA-binding protein is an essential component of gammac basal promoter activity. Although GABP expression is not restricted to the hematopoietic lineage, its interaction with other specific factors may contribute to the tissue-specific expression of the gammac gene. 


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Transcriptional activation of the vascular cell adhesion molecule-1 gene in T lymphocytes expressing human T-cell leukemia virus type 1 Tax protein. 
Recruitment and extravasation of T cells through the blood-brain barrier are favored by adhesion molecule-mediated interactions of circulating T cells with endothelial cells. Since a common pathological finding in human T-cell leukemia virus type 1 (HTLV-1)-associated diseases is the infiltration of HTLV-1-infected T lymphocytes into various organs, we have looked for the profile of adhesion molecules expressed by HTLV-1-transformed T cells. Flow cytometry analysis indicated that these cells were expressing high levels of vascular cell adhesion molecule 1 (VCAM-1 [CD106]), a 110-kDa member of the immunoglobulin gene superfamily, first identified on endothelial cells stimulated with inflammatory cytokines. This adhesion molecule was also expressed by T cells obtained from one patient with HTLV-1-associated myelopathy/tropical spastic paraparesis but not by activated T cells isolated from one normal blood donor. The role of the viral trans-activator Tax protein in the induction of VCAM-1 was first indicated by the detection of this adhesion molecule on Jurkat T-cell clones stably expressing the tax gene. The effect of Tax on VCAM-1 gene transcription was next confirmed in JPX-9 cells, a subclone of Jurkat cells, carrying the tax sequences under the control of an inducible promoter. Furthermore, deletion and mutation analyses of the VCAM-1 promoter performed with chloramphenicol acetyltransferase constructs revealed that Tax was trans activating the VCAM-1 promoter via two NF-kappaB sites present at bp -72 and -57 in the VCAM-1 gene promoter, with both of them being required for the Tax-induced expression of this adhesion molecule. Finally, gel mobility shift assays demonstrated the nuclear translocation of proteins specifically bound to these two NF-kappaB motifs, confirming that VCAM-1 was induced on Tax-expressing cells in a kappaB-dependent manner. Collectively, these results therefore suggest that the exclusive Tax-induced expression of VCAM-1 on T cells may represent a pivotal event in the progression of HTLV-1-associated diseases. 


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Rel/NF-kappa B transcription factors and the control of apoptosis. 
The process of apoptosis is used to eliminate unwanted cells from a wide variety of organisms. Various extracellular signals, often converging in common intracellular pathways, can induce apoptosis in a cell-type-specific fashion. Recent work from several laboratories has demonstrated that Rel/NF-kappa B transcription factors regulate apoptosis in many cell types. In most cells, Rel/NF-kappa B transcription factors appear to mediate survival signals that protect cells from apoptosis; however, under some circumstances, activation of these factors may also promote apoptosis. 


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Reactive oxygen species and antioxidants in inflammatory diseases. 
This paper aims to review the role of free radical-induced tissue damage and antioxidant defence mechanisms in inflammatory diseases that involve pathogenic processes similar to the periodontal diseases. There is a clearly defined and substantial role for free radicals or reactive oxygen species (ROS) in periodontitis, but little research has been performed in this area. This paper reviews the considerable data available relating ROS activity and antioxidant defence to inflammatory diseases and attempts to draw parallels with periodontitis, in an effort to stimulate more periodontal research in this important area. The recent discovery of the transcription factor nuclear factor kappa B (NF-kappa B) is reviewed and several potential pathways for cytokine-induced periodontal tissue damage, mediated by NF-kappa B1 are discussed. Emphasis is placed on cytokines that have been studied in periodontitis, principally TNF-alpha, IL-1, IL-6, IL-8 and beta-interferon. The link between cellular production of such important mediators of inflammation and the antioxidant (AO) thiols, cysteine and reduced glutathione (GSH), is discussed and it is hypothesised that NF-kappa B antagonists may offer important therapeutic benefits. 


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A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes. 
We have isolated a novel cDNA clone encoding interferon (IFN) consensus sequence-binding protein in adult T-cell leukemia cell line or activated T cells (ICSAT); this protein is the human homolog of the recently cloned Pip/LSIRF. ICSAT is structurally most closely related to the previously cloned ICSBP, a member of the IFN regulatory factor (IRF) family of proteins that binds to interferon consensus sequences (ICSs) found in many promoters of the IFN-regulated genes. Among T-cell lines investigated, ICSAT was abundantly expressed in human T-cell leukemia virus type 1 (HTLV-1)-infected T cells. When the HTLV-1 tax gene was expressed or phorbol myristake acetate-A23187 stimulation was used, ICSAT expression was induced in Jurkat cells which otherwise do not express ICSAT. When the binding of ICSAT to four different ICSs was tested, the relative differences in binding affinities for those ICSs were determined. To study the functional role of ICSAT, we performed cotransfection experiments with the human embryonal carcinoma cell line N-Tera2. ICSAT was demonstrated to possess repressive function over the gene activation induced by IFN stimulation or by IRF-1 cotransfection. Such repressive function is similar to that seen in IRF-2 or ICSBP. However, we have found that ICSAT has a different repressive effect from that of IRF-2 or ICSBP in some IFN-responsive reporter constructs. These results suggest that a novel mechanism of gene regulation by "differential repression" is used by multiple members of repressor proteins with different repressive effects on the IFN-responsive genes. 


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Transcriptional regulation during myelopoiesis. 
The coordinated production of all blood cells from a common stem cell is a highly regulated process involving successive stages of commitment and differentiation. From analyses of mice deficient in transcription factor genes and from the characterizations of chromosome breakpoints in human leukemias, it has become evident that transcription factors are important regulators of hematopoiesis. During myelopoiesis, which includes the development of granulocytic and monocytic lineages, transcription factors from several families are active, including AML1/CBF beta, C/EBP, Ets, c-Myb, HOX, and MZF-1. Few of these factors are expressed exclusively in myeloid cells; instead it appears that they cooperatively regulate transcription of myeloid-specific genes. Here we discuss recent advances in transcriptional regulation during myelopoiesis. 


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The role of Rel/NF-kappa B proteins in viral oncogenesis and the regulation of viral transcription. 
Rel/NF-kappa B is a ubiquitous transcription factor that consists of multiple polypeptide subunits, and is subject to complex regulatory mechanisms that involve protein-protein interactions, phosphorylation, ubiquitination, proteolytic degradation, and nucleocytoplasmic translocation. The sophisticated control of Rel/NF-kappa B activity is not surprising since this transcription factor is involved in a wide array of cellular responses to extracellular cues, associated with growth, development, apoptosis, and pathogen invasion. Thus, it is not unexpected that this versatile cellular homeostatic switch would be affected by a variety of viral pathogens, which have evolved mechanisms to utilize various aspects of Rel/NF-kappa B activity to facilitate their replication, cell survival and possibly evasion of immune responses. This review will cover the molecular mechanisms that are utilized by mammalian oncogenic viruses to affect the activity of Rel/NF-kappa B transcription factors and the role of Rel/NF-kappa B in the regulation of viral gene expression and replication. 


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Inhibition of cyclooxygenase-2 expression by 4-trifluoromethyl derivatives of salicylate, triflusal, and its deacetylated metabolite, 2-hydroxy-4-trifluoromethylbenzoic acid. 
The therapeutic potential of drugs that block the induction of cyclooxygenase-2 has been emphasized. When two 4-trifluoromethyl salicylate derivatives [2-acetoxy-4-trifluoromethyl-benzoic acid (triflusal) and its deacetylated metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB)] were compared with aspirin and sodium salicylate as cyclooxygenase-2 (COX-2) inhibitors, we observed that in bacterial lipopolysaccharide-activated human blood, triflusal, aspirin, and HTB, but not sodium salicylate, inhibited COX-2-mediated prostaglandin E2 (PGE2) production (IC50 = 0.16, 0.18, 0.39, and >10 mM, respectively). However, only triflusal and aspirin inhibited purified COX-2 enzyme. To test this apparent discrepancy, we realized that HTB and triflusal (but neither aspirin nor salicylate) produced a concentration-dependent inhibition of COX-2 protein expression in peripheral human mononuclear cells. This observation was further confirmed in a rat air pouch model in vivo, in which both aspirin and triflusal inhibited PGE2 production (ID50 = 18.9 and 11.4 mg/kg p.o., respectively) but only triflusal-treated animals showed a decrease in COX-2 expression. This different behavior may be, at least in part, due to the ability of HTB and triflusal to block the activation of the transcription factor nuclear factor-kappaB to a higher extent than aspirin and sodium salicylate. Thus, in addition to inhibiting the COX-2 activity at therapeutic concentrations, triflusal is able to block through its metabolite HTB the expression of new enzyme, and hence the resumption of PGE2 synthesis. Triflusal and HTB may exert beneficial effects in processes in which de novo COX-2 expression is involved and, in a broader sense, in pathological situations in which genes under nuclear factor-kappaB control are up-regulated. 


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Transcriptional regulation of T lymphocyte development and function. 
The development and function of T lymphocytes are regulated tightly by signal transduction pathways that include specific cell-surface receptors, intracellular signaling molecules, and nuclear transcription factors. Since 1988, several families of functionally important T cell transcription factors have been identified. These include the Ikaros, LKLF, and GATA3 zinc-finger proteins; the Ets, CREB/ATF, and NF-kappa B/Rel/NFAT transcription factors; the Stat proteins; and HMG box transcription factors such as LEF1, TCF1, and Sox4. In this review, we summarize our current understanding of the transcriptional regulation of T cell development and function with particular emphasis on the results of recent gene targeting and transgenic experiments. In addition to increasing our understanding of the molecular pathways that regulate T cell development and function, these results have suggested novel targets for genetic and pharmacological manipulation of T cell immunity. 


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A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site. 
A Rel-related, mitogen-inducible, kappa B-binding protein has been cloned as an immediate-early activation gene of human peripheral blood T cells. The cDNA has an open reading frame of 900 amino acids capable of encoding a 97-kDa protein. This protein is most similar to the 105-kDa precursor polypeptide of p50-NF-kappa B. Like the 105-kDa precursor, it contains an amino-terminal Rel-related domain of about 300 amino acids and a carboxy-terminal domain containing six full cell cycle or ankyrin repeats. In vitro-translated proteins, truncated downstream of the Rel domain and excluding the repeats, bind kappa B sites. We refer to the kappa B-binding, truncated protein as p50B by analogy with p50-NF-kappa B and to the full-length protein as p97. p50B is able to form heteromeric kappa B-binding complexes with RelB, as well as with p65 and p50, the two subunits of NF-kappa B. Transient-transfection experiments in embryonal carcinoma cells demonstrate a functional cooperation between p50B and RelB or p65 in transactivation of a reporter plasmid dependent on a kappa B site. The data imply the existence of a complex family of NF-kappa B-like transcription factors. 


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MEK1 and the extracellular signal-regulated kinases are required for the stimulation of IL-2 gene transcription in T cells. 
TCR engagement stimulates the activation of the protein kinase Raf-1. Active Raf-1 phosphorylates and activates the mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase 1 (MEK1), which in turn phosphorylates and activates the MAP kinases/extracellular signal regulated kinases, ERK1 and ERK2. Raf-1 activity promotes IL-2 production in activated T lymphocytes. Therefore, we sought to determine whether MEK1 and ERK activities also stimulate IL-2 gene transcription. Expression of constitutively active Raf-1 or MEK1 in Jurkat T cells enhanced the stimulation of IL-2 promoter-driven transcription stimulated by a calcium ionophore and PMA, and together with a calcium ionophore the expression of each protein was sufficient to stimulate NF-AT activity. Expression of MEK1-interfering mutants inhibited the stimulation of IL-2 promoter-driven transcription and blocked the ability of constitutively active Ras and Raf-1 to costimulate NF-AT activity with a calcium ionophore. Expression of the MAP kinase-specific phosphatase, MKP-1, which blocks ERK activation, inhibited IL-2 promoter and NF-AT-driven transcription stimulated by a calcium ionophore and PMA, and in addition, MKP-1 neutralized the transcriptional enhancement caused by active Raf-1 and MEK1 expression. We conclude that the MAP kinase signal transduction pathway consisting of Raf-1, MEK1, and ERK1 and ERK2 functions in the stimulation IL-2 gene transcription in activated T lymphocytes. 


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Function and activation of NF-kappa B in the immune system. 
NF-kappa B is a ubiquitous transcription factor. Nevertheless, its properties seem to be most extensively exploited in cells of the immune system. Among these properties are NF-kappa B's rapid posttranslational activation in response to many pathogenic signals, its direct participation in cytoplasmic/nuclear signaling, and its potency to activate transcription of a great variety of genes encoding immunologically relevant proteins. In vertebrates, five distinct DNA binding subunits are currently known which might extensively heterodimerize, thereby forming complexes with distinct transcriptional activity, DNA sequence specificity, and cell type- and cell stage-specific distribution. The activity of DNA binding NF-kappa B dimers is tightly controlled by accessory proteins called I kappa B subunits of which there are also five different species currently known in vertebrates. I kappa B proteins inhibit DNA binding and prevent nuclear uptake of NF-kappa B complexes. An exception is the Bcl-3 protein which in addition can function as a transcription activating subunit in th nucleus. Other I kappa B proteins are rather involved in terminating NF-kappa B's activity in the nucleus. The intracellular events that lead to the inactivation of I kappa B, i.e. the activation of NF-kappa B, are complex. They involve phosphorylation and proteolytic reactions and seem to be controlled by the cells' redox status. Interference with the activation or activity of NF-kappa B may be beneficial in suppressing toxic/septic shock, graft-vs-host reactions, acute inflammatory reactions, acute phase response, and radiation damage. The inhibition of NF-kappa B activation by antioxidants and specific protease inhibitors may provide a pharmacological basis for interfering with these acute processes. 


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A novel genetic system to isolate a dominant negative effector on DNA-binding activity of Oct-2. 
Recent studies have revealed that interactions between transcription factors play an important role in regulation of gene expression in eukaryotic cells. To isolate cDNA clones that dominantly inhibit the DNA-binding activity of Oct-2, chosen as a representative factor, we have developed a novel screening system. This employs an Escherichia coli tester strain carrying a modified lac operon as a reporter gene, with the lac operator sequence replaced by an octamer sequence. Oct-2 expressed in this tester strain represses the expression of the reporter gene and changes the phenotype of the cell from Lac+to Lac-. Introduction of a cDNA expression library prepared from a human T-cell line into the Oct-2-harboring tester strain allowed selection of three Lac+clones out of 1 x 10(5) transformants. One of them, hT86, encoding a putative zinc finger protein was found to derepress beta-galactosidase activity in the Oct-2-harboring tester strain at the transcriptional level. In gel mobility shift assays, hT86 attenuated the intensity of the retarded band composed of the octamer probe and Oct-2, suggesting a dominant negative effect on the DNA-binding activity of Oct-2. The strategy described here provides a new approach for studying protein-protein interactions that govern the complex regulation of gene expression. 


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Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-kappaB. 
The Tpl-2 kinase activates the nuclear factor of activated T cells (NFAT) and induces IL-2 expression in T-cell lines. Here we show that the activation of the IL-2 promoter by Tpl-2 is inhibited by mutant signaling molecules that inhibit the mitogen-activated protein kinase (MAPK) or the calcineurin/NFAT pathways and is promoted by combinations of signaling molecules that activate these pathways. We, therefore, conclude that signals generated by the convergence of the MAPK and the calcineurin/NFAT pathway are necessary and sufficient for the activation of the IL-2 promoter by Tpl-2. The activation of both the IL-2 promoter and an NFAT-driven minimal promoter were shown to depend on signals transduced by Raf1. However, it was only the IL-2 promoter whose activation by Tpl-2 was fully blocked by the dominant negative mutant MEK1S218/222A and the MEK1/MEK2 inhibitor PD098059. Since the activation of NFAT is MAPK-dependent these findings suggested that the activation of MAPK by Tpl-2 is either independent or only partially dependent on MEK1 and MEK2. In addition, they suggested that the activation of the IL-2 promoter is under the control of not only NFAT but also a second factor whose activation is MEK-dependent. Experiments in COS-1 and EL-4 cells confirmed both hypotheses and revealed that the second factor activated by Tpl-2 is NF-kappaB. While the activation of the IL-2 promoter and an NFAT-driven minimal promoter by Tpl-2 was fully blocked by the dominant negative mutant NFAT delta418, it was only partially blocked by the calcineurin inhibitor cyclosporin A suggesting that the Tpl-2-mediated NFAT activation is under the control of a combination of calcineurin-dependent and independent pathways. Both pathways were fully blocked by Bcl-2 or Bcl-X(L). 


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Regulation of cytokine and cytokine receptor expression by glucocorticoids. 
Glucocorticoids (GCS) profoundly inhibit several aspects of T cell immunity largely through inhibition of cytokine expression at the transcriptional and posttranscriptional levels. GCS were also reported to act indirectly by inducing transforming growth factor-beta expression, which in turn blocks T cell immunity. In exerting their antiproliferative effects, GCS diffuse into target cells where they bind their cytoplasmic receptor, which in turn translocates to the nucleus where it inhibits transcription of cytokine genes through direct binding to the glucocorticoid response elements (GRE), which are located in the promoter region of cytokine genes or, alternatively, through antagonism of the action of transcription factors required for optimal transcriptional activation. In contrast to their inhibitory effects on cytokine expression, GCS up-regulate cytokine receptor expression that correlates with enhanced cytokine effects on target cells. In this review, we summarize the current state of knowledge of the mechanism of action of GCS, including the phenomenon of steroid-induced rebound, which ensues upon GCS withdrawal. 


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Selenium-mediated inhibition of transcription factor NF-kappa B and HIV-1 LTR promoter activity. 
The eukaryotic transcription factor NF-kappa B is involved in the inducible expression of various inflammatory genes as well as in HIV-1 replication. Activation of NF-kappa B is induced by prooxidants and several stimuli eliciting oxidative stress, such as cytokines, lipopolysaccharide, UV irradiation and other mediators. Various antioxidants inhibit NF-kappa B activation in response to these stimuli. In this study, we have investigated the effects of selenium, an integral component of glutathione peroxidase (GPX), on NF-kappa B activation. In selenium-deprived Jurkat and ESb-L T lymphocytes, supplementation of selenium led to a substantial increase of GPX activity. Analysis of DNA binding revealed that NF-kappa B activation in response to TNF was significantly inhibited under these conditions. Likewise, reporter gene assays using luciferase constructs driven by the HIV-1 long terminal repeat showed a dose-dependent inhibition of NF-kappa B controlled gene expression by selenium. The effects of selenium were specific for NF-kappa B, since the activity of the transcription factor AP-1 was not suppressed. These data suggest that selenium supplementation may be used to modulate the expression of NF-kappa B target genes and HIV-1. 


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Soluble factors secreted by activated T-lymphocytes modulate the transcription of the immunosuppressive cytokine TGF-beta 2 in glial cells. 
Coordination of the immune response to injury or disease in the brain is postulated to involve bi-directional discourse between the immune system and the central nervous system. This cross communication involves soluble mediators, including various growth factors, cytokines, and neuropeptides. In this report, we demonstrate that the supernatant from activated T-lymphocytes is able to induce the transcription of a potent cytokine, TGF-beta 2 in glial cells. The activating stimulus invokes signaling mechanisms distinct from known kinase or protease pathways. Activation of TGF-beta 2 transcription correlates with the loss of binding activity for an 80 kDA glial labile repressor protein, GLRP, to a responsive region within the TFG-beta 2 promoter. Although GLRP shares some characteristics with the inducible transcription factor AP-1, it appears to be distinct from known AP-1 family members. These data along with previous observations demonstrating the potent immunosuppressive activity of TGF-beta 2, support a model for a feedback mechanism between the activated T-lymphocytes and astrocytes via TGF-beta 2 to regulate the immune response. 


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Distinct DNase-I hypersensitive sites are associated with TAL-1 transcription in erythroid and T-cell lines. 
The tal-1 gene, frequently activated in human T-cell acute lymphoblastic leukemia (T-ALL), is expressed in the erythroid, megakaryocytic, and mast cell lineages during normal hematopoiesis. To gain further insight into the molecular mechanisms that control tal-1 expression, we investigated tal-1 chromatin structure in erythroid/megakaryocytic cell lines and in T-cell lines either with or without tal-1 rearrangements. Tal-1 transcription was shown to be monoallelic in Jurkat, a T-cell line that expresses tal-1 in the absence of apparent genomic alteration of the locus. Methylation studies indicated that the tal-15' GC-rich region behaves like a CpG island, hypomethylated in normal cells, and methylated de novo on transcriptionally inactive alleles in established cell lines. Five major DNase-I hypersensitive sites (HS) were mapped in the tal-1 locus. HS I, IV, and V were exclusively observed in the erythroid/megakaryocytic cell lines that express tal-1 from the promoters 1a and 1b. HS II was weak in hematopoietic cell lines, absent in Hela, and greatly enhanced in Jurkat, suggesting that this region might be implicated in the cis-activation of tal-1 promoter 1b in this cell line. HS III was weak in HEL and Jurkat, and greatly enhanced in DU528, a T-cell line that bears a t (1;14) and initiates tal-1 transcription within exon 4. These results suggest that distinct regulatory elements are associated with the use of the different tal-1 promoters. 


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The tax protein of human T-cell leukemia virus type 1 mediates the transactivation of the c-sis/platelet-derived growth factor-B promoter through interactions with the zinc finger transcription factors Sp1 and NGFI-A/Egr-1. 
Transcriptional up-regulation of the c-sis/platelet-derived growth factor-B (PDGF-B) proto-oncogene by the Tax protein of human T-cell leukemia virus type 1 has been implicated as one possible mechanism of cellular transformation by human T-cell leukemia virus type 1. In previous work, we identified an essential site in the c-sis/PDGF-B promoter, Tax-responsive element 1 (TRE1), necessary for transactivation by Tax. We also identified Sp1, Sp3, and NGFI-A/Egr-1 as the primary nuclear transcription factors binding to TRE1 which mediate Tax responsiveness. In the present work, we have investigated the mechanism(s) whereby Tax transactivates the c-sis/PDGF-B proto-oncogene. In vitro transcription assays showed that Tax was able to significantly increase the transcriptional activity of a template containing the -257 to +74 region of the c-sis/PDGF-B promoter. Electrophoretic mobility shift assay analysis showed that Tax increased the DNA binding activity of both Sp1 and NGFI-A/Egr-1 using a TRE1 probe. Analysis of Tax mutants showed that two mutants, IEXC29S and IEXL320G, were unable to significantly transactivate the c-sis/PDGF-B promoter. Finally, co-immunoprecipitation analysis revealed that Tax is able to stably bind to both Sp1 and NGFI-A/Egr-1. Interestingly, co-immunoprecipitation analysis also revealed that Tax mutant IEXC29S is unable to interact with NGFI-A/Egr-1, whereas Tax mutant IEXL320G is able to interact with NGFI-A/Egr-1. 


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An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes. 
Lymphocytes are particularly susceptible to DNA damage-induced apoptosis, a response which may serve as a form of 'altruistic suicide' to counter their intrinsic high potential for mutation and clonal expansion. The tumour suppressor p53 has been shown to regulate this type of apoptosis in thymocytes, but an as yet unknown, p53-independent pathway(s) appears to mediate the same event in mitogen-activated mature T lymphocytes. Here we show DNA damage-induced apoptosis in these T lymphocytes is dependent on the antioncogenic transcription factor interferon regulatory factor (IRF)-1. Thus two different anti-onco-genic transcription factors, p53 and IRF-1, are required for distinct apoptotic pathways in T lymphocytes. We also show that mitogen induction of the interleukin-1 beta converting enzyme (ICE) gene, a mammalian homologue of the Caenorhabditis elegans cell death gene ced-3, is IRF-1-dependent. Ectopic overexpression of IRF-1 results in the activation of the endogenous gene for ICE and enhances the sensitivity of cells to radiation-induced apoptosis. 


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Effects of CD45 on NF-kappa B. Implications for replication of HIV-1. 
Increased levels of replication of the HIV type 1 are observed after the activation of infected T cells through the TCR. However, anti-CD45 antibodies inhibit these effects in cells from infected individuals. In this study, we examined interrelationships between CD45 and HIV-1 further. We measured effects on the HIV-1 LTR in T cell lines that were stimulated with antibodies against CD45 and in those that lacked the expression of CD45 on their surfaces. First, anti-CD45 antibodies did not affect basal but decreased activated levels of expression from the HIV-1 LTR. Second, T cells, which lack CD45 and cannot signal via the TCR, supported higher levels of viral replication and gene expression. This was due to the presence of active NF-kappa B complexes in the nucleus of CD45- T cells. Additionally, infected T cells displayed lower levels of CD45 on their surfaces. Thus, CD45 plays an active role in the physiology of T cells and in the replication of HIV-1. 


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Astrocytes and glioblastoma cells express novel octamer-DNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins. 
The 'octamer' sequence, ATGCAAAT or its complement ATTTGCAT, is a key element for the transcriptional regulation of immunoglobulin genes in B-lymphocytes as well as a number of housekeeping genes in all cell types. In lymphocytes, the octamer-binding protein Oct-2A and variants thereof are thought to contribute to the B-cell specific gene expression, while the ubiquitous protein Oct-1 seems to control general octamer site-dependent transcription. Various other genes, for example interleukin-1 and MHC class II genes, contain an octamer sequence in the promoter and are expressed in cells of both the immune and nervous systems. This prompted us to analyze the octamer-binding proteins in the latter cells. Using the electrophoretic mobility shift assay, at least six novel octamer binding proteins were detected in nuclear extracts of cultured mouse astrocytes. These proteins are differentially expressed in human glioblastoma and neuroblastoma cell lines. The nervous system-derived (N-Oct) proteins bound to the octamer DNA sequence in a manner which is indistinguishable from the Oct-1 and Oct-2A proteins. The relationship of the N-Oct proteins to Oct-1 and Oct-2A was analyzed by proteolytic clipping bandshift assays and by their reactivity towards antisera raised against recombinant Oct-1 and Oct-2A proteins. On the basis of these assays, all N-Oct-factors were found to be distinct from the ubiquitous Oct-1 and the lymphoid-specific Oct-2A proteins. In melanoma cells that contain the N-Oct-3 factor, a transfected lymphocyte-specific promoter was neither activated nor was it repressed upon contransfection with an Oct-2A expression vector. We therefore speculate that N-Oct-3 and other N-Oct factors have a specific role in gene expression in cells of the nervous system. 


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CD40 is a functional activation antigen and B7-independent T cell costimulatory molecule on normal human lung fibroblasts. 
CD40 is an important signaling and activation Ag found on certain bone marrow-derived cells. Recently, CD40 also has been shown to be expressed by mesenchymal cells, including human fibroblasts. Little is known about the role of CD40 in fibroblasts. The current study investigates the hypothesis that CD40 expressed on lung fibroblasts is an activation structure and mechanism for interaction with hemopoietic cells. Communication between resident tissue fibroblasts and T cells is necessary for normal wound healing, and can be pathologic, resulting in tissue fibrosis. Signaling through CD40 with soluble CD40 ligand stimulated fibroblast activation, as evidenced by mobilization of nuclear factor-kappaB and by induction of the proinflammatory and chemoattractant cytokines IL-6 and IL-8. IFN-gamma-primed lung fibroblasts costimulate T lymphocyte proliferation utilizing CD40, but not the well-studied costimulatory molecules B7-1 and B7-2. Data reported herein support the hypothesis that cognate interactions between tissue fibroblasts and infiltrating T lymphocytes, via the CD40/CD40L pathway, augment inflammation and may promote fibrogenesis by activating both cell types. 


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A factor that regulates the class II major histocompatibility complex gene DPA is a member of a subfamily of zinc finger proteins that includes a Drosophila developmental control protein. 
A novel DNA sequence element termed the J element involved in the regulated expression of class II major histocompatibility complex genes was recently described. To study this element and its role in class II gene regulation further, a cDNA library was screened with oligonucleotide probes containing both the S element and the nearby J element of the human DPA gene. Several DNA clones were obtained by this procedure, one of which, clone 18, is reported and characterized here. It encodes a protein predicted to contain 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Kruppel-like in the conservation of the H/C link sequence connecting them. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Kruppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box) here. One of the Drosophila sequences, ttk, is a developmental control gene, while a second does not contain a zinc finger region but encodes a structure important in oocyte development. An acidic activation domain is located between the N-terminal conserved region of clone 18 and its zinc fingers. This protein appears to require both the S and J elements, which are separated by 10 bp for optimal binding. Antisense cDNA to clone 18 inhibited the expression of a reporter construct containing the DPA promoter, indicating its functional importance in the expression of this class II gene. 


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Alpha 4 beta 1 (CD49d/CD29) integrin costimulation of human T cells enhances transcription factor and cytokine induction in the absence of altered sensitivity to anti-CD3 stimulation. 
The integrin alpha 4 beta 1 can provide a costimulus to induce IL-2 secretion and IL-2R expression leading to enhanced proliferation of purified, peripheral blood T cells. Similar to expression of IL-2, we demonstrated that recombinant vascular-cell adhesion molecule-1, when co-immobilized with anti-CD3 mAb, significantly enhanced the induction of transcription factors NF-AT, AP-1, and NF-kappa B as determined by electromobility shift assays. alpha 4 beta 1 ligation alone had no effect on transcription factor binding. The requirements for induction of transcription factors reflected the requirements for the secretion of multiple cytokines, including IL-2, TNF-alpha, IFN-gamma, and granulocyte macrophage-CSF. In contrast to freshly isolated T cells, in vitro-cultured T cells did not require costimulation for cytokine secretion in response to anti-CD3 alone. Comparison of the dose response to anti-CD3 stimulation demonstrated that half-maximal induction of IL-2 was achieved using the same dose of anti-CD3 for both freshly isolated and cultured T cells. Furthermore, the dose of OKT3 required to achieve half-maximal activation was the same using PMA or different concentrations of alpha 4 beta 1 ligands. Therefore, costimulation by alpha 4 beta 1 ligands was not due to stabilization of the interaction of the cells with its substrate. We conclude, rather, that alpha 4 beta 1 in freshly isolated T cells delivers a distinct signal that synergizes early with signals initiated by TCR/CD3 ligation to induce DNA binding of multiple transcription factors required for cytokine gene induction. 


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Activation of nuclear factor-kappa B by beta-amyloid peptides and interferon-gamma in murine microglia. 
An increasing body of evidence suggests that amyloid-beta (A beta) peptides and microglia are crucially involved in the pathogenesis of Alzheimer's disease. In an effort to further elucidate the biological effects of A beta towards microglia, we investigated the ability of A beta peptides to activate nuclear factor (NF)-kappa B in the N9 murine microglial cell line. Co-stimulation of microglia with suboptimal concentrations of A beta(25-35) and 100 U/ml IFN gamma resulted in the detection of a specific NF-kappa B DNA-binding activity in nuclear extracts, as determined in gel mobility shift assays. This response required at least 120 min to be evident and supershift experiments revealed that the NF-kappa B complex contains both RelA and p50. Accordingly, immunoblot experiments showed that amongst NF-kappa B/Rel proteins, RelA and p50 are mobilized to the nucleus following microglial cell stimulation with A beta(25-35) plus IFN gamma. Higher concentrations of A beta(25-35) were effective by themselves in inducing NF-kappa B activation, both in the N9 microglial cell line and in rat primary microglia, as well as in human monocytes. For purposes of comparison, microglia were also stimulated with bacterial LPS, a known NF-kappa B inducer. As expected, LPS strongly induced the formation of two NF-kappa B DNA-binding activities, one of which was identified as RelA/p50. The LPS response was also more rapid, as it was already evident by 40 min and remained sustained for up to 3 h. Collectively, these findings indicate that NF-kappa B activation might constitute one of the mechanisms underlying the inducible expression of kappa B-dependent genes in microglia stimulated by A beta peptides and IFN gamma, or by LPS. 


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Multiple prolactin-responsive elements mediate G1 and S phase expression of the interferon regulatory factor-1 gene. 
The interferon regulatory factor-1 (IRF-1) gene is both an immediate-early G1 phase gene and an S phase gene inducible by PRL in rat Nb2 T lymphocytes. To understand the mechanism by which PRL regulates the biphasic expression of IRF-1, we cloned the rat IRF-1 gene and functionally characterized the IRF-1 promoter. Upon transfection into Nb2 T cells, 1.7 kilobases (kb) of IRF-1 5'-flanking DNA linked to a chloramphenicol acetyl transferase (CAT) reporter gene mediated a 30-fold induction of CAT enzyme activity in response to 24 h of PRL stimulation. Deletion mutants containing 1.3, 0.6, and 0.2 kb 5'-flanking DNA were incrementally less transcriptionally active, although 0.2 kb still mediated a 12-fold induction by PRL. The sequence between -1.7 and -0.2 kb linked to a heterologous thymidine kinase promoter failed to respond to PRL stimulation, suggesting that the activity of upstream PRL response elements may require an interaction with promoter-proximal elements. By assaying CAT enzyme activity across a 24-h PRL induction time course, we were able to assign G1 vs. S phase PRL responses of the IRF-1 gene to different regions of the IRF-1 5'-flanking and promoter DNA. The 0.2-kb IRF-CAT construct was induced by PRL stimulation during the G1 phase of the cell cycle. In contrast, the 1.7-kb IRF-CAT construct was inducible by PRL during both G1 and S phase of the cell cycle. Hence, the PRL-induced biphasic expression of the IRF-1 gene appears to be controlled by separate PRL-responsive elements: elements in the first 0.2 kb of the IRF-1 promoter region act during early activation, and elements between 0.2 and 1.7 kb act in concert with the proximal 0.2-kb region during S phase progression. 


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Peripheral T lymphocytes from women with breast cancer exhibit abnormal protein expression of several signaling molecules. 
We examined signaling molecules of peripheral blood T lymphocytes obtained from women with breast cancer. In 6 of 14 patients, T lymphocytes displayed an impaired ability to translocate NFeB p65 (Rel-A) following activation by anti-CD3 and IL-2. This observation was made despite normal cytoplasmic levels of the Rel-A protein. We also detected abnormally low levels of the signaling molecules T-cell receptor (TCR)-zeta, ZAP-70 and p56lck in 4 of 14 breast cancer patients, i.e., defects in T-cell signaling molecules. T lymphocytes from 6 of the 14 patients also exhibited an increased expression of the dual specificity phosphatase, map kinase phosphatase-1 (MKP-1). MKP-1 inactivates MAP kinase and therefore may interfere with the activation of c-jun and c-fos. Abnormalities of I or more signaling molecules were found in 9 of 14 patients; however, only 3 patients had T cells that exhibited all 5 defects. Our data have implications for the detection of potentially dysfunctional T cells in patients with cancer. For example, the analysis of only 1 signaling molecule may allow patients with significant defects in T-cell signaling to go unnoticed. Finally, despite impaired Rel-A translocation, T cells were capable of transcribing IL-2. Impairments in the translocation of Rel-B and c-Rel further suggest that the NFKB family members Rel-A, Rel-B and c-Rel are not required for the transcription of IL-2 in the peripheral T lymphocytes of patients with breast cancer. 


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Potent inhibition of HIV type 1 replication by an antiinflammatory alkaloid, cepharanthine, in chronically infected monocytic cells. 
Cepharanthine is a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata and has been shown to have antiinflammatory, antiallergic, and immunomodulatory activities in vivo. As several inflammatory cytokines and oxidative stresses are involved in the pathogenesis of HIV-1 infection, we investigated the inhibitory effects of cepharanthine on tumor necrosis factor alpha (TNF-alpha)- and phorbol 12-myristate 13-acetate (PMA)-induced HIV-1 replication in chronically infected cell lines. Two chronically HIV-1-infected cell lines, U1 (monocytic) and ACH-2 (T lymphocytic), were stimulated with TNF-alpha or PMA and cultured in the presence of various concentrations of the compound. HIV-1 replication was determined by p24 antigen level. The inhibitory effects of cepharanthine on HIV-1 long terminal repeat (LTR)-driven gene expression and nuclear factor kappaB (NF-kappaB) activation were also examined. Cepharanthine dose dependently inhibited HIV-1 replication in TNF-alpha- and PMA-stimulated U1 cells but not in ACH-2 cells. Its 50% effective and cytotoxic concentrations were 0.016 and 2.2 microg/ml in PMA-stimulated U1 cells, respectively. Cepharanthine was found to suppress HIV-1 LTR-driven gene expression through the inhibition of NF-kappaB activation. These results indicate that cepharanthine is a highly potent inhibitor of HIV-1 replication in a chronically infected monocytic cell line. Since biscoclaurine alkaloids, containing cepharanthine as a major component, are widely used for the treatment of patients with various inflammatory diseases in Japan, cepharanthine should be further pursued for its chemotherapeutic potential in HIV-1-infected patients. 


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Activation of Stat 5b in erythroid progenitors correlates with the ability of ErbB to induce sustained cell proliferation. 
Self renewal of normal erythroid progenitors is induced by the receptor tyrosine kinase c-ErbB, whereas other receptors (c-Kit/Epo-R) regulate erythroid differentiation. To address possible mechanisms that could explain this selective activity of c-ErbB, we analyzed the ability of these receptors to activate the different members of the Stat transcription factor family. Ligand activation of c-ErbB induced the tyrosine phosphorylation, DNA-binding, and reporter gene transcription of Stat 5b in erythroblasts. In contrast, ligand activation of c-Kit was unable to induce any of these effects in the same cells. Activation of the erythropoietin receptor caused specific DNA-binding of Stat 5b, but failed to induce reporter gene transcription. These biochemical findings correlate perfectly with the selective ability of c-ErbB to cause sustained self renewal in erythroid progenitors. 


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Identification of human TR2 orphan receptor response element in the transcriptional initiation site of the simian virus 40 major late promoter [published erratum appears in J Biol Chem 1995 Nov 3;270(44):26721] 
A DNA response element (TR2RE-SV40) for the TR2 orphan receptor, a member of the steroid-thyroid hormone receptor superfamily, has been identified in the simian virus 40 (SV40) +55 region (nucleotide numbers 368-389, 5'-GTTAAGGTTCGTAGGTCATGGA-3'). Electrophoretic mobility shift assay, using in vitro translated TR2 orphan receptor with a molecular mass of 67 kilodaltons, showed a specific binding with high affinity (dissociation constant = 9 nM) for this DNA sequence. DNA-swap experiments using chloramphenicol acetyl-transferase assay demonstrated that androgen can suppress the transcriptional activities of SV40 early promoter via the interaction between this TR2RE-SV40 and the chimeric receptor AR/TR2/AR with the DNA-binding domain of the TR2 orphan receptor flanked by the N-terminal and androgen-binding domains of the androgen receptor. In addition, this TR2RE-SV40 can function as a repressor to suppress the transcriptional activities of both SV40 early and late promoters. Together, these data suggest the TR2RE-SV40 may represent the first identified natural DNA response element for the TR2 orphan receptor that may function as a repressor for the SV40 gene expression. 


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T cell priming enhances IL-4 gene expression by increasing nuclear factor of activated T cells. 
The repetitive activation of T cells (priming) enhances the expression of many cytokines, such as IL-4, but not others, such as IL-2. Molecular mechanisms underlying selective expression of cytokines by T cells remain poorly understood. Here we show that priming of CD4 T cells selectively enhances IL-4 expression relative to IL-2 expression by a transcriptional mechanism involving nuclear factor of activated T cells (NFAT) proteins. As detected by in vivo footprinting, priming markedly increases the activation-dependent engagement of the P0 and P1 NFAT-binding elements of the IL-4 promoter. Moreover, each proximal P element is essential for optimal IL-4 promoter activity. Activated primed CD4 T cells contain more NFAT1 and support greater NFAT-directed transcription than unprimed CD4 T cells, while activator protein 1 binding and activator protein 1-mediated transcription by both cell types is similar. Increased expression of wild-type NFAT1 substantially increases IL-4 promoter activity in unprimed CD4 T cells, suggesting NFAT1 may be limiting for IL-4 gene expression in this cell type. Furthermore, a truncated form of NFAT1 acts as a dominant-negative, reducing IL-4 promoter activity in primed CD4 T cells and confirming the importance of endogenous NFAT to increased IL-4 gene expression by effector T cells. NFAT1 appears to be the major NFAT family member responsible for the initial increased expression of IL-4 by primed CD4 T cells. 


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Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors. 
The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (IL-12), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL-12 production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of IL-12 production by human macrophages, and that induction is mediated by Toll-like receptors (TLRs). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens. 


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Interleukin (IL)-10 inhibits nuclear factor kappa B (NF kappa B) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. 
Our previous studies in human monocytes have demonstrated that interleukin (IL)-10 inhibits lipopolysaccharide (LPS)-stimulated production of inflammatory cytokines, IL-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha by blocking gene transcription. Using electrophoretic mobility shift assays (EMSA), we now show that, in monocytes stimulated with LPS or TNF alpha, IL-10 inhibits nuclear stimulation of nuclear factor kappa B (NF kappa B), a transcription factor involved in the expression of inflammatory cytokine genes. Several other transcription factors including NF-IL-6, AP-1, AP-2, GR, CREB, Oct-1, and Sp-1 are not affected by IL-10. This selective inhibition by IL-10 of NF kappa B activation occurs rapidly and in a dose-dependent manner and correlates well with IL-10's cytokine synthesis inhibitory activity in terms of both kinetics and dose responsiveness. Furthermore, compounds such as tosylphenylalanyl chloromethyl ketone and pyrrolidinedithiocarbamate that are known to selectively inhibit NF kappa B activation block cytokine gene transcription in LPS-stimulated monocytes. Taken together, these results suggest that inhibition of NF kappa B activation may be an important mechanism for IL-10 suppression of cytokine gene transcription in human monocytes. IL-4, another cytokine that inhibits cytokine mRNA accumulation in monocytes, shows little inhibitory effect on LPS-induced NF kappa B activation. Further examination reveals that, unlike IL-10, IL-4 enhances mRNA degradation and does not suppress cytokine gene transcription. These data indicate that IL-10 and IL-4 inhibit cytokine production by different mechanisms. 


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Activation of lymphokine genes in T cells: role of cis-acting DNA elements that respond to T cell activation signals. 
Activation of T cells is initiated by the recognition of antigen on antigen presenting cells to exert the effector functions in immune and inflammatory responses. Two types of helper T cell (Th) clones (Th1 and Th2) are defined on the basis of different patterns of cytokine (lymphokine) secretion. They determine the outcome of an antigenic response toward humoral or cell-mediated immunity. Although lymphokine genes are coordinately regulated upon antigen stimulation, they are regulated by the mechanisms common to all as well as those which are unique to each gene. For most lymphokine genes, a combination of phorbol esters (phorbol 12-myristate 13 acetate, PMA) and calcium ionophores (A23187) is required for their maximal induction. Yet phorbol ester alone or calcium ionophore alone produce several lymphokines. The production of the granulocyte-macrophage colony stimulating factor (GM-CSF) is completely dependent on the two signals. We have previously found a cis-acting region spanning the GM-CSF promoter region (positions -95 to +27) that confers inducibility to reporter genes in transient transfection assays. Further analysis identified three elements required for efficient induction, referred to as GM2, GC-box and conserved lymphokine element (CLE0). GM2 defines a binding site for protein(s) whose binding is inducible by PMA. One protein, NF-GM2 is similar to the transcription factor NF-kB. GC-box is a binding site for constitutively bound proteins. CLEO defines a binding site for protein(s) whose optimum binding is stimulated by PMA and A23187. Viral trans-activators such as Tax (human T cell leukemia virus-1, HTLV-1) and E2 (bovine papilloma virus, BPV) proteins are other agents which activate lymphokine gene expression by bypassing T cell receptor (TCR) mediated signaling. The trans-activation domain of E2 and Tax is interchangeable although they have no obvious sequence homology between them. The viral trans-activators appear to target specific DNA binding protein such as NF-kB and Sp1 to cis-acting DNA site and promote lymphokine gene expression without TCR-mediated stimulation. 


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Cloning of ARE-containing genes by AU-motif-directed display. 
A procedure suitable for cloning labile mRNAs that contain AU motifs is presented (AU-DD). These motifs are regulatory sequences within the so-called AU-rich elements (AREs) often found in 3' untranslated regions of genes such as cytokines, proto-oncogenes, and transcription factors. AU-DD is an AU-motif-directed differential display that permits the identification of ARE-containing genes differentially expressed after cell activation. It has been applied to peripheral blood monocytes and a T cell clone to isolate 59 cDNA fragments associated to activation. Fourteen percent of isolated fragments belong to already known genes that certainly are cytokines and transduction/transcription factors. The remaining 86% correspond to unknown genes of which 92% have been confirmed to be differentially expressed. These data demonstrate the efficiency of the system and support the notion that numerous genes falling into those categories remain unidentified and that they can be cloned by this method. Copyright 1998 Academic Press. 


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Role of ascorbate in the activation of NF-kappaB by tumour necrosis factor-alpha in T-cells. 
The first product of ascorbate oxidation, the ascorbate free radical (AFR), acts in biological systems mainly as an oxidant, and through its role in the plasma membrane redox system exerts different effects on the cell. We have investigated the role of ascorbate, AFR and dehydroascorbate (DHA) in the activation of the NF-kappaB transcription factor in Jurkat T-cells stimulated by tumour necrosis factor-alpha (TNF-alpha). Here we show, by electrophoretic mobility shift assays, that ascorbate increases the binding of NF-kappaB to DNA in TNF-alpha-stimulated Jurkat cells. The ability of ascorbate to enhance cytoplasmic inhibitory IkBalpha protein degradation correlates completely with its capacity to induce NF-kappaB binding to DNA and to potentiate NF-kappaB-mediated transactivation of the HIV-1 long terminal repeat promoter in TNF-alpha-stimulated Jurkat cells but not in cells stimulated with PMA plus ionomycin. AFR behaves like ascorbate, while DHA and ascorbate phosphate do not affect TNF-alpha-mediated NF-kappaB activation. These results provide new evidence for a possible relationship between the activation of the electron-transport system at the plasma membrane by ascorbate or its free radical and redox-dependent gene transcription in T-cells. 


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Stimulation of interferon beta gene transcription in vitro by purified NF-kappa B and a novel TH protein. 
The human interferon beta (IFN-beta) regulatory element consists of multiple enhanson domains which are targets for transcription factors involved in inducible expression of the promoter. To further characterize the protein-DNA interactions mediating IFN-beta induction, positive regulatory domain (PRD) II binding proteins were purified from phorbol ester induced Jurkat T-cells and from IFN primed, cycloheximide/polyinosinic-polycytidylic acid treated HeLa S3 cells. From HeLa cells, two major proteins of 52 and 45 kilodaltons (kD) copurified with DNA binding activity, whereas from T-cells, four proteins--a major protein of 52 kD and three minor proteins of 82, 67, and 43-47 kD--were purified. Also, an induction specific DNA binding protein was purified from HeLa cells that interacted with the (AAGTGA)4 tetrahexamer sequence and the PRDI domain. This protein is immunologically distinct from IRF-1/ISGF2. Uninduced or Sendai virus induced HeLa extracts were used to examine transcription in vitro using a series of IFN beta promoter deletions. Deletions upstream of the PRDII element increased transcription in the uninduced extract, indicating predominantly negative regulation of the promoter. A 2-4-fold increase in IFN-beta promoter transcription was observed in Sendai virus induced extracts, and deletion of PRDI and PRDII elements decreased this induced level of transcription. When purified PRDII and tetrahexamer binding proteins were added to the induced extract, a 4-fold increase in transcription was observed. These experiments demonstrate that it is possible to modulate IFN-beta transcription in vitro but indicate that additional proteins may be required to fully activate IFN-beta transcription. 


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Cross-linking CD40 on B cells rapidly activates nuclear factor-kappa B. 
The B cell-associated surface molecule CD40 functions to regulate B cell responses. Cross-linking CD40 on B cells can lead to homotypic cell adhesion, IL-6 production, and, in combination with cytokines, to Ig isotype switching. Tyrosine kinase activity is increased shortly after engagement of this receptor. Little is known about how the very early events induced by CD40 cross-linking link to cellular responses. In this study, we demonstrate that nuclear factor (NF)-kappa B and NF-kappa B-like transcription factors are activated after cross-linking CD40 on resting human tonsillar B cells and on B cell lines. The activation is rapid and is mediated through a tyrosine kinase-dependent pathway. The complexes detected in electrophoretic mobility shift assays contain p50, p65 (RelA), c-Rel, and most likely other components. By using transient transfection assays, we found that cross-linking CD40 supports NF-kappa B-dependent gene expression. Our results define the NF-kappa B system as an intermediate event in CD40 signaling and suggest that the CD40 pathway can influence the expression of B cell-associated genes with NF-kappa B consensus sites. 


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Intranuclear targeted delivery of functional NF-kappaB by 70 kDa heat shock protein. 
The 70 kDa heat shock protein (Hsp70) is a highly conserved, ubiquitous protein involved in chaperoning proteins to various cellular organelles. Here we show that when added exogenously to cells, Hsp70 is readily imported into both cytoplasmic and nuclear compartments in a cell-type-specific fashion. We exploited this ability of Hsp70 to deliver NF-kappaB, a key transcriptional regulator of inflammatory responses. We demonstrate that a fusion protein composed of a C-terminal Hsp70 peptide and the p50 subunit of NF-kappaB was directed into the nucleus of cells, could bind DNA specifically, and activated Igkappa expression and TNFalpha production. We therefore propose that Hsp70 can be used as a vehicle for intracytoplasmic and intranuclear delivery of proteins or DNA to modulate gene expression and thereby control immune responses. 


Document 0030011899 ends.


A negative regulatory region containing a glucocorticosteroid response element (nGRE) in the human interleukin-1beta gene. 
Interleukin-1 beta (IL-1beta) is one of the most important inflammatory mediators in human inflammatory and immunological diseases. The regulation of human IL-1beta gene expression has been studied for several years, and a few regulatory elements have been discovered in the promoter region. However, little is known about negative regulation of IL-1beta expression at the transcriptional level, which may play an important role in anti-inflammatory and immunosuppressive effects. We have identified a negative regulatory element located in the region between -685 and -395. Within this region, a 19-bp nuclear factor binding site (-570 to -552) was characterized by DNase I footprinting and electromobility shift assay. A consensus sequence for a negative glucocorticoid response element (nGRE) and a transcription activator protein-2 binding site were noted within this footprint. Functional studies showed a 2.5-fold increase in promoter activity when this 19-bp binding site was deleted in the reporter constructs IL-1beta/CAT and IL-1beta/SV40 promoter/CAT. Dexamethasone (10(-8) M) repressed chloramphenicol acetyltransferase (CAT) production by 75% in the wild-type fragment but not in a deletion mutant lacking the 19-bp site. A protein of about 150 kD that bound to this negative regulatory sequence was identified by UV cross-linking. This is the first description of a negative regulatory region responsive to glucocorticoids in a cytokine gene. 


Document 0030011900 ends.


Differences in transcriptional enhancers of HIV-1 and HIV-2. Response to T cell activation signals. 
T cell activation results in high levels of HIV replication and is thought to be one mechanism leading to the conversion from latent to active viral infection. In HIV-1, the sequences that respond to these signaling events are found in the long terminal repeat (LTR) and comprise the transcriptional enhancer, which contains two conserved binding sites for the nuclear factor kappa B (NF kappa B). The corresponding region in the second AIDS retrovirus, HIV-2, contains a conserved and a divergent NF kappa B binding site. We demonstrate that the HIV-1 LTR responds better than the HIV-2 LTR to T cell activation signals. These qualitative differences in the response to T cell activation are reproduced not only when HIV-1 or HIV-2 enhancers are placed upstream of a heterologous promoter but also when these enhancers are switched between their respective LTR. In electrophoretic mobility shift assays, NF kappa B binds to both conserved sites in the HIV-1 transcriptional enhancer and only to the single conserved site in the HIV-2 transcriptional enhancer. Instead of NF kappa B, the activator protein 3 binds to the divergent site in HIV-2. In conclusion, HIV-1 and HIV-2 are differentially regulated by T cell activation signals, and this difference may account for the longer period of viral latency observed with HIV-2 than with HIV-1 infection. 


Document 0030011907 ends.


Involvement of NF-kappaB p50/p65 heterodimer in activation of the human pro-interleukin-1beta gene at two subregions of the upstream enhancer element. 
A region between-3134 and -2729 bp upstream from the transcription site of the human pro-interleukin 1beta (proIL-1beta) gene was identified as an LPS-responsive enhancer element. In this study, the influence of the sequences located between -3134 and -2987 on the transcriptional activity of the proIL-1beta gene in LPS-stimulated Raw 264.7 cells was examined in detail. The results obtained by transient transfection of fos -CAT constructs that contained serial 5'-deletion mutations showed that the region between -3134 and -3059 appears to be required for the induction of transcription by LPS. Gel shift assay studies with synthetic oligonucleotides corresponding to partial sequences of the latter region and nuclear extracts from stimulated cells revealed specific protein binding sites between -3110 and -3090 and between -3079 and -3059. These specific bindings were time and LPS dose dependent. The results of supershift analysis using specific antibodies against transcription factors suggested that both binding complexes contained the NF-kappaB components p50 and p65, and did not contain other NF-kappaB proteins (p52, c-Rel, Rel B), AP-1 proteins (c-Fos, C-Jun), CREB or C/EBPbeta (NF-IL6). Mutation of either of the putative NF-kappaB-binding sites in the enhancer element decreased the LPS-stimulated transcriptional activity. These data indicated that two NF-kappaB-binding sites, which are located between -3134 and -3059, are critical for the activation of proIL-1beta gene transcription. Copyright 1999 Academic Press. 


Document 0030011914 ends.


The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells. 
Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) infection is associated with increased levels of viral replication and burden in the peripheral blood and lymphoid organs. T cell activation and ensuing cellular gene activation can be critical for HIV-1 replication. The hypothesis that the nuclear factor of activated T cells (NF-AT) may influence HIV-1 replication is therefore compelling given the tight correlation of HIV-1 transcriptional induction to T cell activation. We report that certain NF-AT(Rel) family members productively bind the kappaB regulatory elements, synergize with NF-kappaB and Tat in transcriptional activation of HIV-1, and enhance HIV-1 replication in T cells. These results link regulatory factors critical to T cell commitment directly to HIV-1 replication. 


Document 0030011921 ends.


Studies into the effect of tyrosine phosphatase inhibitor phenylarsine oxide on NFkappaB activation in T lymphocytes during aging: evidence for altered IkappaB-alpha phosphorylation and degradation. 
Nuclear Factor kappa B (NFkappaB) is a critical regulator of several genes involved in immune and inflammatory responses. Treatment of T cells with a variety of stimuli, including TNF-alpha, leads to the translocation of the active p65-50 heterodimer to the nucleus, albeit at a lower level in T cells from the elderly. We demonstrate here that pretreatment with PAO results in the inhibition of NFkappaB induction in TNF-alpha treated T cells, suggesting a role for PAO-sensitive phosphatase in the activation of the NFkappaB via this pathway in human T cells. Furthermore, it demonstrates that aging does not influence the sensitivity of this phosphatase. Treatment with DMP prior to treatment with PAO and TNF abolishes the inhibition induced by PAO, in T cells from both young and old donors, alike. Finally, we demonstrate that a failure to degrade IkappaB-alpha in cytosols of TNF-treated T cells pretreated with PAO is due to its interference with the phosphorylation of IkappaB-alpha and not due to its inhibitory effect on proteasomal degradation. These data collectively suggest that PAO interferes with the phosphorylation and the regulated degradation of IkappaB-alpha, induced by TNF, without affecting the chymotryptic activity of the proteasome, independent of age. 


Document 0030011923 ends.


Defining therapeutic targets by using adenovirus: blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators. 
The role of the transcription factor NF-kappaB in the pathogenesis of rheumatoid arthritis has long been a subject of controversy. We used an adenoviral technique of blocking NF-kappaB through overexpression of the inhibitory subunit IkappaBalpha, which has the advantage that it can be used in the diseased tissue itself, with >90% of the synovial macrophages, fibroblasts, and T cells infected. We found that the spontaneous production of tumor necrosis factor alpha and other pro-inflammatory cytokines is NF-kappaB-dependent in rheumatoid synovial tissue, in contrast to the main anti-inflammatory mediators, like IL-10 and -11, and the IL-1 receptor antagonist. Of even more interest, IkappaBalpha overexpression inhibited the production of matrix metalloproteinases 1 and 3 while not affecting their tissue inhibitor. Blocking NF-kappaB in the rheumatoid joint thus has a very beneficial profile, reducing both the inflammatory response and the tissue destruction. The adenoviral technique described here has widespread applicability, allowing rapid testing of the effects of blocking a potential therapeutic target in either cultures of normal cells or in the diseased tissue itself. 


Document 0030011927 ends.


The control of lytic replication of Epstein-Barr virus in B lymphocytes (Review). 
Uncontrolled replication of a virus, which is harmful to the host is also disadvantageous to the virus. Most viruses cannot compete with the various immune mechanisms and become eliminated in the course of infection. Therefore, only the time between infection and eradication remains for these viruses to proliferate. A few viruses, like the Herpesviruses or the papillomaviruses, however, have developed a sophisticated strategy for persisting lifelong, usually asymptomatically in the host, hiding from the immune system and producing infectious progeny at the same time. This strategy depends on a separation of latency and the lytic replication, either by time due to differentiation-dependent mechanisms or by spatial separation as the result of different host cell types. Both are true for the Epstein-Barr virus (EBV). B cells and epithelial cells have a pivotal role in the life cycle of the virus. The former can become latently infected and are thought to be the virus reservoir in vivo, whereas the latter were shown to be permissive for lytic replication. However, replication of EBV in vivo is controlled primarily by host immune mechanisms selecting for cells that are not permissive for viral replication as the result of a particular set of transcription factors. These factors control the activity of the regulatory immediate-early genes and, in addition, lytic and latent cycle regulatory genes negatively interfere with each other and thus link cellular and viral gene regulatory mechanisms. Disturbance of both the immune surveillance as well as viral gene regulation may result in EBV-associated disease. 


Document 0030011933 ends.


Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1). 
The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E.Zhang, C.J.Hetherington, H.-M.Chen, and D.G.Tenen, Mol.Cell. Biol.14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to - 59), which lies 10 bp upstream from the PU.1-binding site. When analyzed by DNase footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family, which includes the AML1 gene product, while Mono A is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor PU.1 and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the CSF-1 receptor. 


Document 0030011936 ends.


IL-10 cooperates with TNF-alpha to activate HIV-1 from latently and acutely infected cells of monocyte/macrophage lineage. 
IL-10 is elevated in HIV-1-infected individuals and has been implicated in disease progression. In this study, we investigated the effects of IL-10 on the activation of HIV-1 from infected monocytes and macrophages. Although IL-10 alone did not induce HIV-1 replication, in the presence of TNF-alpha, IL-10 markedly enhanced virion production from a chronically infected promonocytic cell line (U1) and in acutely infected monocyte-derived macrophages. Neutralizing mAbs to IL-10 and TNF-alpha indicated that both cytokines were essential for the induction and were required to generate a synergistic increase in virus expression. The effects of the two cytokines were distinguishable functionally since pretreatment with TNF-alpha attenuated the cytokine cooperativity, while pretreatment with IL-10 potentiated their cooperativity, suggesting that IL-10 and TNF-alpha play different roles in the activation of virus. Northern blot analysis as well as Ab blocking and cytokine secretion studies indicated that the induction of either endogenous TNF-alpha or IL-10 was not involved in the cooperativity, nor was an up-regulation of TNF-alpha receptors. In combination with TNF-alpha, IL-10 stimulated activating protein-1 (AP-1) and nuclear factor (NF)-kappa B binding activities and cooperated to increase HIV-1 steady-state mRNA levels and enhance long terminal repeat-directed transcription through activation of the NF-kappa B binding sites, suggesting the IL-10 effect occurs at least in part at the transcriptional level. These results indicate that IL-10, in addition to down-regulating the cellular immune response to HIV-1, may also play a role in TNF-alpha-mediated activation of HIV-1 replication in the monocyte/macrophage lineage. 


Document 0030011937 ends.


Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor. 
T-lymphocyte proliferation is suppressed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, and is associated with a decrease in interleukin 2 (IL-2), gamma interferon, and granulocyte-macrophage colony-stimulating factor mRNA levels. We report here that 1,25(OH)2D3-mediated repression in Jurkat cells is cycloheximide resistant, suggesting that it is a direct transcriptional repressive effect on IL-2 expression by the vitamin D3 receptor (VDR). We therefore examined vitamin D3-mediated repression of activated IL-2 expression by cotransfecting Jurkat cells with IL-2 promoter/reporter constructs and a VDR overexpression vector and by DNA binding. We delineated an element conferring both DNA binding by the receptor in vitro and 1,25(OH)2D3-mediated repression in vivo to a short 40-bp region encompassing an important positive regulatory element, NF-AT-1, which is bound by a T-cell-specific transcription factor, NFATp, as well as by AP-1. VDR DNA-binding mutants were unable to either bind to this element in vitro or repress in vivo; the VDR DNA-binding domain alone, however, bound the element but also could not repress IL-2 expression. These results indicate that DNA binding by VDR is necessary but not sufficient to mediate IL-2 repression. By combining partially purified proteins in vitro, we observed the loss of the bound NFATp/AP-1-DNA complex upon inclusion of VDR or VDR-retinoid X receptor. Order of addition and off-rate experiments indicate that the VDR-retinoid X receptor heterodimer blocks NFATp/AP-1 complex formation and then stably associates with the NF-AT-1 element. This direct inhibition by a nuclear hormone receptor of transcriptional activators of the IL-2 gene may provide a mechanistic explanation of how vitamin derivatives can act as potent immunosuppressive agents. 


Document 0030011940 ends.


Leukotriene B4 stimulates c-fos and c-jun gene transcription and AP-1 binding activity in human monocytes. 
We have examined the effect of leukotriene B4 (LTB4), a potent lipid proinflammatory mediator, on the expression of the proto-oncogenes c-jun and c-fos. In addition, we looked at the modulation of nuclear factors binding specifically to the AP-1 element after LTB4 stimulation. LTB4 increased the expression of the c-fos gene in a time- and concentration-dependent manner. The c-jun mRNA, which is constitutively expressed in human peripheral-blood monocytes at relatively high levels, was also slightly augmented by LTB4, although to a much lower extent than c-fos. The kinetics of expression of the two genes were also slightly different, with c-fos mRNA reaching a peak at 15 min after stimulation and c-jun at 30 min. Both messages rapidly declined thereafter. Stability of the c-fos and c-jun mRNA was not affected by LTB4, as assessed after actinomycin D treatment. Nuclear transcription studies in vitro showed that LTB4 increased the transcription of the c-fos gene 7-fold and the c-jun gene 1.4-fold. Resting monocytes contained nuclear factors binding to the AP-1 element, but stimulation of monocytes with LTB4 induced greater AP-1-binding activity of nuclear proteins. These results indicate that LTB4 may regulate the production of different cytokines by modulating the yield and/or the function of transcription factors such as AP-1-binding proto-oncogene products. 


Document 0030011942 ends.


Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. 
Hydrogen peroxide and oxygen radicals are agents commonly produced during inflammatory processes. In this study, we show that micromolar concentrations of H2O2 can induce the expression and replication of HIV-1 in a human T cell line. The effect is mediated by the NF-kappa B transcription factor which is potently and rapidly activated by an H2O2 treatment of cells from its inactive cytoplasmic form. N-acetyl-L-cysteine (NAC), a well characterized antioxidant which counteracts the effects of reactive oxygen intermediates (ROI) in living cells, prevented the activation of NF-kappa B by H2O2. NAC and other thiol compounds also blocked the activation of NF-kappa B by cycloheximide, double-stranded RNA, calcium ionophore, TNF-alpha, active phorbol ester, interleukin-1, lipopolysaccharide and lectin. This suggests that diverse agents thought to activate NF-kappa B by distinct intracellular pathways might all act through a common mechanism involving the synthesis of ROI. ROI appear to serve as messengers mediating directly or indirectly the release of the inhibitory subunit I kappa B from NF-kappa B. 


Document 0030011945 ends.


The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites. 
The interleukin-2 IL-2 receptor beta-chain (IL-2Rbeta) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rbeta is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). We have now characterized factors that bind to an enhancer region located between nucleotides -170 and -139 of the human IL-2Rbeta promoter. Both Sp1 and Sp3 bound to the 5' portion of this region, whereas a PMA-inducible factor (PIF) mainly bound to its 3' portion and bound to the Sp binding motifs as well. In Jurkat T cells, induction of PIF DNA binding activity was rapidly induced, required de novo protein synthesis, and was sustained at a high level for at least 23 h. Interestingly, PIF was constitutively activated in human T-cell leukemia virus type 1-transformed MT-2 cells. In this paper, we demonstrate that PIF is Egr-1 based on its recognition by anti-Egr-1 antisera in gel mobility shift assays, even though the IL-2Rbeta DNA binding motif differed substantially from the canonical Egr-1 binding site. In addition, Egr-1 bound to the Sp binding site. In Jurkat cells, both sites were required for maximal IL-2Rbeta promoter activity, and in HeLaS3 cells, transfection of Egr-1 could drive activity of a reporter construct containing both sites. Moreover, Sp1 and Egr-1 could form a complex with kinetics that correlated with the production of Egr-1 in Jurkat cells upon PMA stimulation. Thus, Sp1 and Egr-1 physically and functionally cooperate to mediate maximal IL-2Rbeta promoter activity. 


Document 0030011947 ends.


Processing of the precursor of NF-kappa B by the HIV-1 protease during acute infection. 
Transcription of the human immunodeficiency virus type-1 (HIV-1) genome is regulated in part by cellular factors and is stimulated by activation of latently infected T cells. T-cell activation also correlates with the induction of the factor NF-kappa B which binds to two adjacent sites in the HIV-1 long terminal repeat. This factor consists of two DNA-binding subunits of relative molecular mass 50,000 (50K) associated with two 65K subunits. It is located in the nucleus in mature B cells, but is present in other cell types as an inactive cytoplasmic complex. External stimuli, including those that activate T cells, result in nuclear translocation of active NF-kappa B. The cloning of the complementary DNA for the 50K subunit helped to identify an exclusively cytoplasmic 105K precursor (p105) (V.B., P.K. and A.I., manuscript submitted). The expression of active NF-kappa B might therefore also be regulated by the extent of processing of p105. Because HIV-1 requires active NF-kappa B for efficient transcription, we tested the effect of HIV-1 infection on the processing of the human 105K precursor. We show here that the HIV-1 protease can process p105 and increases levels of active nuclear NF-kappa B complex. 


Document 0030011949 ends.


Transcription specific differences visualized by fluorescence in situ hybridization pattern on interphase nuclei of different cell types. 
Application of a "formamide free" and thus "material preserving" in situ hybridization technique using the cDNA of the myf3 gene revealed the following results: Human rhabdomyosarcoma cells, characterized by a high expression of myf3 show intensive hybridization signals in their interphase. RNase treatment prior to hybridization considerably reduces the size of this signals. In comparison, isolated nuclei of human lymphocytes in which no need for the expression of this gene exists, show barely hybridization signals. Correspondingly, RNase treatment had no effect on hybridization pattern at all. In conclusion an increased transcription efficiency of a cell type specific gene is accompanied by a higher hybridization accessibility in the corresponding cell nuclei. 


Document 0030011953 ends.


Protease treatment of nuclear extracts distinguishes between class II MHC X1 box DNA-binding proteins in wild-type and class II-deficient B cells. 
The X box region is critical for directing the expression of class II major histocompatibility complex genes in B lymphocytes. Although several class II promoter-specific DNA binding factors have been described, only the X box region factor, RFX, shows a genetic correlation with class II expression, being deficient in some B cell lines derived from patients with class II-deficient congenital immunodeficiency. To further evaluate the role of X box DNA-binding proteins in class II gene expression, the role of the X box region was examined in both class II-positive and -negative lymphoid cells. In addition to the wild-type B cell line Raji, two class II transcriptional mutant cell lines, SJO and RJ2.2.5, and Jurkat, a class II negative T cell line, were examined. In contrast to wild-type B cells, neither of the class II mutant cell lines could use the X box region to direct the expression of a transiently transfected reporter gene, indicating that the X box-dependent transcriptional pathway is defective in these cells. The binding activity of the X1 box DNA-binding protein RFX was examined and found to be present in wild-type B cells and the mutant RJ2.2.5 but was absent in SJO and Jurkat. However, other X1 box-specific activities were detected in all these cell lines. To determine whether these different X1 box activities represented distinct DNA binding proteins or multimeric forms of the same factor(s), protease treatment of the crude nuclear extracts followed by DNA-binding assays were carried out and demonstrated that B cell extracts contain at least two X1-specific factors. One of these cleaved products (band 1 pk) correlates with RFX activity. A similar comparison with protease-treated extracts prepared from Jurkat cells demonstrated the presence of the band 1pk activity despite an absence of the native RFX activity. In contrast, protease treatment and analysis of SJO extracts showed no detectable levels of the band 1pk activity. These results demonstrate that multiple X1 box-specific DNA-binding activities exist in all lymphoid cells, but the presence of an actively binding RFX species correlates with class II transcription. 


Document 0030011958 ends.


Differential RNA display identifies novel genes associated with decreased vitamin D receptor expression. 
To characterize further the function of the intracellular vitamin D receptor (VDR), we have developed stable transfectant variants of a vitamin D-responsive cell line (U937) which express either decreased or increased numbers of VDR. In this study we have analyzed changes in gene expression associated with this variable VDR expression. Initial experiments indicated that a 50% decrease in VDR levels was associated with a 2-fold increase in cell proliferation and a similar rise in c-myc mRNA expression. Further studies were carried out using differential RNA display (DD). Sequence analysis of DD products revealed two cDNAs with identity to known gene products: the catalytic sub-unit of DNA-protein kinase (DNA-PK(CS)), and the peroxisomal enzyme 17beta-hydroxysteroid dehydrogenase type IV (17beta-HSD IV). Northern analysis confirmed that expression of both mRNAs was reduced in cells with decreased numbers of VDR. Down-regulation of 17beta-HSD IV mRNA expression was associated with enhanced estradiol inactivation by U937 cells, suggesting a link between estrogenic pathways and cell proliferation. Further Northern analyses indicated that there was no significant change in 17beta-HSD IV or DNA-PK(CS) mRNA levels following treatment with 1,25(OH)2D3, although expression of both genes varied with changes in cell proliferation. These data suggest that, in addition to its established role as a hormone-dependent trans-activator, VDR may influence gene expression by ligand-independent mechanisms. 


Document 0030011959 ends.


Mapping of the transcriptional repression domain of the lymphoid-specific transcription factor oct-2A. 
The lymphoid-specific transcription factor Oct-2a is implicated in B cell-specific transcriptional activity via the octamer motif. Structure/function analysis of various Oct-2a effector regions in the context of the GAL4 DNA-binding domain revealed that Oct-2a contains two functionally different activation domains at the N and the C termini. The transcriptional activity of both domains is strongly potentiated by interactions with distinct B cell-specific coactivators. Recently, we have identified a repression domain located within the N terminus of Oct-2a (amino acids 2-99). When this domain was transferred to a potent activator, transcription was strongly inhibited. In this study we present a deletion analysis of the N-terminal region of Oct-2a to determine the minimal repression domain. We identified a stretch of 23 amino acids, rich in serine and threonine residues, which was responsible for most of the repression activity. We show that repression is strongly dependent on the type of enhancer present in the reporter plasmid as well as on the cell line tested. The possibility that Oct-2a can act as an activator and/or a repressor may have important consequences for the function of Oct-2a in B cell differentiation and other developmental processes. 


Document 0030011964 ends.


The tumour associated cell surface antigen A6H is costimulatory for human CD4+ but not CD8+ T cells. 
The A6H monoclonal antibody (mAb) recognizes a 120,000-140,000 MW antigen that is expressed at similar densities on 85-90% of human CD4+ and CD8+ T cells and on renal cell carcinomas. The binding of the A6H mAb induced a costimulatory signal in anti-CD3 activated T cells. In the present report, we show that A6H costimulated cell proliferation and cytokine production in purified CD4+ T cells. Unexpectedly, the CD8+ T-cell subpopulation failed to respond. CD4+ T cells costimulated with the A6H mAb upregulated CD80, CD86, CD71, interleukin-2 (IL-2)R alpha, IL-2R beta and IL-2R gamma, while no corresponding up-regulation of these cell surface molecules was seen in CD8+ T cells. In order to investigate the nature of the A6H mAb costimulus at the transcriptional level we have examined induction of the transcription factors OCT-1, AP-1 and NF-kappa B which are known to be transcriptional regulators of several cytokine and cytokine receptor genes, including the IL-2 and IL-2R genes. Co-ligation of the A6H antigen and the CD3 complex induced expression of the transcription factor AP-1 in CD4+ T cells, whereas no increase in NF-kappa B and octamer-binding (Oct) proteins was seen compared to T cells stimulated with anti-CD3 alone. Furthermore, no induction of AP-1 was seen in A6H costimulated CD8+ T cells. These results suggests that both proximal steps in CD8+ T-cell activation as well as the later phases are unresponsive to A6H ligation. Molecular differences of the A6H molecule or distinct regulation of the A6H transduced AP-1 activation pathway may exist in CD4+ and CD8+ T cell subpopulations. 


Document 0030011965 ends.


Regulation of cell-type-specific interleukin-2 receptor alpha-chain gene expression: potential role of physical interactions between Elf-1, HMG-I(Y), and NF-kappa B family proteins. 
The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is rapidly and potently induced in T cells in response to mitogenic stimuli. Previously, an inducible enhancer between nucleotides -299 and -228 that contains NF-kappa B and CArG motifs was identified. We now report the characterization of a second essential positive regulatory element located between nucleotides -137 and -64 that binds Elf-1 and HMG-I(Y). This element had maximal activity in lymphoid cells, paralleling the cell type specificity of Elf-1 expression. Transcription from the IL-2R alpha promoter was inhibited when either the Elf-1 or the HMG-I(Y) binding site was mutated. Coexpression of both proteins activated transcription of the -137 to -64 element in COS-7 cells. Elf-1 physically associated with HMG-I and with NF-kappa B p50 and c-Rel in vitro, suggesting that protein-protein interactions might functionally coordinate the actions of the upstream and downstream positive regulatory elements. This is the first report of a physical interaction between an Ets family member and NF-kappa B family proteins. These findings provide significant new insights into the protein-protein and protein-DNA interactions that regulate cell-type-specific and inducible IL-2R alpha gene expression and also have implications for other genes regulated by Elf-1 and NF-kappa B family proteins. 


Document 0030011968 ends.


LMP-1 activates NF-kappa B by targeting the inhibitory molecule I kappa B alpha. 
LMP-1, an Epstein-Barr virus membrane protein expressed during latent infection, has oncogenic properties, as judged from its ability to transform B lymphocytes and rodent fibroblasts. LMP-1 induces the expression of bcl2, an oncogene which protects cells from apoptosis, as well as of genes encoding other proteins involved in cell regulation and growth control. The mechanisms by which LMP-1 upregulates these proteins is unknown, but it is plausible that LMP-1 modifies signal transduction pathways that result in the activation of one or more transcription factors that ultimately regulate transcription of oncogenic genes. NF-kappa B, a transcription factor controlling the expression of genes involved in cell activation and growth control, has been shown to be activated by LMP-1. The mechanism(s) regulating this activation remains unknown. Our data indicate that increased NF-kappa B DNA binding and functional activity are present in B-lymphoid cells stably or transiently expressing LMP-1. I kappa B alpha is selectively modified in LMP-1-expressing B cells. A phosphorylated form of I kappa B alpha and increased protein turnover-degradation correlate with increased NF-kappa B nuclear translocation. This results in increased transcription of NF-kappa B-dependent-genes, including those encoding p105 and I kappa B alpha (MAD3). These results indicate that LMP-1 activates NF-kappa B in B-cell lines by targeting I kappa B alpha. Identification of the pathways activated by LMP-1 to result in posttranslational modifications of I kappa B alpha will aid in determining the role of this virus-host cell protein interaction in Epstein-Barr virus-mediated oncogenesis. 


Document 0030011975 ends.


Granulocyte colony-stimulating factor activates a 72-kDa isoform of STAT3 in human neutrophils. 
Granulocyte colony-stimulating factor (G-CSF) signaling involves activation of STATs, proteins that serve the dual function of signal transduction and activation of transcription. We previously demonstrated that G-CSF activated a distinct Stat3-like protein in immature and mature normal myeloid cells, StatG. StatG in normal immature human myeloid cells, i.e. adult CD34+ bone marrow cells, was composed of Stat3beta. This investigation was undertaken to determine the composition of StatG in mature normal human myeloid cells, i.e. polymorphonuclear neutrophilic granulocytes (PMN). These studies revealed that the major protein in extracts of PMN activated by G-CSF to bind the high-affinity serum-inducible element (hSIE) is a 72-kDa protein that cross-reacts with Stat3 monoclonal antibody, which we have designated Stat3gamma. Stat3gamma is derived from Stat3alpha by limited proteolysis and lacks the carboxyl-terminal portion of Stat3alpha. Because this region of Stat3alpha is involved in transcriptional activation, our findings suggest the possibility that Stat3gamma may be transcriptionally inactive and may compete with Stat3alpha for Stat3 binding sites in these terminally differentiated myeloid cells. 


Document 0030011978 ends.


NF-kappa B-independent suppression of HIV expression by ascorbic acid. 
Ascorbic acid (ascorbate or vitamin C) has been shown to suppress the induction of HIV in latently infected T lymphocytic cells following stimulation with a tumor promoter (PMA) and inflammatory cytokine (TNF-alpha). To assess whether this inhibition was mediated via modulation of the cellular transcription factor, NF-kappa B, we carried out gel shift analysis on nuclear extracts prepared under different conditions of cell stimulation in the presence or absence of ascorbate, N-acetylcysteine (NAC), or zidovudine (AZT). Pretreatment of ACH-2 T cells by NAC followed by stimulation with PMA, TNF-alpha, or hydrogen peroxide (H2O2) resulted in strong suppression of NF-kappa B activation. In contrast, neither ascorbate nor AZT affected NF-kappa B activity under all three induction conditions in the ACH-2 cell line. Ascorbate and AZT also had no effect on NF-kappa B activation following TNF-alpha- or PMA-induced stimulation of U1 promonocytic cells. These results suggest that the molecular mechanism of HIV inhibition by ascorbate is not mediated via NF-kappa B inhibition, unlike that seen with other antioxidants. 


Document 0030011989 ends.


Stat6 inhibits human interleukin-4 promoter activity in T cells. 
The differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4-secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4-activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner. 


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Paradoxical priming effects of IL-10 on cytokine production. 
IL-10 is a well-known immunosuppressive and/or anti-inflammatory cytokine. However, we report in vitro experimental studies in which IL-10 primed leukocytes and led to an enhanced production of tumor necrosis factor (TNF) upon further stimulation by lipopolysaccharide (LPS). Monocytes and peripheral blood mononuclear cells (PBMC) prepared from whole blood maintained for 20 h at 37 degrees C in the presence of recombinant human IL-10 had an enhanced capacity to produce TNF in response to LPS. In addition to TNF, LPS-induced IL-6 and spontaneous IL-1ra production were also enhanced. When isolated PBMC were first cultured for 20 h in the presence of IL-10 on Teflon to prevent adherence, washed to remove IL-10 and then further cultured in plastic dishes for an additional 20 h in the presence of LPS or IL-1beta, an enhanced release of TNF was observed. This was not the case when PBMC were pre-cultured in plastic multidishes in the presence of IL-10. TNF mRNA expression induced by LPS was decreased when the pre-treatment of PBMC with IL-10 was performed on plastic, whereas this was not the case when cells were pre-cultured with IL-10 on Teflon. Furthermore, NFkappaB translocation following LPS activation was higher after IL-10 pre-treatment on Teflon than on plastic. Interestingly, an enhanced frequency of CD16 and CD68(+) cells among the CD14(+) cells was observed in the presence of IL-10, independently of the pre-culture conditions of the PBMC. Altogether, these results indicate that the IL-10-induced up-regulation of cytokine production depends on the prevention of monocyte adherence by red cells in the whole blood assays or by cultures of PBMC on Teflon. In contrast, the adherence parameter has no effect on the IL-10-induced modulation of some monocyte surface markers. 


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Sequence analysis and expression in cultured lymphocytes of the human FOSB gene (G0S3). 
G0S3 is a member of a set of putative G0/G1 switch regulatory genes (G0S genes) selected by screening cDNA libraries prepared from human blood mononuclear cells cultured for 2 hr with lectin and cycloheximide. The sequence shows high homology with the murine FOSB gene, which encodes a component of the AP1 transcriptional regulator. Comparison of cDNA and genomic sequences reveals a 4-exon structure characteristic of the FOS family of genes. Freshly isolated cells show high levels of FOSB/G0S3 and FOS/G0S7 mRNAs, which decline rapidly during incubation in culture medium. The kinetics of expression suggest that the high initial levels are caused by the isolation procedure, and do not reflect constitutive expression. In cells preincubated for a day, levels of FOS mRNA reach a maximum 20 min after the addition of lectin and decline to control levels over the next 3 hr. Levels of FOSB mRNA reach a maximum 40 min after the addition of lectin and decline to control levels over the next 6 hr. In freshly isolated cells, both FOS and FOSB mRNAs increase dramatically in response to the protein synthesis inhibitor cycloheximide. In preincubated cells, the cycloheximide response is decreased, especially in the case of FOSB. These differences in expression of FOS and FOSB suggest different roles and regulation. Regions of low base order-dependent stem-loop potential in the region of the gene are defined. These indicate where base order has been adapted for purposes other than stem-loop stability (e.g., encoding proteins or gene regulation). Regions of low potential in a 68.5-kb genomic segment containing the FOSB gene suggest that the potential may help locate genes in uncharted DNA sequences. 


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Transcriptional regulation of the ferritin heavy-chain gene: the activity of the CCAAT binding factor NF-Y is modulated in heme-treated Friend leukemia cells and during monocyte-to-macrophage differentiation. 
The ferritin H-chain gene promoter regulation was analyzed in heme-treated Friend leukemia cells (FLCs) and during monocyte-to-macrophage differentiation. In the majority of cell lines studied, the regulation of ferritin expression was exerted mostly at the translational level. However, in differentiating erythroid cells, which must incorporate high levels of iron to sustain hemoglobin synthesis, and in macrophages, which are involved in iron storage, transcriptional regulation seemed to be a relevant mechanism. We show here that the minimum region of the ferritin H-gene promoter that is able to confer transcriptional regulation by heme in FLCs to a reporter gene is 77 nucleotides upstream of the TATA box. This cis element binds a protein complex referred to as HRF (heme-responsive factor), which is greatly enhanced both in heme-treated FLCs and during monocyte-to-macrophage differentiation. The CCAAT element present in reverse orientation in this promoter region of the ferritin H-chain gene is necessary for binding and for gene activity, since a single point mutation is able to abolish the binding of HRF and the transcriptional activity in transfected cells. By competition experiments and supershift assays, we identified the induced HRF as containing at least the ubiquitous transcription factor NF-Y. NF-Y is formed by three subunits, A, B, and C, all of which are necessary for DNA binding. Cotransfection with a transdominant negative mutant of the NF-YA subunit abolishes the transcriptional activation by heme, indicating that NF-Y plays an essential role in this activation. We have also observed a differential expression of the NF-YA subunit in heme-treated and control FLCs and during monocyte-to-macrophage differentiation. 


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Biphasic control of NF-kappa B activation induced by the triggering of HLA-DR antigens expressed on B cells. 
The regulation of NF-kappa B activation following the triggering of HLA-DR antigens by mAb L243 has been studied at various times in Raji cells. Electrophoretic mobility shift assays demonstrated a strong increase of NF-kappa B DNA binding after triggering of HLA-DR antigens. Using TNF-alpha-activity neutralizing antibodies, the authors demonstrated that the upregulation of NF-kappa B was found to depend, at later time point, on an autocrine effect of TNF-alpha secreted following triggering of HLA-DR antigens. In contrast, it was found to be TNF-alpha independent in the early time point. Moreover, the upregulation of NF-kappa B binding activity is regulated by the triggering of selected epitopes of HLA-DR antigens. In fact, mAb L243 but not the staphylococcal superantigens, staphylococcal exotoxin toxic shock syndrome toxin-I or staphylococcal enterotoxin B, regulate the NF-kappa B binding activity. 


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Tissue-specific regulation of the ecto-5'-nucleotidase promoter. Role of the camp response element site in mediating repression by the upstream regulatory region. 
We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity. 


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Characterization of a new isoform of the NFAT (nuclear factor of activated T cells) gene family member NFATc [published erratum appears in J Biol Chem 1996 Dec 27;271(52):33705] 
The cyclosporin A (CsA)/FK506-sensitive nuclear factor of activated T cells (NFAT) plays a key role in the inducible expression of cytokine genes in T cells. Although NFAT has been recently shown to be inducible in several non-T immune cells, the NFAT gene family members characterized to date have been isolated only from T cells. To further characterize NFAT function in human B cells and to demonstrate cytokine gene specificity of NFAT proteins, we report here the isolation and characterization of a cDNA clone from the Raji B cell line. The cDNA clone encodes a new isoform, NFATc.beta, of the NFAT gene family member NFATc (designated here NFATc.alpha). The amino acid sequence of NFATc.beta differs from that of NFATc.alpha in the first NH2-terminal 29 residues and contains an additional region of 142 residues at the COOH terminus. Northern analysis using a probe encompassing a common region of both isoforms showed two mRNA species of 2.7 and 4.5 kilobase pairs, while an NFATc.beta-specific probe detected only the 4.5-kilobase pair mRNA which was preferentially expressed in the spleen. Transient expression of NFATc.beta was capable of activating an interleukin-2 NFAT-driven reporter gene in stimulated Jurkat cells in a CsA-sensitive manner. However, NFATc.beta neither bound to the kappa3 element ( an NFAT-binding site ) in the tumor necrosis factor-alpha promoter nor activated the tumor necrosis factor-alpha promoter in cotransfection assays. These data suggest that different members or isoforms of NFAT gene family may regulate inducible expression of different cytokine genes. 


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Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. 
Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells. 


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The human toll signaling pathway: divergence of nuclear factor kappaB and JNK/SAPK activation upstream of tumor necrosis factor receptor-associated factor 6 (TRAF6). 
The human homologue of Drosophila Toll (hToll) is a recently cloned receptor of the interleukin 1 receptor (IL-1R) superfamily, and has been implicated in the activation of adaptive immunity. Signaling by hToll is shown to occur through sequential recruitment of the adapter molecule MyD88 and the IL-1R-associated kinase. Tumor necrosis factor receptor-activated factor 6 (TRAF6) and the nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK) are both involved in subsequent steps of NF-kappaB activation. Conversely, a dominant negative version of TRAF6 failed to block hToll-induced activation of stress-activated protein kinase/c-Jun NH2-terminal kinases, thus suggesting an early divergence of the two pathways. 


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Cell specific expression of human Bruton's agammaglobulinemia tyrosine kinase gene (Btk) is regulated by Sp1- and Spi-1/PU.1-family members. 
Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase involved in the human disease X-linked agammaglobulinemia (XLA). The gene is expressed in all hematopoietic cells with the exception of T-cells and plasma cells. For this expression pattern the first 280 bp upstream of the major transcriptional start site seems to be sufficient. In vitro footprinting analysis within this part of the promoter revealed two Sp1 binding sites as well as a PU-box. The transcription factor Spi-1/PU.1 as well as the closely related factor Spi-B bound to the PU-box in B-cells. In the erythroleukemia cell line K562, due to the absence of Spi-B, only PU.1 bound to the Btk promoter. Mutation of either site reduced the expression in transient transfection experiments. However, mutation of the PU box had no effect in the T-cell line Jurkat, where none of the Spi-1 family members is expressed. In addition Spi-B as well as PU.1 were able to transactivate Btk expression. In fetal liver of PU.1-/- mice, which lack lymphoid and myeloid cells, expression of Btk was reduced two- to threefold but not abolished. Collectively this study shows that expression of the Btk gene is regulated by the combined action of Sp1- and PU.1-family members. 


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Activation of nuclear factor kappa B in human neuroblastoma cell lines. 
The nuclear factor kappa B (NF-kappa B) is a eukaryotic transcription factor. In B cells and macrophages it is constitutively present in cell nuclei, whereas in many other cell types, NF-kappa B translocates from cytosol to nucleus as a result of transduction by tumor necrosis factor alpha (TNF alpha), phorbol ester, and other polyclonal signals. Using neuroblastoma cell lines as models, we have shown that in neural cells NF-kappa B was present in the cytosol and translocated into nuclei as a result of TNF alpha treatment. The TNF alpha-activated NF-kappa B was transcriptionally functional. NF-kappa B activation by TNF alpha was not correlated with cell differentiation or proliferation. However, reagents such as nerve growth factor (NGF) and the phorbol ester phorbol 12-myristate 13-acetate (PMA), which induce phenotypical differentiation of the SH-SY5Y neuroblastoma cell line, activated NF-kappa B, but only in that particular cell line. In a NGF-responsive rat pheochromocytoma cell line, PC12, PMA activated NF-kappa B, whereas NGF did not. In other neuroblastoma cell lines, such as SK-N-Be(2), the lack of PMA induction of differentiation was correlated with the lack of NF-kappa B activation. We found, moreover, that in SK-N-Be(2) cells protein kinase C (PKC) enzymatic activity was much lower compared with that in a control cell line and that the low PKC enzymatic activity was due to low PKC protein expression. NF-kappa B was not activated by retinoic acid, which induced morphological differentiation of all the neuroblastoma cell lines used in the present study. Thus, NF-kappa B activation was not required for neuroblastoma cell differentiation. Furthermore, the results obtained with TNF alpha proved that NF-kappa B activation was not sufficient for induction of neuroblastoma differentiation. 


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Specific NF-kappa B subunits act in concert with Tat to stimulate human immunodeficiency virus type 1 transcription. 
NF-kappa B is a protein complex which functions in concert with the tat-I gene product to stimulate human immunodeficiency virus (HIV) transcription. To determine whether specific members of the NF-kappa B family contribute to this effect, we have examined the abilities of different NF-kappa B subunits to act with Tat-I to stimulate transcription of HIV in Jurkat T-leukemia cells. We have found that the p49(100) DNA binding subunit, together with p65, can act in concert with Tat-I to stimulate the expression of HIV-CAT plasmid. Little effect was observed with 50-kDa forms of p105 NF-kappa B or rel, in combination with p65 or full-length c-rel, which do not stimulate the HIV enhancer in these cells. These findings suggest that the combination of p49(100) and p65 NF-kappa B can act in concert with the tat-I gene product to stimulate the synthesis of HIV RNA. 


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Cobalt chloride-induced signaling in endothelium leading to the augmented adherence of sickle red blood cells and transendothelial migration of monocyte-like HL-60 cells is blocked by PAF-receptor antagonist. 
In response to hypoxia, sickle red blood cells (SS RBC) and leukocytes exhibit increased adherence to the vascular endothelium, while diapedesis of leukocytes through the blood vessel increases. However, the cellular signaling pathway(s) caused by hypoxia is poorly understood. We utilized CoCl2 as a mimetic molecule for hypoxia to study cellular signaling pathways. We found that in human umbilical vein endothelial cells (HUVEC), CoCl2 at 2 mM concentration induced the surface expression of a subset of CAMs (VCAM-1) and activation of transcription factor NF-kappaB in the nuclear extracts of HUVEC. Furthermore, CoCl2 also caused time-dependent tyrosine phosphorylation of mitogen-activated protein (MAP) kinase isoform ERK2 without significantly affecting ERK1, indicating ERK2 is the preferred substrate for upstream kinase of the MAPK pathway. Inhibitors of MAP kinase (PD98059) or platelet-activating factor (PAF)- receptor antagonist (CV3988) inhibited the CoCl2-induced NF-kappaB activation and VCAM-1 expression. Augmented expression of VCAM-1 led to increased SS RBC adhesion, inhibitable by a VCAM-1 antibody. Additionally, CoCl2 caused a two- to threefold increase in the rate of transendothelial migration of monocyte-like HL-60 cells and a twentyfold increase in phosphorylation of platelet endothelial cell adhesion molecules (PECAM-1). The transendothelial migration of monocytes was inhibited by an antibody to PECAM-1. Both phosphorylation of PECAM-1 and transendothelial migration of monocytes in response to CoCl2 were inhibited by protein kinase inhibitor (GF109203X) and augmented by protein phosphatase inhibitor (Calyculin A). Our data suggests that CoCl2-induced cellular signals directing increased expression of VCAM-1 in HUVEC involve downstream activation of MAP kinase and NF-kappaB, while the phosphorylation of PECAM-1 occurs as a result of activation of PKC. We conclude that PAF-receptor antagonist inhibits the CoCl2- or hypoxia-induced increase in the adhesion of SS RBC, PECAM-1 phosphorylation, and the concomitant transendothelial migration of monocytes. 


Document 0030012061 ends.


Tap: a novel cellular protein that interacts with tip of herpesvirus saimiri and induces lymphocyte aggregation. 
Tip of herpesvirus saimiri associates with Lck and down-regulates Lck-mediated activation. We identified a novel cellular Tip-associated protein (Tap) by a yeast two-hybrid screen. Tap associated with Tip following transient expression in COS-1 cells and stable expression in human Jurkat-T cells. Expression of Tip and Tap in Jurkat-T cells induced dramatic cell aggregation. Aggregation was likely caused by the up-regulated surface expression of adhesion molecules including integrin alpha, L-selectin, ICAM-3, and H-CAM. Furthermore, NF-kappaB transcriptional factor of aggregated cells had approximately 40-fold higher activity than that of parental cells. Thus, Tap is likely to be an important cellular mediator of Tip function in T cell transformation by herpesvirus saimiri. 


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Cell-type-specific regulation of the human tumor necrosis factor alpha gene in B cells and T cells by NFATp and ATF-2/JUN. 
The human tumor necrosis factor alpha (TNF-alpha) gene is one of the earliest genes transcribed after the stimulation of a B cell through its antigen receptor or via the CD-40 pathway. In both cases, induction of TNF-alpha gene transcription can be blocked by the immunosuppressants cyclosporin A and FK506, which suggested a role for the NFAT family of proteins in the regulation of the gene in B cells. Furthermore, in T cells, two molecules of NFATp bind to the TNF-alpha promoter element kappa 3 in association with ATF-2 and Jun proteins bound to an immediately adjacent cyclic AMP response element (CRE) site. Here, using the murine B-cell lymphoma cell line A20, we show that the TNF-alpha gene is regulated in a cell-type-specific manner. In A20 B cells, the TNF-alpha gene is not regulated by NFATp bound to the kappa 3 element. Instead, ATF-2 and Jun proteins bind to the composite kappa 3/CRE site and NFATp binds to a newly identified second NFAT site centered at -76 nucleotides relative to the TNF-alpha transcription start site. This new site plays a critical role in the calcium-mediated, cyclosporin A-sensitive induction of TNF-alpha in both A20 B cells and Ar-5 cells. Consistent with these results, quantitative DNase footprinting of the TNF-alpha promoter using increasing amounts of recombinant NFATp demonstrated that the -76 site binds to NFATp with a higher affinity than the kappa 3 site. Two other previously unrecognized NFATp-binding sites in the proximal TNF-alpha promoter were also identified by this analysis. Thus, through the differential use of the same promoter element, the composite kappa 3/CRE site, the TNF-alpha gene is regulated in a cell-type-specific manner in response to the same extracellular signal. 


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Regulation of human epsilon germline transcription: role of B-cell-specific activator protein. 
Germline transcripts initiate from promoters upstream of the immunoglobulin switch region, and are necessary to target the appropriate switch region for recombination and switching. Different cytokines activate transcription at the appropriate germline promoter. Because binding sites for B-cell-specific activator protein (BSAP) are located upstream of several switch regions in the immunoglobulin heavy chain gene cluster, BSAP might play a role in the regulation of germline transcription and isotype switching. We investigated whether BSAP plays a role in the transcriptional regulation of the epsilon germline promoter in human B cells. Our results showed that BSAP plays a role in both IL-4-dependent induction and CD40-mediated upregulation of human epsilon germline transcription. BSAP is unique among the transcription factors that regulate epsilon germline expression, because it is B cell specific, and is at the merging point of two signalling pathways that are critical for IgE switching. 


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Activation of the human delta-globin gene promoter in primary adult erythroid cells. 
Restoration of the CCAAT box or insertion of an erythroid Kruppel-like factor (EKLF) binding site in the delta promoter activates its expression in several erythroid cell lines. We extended these studies using a novel primary human adult erythroid cell (hAEC) system to investigate these effects at the late erythroblast stage. Restoration of the CCAAT box at -70 bp, or insertion of an EKLF binding site at -85 bp or -95 bp in the promoter significantly increased delta globin gene expression in hAEC. Our results demonstrate that the altered CCAAT box (CCAAC) and the lack of an EKLF binding site in delta-globin contribute to its low level of expression in the hAEC model as well. 


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Induction of a functional vitamin D receptor in all-trans-retinoic acid-induced monocytic differentiation of M2-type leukemic blast cells. 
Different types of acute myeloid leukemia blast cells were induced to differentiate in vitro with all-trans-retinoic acid (ATRA) and vitamin D3 (VD). M0/M1 leukemic cells are not sensitive to differentiating agents, whereas M3 leukemic cells are induced to undergo granulocytic differentiation after ATRA treatment but are not sensitive to VD. M2 leukemic blast cells behave differently because they undergo monocytic differentiation with both the differentiation inducers. To gain some insight into the maturation of M2-type leukemic cells, we studied the molecular mechanisms underlying monocytic differentiation induced by ATRA and VD in spontaneous M2 blast cells as well as in Kasumi-1 cells (an acute myeloid leukemia M2-type cell line). Our results indicate that ATRA as well as VD efficiently increases the nuclear abundance of VD receptor (VDR) and promotes monocytic differentiation. VDR is functionally active in ATRA-treated Kasumi-1 cells because it efficiently heterodimerizes with retinoid X receptor, binds to a DR3-type vitamin D-responsive element, and activates the transcription of a vitamin D-responsive element-regulated reporter gene. Consistent with these findings, VD-responsive genes are induced by ATRA treatment of Kasumi-1 cells, suggesting that the genetic program underlying monocytic differentiation is activated. The molecular mechanism by which ATRA increases the nuclear abundance of a functional VDR is still unknown, but our data clearly indicate that the M2 leukemic cell context is only permissive of monocytic differentiation. 


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Cell type specificity and activation requirements for NFAT-1 (nuclear factor of activated T-cells) transcriptional activity determined by a new method using transgenic mice to assay transcriptional activity of an individual nuclear factor. 
Nuclear factor of activated T-cells (NFAT-1) is a transcription factor which is considered to be an important regulator in early T-cell activation. We have developed a system to monitor the transcriptional activity of NFAT-1 at the single cell level in whole animals. The system is based on the use of an oligomerized NFAT-1 binding motif that directs transcription of SV40 T-antigen in transgenic mice. This report represents the first demonstration that a multimerized short binding motif can function appropriately in transgenic mice. NFAT-1 activity had previously been thought to be confined to activated T-lymphocytes upon release of intracellular calcium. By targeting NFAT-1-dependent gene expression in transgenic mice we discovered new sites of NFAT-1 activity. Besides in T-lymphocytes NFAT-1 activity could also be induced in T-lymphocyte-depleted spleen cells and purified B-lymphocytes and requires agents that both release intracellular calcium and activate protein kinase C. A difference in the time course of appearance of NFAT-1 activity between T-lymphocytes and non-T-lymphocytes was revealed. Constitutive expression was observed in a small population of cells in the dermis and some mice have developed skin lesions. Interestingly, the tissue pattern of expression of the NFAT-1 activity resembles the expression pattern described for HIV-LTR/tat transgenic mice (Vogel, J., Hinrichs, S. H., Reynolds, R. K., Luciw, P. A., and Jay, G. (1988) Nature 335, 606-611). This similarity in expression and the fact that NFAT-1 has been shown to bind functional sequences in HIV-LTR suggest a role for NFAT-1 in dermal activation of the HIV-LTR. 


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The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes. 
All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression. 


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alpha-Tocopheryl succinate inhibits monocytic cell adhesion to endothelial cells by suppressing NF-kappa B mobilization. 
The adherence of monocytes to activated endothelium is an early event in atherogenesis. Because antioxidants have been considered to be of antiatherosclerotic potential, we investigated the effects of alpha-tocopherol (TCP) and its acetate and succinate esters on monocyte adhesion to cytokine-stimulated human umbilical vein endothelial cells (HUVEC). Endothelial cells were treated with TCP, alpha-tocopherol acetate (TCP acetate), or alpha-tocopheryl succinate (TCP succinate) before stimulation with tumor necrosis factor-alpha (TNF-alpha; 10 U/ml, 6 h) or interleukin-1 beta (IL-1 beta; 10 U/ml, 6 h). Cytokine-stimulated cell surface expression of vascular cell adhesion molecule-1 (VCAM-1, CD106) and E-selectin (ELAM-1, CD62E), but not of intercellular adhesion molecule-1 (ICAM-1, CD54), was time- and dose-dependently inhibited by TCP succinate but not by TCP or TCP acetate. TCP succinate (200 microM, 24 h) reduced TNF-induced VCAM-1 and E-selectin expression from a specific mean fluorescence intensity of 151 +/- 28 to 12 +/- 4 channels and from 225 +/- 38 to 79 +/- 21 channels, respectively. Succinate alone had no effect. Decreased adhesion molecule expression was associated with a reduction of monocytic cell adhesion. TCP succinate (20 microM, 72 h), but not TCP (200 microM, 72 h), reduced U-937 cell adhesion to TNF-alpha-stimulated (10 U/ml, 6 h) HUVEC by 30% (P < 0.025) and to IL-1 beta-stimulated HUVEC by 56% (P < 0.010). Electrophoretic mobility-shift assays of HUVEC nuclear proteins revealed a decrease in TNF-alpha-stimulated nuclear factor-kappa B (NF-kappa B) activation after pretreatment of HUVEC with TCP succinate but not with TCP, TCP acetate, or succinate alone. In conclusion, we demonstrate that the vitamin E derivative TCP succinate prevents monocytic cell adhesion to cytokine-stimulated endothelial cells by inhibiting the activation of NF-kappa B, further emphasizing the antiatherosclerotic potential of lipid soluble antioxidants. 


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Lineage- and stage-specific expression of runt box polypeptides in primitive and definitive hematopoiesis. 
Translocations involving the human CBFA2 locus have been associated with leukemia. This gene, originally named AML1, is a human homologue of the Drosophila gene runt that controls early events in fly embryogenesis. To clarify the role of mammalian runt products in normal and leukemic hematopoiesis, we have studied their pattern of expression in mouse hematopoietic tissues in the adult and during ontogeny using an anti-runt box antiserum. In the adult bone marrow, we found expression of runt polypeptides in differentiating myeloid cells and in B lymphocytes. Within the erythroid lineage, runt expression is biphasic, clearly present in the erythroblasts of early blood islands and of the fetal liver, but absent in the adult. Biochemical analysis by Western blotting of fetal and adult hematopoietic populations shows several runt isoforms. At least one of them appears to be myeloid specific. 


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Some antioxidants inhibit, in a co-ordinate fashion, the production of tumor necrosis factor-alpha, IL-beta, and IL-6 by human peripheral blood mononuclear cells. 
Some antioxidants, including butylated hydroxyanisole (BHA), tetrahydropapaveroline (THP), nordihydroguiauretic acid, and 10,11-dihydroxyaporphine (DHA), were found to be potent inhibitors of the production of tumor necrosis factor (TNF)-alpha, IL-1 beta, and IL-6 by human peripheral blood mononuclear cells (PBMC) stimulated by lipopolysaccharide (LPS) (IC50s in the low micromolar range). Inhibition of cytokine production was gene selective and not due to general effects on protein synthesis. Inhibition of cytokine production by PBMC was observed also when other inducers were used (staphylococci, silica, zymosan). Much higher concentrations of other antioxidants--including ascorbic acid, trolox, alpha-tocopherol, butylated hydroxytoluene, and the 5-lipoxygenase inhibitor zileuton--did not affect the production of these cytokines. The active compounds did not inhibit IL-1-induced production of IL-6 in fibroblasts, showing the cell selectivity of the effect. Antioxidant-mediated inhibition of cytokine production was correlated with low levels of the corresponding messenger RNAs. Nuclear run-on experiments showed that THP inhibited transcription of the IL-1 beta gene. THP decreased the concentration of the transcription factors NF-kappa B and AP-1 detected in nuclear extracts of PBMC cultured in the presence or absence of LPS. THP and DHA markedly decreased the levels of TNF-alpha and IL-1 beta in the circulation of mice following LPS injection. Thus antioxidants vary widely in potency as inhibitors of the activation of transcription factors and of the transcription of genes for pro-inflammatory cytokines. Coordinate inhibition of the transcription of genes for inflammatory cytokines could provide a strategy for therapy of diseases with inflammatory pathogenesis and for septic shock. 


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Activation of nuclear factor-kappaB by lipopolysaccharide in mononuclear leukocytes is prevented by inhibitors of cytosolic phospholipase A2. 
In monocytes, lipopolysaccharide induces synthesis and activity of the 85-kDa cytosolic phospholipase A2. This enzyme releases arachidonic acid and lyso-phospholipids from membranes which are metabolized to eicosanoids and platelet-activating-factor. These lipid mediators increase activity of transcription factors and expression of cytokine genes indicating a function for cytosolic phospholipase A2 in signal transduction and inflammation. We have shown previously that trifluoromethylketone inhibitors of cytosolic phospholipase A2 suppressed interleukin-1beta protein and steady-state mRNA levels in human lipopolysaccharide-stimulated peripheral blood mononuclear leukocytes. In this study, the subcellular mechanisms were analyzed by which trifluoromethylketones interfere with gene expression. We found that they reduced the initial interleukin-1beta mRNA transcription rate through prevention of degradation of inhibitor-kappaB alpha. Consequently, cytosolic activation, nuclear translocation and DNA-binding of nuclear factor-kappaB were decreased. Trifluoromethylketones ameliorate chronic inflammation in vivo. Thus, this therapeutic potency may reside in retention of inactive nuclear factor-kappaB in the cytosol thereby abrogating interleukin-1beta gene transcription. 


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C/EBP activators are required for HIV-1 replication and proviral induction in monocytic cell lines. 
Previous work has shown that C/EBP sites and C/EBP transcriptional activators are necessary for HIV-1 LTR activity in monocytes/macrophages. We have investigated the role that C/EBP proteins play in induction and replication of HIV-1. Ectopic expression of the dominant negative C/EBP protein LIP inhibited HIV-1 mRNA and virus production in activated U1 cells, demonstrating that C/EBP proteins are required for provirus induction. U1 lines overexpressing C/EBP activator NF-IL-6 produced more viral mRNA and virus particles following cellular activation than control lines, demonstrating that C/EBP proteins are limiting for virus transcription. HIV-1 harboring mutations within two C/EBP sites were crippled in their ability to replicate in U937 promonocytic cells, indicating that these sites are required for replication. These data identify C/EBP proteins as regulators of HIV-1 expression in monocytes/macrophages. 


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The ability of BHRF1 to inhibit apoptosis is dependent on stimulus and cell type. 
The development of resistance to host defense mechanisms such as tumor necrosis factor (TNF)- and Fas-mediated apoptosis of transformed or virus-infected cells may be a critical component in the development of disease. To find genes that protect cells from apoptosis, we used an expression cloning strategy and identified BHRF1, an Epstein-Barr virus (EBV) early-lytic-cycle protein with distant homology to Bcl-2, as an anti-apoptosis protein. Expression of BHRF1 in MCF-Fas cells conferred nearly complete resistance against both anti-Fas antibody and TNF-mediated apoptosis. In addition, BHRF1 protected these cells from monocyte-mediated killing but failed to protect them from killing mediated by lymphokine-activated killer cells. The ability of BHRF1 to protect MCF-Fas cells from apoptosis induced by various stimuli was identical to that of Bcl-2 and Bcl-xL. Moreover, the mechanism of action of BHRF1 resembled that of Bcl-2 and Bcl-xL as it inhibited TNF- and anti-Fas-induced activation of two enzymes participating in the apoptosis pathway, cytosolic phospholipase A2 and caspase-3/CPP32, but did not interfere with the activation of NF-kappaB-like transcription factors. A putative function of BHRF1 in EBV-infected epithelial cells may be to protect virus-infected cells from TNF- and/or anti-Fas- induced cell death in order to maximize virus production. Surprisingly, expression of neither BHRF1 nor Bcl-2 in a B-cell line, BJAB, protected the cells from anti-Fas-mediated apoptosis even though they increased the survival of serum-starved cells. Thus, the protective role of BHRF1 against apoptosis resembles that of Bcl-2 in being cell type specific and dependent on the apoptotic stimulus. 


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In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1. 
Cellular pathways for induction of programmed cell death (PCD) have been identified, but little is known about specific extracellular matrix processes that may affect apoptosis along those pathways. In this study, a series of Burkitt's lymphoma (BL) cell lines were assayed for their expression of tissue inhibitor of metalloproteinases (TIMP)-1. Results indicate that TIMP-1-positive BL lines show resistance to cold-shock-induced apoptosis. Furthermore, recombinant TIMP-1, but not TIMP-2 or a synthetic metalloproteinase inhibitor (BB-94), confers resistance to apoptosis induced by both CD95-dependent and -independent (cold shock, serum deprivation, and gamma-radiation) pathways in TIMP-1-negative BL lines. TIMP-1 suppression of PCD is not due to metalloproteinase inhibition, as reduction and alkylation of the TIMP-1 did not abolish this activity. Retroviral induction of TIMP-1 not only resulted in cell survival but also in continued DNA synthesis for up to 5 d in the absence of serum, while controls underwent apoptosis. This resistance to apoptosis is reversed by anti-TIMP-1 antibodies, demonstrating that secreted TIMP-1 is active in blocking apoptosis. Furthermore, TIMP-1 upregulation induced expression of Bcl-XL but not Bcl-2 as well as decreased NF-kappaB activity as compared with controls. These results demonstrate that TIMP-1 suppresses apoptosis in B cells and suggests a novel activity for TIMP-1 in tissue homeostasis. 


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Cooperation between core binding factor and adjacent promoter elements contributes to the tissue-specific expression of interleukin-3. 
Tissue-specific expression of interleukin-3 (IL-3) is mediated via cis-acting elements located within 315 base pairs of the transcription start. This is achieved in part through the positive activities of the AP-1 and Elf-1 sites in the IL-3 promoter. The contribution to T cell-specific expression by other promoter sites was assessed in a transient expression assay with IL-3 promoter constructs linked to a luciferase gene, focusing initially on the core binding factor (CBF) site, which is footprinted in vivo upon T cell activation. Activity of the CBF site is shown to be critically dependent on the adjacent activator site Act-1. Together the Act-1 and CBF sites form a functional unit (AC unit) with dual activity. The AC unit is demonstrated to enhance basal activity of promoters both in fibroblasts and T cells. This activity is further inducible in activated T cells, but not in fibroblasts. In addition to the already identified NIP repressor site, evidence is presented for a second repressor region that restricts promoter activity in fibroblasts. Finally, a novel positive regulatory element has been mapped in the IL-3 promoter between nucleotide -180 and -210 that leads to increased expression in T cells. Together these results demonstrate that T cell expression of IL-3 is not specified by the activity of a single tissue-specific element, but instead involves multiple interacting elements that provide both specific positive regulation in T cells and specific negative regulation in fibroblasts. 


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Relief of cyclin A gene transcriptional inhibition during activation of human primary T lymphocytes via CD2 and CD28 adhesion molecules. 
Cyclin A transcription is cell cycle regulated and induced by cell proliferative signals. To understand the mechanisms underlined in this regulation in normal human cells, we have analysed in vivo protein-DNA interactions at the Cyclin A locus in primary T lymphocytes. Stimulation of purified T lymphocytes by a combination of monoclonal antibodies directed at CD2 and CD28 adhesion molecules gives rise to a long lasting proliferation in the absence of accessory cells. Cyclin A was observed after 4 days of costimulation with anti CD2 + CD28 whereas stimulation by anti CD2 or anti CD28 alone was not effective. In vivo genomic DMS footprinting revealed upstream of the major transcription initiation sites, the presence of at least three protein binding sites, two of which were constitutively occupied. They bind in vitro respectively ATF-1 and NF-Y proteins. The third site was occupied in quiescent cells or in cells stimulated by anti CD2 or anti CD28 alone. The mitogenic combination of anti CD2 + anti CD28 released the footprint as cells were committed to proliferation. Consistent with theses results, nuclear extracts prepared from quiescent cells formed a specific complex with this element, whereas extracts prepared from cells treated with anti CD2 + anti CD28 failed to do so after cells entered a proliferative state. 


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A factor known to bind to endogenous Ig heavy chain enhancer only in lymphocytes is a ubiquitously active transcription factor. 
The transcriptional enhancer located in the first intron of the immunoglobulin heavy chain constant region is a major determinant of B-cell-specific expression of immunoglobulin genes. Like other enhancers, the Ig heavy chain enhancer contains several short sequence motifs that bind specific transcription factors. Each binding site contributes to the overall activity of the enhancer, however no single element seems absolutely required for activity. For a better understanding of the Ig heavy chain enhancer components, we have cloned and analyzed individual sequence elements. We find that the factor that binds to the E3 enhancer motif, CATGTGGC, is a ubiquitous transcription factor. It is present in an active form in both B cells and non-B cells, where it can mediate transcriptional activation in vitro and in vivo. However, despite its ability to activate transcription of a transfected reporter gene, the factor is apparently unable to bind to the endogenous Ig heavy chain enhancer in non-lymphoid cells: In previous experiments by others, the characteristic in vivo footprint of this factor, designated NF-muE3, was detected in B cells but not in non-B cells. From this and other findings the picture emerges that there are at least three categories of factors which mediate cell-type-specific transcription in B lymphocytes: (a) cell-specific factors such as Oct-2A and Oct-2B that are not expressed in most other cell types: (b) ubiquitous factors such as NF-kappa B that are constitutively active in B cells but are sequestered in an inactive form in other cells; (c) ubiquitously active factors, exemplified by the one binding to the E3 sequence motif. This factor is present in an active form in a variety of cell types but is apparently unable to bind to the endogenous Ig heavy chain enhancer in non-B cells, perhaps due to a non-permissive chromatin structure of the Ig heavy chain locus. 


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Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells. 
Malignant transformation usually inhibits terminal cell differentiation but the precise mechanisms involved are not understood. PU.1 is a hematopoietic-specific Ets family transcription factor that is required for development of some lymphoid and myeloid lineages. PU.1 can also act as an oncoprotein as activation of its expression in erythroid precursors by proviral insertion or transgenesis causes erythroleukemias in mice. Restoration of terminal differentiation in the mouse erythroleukemia (MEL) cells requires a decline in the level of PU.1, indicating that PU.1 can block erythroid differentiation. Here we investigate the mechanism by which PU.1 interferes with erythroid differentiation. We find that PU.1 interacts directly with GATA-1, a zinc finger transcription factor required for erythroid differentiation. Interaction between PU.1 and GATA-1 requires intact DNA-binding domains in both proteins. PU.1 represses GATA-1-mediated transcriptional activation. Both the DNA binding and transactivation domains of PU.1 are required for repression and both domains are also needed to block terminal differentiation in MEL cells. We also show that ectopic expression of PU.1 in Xenopus embryos is sufficient to block erythropoiesis during normal development. Furthermore, introduction of exogenous GATA-1 in both MEL cells and Xenopus embryos and explants relieves the block to erythroid differentiation imposed by PU.1. Our results indicate that the stoichiometry of directly interacting but opposing transcription factors may be a crucial determinant governing processes of normal differentiation and malignant transformation. 


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Phosphorylation of TRAF2 inhibits binding to the CD40 cytoplasmic domain. 
TRAF2 is a signal transducing adaptor molecule which binds to the CD40 cytoplasmic domain. We have found that it is phosphorylated, predominantly on serine residues, when transiently overexpressed in 293 cells. The phosphorylation appears to be related to the signaling events that are activated by TRAF2 under these circumstances, since two nonfunctional mutants were found to be phosphorylated significantly less than the wild-type protein. Furthermore, the phosphorylation status of TRAF2 had significant effects on the ability of the protein to bind to CD40, as evidenced by our observations that the CD40 cytoplasmic domain interacted preferentially with underphosphorylated TRAF2 and that phosphatase treatment significantly enhanced the binding of TRAF2 to CD40. We conclude from these studies that the phosphorylation of TRAF2 is likely to play an important role in regulating signaling by virtue of its ability to influence the CD40-TRAF2 interaction. Copyright 1999 Academic Press. 


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Signaling pathways mediated by the TNF- and cytokine-receptor families target a common cis-element of the IFN regulatory factor 1 promoter. 
CD40 activation of B cells is strongly influenced by the presence of cytokines. However, the molecular basis for the interplay between these distinct stimuli is not clearly delineated. IFN regulatory factor 1 (IRF-1) is a transcription factor activated by either CD40 or cytokines. We have found that these different sets of signals target a common cis-acting element in the promoter of this gene, the IRF-1 gamma-activated site (GAS). Targeting of the IRF-1 GAS is not confined to activation via CD40 but extends to other stimuli that mimic the CD40 signaling cascade, like TNF-alpha and EBV. In contrast to induction of STATs by cytokines, the IRF-1 GAS-binding complex activated by CD40, TNF-alpha, or EBV contains Rel proteins, specifically p50 and p65. In this system, simultaneous exposure to CD40L together with either IL-4 or IFN-gamma does not lead to the activation of novel Rel/STAT complexes. Given the importance of IRF-1 in a variety of biologic functions from proliferation to apoptosis, our findings support the notion that modulation of IRF-1 levels may be a critical control point in B cell activation. 


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Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae. 
Chlamydia pneumoniae is an important respiratory pathogen. Recently, its presence has been demonstrated in atherosclerotic lesions. In this study, we characterized C. pneumoniae-mediated activation of endothelial cells and demonstrated an enhanced expression of endothelial adhesion molecules followed by subsequent rolling, adhesion, and transmigration of leukocytes (monocytes, granulocytes). These effects were blocked by mAbs against endothelial and/or leukocyte adhesion molecules (beta1 and beta2 integrins). Additionally, activation of different signal transduction pathways in C. pneumoniae-infected endothelial cells was shown: protein tyrosine phosphorylation, up-regulation of phosphorylated p42/p44 mitogen-activated protein kinase, and NF-kappaB activation/translocation occurred within 10-15 min. Increased mRNA and surface expression of E-selectin, ICAM-1, and VCAM-1 were noted within hours. Thus, C. pneumoniae triggers a cascade of events that could lead to endothelial activation, inflammation, and thrombosis, which in turn may result in or may promote atherosclerosis. 


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A functional T-cell receptor signaling pathway is required for p95vav activity. 
Stimulation of the T-cell antigen receptor (TCR) induces activation of multiple tyrosine kinases, resulting in phosphorylation of numerous intracellular substrates. One substrate is p95vav, which is expressed exclusively in hematopoietic and trophoblast cells. It contains a number of structural motifs, including Src homology 2, Src homology 3, and pleckstrin homology domains and a putative guanine nucleotide exchange domain. The role of p95vav in TCR-mediated signaling processes is unclear. Here, we show that overexpression of p95vav alone in Jurkat T cells leads to activation of the nuclear factors, including NFAT, involved in interleukin-2 expression. Furthermore, p95vav synergizes with TCR stimulation in inducing NFAT- and interleukin-2-dependent transcription. In contrast, NFAT activation by a G-protein-coupled receptor is not modulated by p95vav overexpression, suggesting that the effect is specific to the TCR signaling pathways. Although removal of the first 67 amino acids of p95vav activates its transforming potential in NIH 3T3 cells, this region appears to be required for its function in T cells. We further demonstrate that the p95vav-induced NFAT activation is not mimicked by Ras activation, though its function is dependent upon Ras and Raf. Furthermore, the activating function of p95vav is blocked by FK506, suggesting that its activity also depends on calcineurin. To further dissect p95vav involvement in TCR signaling, we analyzed various Jurkat mutants deficient in TCR signaling function or TCR expression and showed that an intact TCR signaling pathway is required for p95vav to function. However, overexpression of p95vav does not appear to influence TCR-induced protein tyrosine phosphorylation or increases in cytoplasmic free calcium. Taken together, our data suggest that p95vav plays an important role at an yet unidentified proximal position in the TCR signaling cascade. 


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Transcription factors in immune-mediated disease. 
A large amount of detailed information about the intracellular proteins regulating NF-kappa B activation and the cellular response to NF-kappa B activation has emerged recently. Several small molecules, an antisense oligonucleotide, and gene therapeutic agents that inhibit NF-kappa b activation have been described. Despite this, there are still significant gaps in our understanding of this process and its consequences. In contrast, the characterization of transcription factors selectively regulating cytokine production by CD4+ T cell subsets is at a very early stage. Three interacting proteins have recently been shown to contribute to subset-restricted expression of the IL-4 gene. There are other elements regulating IL-4 gene expression, however, and the relative importance of these recently identified proteins has yet to be determined. 


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Separation of oxidant-initiated and redox-regulated steps in the NF-kappa B signal transduction pathway. 
Studies presented here show that overall NF-kappa B signal transduction begins with a parallel series of stimuli-specific pathways through which cytokines (tumor necrosis factor alpha), oxidants (hydrogen peroxide and mitomycin C), and phorbol ester (phorbol 12-myristate 13-acetate) individually initiate signaling. These initial pathways culminate in a common pathway through which all of the stimulating agents ultimately signal NF-kappa B activation. We distinguish the stimuli-specific pathways by showing that the oxidative stimuli trigger NF-kappa B activation in only one of two human T-cell lines (Wurzburg but not Jurkat), whereas tumor necrosis factor alpha and phorbol 12-myristate 13-acetate readily stimulate in both lines. We propose the common pathway as the simplest way of accounting for the common requirements and properties of the signaling pathway. We include a redox-regulatory mechanism(s) in this common pathway to account for the previously demonstrated redox regulation of NF-kappa B activation in Jurkat cells (in which oxidants don't activate NF-kappa B); we put tyrosine phosphorylation in the common pathway by showing that kinase activity (inhibitable by herbimycin A and tyrphostin 47) is required for NF-kappa B activation by all stimuli tested in both cell lines. Since internal sites of oxidant production have been shown to play a key role in the cytokine-stimulated activation of NF-kappa B, and since tyrosine kinase and phosphatase activities are known to be altered by oxidants, these findings suggest that intracellular redox status controls NF-kappa B activation by regulating tyrosine phosphorylation event(s) within the common step of the NF-kappa B signal transduction pathway. 


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CD30 ligation induces nuclear factor-kappa B activation in human T cell lines. 
CD30 is a recently described member of the tumor necrosis factor/nerve growth factor receptor superfamily. In this report, we show that following incubation of L540 cells (Hodgkin's disease-derived, T cell-like, CD30+ cells) with the agonistic anti-CD30 monoclonal antibodies (mAb) M44 and M67, two nuclear factor (NF)-kappa B DNA binding activities were induced in nuclear extracts, as determined in gel retardation assays. The effect of the mAb towards NF-kappa B activation was rapid, as it occurred within 20 min, and was sustained for up to 6 h. By comparison, an isotype-matched antibody had no effect on NF-kappa B activation. Moreover, in human T helper (Th) clones functionally characterized as being of the type 0, type 1 and type 2 (28%, < 1% und 93% CD30+, respectively), the extent of CD30-mediated NF-kappa B activation correlated with the proportion of CD30+ cells. In all cell lines investigated, the NF-kappa B complexes induced following CD30 engagement were shown to contain p50 NF-kappa B1, p65 RelA, and possibly other transcription factors. Collectively, our results demonstrate that nuclear translocation and activation of NF-kappa B rank among the short-term cellular responses elicited following CD30 ligation. 


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Downregulation of Wilms' tumor gene (WT1) is not a prerequisite for erythroid or megakaryocytic differentiation of the leukemic cell line K562. 
The Wilms' tumor gene (WT1) encodes a transcription factor of the zinc finger type. A high expression of WT1 has been detected in a range of acute leukemias, and WT1 is downregulated during induced differentiation of some leukemic cell lines. Overexpression of WT1 in some myeloid cell lines confers resistance to differentiation induction. These observations suggest that a high WT1 expression in hematopoietic cells is incompatible with differentiation. In this study, each of the four different isoforms of WT1 was constitutively overexpressed in the leukemic cell line K562. K562 cells express endogenous WT1, which is downregulated as a response to induced differentiation along the erythroid and megakaryocytic pathways. We now demonstrate that a forced exogenous expression of the four different isoforms of WT1 in K562 does not affect the differentiation response, as judged by accumulation of hemoglobin in response to hemin or the expression of megakaryocytic cell surface markers in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). We conclude that downregulation of WT1 during induced differentiation of K562 cells is not a prerequisite for erythroid or megakaryocytic differentiation of these cells. 


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A critical role of the p75 tumor necrosis factor receptor (p75TNF-R) in organ inflammation independent of TNF, lymphotoxin alpha, or the p55TNF-R. 
Despite overwhelming evidence that enhanced production of the p75 tumor necrosis factor receptor (p75TNF-R) accompanies development of specific human inflammatory pathologies such as multi-organ failure during sepsis, inflammatory liver disease, pancreatitis, respiratory distress syndrome, or AIDS, the function of this receptor remains poorly defined in vivo. We show here that at levels relevant to human disease, production of the human p75TNF-R in transgenic mice results in a severe inflammatory syndrome involving mainly the pancreas, liver, kidney, and lung, and characterized by constitutively increased NF-kappaB activity in the peripheral blood mononuclear cell compartment. This process is shown to evolve independently of the presence of TNF, lymphotoxin alpha, or the p55TNF-R, although coexpression of a human TNF transgene accelerated pathology. These results establish an independent role for enhanced p75TNF-R production in the pathogenesis of inflammatory disease and implicate the direct involvement of this receptor in a wide range of human inflammatory pathologies. 


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A novel Ets-related transcription factor, Elf-1, binds to human immunodeficiency virus type 2 regulatory elements that are required for inducible trans activation in T cells. 
Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are structurally related retroviruses which both cause AIDS in humans. Although both viruses establish latency in quiescent human-peripheral-blood T cells, the asymptomatic phase of HIV-2 infection may be more prolonged than that of HIV-1. The latent phases of both HIV-1 and HIV-2 infection have been shown to be disrupted by T-cell activation, a process that requires host cell transcription factors. In the case of HIV-1, the transcription factor NF-kappa B is sufficient for inducible transcriptional activation. In contrast, factors in addition to NF-kappa B are required to activate HIV-2 transcription in infected T cells. In this report, we demonstrate that a novel Ets-related transcription factor, Elf-1, binds specifically to two purine-rich motifs in the HIV-2 enhancer. Mutagenesis experiments demonstrated that these Elf-1 binding sites are required for induction of HIV-2 transcription following T-cell-receptor-mediated T-cell activation. Moreover, Elf-1 is the only factor present in activated T-cell nuclear extracts that binds to these sites in electrophoretic mobility shift assays. Thus, Elf-1 is a novel transcription factor that appears to be required for the T-cell-receptor-mediated trans activation of HIV-2 gene expression. These results may explain differences in the clinical spectra of diseases caused by HIV-1 and HIV-2 and may also have implications for the design of therapeutic approaches to HIV-2 infection. 


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A T cell-specific enhancer in the interleukin-3 locus is activated cooperatively by Oct and NFAT elements within a DNase I-hypersensitive site. 
Interleukin-3 (IL-3) is a cytokine that is expressed primarily in activated T cells. Here we identified an inducible T cell-specific enhancer 14 kb upstream of the IL-3 gene that responded to activation of T cell receptor signaling pathways. The IL-3 enhancer spanned an inducible cyclosporin A-sensitive DNase I-hypersensitive site found only in T cells. Four NFAT-like elements exist within the enhancer. The two most active NFAT-like elements were located at the center of the DNase I-hypersensitive site. One of these NFAT-like elements encompassed overlapping Oct- and NFATp/c-binding sites, which functioned in a highly synergistic manner. We suggest that the T cell-specific expression of the IL-3 gene is partly controlled through the enhancer by cooperation between Oct and NFAT family proteins. 


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An allosteric drug, o,o'-bismyristoyl thiamine disulfide, suppresses HIV-1 replication through prevention of nuclear translocation of both HIV-1 Tat and NF-kappa B. 
The efficacy of o,o'-bismyristoyl thiamine disulfide (BMT) was examined in detail against HIV-1 laboratory isolates (HTLV-IIIB, JRFL, and MN), primary isolates (KMT and KMO), and simian immunodeficiency virus (SIVmac251) in vitro. BMT inhibited the replication of HIV-1 in both laboratory and primary isolates in vitro. In addition, BMT exhibited antiviral activity against SIVmac251. Minimizing energy studies of BMT structure reveal that a trans-disulfide of thiamine (holo drug) disulfide (TDS, protodrug) is allosterically transited to the reactive twisted disulfide of BMT (allo drug) by o,o'-bismyristoyl esterification of TDS. BMT inhibits nuclear translocation of both HIV-1 transactivator (TAT) and the cellular transcriptional nuclear factor-KB (NF-kappa B), resulting in the suppression of HIV-1 replication. 


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Activation of nuclear factor kappa B in human lymphoblastoid cells by low-dose ionizing radiation. 
Nuclear factor kappa B (NF-kappa B) is a pleiotropic transcription factor which is involved in the transcriptional regulation of several specific genes. Recent reports demonstrated that ionizing radiation in the dose range of 2-50 Gy results in expression of NF-kappa B in human KG-1 myeloid leukemia cells and human B-lymphocyte precursor cells; the precise mechanism involved and the significance are not yet known. The present report demonstrates that even lower doses of ionizing radiation, 0.25-2.0 Gy, are capable of inducing expression of NF-kappa B in EBV-transformed 244B human lymphoblastoid cells. These results are in a dose range where the viability of the cells remains very high. After exposure to 137Cs gamma rays at a dose rate of 1.17 Gy/min, a maximum in expression of NF-kappa B was seen at 8 h after a 0.5-Gy exposure. Time-course studies revealed a biphasic time-dependent expression after 0.5-, 1- and 2-Gy exposures. However, for each time examined, the expression of NF-kappa B was maximum after the 0.5-Gy exposure. The expression of the p50 and p65 NF-kappa B subunits was also shown to be regulated differentially after exposures to 1.0 and 2.0 Gy. 


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Interleukin-10 stabilizes inhibitory kappaB-alpha in human monocytes. 
Interleukin-10 (IL-10) protects animals from lethal endotoxemia. This beneficial effect is mediated, in part, by inhibition of inflammatory cytokine production, including tumor necrosis factor-alpha (TNF-alpha). Evidence suggests that IL-10 may inhibit activation of the transcription factor nuclear factor-kappaB (NF-kappaB) through an unknown mechanism. NF-kappaB activation in response to inflammatory signals is dependent upon degradation of its associated inhibitory peptide, inhibitory kappaB-alpha (IkappaB-alpha). We hypothesized that IL-10 prevents human monocyte NF-kappaB activation and resultant TNF-alpha production by stabilization of IkappaB-alpha. The purpose of this study was to determine the effect of IL-10 on lipopolysaccharide (LPS)-induced human monocyte TNF-alpha production, NF-kappaB activation, and IkappaB-alpha degradation. Monocytes were isolated from human donors. Cells were stimulated with endotoxin (LPS, 100 ng/mL) with and without human IL-10 (10 ng/mL). Following stimulation, TNF-alpha was measured in cell supernatants by ELISA, NF-kappaB activity by electrophoretic mobility shift assay, and IkappaB-alpha levels by Western blot. We observed that after LPS stimulation of human monocytes, TNF-alpha increased to 798+/-67 pg/mL (p < .001 versus control). IL-10 attenuated LPS-stimulated TNF-alpha production (297+/-54; p < .001 versus LPS alone). After LPS stimulation in human monocytes, IkappaB-alpha protein levels decreased, and NF-kappaB DNA binding increased. IL-10 pretreatment prevented LPS-induced decreases in IkappaB-alpha protein levels and attenuated NF-kappaB DNA binding. IL-10 appears to prevent activation of NF-kappaB by preserving IkappaB-alpha protein levels, leading to a reduction in TNF-alpha release. 


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Human alveolar macrophages are markedly deficient in REF-1 and AP-1 DNA binding activity. 
Although many functions of human alveolar macrophages are altered compared with their precursor cell, the blood monocyte (monocyte), the reason(s) for these functional changes have not been determined. We recently reported that human alveolar macrophages do not express AP-1 DNA binding activity (Monick, M. M., Carter, A. B., Gudmundsson, G., Geist, L. J., and Hunninghake, G. W. (1998) Am. J. Physiol. 275, L389-L397). To determine why alveolar macrophages do not express AP-1 DNA binding activity, we first showed that there was not a decrease in expression of the FOS and JUN proteins that make up the AP-1 complex. There was, however, a significant difference in the amounts of the nuclear protein, REF-1 (which regulates AP-1 DNA binding by altering the redox status of FOS and JUN proteins), in alveolar macrophages compared with monocytes. In addition, in vitro differentiation of monocytes to a macrophage-like cell resulted in decreased amounts of REF-1. Finally, addition of REF-1 from activated monocytes to alveolar macrophage nuclear proteins resulted in a marked increase in AP-1 DNA binding. These studies strongly suggest that the process of differentiation of monocytes into alveolar macrophages is associated with a loss of REF-1 and AP-1 activity. This observation may explain, in part, some of the functional differences observed for alveolar macrophages compared with monocytes. 


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Control of cell cycle entry and apoptosis in B lymphocytes infected by Epstein-Barr virus. 
Infection of human B cells with Epstein-Barr virus (EBV) results in activation of the cell cycle and cell growth. To interpret the mechanisms by which EBV activates the cell, we have assayed many proteins involved in control of the G0 and G1 phases of the cell cycle and regulation of apoptosis. In EBV infection most of the changes, including the early induction of cyclin D2, are dependent on expression of EBV genes, but an alteration in the E2F-4 profile was partly independent of viral gene expression, presumably occurring in response to signal transduction activated when the virus binds to its receptor, CD21. By comparing the expression of genes controlling apoptosis, including those encoding several members of the BCL-2 family of proteins, the known relative resistance of EBV-immortalized B-cell lines to apoptosis induced by low serum was found to correlate with expression of both BCL-2 and A20. A20 can be regulated by the NF-kappaB transcription factor, which is known to be activated by the EBV LMP-1 protein. Quantitative assays demonstrated a direct temporal relationship between LMP-1 protein levels and active NF-kappaB during the time course of infection. 


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Induction of immediate early response genes by macrophage colony-stimulating factor in normal human monocytes. 
A group of coordinately induced protooncogenes, cytoskeletal, and extracellular matrix genes have been termed immediate early response genes, and their induction has been associated with growth factor-stimulated cell proliferation. We have investigated the induction of these genes by macrophage-CSF (M-CSF) in human monocytes that do not proliferate in response to M-CSF but require the factor for optimal cell differentiation. Normal human monocytes were isolated, carefully washed, and incubated for 36 to 48 h in fetal bovine serum-containing medium. At the end of this incubation the resting cells were stimulated with M-CSF, and RNA was isolated for analysis by Northern blotting. RNA from control resting cells contained low to undetectable levels of c-jun, fibronectin receptor, and actin mRNA. Within 15 to 30 min of addition of M-CSF, however, there was a dramatic coordinate induction of these genes. The c-jun gene expression was very transient and was not detectable by 60 min after M-CSF addition. In contrast, the expression of actin and fibronectin receptor mRNA was more sustained, and the expression of these genes remained elevated at 24 to 48 h after M-CSF addition. We also observed the induction of the myelomonocytic specific tyrosine kinase hck gene simultaneously with the other immediate early response genes. The protein synthesis inhibitor cycloheximide did not block the induction of any of these genes, and in fact, super-induced the expression of c-jun and hck. Nuclear run on transcription of the c-jun, hck, and actin genes. Therefore, in normal human monocytes M-CSF induces immediate early response genes without inducing cell proliferation. These genes may then play a role in altering the physiologic status of the cells in response to CSF. 


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An active v-abl protein tyrosine kinase blocks immunoglobulin light-chain gene rearrangement. 
Lymphoid cells transformed by Abelson murine leukemia virus have provided one of the classic models for study of early B-cell development and immunoglobulin rearrangement. Most of these cells have rearranged their heavy-chain locus but not their light chain genes, suggesting that an active v-abl protein interferes with this differentiation step. To test this hypothesis, light-chain gene structure was examined in pre-B cells transformed by temperature-sensitive mutants of the Abelson virus and in derivatives that survive at the nonpermissive temperature because they express a human BCL-2 gene. Our studies reveal that inactivation of the v-abl protein tyrosine kinase triggers high-frequency rearrangement of kappa and lambda light-chain genes. These events are accompanied by marked increases in the expression of RAG-1 and RAG-2 RNAs. These increases occur in the absence of protein synthesis but are dependent on inactivation of the v-abl protein tyrosine kinase. As documented in the accompanying paper (Klug et al., this issue), an active v-abl protein also suppresses the activity of NF-kappa B/rel and expression controlled by the kappa intron enhancer. Together these data demonstrate that the v-abl protein specifically interferes with light-chain gene rearrangement by suppressing at least two pathways essential for this stage of B-cell differentiation and suggest that tyrosine phosphorylation is important in regulating RAG gene expression. 


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Concomitant downregulation of IgH 3' enhancer activity and c-myc expression in a plasmacytoma x fibroblast environment: implications for dysregulation of translocated c-myc. 
Regulation of immunoglobulin heavy chain (IgH) gene expression is controlled by a B cell-specific promoter, intronic enhancer and additional B cell-specific enhancer elements identified recently in the 3' end of the IgH locus. One of the latter elements, the IgH 3' enhancer, is of particular interest: (1) it is B cell-specific and active only in late B cell development; (2) in rodent plasmacytomas and in some human Burkitt's lymphomas it is part of a locus control region (LCR) that is involved in deregulation of the c-myc oncogene as a result of translocation into the IgH locus; and (3) it has been implicated in the mechanisms that control Ig gene class switch recombination. We have used a somatic cell hybridization approach to genetically analyse regulation of the activity of the IgH 3' enhancer. When mouse MPC11 plasmacytoma cells, in which the IgH 3' enhancer is active, are fused with fibroblasts, Ig expression is extinguished at the level of transcription. Here we show that in a MPC11 plasmacytoma x fibroblast environment, the IgH 3' enhancer is transcriptionally inactive. Furthermore, we demonstrate that binding of several B cell-specific transcription factors, essential for IgH 3' enhancer activity, is lacking, which may explain 3' enhancer inactivity, although the binding of repressors cannot be excluded. Moreover, the high expression level of c-myc, characteristic of the parental MPC11 cells carrying the t(12;15) translocation, is down-regulated in the hybrids to that in unfused fibroblasts. Therefore, inactivation of the IgH 3' enhancer is a multifactorial process affecting several transcription factors that control the cell-specific and developmental activity of the enhancer. 


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Sp1 is a critical factor for the monocytic specific expression of human CD14. 
CD14 is a membrane glycoprotein expressed specifically on monocytes and macrophages, and its expression is markedly increased during the process of monocyte differentiation. In order to study CD14 gene regulation, the human CD14 gene was cloned from a partial EcoRI digested chromosome 5 library. A 5.5-kilobase genomic clone contained the full-length CD14 coding sequence and 4.2 kilobases of 5'-upstream sequence. One major and one minor transcription start site were identified 101 and 130 base pairs (bp) upstream, respectively, from the protein translation start ATG. A DNA fragment containing 128 bp of upstream sequence had strong, monocyte-specific promoter activity in the CD14 positive monocytic cell line Mono Mac 6 as compared to the nonmonocytic cell lines HeLa and REX. Four regions in this DNA fragment interact with nuclear proteins isolated from monocytic cells. The Sp1 transcription factor bound to three different regions in the CD14 promoter. Mutation of the major Sp1 binding site (-110 bp) decreased tissue-specific promoter activity, and these results, together with transactivation experiments, demonstrate that Sp1 plays a critical role in the tissue-specific expression of CD14 in monocytic cells. CD14 Sp1 site oligonucleotides bound preferentially to a 105-kDa Sp1 species, which is present in higher relative levels in monocytic than non-monocytic cells, suggesting that modification of Sp1, such as phosphorylation, may explain how the Sp1 site mediates monocytic specific promoter activity. 


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Redox signals and NF-kappaB activation in T cells. 
Accumulating data from a number of laboratories have recently indicated that the response of transcription factor NF-kappaB to alterations in the redox homeostasis of cells may play an important role in modulating immune function. The activation of NF-kappaB has been recognized to regulate a number of genes necessary for normal T cell responses including IL-2, IL-6, IL-8, and several T cell surface receptors. Diminished NF-kappaB activity has been shown to occur in T cells with aging, suggesting that impaired activation of NF-kappaB might occur during cellular senescence. In addition, aberrancies in NF-kappaB activity have been implicated in the immunopathogenesis of diseases involving immune or inflammatory processes such as atherosclerosis and HIV-1 infection. The role of H2O2 and other reactive oxygen species (ROS) as an integratory secondary messenger for divergent T cell signals has been complicated by the fact that various T cell lines and peripheral blood T cells differ markedly in the levels of NF-kappaB activation induced by oxidant stress. Additionally, proposed pathways of NF-kappaB activation have been based on indirect evidence provided by experiments which used antioxidants to inhibit active NF-kappaB formation. Further, complete activation of T cells requires at least two signals, one that stimulates an increase in intracellular calcium and one that stimulates enzymatic processes including kinases. Similarly, substantial evidence indicates that full activation of NF-kappaB requires dual signals. The ability of H2O2 or other ROS to induce T cell signals and functional responses by these two mechanisms is reviewed and the specific response of NF-kappaB to redox changes in T cells is examined. Data are also presented to suggest that the redox regulation in NF-kappaB activation may be relevant to immune-related diseases and to aging. 


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PGG-glucan, a soluble beta-(1,3)-glucan, enhances the oxidative burst response, microbicidal activity, and activates an NF-kappa B-like factor in human PMN: evidence for a glycosphingolipid beta-(1,3)-glucan receptor. 
PGG-Glucan, a soluble beta-(1,6)-branched beta-(1,3)-linked glucose homopolymer derived from the cell wall of the yeast Saccharomyces cerevisiae, is an immunomodulator which enhances leukocyte anti-infective activity and enhances myeloid and megakaryocyte progenitor proliferation. Incubation of human whole blood with PGG-Glucan significantly enhanced the oxidative burst response of subsequently isolated blood leukocytes to both soluble and particulate activators in a dose-dependent manner, and increased leukocyte microbicidal activity. No evidence for inflammatory cytokine production was obtained under these conditions. Electrophoretic mobility shift assays demonstrated that PGG-Glucan induced the activation of an NF-kappaB-like nuclear transcription factor in purified human neutrophils. The binding of 3H-PGG-Glucan to human leukocyte membranes was specific, concentration-dependent, saturable, and high affinity (Kd approximately 6 nM). A monoclonal antibody specific to the glycosphingolipid lactosylceramide was able to inhibit activation of the NF-kappaB-like factor by PGG-Glucan, and ligand binding data, including polysaccharide specificity, suggested that the PGG-Glucan binding moiety was lactosylceramide. These results indicate that PGG-Glucan enhances neutrophil anti-microbial functions and that interaction between this beta-glucan and human neutrophils is mediated by the glycosphingolipid lactosylceramide present at the cell surface. 


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Cloning of the novel human myeloid-cell-specific C/EBP-epsilon transcription factor. 
Chicken NF-M transcription factor, in cooperation with either c-Myb or v-Myb, is active in the combinatorial activation of myeloid-cell-specific genes in heterologous cell types, such as embryonic fibroblasts. In humans, similar effects were observed with homologous members of the CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators, especially the human homolog of chicken NF-M, C/EBP-beta (NF-IL6). However, the NF-IL6 gene is expressed in a variety of nonmyeloid cell types and is strongly inducible in response to inflammatory stimuli, making it an unlikely candidate to have an exclusive role as a combinatorial differentiation switch during myelopoiesis in human cells. By using a reverse transcription-PCR-based approach and a set of primers specific for the DNA-binding domains of highly homologous members of the C/EBP family of transcriptional regulators, we have cloned a novel human gene encoding a member of the C/EBP gene family, identified as the human homolog of CRP1, C/EBP-epsilon. A 1.2-kb cDNA encoding full-length human C/EBP-epsilon was cloned from a promyelocyte-late myeloblast-derived lambda gt11 library. Molecular analysis of the cDNA and genomic clones indicated the presence of two exons encoding a protein with an apparent molecular mass of 32 kDa and a pI of 9.5. Primer extension analysis of C/EBP-epsilon mRNA detected a single major transcription start site approximately 200 bp upstream of the start codon. The putative promoter area is similar to those of several other myeloid-cell-specific genes in that it contains no TATAAA box but has a number of purine-rich stretches with multiple sites for the factors of the Ets family of transcriptional regulators. Northern blot analyses indicated a highly restricted mRNA expression pattern, with the strongest expression occurring in promyelocyte and late-myeloblast-like cell lines. Western blot and immunoprecipitation studies using rabbit anti-C/EBP-epsilon antibodies raised against the N-terminal portion of C/EBP-epsilon (amino acids 1 to 115) showed that C/EBP-epsilon is a 32-kDa nuclear phosphoprotein. The human C/EBP-epsilon protein exhibited strong and specific binding to double-stranded DNA containing consensus C/EBP sites. Cotransfection of the C/EBP-epsilon sense and antisense expression constructs together with chloramphenicol acetyltransferase reporter vectors containing myeloid-cell-specific c-mim and human myeloperoxidase promoters suggested a role for C/EBP-epsilon transcription factor in the regulation of a subset of myeloid-cell-specific genes. Transient tranfection of a promyelocyte cell line (NB4) with a C/EBP-epsilon expression plasmid increased cell growth by sevenfold, while antisense C/EBP-epsilon caused a fivefold decrease in clonal growth of these cells. 


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Regulation of the Ets-related transcription factor Elf-1 by binding to the retinoblastoma protein. 
The retinoblastoma gene product (Rb) is a nuclear phosphoprotein that regulates cell cycle progression. Elf-1 is a lymphoid-specific Ets transcription factor that regulates inducible gene expression during T cell activation. In this report, it is demonstrated that Elf-1 contains a sequence motif that is highly related to the Rb binding sites of several viral oncoproteins and binds to the pocket region of Rb both in vitro and in vivo. Elf-1 binds exclusively to the underphosphorylated form of Rb and fails to bind to Rb mutants derived from patients with retinoblastoma. Co-immunoprecipitation experiments demonstrated an association between Elf-1 and Rb in resting normal human T cells. After T cell activation, the phosphorylation of Rb results in the release of Elf-1, which is correlated temporally with the activation of Elf-1-mediated transcription. Overexpression of a phosphorylation-defective form of Rb inhibited Elf-1-dependent transcription during T cell activation. These results demonstrate that Rb interacts specifically with a lineage-restricted Ets transcription factor. This regulated interaction may be important for the coordination of lineage-specific effector functions such as lymphokine production with cell cycle progression in activated T cells. 


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Human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1- and NF-kappaB-binding sites of the interleukin-8 gene. 
Cytomegalovirus (CMV) infection induced interleukin-8 (IL-8) gene transcription in a human monocytic cell line, THP-1 cells, leading to IL-8 secretion. The functional analysis of the IL-8 gene revealed that both AP-1- and NF-kappaB factor-binding elements were involved in conferring the responsiveness to CMV. Moreover, electrophoretic mobility shift assays demonstrated that CMV induced the formation of NF-kappaB and AP-1 complexes. These results suggest that CMV activates these transcriptional factors, resulting in IL-8 gene expression. 


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The NF kappa B independent cis-acting sequences in HIV-1 LTR responsive to T-cell activation. 
The rate of transcription initiation directed by the long terminal repeat (LTR) of HIV-1 increases in response to mitogenic stimuli of T cells. Here we show that the response of the HIV-1 LTR may be governed by two independent sequences located 5' to the site of transcription initiation sequences that bind either NFAT-1 or NF kappa B. The rate of LTR-directed gene expression increased in response to treatment with either a phorbol ester or tumor necrosis factor alpha if either the NFAT-1 or NF kappa B binding sites were deleted, but failed to respond to these mitogenic stimuli if both sequences were absent. The HIV-1 mutant virus containing both NF kappa B and NFAT-1 deletion was able to replicate although at a much decreased growth rate, while the deletion of NFAT-1 alone increased the viral growth rate in Jurkat cells. Neither deletion of NF kappa B nor deletion of NFAT-1 decreased activation of viral replication by phorbol ester. 


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Control of NF-kappa B activity by the I kappa B beta inhibitor. 
The transcription factor NF-kappa B is maintained in an inactive cytoplasmic state by I kappa B inhibitors. In mammalian cells, I kappa B alpha and I kappa B beta proteins have been purified and shown to be the inhibitors of NF-kappa B through their association with the p65 or c-Rel subunits. In addition, we have isolated a third NF-kappa B inhibitor, I kappa B epsilon (1). Upon treatment with a large variety of inducers, I kappa B alpha, I kappa B beta are proteolytically degraded, resulting in NF-kappa B translocation into the nucleus. Here we show that in E29.1 T cell hybridoma I kappa B alpha and I kappa B beta are equally associated with p65 and that I kappa B beta is degraded in response to TNF alpha in contrast to what has been originally published. Our data also suggest that, unlike I kappa B alpha, I kappa B beta is constitutively phosphorylated and resynthesized as a hypophosphorylated form. The absence of slow migrating forms of I kappa B beta following stimulation suggests that the phosphorylation does not necessarily constitute the signal-induced event which targets the molecule for proteolysis. 


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Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site. 
Exposure of monocytic cells to bacterial lipopolysaccharide (LPS) activates the NF-kappa B/Rel family of proteins and leads to the rapid induction of inflammatory gene products, including tissue factor (TF). TF is the primary cellular initiator of the coagulation protease cascades. Here we report the characterization of a nuclear complex from human monocytic cells that bound to a kappa B-like site, 5'-CGGAGTTTCC-3', in the 5'-flanking region of the human TF gene. This nuclear complex was activated by LPS with kinetics that preceded induction of the TF gene. In vitro binding studies demonstrated that the TF site bound translated c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 homodimers. Base-pair substitutions in the TF site indicated that the presence of a cytosine at position 1 precluded binding of NF-kappa B. In fact, under low-ionic-strength conditions, the TF complex did not migrate with translated p50/p65 dimers but instead comigrated with c-Rel/p65 dimers. Antibodies against the NF-kappa B and Rel proteins and UV cross-linking studies revealed the presence of c-Rel and p65 and the absence of p50 in the TF complex and further showed that c-Rel/p65 heterodimers selectively bound to the TF kappa B-like site. Functional studies indicated that the TF site conferred LPS inducibility on a heterologous promoter and was transactivated by c-Rel or p65. Taken together, our results demonstrated that binding of c-Rel/p65 heterodimers to a novel kappa B-like site mediated LPS induction of TF gene expression in monocytic cells. 


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A thiol antioxidant regulates IgE isotype switching by inhibiting activation of nuclear factor-kappaB. 
The binding site for nuclear factor-kappaB (NF-kappaB) is present at the promoter region of the germline Cepsilon gene, but there is little information on whether this factor is involved in regulating IgE synthesis by human B cells. Accordingly, we studied the role of NF-kappaB in germline Cepsilon transcription by using two human Burkitt's lymphoma B cell lines, DND39 and DG75. In both cell lines, n-acetyl-L-cysteine (NAC), a potent thiol antioxidant, inhibited the triggering of the nuclear expression of NF-kappaB by IL-4 and by anti-CD40 monoclonal antibody. Although IL-4 activated signal transducers and activators of transcription (STAT) 6 in addition to NF-kappaB, NAC treatment or the transfection of decoy oligodeoxynucleotides for NF-kappaB or STAT6 only partly blocked IL-4-induced germline Cepsilon transcription. However, these two decoy oligodeoxynucleotides together almost completely abrogated IL-4-induced germline Cepsilon transcription. Of note, CD40-mediated enhancement of IL-4-driven germline Cepsilon transcription was markedly decreased by NAC or by a decoy oligodeoxynucleotide for NF-kappaB. The effect of NAC was also examined on deletional switch recombination underlying the isotype switch to IgE. NAC inhibited the generation of Smu/Sepsilon switch fragments in normal human B cells costimulated with IL-4 and anti-CD40 monoclonal antibody. It also abolished IL-4-induced upregulation of CD40 but promoted upregulation of CD23. These results suggest that coordination of NF-kappaB and STAT6 may be required for induction of germline Cepsilon transcription by IL-4, and that CD40-mediated NF-kappaB activation may be important in regulating both enhancement of germline Cepsilon transcription and class switching to IgE. 


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Two distinct pathways of interleukin-5 synthesis in allergen-specific human T-cell clones are suppressed by glucocorticoids. 
Glucocorticoids (GC) have long been used as the most effective agents for the treatment of allergic diseases accompanied by eosinophilia such as chronic asthma and atopic dermatitis. The development of chronic eosinophilic inflammation is dependent on interleukin-5 (IL-5), a selective eosinophil-activating factor, produced by helper T cells. To delineate the regulatory mechanisms of human IL-5 synthesis, we established allergen-specific CD4+ T-cell clones from asthmatic patients. GC efficiently suppressed IL-5 synthesis of T-cell clones activated via either T-cell receptor (TCR) or IL-2 receptor (IL-2R). Induction of IL-5 mRNA upon TCR and IL-2R stimulation was totally inhibited by dexamethasone. Human IL-5 promoter/enhancer-luciferase gene construct transfected to T-cell clones was transcribed on either TCR or IL-2R stimulation and was clearly downregulated by dexamethasone, indicating that the approximately 500-bp human IL-5 gene segment located 5' upstream of the coding region contains activation-inducible enhancer elements responsible for the regulation by GC. Electrophoretic mobility shift assay analysis suggested that AP-1 and NF-kappaB are among the possible targets of GC actions on TCR-stimulated T cells. NF-AT and NF-kappaB were not significantly induced by IL-2 stimulation. Our results showing that GC suppressed IL-5 production by human CD4+ T cells activated by two distinct stimuli, TCR and IL-2R stimulation, underscore the efficacy of GC in the treatment of allergic diseases via suppression of T-cell IL-5 synthesis. 


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Transcription of a minimal promoter from the NF-IL6 gene is regulated by CREB/ATF and SP1 proteins in U937 promonocytic cells. 
NF-IL6 is an important transcriptional regulator of genes induced in activated monocytes/macrophages, and NF-IL6 is the only CCAAT/enhancer-binding protein (C/EBP) family member whose steady-state mRNA levels increase upon activation of monocytes (1). We show that increased transcription of the NF-IL6 gene is responsible, at least in part, for induction of NF-IL6 mRNA following activation of U937 promonocytic cells. We have identified a 104-bp minimal promoter region of the NF-IL6 gene that is sufficient for basal and activation-dependent induction of transcription in U937 cells. This region contains binding sites for the cAMP response element-binding protein/activation transcription factor (CREB/ATF) and Sp1 families of transcription factors. Each site is functionally important and contributes independently to transcription of the NF-IL6 gene in U937 cells. 


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Identification of nucleotide sequences that regulate transcription of the MCF13 murine leukemia virus long terminal repeat in activated T cells. 
The region downstream of the enhancer (DEN) of the long terminal repeat of the mink cell focus-forming murine leukemia virus is important for viral pathogenicity. Another important activity of DEN is its control of transcription in activated T cells, and we have determined that an NF-kappaB site is critical for this activity. 


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Lymphoid specific gene expression of the adenovirus early region 3 promoter is mediated by NF-kappa B binding motifs. 
A primary site of infection by human adenoviruses is lymphoid cells. However, analysis of the viral control elements and the cellular factors that regulate adenoviral gene expression in lymphocytes has not been reported. The adenovirus early region 3 (ES) gene products are involved in the maintenance of viral persistence by complexing with the class I MHC antigens, thus preventing their cell surface expression with a resultant decrease in host immunologic destruction. To determine whether different cellular factors were involved in E3 regulation in lymphocytes as compared with HeLa cells, both DNA binding and transfection analysis with the E3 promoter in both cell types were performed. These studies detected two novel domains referred to as L1 and L2 with a variety of lymphoid but not HeLa extracts. Each of these domains possessed strong homology to motifs previously found to bind the cellular factor NF-kappa B. Transfections of E3 constructs linked to the chloramphenicol acetyltransferase gene revealed that mutagenesis of the distal NF-kappa B motif (L2) had minimal effects on promoter expression in HeLa cells, but resulted in dramatic decreases in expression by lymphoid cells. In contrast, mutagenesis of proximal NF-kappa B motif (L1) had minimal effects on gene expression in both HeLa cells and lymphoid cells but resulted in a small, but reproducible, increase in gene expression in lymphoid cells when coupled to the L2 mutation. Reversing the position and subsequent mutagenesis of the L1 and L2 domains indicated that the primary sequence of these motifs rather than their position in the E3 promoter was critical for regulating gene expression. (ABSTRACT TRUNCATED AT 250 WORDS) 


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The Megakaryocyte/Platelet-specific enhancer of the alpha2beta1 integrin gene: two tandem AP1 sites and the mitogen-activated protein kinase signaling cascade. 
The alpha2beta1 integrin, a collagen receptor on platelets and megakaryocytes, is required for normal platelet function. Transcriptional regulation of the alpha2 integrin gene in cells undergoing megakaryocytic differentiation requires a core promoter between bp -30 and -92, a silencer between bp -92 and -351, and megakaryocytic enhancers in the distal 5' flank. We have now identified a 229-bp region of the distal 5' flank of the alpha2 integrin gene required for high-level enhancer activity in cells with megakaryocytic features. Two tandem AP1 binding sites with dyad symmetry are required for enhancer activity and for DNA-protein complex formation with members of the c-fos/c-jun family. The requirement for AP1 activation suggested a role for the mitogen-activated protein kinase (MAPK) signaling pathway in regulating alpha2 integrin gene expression. Inhibition of the MAP kinase cascade with PD98059, a specific inhibitor of MAPK kinase 1, prevented the expression of the alpha2 integrin subunit in cells induced to become megakaryocytic. We provide a model of megakaryocytic differentiation in which expression of the alpha2 integrin gene requires signaling via the MAP kinase pathway to activate two tandem AP1 binding sites in the alpha2 integrin enhancer. 


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Constitutive NF-kappa B activation, enhanced granulopoiesis, and neonatal lethality in I kappa B alpha-deficient mice. 
Transcription factors belonging to the NF-kappa B family are controlled by inhibitory I kappa B proteins, mainly I kappa B alpha and I kappa B beta. Apparently normal at birth, I kappa B alpha-/- mice exhibit severe runting, skin defects, and extensive granulopoiesis postnatally, typically dying by 8 days. Hematopoietic tissues from these mice display elevated levels of both nuclear NF-kappa B and mRNAs of some, but not all, genes thought to be regulated by NF-kappa B. NF-kappa B elevation results in these phenotypic abnormalities because mice lacking both I kappa B alpha and the p50 subunit of NF-kappa B show a dramatically delayed onset of abnormalities. In contrast to hematopoietic cells, I kappa B alpha-/- embryonic fibroblasts show minimal constitutive NF-kappa B, as well as normal signal-dependent NF-kappa B activation that is concomitant with I kappa B beta degradation. Our results indicate that I kappa b beta, but not I kappa B alpha, is required for the signal-dependent activation of NF-kappa B in fibroblasts. However, I kappa B alpha is required for the postinduction repression of NF-kappa B in fibroblasts. These results define distinct roles for the two forms of I kappa B and demonstrate the necessity for stringent control of NF-kappa B. 


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The evolutionarily conserved sequence upstream of the human Ig heavy chain S gamma 3 region is an inducible promoter: synergistic activation by CD40 ligand and IL-4 via cooperative NF-kappa B and STAT-6 binding sites. 
Germline C gamma gene transcription is a crucial event in the process that leads to switch DNA recombination to IgG, but its regulation in the human is poorly understood. We took advantage of our monoclonal model of germinal center B cell differentiation, IgM+ IgD+ CL-01 cells, to define the role of the I gamma 3 evolutionarily conserved sequence (ECS) in the germline transcriptional activation of the human C gamma 3 gene. The I gamma 3 ECS lies upstream of the major I gamma 3 transcription initiation site and displays more than 90% identity with the corresponding human I gamma 1, I gamma 2, and I gamma 4 regions. Reporter luciferase gene vectors containing the human gamma 3 ECS were used to transfect CL-01 cells, which have been shown to undergo Smu-->S gamma 3 DNA recombination, upon engagement of CD40 by CD40 ligand (CD40L) and exposure to IL-4. In these transfected CL-01 cells, CD40:CD40L engagement and exposure to IL-4 synergistically induced gamma 3 ECS-dependent luciferase reporter gene activation. Targeted mutational analysis demonstrated that a tandem NF-kappa B/Rel binding motif is critical for the gamma 3 ECS responsiveness to both CD40L and IL-4, while a STAT-6-binding site is additionally required for IL-4 inducibility. Electrophoretic mobility shift assays showed that p50/p65/c-Rel and STAT-6 are effectively induced by CD40L and IL-4, respectively, and bind to specific DNA motifs within the ECS. These partially overlapping CD40L and IL-4 responsive elements are functionally cooperative as the disruption of one of them prevents synergistic promoter activation. Thus, the gamma 3 ECS is an inducible promoter containing cis elements that critically mediate CD40L and IL-4-triggered transcriptional activation of the human C gamma 3 gene. 


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Transcriptional control of the IL-5 gene by human helper T cells: IL-5 synthesis is regulated independently from IL-2 or IL-4 synthesis. 
BACKGROUND: IL-5 is fundamentally involved in eosinophilic inflammation. Control of IL-5 production may be effective for the management of allergic diseases. OBJECTIVE: We aimed to find the transcriptional mechanisms that regulate the IL-5 gene to selectively control IL-5 synthesis. METHODS: Allergen-specific T-cell clones and T-cell hybridomas were established from the peripheral blood lymphocytes of patients with asthma, and the transcriptional regulation of the IL-5 gene was investigated with transient transfection and electrophoretic mobility shift analysis. RESULTS: A human IL-5 promoter/enhancer-luciferase gene construct, pIL-5(-511)Luc, was transcribed on activation of IL-5-producing T-cell clones, but not IL-5-nonproducing clones. pIL-5(-511)Luc was transcribed by T-cell hybridomas derived from fusion between IL-5-producing T-cell clones and an IL-5 gene-nonexpressing T-cell line, but not by hybridomas derived from IL-5-nonproducing T-cell clones. IL-5 synthesis was not only induced by T-cell receptor stimulation but also by IL-2 receptor stimulation. Binding of NF-AT, NF-kappaB, and AP-1 was induced by T-cell receptor (TcR) stimulation, although there was no significant upregulation of binding by IL-2 stimulation. CONCLUSION: IL-5 synthesis by human helper T cells is regulated at the transcriptional level. A unique transcriptional mechanism distinct from those regulating the IL-2 or IL-4 genes seems to control the IL-5 gene. Selective regulation of IL-5 gene transcription may be useful for treating eosinophlic inflammation. 


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Monochloramine inhibits phorbol ester-inducible neutrophil respiratory burst activation and T cell interleukin-2 receptor expression by inhibiting inducible protein kinase C activity. 
Monochloramine derivatives are long lived physiological oxidants produced by neutrophils during the respiratory burst. The effects of chemically prepared monochloramine (NH2Cl) on protein kinase C (PKC) and PKC-mediated cellular responses were studied in elicited rat peritoneal neutrophils and human Jurkat T cells. Neutrophils pretreated with NH2Cl (30-50 microM) showed a marked decrease in the respiratory burst activity induced by phorbol 12-myristate 13-acetate (PMA), which is a potent PKC activator. These cells, however, were viable and showed a complete respiratory burst upon arachidonic acid stimulation, which induces the respiratory burst by a PKC-independent mechanism. The NH2Cl-treated neutrophils showed a decrease in both PKC activity and PMA-induced phosphorylation of a 47-kDa protein, which corresponds to the cytosolic factor of NADPH oxidase, p47(phox). Jurkat T cells pretreated with NH2Cl (20-70 microM) showed a decrease in the expression of the interleukin-2 receptor alpha chain following PMA stimulation. This was also accompanied by a decrease in both PKC activity and nuclear transcription factor-kappaB activation, also without loss of cell viability. These results show that NH2Cl inhibits PKC-mediated cellular responses through inhibition of the inducible PKC activity. 


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Expression of IkappaBalpha in the nucleus of human peripheral blood T lymphocytes. 
According to current models the inhibitory capacity of I(kappa)B(alpha) would be mediated through the retention of Rel/NF-kappaB proteins in the cytosol. However, I(kappa)B(alpha) has also been detected in the nucleus of cell lines and when overexpressed by transient transfection. To gain better insight into the potential role of nuclear I(kappa)B(alpha) in a physiological context we have analysed its presence in the nucleus of human peripheral blood T lymphocytes (PBL). We demonstrate the nuclear localization of I(kappa)B(alpha) in PBL by different techniques: Western blot, indirect immunofluorescence and electron microscopy. Low levels of nuclear I(kappa)B(alpha) were detected in resting cells whereas a superinduction was obtained after PMA activation. The nuclear pool of I(kappa)B(alpha) showed a higher stability than cytosolic I(kappa)B(alpha) and was partially independent of the resynthesis of the protein. Unexpectedly, the presence of nuclear I(kappa)B(alpha) did not inhibit NF-kappaB binding to DNA and this phenomenon was not due to the presence of IkappaBbeta at the nuclear level. Immunoprecipitation experiments failed to demonstrate an association between nuclear I(kappa)B(alpha) and NF-kappaB proteins. Our results demonstrate that in resting and PMA-activated human PBL, I(kappa)B(alpha) is present in the nucleus in an apparently inactive form unable to disrupt NF-kappaB binding from DNA. 


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Tolerance to lipopolysaccharide involves mobilization of nuclear factor kappa B with predominance of p50 homodimers. 
Stimulation of the human monocytic cell line Mono Mac 6 with lipopolysaccharide (LPS) leads to rapid and transient expression of cytokines like tumor necrosis factor (TNF). When such cells are precultured for 2 days with a low dose of LPS (20 ng/ml) followed by stimulation with a high dose of LPS (1 microgram/ml), expression of the TNF gene is minimal, i.e. the cells are tolerant. In nuclear run-on analysis, such tolerant cells show only a low degree of transcription, indicating that tolerance operates at or upstream of the transcription level. The CD14 LPS receptor is, however, up-regulated (not down-regulated) in tolerant cells, and LPS can, in fact, still lead to activation of tolerant cells as evidenced by mobilization of the transcription factor nuclear factor kappa B (NF-kappa B). Resolution of the NF-kappa B complex in gel shift analysis shows that the binding protein, mobilized in naive Mono Mac 6 cells, consists mainly of p50-p65 heterodimers, while in tolerant cells, the p50 homodimer is predominant. This increase in p50 homodimers coincides with an increase in p105 mRNA, suggestive of a transcriptional up-regulation of p50. Reporter gene analysis reveals that the NF-kappa B complex mobilized in tolerant cells is functionally inactive in that NF-kappa B-dependent luciferase constructs containing the human immunodeficiency virus long terminal repeat or the TNF 5'-region show only minimal transactivation after LPS stimulation. Similar to Mono Mac 6 cells, primary blood monocytes, when precultured with a low dose of LPS, also become tolerant and produce little TNF after LPS stimulation. The tolerant blood monocytes also up-regulate CD14, and they mobilize NF-kappa B with a predominance of p50 homodimers. Taken together, these results demonstrate that tolerance to LPS is determined by post-receptor mechanisms that involve an altered composition of the NF-kappa B complex. 


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Evidence for suppressed activity of the transcription factor NFAT1 at its proximal binding element P0 in the IL-4 promoter associated with enhanced IL-4 gene transcription in T cells of atopic patients. 
Allergen-specific T cells in atopic patients are polarized IL-4-producing Th2 cells, promoting IgE synthesis by B cells. The molecular basis for increased IL-4 gene expression in atopy is not fully understood. IL-4 gene regulation in general involves the nuclear factor of activated T cells (NFAT) family of transcription factors, of which NFAT1 and NFAT2 are most prominent in peripheral T cells. Recently, a unique inhibitory role of NFAT1 in IL-4 gene control was shown in the mouse. In a series of electrophoretic mobility shift assays with protein extracts of highly polarized Th2 clones from atopics and Th1 clones from controls we compared DNA-binding activities at the two NFAT-binding elements P0 and P1 of the crucial proximal human IL-4 promoter. At the most proximal P0 site, NFAT-containing complexes devoid of NFAT2 were readily inducible in the Th1 clones, but hardly or not in the Th2 clones. In contrast, both in Th1 and Th2 clones NFAT-containing complexes were strongly inducible at the P1 site, consisting of NFAT2 and a P0-compatible NFAT activity, without apparent differences between Th1 and Th2 clones. Like in Th2 clones, suppressed NFAT-P0 complex formation was observed also at the polyclonal level in peripheral blood mononuclear cells (PBMC) of three of five severe atopic dermatitis patients with strongly elevated serum IgE levels, but not in control PBMC. These findings suggest that high-level IL-4 production in atopic Th2 cells is associated with selective reduction of suppressive NFAT1 activity at the IL-4 P0 element and that some patients with this multifactorial disease may have a putative systemic disorder at this level. 


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Platelet-activating factor induces phospholipid turnover, calcium flux, arachidonic acid liberation, eicosanoid generation, and oncogene expression in a human B cell line. 
Platelet-activating factor is a potent mediator of the inflammatory response. Studies of the actions of platelet-activating factor have centered mainly around neutrophils, monocytes, and platelets. In this report we begin to uncover the influence of platelet-activating factor on B lymphocytes. Employing the EBV-transformed human B cell line SKW6.4, we demonstrate that platelet-activating factor significantly alters membrane phospholipid metabolism indicated by the incorporation of 32P into phosphatidylcholine, phosphatidylinositol, and phosphatidic acid but not significantly into phosphatidylethanolamine at concentrations ranging from 10(-9) to 10(-6) M. The inactive precursor, lyso-platelet-activating factor, at a concentration as high as 10(-7) M had no effect on any of the membrane phospholipids. We also show that platelet-activating factor from 10(-12) to 10(-6) M induced rapid and significant elevation in intracellular calcium levels, whereas lyso-platelet-activating factor was again ineffective. We further demonstrate the impact of platelet-activating factor binding to B cells by measuring platelet-activating factor induced arachidonic acid release and 5-hydroxyeicosatetraenoic acid production. Moreover, platelet-activating factor was capable of inducing transcription of the nuclear proto-oncogenes c-fos and c-jun. Finally we explored the possible role of 5-hydroxyeicosatetraenoic acid as a regulator of arachidonic acid liberation demonstrating that endogenous 5-lipoxygenase activity modulates platelet-activating factor induced arachidonic acid release perhaps acting at the level of phospholipase A2. In summary, platelet-activating factor is shown here to have a direct and profound effect on a pure B cell line. 


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Molecular mechanisms of neutrophil-endothelial cell adhesion induced by redox imbalance. 
Previous studies have implicated a role for intracellular thiols in the activation of nuclear factor-kappaB and transcriptional regulation of endothelial cell adhesion molecules. This study was designed to determine whether changes in endothelial cell glutathione (GSH) or oxidized glutathione (GSSG) can alter neutrophil adhesivity and to define the molecular mechanism that underlies this GSSG/GSH-induced adhesion response. Treatment of human umbilical vein endothelial cell (HUVEC) monolayers for 6 hours with 0.2 mmol/L diamide and 1 mmol/L buthionine sulfoximine (BSO) decreased GSH levels and increased the ratio of GSSG to GSH without cell toxicity. These redox changes are similar to those observed with anoxia/reoxygenation. Diamide plus BSO-induced thiol/disulfide imbalance was associated with a biphasic increase in neutrophil adhesion to HUVECs with peak responses observed at 15 minutes (phase 1) and 240 minutes (phase 2). N-Acetylcysteine treatment attenuated neutrophil adhesion in both phases, which indicated a role for GSH in the adhesion responses. Interestingly, phase 1 adhesion was inversely correlated with GSH levels but not with the GSSG/GSH ratio, whereas phase 2 neutrophil adhesion was positively correlated with GSSG/GSH ratio but not with GSH levels. Intercellular adhesion molecule-1 and P-selectin-specific monoclonal antibodies attenuated the increased neutrophil adhesion during both phases, whereas an anti-E-selectin monoclonal antibody also attenuated the phase 2 response. Pretreatment with actinomycin D and cycloheximide or with competing ds-oligonucleotides that contained nuclear factor-kappaB or activator protein-1 cognate DNA sequences significantly attenuated the phase 2 response, which implicated a role for de novo protein synthesis. Surface expression of intercellular adhesion molecule-1, P-selectin, and E-selectin on HUVECs correlated with the phase 1 and 2 neutrophil adhesion responses. This study demonstrates that changes in endothelial cell GSSG/GSH cause transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules, which leads to a 2-phase neutrophil-endothelial adhesion response. 


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Alpha-tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells. 
Antioxidants have been proposed to be anti-atherosclerotic agents; however, the mechanisms underlying their beneficial effects are poorly understood. We have examined the effect of alpha-tocopherol (alpha-tcp) on one cellular event in atherosclerotic plaque development, monocyte adhesion to stimulated endothelial cells (ECs). Human umbilical vein ECs were pretreated with alpha-tcp before stimulation with known agonists of monocyte adhesion: IL-1 (10 ng/ml), LPS (10 ng/ml), thrombin (30 U/ml), or PMA (10 nM). Agonist-induced monocytic cell adhesion, but not basal adhesion, was inhibited in a time- and concentration-dependent manner by alpha-tcp. The IC50 of alpha-tcp on an IL-1-induced response was 45 microM. The inhibition correlated with a decrease in steady state levels of E-selectin mRNA and cell surface expression of E-selectin which is consistent with the ability of a monoclonal antibody to E-selectin to inhibit monocytic cell adhesion in this system. Probucol (50 microM) and N-acetylcysteine (20 mM) also inhibited agonist-induced monocytic cell adhesion; whereas, several other antioxidants had no significant effect. Protein kinase C (PKC) does not appear to play a role in the alpha-tcp effect since no suppression of phosphorylation of PKC substrates was observed. Activation of the transcription factor NF-kappa B is reported to be necessary but not sufficient for E-selectin expression in EC. Electrophoretic mobility shift assays failed to show an alpha-tcp-induced decrease in activation of this transcription factor after cytokine stimulation. It has been hypothesized that alpha-tcp acts as an anti-atherosclerotic molecule by inhibiting generation of oxidized LDL--a putative triggering molecule in the atherosclerotic process. Our results point to a novel alternative mechanism of action of alpha-tcp. 


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CD14-mediated translocation of nuclear factor-kappa B induced by lipopolysaccharide does not require tyrosine kinase activity. 
During the course of serious bacterial infections, lipopolysaccharide (LPS) is believed to interact with macrophage receptors, resulting in the generation of inflammatory mediators and systemic symptoms including hemodynamic instability and shock. CD14, a glycosylphosphatidylinositol-linked antigen, functions as an LPS signaling receptor. A critical issue concerns the mechanism by which CD14, which has no transmembrane domain, transduces its signal following LPS binding. Recently, investigators have hypothesized that CD14-mediated signaling is effected through a receptor-associated tyrosine kinase (TK), suggesting a multicomponent receptor model of LPS signaling. Wild-type Chinese hamster ovary (CHO)-K1 cells can be activated by endotoxin to release arachidonate following transfection with human CD14 (CHO/CD14). Nuclear translocation of cytosolic NF-kappa B is correlated with a number of LPS-inducible responses. We sought to determine if this pathway were present in CHO/CD14 cells and to elucidate the relationship of NF-kappa B activation to the CD14 receptor system. LPS-stimulated translocation of NF-kappa B in CHO/CD14 cells resembled the same response in the murine macrophage-like cell line RAW 264.7. Protein synthesis inhibitors and corticosteroids, which suppress arachidonate release and the synthesis of proinflammatory cytokines, had no effect on translocation of NF-kappa B in CHO/CD14 or RAW 264.7 cells, demonstrating that NF-kappa B translocation is an early event. Although TK activity was consistently observed by immunoblotting extracts from activated RAW 264.7 cells, LPS-induced phosphotyrosine residues were not observed from similarly treated CHO/CD14 cells. Furthermore, the TK inhibitors herbimycin A and genistein failed to inhibit translocation of NF-kappa B in CHO/CD14 or RAW 264.7 cells, although both of these agents inhibited LPS-induced TK activity in RAW 264.7 cells. These results imply that TK activity is not obligatory for CD14-mediated signal transduction to occur in response to LPS. 


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Itk, a T cell-specific tyrosine kinase, is required for CD2-mediated interleukin-2 promoter activation in the human T cell line Jurkat. 
We investigated the functional role of Itk, a member of the cytoplasmic tyrosine kinase Tec family, in T cell activation. Stimulation of either CD2 or T cell receptor (TCR)/CD3 on Tcells by monoclonal antibody-mediated cross-linking induced tyrosine phosphorylation of Itk, which was maximal as early as 1 min after stimulation. The tyrosine kinase activity in the anti-Itk immunoprecipitate was significantly activated upon these stimulations. Interleukin-2 (IL-2) promoter activity stimulated by cross-linking of CD2, TCR/CD3, and CD28 with antibodies was significantly reduced by transient expression of an Itk mutant lacking the kinase activity. The reduction paralleled a decrease in tyrosine phosphorylation of endogenous wild-type Itk. Stimulation of CD2 or TCR/CD3 induced activation of the nuclear factor of activated T cells (NFAT), the binding site of which is included in the IL-2 gene promoter. The activation of NFAT was also impaired by expression of the Itk mutant. These results demonstrate that Itk plays a role in IL-2 production, indicating a critical involvement of Itk in the initial stage of T cell activation by mediating signals from the TCR/CD3 complex, CD2, and CD28. 


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Involvement of a second lymphoid-specific enhancer element in the regulation of immunoglobulin heavy-chain gene expression. 
To determine whether enhancer elements in addition to the highly conserved octamer (OCTA)-nucleotide motif are important for lymphoid-specific expression of the immunoglobulin heavy-chain (IgH) gene, we have investigated the effect of mutating the binding site for a putative additional lymphoid-specific transcription factor, designated NF-microB, in the murine IgH enhancer. We demonstrate that the NF-microB-binding site plays a critical role in the IgH enhancer, because mutation of the microB DNA motif decreased transcriptional activity of the IgH enhancer in cells of the B-cell lineage but not in nonlymphoid cells. This effect was comparable to or even stronger than the effect of a mutation in the OCTA site. Moreover, combined mutation of both microB and OCTA sites further reduced enhancer activity in lymphoid cells. Interestingly, alteration of either the microB or E3 site in a 70-base-pair fragment of the IgH enhancer that lacks the binding site for OCTA abolished enhancer activity in lymphoid cells completely. Nevertheless, a multimer of the microB motif alone showed no enhancer activity. DNase footprinting analysis corroborated the functional data showing that a lymphoid-specific protein binds to the microB DNA motif. Our results suggest that the microB element is a new crucial element important for lymphoid-specific expression of the IgH gene but that interaction with another enhancer element is essential for its activity. 


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Molecular regulation of cytokine gene expression during the immune response. 
Cytokine expression by immune system cells plays an important role in the regulation of the immune response. On first encounter with antigen, naive CD4+ T helper (Th) cells differentiate into cytokine-producing effector cells. Two types of effector cells characterized by their distinct expression of cytokine profiles have been described. Th1 cells produce IL-2 and IFN-gamma, whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13. In many pathological situations, the balance between Th1 and Th2 immune responses determines the outcome of diverse immunologically mediated clinical syndromes including infectious, autoimmune, and allergic diseases. However, the molecular basis for the tissue-specific expression of Th1/Th2-like cytokines has remained elusive. In this review we evaluate the possible in vivo role of different transcription factors and transcriptional mechanisms in T cell differentiation and the immune response. 


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Characterization of NF(P), the nuclear factor that interacts with the regulatory P sequence (5'-CGAAAATTTCC-3') of the human interleukin-4 gene: relationship to NF-kappa B and NF-AT. 
The P sequence of the human interleukin-4 (IL-4) gene, which was defined as a responsive element for phorbol 12-myristate 13-acetate and calcium ionophore (A23187) in Jurkat T cells, shares sequence similarity with the NF-kappa B and the NF-AT binding sites. We examined whether NF(P), a nuclear factor specific for the P sequence, is related to NF-kappa B and NF-AT. NF-kappa B (P65 or P65/P50 heterodimer) bound to the P sequence in electrophoretic mobility shift assays (EMSA) and activated transcription through the P sequence when expression plasmids were cotransfected with P sequence-driven reporter plasmids in Jurkat T cells. In EMSAs, NF(P) binding was inhibited by the unlabeled NF-AT binding site but not by the unlabeled AP1 binding site and purified NF-AT contained an activity that bound to the P sequence. Both mobility shift and sequence specificity of NF-AT were similar to those of NF(P) and only a small amount of P65 was detected in NF(P) in crude nuclear extracts. These results indicate that the component(s) of NF-AT has the potential to reconstitute NF(P) whereas NF-kappa B alone cannot account for NF(P) in crude extracts. Unlike NF-AT, NF(P) does not contain AP1 as its DNA binding component. 


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Activation of early growth response 1 gene transcription and pp90rsk during induction of monocytic differentiation. 
The present work has studied mechanisms responsible for induction of early growth response 1 (EGR-1) gene expression during monocytic differentiation of U-937 myeloid leukemia cells. Differentiation of U-937 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of the serine/threonine protein kinase C, was associated with transcriptional activation of EGR-1 promoter-reporter constructs. The EGR-1 promoter contains six CC(A/T)6GG (CArG) motifs. The two 5'-most distal CArG sequences conferred TPA inducibility. In contrast, there was little effect of TPA on EGR-1 transcription in a TPA-resistant U-937 cell variant, designated TUR. Treatment of both U-937 and TUR cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A, was associated with induction of monocytic differentiation and EGR-1 transcription through the 5'-most CArG element. Since these findings supported the involvement of serine/threonine protein phosphorylation in the regulation of EGR-1 expression, we studied activation of the 40S ribosomal protein S6 serine/threonine kinases, pp70S6K and pp90rsk. Although both kinases participate in regulating cell growth, there was no detectable activation of pp70S6K during TPA- or okadaic acid-induced monocytic differentiation. Moreover, rapamycin, an inhibitor of pp70S6K activation, had no effect on induction of EGR-1 expression. In contrast, analysis of pp90rsk activity by phosphorylation of a peptide derived from S6 protein demonstrated stimulation of this kinase in TPA-treated U-937, and not TUR, cells. Okadaic acid treatment of both cell types was associated with activation of pp90rsk. 


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Human immunodeficiency virus type 1 Tat upregulates interleukin-2 secretion in activated T cells. 
Dysregulation of cytokines secreted by T cells may play an important role in the pathogenesis of AIDS. To investigate the effects of human immunodeficiency virus type 1 (HIV-1) Tat on interleukin-2 (IL-2) expression, we used IL-2 promoter-chloramphenicol acetyltransferase constructs and IL-2-secreting Jurkat T cells as a model system. Transient expression of HIV-1 Tat induced a five- to eightfold increase in IL-2 promoter activity in Jurkat T cells stimulated with phytohemagglutinin and phorbol myristate acetate. IL-2 secretion was increased more than twofold in both Jurkat T cells and primary T cells stimulated by extracellular HIV-1 Tat protein. Analysis of mRNA suggested that Tat exerts its effect on IL-2 primarily at the transcriptional level. The NF-kappa B site at positions -206 to -195 of the IL-2 promoter was required but not sufficient for the Tat effect. The Tat-mediated increase in IL-2 promoter activity could selectively be blocked by antisense tat or-unlike the analogous effect of human T-cell lymphotropic virus type 1 Tax-by cyclosporin A. The observed increase in IL-2 levels might facilitate virus spread from or to T cells. Furthermore, it might contribute to the hypergammaglobulinemia or, together with other cytokines found to be dysregulated, the T-helper cell dysfunctions observed in AIDS patients. 


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Pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor kappa B (NF-kappa B) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes. 
We examined the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor kappa B (NF-kappa B), on the induction of apoptosis by a variety of agents. Treatment of a human promyelocytic leukemia cell line, HL-60, with 10 micrograms/mL etoposide or 2 microM 1-beta-D-arabinofuranosylcytosine induced NF-kappa B activation within 1 hr and subsequently caused apoptosis within 3-4 hr. The simultaneous addition of 50-500 microM PDTC with these agents blocked NF-kappa B activation and completely abrogated both morphologically apoptotic changes and internucleosomal DNA fragmentation for up to 6 hr. However, PDTC failed to inhibit the endonuclease activity contained in the whole cell lysates. The inhibitory effect of PDTC was also observed in etoposide- and dexamethasone-induced apoptosis in human thymocytes at a concentration of 1-10 microM. Since PDTC has both antioxidant and metal-ion chelating activities, we tested the effects of N-acetyl-L-cysteine (NAC) (antioxidant) or o-phenanthroline (OP) (metal-ion chelator) on the induction of apoptosis. Pretreatment of HL-60 cells or thymocytes with 100-500 microM OP for 2 hr, but not 10-60 mM NAC, suppressed subsequent occurrence of apoptosis induced by etoposide. These results suggest that the activation of NF-kappa B plays an important role in the apoptotic process of human hematopoietic cells. 


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The development of functionally responsive T cells. 
The work reviewed in this article separates T cell development into four phases. First is an expansion phase prior to TCR rearrangement, which appears to be correlated with programming of at least some response genes for inducibility. This phase can occur to some extent outside of the thymus. However, the profound T cell deficit of nude mice indicates that the thymus is by far the most potent site for inducing the expansion per se, even if other sites can induce some response acquisition. Second is a controlled phase of TCR gene rearrangement. The details of the regulatory mechanism that selects particular loci for rearrangement are still not known. It seems that the rearrangement of the TCR gamma loci in the gamma delta lineage may not always take place at a developmental stage strictly equivalent to the rearrangement of TCR beta in the alpha beta lineage, and it is not clear just how early the two lineages diverge. In the TCR alpha beta lineage, however, the final gene rearrangement events are accompanied by rapid proliferation and an interruption in cellular response gene inducibility. The loss of conventional responsiveness is probably caused by alterations at the level of signaling, and may be a manifestation of the physiological state that is a precondition for selection. Third is the complex process of selection. Whereas peripheral T cells can undergo forms of positive selection (by antigen-driven clonal expansion) and negative selection (by abortive stimulation leading to anergy or death), neither is exactly the same phenomenon that occurs in the thymic cortex. Negative selection in the cortex appears to be a suicidal inversion of antigen responsiveness: instead of turning on IL-2 expression, the activated cell destroys its own chromatin. The genes that need to be induced for this response are not yet identified, but it is unquestionably a form of activation. It is interesting that in humans and rats, cortical thymocytes undergoing negative selection can still induce IL-2R alpha expression and even be rescued in vitro, if exogenous IL-2 is provided. Perhaps murine thymocytes are denied this form of rescue because they shut off IL-2R beta chain expression at an earlier stage or because they may be uncommonly Bcl-2 deficient (cf. Sentman et al., 1991; Strasser et al., 1991). Even so, medullary thymocytes remain at least partially susceptible to negative selection even as they continue to mature . 


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LPS tolerance in monocytes/macrophages: three 3' cytosins are required in the DNA binding motif for detection of upregulated NF-kappa B p50 homodimers. 
When monocytes are stimulated with LPS (lipopolysaccharide) repeatedly then the initially high expression of the TNF (tumor necrosis factor) gene is only very low, i.e. the cells are tolerant to LPS. Tolerant cells still express the CD14 receptor and they can still be activated to mobilize NF-kappa B into nucleus. Analysis of the binding proteins employing the -605 motif of the human TNF promoter (GGGGCTGTCCC) revealed that in tolerant cells of the human monocytic cell line Mono Mac 6 there is a predominance of p50p50 of NF-kappa B. We now show that a mutant motif that exchanges the terminal 3' C for a G fails to bind the p50 homodimer that is upregulated in LPS toler ant human Mono Mac 6 cells. The same is true for nuclear extracts taken from the murine P388D1 macrophage cell line when tested with the -516 motif of the murine TNF promoter (GGGGGCTTTCCC). Here the wild type motif gives efficient binding of p50p50 that again is upregulated in tolerant cells whereas a mutant with a 3' G shows hardly any binding of p50p50. Conversely, the murine kappa light chain enhancer motif (GGGGACTTTCCG) does not efficiently bind the nuclear p50p50 from tolerant murine P388 macrophages. Binding is, however, readily detected when the 3' G is replaced by a C. These data show that the detection of upregulated p50 homodimers in LPS tolerant cells is dependent on subtle differences in the sequence of the DNA binding motif. 


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Cellular disposition of sulphamethoxazole and its metabolites: implications for hypersensitivity. 
1. Bioactivation of sulphamethoxazole (SMX) to chemically-reactive metabolites and subsequent protein conjugation is thought to be involved in SMX hypersensitivity. We have therefore examined the cellular metabolism, disposition and conjugation of SMX and its metabolites in vitro. 2. Flow cytometry revealed binding of N-hydroxy (SMX-NHOH) and nitroso (SMX-NO) metabolites of SMX, but not of SMX itself, to the surface of viable white blood cells. Cellular haptenation by SMX-NO was reduced by exogenous glutathione (GSH). 3. SMX-NHOH and SMX-NO were rapidly reduced back to the parent compound by cysteine (CYS), GSH, human peripheral blood cells and plasma, suggesting that this is an important and ubiquitous bioinactivation mechanism. 4. Fluorescence HPLC showed that SMX-NHOH and SMX-NO depleted CYS and GSH in buffer, and to a lesser extent, in cells and plasma. 5. Neutrophil apoptosis and inhibition of neutrophil function were induced at lower concentrations of SMX-NHOH and SMX-NO than those inducing loss of membrane viability, with SMX having no effect. Lymphocytes were significantly (P<0.05) more sensitive to the direct cytotoxic effects of SMX-NO than neutrophils. 6. Partitioning of SMX-NHOH into red blood cells was significantly (P<0.05) lower than with the hydroxylamine of dapsone. 7. Our results suggest that the balance between oxidation of SMX to its toxic metabolites and their reduction is an important protective cellular mechanism. If an imbalance exists, haptenation of the toxic metabolites to bodily proteins including the surface of viable cells can occur, and may result in drug hypersensitivity. 


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Induction of Mn SOD in human monocytes without inflammatory cytokine production by a mutant endotoxin. 
Endotoxin selectively induces monocyte Mn superoxide dismutase (SOD) without affecting levels of Cu,Zn SOD, catalase, or glutathione peroxidase. However, little is known about the structure-activity relationship and the mechanism by which endotoxin induces Mn SOD. In this study we demonstrated that a mutant Escherichia coli endotoxin lacking myristoyl fatty acid at the 3' R-3-hydroxymyristate position of the lipid A moiety retained its full capacity to coagulate Limulus amoebocyte lysate compared with the wild-type E. coli endotoxin and markedly stimulated the activation of human monocyte nuclear factor-kappaB and the induction of Mn SOD mRNA and enzyme activity. However, in contrast to the wild-type endotoxin, it failed to induce significant production of tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha by monocytes and did not induce the phosphorylation and nuclear translocation of mitogen-activated protein kinase. These results suggest that 1) lipid A myristoyl fatty acid, although it is important for the induction of inflammatory cytokine production by human monocytes, is not necessary for the induction of Mn SOD, 2) endotoxin-mediated induction of Mn SOD and inflammatory cytokines are regulated, at least in part, through different signal transduction pathways, and 3) failure of the mutant endotoxin to induce tumor necrosis factor-alpha production is, at least in part, due to its inability to activate mitogen-activated protein kinase. 


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Of the GATA-binding proteins, only GATA-4 selectively regulates the human IL-5 gene promoter in IL-5 producing cells which express multiple GATA-binding proteins. 
Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region, within the human IL-5 gene promoter, that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of these family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and PMA/A23187 stimulation are necessary for the IL-5 promoter activation. The requirement of another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNA of three GATA-binding proteins, hGATA-2, hGATA-3 and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/ A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms specific DNA-protein complex with the -70 GATA site. The electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity to the -70 GATA site among the three GATA-binding proteins. When the transactivation ability was compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed. 


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Transcriptional and posttranscriptional regulation of erythroid gene expression in anthracycline-induced differentiation of human erythroleukemic cells. 
Aclacinomycin (ACLA) and doxorubicin (DOX) were used at subtoxic concentrations to induce erythroid differentiation in the human leukemic cell line K562. Cell hemoglobinization was accompanied by the increased expression of genes encoding gamma-globin and porphobilinogen deaminase (PBGD), an enzyme of heme synthesis. By using run-on assays, ACLA was shown to induce an enhancement of the transcription of erythroid genes, including gamma-globin, PBGD, erythropoietin receptor, and GATA-1 transcription factor. In contrast, in DOX-treated cells, the transcription rate of these genes was unchanged in comparison with control cells. In addition, inhibition of mRNA synthesis with actinomycin D indicated that DOX induced an increased stability of PBGD and GATA-1 mRNAs, whereas ACLA did not affect the half-lives of these mRNAs. Because the increase in erythroid mRNA steady-state level in anthracycline-treated cells was inhibited by cycloheximide, this suggests that transcriptional activation in ACLA-treated cells and mRNA stabilization in DOX-treated cells were dependent on de novo protein synthesis. Finally, GATA-1 protein level was shown to be increased in ACLA-treated but not in DOX-treated cells. These two anthracyclines, although closely related in their structures, appeared to act as differentiation inducers by distinct mechanisms. Indeed, erythroid gene expression was demonstrated to be regulated transcriptionally by ACLA and mainly posttranscriptionally by DOX. 


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Expression of erythroid-specific genes in megakaryoblastic disorders. 
Currently available data indicate that erythroid and megakaryocytic differentiation pathways are closely related to each other, and there may exist progenitor cells common to those two lineages may exist. Acute megakaryoblastic leukemia (AML-M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. These blast cells express lineage-specific transcription factors such as GATA-1 common to these lineages and frequently express erythroid-specific mRNAs such as gamma-globin and erythroid delta-aminolevulinate synthase (ALAS-E), indicating that most of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. These results suggest that blasts in M7 and TMD may correspond to progenitors of both erythroid and megakaryocytic lineages. 


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Replication of human immunodeficiency virus-1 in primary human T cells is dependent on the autocrine secretion of tumor necrosis factor through the control of nuclear factor-kappa B activation. 
Tumor necrosis factor (TNF)-alpha controls T-cell activation and is a major inducer of human immunodeficiency virus (HIV)-1 replication in chronically infected cells. Therefore, we have investigated its role in primary cultures of HIV-infected human T lymphocytes by using neutralizing anti-TNF-alpha antibodies or TNF-alpha. Primary resting T lymphocytes produced TNF-alpha and supported HIV replication after T-cell receptor activation. Addition of neutralizing anti-TNF-alpha antibodies drastically reduced p24 antigen release and prevented CD4+ cell depletion associated with infection. Anti-TNF-alpha also prevented nuclear factor-kappa B (NF-kappa B) activation, and a good correlation between this inhibition and inhibition of HIV replication was observed. Moreover, supplementing the cultures with high doses of IL-2 reverted anti-TNF-alpha inhibition of cell proliferation but did not affect the inhibition of HIV p24 antigen release or NF-kappa B activation in the same cultures. Moreover, anti-TNF-alpha inhibited HIV-1 long terminal repeat (LTR)-driven transcription of a reporter gene in primary T cells in response to activation, either in the presence or the absence of HIV-1 Tat. Our results support an important role for autocrine TNF-alpha secretion in controlling HIV replication in primary T cells because of its ability to maintain NF-kappa B elevated in the nucleus of T cells. 


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A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor alpha gene expression: molecular cloning, sequencing, characterization, and chromosomal assignment. 
Lipopolysaccharide (LPS) is a potent stimulator of monocytes and macrophages, causing secretion of tumor necrosis factor alpha (TNF-alpha) and other inflammatory mediators. Given the deleterious effects to the host of TNF-alpha, it has been postulated that TNF-alpha gene expression must be tightly regulated. The nature of the nuclear factor(s) that control TNF-alpha gene transcription in humans remains obscure, although NF-kappaB has been suggested. Our previous studies pertaining to macrophage response to LPS identified a novel DNA-binding domain located from -550 to -487 in the human TNF-alpha promoter that contains transcriptional activity, but lacks any known NF-kappaB-binding sites. We have used this DNA fragment to isolate and purify a 60-kDa protein binding to this fragment and obtained its amino-terminal sequence, which was used to design degenerate probes to screen a cDNA library from THP-1 cells. A novel cDNA clone (1.8 kb) was isolated and fully sequenced. Characterization of this cDNA clone revealed that its induction was dependent on LPS activation of THP-1 cells; hence, the name LPS-induced TNF-alpha factor (LITAF). Inhibition of LITAF mRNA expression in THP-1 cells resulted in a reduction of TNF-alpha transcripts. In addition, high level of expression of LITAF mRNA was observed predominantly in the placenta, peripheral blood leukocytes, lymph nodes, and the spleen. Finally, chromosomal localization using fluorescence in situ hybridization revealed that LITAF mapped to chromosome 16p12-16p13.3. Together, these findings suggest that LITAF plays an important role in the activation of the human TNF-alpha gene and proposes a new mechanism to control TNF-alpha gene expression. 


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BCL-6, a POZ/zinc-finger protein, is a sequence-specific transcriptional repressor. 
Approximately 40% of diffuse large cell lymphoma are associated with chromosomal translocations that deregulate the expression of the BCL6 gene by juxtaposing heterologous promoters to the BCL-6 coding domain. The BCL6 gene encodes a 95-kDa protein containing six C-terminal zinc-finger motifs and an N-terminal POZ domain, suggesting that it may function as a transcription factor. By using a DNA sequence selected for its ability to bind recombinant BCL-6 in vitro, we show here that BCL-6 is present in DNA-binding complexes in nuclear extracts from various B-cell lines. In transient transfectin experiments, BCL6 can repress transcription from promoters linked to its DNA target sequence and this activity is dependent upon specific DNA-binding and the presence of an intact N-terminal half of the protein. We demonstrate that this part of the BCL6 molecule contains an autonomous transrepressor domain and that two noncontiguous regions, including the POZ motif, mediate maximum transrepressive activity. These results indicate that the BCL-6 protein can function as a sequence-specific transcriptional repressor and have implications for the role of BCL6 in normal lymphoid development and lymphomagenesis. 


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Expression of c-fos and c-jun proteins and AP-1 binding activity during cell cycle progression of HL60 cells and phytohemagglutinin-stimulated lymphocytes. 
The protein products of the c-fos (p62c-fos) and c-jun (p39c-jun) genes are members of the AP-1 transcription factor family and are thought to play important roles in the regulation of gene expression during the cell cycle. Most studies on the expression of these proteins in relation to the cell cycle have been performed at the mRNA level, and therefore do not give direct information about the presence of the proteins during the cell cycle. We have used Western blotting to investigate the presence of these proteins during the cell cycles of two different cellular systems: a continuously growing myeloid leukemic cell line, HL60, and normal cells stimulated into cycle, phyto- hemagglutinin (PHA)-stimulated normal human peripheral blood lymphocytes (PBL). The binding activity of transcription factor AP-1, which consists of dimers of Fos and Jun family proteins, was also studied using a gel shift assay. We found nuclear p62c-fos, p39c-jun, and AP-1 binding activity throughout the cell cycle both in HL60 cells and in PHA-stimulated PBL, and we postulate that these proteins are required throughout the cell cycle and not transiently in the G0 to G1 transition as previous mRNA studies have indicated. We demonstrated an uncoupling of AP-1 binding activity from p62c-fos, and p39c-jun AP-1 activity was expressed more strongly in the G1- and G2/M-phase enriched samples than in the S-phase enriched samples of HL60 cells, while levels of nuclear p62c-fos and p39c-jun were constant. Nuclei of unstimulated PBL from different donors expressed p62c-fos and p39c-jun, but AP-1 was not detected in the majority of samples. Following PHA stimulation of PBL, the increase in AP-1 activity was delayed with respect to the augmentation of p39c-jun expression. We also observed that cytoplasmic p62c-fos and p39c-jun were present in HL60 cells and PHA-stimulated PBL. However, no cytoplasmic p62c-fos was detected in unstimulated PBL, although in some cases cytoplasmic p39c-jun was detected, suggesting that subcellular compartmentalization of these proteinsmay occur under certain circumstances. 


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Inhibition of T cell signaling by mitogen-activated protein kinase-targeted hematopoietic tyrosine phosphatase (HePTP). 
Activation of T lymphocytes to produce cytokines is regulated by the counterbalance of protein-tyrosine kinases and protein-tyrosine phosphatases, many of which have a high degree of substrate specificity because of physical association with their targets. Overexpression of hematopoietic protein-tyrosine phosphatase (HePTP) results in suppression of T lymphocyte activation as measured by T cell antigen receptor-induced activation of transcription factors binding to the 5' promoter of the interleukin-2 gene. Efforts to pinpoint the exact site of action and specificity of HePTP in the signaling cascade revealed that HePTP acts directly on the mitogen-activated protein (MAP) kinases Erk1 and 2 and consequently reduces the magnitude and duration of their catalytic activation in intact T cells. In contrast, HePTP had no effects on N-terminal c-Jun kinase or on events upstream of the MAP kinases. The specificity of HePTP correlated with its physical association through its noncatalytic N terminus with Erk and another MAP kinase, p38, but not Jnk or other proteins. We propose that HePTP plays a negative role in antigen receptor signaling by specifically regulating MAP kinases in the cytosol and at early time points of T cell activation before the activation-induced expression of nuclear dual-specific MAP kinase phosphatases. 


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Human T cell activation through the activation-inducer molecule/CD69 enhances the activity of transcription factor AP-1. 
The induction of the AP-1 transcription factor has been ascribed to the early events leading to T cell differentiation and activation. We have studied the regulation of AP-1 activity in human peripheral blood T lymphocytes stimulated through the activation inducer molecule (AIM)/CD69 activation pathway. Phorbol esters are required to induce AIM/CD69 cell-surface expression as well as for triggering the proliferation of T cells in conjunction with anti-AIM mAb. Mobility shift assays showed that addition of anti-AIM mAb to PMA-treated T lymphocytes markedly enhanced the binding activity of AP-1 to its cognate sequence, the phorbol ester response element. In contrast, anti-AIM mAb did not induce any change in the binding activity of NF-kappa B, a transcription factor whose activity is also regulated by protein kinase C. The increase in AP-1-binding activity was accompanied by the marked stimulation of the transcription of c-fos but not that of c-jun. Blockade of the DNA-binding complexes with an anti-Fos mAb demonstrated a direct participation of c-Fos in the AP-1 complexes induced by anti-AIM mAb. Most of the AP-1 activity could be eliminated when the anti-AIM mAb was added to the culture medium in the presence of cycloheximide, suggesting that de novo protein synthesis is crucial for the induction of AP-1-binding activity. These data provide the evidence that activation of human peripheral blood T cells through the AIM activation pathway regulate the activity of AP-1. Therefore, this pathway appears as a crucial step in the initiation of early T cell activation events. 


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A critical role of Sp1- and Ets-related transcription factors in maintaining CTL-specific expression of the mouse perforin gene. 
This study was designed to determine the potential cis-elements involved in transcriptional regulation of the mouse perforin gene. DNase I hypersensitive site (DHS) mapping revealed that the perforin locus contained six DHS within 7.0 kb of the 5' upstream sequence (-7.0 kb) and two DHS in intron 2. The six 5' upstream and one intronic DHS were detected in only perforin-expressing lymphocytes. Chloramphenicol acetyltransferase (CAT) activities directed by 5' upstream promoter were detected preferentially in perforin-expressing cell lines. A construct termed PFP5a containing -795 bp exhibited the highest CAT activity, and PFP9a20 containing only -73 bp also produced significantly high CAT activity in CTLL-R8 cells. The proximal region in PFP9a20 contained two potential Sp1 binding sites (GC box and GT box) and one Ets binding site (EBS). Electrophoretic mobility shift assay showed that each of the cis-elements bound specific protein factors. When single-point mutation was introduced to each GC box, EBS, and GT box in PFP9a20, at least 3-fold less CAT activity was observed in CTLL-R8 cells. To confirm the importance of the three cis-acting elements in the perforin gene expression, point mutation was introduced again to each proximal GC box, EBS, and GT box of PFP5a. The point mutations resulted in a 2.5- to 3-fold reduction of CAT activity. The results suggest that a combination of the three proximal cis-acting elements may constitute a minimal region responsible for CTL-specific expression of perforin. 


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Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture. 
All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid-suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation. 


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STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients. 
A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis. 


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Glucocorticoid-induced apoptosis of lymphoid cells. 
The induction of cell death in lymphoid cells by glucocorticoids is one of the earliest and most thoroughly studied models of apoptosis. Although the exact mechanism by which apoptosis occurs in lymphocytes is unknown many biochemical and molecular changes have been shown to occur in these cells in response to glucocorticoids. The role of chromatin degradation and endonucleases in the apoptotic process has been closely studied, as well as the involvement of several oncogenes in glucocorticoid-induced cell lysis. In addition, the clinical importance of glucocorticoid-induced apoptosis in the treatment of lymphoid neoplasms has recently received increased attention. 


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Targeted degradation of c-Fos, but not v-Fos, by a phosphorylation-dependent signal on c-Jun. 
The proto-oncogene products c-Fos and c-Jun heterodimerize through their leucine zippers to form the AP-1 transcription factor. The transcriptional activity of the heterodimer is regulated by signal-dependent phosphorylation and dephosphorylation events. The stability of c-Fos was found to also be controlled by intracellular signal transduction. In transient expression and in vitro degradation experiments, the stability of c-Fos was decreased when the protein was dimerized with phosphorylated c-Jun. c-Jun protein isolated from phorbol ester-induced cells did not target c-Fos for degradation, which suggests that c-Fos is transiently stabilized after stimulation of cell growth. v-Fos protein, the retroviral counterpart of c-Fos, was not susceptible to degradation targeted by c-Jun. 


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Functional antagonism between vitamin D3 and retinoic acid in the regulation of CD14 and CD23 expression during monocytic differentiation of U-937 cells. 
1,25 alpha-Dihydroxicholecalciferol (VitD3) and retinoic acid (RA) are important regulators of the proliferation and differentiation of several cell types. This paper describes how the expression of the monocyte-macrophage Ag, CD14, and the low affinity Fc receptor for IgE, CD23, were inversely regulated during VitD3- and RA-induced monocytic differentiation of human U-937 monoblasts. PMA induced the expression of both CD14 and CD23 mRNA and protein. Exposure to VitD3 rapidly induced the de novo expression of CD14 mRNA and protein. The addition of cycloheximide completely blocked the VitD3 induction of CD14 mRNA expression, indicating that the induction was dependent on ongoing protein synthesis. While inducing CD14 expression, VitD3 concomitantly suppressed the basal, PMA-, and RA-inducible CD23 expression in a dose-dependent manner. In contrast, U-937 cells induced by RA strongly increased their expression of CD23 mRNA and protein, whereas they completely lacked detectable CD14 cell surface or mRNA expression. Furthermore, the VitD3- and the PMA-induced CD14 expression was inhibited as a temporal consequence of the RA-induced differentiation. The results suggest that there exists a functional antagonism between VitD3 and RA that may have important implications for the regulation of certain immune and inflammatory responses through their inverse effects on CD14 and CD23 gene expression. 


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Peripheral blood mononuclear cells isolated from patients with diabetic nephropathy show increased activation of the oxidative-stress sensitive transcription factor NF-kappaB. 
Increased oxidative stress and subsequent activation of the transcription factor NF-kappaB has been linked to the development of late diabetic complications. To determine whether oxidative stress dependent NF-kappaB activation is evident in patients with diabetic nephropathy we used an Electrophoretic Mobility Shift Assay based semiquantitative detection system which enabled us to determine NF-kappaB activation in ex vivo isolated peripheral blood mononuclear cells. We examined 33 patients with diabetes mellitus (Type I and Type II). Patients with diabetic nephropathy showed higher NF-kappaB binding activity in Electrophoretic Mobility Shift Assays and stronger immunohistological staining for activated NF-kappaBp65 than patients without renal complications. NF-kappaB binding activity correlated with the degree of albuminuria (r = 0.316) and with thrombomodulin plasma concentrations (r = 0.33), indicative for albuminuria associated endothelial dysfunction. In a 3 day intervention study in which 600 mg of the antioxidant thioctic acid (alpha-lipoic acid) per day were given to nine patients with diabetic nephropathy oxidative stress in plasma samples was decreased by 48% and NF-kappaB binding activity in ex vivo isolated peripheral blood mononuclear cells by 38%. In conclusion, activation of the transcription factor NF-kappaB in ex vivo isolated peripheral blood mononuclear cells of patients with diabetes mellitus correlates with the degree of diabetic nephropathy. NF-kappaB activation is at least in part dependent on oxidative stress since thioctic acid (alpha-lipoic acid) reduced NF-kappaB binding activity. 


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Oxidative stress suppresses transcription factor activities in stimulated lymphocytes. 
Effects of oxidative stress on stimulation-dependent signal transduction, leading to IL-2 expression, were studied. Purified quiescent human blood T lymphocytes were subjected to: (i) acute exposure to hydrogen peroxide; (ii) chronic exposure to hydrogen peroxide; and (iii) acute exposure to ionizing radiation. The cells were then stimulated for 6 h. DNA-binding activities (determined by the electrophoretic mobility shift assay) of three transcription factors: NFkappaB, AP-1 and NFAT, were abolished in the lymphocytes by all three modes of oxidative stress. The lymphocytes exhibited lipid peroxidation only upon exposure to the lowest level of hydrogen peroxide used (20 microM). All three modes of oxidative stress induced catalase activity in the lymphocytes. The only exception was hydrogen peroxide at 20 microM, which did not induce catalase activity. We conclude that: (i) suppression of specific transcription factor functions can potentially serve as a marker of exposure to oxidative stress and its effects on human lymphocytes; (ii) lipid peroxidation is only detectable in human lymphocytes upon exposure to weak oxidative stress which does not induce catalase activity; (iii) therefore, transcription factor DNA-binding activities are more sensitive to oxidative stress than lipid peroxidation. 


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Induction of NF-kappa B during monocyte differentiation is associated with activation of HIV-gene expression. 
Cells of the monocyte-macrophage lineage are important targets of HIV infection. We report here that the phenotypic differentiation of monocyte cell lines induced by phorbol esters or tumour necrosis factor alpha (TNF alpha) is associated with expression of nuclear factor kappa B (NF-kappa B). In parallel with such differentiation, HIV transcription, monitored using an HIV long terminal repeat reporter gene construct, is activated in such cells under the influence of enhanced NF-kappa B expression. Also, in a promonocyte cell line chronically infected with HIV, NF-kappa B expression and HIV transcription were enhanced on stimulation with phorbol ester or TNF alpha. Thus, stimulation of monocyte cell lines by phorbol esters or TNF alpha induces cell differentiation and activates HIV transcription. Such a process may have fundamental implications in AIDS pathogenesis in vivo and may be important in disease progression induced by opportunistic infections directly or indirectly involving macrophages. 


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Hypoxia causes the activation of nuclear factor kappa B through the phosphorylation of I kappa B alpha on tyrosine residues. 
The response of mammalian cells to stress is controlled by transcriptional regulatory proteins such as nuclear factor kappa B (NF-kappa B) to induce a wide variety of early response genes. In this report, we show that exposure of cells to hypoxia (0.02% O2) results in I kappa B alpha degradation, increased NF-kappa B DNA binding activity, and transactivation of a reporter gene construct containing two NF-kappa B DNA binding sites. Pretreatment of cells with protein tyrosine kinase inhibitors and the dominant negative allele of c-Raf-1 (Raf 301) inhibited I kappa B alpha degradation, NF-kappa B binding, and transactivation of kappa B reporter constructs by hypoxia. To demonstrate a direct link between changes in the phosphorylation pattern of I kappa B alpha with NF-kappa B activation, we immunoprecipitated I kappa B alpha after varying times of hypoxic exposure and found that its tyrosine phosphorylation status increased during hypoxic exposure. Inhibition of the transfer of tyrosine phosphoryl groups onto I kappa B alpha prevented I kappa B alpha degradation and NF-kappa B binding. In comparison to other activators of NF-kappa B such as phorbol myristate acetate or tumor necrosis factor, we did not detect changes in the tyrosine phosphorylation status of I kappa B alpha following treatment with either of these agents. These results suggest that tyrosine phosphorylation of I kappa B alpha during hypoxia is an important proximal step which precedes its dissociation and degradation from NF-kappa B. 


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A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria [see comments] 
Genetic variation in cytokine promoter regions is postulated to influence susceptibility to infection, but the molecular mechanisms by which such polymorphisms might affect gene regulation are unknown. Through systematic DNA footprinting of the TNF (encoding tumour necrosis factor, TNF) promoter region, we have identified a single nucleotide polymorphism (SNP) that causes the helix-turn-helix transcription factor OCT-1 to bind to a novel region of complex protein-DNA interactions and alters gene expression in human monocytes. The OCT-1-binding genotype, found in approximately 5% of Africans, is associated with fourfold increased susceptibility to cerebral malaria in large case-control studies of West African and East African populations, after correction for other known TNF polymorphisms and linked HLA alleles. 


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NF-kappaB activation is required for C5a-induced interleukin-8 gene expression in mononuclear cells. 
C5a, a potent peptide chemoattractant, stimulates interleukin-8 (IL-8) secretion from peripheral blood mononuclear cells (PBMC). Experiments were conducted to understand the mechanisms for C5a-induced IL-8 production, which was 14-fold greater than that in unstimulated cells by 2 hours. IL-8 secretion was accompanied by accumulation of IL-8 mRNA in the cytosol and by nuclear expression of a kappaB DNA binding activity within 30 minutes. AP-1 but not NF-IL-6 DNA binding activity was also detected in C5a-stimulated PBMC; however, its delayed expression (maximal at 4 hours) suggested a less important role in the rapid production of IL-8. The correlation between C5a-induced kappaB binding activity and IL-8 gene expression was examined in the RAW264.7 macrophage cells using reporter genes directed by the kappaB sequence from IkappaBalpha and IL-8 promoter regions. C5a-induced reporter gene expression was abolished by introducing mutations into the kappaB sites and by coexpression of a dominant negative IkappaBalpha construct resistant to agonist-induced phosphorylation. Pertussis toxin, which ADP-ribosylates the Gi proteins known to couple to the C5a receptor, produced minimal inhibition of C5a-induced IL-8 expression and had little effect on C5a-induced calcium mobilization in RAW264.7 cells. These results suggest that NF-kappaB activation is required for C5a-induced IL-8 gene expression and that this response is mediated primarily through a pertussis toxin-insensitive pathway. 


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CIITA-induced occupation of MHC class II promoters is independent of the cooperative stabilization of the promoter-bound multi-protein complexes. 
Precise regulation of MHC class II expression plays a crucial role in the control of the immune response. The transactivator CIITA behaves as a master controller of constitutive and inducible MHC class II gene activation, but its exact mechanism of action is not known. Activation of MHC class II promoters requires binding of at least three distinct multi-protein complexes (RFX, X2BP and NF-Y). It is known that the stability of this binding results from cooperative interactions between these proteins. We show here that expression of CIITA in MHC class II- cells triggers occupation of the promoters by these complexes. This observation raised the possibility that the effect of CIITA on promoter occupation is mediated by an effect on the cooperative stabilization of the DNA-bound multi-protein complexes. We show, however, that the presence of CIITA does not affect the stability of the higher-order protein complex formed on DNA by RFX, X2BP and NF-Y. This suggests other mechanisms for CIITA-induced promoter occupancy, such as an effect on chromatin structure leading to increased accessibility of MHC class II promoters. This ability of CIITA to facilitate promoter occupation is undissociable from its transactivation potential. Finally, we conclude that this effect of CIITA is cell-type specific, since expression of CIITA is not required for normal occupation of MHC class II promoters in B lymphocytes. 


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Activation of distinct transcription factors in neutrophils by bacterial LPS, interferon-gamma, and GM-CSF and the necessity to overcome the action of endogenous proteases. 
Human neutrophils can be induced to actively transcribe a number of early-response genes, in particular those encoding cytokines, chemokines, and the high-affinity surface receptor for IgG, FcgammaRI. Although little is known to date about the regulation of gene transcription in neutrophils, several indications point to a role for distinct transcription factors, such as members of the NF-kappaB and STAT families. In this study, we investigated whether these transcription factors become activated under stimulatory conditions which are known to induce gene transcription in neutrophils. Unexpectedly, we found that conventional procedures employed to prepare cellular extracts cause the release of proteolytic activities that are normally stored in intracellular granules, resulting in the degradation of various NF-kappaB/Rel and STAT proteins. To circumvent this problem, we developed an alternative procedure which allowed us to show that in neutrophils, LPS and TNFalpha induce a NF-kappaB DNA-binding activity which essentially consists of p50/RelA dimers, and that IFNgamma promotes the binding of STAT1 homodimers to the IFNgamma response region of the FcgammaRI promoter. Moreover, we report that neutrophil stimulation with GM-CSF results in the formation of a STAT5-containing DNA-binding activity. Collectively, the current findings open new perspectives about mechanisms that are likely to regulate gene transcription in neutrophils. In addition, the procedure described herein could prove useful in other cell types that express high levels of endogenous proteases. 


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Transcription of the hypersensitive site HS2 enhancer in erythroid cells. 
In the human genome, the erythroid-specific hypersensitive site HS2 enhancer regulates the transcription of the downstream beta-like globin genes 10-50 kilobases away. The mechanism of HS2 enhancer function is not known. The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids. In erythroid K562 cells in which the HS2 enhancer is active, the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene. In nonerythroid HL-60 cells in which the HS2 enhancer is inactive, long enhancer transcripts are not detectable. Splitting the HS2 enhancer between two tandem Ap1 sites abolishes the synthesis of a group of long enhancer transcripts and results in loss of enhancer function and transcriptional silencing of the cis-linked CAT gene. In directing the synthesis of RNA through the intervening DNA and the gene by a tracking and transcription mechanism, the HS2 enhancer may (i) open up the chromatin structure of a gene domain and (ii) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site. 


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Low CD3+CD28-induced interleukin-2 production correlates with decreased reactive oxygen intermediate formation in neonatal T cells. 
The capacity of neonatal T cells to secrete interleukin-2 (IL-2) has been reported to be variable. We analysed IL-2 production in purified neonatal and adult T cells using polyclonal activator phorbol ester + calcium ionophore (PDBu + iono) or receptor-mediated anti-CD3/anti-CD3+ anti-CD28 stimulation. PDBu + iono induced equally high IL-2 levels in both groups and, when stimulated with plate-bound anti-CD3 monoclonal antibody (mAb), the IL-2 secretion by neonatal cells was undetectable and adult cells produced low amounts of IL-2 (mean 331 +/- 86 pg/ml). The addition of anti-CD28 mAb to anti-CD3-stimulated cells markedly increased IL-2 production in both cell types, but levels of IL-2 in neonatal T cells remained clearly lower than those of adult T cells (respective mean values: 385 +/- 109 pg/ml and 4494 +/- 1199 pg/ml). As NF-kappa B is a critical transcription factor in the control of IL-2 expression, we next analysed its nuclear translocation in neonatal and adult T cells using the electrophoretic mobility shift assay and, because induction of reactive oxygen intermediates (ROI) is required for the activation of NF-kappa B, we also analysed levels of intracellular ROI in these cells using the ROI-reactive fluorochrome DCFH-DA and flow cytometry. In neonatal T cells NF-kappa B activation and ROI formation after anti-CD3 stimulation were low compared with adult T cells and, although addition of anti-CD28 mAb increased induction of NF-kappa B and ROI formation, levels similar to those of adults were not achieved. After PDBu + iono stimulation, the cells showed similar ROI formation and IL-2 secretion. Our results suggest that reduced IL-2 production by neonatal T cells is specific for anti-CD3 and anti-CD3+ anti-CD28-mediated stimulation and that these activators cannot effectively activate the ROI-NF-kappa B signalling pathway in neonatal T cells. 


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Control of NFATx1 nuclear translocation by a calcineurin-regulated inhibitory domain. 
The nuclear factor of activated T cells (NFAT) regulates cytokine gene expression in T cells through cis-acting elements located in the promoters of several cytokine genes. NFATx1, which is preferentially expressed in the thymus and peripheral blood leukocytes, is one of four members of the NFAT family of transcription factors. We have performed domain analysis of NFATx1 by examining the effects of deletion mutations. We found that NFATx1 DNA binding activity and interaction with AP-1 polypeptides were dependent on its central Rel similarity region and that transcriptional activation was reduced by deletions of either its N-terminal domain or its C-terminal domain, suggesting the presence of intrinsic transcriptional activation motifs in both regions. We also identified a potent inhibitory sequence within its N-terminal domain. We show that the inactivation of the inhibition was dependent on the activity of calcineurin, a calcium-calmodulin-dependent phosphatase. We also show that calcineurin associated with the N-terminal domain of NFATx1 at multiple docking sites and caused a reduction of size, indicative of dephosphorylation, in NFATx1. We have mapped the inhibitory activity to less than 60 residues, containing motifs that are conserved in all NFAT proteins. Finally, we demonstrate that deletion in NFATx1 of the mapped 60 residues leads to its nuclear translocation independent of calcium signaling. Our results support the model proposing that the N-terminal domain confers calcium-signaling dependence on NFATx1 transactivation activity by regulating its intracellular localization through a protein module that associates with calcineurin and is a target of its phosphatase activity. 


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Precise alignment of sites required for mu enhancer activation in B cells. 
The lymphocyte-specific immunoglobulin mu heavy-chain gene intronic enhancer is regulated by multiple nuclear factors. The previously defined minimal enhancer containing the muA, muE3, and muB sites is transactivated by a combination of the ETS-domain proteins PU.1 and Ets-1 in nonlymphoid cells. The core GGAAs of the muA and muB sites are separated by 30 nucleotides, suggesting that ETS proteins bind to these sites from these same side of the DNA helix. We tested the necessity for appropriate spatial alignment of these elements by using mutated enhancers with altered spacings. A 4- or 10-bp insertion between muE3 and muB inactivated the mu enhancer in S194 plasma cells but did not affect in vitro binding of Ets-1, PU.1, or the muE3-binding protein TFE3, alone or in pairwise combinations. Circular permutation and phasing analyses demonstrated that PU.1 binding but not TFE3 or Ets-1 bends mu enhancer DNA toward the major groove. We propose that the requirement for precise spacing of the muA and muB elements is due in part to a directed DNA bend induced by PU.1. 


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Regulation of the megakaryocytic glycoprotein IX promoter by the oncogenic Ets transcription factor Fli-1. 
Glycoprotein (GP) IX is a subunit of the von Willebrand receptor, GPIb-V-IX, which mediates adhesion of platelets to the subendothelium of damaged blood vessels. Previous characterization of the GPIX promoter identified a functional Ets site that, when disrupted, reduced promoter activity. However, the Ets protein(s) that regulated GPIX promoter expression was unknown. In this study, transient cotransfection of several GPIX promoter/reporter constructs into 293T kidney fibroblasts with a Fli-1 expression vector shows that the oncogenic protein Fli-1 can transactivate the GPIX promoter when an intact GPIX Ets site is present. In addition, Fli-1 binding of the GPIX Ets site was identified in antibody supershift experiments in nuclear extracts derived from hematopoietic human erythroleukemia cells. Comparative studies showed that Fli-1 was also able to transactivate the GPIbalpha and, to a lesser extent, the GPIIb promoter. Immunoblot analysis identified Fli-1 protein in lysates derived from platelets. In addition, expression of Fli-1 was identified immunohistochemically in megakaryocytes derived from CD34(+) cells treated with the megakaryocyte differentiation and proliferation factor, thrombopoietin. These results suggest that Fli-1 is likely to regulate lineage-specific genes during megakaryocytopoiesis. 


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Aspirin inhibits nuclear factor-kappa B mobilization and monocyte adhesion in stimulated human endothelial cells. 
BACKGROUND: The induction of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by tumor necrosis factor-alpha (TNF) is mediated by mobilization of the transcription factor nuclear factor-kappa B (NF-kappa B). Since salicylates have been reported to inhibit NF-kappa B activation by preventing the degradation of its inhibitor I kappa B, we studied a potential inhibition of this pathway by acetylsalicylate (aspirin) in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Gel-shift analyses demonstrated dose-dependent inhibition of TNF-induced NF-kappa B mobilization by aspirin at concentrations ranging from 1 to 10 mmol/L. Induction of VCAM-1 and E-selectin surface expression by TNF was dose-dependently reduced by aspirin over the same range, while induction of intercellular adhesion molecule-1 (ICAM-1) was hardly affected. Aspirin appeared to prevent VCAM-1 transcription, since it dose-dependently inhibited induction of VCAM-1 mRNA by TNF. As a functional consequence, adhesion of U937 monocytes to TNF-stimulated HUVECs was markedly reduced by aspirin due to suppression of VCAM-1 and E-selectin upregulation. These effects of aspirin were not related to the inhibition of cyclooxygenase activity, since indomethacin was ineffective. CONCLUSIONS: Our data suggest that aspirin inhibits NF-kappa B mobilization, induction of VCAM-1 and E-selectin, and subsequent monocyte adhesion in endothelial cells stimulated by TNF, thereby providing an additional mechanism for therapeutic effects of aspirin. 


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The role of early growth response gene 1 (egr-1) in regulation of the immune response. 
The induction of immediate early genes in cells of the immune system is critical to determining the ultimate outcome of exposure to antigen. The importance of many of these genes relates to the role their transcription factor products play in dictating patterns of expression of downstream, function-related genes. Evidence from several systems indicates that the immediate early gene, egr-1 may be of particular importance in the immune system. Recently, the egr-1 promoter has been shown to be highly responsive to the diverse biochemical signals generated by antigen and cytokines in cells of the immune system. Furthermore, an important role for egr-1 in determining the differentiation pathway of myeloid cell precursors has been recently elaborated. Finally, potential targets of regulation by the zinc-finger transcription factor encoded by egr-1 include the interleukin-2, CD44, ICAM-1, and tumor necrosis factor genes. The role of egr-1 in regulation of the immune response will be discussed in the context of these recent studies. 


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T-cell functional regions of the human IL-3 proximal promoter. 
The human interleukin-3 (IL-3) gene is expressed almost exclusively in activated T cells. Its expression is regulated at both the transcriptional and post-transcriptional level. We have previously shown that treatment of Jurkat T cells with phytohemaglutinin (PHA) and the phorbol ester, PMA, activated transcription initiation from the IL-3 gene. To define the regions of the gene required for transcription activation, we generated a series of reporter constructs containing different regions of the IL-3 gene 5' and 3' flanking sequences. Both positive and negative regulatory elements were identified in the proximal 5' flanking region of the IL-3 gene. The promoter region between -173 and -60 contained the strongest activating elements. The transcription factor AP-1 could bind to this positive activator region of the promoter. We also examined the function of the IL-3 CK-1/CK-2 elements that are present in many cytokine genes and found that they acted as a repressor of basal level expression when cloned upstream of a heterologous promoter but were also inducible by PMA/PHA. 


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Negative regulation of human immunodeficiency virus type 1 expression in monocytes: role of the 65-kDa plus 50-kDa NF-kappa B dimer. 
Although monocytic cells can provide a reservoir for viral production in vivo, their regulation of human immunodeficiency virus type 1 (HIV-1) transcription can be either latent, restricted, or productive. These differences in gene expression have not been molecularly defined. In THP-1 cells with restricted HIV expression, there is an absence of DNA-protein binding complex formation with the HIV-1 promoter-enhancer associated with markedly less viral RNA production. This absence of binding was localized to the NF-kappa B region of the HIV-1 enhancer; the 65-kDa plus 50-kDa NF-kappa B heterodimer was preferentially lost. Adding purified NF-kappa B protein to nuclear extracts from cells with restricted expression overcomes this lack of binding. In addition, treatment of these nuclear extracts with sodium deoxycholate restored their ability to form the heterodimer, suggesting the presence of an inhibitor of NF-kappa B activity. Furthermore, treatment of nuclear extracts from these cells that had restricted expression with lipopolysaccharide increased viral production and NF-kappa B activity. Antiserum specific for NF-kappa B binding proteins, but not c-rel-specific antiserum, disrupted heterodimer complex formation. Thus, both NF-kappa B-binding complexes are needed for optimal viral transcription. Binding of the 65-kDa plus 50-kDa heterodimer to the HIV-1 enhancer can be negatively regulated in monocytes, providing one mechanism restricting HIV-1 gene expression. 


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Cooperation of binding sites for STAT6 and NF kappa B/rel in the IL-4-induced up-regulation of the human IgE germline promoter. 
Ig heavy chain class switching is directed by cytokines inducing transcription from unrearranged CH genes. Subsequently, such primed cells can undergo switch recombination to express the selected new isotype. In the case of IgE class switching, IL-4 activates the IgE germline promoter by inducing the interaction of the transcription factor STAT6 (IL-4STAT) with a responsive DNA element in the proximal region of the promoter. This study describes the characterization of two additional cis-acting elements that interact with members of the NF kappa B/rel transcription factor family in an IL-4-independent fashion. Electrophoretic mobility shift assays show that the nucleoprotein complex formed on the upstream site (NF kappa B1) contains the classical p50/p65 heterodimer. The complex on the proximal site (NF kappa B2) appears to be composed of p50 and relB. IgE germline promoter reporter gene constructs carrying point mutations in the NF kappa B2 site were largely unresponsive to IL-4 stimulation in transient transfection experiments, while plasmids with similar mutations in the NF kappa B1 site responded to cytokine stimulation better than the wild-type promoter. The NF kappa B2 effect was dependent on the presence of the STAT6 binding site, demonstrating that the NF kappa B2 motif is necessary but not sufficient for mediating cytokine up-regulation. In addition, the combination of a NF kappa B/rel binding site and the STAT6 response element conferred IL-4 inducibility to a heterologous minimal promoter, while the individual sites had no effect. The available data suggest that the NF kappa B2 nucleoprotein complex may cooperate with DNA-bound STAT6 to achieve IL-4-dependent activation of the human IgE germline gene. 


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Retinoic acid inhibits CD40 + interleukin-4-mediated IgE production in vitro. 
To elucidate the role of retinoic acid (RA) in anti-CD40 + interleukin-4 (IL-4)-mediated B-cell activation, the effect of 10(-12) to 10(-6) mol/L RA was studied in anti-CD40 (1 microgram/mL) + IL-4 (5 ng/mL)-mediated proliferation and Ig synthesis by human peripheral blood mononuclear cells (PBMC) and B cells in healthy donors. Anti-CD40 + IL-4-mediated proliferation of PBMC and B cells was inhibited by RA in a dose-dependent manner, with maximal inhibition of 62% +/- 5% in PBMC and 55% +/- 4.4% in B cells by all-trans RA, and 58% +/- 6.7% and 51% +/- 4.7%, respectively by 13-cis RA. IgE synthesis was even more markedly inhibited by RA starting at concentrations of >10(-14) mol/L for B cells and >10(-10) mol/L for PBMC. Maximal inhibition of IgE production for B cells was at 10(-8) mol/L for all-trans RA (94% +/- 1.8%) and 96% +/- 3.2% for 13-cis RA. Low concentrations of RA inhibiting IgE synthesis (10(-10) mol/L) affected neither B-cell proliferation nor the production of IgA, IgG, and IgM. Elucidation of the mechanism involved in this inhibition of IgE production shows that epsilon germline transcription is decreased by RA, whereas production of interferon-gamma (IFN-gamma) was not enhanced in the presence of RA. To differentiate whether the RA effect was mediated by RA receptors alpha, beta, and gamma, the expression of the retinoic acid receptors (RAR) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The data show that unstimulated human peripheral B cells express mRNA of the RA receptor alpha, beta, and gamma. Using retinoids with different receptor binding specificity (CD336, CD437, CD2019, CD367), dose-dependent inhibition of IgE synthesis was shown by all four derivates, but was most marked by an RA binding the alpha receptor with high specificity. Taken together, this study shows that RA inhibits IgE production of anti-CD40 + IL-4-stimulated B cells in vitro. Copyright 1998 by The American Society of Hematology. 


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Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by inducing SOCS-1 gene expression. 
Interferons (IFNs) inhibit induction by IL-4 of multiple genes in human monocytes. However, the mechanism by which IFNs mediate this inhibition has not been defined. IL-4 activates gene expression by inducing tyrosine phosphorylation, homodimerization, and nuclear translocation of the latent transcription factor, STAT6 (signal transducer and activator of transcription-6). STAT6-responsive elements are characteristically present in the promoters of IL-4-inducible genes. Because STAT6 activation is essential for IL-4-induced gene expression, we examined the ability of type I and type II IFNs to regulate activation of STAT6 by IL-4 in primary human monocytes. Pretreatment of monocytes with IFN-beta or IFN-gamma, but not IL-1, IL-2, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, IL-6, or transforming growth factor beta suppressed activation of STAT6 by IL-4. This inhibition was associated with decreased tyrosine phosphorylation and nuclear translocation of STAT6 and was not evident unless the cells were preincubated with IFN for at least 1 hr before IL-4 stimulation. Furthermore, inhibition by IFN could be blocked by cotreatment with actinomycin D and correlated temporally with induction of the JAK/STAT inhibitory gene, SOCS-1. Forced expression of SOCS-1 in a macrophage cell line, RAW264, markedly suppressed trans-activation of an IL-4-inducible reporter as well as IL-6- and IFN-gamma-induced reporter gene activity. These findings demonstrate that IFNs inhibit IL-4-induced activation of STAT6 and STAT6-dependent gene expression, at least in part, by inducing expression of SOCS-1. 


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Regulation of IkB alpha phosphorylation by PKC- and Ca(2+)-dependent signal transduction pathways. 
The Ca(2+)-dependent phosphatase calcineurin, a target of FK506 and CsA, synergizes with PKC-induced activation of nuclear factor (NF)-kappa B in T cell lines. We have investigated whether this synergy is present in other cell types and the mechanism(s) by which these two pathways lead to NF-kappa B activation. While this synergy is present in other cell types, in the monocytic cell line U937 calcineurin is also sufficient to activate NF-kappa B. Having previously shown that Ca(2+)- and PKC-dependent pathways synergize by accelerating the degradation of IkB alpha, we focused on the regulation of IkB alpha phosphorylation. While PKC-dependent pathways sequentially result in the phosphorylation and in an incomplete degradation of IkB alpha in T cell lines, co-activation of Ca(2+)-dependent pathways accelerates the rate of IkB alpha phosphorylation and results in its complete degradation. Activation of Ca(2+)-dependent pathways alone do not result in the phosphorylation and/or degradation of IkB alpha in Jurkat T or in U937 cells. Treatment of T cells with the selective PKC inhibitor GF109203X abrogates the PMA-induced IkB alpha phosphorylation/degradation irrespective of activation of Ca(2+)-dependent pathways, but not the phosphorylation and degradation of IkB alpha induced by TNF-alpha, a PKC-independent stimulus. Contrary to the interaction with PKC, Ca(2+)-dependent pathways synergize with TNF-alpha not at the level of IkB alpha phosphorylation, but at the level of its degradation. These results indicate that Ca(2+)-dependent pathways, including the phosphatase calcineurin, participate in the regulation of NF-kappa B in a cell specific fashion and synergize with PKC-dependent and -independent pathways at the level of IkB alpha phosphorylation and degradation. 


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Protein kinase C is not a downstream effector of p21ras in activated T cells. 
The aim of this present study was to investigate the role of protein kinase C (PKC), downstream of p21ras, in activating interleukin-2 (IL-2) gene expression. It has been reported that PKC is an effector of p21ras in T cells. Data is presented, using the potent and selective PKC inhibitor Ro 31-8425 and transient expression of a constitutively active ras mutant, which clearly shows that PKC is not downstream of p21ras in the induction of NF-AT and AP-1 transcriptional activity and in the expression of IL-2 in human Jurkat T cells. Reporter gene experiments demonstrated that NF-kappa B transcriptional activity is not affected by expression of activated p21ras. The signaling pathways involving PKC activation, calcium mobilization and ras activation combine to provide the necessary components for production of IL-2 during T cell activation. 


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Involvement of mitogen-activated protein kinase pathways in interleukin-8 production by human monocytes and polymorphonuclear cells stimulated with lipopolysaccharide or Mycoplasma fermentans membrane lipoproteins. 
Interleukin-8 (IL-8) is a chemokine that belongs to the alpha-chemokine or CXC subfamily and is produced by a wide variety of human cells, including monocytes and polymorphonuclear cells (PMN). IL-8 is secreted in response to inflammatory stimuli, notably bacterial products such as lipopolysaccharide (LPS), but little is known about the mechanisms by which these agents mediate IL-8 induction. In this report, we show that Mycoplasma fermentans lipid-associated membrane proteins (LAMPf) induce the production of high levels of IL-8 by THP-1 (human monocyte) cells and PMN at the same extent as LPS. It was previously demonstrated that stimulation of monocytic cells with either LPS or LAMPf led to a series of common downstream signaling events, including the activation of protein tyrosine kinase and of mitogen-activated protein kinase cascades. By using PD-98059 and SB203580, two potent and selective inhibitors of MEK1 (a kinase upstream of ERK1/2) and p38, respectively, we have demonstrated that both ERK1/2 and p38 cascades play a key role in the production of IL-8 by monocytes and PMN stimulated with bacterial fractions. 


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Interleukin-10 and transforming growth factor-beta promoter polymorphisms in allergies and asthma. 
Interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta) are inhibitory for B and T cells, IgE production, and mast cell proliferation, and they induce apoptosis in eosinophils. These cytokines are therefore candidate genes which could contribute to the development of asthma or allergies. We investigated the hypothesis that polymorphic nucleotides within the IL-10 and TGF-beta gene promoters would link to the expression of allergies and asthma. DNA taken from families with an asthmatic proband was examined for base exchanges by single-stranded conformational polymorphism (SSCP). We demonstrated the presence of a polymorphism in the promoter region of the IL-10 gene and four in the TGF-beta gene promoters (3 in TGF-beta1 and 1 in TGF-beta2). The IL-10 gene polymorphism was a C-to-A exchange 571 base pairs upstream from the translation start site and was present between consensus binding sequences for Sp1 and elevated total serum. This polymorphism was associated with elevated total serum IgE in subjects heterozygotic or homozygotic for this base exchange (p < 0.009). The base exchange at -509 (from the transcription initiation site) in the TGF-beta promoter also linked to elevated total IgE (p < 0.01). This polymorphism represented a C-to-T base exchange which induced a YY1 consensus sequence and is present in a region of the promoter associated with negative transcription regulation. 


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Nuclear accumulation of NFAT4 opposed by the JNK signal transduction pathway. 
The nuclear factor of activated T cells (NFAT) group of transcription factors is retained in the cytoplasm of quiescent cells. NFAT activation is mediated in part by induced nuclear import. This process requires calcium-dependent dephosphorylation of NFAT caused by the phosphatase calcineurin. The c-Jun amino-terminal kinase (JNK) phosphorylates NFAT4 on two sites. Mutational removal of the JNK phosphorylation sites caused constitutive nuclear localization of NFAT4. In contrast, JNK activation in calcineurin-stimulated cells caused nuclear exclusion of NFAT4. These findings show that the nuclear accumulation of NFAT4 promoted by calcineurin is opposed by the JNK signal transduction pathway. 


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Stimulation of neutrophil interleukin-8 production by eosinophil granule major basic protein. 
We evaluated the ability of eosinophil granule major basic protein (MBP) to stimulate interleukin (IL)-8 production by neutrophils. MBP over the concentration range of 0.1 to 10 microM stimulated the release of up to approximately 8 ng/ml IL-8. Incubation with 2 microM MBP showed that, after a 1 h lag, the level of IL-8 release increased with time for approximately 10 h. At the 2 microM concentration, eosinophil cationic protein, eosinophil-derived neurotoxin, and eosinophil peroxidase did not stimulate significant levels of IL-8 production. MBP stimulated 2-fold increases in IL-8 messenger RNA (mRNA) after 1 and 3 h of incubation, which were blocked by pretreatment with actinomycin D. However, stimulation with MBP did not produce an increase in the binding activity of nuclear factor (NF)-kappaB or activator protein-1. No NF-IL-6 binding activity was detected in the same nuclear extracts. In addition, stimulation with MBP prolonged the stability of IL-8 mRNA. MBP also induced transient increases in mRNA for macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, but did not stimulate the release of either chemokine. These findings indicate that MBP is selective among the eosinophil granule proteins as a stimulus for neutrophil IL-8 release and, further, that stimulation of neutrophil IL-8 release by MBP involves both transcriptional and posttranscriptional regulation. We postulate that MBP-induced release of IL-8 by neutrophils may contribute to the pathophysiology of acute asthma and other inflammatory lung diseases. 


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Activation and expression of the nuclear factors of activated T cells, NFATp and NFATc, in human natural killer cells: regulation upon CD16 ligand binding. 
The putative factors that couple the signal transduction from surface receptors to the activation of cytokine synthesis in natural killer (NK) cells have not been elucidated. We report here that the nuclear factor of activated T cells (NFATp), a cyclosporin A (CsA)-sensitive factor that regulates the transcription of several cytokines, mediates CD16-induced activation of cytokine genes in human NK cells. CD16 (Fc gamma RIIIA)-induced expression of cytokine mRNA in NK cells occurs via a CsA-sensitive and Ca(2+)-dependent mechanism. Stimulation of NK cells with CD16 ligands induces NFAT-like DNA binding activity in the nuclear extracts from these cells, as detected in electrophoretic mobility shift assays. This occurs with fast kinetics after stimulation, via a CsA-sensitive and Ca(2+)-dependent mechanism that does not require de novo protein synthesis. NK cell NFAT is present in the cytosol of nonstimulated cells, migrates to the nucleus upon stimulation, and can associate with AP-1. Two distinct molecules, NFATp and NFATc, have been reported to mediate NFAT activity. The results of supershift assays using NFATp- and NFATc- specific antibodies indicate that NK cell activation early after CD16 ligand binding involves primarily, if not exclusively, NFATp, and Western blot analysis shows that this has the same electrophoretic mobility (approximately 120 kD) as that of T lymphocytes. NK cells do not express NFATc constitutively, but NFATc mRNA accumulation is induced in these cells within 2 h of stimulation with CD16 ligands. However, supershift assays using the available mAb recognizing the T cell NFATc revealed no detectable NFATc protein in nuclear and cytoplasmic extracts from CD16- or phorbol ester-stimulated cells at any time tested, up to 4 h. These results provide the first direct evidence that both CsA-sensitive transcription factors, NFATp and NFATc, are expressed in human NK cells, and that their activation and/or expression can be regulated in primary cells by a single stimulus, that, in the case of CD16 in NK cells, results in early activation of NFATp and subsequently induced expression of NFATc mRNA. 


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Overexpression of protein kinase C-zeta stimulates leukemic cell differentiation. 
A function for protein kinase C-zeta (PKC-zeta), a member of the phorbol ester nonresponsive atypical protein kinase C subfamily, in modulating differentiation was examined in the leukemic U937 cell. Transfected U937 cells stably overexpressing PKC-zeta displayed a longer doubling time, lower saturation density at confluency, and an increase in adherence to plastic as compared to control cells. PKC-zeta cells expressed a more differentiated phenotype as assessed by changes in morphology, surface antigen expression, and lysosomal enzyme activities and were distinct from parental U937 cells stimulated to differentiate by exposure to phorbol esters. In contrast to parental U937 cells, PKC-zeta cells constitutively expressed mRNA transcripts for c-jun and a low mobility AP-1 binding activity. Thus, PKC-zeta overexpression stimulates a type of phenotypic differentiation that differs significantly from maturation occurring upon activation of other PKC subfamilies induced by phorbol ester treatment. Increased expression of the c-jun protooncogene and an increase in AP-1 binding activity in PKC-zeta cells provides a potential mechanism for explaining the altered differentiation status of this cell. 


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Characterization of a mutant cell line that does not activate NF-kappaB in response to multiple stimuli. 
Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor kappaB (NF-kappaB). Associated with its inhibitor, I kappaB, NF-kappaB resides as an inactive form in the cytoplasm. Upon stimulation by various agents, I kappaB is proteolyzed and NF-kappaB translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to I kappaB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-kappaB in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, I kappaB alpha is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in I kappaB alpha nor a mutation in p50 or relA, the two major subunits of NF-kappaB in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of I kappaB alpha on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce I kappaB alpha degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-kappaB activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other I kappaB molecules, I kappaB beta and the recently identified I kappaB epsilon, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-kappaB. In addition, this analysis suggests a common step in the signaling pathways that trigger I kappaB alpha, I kappaB beta, and I kappaB epsilon degradation. 


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RP1, a new member of the adenomatous polyposis coli-binding EB1-like gene family, is differentially expressed in activated T cells. 
Cross-linking of the CD3 and CD28 molecules on T lymphocytes represents one of the most effective signals for T lymphocyte activation and triggering of their cytotoxic effector function. To identify genes that are expressed in T cells after stimulation, mRNA from T lymphocytes that had been activated by the simultaneous stimulation of the CD3 and CD28 trigger molecules was transcribed for a differential mRNA display analysis into cDNA and was compared with cDNA from CD28- or CD3-activated or resting lymphocytes. Differential expression was confirmed subsequently by Northern blot analysis. One of the cDNA fragments expressed specifically in CD3- and CD28-activated T cells was designated RP1. The predictive protein-coding region of RP1 had a significant homology to members of the recently found adenomatous polyposis coli (APC) protein-binding EB1 gene family, which codes for yet unknown protein(s). Bacterially expressed RP1 protein revealed specific binding to wild-type but not to mutated APC protein. The rapid up-regulation of RP1 mRNA in properly activated T cells suggests that this gene might belong to the immediate/early gene family, which controls the signal transduction cascade downstream of the TCR. As the expression level of the RP1 gene in activated T cells and a spectrum of tumor-derived cell lines correlates with the proliferative status of the cells, members of the EB1-like gene family may not only be involved in the tumorigenesis of colorectal cancers but may also play a role in the proliferative control of normal cells. 


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Non-steroidal anti-inflammatory drugs inhibit the expression of cytokines and induce HSP70 in human monocytes. 
Recent studies have shown that the non-steroidal anti-inflammatory drugs (NSAIDs) activate heat shock transcription factor (HSF1) from a latent cytoplasmic form to a nuclear, DNA binding state. As HSF1 can function as both an activator of heat shock genes and a repressor of non-heat shock genes such as IL1B and c- fos, we have examined the potential role of HSF1 in the effects of NSAIDs on gene expression in a human monocytic cell line THP-1. We found that two members of the NSAIDs, sodium salicylate and sulindac repress the IL1B promoter to similar degree to heat shock or HSF1 overexpression. In addition, sodium salicylate and additional NSAIDs used at concentrations that activate HSF1 also inhibited the expression of other monocytic genes (TNF-alpha, IL-1beta, IL-6, IL-8, IL-10, ICAM-1) activated by exposure to a pro-inflammatory stimulus (lipopolysaccharide, LPS). At least in the case of the IL1B promoter, repression did not seem to involve another factor whose activity is affected by the NSAIDs, NFkappaB as the IL1B promoter fragment used in our studies is not NFkappaB responsive and binds specifically to HSF1. Exposure to NSAIDs had a complex effect on HSP gene expression and while sulindac activated the stress responsive HSP70B promoter, sodium salicylate did not. In addition, only a subset of the NSAIDs induced HSP70 mRNA species. These findings reflect the properties of HSF1 which can be activated to at least two DNA binding forms only one of which activates heat shock promoters and suggest that individual NSAID family members may differentially induce one or other of these forms. Overall therefore, exposure to NSAIDs leads to a profound switch in gene expression in monocytic cells, with suppression of genes involved in macrophage activation and induction of stress genes and HSF1 appears to play a regulatory role in these effects. Copyright 1999 Academic Press. 


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Modulation of mRNA expression of a novel human myeloid-selective CCAAT/enhancer binding protein gene (C/EBP epsilon). 
Human C/EBP epsilon is a newly cloned gene coding for a CCAAT/enhancer binding protein that may be involved in the regulation of myeloid differentiation. Our studies showed that levels of C/EBP epsilon mRNA were markedly increased in NB4 cells (promyelocytic leukemia line), because they were induced by 9-cis retinoic acid (9-cis RA) to differentiate towards granulocytes. Accumulation of C/EBP epsilon mRNA occurred as early as 1 hour after exposure of NB4 cells to 9-cis RA (5 x 10(-7) mol/L); and at 48 hours, levels were increased by 5.1-fold. Dose-response studies showed that 10(-7) to 10(-6) mol/L 9-cis RA (12 hours) resulted in peak levels of C/EBP epsilon mRNA; but even 10(-10) mol/L 9-cis RA increased levels of these transcripts. NB4 cells pulse-exposed (30 minutes) to all-trans retinoic acid (ATRA), washed, and cultured (3 days) with either dimethylsulfoxide (DMSO) or hexamethylene bisacetamide (HMBA) had a prominent increase in levels of C/EBP epsilon mRNA and an increase in granulocytic differentiation, but exposure to either DMSO or HMBA alone had no effect on base levels of C/EBP epsilon and did not induce differentiation. Macrophage-differentiation of NB4 reduced levels of C/EBP epsilon mRNA. Nuclear run-off assays and half-life studies showed that accumulation of C/EBP epsilon mRNA by 9-cis RA was due to enhanced transcription. Furthermore, this C/EBP epsilon mRNA accumulation did not require synthesis of new protein factors because 9-cis RA induced C/EBP epsilon mRNA accumulation in the absence of new protein synthesis. ATRA also induced expression of C/EBP epsilon protein in NB4 cells, as shown by Western blotting. In contrast to the increase of C/EBP epsilon in 9-cis RA-mediated granulocytic differentiation, the DMSO-induced differentiation of HL-60 cells down the granulocytic pathway was associated with an initial reduction of C/EBP epsilon mRNA levels. In summary, we have discovered that expression of C/EBP epsilon mRNA is markedly enhanced as the NB4 promyelocytes are induced by retinoids to differentiate towards granulocytes. This induction of C/EBP epsilon mRNA expression is transcriptionally mediated and occurs in the absence of synthesis of additional protein factors. We suspect that the C/EBP epsilon promoter/enhancer contains a retinoic acid-response element that is directly stimulated by retinoids. 


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Signalling via CD28 of human naive neonatal T lymphocytes. 
Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. We have examined the role of CD28 in modulating the 'naive' neonatal T cell response to anti-CD2-mediated activation. To compare the role of CD28, neonatal and adult T cells were stimulated with a pair of mitogenic anti-CD2 antibodies in the presence or absence of anti-CD28 MoAb. With anti-CD2 alone, neonatal T cells proliferated slightly but produced no detectable IL-2, whereas adult T cells proliferated vigorously, with significant IL-2 production. Costimulation with anti-CD28 MoAb greatly enhanced the proliferative response of neonatal T cells to levels equivalent to those of adult T cells, whereas adult T cells showed only slight increases. Although IL-2 secretion was increased in the presence of anti-CD28 MoAb, neonatal T cell IL-2 production remained lower than in adults. In contrast, enhancement of IL-2 mRNA expression in neonates was similar to adult levels. Anti-CD28 MoAb costimulation increased NF kappa B levels in neonates, albeit to levels lower than that of adults. The cellular mechanism governing the diminished proliferative response of neonatal T lymphocytes to anti-CD2 may therefore be due to decreased NF kappa B induction, reduced IL-2 mRNA expression and deficient IL-2 production. Although anti-CD28 MoAb costimulation enhances all of the above signals, NF kappa B and IL-2 levels remain lower than in adults, suggesting the need for further activation requirements in the neonate. 


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c-Rel and p65 subunits bind to an upstream NF-kappaB site in human granulocyte macrophage-colony stimulating factor promoter involved in phorbol ester response in 5637 cells. 
To further clarify the complex transcriptional regulation of the human GM-CSF gene, which was extensively investigated in activated T cells, we have studied the role of an upstream NF-kappaB like site in the 5637 non-lymphoid cell line, which derives from a bladder carcinoma and constitutively produces GM-CSF. This sequence, named the A element, has an active role on GM-CSF transcription and is responsive to the tumor promoter PMA in transient transfection experiments. We describe here a heterodimeric binding complex of NF-kappaB subunits (c-Rel and p65) which is identical to the one obtained using the HIV-LTR-kappaB site as recognition sequence and different from the one (c-Rel and p50) observed with nuclear extracts from Mo T-lymphoid HTLV-II infected cells. 


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Ubiquitin-mediated processing of NF-kappa B transcriptional activator precursor p105. Reconstitution of a cell-free system and identification of the ubiquitin-carrier protein, E2, and a novel ubiquitin-protein ligase, E3, involved in conjugation. 
In most cases, the transcriptional factor NF-kappa B is a heterodimer consisting of two subunits, p50 and p65, which are encoded by two distinct genes of the Rel family. p50 is translated as a precursor of 105 kDa. The C-terminal domain of the precursor is rapidly degraded, forming the mature p50 subunit consisted of the N-terminal region of the molecule. The mechanism of generation of p50 is not known. It has been suggested that the ubiquitin-proteasome system is involved in the process; however, the specific enzymes involved and the mechanism of limited proteolysis, in which half of the molecule is spared, have been obscure. Palombella and colleagues (Palombella, V.J., Rando, O.J., Goldberg, A.L., and Maniatis, T.(1994) Cell 78, 773-785) have shown that ubiquitin is required for the processing in a cell-free system of a truncated, artificially constructed, 60-kDa precursor. They have also shown that proteasome inhibitors block the processing both in vitro and in vivo. In this study, we demonstrate reconstitution of a cell-free processing system and demonstrate directly that: (a) the ubiquitin-proteasome system is involved in processing of the intact p105 precursor, (b) conjugation of ubiquitin to the precursor is an essential intermediate step in the processing, (c) the recently discovered novel species of the ubiquitin-carrier protein, E2-F1, that is involved in the conjugation and degradation of p53, is also required for the limited processing of the p105 precursor, and (d) a novel, approximately 320-kDa species of ubiquitin-protein ligase, is involved in the process. This novel enzyme is distinct from E6-AP, the p53-conjugating ligase, and from E3 alpha, the "N-end rule" ligase. 


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The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor. 
The macrophage colony-stimulating factor (M-CSF) receptor is expressed in a tissue-specific fashion from two distinct promoters in monocytes/macrophages and the placenta. In order to further understand the transcription factors which play a role in the commitment of multipotential progenitors to the monocyte/macrophage lineage, we have initiated an investigation of the factors which activate the M-CSF receptor very early during the monocyte differentiation process. Here we demonstrate that the human monocytic M-CSF receptor promoter directs reporter gene activity in a tissue-specific fashion. Since one of the few transcription factors which have been implicated in the regulation of monocyte genes is the macrophage- and B-cell-specific PU.1 transcription factor, we investigated whether PU.1 binds and activates the M-CSF receptor promoter. Here we demonstrate that both in vitro-translated PU.1 and PU.1 from nuclear extracts bind to a specific site in the M-CSF receptor promoter just upstream from the major transcription initiation site. Mutations in this site which eliminate PU.1 binding decrease M-CSF receptor promoter activity significantly in macrophage cell lines only. Furthermore, PU.1 transactivates the M-CSF receptor promoter in nonmacrophage cells. These results suggest that PU.1 plays a major role in macrophage gene regulation and development by directing the expression of a receptor for a key macrophage growth factor. 


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The severe phenotype of females with tiny ring X chromosomes is associated with inability of these chromosomes to undergo X inactivation. 
Mental retardation and a constellation of congenital malformations not usually associated with Turner syndrome are seen in some females with a mosaic 45,X/46,X,r(X) karyotype. Studies of these females show that the XIST locus on their tiny ring X chromosomes is either not present or not expressed. As XIST transcription is well correlated with inactivation of the X chromosome in female somatic cells and spermatogonia, nonexpression of the locus even when it is present suggests that these chromosomes are transcriptionally active. We examined the transcriptional activity of ring X chromosomes lacking XIST expression (XISTE-), from three females with severe phenotypes. The two tiny ring X chromosomes studied with an antibody specific for the acetylated isoforms of histone H4 marking transcribed chromatin domains were labeled at a level consistent with their being active. We also examined tow of the XISTE- ring chromosomes to determine whether genes that are normally silent on an inactive X are expressed from these chromosomes. Analyses of hybrid cells show that TIMP, ZXDA, and ZXDB loci on the proximal short arm, and AR and PHKA1 loci on the long arm, are well expressed from the tiny ring X chromosome lacking XIST DNA. Studies of the ring chromosome that has XIST DNA but does not transcribe it show that its AR allele is transcribed along with the one on the normal X allele. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Various modes of basic helix-loop-helix protein-mediated regulation of murine leukemia virus transcription in lymphoid cell lines. 
The transcriptionally regulatory regions of the lymphomagenic Akv and SL3-3 murine leukemia retroviruses (MLVs) contain two types of E-box consensus motifs, CAGATG. One type, EA/S, is located in the upstream promoter region, and the other, E(gre), is located in a tandem repeat with enhancer properties. We have examined the requirements of the individual E-boxes in MLV transcriptional regulation. In lymphoid cell lines only, the E(gre)-binding protein complexes included ALF1 or HEB and E2A basic helix-loop-helix proteins. Ectopic ALF1 and E2A proteins required intact E(gre) motifs for mediating transcriptional activation. ALF1 transactivated transcription of Akv MLV through the two E(gre) motifs equally, whereas E2A protein required the promoter-proximal E(gre) motif. In T- and B-cell lines, the E(gre) motifs were of major importance for Akv MLV transcriptional activity, while the EA/S motif had some effect. In contrast, neither E(gre) nor EA/S motifs contributed pronouncedly to Akv MLV transcription in NIH 3T3 cells lacking DNA-binding ALF1 or HEB and E2A proteins. The Id1 protein was found to repress ALF1 activity in vitro and in vivo. Moreover, ectopic Id1 repressed E(gre)-directed but not EA/S-directed MLV transcription in lymphoid cell lines. In conclusion, E(gre) motifs and interacting basic helix-loop-helix proteins are important determinants for MLV transcriptional activity in lymphocytic cell lines. 


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Inhibition of IL-4-inducible gene expression in human monocytes by type I and type II interferons. 
The Th2-type cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13), induce expression of a distinct subset of genes in human monocytes, including FcepsilonRIIb (CD23), 15-lipoxygenase, IL-1 receptor antagonist (IL-1ra), and type I and type II IL-1 receptors (IL-1R). Type I interferons (IFN-alpha and IFN-beta) and type II interferon (IFN-gamma) inhibit induction of these genes by IL-4 and IL-13. However, the mechanism by which IFNs mediate this inhibition has not been defined. In this overview, we discuss the role of the transcription factor, STAT6 (signal transducer and activator of transcription-6) in mediating IL-4- and IL-13-induced gene expression in monocytes. We also discuss our recent findings that type I and type II IFNs suppress IL-4/IL-13-inducible gene expression by inhibiting tyrosine phosphorylation and nuclear translocation of STAT6. The ability of type I and type II IFNs to inhibit IL-4/IL-13-induced STAT6 activity is dose- and time-dependent, and is not unique to monocytes because IFNs induce the same effects in fibroblasts. Inhibition of STAT6 activity is not evident unless cells are preincubated with IFN for at least 1 h before IL-4 stimulation. Furthermore, inhibition can be blocked by actinomycin D, indicating a requirement for de novo transcription. We propose a model in which stimulation of monocytes by IFN activates de novo synthesis of an inhibitory factor, possibly one or more members of the SOCS/ SSI/CIS gene family, capable of suppressing activation of STAT6 by IL-4 and IL-13. Because STAT6 activation plays an essential role in IL-4/IL-13-induced gene expression, the ability of IFN-beta and IFN-gamma to inhibit STAT6 activity provides an explanation for how IFNs can suppress IL-4/IL-13-inducible gene expression. 


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Positive and negative regulation of IL-2 gene expression: role of multiple regulatory sites. 
Interleukin 2 (IL-2) is an important lymphokine required in the process of T cell activation, proliferation, clonal expansion and differentiation. The IL-2 gene displays both T cell specific and inducible expression: it is only expressed in CD4+ T cells after antigenic or mitogenic stimulation. Several cis-acting regulatory sites are required for induction of the IL-2 gene after stimulation. In this study, we have analysed the function of these cis-acting regulatory sites in the context of the native IL-2 enhancer and promoter sequence. The results of this study suggest that the NFAT (-276 to -261), the distal octamer (-256 to -248) and the proximal octamer (-75 to -66) sites not only act as enhancers of IL-2 gene transcription in the presence of cellular stimulation, but also have a silencing effect on IL-2 gene expression in resting cells. Two other sites display disparate effects on IL-2 gene expression in different T leukemia cell lines: the distal purine box (-291 to -277) and the proximal purine box sites (-145 to -128). Finally, the AP-1 (-186 to -176) and the kappa B sites (-206 to -195) respond to different cellular activation in EL4 cells. The AP-1 site mediated the response to PMA stimulation while the kappa B site responded to IL-1 stimulation. These data suggest that the regulation of IL-2 gene expression is a complex process and multiple cis-acting regulatory sites interact to exert different effects in T cells representative of alternative stages of differentiation. 


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Nuclear transcription factors that bind to elements of the IL-2 promoter. Induction requirements in primary human T cells. 
Prior studies have identified several elements that contribute to the activity of the IL-2 promoter in the stimulated T cell line, Jurkat. The sites and their corresponding nuclear binding factors include: NF-kappa B, AP-1, AP-3, OCT-1, and NF-AT. The latter "nuclear factor for activated T cells" likely contributes to the tissue specificity of IL-2 gene expression. Using electrophoretic mobility shift assays, we have studied these transcription factors in primary T cells from human blood to verify their presence in a physiologic setting and to identify the signals that stimulate factor activity. All factors are induced in the nuclei of T cells upon activation with mitogens but not with exogenous IL-2 growth factor. However, the signaling requirements and sensitivity to protein synthesis inhibitors differ considerably. Only the activities for NF-AT and AP-1 sites require two signals for optimal induction, i.e., PMA plus either lectin or antibody to the CD3 or CD28 surface molecules. Other factors are induced by lectin, antibody, and/or PMA alone. After appropriate stimulation, both NF-AT and AP-1 are peculiarly sensitive to the protein synthesis inhibitor anisomycin. Our data correlate the activity of NF-AT and AP-1 in gel shift assays with the two signals requirements for IL-2 gene expression. 


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Cytokine-modulating activity of tepoxalin, a new potential antirheumatic. 
Tepoxalin is a new dual cyclooxygenase/5-lipoxygenase anti-inflammatory compound currently under clinical investigation. It has been shown to possess anti-inflammatory activity in a variety of animal models and more recently to inhibit IL-2 induced signal transduction. The current study was conducted to evaluate the cytokine modulating activity of tepoxalin and the role of iron in these effects. In human peripheral blood mononuclear cells (PBMC) stimulated with OKT3/PMA, tepoxalin inhibited lymphocyte proliferation with an IC50 of 6 microM. Additionally, it inhibited the production of LTB4 (IC50 = 0.5 microM) and the cytokines IL-2, IL-6 and TNF alpha (IC50 = 10-12 microM). Cytotoxicity was not demonstrated at these concentrations. Add-back experiments with either cytokines (IL-2 or IL-6), LTB4 or conditioned media failed to restore the proliferative response in the presence of tepoxalin. However, the concurrent addition of iron (in the form of ferrous or ferric chloride and other iron salts) reversed the inhibition of proliferation caused by tepoxalin. Tepoxalin also inhibits the activation of NF kappa B, a transcription factor which acts on several cytokine genes. Tepoxalin's effect on NF kappa B is also reversed by the addition of iron salts. These data suggest that the action of tepoxalin to inhibit proliferation in PBMC may be at least in part due to its ability to reduce the amount of available iron resulting in decreased activation of NF kappa B and subsequent inhibition of cytokine production. 


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Role of the X2 box in activated transcription from the DRA promoter in B cells. 
We investigated the function of the evolutionary conserved X2 box in the promoter of the HLA-DRA gene from the human major histocompatibility complex (MHC) in resting and activated B cells. NF-X2, which contains members of the AP-1/ATF/CREB families of transcription factors, interacts with the X2 box (5'-TGCGTCA-3') from positions -97 to -91 in the DRA promoter. In resting Raji cells, little to no binding to the X2 box was observed. In sharp contrast, in B cells treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), strong interactions between the X2 box and NF-X2 containing c-Fos were observed. As determined by transient expression and RNA analyses, the activation of protein kinase C (PKC) also increased rates of transcription from the wild-type DRA promoter but not from a DRA promoter bearing clustered point mutations in the X2 box. Since the co-expression with a dominant negative c-Fos abolished the responsiveness to TPA, we conclude that activated transcription of the DRA gene depends on interactions between the X2 box and NF-X2, which contains c-Fos. 


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Expression of transcription factor genes after influenza A virus infection. 
Infection of human monocytes with influenza A virus induces a broad range of proinflammatory cytokines and mononuclear cell attracting chemokines before the infected cells undergo apoptosis. The underlying mechanisms by which the corresponding genes are transcriptionally initiated after virus infection are still poorly understood. Activation of NF-kappa B seems to play an important role in the regulation of many proinflammatory cytokine genes, but cannot be the only mechanism, since several cytokine genes lack respective binding sites in their promoter regions. Therefore, we additionally investigated other transcription factors of possible importance such as CREB, CTF, OTF-1, and OTF-2. To explore long-term regulatory mechanisms, we investigated the induction of transcription factors on the gene expression level which may be important to substitute for metabolized transcription factor proteins after their activation. We identified a cell-type-specific differential response: CREB, CTF, OTF-1, OFT-2, and NF-kappa B genes were strongly induced 1 to 4 hours after influenza A virus infection in the monocytic cell line Mono Mac 6, while in freshly prepared human monocytes no significant changes were detected. In infected monocytes, which die by apoptosis, the expression of CREB, CTF, and OTF-2 was rather suppressed 8 hours after infection. In conclusion, the long-term regulation of transcription factor gene expression in non-proliferating cells seems to be of minor importance after influenza infection since in apoptosisprone cells an immediate availability of transcription factor proteins is required. 


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Cell-specific expression of helix-loop-helix transcription factors encoded by the E2A gene. 
The E2A gene encodes transcription factors of the helix-loop-helix family that are implicated in cell-specific gene expression as part of dimeric complexes that interact with E box enhancer elements. It has previously been shown that transcripts of the E2A gene can be detected in a wide range of cell types. We have now examined expression of the mouse E2A gene at the protein level using polyclonal antisera directed against distinct portions of the E2A protein to probe blots of cellular extracts. A 73 kDa protein was identified by this analysis: this protein is highly enriched in cell lines of B lymphoid origin as compared to pancreatic beta-cells and fibroblast cells. The detection of this protein selectively in extracts of lymphoid cells correlates with the presence of the E box-binding activity LEF1/BCF1 in these cells; this binding activity was previously shown to be efficiently recognized by antiserum directed against E2A gene products. Transfection of cells with full length E2A cDNA leads to appearance of protein co-migrating with the 73 kDa protein on SDS gel electrophoresis and co-migrating with LEF1/BCF1 on mobility shift analysis. Our results are consistent with the view that the DNA-binding activity LEF1/BCF1 is a homodimer of E2A proteins; the selective appearance of this putative cell-specific transcription factor in B lymphoid cells seems to be attributable, at least in part, to the elevated E2A protein concentrations in these cells. 


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Inducible nitric oxide: an autoregulatory feedback inhibitor of vascular inflammation. 
Inducible nitric oxide (iNO) is produced at sites of vascular inflammation by resident and nonresident vascular wall cells, but its role in the inflammatory process is not known. In this study, we show that a novel function of iNO is to terminate inflammatory processes. We find that iNO produced by murine macrophage-like cells, RAW264.7, can inhibit cytokine-induced endothelial cell activation in a separated and mixed endothelial-RAW264.7 coculture system. Both iNO production and endothelial VCAM-1 expression were induced simultaneously with bacterial LPS and murine-specific IFN-gamma. Inhibition of iNO synthase (iNOS) activity with N omega-monomethyl-L-arginine in endothelial-RAW264.7 cocultures, stimulated with murine-specific IFN-gamma and LPS, decreased iNO production by 86%, augmented VCAM-1 and iNOS expression in endothelial and RAW264.7 cells, respectively, and increased monocyte adhesion to the endothelial cell surface. Transient transfection studies using various VCAM-1 promoter constructs demonstrated that inhibitory effects of iNO on VCAM-1 gene transcription were mediated, in part, by inhibitory effects of iNO on kappa B cis-acting elements. Immunofluorescence studies using an Ab to the RelA (p65) subunit of nuclear factor-kappa B revealed that iNO inhibited the activation of nuclear factor-kappa B. These studies indicate that iNO attenuates iNOS expression in macrophages and inhibits monocyte adhesion to endothelial cells, and suggest that endogenously derived iNO may be an important autoregulatory inhibitor of vascular inflammation. 


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Human cytomegalovirus binding to human monocytes induces immunoregulatory gene expression. 
To continue our investigation of the cellular events that occur following human CMV (HCMV) infection, we focused on the regulation of cellular activation following viral binding to human monocytes. First, we showed that viral binding induced a number of immunoregulatory genes (IL-1beta, A20, NF-kappaB-p105/p50, and IkappaBalpha) in unactivated monocytes and that neutralizing Abs to the major HCMV glycoproteins, gB (UL55) and gH (UL75), inhibited the induction of these genes. Next, we demonstrated that these viral ligands directly up-regulated monocyte gene expression upon their binding to their appropriate cellular receptors. We then investigated if HCMV binding also resulted in the translation and secretion of cytokines. Our results showed that HCMV binding to monocytes resulted in the production and release of IL-1beta protein. Because these induced gene products have NF-kappaB sites in their promoter regions, we next examined whether there was an up-regulation of nuclear NF-kappaB levels. These experiments showed that, in fact, NF-kappaB was translocated to the nucleus following viral binding or purified viral ligand binding. Changes in IkappaBalpha levels correlated with the changes in NF-kappaB translocation. Lastly, we demonstrated that p38 kinase activity played a central role in IL-1beta production and that it was rapidly up-regulated following infection. These results support our hypothesis that HCMV initiates a signal transduction pathway that leads to monocyte activation and pinpoints a potential mechanism whereby HCMV infection of monocytes can result in profound pathogenesis, especially in chronic inflammatory-type conditions. 


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Evidence that calcineurin is rate-limiting for primary human lymphocyte activation. 
Cyclosporine (CsA) is both a clinical immunosuppressive drug and a probe to dissect intracellular signaling pathways. In vitro, CsA inhibits lymphocyte gene activation by inhibiting the phosphatase activity of calcineurin (CN). In clinical use, CsA treatment inhibits 50-75% of CN activity in circulating leukocytes. We modeled this degree of CN inhibition in primary human leukocytes in vitro in order to study the effect of partial CN inhibition on the downstream signaling events that lead to gene activation. In CsA-treated leukocytes stimulated by calcium ionophore, the degree of reduction in CN activity was accompanied by a similar degree of inhibition of each event tested: dephosphorylation of nuclear factor of activated T cell proteins, nuclear DNA binding, activation of a transfected reporter gene construct, IFN-gamma and IL-2 mRNA accumulation, and IFN-gamma production. Furthermore, the degree of CN inhibition was reflected by a similar degree of reduction in lymphocyte proliferation and IFN-gamma production in the allogeneic mixed lymphocyte cultures. These data support the conclusion that CN activity is rate-limiting for the activation of primary human T lymphocytes. Thus, the reduction of CN activity observed in CsA-treated patients is accompanied by a similar degree of reduction in lymphocyte gene activation, and accounts for the immunosuppression observed. 


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Activation of transcription by binding of NF-E1 (YY1) to a newly identified element in the first exon of the human DR alpha gene. 
A previously unrecognized element, located downstream of the start site of transcription in the first exon of the DR alpha gene, has been defined that enhances promoter activity up to eightfold in a position-dependent manner. Mutations in this DNA-binding site abolished binding of a nuclear factor in human B cell nuclear extract and decreased the activity of the DR alpha promoter to a basal level. Significant sequence homology of this element was found in the DNA of the DR beta, DP alpha and -beta, and DQ alpha genes, always located downstream of the transcriptional start site. The nuclear factor binds to the DR alpha and DP alpha element but not to the element in the DQ alpha gene. It was identified as NF-E1 (YY1). This protein, previously identified by its binding to the Ig kappa 3' enhancer and the Ig heavy chain mu E1 site, thus also appears to be quite important in the regulation of MHC class II gene expression. 


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Identification of upstream regulatory elements that repress expression of adult beta-like globin genes in a primitive erythroid environment. 
Our investigations have focused on localizing cis-elements responsible for the down regulation of the adult beta-like globin genes (delta and beta) in immature, or primitive erythroid tissues. We studied their activity after transfection into K562 cells, an erythroleukemia cell line with an embryonic-fetal phenotype. Analyzed DNA sequences included delta and beta 5' flanking regions extending from approximately -500 to +50bp (promoter regions), truncated delta and beta 5' flanking regions extending from approximately -250 to +50 bp, and chimeric promoter constructions, which consisted of a distal delta or beta fragment fused to a proximal beta or delta sequence. In CAT reporter constructions no appreciable level of CAT activity was supported by the beta globin promoter, and only low level activity by the delta promoter. Truncation of the beta globin promoter led to a 2-3 fold increase in promoter activity. In contrast, deletion of the upstream portion of the delta promoter led to a 10 fold decrease in expression. Coupling of the upstream beta globin sequence from approximately -500 to -250 bp to the truncated delta promoter fragment led to complete extinction of transcription activity, consistent with a negative regulatory effect of the beta globin gene upstream element(s). Fusion of the upstream portion of the delta promoter to the truncated beta globin promoter yielded a modest increase in promoter strength relative to the truncated beta gene promoter, indicating the presence of a positive transcriptional element(s) in the upstream delta globin regulatory region. Site-directed mutagenesis of binding sites for the repressor proteins BP1 and BP2 in the upstream portion of the beta globin gene flanking region led to a 4-6 fold increase in promoter activity. DNase I footprinting of the upstream delta-globin region revealed protected sequences corresponding to consensus binding sites for GATA-1 and BP2. These results confirm that sequences in the upstream promoter region of the adult beta globin gene contribute to its factor-mediated suppression early in development and then may modulate its expression at a later stage. 


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IL-2 and IL-7 induce heterodimerization of STAT5 isoforms in human peripheral blood T lymphoblasts. 
Despite differences in T cell responses induced by interleukin (IL)-2 and IL-7, both cytokines modulate T cell functions by activation of signal transducers and activators of transcription (STAT) proteins. We examined the contribution of the two isoforms of STAT5, STAT5A and STAT5B, to IL-2- and IL-7-induced activation of human peripheral blood T lymphoblasts. Both cytokines induced assembly of STAT5A and STAT5B containing complexes capable of binding to the interferon-gamma activation sequence (GAS), and these complexes rapidly translocated (within 1 min) into the nucleus of IL-2- or IL-7-treated cells. The kinetics of this translocation were delayed in IL-7-treated as compared to IL-2-treated cells. IL-2 and IL-7 were equivalent in their ability to induce tyrosine phosphorylation of STAT5A and STAT5B and to facilitate binding of these STATs to an immobilized GAS element. Both IL-2 and IL-7 induced substantial amounts of STAT5A/STAT5B heterodimerization. Moreover, we observed constitutive association of STAT3 with each STAT5 isomer. These data suggest that IL-2 and IL-7 induce assembly of STAT heterodimers in a similar manner and that subsequent cellular responses may be driven by induction of similar sets of genes. 


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Characterization of the human immunodeficiency virus type 1 enhancer-binding proteins from the human T-cell line Jurkat. 
The transcription of the human immunodeficiency virus type 1 (HIV-1) is under the control of cellular proteins that bind to the viral long terminal repeat (LTR). Among the protein-binding regions of the HIV-1 LTR is the transcription-enhancer region. We show that at least one inducible, C1, and one constitutive, C2, protein can bind to the HIV enhancer in Jurkat cells. The two proteins differ in their surface charge, since they are separable by anion-exchange chromatography. Bivalent cations such as Mg2+ and Zn2+ differentially affect their binding to oligonucleotides which contain the HIV-enhancer domain. Both C1 and C2 proteins also bind to a similar sequence found in the interleukin-2-receptor alpha-subunit enhancer. The inducible C1 protein was partially purified by three chromatographic steps and characterized by u.v. cross-linking as a 47 kDa protein. 


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Cyclosporin A-resistant transactivation of the IL-2 promoter requires activity of okadaic acid-sensitive serine/threonine phosphatases. 
Expression of the IL-2 gene requires activation of T cells through stimulation of the TCR and costimulation through accessory receptors. We have found recently that okadaic acid-sensitive Ser/Thr phosphatases are involved in a cyclosporin A-insensitive pathway that selectively transmits costimulatory signals. In this study, we analyzed whether activities of these phosphatases are necessary for the expression of the IL-2 gene. In both activated peripheral blood T lymphocytes and activated tumorigenic T cell lines, IL-2 gene expression was blocked at the transcriptional level by okadaic acid. The transcription factors active at the IL-2 promoter were differentially influenced: upon down-modulation of okadaic acid-sensitive phosphatases, transactivation by octamer, NF-kappa B, and NF of activated T cells proteins was abrogated, while transactivation by AP-1 proteins was even enhanced. 


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TCL1 oncogene activation in preleukemic T cells from a case of ataxia-telangiectasia. 
The TCL1 oncogene on human chromosome 14q32.1 is involved in chromosome translocations [t(14;14)(q11;q32.1) and t(7;14)(q35;q32.1)] and inversions [inv14(q11;q32.1)] with TCR alpha/beta loci in T-cell leukemias, such as T-prolymphocytic (T-PLL). It is also involved in T- acute and- chronic leukemias arising in cases of ataxia-telangiectasia (AT), an immunodeficiency syndrome. Similar chromosomal rearrangements occur also in the clonally expanded T cells in AT patients before the appearance of the overt leukemia. We have analyzed the expression of TCL1 mRNA and protein in peripheral blood lymphocytes (PBLs) from four AT cases and from healthy controls. We found that the TCL1 gene was overexpressed in the PBLs of an AT patient with a large clonal T-cell population exhibiting the t(14;14) translocation but not in the lymphocytes of the other cases. Fluorescence in situ hybridization of the TCL1 genomic locus to lymphocyte metaphases from the AT patient with the T-cell clonal expansion showed that the breakpoint of the t(14;14) translocation lies within the TCL1 locus and is accompanied by an inverted duplication of the distal part of chromosome 14. These data indicate that TCL1 is activated in preleukemic clonal cells as a consequence of chromosome translocation involving sequences from the TCR locus at 14q11. Deregulation of TCL1 is the first event in the initiation of malignancy in these types of leukemias and represents a potential tool for clinical evaluation. 


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Arrested development: understanding v-abl. 
The protein tyrosine kinase activity of the v-abl oncogene has been demonstrated to subvert the normal second messenger systems used by lymphoid cells for growth and differentiation. Transformation of bone marrow with the Abelson murine leukemia virus results in the appearance of B cell lineage cells arrested at the pre-B cell stage. Recent reports have characterized these cells expressing high v-abl kinase activity as deficient in detectable NF-kappaB DNA binding activity and low level RAG gene expression. These observations suggest that v-abl may be inhibiting the differentiation of B cells by blocking these two crucial elements in the maturation pathway. 


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Mechanisms of transactivation by nuclear factor of activated T cells-1. 
Nuclear factor of activated T cells-family proteins (NFAT1/NFATp, NFATc, NFAT3, and NFAT4/NFATx/NFATc3) play a key role in the transcription of cytokine genes and other genes during the immune response. We have defined the mechanisms of transactivation by NFAT1. NFAT1 possesses two transactivation domains whose sequences are not conserved in the other NFAT-family proteins, and a conserved DNA-binding domain that mediates the recruitment of cooperating nuclear transcription factors even when it is expressed in the absence of other regions of the protein. The activity of the NH2-terminal transactivation domain is modulated by an adjacent regulatory region that contains several conserved sequence motifs represented only in the NFAT family. Our results emphasize the multiple levels at which NFAT-dependent transactivation is regulated, and predict significant differences in the architecture of cooperative transcription complexes containing different NFAT-family proteins. 


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Inhibition of transcription factor Stat1 activity in mononuclear cell cultures and T cells by the cyclic AMP signaling pathway. 
Activation of T cells results in a cascade of gene activation and subsequent proliferation and differentiation into effector phenotypes. The regulation of transcription factors belonging to the signal transducer and activator of transcription (STAT) family was analyzed in PHA-activated mononuclear cells and in purified T cells activated by cross-linking cell surface CD3. Cell activation resulted in a delayed induction of STAT DNA-binding activity, which was sustained for several days, was composed predominantly of Stat1 and Stat3, and was blocked by cycloheximide and actinomycin D. Increased Stat1 and Stat3 mRNA and protein levels were detected, respectively 4 and 24 h after activation. Stimulation of the cAMP signal transduction pathway, which skews cytokine production toward a Th2 pattern, resulted in the preferential suppression of Stat1 activity. cAMP inhibited the induction of expression of IL-2 receptor components, but did not inhibit IL-4 receptor alpha-chain and CD69 expression or the induction of activator protein 1 transcription factors. cAMP signaling inhibited Stat1 at several different levels, including suppression of DNA binding and down-regulation of Stat1 protein and mRNA levels. Our results demonstrate the regulation of STAT activity by a signaling pathway that regulates the T cell functional phenotype and is distinct from the cytokine-activated Janus kinase-STAT signaling pathway. 


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Neutrophil maturation and the role of retinoic acid. 
Neutrophil maturation occurs in well defined morphological stages that correlate with the acquisition of molecular markers associated with neutrophil function. A variety of factors are known to play a role in terminal neutrophil maturation, including the vitamin A derivative, retinoic acid. Retinoic acid can directly modulate gene expression via binding to its nuclear receptors, which can, in turn, activate transcription of target genes. A role for retinoic acid during neutrophil maturation has been suggested from a variety of sources. Here we present a review of the mechanism of retinoic acid receptor action and the major evidence showing that normal retinoid signaling is required for neutrophil maturation. 


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Activation protein 1-dependent transcriptional activation of interleukin 2 gene by Ca2+/calmodulin kinase type IV/Gr. 
The Ca2+/calmodulin-dependent protein kinase (CaMK) type IV/Gr is selectively expressed in T lymphocytes and is activated after signaling via the T cell antigen receptor (TCR), indicating that it mediates some of the Ca(2+)-dependent transcriptional events that follow TCR engagement. Here we show that CaMKIV/Gr induces the transcription factor activation protein 1 (AP-1) alone or in synergy with T cell mitogens and with the p21ras oncoprotein. CaMKIV/ Gr signaling is associated with transcriptional activation of c-fos but is independent of p21ras or calcineurin. AP-1 is an integral component of the nuclear factor of activated T cells (NFAT) transcriptional complex, which is required for interleukin 2 gene expression in T cells. We demonstrate that CaMKIV/Gr reconstitutes the capacity of the cytosolic component of NFAT to direct transcription from NFAT sites in non-T cells. These results reveal a central role for CaMKIV/Gr as a Ca(2+)-regulated activator of gene transcription in T lymphocytes. 


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Transactivation of the interleukin-1alpha promoter by human T-cell leukemia virus type I and type II Tax proteins. 
Human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of interleukin-1alpha (IL-1alpha). To analyze the mechanisms that lead to the expression of IL-1alpha in HTLV-I-infected cell lines, we studied regulatory regions of the human IL-1alpha promoter involved in activation of the IL-1alpha gene. IL-1alpha promoter constructs drive transcription of the chloramphenicol acetyltransferase (CAT) reporter gene in HTLV-I-positive MT-2 cells, which constitutively produce IL-1alpha. In a cotransfection assay, the Tax protein of both HTLV-I and HTLV-II specifically activated transcription from the IL-1alpha promoter in an uninfected Jurkat cell line. A mutant Tax protein deficient in transactivation of genes by the nuclear factor (NF)-kappaB pathway was unable to induce transcriptional activity of IL-1alpha promoter-CAT constructs, but was rescued by exogenous provision of p65/p50 NF-kappaB. We found that two IL-1alpha kappaB-like sites (positions -1,065 to -1,056 and +646 to +655) specifically formed a complex with NF-kappaB-containing nuclear extract from MT-2 cells and that NF-kappaB bound with higher affinity to the 3' NF-kappaB binding site than to the 5' NF-kappaB site. Moreover, deletion of either 5' or 3' NF-kappaB sites reduced IL-1alpha promoter activity in MT-2 cells and transactivation of the IL-1alpha promoter by exogenous NF-kappaB and Tax in Jurkat cells. These data suggest a general role for Tax induction of IL-1alpha gene transcription by the NF-kappaB pathway. Expression of IL-1alpha by HTLV-I productively infected cells may be important in the hypercalcemia, osteolytic bone lesions, neutrophilia, elevation of C-reactive protein, and fever frequently seen in patients with HTLV-I-induced adult T-cell leukemia/lymphoma. 


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IL-12 induces IFN regulating factor-1 (IRF-1) gene expression in human NK and T cells. 
IL-12 is a critical immunoregulatory cytokine that promotes cell-mediated immune responses and the differentiation of naive CD4+ cells to Th1 cells; however, relatively few IL-12 target genes have been identified. To better clarify the molecular basis of IL-12 action, we set out to characterize genes up-regulated by IL-12, first by contrasting IL-12- and IFN-alpha-inducible genes. We identified several genes up-regulated by IL-12, namely, MIP-1alpha, MIP-1beta, IL-1RA, and IFN regulatory factor-1 (IRF-1). IRF-1 is a transcription factor regulated by IFNs that is also essential for Th1 responses. We demonstrated that IL-12 directly up-regulates IRF-1 to the same extent as IFN-alpha in normal human T cells and in NK cells. We showed that IL-12 had a direct effect on IRF-1, an effect not mediated indirectly by the induction of IFN-gamma production. Furthermore, IL-2 and IL-12 synergistically induced IRF-1, whereas IFN-alpha and IL-12 did not. The participation of STAT4 in the regulation of IRF-1 was demonstrated in two ways. First, STAT4 was required for the IL-12-dependent transactivation of an IRF-1 reporter construct, and second, STAT4 binding to the IRF-1 promoter was shown using EMSA. In contrast to IL-12, no up-regulation of IRF-1 was found in IL-4-stimulated cells, and IL-4 did not block IL-12-dependent up-regulation of IRF-1. Therefore, IRF-1 may be an important contributor to IL-12 signaling, and we speculate that the defective IL-12 responses seen in IRF-1-/- mice might be attributable, in part, to the absence of this transcription factor. 


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USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1. 
The transcription factor HIV-TF1, which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 (HIV-1), was purified from human B cells. HIV-TF1 had a molecular weight of 39,000. Binding of HIV-TF1 to the HIV long terminal repeat (LTR) activated transcription from the HIV promoter in vitro. The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor (USF) in the adenovirus major late promoter. DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF. Interestingly, treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 was essential for DNA binding. The disruption of HIV-TF1-binding site induced a 60% decrease in the level of transcription from the HIV promoter in vivo. These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1. 


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An essential role for NF-kappaB in human CD34(+) bone marrow cell survival. 
The transcription factor, NF-kappaB, is important for T-cell activation, B-cell maturation, and human immunodeficiency virus transcription and plays a role in alternatively mediating and protecting against apoptosis in a variety of cell types. However, a role for NF-kappaB in human CD34(+) bone marrow cells has not been described. We provide evidence here that virtually all human CD34(+) bone marrow cells express NF-kappaB that can be activated by exposure to phorbol 12-myristate 13-acetate and a variety of cytokines, eg, tumor necrosis factor alpha, interleukin-3, and granulocyte-macrophage colony-stimulating factor. In addition, we demonstrate that NF-kappaB may be required for human CD34(+) bone marrow cell clonogenic function and survival. These results offer insight into a new role for NF-kappaB in maintaining survival and function in hematopoietic stem and progenitor cells and suggest that proposed strategies involving inhibition of NF-kappaB activation as an adjunct to cancer chemotherapy should be approached with caution. 


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Induction of Bcl-x(L) expression by human T-cell leukemia virus type 1 Tax through NF-kappaB in apoptosis-resistant T-cell transfectants with Tax. 
Human T-cell leukemia virus type 1 (HTLV-1) Tax is thought to play a pivotal role in immortalization of T cells. We have recently shown that the expression of Tax protected the mouse T-cell line CTLL-2 against apoptosis induced by interleukin-2 (IL-2) deprivation and converted its growth from being IL-2 dependent to being IL-2 independent. In this study, we demonstrate that constitutive expression of bcl-xl but not bcl-2, bcl-xs, bak, bad, or bax was associated with apoptosis resistance after IL-2 deprivation in CTLL-2 cells that expressed Tax. Transient-transfection assays showed that bcl-x promoter was transactivated by wild-type Tax. Similar effects were observed in mutant Tax retaining transactivating ability through NF-kappaB. Deletion or substitution of a putative NF-kappaB binding site identified in the bcl-x promoter significantly decreased Tax-induced transactivation. This NF-kappaB-like element was able to form a complex with NF-kappaB family proteins in vitro. Furthermore, Tax-induced transactivation of the bcl-x promoter was also diminished by the mutant IkappaBalpha, which specifically inhibits NF-kappaB activity. Our findings suggest that constitutive expression of Bcl-x(L) induced by Tax through the NF-kappaB pathway contributes to the inhibition of apoptosis in CTLL-2 cells after IL-2 deprivation. 


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Reactivation of Kaposi's sarcoma-associated herpesvirus infection from latency by expression of the ORF 50 transactivator, a homolog of the EBV R protein. 
Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), or human herpesvirus 8, is a lymphotropic virus strongly linked to several AIDS-related neoplasms. The primary reservoir of infection consists of latently infected B lymphocytes and possibly other mononuclear cells. Viral reactivation from latency and spread from this lymphoid reservoir is presumably required for development of nonlymphoid tumors like KS. Here we show that deregulated expression of a single viral gene, ORF 50, which encodes a transactivator able to selectively upregulate delayed-early viral genes, suffices to disrupt latency and induce the lytic gene cascade in latently infected B cells. The identification of this gene opens the way to studies of the physiologic mechanisms controlling reactvation of KSHV from latency. Copyright 1998 Academic Press. 


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Interleukin-2 promoter activity in Epstein-Barr virus-transformed B lymphocytes is controlled by nuclear factor-chi B. 
The regulation of interleukin (IL)-2 gene expression has been investigated mainly in T lymphocytes, the predominant producers of IL-2. However, B cells can also synthesize IL-2. In the present study we analyzed the control of IL-2 promoter activity in Epstein-Barr virus (EBV)-transformed B cell clones which are capable of secreting IL-2 at a low level after stimulation with phorbol 12-myristate 13-acetate and the Ca2+ ionophore ionomycin. Transient transfections using reporter constructs with multiples of transcription factor binding sites from the IL-2 promoter [distal nuclear factor (NF)-AT, proximal NF-AT, AP-1/Octamer (UPS) or NF-chi B (TCEd) sites] were performed. In EBV-transformed B clones, the chi B site exerted the strongest inducible activity; the NF-AT binding sites showed either no or only weak activity compared to Jurkat T cells. An IL-2 promoter bearing a defective NF-chi B site was completely inactive in EBV-transformed B cells, while it still had activity in Jurkat T cells. In seven EBV-B cell clones or lines differing in their capacity to secrete IL-2, the activity of the IL-2 promoter correlated well with the status of IL-2 secretion. Similarly, a human immunodeficiency virus promoter, whose activity is controlled through chi B factors, was found to be active in the IL-2 producing EBV-B cells, but inactive in the non-IL-2-producing cells. Electrophoretic mobility shift assays using protein extracts from EBV-B cells and the IL-2 NF-chi B probe revealed the constitutive generation of chi B complexes in IL-2-secreting cells consisting mainly of heterodimeric p50/p65 complexes. A weaker chi B complex formation and faster-migrating complexes were detected in non-IL-2-secreting cells. These results demonstrate that the IL-2 NF-chi B site is indispensable for the activity of the IL-2 promoter in EBV-transformed B cells, whereas other transcription factors appear to be less important for IL-2 expression in these cells. 


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Prostaglandin E2 Up-regulates HIV-1 long terminal repeat-driven gene activity in T cells via NF-kappaB-dependent and -independent signaling pathways. 
Replication of human immunodeficiency virus type-1 (HIV-1) is highly dependent on the state of activation of the infected cells and is modulated by interactions between viral and host cellular factors. Prostaglandin E2 (PGE2), a pleiotropic immunomodulatory molecule, is observed at elevated levels during HIV-1 infection as well as during the course of other pathogenic infections. In 1G5, a Jurkat-derived T cell line stably transfected with a luciferase gene driven by HIV-1 long terminal repeat (LTR), we found that PGE2 markedly enhanced HIV-1 LTR-mediated reporter gene activity. Experiments have been conducted to identify second messengers involved in this PGE2-dependent up-regulating effect on the regulatory element of HIV-1. In this study, we present evidence indicating that signal transduction pathways induced by PGE2 necessitate the participation of cyclic AMP, protein kinase A, and Ca2+. Experiments conducted with different HIV-1 LTR-based vectors suggested that PGE2-mediated activation effect on HIV-1 transcription was transduced via both NF-kappaB-dependent and -independent signaling pathways. The involvement of NF-kappaB in the PGE2-dependent activating effect on HIV-1 transcription was further confirmed using a kappaB-regulated luciferase encoding vector and by electrophoretic mobility shift assays. Results from Northern blot and flow cytometric analyses, as well as the use of a selective antagonist indicated that PGE2 modulation of HIV-1 LTR-driven reporter gene activity in studied T lymphoid cells is transduced via the EP4 receptor subtype. These results suggest that secretion of PGE2 by macrophages in response to infection or inflammatory activators could induce signaling events resulting in activation of proviral DNA present into T cells latently infected with HIV-1. 


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Suppression of signals required for activation of transcription factor NF-kappa B in cells constitutively expressing the HTLV-I Tax protein. 
Transient short-term expression of the Tax protein of human T-cell leukemia virus type-I (HTLV-I) leads to activation of the pleiotropic transcription factor NF-kappa B. Consistent with findings obtained with transient expression assays, we observed marked accumulation of the transcription factor NF-kappa B in the nucleus of Namalwa B lymphoid cells, which constitutively express Tax. In contrast, NF-kappa B activity was not detected in the nucleus following long-term expression of Tax in Jurkat T lymphocytes. The ability of both mitogens and cytokines to activate NF-kappa B was also blocked in Jurkat cells constitutively expressing Tax. However, the activation of other mitogen-inducible transcription factors, such as Fos and Jun, was unaffected. Thus, depending on the cellular environment, the short- and long-term effects of Tax expression can be quite different. Consequently, one function of Tax in cells infected with HTLV-I might involve cell-type-specific suppression, as opposed to activation, of distinct signal pathways. The cells lines described here should be useful for the delineation of signaling pathways utilized in the selective regulation of gene expression. 


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Upregulation of bcl-2 by the Epstein-Barr virus latent membrane protein LMP1: a B-cell-specific response that is delayed relative to NF-kappa B activation and to induction of cell surface markers. 
An ability of the Epstein-Barr virus latent membrane protein LMP1 to enhance the survival of infected B cells through upregulation of the bcl-2 oncogene was first suggested by experiments involving gene transfection and the selection of stable LMP1+ clones (S.Henderson, M. Rowe, C.Gregory, F.Wang, E.Kieff, and A.Rickinson, Cell 65:1107-1115, 1991). However, it was not possible to ascertain whether Bcl-2 upregulation was a specific consequence of LMP1 expression or an artifact of the selection procedure whereby rare Bcl-2+ cells already present in the starting population might best be able to tolerate the potentially toxic effects of LMP1. We therefore reexamined this issue by using two different experimental approaches that allowed LMP1- induced effects to be monitored immediately following expression of the viral protein and in the absence of selective pressures; activation of the NF-kappa B transcription factor and upregulation of the cell adhesion molecule ICAM-1 were used as early indices of LMP1 function. In the first approach, stable clones of two B-cell lines carrying an LMP1 gene under the control of an inducible metallothionein promoter were induced to express LMP1 in all cells. Activation of NK-kappa B and upregulation of ICAM-1 occurred within 24 h and were followed at 48 to 72 h by upregulation of Bcl-2. In the second approach, we tested the generality of this phenomenon by transiently expressing LMP1 from a strong constitutively active promoter in a range of different cell types. All six B-cell lines tested showed NF-kappa B activation in response to LMP1 expression, and this was followed in five of six lines by expression of ICAM-1 and Bcl-2. In the same experiments, all three non-B-cell lines showed NF-kappa B activation and ICAM-1 upregulation but never any effect upon Bcl-2. We therefore conclude that Bcl-2 upregulation is part of the panoply of cellular changes induced by LMP1 but that the effect is cell type specific. Our data also suggest that whilst NF-kappa B may be an essential component of LMP1 signal transduction, other cell-specific factors may be required to effect some functions of the viral protein. 


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Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives. 
HIV-1 proviral DNA contains two binding sites for the transcription factor NF-kappa B. HIV-1-infected individuals have, on average, abnormally high levels of tumour necrosis factor alpha (TNF alpha) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-kappa B expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with GSH levels. Cysteine and NAC also inhibit NF-kappa B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase (CAT) gene expression under control of NF-kappa B binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-kappa B-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals. 


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Stimulation of HIV replication in mononuclear phagocytes by leukemia inhibitory factor. 
This study examined the effects of leukemia inhibitory factor (LIF) on human immunodeficiency virus (HIV) replication in mononuclear phagocytes (MNP). LIF induced a dose-dependent increase in p24 antigen production in the chronically infected promonocytic cell line U1. The magnitude and time kinetics of the LIF effects were similar to interleukin 1 (IL-1), IL-6, and tumor necrosis factor (TNF), other cytokines known to induce HIV replication in this cell line. To characterize mechanisms responsible for these LIF effects, levels of HIV mRNA, activation of the DNA binding protein nuclear factor (NF)-kB, signal transduction pathways, and potential interactions with other cytokines were analyzed. LIF increased steady-state levels of HIV mRNA at 2.0, 4.3, and 9.2 kB. This was detectable by 24 h and persisted until 72 h. The DNA binding protein NF-kB is a central mediator in cytokine activation of HIV transcription. NF-kB levels were higher in unstimulated U1 cells as compared to the parent cell line U937. In both cell lines LIF increased NF-kB activity. Induction of NF-kB and HIV replication by cytokines are at least in part dependent on reactive oxygen intermediates. The oxygen radical scavenger N-acetyl-L-cysteine, but not an inhibitor of nitric oxide synthase, inhibited LIF-induced HIV replication. LIF induces the production of other cytokines in monocytes but its effects on HIV replication were not inhibited by antibodies to IL-1, TNF, or IL-6. These results identify LIF as a stimulus of HIV replication. (ABSTRACT TRUNCATED AT 250 WORDS) 


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NF-kappa B-dependent induction of the NF-kappa B p50 subunit gene promoter underlies self-perpetuation of human immunodeficiency virus transcription in monocytic cells. 
The molecular mechanisms underlying the sustained nuclear translocation of NF-kappa B observed in U937 monocytic cells chronically infected with human immunodeficiency virus (HIV) were studied. The activity of the promoter regulating the synthesis of the p105 precursor of the NF-kappa B p50 subunit was enhanced in these cells. Deletions in this promoter indicated that this upregulation was mediated through the NF-kappa B- but not the AP-1-binding motif, by bona fide p50/p65 heterodimers. Analysis of cytosolic extracts indicated that NF-kappa B levels were increased in HIV-infected cells. In contrast to the transient NF-kappa B activation induced by phorbol ester, the permanent NF-kappa B translocation induced by HIV infection was not dependent on PKC isoenzymes alpha and beta as shown by the use of a specific inhibitor (GF 109203X). These observations indicate that during chronic HIV infection of U937 cells, continuous NF-kappa B (p50/p65) translocation results in p105 promoter upregulation with subsequent cytosolic NF-kappa B accumulation, ready for further translocation. This HIV-mediated mechanism results in a self-perpetuating loop of NF-kappa B production. 


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Characterization of the murine cyclin-dependent kinase inhibitor gene p27Kip1. 
The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in regulating cell-cycle progression. p27Kip1 directly inhibits the catalytic activity of cyclin/cdks (cyclin-dependent kinase) complexes and/or interferes physically with cyclin/cdks activation by CAK. Interestingly, the expression level of p27Kip1 mRNA was maximal in resting Go T-cells and rapidly declined following anti-CD3 activation. We report here the cloning of p27Kip1 gene from murine genomic DNA and the functional analysis of the promoter of the p27Kip1 gene. The gene consists of at least three exons and spans more than 5.6 kb of DNA. Primer extension and nuclease S1 protection analysis revealed two major transcription initiation sites. The promoter region lacked a TATA box but contained potential binding sites for the transcriptional factors including two Sp1, CRE, Myb and NFkB located at positions -153, -178, -286, -875, and -1011, respectively. To analyze the regulatory mechanisms controlling p27Kip1 gene expression, we characterized the 5'-flanking region from nt -1609 to +178. The -326 to -615 region contained positive regulatory elements. 


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CTLA-4-Mediated inhibition of early events of T cell proliferation. 
CTLA-4 engagement by mAbs inhibits, while CD28 enhances, IL-2 production and proliferation upon T cell activation. Here, we have analyzed the mechanisms involved in CTLA-4-mediated inhibition of T cell activation of naive CD4+ T cells using Ab cross-linking. CTLA-4 ligation inhibited CD3/CD28-induced IL-2 mRNA accumulation by inhibiting IL-2 transcription, which appears to be mediated in part through decreasing NF-AT accumulation in the nuclei. However, CTLA-4 ligation did not appear to affect the CD28-mediated stabilization of IL-2 mRNA. Further, CTLA-4 engagement inhibited progression through the cell cycle by inhibiting the production of cyclin D3, cyclin-dependent kinase (cdk)4, and cdk6 when the T cells were stimulated with anti-CD3/CD28 and with anti-CD3 alone. These results indicate that CTLA-4 signaling inhibits events early in T cell activation both at IL-2 transcription and at the level of IL-2-independent events of the cell cycle, and does not simply oppose CD28-mediated costimulation. 


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oriP is essential for EBNA gene promoter activity in Epstein-Barr virus-immortalized lymphoblastoid cell lines. 
During Epstein-Barr virus latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities are mutually exclusive. Upon infection, Wp is initially active, followed by a switch to Cp for the duration of latency. In this study, the region upstream of Cp was analyzed for the presence of cis elements involved in regulating the activities of the EBNA gene promoters in established in vitro immortalized lymphoblastoid cell lines (LCLs). It was determined that oriP, the origin for episomal maintenance during latency, is essential for efficient transcription initiation from either Cp or Wp in LCLs, as well as in some Burkitt's lymphoma cell lines. Deletion of the EBNA2-dependent enhancer located upstream of Cp resulted in a ca. two- to fivefold reduction in Cp activity in the LCLs assayed. More extensive deletion of sequences upstream of Cp, including the EBNA2-dependent enhancer, resulted in nearly complete loss of Cp activity. This loss of activity was shown to correlate with deletion of two CCAAT boxes, a proximal CCAAT box located at bp -61 to -65 and a distal CCAAT box located at bp -253 to -257, upstream of Cp. Site-directed mutagenesis of these cis elements demonstrated that Cp activity is highly dependent on the presence of a properly positioned CCAAT box, with the dependence on the distal CCAAT box apparent only when the proximal CCAAT box was deleted or mutated. Deletion of the glucocorticoid response elements located at ca. bp -850 upstream of Cp did not result in a significant loss in activity. In general, deletions which diminished Cp activity resulted in induction of Wp activity, consistent with suppression of Wp activity by transcriptional interference from Cp. The identification of oriP and the EBNA2-dependent enhancer as the major positive cis elements involved in regulating Cp activity in LCL suggests that EBNA gene transcription is largely autoregulated by EBNA 1 and EBNA 2. 


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Nuclear factor of activated T cells and AP-1 are insufficient for IL-2 promoter activation: requirement for CD28 up-regulation of RE/AP. 
IL-2 gene transcription in T cells requires both TCR and costimulatory signals. IL-2 promoter activation in Jurkat T cells stimulated with superantigen presented by Raji B cells requires CD28 activation. The addition of rCTLA4Ig, which blocks CD28 binding to its ligand, to the cultures decreased IL-2 promoter activation by >80%. Interestingly, CTLA4Ig did not significantly inhibit the activation of either NF of activated T cells (NFAT) or AP-1 reporters. Therefore, activation of NFAT and AP-1 is insufficient for IL-2 promoter activation. In contrast, an RE/AP reporter was blocked by CTLA4Ig by >90%. Thus, the requirement for CD28 in IL-2 promoter activation appears to be due to RE/AP and not the NFAT or AP-1 sites. In addition, these data suggest that transcriptional activation of RE/AP is not mediated by NFAT, because activation of a NFAT reporter is not affected by the addition of CTLA4Ig. 


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Anoxia/reoxygenation-induced tolerance with respect to polymorphonuclear leukocyte adhesion to cultured endothelial cells. A nuclear factor-kappaB-mediated phenomenon. 
Exposing human umbilical vein endothelial cells (HUVECs) to anoxia/reoxygenation (A/R) results in an increase in polymorphonuclear leukocyte (PMN) adhesion to HUVECs. This A/R-induced hyperadhesion is completely prevented by a previous (24 hours earlier) exposure of HUVECs to A/R. This phenomenon has been termed "A/R tolerance." Exposing HUVECs to A/R induces an increase in nuclear factor kappaB (NF-kappaB) in HUVEC nuclei within 4 hours. Interfering with either NF-kappaB activation (proteasome inhibitor) or translocation (double-stranded oligonucleotides containing NF-kappaB binding sequence) prevents the development of A/R tolerance (ie, the increase in A/R-induced PMN adhesion to HUVECs is the same after the first and second A/R challenges). NO production by HUVECs is increased after the second A/R challenge, but not after the first A/R challenge. Inhibition of NO synthase (NOS) during the second A/R challenge prevents the development of A/R tolerance with respect to PMN adhesion. However, while HUVECs contained endothelial NOS protein, no inducible NOS was detected in either tolerant or nontolerant cells. Further studies indicated that inhibition of GTP-cyclohydrolase I (an enzyme involved in de novo synthesis of an important cofactor for NOS activity, tetrahydrobiopterin) prevented the generation of NO in A/R-tolerant cells. Extracellular generation of NO (NO donor) did not effect the hyperadhesion response induced by the initial A/R challenge. A/R also induced an oxidant stress in naive HUVECs, but not in A/R-tolerant HUVECs. Inhibition of NOS during the second A/R insult results in the generation of an oxidant stress similar to that observed after the first A/R challenge. Taken together, the findings of the present study are consistent with a role for NF-kappaB in the development of A/R tolerance (with respect to PMN adhesion), perhaps by transcriptional regulation of GTP-cyclohydrolase. The increased NO production during the second A/R insult reduces PMN adhesion most likely by reducing the intracellular oxidant stress induced by A/R. 


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Hypoxic induction of interleukin-8 gene expression in human endothelial cells. 
Because leukocyte-mediated tissue damage is an important component of the pathologic picture in ischemia/reperfusion, we have sought mechanisms by which PMNs are directed into hypoxic tissue. Incubation of human endothelial cells (ECs) in hypoxia, PO2 approximately 14-18 Torr, led to time-dependent release of IL-8 antigen into the conditioned medium; this was accompanied by increased chemotactic activity for PMNs, blocked by antibody to IL-8. Production of IL-8 by hypoxic ECs occurred concomitantly with both increased levels of IL-8 mRNA, based on polymerase chain reaction analysis, and increased IL-8 transcription, based on nuclear run-on assays. Northern analysis of mRNA from hypoxic ECs also demonstrated increased levels of mRNA for macrophage chemotactic protein-1, another member of the chemokine superfamily of proinflammatory cytokines. IL-8 gene induction was associated with the presence of increased binding activity in nuclear extracts from hypoxic ECs for the NF-kB site. Studies with human umbilical vein segments exposed to hypoxia also demonstrated increased elaboration of IL-8 antigen compared with normoxic controls. In mice exposed to hypoxia (PO2 approximately 30-40 Torr), there was increased pulmonary leukostasis, as evidenced by increased myeloperoxidase activity in tissue homogenates. In parallel, increased levels of transcripts for IP-10, a murine homologue in the chemokine family related to IL-8, were observed in hypoxic lung tissue. Taken together, these data suggest that hypoxia constitutes a stimulus for leukocyte chemotaxis and tissue leukostasis. 


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Coactivation by OCA-B: definition of critical regions and synergism with general cofactors. 
Molecular dissection of the B-cell-specific transcription coactivator OCA-B has revealed distinct regions important, respectively, for recruitment to immunoglobulin promoters through interaction with octamer-bound Oct-1 and for subsequent coactivator function. Further analysis of general coactivator requirements showed that selective removal of PC4 from the essential USA fraction severely impairs Oct-1 and OCA-B function in a cell-free system reconstituted with partially purified factors. Full activity can be restored by the combined action of recombinant PC4 and the PC4-depleted USA fraction, thus suggesting a joint requirement for PC4 and another, USA-derived component(s) for optimal function of Oct-1/OCA-B in the reconstituted system. Indeed, USA-derived PC2 was found to act synergistically with PC4 in reproducing the function of intact USA in the assay system. Consistent with the requirement for PC4 in the reconstituted system, OCA-B was found to interact directly with PC4. Surprisingly, however, removal of PC4 from the unfractionated nuclear extract has no detrimental effect on OCA-B/Oct-1-dependent transcription. These results lead to a general model for the synergistic function of activation domains in Oct-1 and OCA-B (mediated by the combined action of the multiple USA components) and, further, suggest a functional redundancy in general coactivators. 


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Regulation of c-jun expression during induction of monocytic differentiation by okadaic acid. 
The present work has examined the effects of okadaic acid, an inhibitor of type 1 and 2A protein phosphatases, on the regulation of c-jun expression during monocytic differentiation of U-937 leukemia cells. The results demonstrate that okadaic acid treatment is associated with induction of a differentiated monocyte phenotype characterized by: (a) growth arrest; (b) increases in Mac-1 cell surface antigen expression; (c) down-regulation of c-myc transcripts; and (d) induction of tumor necrosis factor gene expression. This induction of monocytic differentiation was associated with transient increases in c-jun mRNA levels, which were maximal at 6 h. Similar effects were obtained for the c-fos gene. Run-on analysis demonstrated detectable levels of c-jun transcription in U-937 cells and that this rate is increased approximately 40-fold following okadaic acid exposure. c-jun mRNA levels were superinduced in cells treated with both okadaic acid and cycloheximide, whereas inhibition of protein synthesis had little, if any, effect on okadaic acid-induced c-jun transcription. The half-life of c-jun mRNA was similar (45-50 min) in both untreated and okadaic acid-induced cells. In contrast, treatment with both okadaic acid and cycloheximide was associated with stabilization (t 1/2 = 90 min) of c-jun transcripts. Taken together, these findings indicate that the induction of c-jun transcription by okadaic acid is controlled primarily by a transcriptional mechanism. Since previous studies have demonstrated that the c-jun gene is autoinduced by Jun/AP-1, we also studied transcription of c-jun promoter (positions -132/+170)-reporter gene constructs with and without a mutated AP-1 element. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Occurrence of a silencer of the interleukin-2 gene in naive but not in memory resting T helper lymphocytes. 
In the immune system the first activation of a naive T cell by antigen is a key step in the shaping of the peripheral T cell specificity repertoire and maintenance of self-tolerance. In the present study, analysis of the interleukin-2 (IL-2) gene activation shows that naive human helper T cells (cord blood CD4+ T cells, adult CD4+CD45RO- T cells) regulate IL-2 transcription by a mechanism involving both a silencer and an activator acting on the purine-rich IL-2 promoter elements (NF-AT binding sites). By contrast, memory cells, either in vitro activated helper T cells reverting to a resting state, or CD4+ T (memory) clones, or CD4+CD45RO+ T cells isolated ex vivo, no longer have a silencer. Their IL-2 transcription seems to be controlled solely by the transition from inactive to active functional state of a positive transcription factor binding to these promoter elements as well as its cytoplasmic or nuclear location: in resting memory T cells the activator is located in the cytoplasm and is inactive, whereas in stimulated cells it is functional in promoting transcription and now resides in the nucleus. Thus, the regulation of the gene coding for the main T cell growth factor changes irreversibly after the first encounter of T cells with antigen. It is most likely that the presence of a silencer contributes to the more stringent activation requirements of naive CD4+ T cells. 


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Permanent occupancy of the human immunodeficiency virus type 1 enhancer by NF-kappa B is needed for persistent viral replication in monocytes. 
This work aimed to ascertain the role of kappaB-responsive elements of the human immunodeficiency virus type 1 (HIV-1) enhancer not only in early initiation but also in long-term maintenance of proviral transcription in cells of the monocytic lineage. For this purpose, we used three main approaches. The first was to abruptly terminate tumor necrosis factor-induced NF-kappaB binding to the enhancer sequences in U1 monocytic cells, using a short pulse of exogenous tumor necrosis factor. This resulted in concomitant decrease in nuclear NF-kappaB DNA-binding activity and endogenous long terminal repeat transcriptional activity. The second was to suppress the permanent NF-kappaB translocation induced by HIV-1 replication itself in chronically infected U937 cells, using a specific proteasome inhibitor (Z-LLL-H). As early as 2 h after addition of the inhibitor to the culture medium, there was an inhibition of both constitutive activation of NF-kappaB and HIV-1 genome expression. The third approach was to monitor the replication competence in U937 cells of an infectious HIV-1 provirus carrying point mutations in the kappaB-responsive elements of both long terminal repeats. Compared with its wild-type counterpart, this mutated provirus showed a profoundly decreased, Z-LLL-H-insensitive transcriptional and replicative activity in U937 monocytes. Together, our results indicate that occupancy of the viral enhancer by NF-kappaB (p50/p65) heterodimers is required for ongoing transcription of integrated HIV provirus in monocytes, even in cells chronically infected and permanently producing functional HIV Tat protein. Thus, the ability of HIV-1 replication to activate NF-kappaB is crucial to the intense self-perpetuated viral transcription observed in cells of the monocytic lineage. 


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Efficient transcription and replication of simian immunodeficiency virus in the absence of NF-kappaB and Sp1 binding elements. 
Ten mutants of the simian immunodeficiency virus (SIV) SIVmac239 bearing deletions (delta) or substitutions (subst) in the NF-kappaB and/or Sp1 binding elements were created, and the replicative capacities of the mutants were analyzed. All mutants, including one extensively mutagenized strain entirely missing the NF-kappaB and four Spl binding elements, replicated with wild-type kinetics and to a wild-type level in peripheral blood mononuclear cell cultures in 50 to 100% of the experiments. One group of mutants replicated very similarly to SIVmac239 in kinetics and yield in CEMxl74 cells (2xNFKappaB > or = SlVmac239 approximately deltaNFkappaB approximately deltaSpl234 approximately substNFkappaB approximately substSpl2 approximately substSp23), while a second group replicated with delayed or slightly delayed kinetics in CEMxl74 cells (SIVmac239 > substSp34 > deltaNFkappaBdeltaSpl234 approximately deltaNFkappaBdeltaSp1 > substSpl234). Reversions or additional mutations were not detected in the U3 and R regions of proviral DNA from CEMxl74 cells infected with the SIVmac239 mutants. Similar results were obtained when mutants of SIVmacMER (a macrophage-competent derivative of SIVmac239) were tested in peripheral blood mononuclear cell and CEMx174 cultures. However, the growth of most mutated viruses was suppressed in primary rhesus monkey alveolar macrophages (SIVmacMER approximately 2xNFkappaB approximately substNFkappaB > deltaNFkappaB > deltaNFkappaBdeltaSpl234 approximately deltaNFkappaBdeltaSpl > deltaSpl234 approximately substSpl2 > substSp23 approximately substSp34 approximately substSpl234 > or = SIVmac239). Thus, changes in the Sp1 binding sites had the most dramatic effects on SIVmac replication in primary macrophage cultures. Analysis of long terminal repeat-driven secreted alkaline phosphatase activity in transient assays showed that, unlike human immunodeficiency virus type 1, the SIV long terminal repeat possesses an enhancer region just upstream of the NF-kappaB element which maintains significant levels of basal transcription in the absence of NF-kappaB and Sp1 sites. This region is responsive to transactivation by Tat. In addition, the SIV TATA box was shown to be stronger than that of human immunodeficiency virus type 1. Therefore, the surprisingly high replicative capacity of NF-kappaB and Sp1 binding site mutants of SIVmac is due to unique features or the enhancer/promoter region. 


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Regulation of interleukin-2 receptor alpha chain expression and nuclear factor.kappa B activation by protein kinase C in T lymphocytes. Autocrine role of tumor necrosis factor alpha. 
The regulation of interleukin-2 receptor alpha chain (IL-2R alpha) expression and nuclear factor (NF) activation by protein kinase C (PKC) in resting T cells, has been studied. Treatment of human resting T cells with phorbol esters strongly induced the expression of IL-2R alpha and the activation of NF.kappa B. This activation was due to the translocation of p65 and c-Rel NF.kappa B proteins from cytoplasmic stores to the nucleus, where they bound the kappa B sequence of the IL-2R alpha promoter either as p50.p65 or as p50.c-Rel heterodimers. Interestingly, all of those events were largely indirect and mediated by endogenously secreted tumor necrosis factor alpha (TNF alpha), as they were strongly inhibited by a neutralizing anti-TNF alpha monoclonal antibody. Furthermore, cyclosporin A, which blocked TNF alpha production induced by PKC, strongly inhibited IL-2R alpha and NF.kappa B activation. The addition of either TNF alpha or IL-2 partially recovered cyclosporin A-induced IL-2R alpha inhibition, but only TNF alpha completely recovered NF.kappa B activation. Those results indicate that, in resting T cells, PKC activation has only a triggering role, whereas the endogenously secreted TNF alpha plays an essential role in the quantitative control of the expression of IL-2R alpha chain or NF.kappa B activation. 


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Octamer binding factors and their coactivator can activate the murine PU.1 (spi-1) promoter. 
PU.1 (spi-1), a member of the Ets transcription factor family, is predominantly expressed in myeloid and B cells, activates many B cell and myeloid genes, and is critical for development of both of these lineages. Our previous studies (Chen, H.M., Ray-Gallet, D., Zhang, P., Hetherington, C.J., Gonzalez, D.A., Zhang, D.-E., Moreau-Gachelin, F., and Tenen, D.G.(1995) Oncogene 11, 1549-1560) demonstrate that the PU.1 promoter directs cell type-specific reporter gene expression in myeloid cell lines, and that PU.1 activates its own promoter in an autoregulatory loop. Here we show that the murine PU.1 promoter is also specifically and highly functional in B cell lines as well. Oct-1 and Oct-2 can bind specifically to a site at base pair -55 in vitro, and this site is specifically protected in B cells in vivo. We also demonstrate that two other sites contribute to promoter activity in B cells; an Sp1 binding site adjacent to the octamer site, and the PU.1 autoregulatory site. Finally, we show that the B cell coactivator OBF-1/Bob1/OCA-B is only expressed in B cells and not in myeloid cells, and that OBF-1/Bob1/OCA-B can transactivate the PU.1 promoter in HeLa and myeloid cells. This B cell restricted coactivator may be responsible for the B cell specific expression of PU.1 mediated by the octamer site. 


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An interferon-gamma activation sequence mediates the transcriptional regulation of the IgG Fc receptor type IC gene by interferon-gamma. 
Expression of the IgG Fc receptor type I (Fc gamma RI) on myeloid cells is dramatically increased by treatment with interferon-gamma (IFN-gamma). We observed that Fc gamma RI transcript levels in monoblast-like U937 cells were elevated within 3 hr and peaked 12 hr after exposure to IFN-gamma. Treatment of U937 with IFN-gamma for 9 hr in the presence of cycloheximide led to super-induction of Fc gamma RI expression. Nuclear run-on analysis revealed that the rate of Fc gamma RI transcription was increased by IFN-gamma. Genomic sequence upstream of the Fc gamma RIC gene was cloned and subjected to primer extension analysis, which demonstrated a single transcription initiation site without a TATA box. Transient transfections of CAT reporter gene constructs containing various Fc gamma RIC promoter sequences into U937 cells revealed that a 20-bp region surrounding the transcription start site (-7 to +13) was capable of mediating transcription initiation and that an IFN-gamma responsive element (GIRE) was present within 74 bp upstream of the transcription initiation site. A 17-bp sequence between positions -51 and -35 conferred IFN-gamma responsiveness on a heterologous promoter. Double-stranded GIRE sequence, but not a scrambled sequence, was specifically bound by nuclear proteins from IFN-gamma treated U937 cells. Gel shift experiments further showed that the STAT1 alpha protein bound to the Fc gamma RIC GIRE in response to IFN-gamma treatment of U937 cells. The Fc gamma RIC GIRE is homologous to the IFN-gamma activation sequence (GAS) of the guanylate binding protein and to X box elements of class II MHC genes. Our results demonstrate that transcriptional regulation of the Fc gamma RIC gene by IFN-gamma involves the binding of STAT1 alpha to a 17-bp GAS homology in the proximal promoter. 


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Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat. 
The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat. This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes. Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed. 


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Binding of YY1 and Oct1 to a novel element that downregulates expression of IL-5 in human T cells. 
BACKGROUND: IL-5 controls development of eosinophilia and has been shown to be involved in the pathogenesis of allergic diseases. In both atopic and nonatopic asthma, elevated IL-5 has been detected in peripheral blood and the airways. IL-5 is produced mainly by activated T cells, and its expression is regulated at the transcriptional level. OBJECTIVE: This study focuses on the functional analysis of the human IL-5 (hIL-5) promoter and characterization of cis -regulatory elements and transcription factors involved in the suppression of IL-5 transcription in T cells. METHODS: Methods used in this study include DNase I footprint assays, electrophoretic mobility shift assays, and functional analysis by mammalian cell transfection involving deletion analysis and site-directed mutagenesis. RESULTS: We identified 5 protein binding regions (BRs) located within the proximal hIL-5 promoter. Functional analysis indicates that the BRs are involved in control of hIL-5 promoter activity. Two of these regions, BR3 and BR4 located at positions -102 to -73, have not previously been described as regulators of IL-5 expression in T cells. We show that the BR3 sequence contains a novel negative regulatory element located at positions -90 to -79 of the hIL-5 promoter, which binds Oct1, octamer-like, and YY1 nuclear factors. Substitution mutations, which abolished binding of these proteins to the BR3 sequence, significantly increased hIL-5 promoter activity in activated T cells. CONCLUSION: We suggest that Oct1, YY1, and octamer-like factors binding to the -90/-79 sequence within the proximal IL-5 promoter are involved in suppression of IL-5 transcription in T cells. 


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The role of BSAP (Pax-5) in B-cell development. 
The hierarchy of transcriptional control in B-cell development has recently been analyzed by targeted gene inactivation in the mouse. In this manner, the paired box containing gene Pax-5, encoding the B cell specific transcription factor BSAP, has been shown to play a key role in early B lymphopoiesis. Other experimental strategies have implicated BSAP in the control of cell proliferation, isotype switching and transcription of the immunoglobulin heavy-chain gene at late stages of B-cell differentiation. 


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Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene [published erratum appears in Mol Cell Biol 1997 Apr;17(4):2351] 
The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor. 


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Identification of an inducible regulator of c-myb expression during T-cell activation. 
Resting T cells express very low levels of c-Myb protein. During T-cell activation, c-myb expression is induced and much of the increase in expression occurs at the transcriptional level. We identified a region of the c-myb 5' flanking sequence that increased c-myb expression during T-cell activation. In vivo footprinting by ligation-mediated PCR was performed to correlate in vivo protein binding with functional activity. A protein footprint was visible over this region of the c-myb 5' flanking sequence in activated T cells but not in unactivated T cells. An electrophoretic mobility shift assay (EMSA) with nuclear extract from activated T cells and an oligonucleotide of this binding site demonstrated a new protein-DNA complex, referred to as CMAT for c-myb in activated T cells; this complex was not present in unactivated T cells. Because the binding site showed some sequence similarity with the nuclear factor of activated T cells (NFAT) binding site, we compared the kinetics of induction of the two binding complexes and the molecular masses of the two proteins. Studies of the kinetics of induction showed that the NFAT EMSA binding complex appeared earlier than the CMAT complex. The NFAT protein migrated more slowly in a sodium dodecyl sulfate-polyacrylamide gel than the CMAT protein did. In addition, an antibody against NFAT did not cross-react with the CMAT protein. The appearance of the CMAT binding complex was inhibited by both cyclosporin A and rapamycin. The CMAT protein appears to be a novel inducible protein involved in the regulation of c-myb expression during T-cell activation. 


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Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level. 
In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34(+) cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34(+) CD38(-) cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development. 


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Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis. 
Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during the rescue of immature B cells from apoptosis. It is possible that the CRE site is responsible for induction of bcl-2 expression in other cell types, particularly those in which protein kinase C is involved. 


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Nuclear factor-kappaB-dependent induction of interleukin-8 gene expression by tumor necrosis factor alpha: evidence for an antioxidant sensitive activating pathway distinct from nuclear translocation. 
Tumor necrosis factor alpha (TNFalpha) is a pluripotent activator of inflammation by inducing a proinflammatory cytokine cascade. This phenomenon is mediated, in part, through inducible expression of the CXC chemokine, interleukin-8 (IL-8). In this study, we investigate the role of TNFalpha-inducible reactive oxygen species (ROS) in IL-8 expression by "monocyte-like" U937 histiocytic lymphoma cells. TNFalpha is a rapid activator of IL-8 gene expression by U937, producing a 50-fold induction of mRNA within 1 hour of treatment. In gene transfection assays, the effect of TNFalpha requires the presence of an inducible nuclear factor-kappaB (NF-kappaB) (Rel A) binding site in the IL-8 promoter. TNFalpha treatment induces a rapid translocation of the 65 kD transcriptional activator NF-kappaB subunit, Rel A, whose binding in the nucleus occurs before changes in intracellular ROS. Pretreatment (or up to 15 minutes posttreatment) relative to TNFalpha with the antioxidant dimethyl sulfoxide (DMSO) (2% [vol/vol]) blocks 80% of NF-kappaB-dependent transcription. Surprisingly, however, DMSO has no effect on inducible Rel A binding. Similar selective effects on NF-kappaB transcription are seen with the unrelated antioxidants, N-acetylcysteine (NAC) and vitamin C. These data indicate that TNFalpha induces a delayed ROS-dependent signalling pathway that is required for NF-kappaB transcriptional activation and is separable from that required for its nuclear translocation. Further definition of this pathway will yield new insights into inflammation initiated by TNFalpha signalling. 


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Inducible nuclear factor binding to the kappa B elements of the human immunodeficiency virus enhancer in T cells can be blocked by cyclosporin A in a signal-dependent manner. 
Cyclosporin A (CsA) is thought to exert its immunosuppressive effects by inhibiting the expression of a distinct set of lymphokine genes which are induced upon T-cell activation, among them the gene coding for interleukin-2. In addition, the activation of the human immunodeficiency virus (HIV) is partially suppressed. To better understand the molecular mechanisms underlying suppression by CsA, we have investigated the effects of this drug on transcription factors in T cells. Here we report that the formation of two distinct mitogen-inducible DNA-binding complexes, the kappa B complex within the HIV enhancer and the NFAT-1 complex within the interleukin-2 enhancer, is inhibited in the presence of CsA. The kappa B-binding activity with the HIV enhancer is inhibited only if it is activated via the mitogen phytohemagglutinin whereas phorbol myristate acetate-mediated activation is completely insensitive to the drug. This suggests a model in which functionally indistinguishable kappa B complexes can be activated via two separate pathways of signal transduction distinguishable by CsA. 


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GrpL, a Grb2-related adaptor protein, interacts with SLP-76 to regulate nuclear factor of activated T cell activation. 
Propagation of signals from the T cell antigen receptor (TCR) involves a number of adaptor molecules. SH2 domain-containing protein 76 (SLP-76) interacts with the guanine nucleotide exchange factor Vav to activate the nuclear factor of activated cells (NF-AT), and its expression is required for normal T cell development. We report the cloning and characterization of a novel Grb2-like adaptor molecule designated as Grb2-related protein of the lymphoid system (GrpL). Expression of GrpL is restricted to hematopoietic tissues, and it is distinguished from Grb2 by having a proline-rich region. GrpL can be coimmunoprecipitated with SLP-76 but not with Sos1 or Sos2 from Jurkat cell lysates. In contrast, Grb2 can be coimmunoprecipitated with Sos1 and Sos2 but not with SLP-76. Moreover, tyrosine-phosphorylated LAT/pp36/38 in detergent lysates prepared from anti-CD3 stimulated T cells associated with Grb2 but not GrpL. These data reveal the presence of distinct complexes involving GrpL and Grb2 in T cells. A functional role of the GrpL-SLP-76 complex is suggested by the ability of GrpL to act alone or in concert with SLP-76 to augment NF-AT activation in Jurkat T cells. 


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Epithelial cell-initiated inflammation plays a crucial role in early tissue damage in amebic infection of human intestine. 
BACKGROUND & AIMS: Entamoeba histolytica infection of the intestine can induce severe gut inflammation. The aims of this study were to assess the role of the host inflammatory response in the tissue damage observed with amebiasis and the role of the intestinal epithelial cell in initiating that response. METHODS: E. histolytica infection was established in human intestinal xenografts in severe combined immunodeficient (SCID-HU-INT) mice. Human intestinal epithelial cell inflammatory responses to amebic infection were inhibited by the intraluminal administration of an antisense oligonucleotide to the human p65 subunit of nuclear factor kappaB, and the role of neutrophils in tissue damage observed with amebiasis was studied by depleting neutrophils from SCID-HU-INT mice. RESULTS: Administration of the antisense oligonucleotide blocked the production of human interleukin 1beta and interleukin 8 by intestinal epithelial cells and inhibited neutrophil influx into the E. histolytica-infected intestinal xenografts. Inhibition of the gut inflammatory response by the antisense oligonucleotide or the depletion of neutrophils from SCID-HU- INT mice blocked the increase in intestinal permeability observed with amebic infection. CONCLUSIONS: Intestinal epithelial cells initiate an inflammatory response with resulting neutrophil-mediated tissue damage in response to E. histolytica infection; this inflammatory cascade can be blocked by inhibiting the transcription of genes regulated by nuclear factor kappaB. 


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Functional Myc-Max heterodimer is required for activation-induced apoptosis in T cell hybridomas. 
T cell hybridomas respond to activation signals by undergoing apoptotic cell death, and this is likely to represent comparable events related to tolerance induction in immature and mature T cells in vivo. Previous studies using antisense oligonucleotides implicated the c-Myc protein in the phenomenon of activation-induced apoptosis. This role for c-Myc in apoptosis is now confirmed in studies using a dominant negative form of its heterodimeric binding partner, Max, which we show here inhibits activation-induced apoptosis. Further, coexpression of a reciprocally mutant Myc protein capable of forming functional heterodimers with the mutant Max can compensate for the dominant negative activity and restore activation-induced apoptosis. These results imply that Myc promotes activation-induced apoptosis by obligatory heterodimerization with Max, and therefore, by regulating gene transcription. 


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A novel heterodimerization partner for thyroid hormone receptor. Peroxisome proliferator-activated receptor. 
Retinoid-like receptors play a central role in hormonal responses by forming heterodimers with other nuclear hormone receptors. In this study we have identified the peroxisome proliferator-activated receptor (PPAR) as a new thyroid hormone receptor (THR) auxiliary nuclear protein, heterodimerizing with THR in solution. Although these heterodimers do not recognize a classical thyroid hormone response element (TRE) characterized by direct repeat separated by four nucleotides (DR+4), PPAR behaves as a dominant negative regulator of thyroid hormone (TH) action. However, a TH-dependent positive effect is elicited by selective interaction of the THR beta-PPAR but not the THR alpha-PPAR heterodimer with a novel TRE (DR+2). The critical region of THR beta was mapped to 3 amino acids in the distal box of the DNA binding domain. Hence, PPAR can positively or negatively influence TH action depending on TRE structure and THR isotype. 


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Purification of TCF-1 alpha, a T-cell-specific transcription factor that activates the T-cell receptor C alpha gene enhancer in a context-dependent manner. 
The differentiation of T cells into functionally diverse subpopulations is controlled in part, by transcriptional activation and silencing; however, little is known in detail about the proteins that influence this developmental process. We have purified a new T-cell-specific factor, TCF-1 alpha, that is implicated in the activation of genes encoding a major component of the human T-cell receptor (TCR). TCF-1 alpha, originally identified and purified through its binding sites on the HIV-1 promoter, was found to bind to the TCR alpha enhancer and to promoters for several genes expressed at significantly earlier stages of T-cell development than the TCR alpha gene (e.g., p56lck and CD3 delta). Sequences related to the TCF-1 alpha binding motif (5'-GGCACCCTTTGA-3') are also found in the human TCR delta (and possibly TCR beta) enhancers. Southwestern and gel renaturation experiments with the use of purified protein fractions revealed that TCF-1 alpha activity is derived from a family of 57- to 53-kD proteins that are abundantly expressed in mature and immature T-cell lines (Jurkat, CCRF-CEM) and not in mature B cells (JY, Namalwa) or nonlymphoid (HeLa) cell lines. A small 95-bp fragment of the TCR alpha control region that contains the TCF-1 alpha binding site juxtaposed between a cAMP-response element (the CRE or T alpha 1 motif) and the binding site for a distinct lymphoid-specific protein (TCF-2 alpha) behaved as a potent T-cell-specific enhancer in vivo. Tandem copies of this enhancer functioned synergistically in mature (Jurkat) T-cell lines as well as resting and activated immature (CCRF-CEM) T-cell lines. Mutation of the TCF-1 alpha binding site diminished enhancer activity and disrupted the synergism observed in vivo between tandem enhancer repeats. The TCF-1 alpha binding site was also required for TCR alpha enhancer activity in transcriptionally active extracts from Jurkat but not HeLa cells, confirming that TCF-1 alpha is a T-cell-specific transcription factor. Curiously, the TCF-1 alpha binding element was inactive in vivo when removed from its neighboring elements on the TCR alpha enhancer and positioned in one or more copies upstream of a heterologous promoter. Thus, the transcriptional activity of TCF-1 alpha appears to depend on the TCF-2 alpha and T alpha 1 (CREB) transcription factors and the context of its binding site within the TCR alpha enhancer. 


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Induction of CIITA and modification of in vivo HLA-DR promoter occupancy in normal thymic epithelial cells treated with IFN-gamma: similarities and distinctions with respect to HLA-DR-constitutive B cells. 
In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantage that MHC class II induction is studied in a "physiologic" cell lineage that, as a result of this expression within the thymus, is thought to participate to the selection and maturation of the T cells. It was found that the MHC class II gene expression was associated with the de novo transcription of the gene encoding the CIITA trans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II DRA promoter and corresponding binding factors was analyzed by in vivo DNAse I footprint. It was found that treatment with IFN-gamma induces changes in the occupancy of the DRA gene regulatory sequences by nuclear factors. The resulting occupancy displays strong similarities with the one observed in the MHC class II-constitutive B cells, represented by both the Burkitt lymphoma line Raji and normal tonsil- derived B cells. However, some peculiar differences were observed between the TEC, either IFN-gamma-induced or not, and the constitutive B cells. These results suggest that both common mechanisms, such as the one mediated by the CIITA trans-activator, and distinct tissue-specific constraints contribute to the transcriptional control of constitutive and IFN-gamma-induced MHC class II gene expression. 


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Glucocorticoid-mediated repression of cytokine gene transcription in human arteritis-SCID chimeras. 
Giant cell arteritis (GCA) is a vasculitic syndrome that preferentially affects medium and large-sized arteries. Glucocorticoid therapy resolves clinical symptoms within hours to days, but therapy has to be continued over several years to prevent disease relapses. It is not known whether and how glucocorticoids affect the function of the inflammatory infiltrate or why the disease persists subclinically despite chronic treatment. GCA is self-sustained in temporal arteries engrafted into SCID mice, providing a model in which the mechanisms of action and limitations of glucocorticoid therapy can be examined in vivo. Administration of dexamethasone to temporal artery-SCID chimeras for 1 wk induced a partial suppression of T cell and macrophage function as indicated by the reduced tissue concentrations of IL-2, IL-1beta, and IL-6 mRNA, and by the diminished expression of inducible NO synthase. In contrast, synthesis of IFN-gamma mRNA was only slightly decreased, and expression of TGF-beta1 was unaffected. These findings correlated with activation of the IkappaBalpha gene and blockade of the nuclear translocation of NFkappaB in the xenotransplanted tissue. Dose-response experiments suggested that steroid doses currently used in clinical medicine are suboptimal in repressing NFkappaB-mediated cytokine production in the inflammatory lesions. Chronic steroid therapy was able to deplete the T cell products IL-2 and IFN-gamma, whereas the activation of tissue-infiltrating macrophages was only partially affected. IL-1beta transcription was abrogated; in contrast, TGF-beta1 mRNA synthesis was steroid resistant. The persistence of TGF-beta1-transcribing macrophages, despite paralysis of T cell function, may provide an explanation for the chronicity of the disease, and may identify a novel therapeutic target in this inflammatory vasculopathy. 


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Transcriptional regulation of the vacuolar H(+)-ATPase B2 subunit gene in differentiating THP-1 cells. 
Monocyte-macrophage differentiation was used as a model system for studying gene regulation of the human vacuolar H(+)-ATPase (V-ATPase). We examined mRNA levels of various V-ATPase subunits during differentiation of both native monocytes and the cell line THP-1, and found that transcriptional and post-transcriptional mechanisms could account for increases in cell V-ATPase content. From nuclear runoff experiments, we found that one subunit in particular, the B2 isoform (Mr = 56,000), was amplified primarily by transcriptional means. We have begun to examine the structure of the B2 subunit promoter region. Isolation and sequencing of the first exon and 5'-flanking region of this gene reveal a TATA-less promoter with a high G + C content. Primer extension and ribonuclease protection analyses indicate a single major transcriptional start site. We transfected promoter-luciferase reporter plasmids into THP-1 cells to define sequences that mediate transcriptional control during monocyte differentiation. We found that sequences downstream from the transcriptional start site were sufficient to confer increased expression during THP-1 differentiation. DNase I footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5'-untranslated and proximal coding regions. 


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Regulation of fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor kappaB. 
T cell receptor engagement activates transcription factors important for cytokine gene regulation. Additionally, this signaling pathway also leads to activation-induced apoptosis in T lymphocytes that is dependent on FasL transcription and expression. Here we demonstrate that nuclear factor kappaB (NF-kappaB), which is involved in the transcriptional regulation of many cytokine genes expressed in activated lymphocytes, also plays a role in T cell activation-induced FasL expression. Inhibition of NF-kappaB activity in a T cell hybridoma leads to decreased FasL expression and apoptosis upon T cell receptor stimulation. We identified the NF-kappaB site in the FasL promoter that contributes to such regulation. Co-expression of p65 (Rel A) with the FasL promoter enhanced its activity, and co-expression of IkappaB dramatically inhibited the inducible promoter activity. In contrast, the transcription factor AP-1 is not required for activation-induced FasL promoter activity. These results define a role for NF-kappaB in mediating FasL expression during T cell activation. 


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Expression of c-jun, jun B and jun D proto-oncogenes in human peripheral-blood granulocytes. 
We have found that purified human peripheral-blood granulocytes express constitutively significant levels of proto-oncogenes c-jun, jun B and jun D mRNA. Upon functional activation of granulocytes by 4 beta-phorbol 12-myristate 13-acetate (PMA), the levels of c-jun, jun B and jun D transcripts were increased. The three jun genes showed a similar time course in their induction by PMA, maximal mRNA levels being reached after 60 min of induction. These results suggest that expression of c-jun, jun B and jun D genes might be involved in terminal granulocyte differentiation or in regulating granulocyte functionality. 


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Oxidants, transcription factors, and intestinal inflammation. 
It is now well appreciated that chronic gut inflammation is characterized by enhanced production of reactive metabolites of oxygen and nitrogen. Some of these oxidants are known to modulate the expression of a variety of genes that are involved in the immune and inflammatory responses. For example, certain oxidants are known to activate the nuclear transcription factor kappa B, which regulates the expression of a variety of different adhesion molecules, cytokines, and enzymes. Oxidants are also known to activate another transcription factor, activator protein-1. This transcription factor is composed of products from the fos and jun proto-oncogene family and is believed to be important in regulating cell growth and proliferation. Finally, oxidants are believed to promote intestinal epithelial cell apoptosis, and the B-cell lymphoma/leukemia-2 gene product is believed to inhibit this phenomenon in an antioxidant-dependent manner. Taken together, these observations suggest that nontoxic concentrations of reactive metabolites of oxygen and nitrogen play an important role in regulating the expression of genes involved in the inflammatory response and in modulating apoptosis. 


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Human tumor necrosis factor alpha gene regulation in phorbol ester stimulated T and B cell lines. 
The minimal region of the human tumor necrosis factor alpha (TNF-alpha) gene promoter necessary for its transcriptional induction by phorbol esters (PMA) in human T and B lymphocyte cell lines has been localized between -52 and +89 nucleotides (nt) relative to the gene's transcriptional start site. Comparison of these sequences to those required to mediate virus or lipopolysaccharide (LPS) induction of the gene reveal significant differences, and thus, the sequence requirements for PMA induction are distinct from those that mediate induction by virus or LPS. Although three sites in the TNF-alpha promoter (kappa 1, kappa 2, and kappa 3) specifically bind the transcription factor NF-kappa B in lymphoid nuclear extracts, TNF-alpha mRNA induction by PMA does not correlate with NF-kappa B binding activities displayed by different T and B cell lines. Moreover, kappa 1-kappa 3 can each be deleted from the TNF-alpha promoter with little effect on the gene's inducibility by PMA. Therefore, TNF-alpha mRNA induction by PMA, like its induction by virus and LPS, is not primarily mediated by NF-kappa B, but rather is mediated through other sequences and protein factors. Surprisingly, multimers of kappa 1-kappa 3 can confer PMA inducibility on a heterologous promoter in a B (Raji), but not a T (HUT78) cell line. However they are not functional on a truncated TNF-alpha promoter, indicating that promoter context and cell type specificity influence the PMA inducible function of these NF-kappa B binding sites. 


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Differential expression and phosphorylation of CTCF, a c-myc transcriptional regulator, during differentiation of human myeloid cells. 
CTCF is a transcriptional repressor of the c-myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down-regulated during terminal differentiation in most cell lines tested; (ii) CTCF down-regulation is retarded and less pronounced than that of c-myc; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post-transcriptional levels. 


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C/EBPbeta and GATA-1 synergistically regulate activity of the eosinophil granule major basic protein promoter: implication for C/EBPbeta activity in eosinophil gene expression. 
Eosinophil granule major basic protein (MBP) is expressed exclusively in eosinophils and basophils in hematopoietic cells. In our previous study, we demonstrated a major positive regulatory role for GATA-1 and a negative regulatory role for GATA-2 in MBP gene transcription. Further analysis of the MBP promoter region identified a C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp upstream of the functional GATA-binding site in the MBP gene. In the cell line HT93A, which is capable of differentiating towards both the eosinophil and neutrophil lineages in response to retinoic acid (RA), C/EBPalpha mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPbeta expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPbeta protein could bind to the potential C/EBP-binding site (bp -90 to -82) in the MBP promoter. Furthermore, we have demonstrated that both C/EBPbeta and GATA-1 can bind simultaneously to the C/EBP- and GATA-binding sites in the MBP promoter. To determine the functionality of both the C/EBP- and GATA- binding sites, we analyzed whether C/EBPbeta and GATA-1 can stimulate the MBP promoter in the C/EBPbeta and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPbeta and GATA-1 expression vectors produced a 5-fold increase compared with cotransfection with the C/EBPbeta or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPbeta. Expression of FOG (riend ATA), which binds to GATA-1 and acts as a cofactor for GATA-binding proteins, decreased transactivation activity of GATA-1 for the MBP promoter in a dose-dependent manner. Our results provide the first evidence that both GATA-1 and C/EBPbeta synergistically transactivate the promoter of an eosinophil-specific granule protein gene and that FOG may act as a negative cofactor for the eosinophil lineage, unlike its positively regulatory function for the erythroid and megakaryocyte lineages. 


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HIV-1 Nef leads to inhibition or activation of T cells depending on its intracellular localization. 
Nef of primate lentiviruses is required for viremia and progression to AIDS in monkeys. Negative, positive, and no effects of Nef have also been reported on viral replication in cells. To reconcile these observations, we expressed a hybrid CD8-Nef protein in Jurkat cells. Two opposite phenotypes were found, which depended on the intracellular localization of Nef. Expressed in the cytoplasm or on the cell surface, the chimera inhibited or activated early signaling events from the T cell antigen receptor. Activated Jurkat cells died by apoptosis, and only cells with mutated nef genes expressing truncated Nefs survived, which rendered Nef nonfunctional. These mutations paralleled those in other viral strains passaged in vitro. Not only do these positional effects of Nef reconcile diverse phenotypes of Nef and suggest a role for its N-terminal myristylation, but they also explain effects of Nef in HIV infection and progression to AIDS. 


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Regulation of nuclear factor-kappa B and its inhibitor I kappa B-alpha/MAD-3 in monocytes by Mycobacterium tuberculosis and during human tuberculosis. 
Blood monocytes from patients with active tuberculosis are activated in vivo, as evidenced by an increase in the stimulated release of proinflammatory cytokines, such as TNF-alpha, and the spontaneous expression of IL-2R. Further, monocytes from patients demonstrate an augmented susceptibility to a productive infection with HIV-1 in vitro. Mycobacterium tuberculosis and its components are strong signals to activate monocytes to production of cytokines. In this study we examined the basis of activation of monocytes during active tuberculosis and by M. tuberculosis. We found a constitutive degradation of I kappa B-alpha, the major cytoplasmic inhibitor of nuclear factor kappa B (NF-kappa B), in freshly isolated PBMC and monocytes from patients with tuberculosis. In contrast, I kappa B-alpha levels in PBMC and monocytes from healthy subjects or from patients with nontuberculous pulmonary conditions were intact. Further, by electrophoretic mobility shift assay, NF-kappa B was activated in monocytes from tuberculous patients. The expression of I kappa B-alpha gene, which is responsive to activation by NF-kappa B, was up-regulated in PBMC and monocytes from patients, but not in mononuclear cells from healthy subjects or those with nontuberculous lung diseases. By contrast, the expression of other adherence-associated early genes, such as IL-8 and IL-1 beta, was not up-regulated in PBMC of tuberculous patients. Further, M. tuberculosis and its tuberculin, purified protein derivative, induced the degradation of I kappa B-alpha and the expression of I kappa B-alpha mRNA, and purified protein derivative induced the activation of NF-kappa B in monocytes. 


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Mapping of the interaction site of the defective transcription factor in the class II major histocompatibility complex mutant cell line clone-13 to the divergent X2-box. 
We have previously described a mutant B lymphoblastoid cell line, Clone-13, that expresses HLA-DQ in the absence of HLA-DR and -DP. Several criteria indicated that the defect in this cell line influences the activity of an isotype-specific transcription factor. Indeed, transient transfection of HLA-DRA and DQB reporter constructs indicated that the affected factor operates via cis-elements located between -141 base pairs and the transcription initiation site. A series of hybrid DRA/DQB reporter constructs was generated to further map the relevant cis-elements in this system. Insertion of oligonucleotides spanning the DQB X-box (but not the DQB-W region or the DQB Y-box) upstream of -141 in a DRA reporter plasmid rescued expression to nearly wild-type levels. Substitution promoters were then generated where the entire X-box, or only the X1- or X2-boxes of HLA-DRA were replaced with the analogous regions of HLA-DQB. The DQB X2-box was able to restore expression to the silent DRA reporter construct. Moreover, replacement of the DQB X2-box with the DRA X2-box markedly diminished the activity of the DQB promoter in the mutant cell. None of the hybrid reporter constructs were defective when transfected into the wild-type, HLA-DR/-DQ positive parental cell line, Jijoye. These studies suggest that the divergent X2-box of the class II major histocompatibility complex promoters plays an important role in influencing differential expression of the human class II isotypes. 


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Multiple transcription factors are required for activation of human interleukin 9 gene in T cells. 
The genetic elements and regulatory mechanisms responsible for human interleukin 9 (IL-9) gene expression in a human T cell leukemia virus type I-transformed human T cell line, C5MJ2, were investigated. We demonstrated that IL-9 gene expression is controlled, at least in part, by transcriptional activation. Transient expression of the luciferase reporter gene linked to serially deleted sequences of the 5'-flanking region of the IL-9 gene has revealed several positive and negative regulatory elements involved in the basal and inducible expression of the IL-9 gene in C5MJ2 cells. An AP-1 site at -146 to -140 was shown to be involved in the expression of the IL-9 gene. A proximal region between -46 and -80 was identified as the minimum sequence for the basal and inducible expression of the IL-9 gene in C5MJ2 cells. Within this region, an NF-kappaB site at -59 to -50 and its adjacent 20-base pair upstream sequence were demonstrated to play a critical role for the IL-9 promoter activity. DNA-protein binding studies indicated that NF-kappaB, c-Jun, and potentially novel proteins (around 35 kDa) can bind to this important sequence. Mutations at different sites within this proximal promoter region abolished the promoter activity as well as the DNA binding. Taken together, these results suggest that the cooperation of different transcription factors is essential for IL-9 gene expression in T cells. 


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AML1 (CBFalpha2) cooperates with B cell-specific activating protein (BSAP/PAX5) in activation of the B cell-specific BLK gene promoter. 
AML1 plays a critical role during hematopoiesis and chromosomal translocations involving AML1 are commonly associated with different forms of leukemia, including pre-B acute lymphoblastic leukemia. To understand the function of AML1 during B cell differentiation, we analyzed regulatory regions of B cell-specific genes for potential AML1-binding sites and have identified a putative AML1-binding site in the promoter of the B cell-specific tyrosine kinase gene, blk. Gel mobility shift assays and transient transfection assays demonstrate that AML1 binds specifically to this site in the blk promoter and this binding site is important for blk promoter activity. Furthermore, in vitro binding analysis revealed that the AML1 runt DNA-binding domain physically interacts with the paired DNA-binding domain of BSAP, a B cell-specific transcription factor. BSAP has been shown previously to be important for B cell-specific regulation of the blk gene. Physical interaction of AML1 with BSAP correlates with functional cooperativity in transfection studies where AML1 and BSAP synergistically activate blk promoter transcription by more than 50-fold. These results demonstrate physical and functional interactions between AML1 and BSAP and suggest that AML1 is an important factor for regulating a critical B cell-specific gene, blk. 


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A shortened life span of EKLF-/- adult erythrocytes, due to a deficiency of beta-globin chains, is ameliorated by human gamma-globin chains. 
Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF-/- ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF-/- colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of beta h1-globin, which continued to be expressed at a very low level. The ratio of adult beta-globin/alpha-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF-/- cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF-/- erythrocytes in adult animals must be short-lived, apparently due to the imbalance of beta- versus alpha-globin chains, leading to the precipitation of excess alpha-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF-/- ES cells of a human LCR/gamma-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals. 


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Activation of NF-kappa B in vivo is regulated by multiple phosphorylations. 
The activation of nuclear factor kappa B (NF-kappa B) in intact cells is mechanistically not well understood. Therefore we investigated the modifications imposed on NF-kappa B/I kappa B components following stimulation and show that the final step of NF-kappa B induction in vivo involves phosphorylation of several members of the NF-kappa B/I kappa B protein families. In HeLa cells as well as in B cells, TNF-alpha rapidly induced nuclear translocation primarily of p50-p65, but not of c-rel. Both NF-kappa B precursors and I kappa B alpha became strongly phosphorylated with the same kinetics. In addition to the inducible phosphorylation after stimulation, B lymphocytes containing constitutive nuclear NF-kappa B revealed constitutively phosphorylated p65 and I kappa B alpha. Phosphorylation was accompanied by induced processing of the precursors p100 and p105 and by degradation of I kappa B alpha. As an in vitro model we show that phosphorylation of p105 impedes its ability to interact with NF-kappa B, as has been shown before for I kappa B alpha. Surprisingly, even p65, but not c-rel, was phosphorylated after induction in vivo, suggesting that TNF-alpha selectively activates only specific NF-kappa B heteromers and that modifications regulate not only I kappa B molecules but also NF-kappa B molecules. In fact, cellular NF-kappa B activity was phosphorylation-dependent and the DNA binding activity of p65-containing NF-kappa B was enhanced by phosphorylation in vitro. Furthermore, we found that the induction by hydrogen peroxide of NF-kappa B translocation to the nucleus, which is assumed to be triggered by reactive oxygen intermediates, also coincided with incorporation of phosphate into the same subunits that were modified after stimulation by TNF-alpha. Thus, phosphorylation appears to be a general mechanism for activation of NF-kappa B in vivo. 


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Identification of transcriptional suppressor proteins that bind to the negative regulatory element of the human immunodeficiency virus type 1. 
Two different proteins which independently bound to neighboring sequences within the negative regulatory element (NRE) of human immunodeficiency virus type 1 (HIV-1) were detected in the nuclear extract of a virus-infected human T cell line. One of the factors bound to a novel dyad symmetrical sequence. This sequence is well conserved in various HIV-1 isolates and partial homology was found with the promoter region of the human retinoblastoma gene. Similar DNA binding activity was detected in a variety of virus-uninfected human T cell lines and HeLa cells by means of a gel mobility shift assay. The other factor bound to a putative AP-1 recognition sequence predicted for the HIV-1 NRE. However, this factor did not bind to a typical AP-1 site. The insertion of multiple copies of the binding site for the former or latter factor into a heterologous promoter reduced the promoter activity to one-tenth or one-third, respectively. Thus, each factor may function as a novel negative regulator of transcription. 


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Transcription factors of T and B lymphocytes--basic research and clinical perspectives for gastroenterology. 
Tissue specific regulation of gene expression by transcription factors is a fascinating new field in molecular immunology. This review summarizes data on specific regulation of promoters and enhancers by nuclear trans-acting factors in lymphocytes. The structural classes of transcription factors are described and basic methods for detection and analysis of transcription factors are detailed. Furthermore, the most important trans-acting factors of T and B lymphocytes (e.g. NF-kB, NF-AT and STAT families) and their functional importance are described. Several methods for specific down-regulation of transcription factors are shown that may be relevant to treatment of human disease. The data are discussed with regard to their potential clinical relevance for gastroenterology. 


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E3, a hematopoietic-specific transcript directly regulated by the retinoic acid receptor alpha. 
Retinoic acid (RA)-induced maturation mediated by the retinoic acid receptor alpha (RAR alpha) has been implicated in myeloid development. We have used differential hybridization analysis of a cDNA library constructed from the murine RA-inducible MPRO promyelocyte cell line to identify immediate-early genes induced by RA during granulocytic differentiation. E3, one of nine sequences identified, was upregulated in an immediate-early manner, with transcript levels peaking after 60 minutes exposure to RA. E3 transcripts were RA-inducible in HL60 cells, but not in an RA-resistant subclone, HL60R, that harbors a mutated RAR alpha gene. However, when HL60R cells were transduced with a functional copy of the RAR alpha gene, RA induced a 10-fold increase in E3 mRNA levels. E3 transcripts are present in the myeloid, B-lymphoid, and erythroid lineages, absent in nonhematopoietic cells, and encode a highly hydrophobic, potentially phosphorylated polypeptide of unknown function with significant homology to a putative protein expressed in myeloid cells. The murine E3 promoter harbors a single bipartite retinoic acid response element which in transient transfection assays conferred RA sensitivity. These results indicate that E3 is a hematopoietic-specific gene that is an immediate target for the activated RAR alpha during myelopoiesis. 


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Signaling through the lymphotoxin-beta receptor stimulates HIV-1 replication alone and in cooperation with soluble or membrane-bound TNF-alpha. 
The level of ongoing HIV-1 replication within an individual is critical to HIV-1 pathogenesis. Among host immune factors, the cytokine TNF-alpha has previously been shown to increase HIV-1 replication in various monocyte and T cell model systems. Here, we demonstrate that signaling through the TNF receptor family member, the lymphotoxin-beta (LT-beta) receptor (LT-betaR), also regulates HIV-1 replication. Furthermore, HIV-1 replication is cooperatively stimulated when the distinct LT-betaR and TNF receptor systems are simultaneously engaged by their specific ligands. Moreover, in a physiological coculture cellular assay system, we show that membrane-bound TNF-alpha and LT-alpha1beta2 act virtually identically to their soluble forms in the regulation of HIV-1 replication. Thus, cosignaling via the LT-beta and TNF-alpha receptors is probably involved in the modulation of HIV-1 replication and the subsequent determination of HIV-1 viral burden in monocytes. Intriguingly, surface expression of LT-alpha1beta2 is up-regulated on a T cell line acutely infected with HIV-1, suggesting a positive feedback loop between HIV-1 infection, LT-alpha1beta2 expression, and HIV-1 replication. Given the critical role that LT-alpha1beta2 plays in lymphoid architecture, we speculate that LT-alpha1beta2 may be involved in HIV-associated abnormalities of the lymphoid organs. 


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Calcium/calmodulin-dependent protein kinase II downregulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells. 
Engagement of the T cell receptor for antigen activates phospholipase C resulting in an increase in intracellular free calcium concentration ([Ca2+]i) and activation of protein kinase C (PKC). Increased [Ca2+]i activates Ca2+/calmodulin-dependent kinases including the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM-K II), as well as calcineurin, a type 2B protein phosphatase. Recent studies have identified calcineurin as a key enzyme for interleukin (IL)-2 and IL-4 promoter activation. However, the role of CaM-K II remains unknown. We have used mutants of these kinases and phosphatases (gamma B*CaM-K and delta CaM-AI, respectively) to explore their relative role in cytokine gene transcription and their interactions with PKC-dependent signaling systems. gamma B*CaM-K and delta CaM-AI, known to exhibit constitutive Ca(2+)-independent activity, were cotransfected (alone or in combination) in Jurkat T cells with a plasmid containing the intact IL-2 promoter driving the expression of the chloramphenicol acetyltransferase reporter gene. Cotransfection of gamma B*CaM-K with the IL-2 promoter construct downregulated its transcription in response to stimulation with ionomycin and phorbol myristate acetate (PMA). The inhibitory effect of CaM-K II on IL-2 promoter was associated with decreased transcription of its AP-1 and NF-AT transactivating pathways. Under the same conditions, delta CaM-AI superinduced IL-2 promoter activity (approximately twofold increase). When both mutants were used in combination, gamma B*CaM-K inhibited the induction of the IL-2 promoter by delta CaM-AI. Similar results were obtained when a construct containing the IL-4 promoter also was used. gamma B*CaM-K also downregulated the activation of AP-1 in response to transfection with a constitutively active mutant of PKC or stimulation with PMA. These results suggest that CaM-K II may exert negative influences on cytokine gene transcription in human T cells, and provide preliminary evidence for negative cross-talk with the calcineurin- and PKC- dependent signaling systems. 


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HIV does not replicate in naive CD4 T cells stimulated with CD3/CD28. 
In this report, we demonstrate that the T cell tropic strain of HIV, LAI, does not replicate in naive CD4 T cells stimulated by cross-linking CD3 and CD28. In contrast, LAI replicates well in memory CD4 T cells stimulated in the same way. Unlike this physiologically relevant stimulation, PHA stimulates productive LAI replication in both naive and memory T cells. These studies were conducted with highly purified (FACS-isolated) subsets of CD4 T cells identified by expression of both CD45RA and CD62L. Remixing of purified T cells showed that naive T cells do not suppress LAI replication in memory T cells and that memory T cells do not restore LAI expression in naive T cells. The suppression of productive LAI replication in naive T cells is not due to differential expression of viral coreceptors, nor is it due to inhibition of activation of the important HIV transcription factors, nuclear factor-kappaB and activator protein-1. The inherent resistance of naive T cells to productive HIV infection, coupled with their proliferative advantage as demonstrated here, provides a sound basis for proposed clinical therapies using ex vivo expansion and reinfusion of CD4 T cells from HIV-infected adults. 


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Activation of the signal transducer and transcription (STAT) signaling pathway in a primary T cell response. Critical role for IL-6. 
The T cell activation is initiated by interaction of specific Ags with TCR, followed by activation of intracellular biochemical events leading to activation of several genes. The activation of signal transducer and activator of transcription (STAT) proteins in a primary TCR-mediated activation of T cells have been explored. In purified human peripheral blood T cells, nuclear STAT proteins were activated approximately 3 h after activation by cross-linked anti-CD3 Abs. These STAT proteins were detected by using the IFN-gamma-activated sequence (GAS) and related oligonucleotides as probes in electrophoretic mobility shift assay. Analysis of the nuclear extracts with anti-STAT Abs indicated that they contained STAT-3 and additional proteins crossreactive with the STAT family. The induction of STAT activity was inhibited completely by pretreatment with either cycloheximide or cyclosporin A, thus indicating that the induction was due to a secondary factor produced by the activated T cells. As neutralizing anti-IL-6 Abs effectively down-regulated the early induction of STAT proteins and as exogenously added IL-6 rapidly activated DNA binding similar to TCR-mediated bindings, it can be concluded that IL-6 is the factor responsible for the activation of STAT proteins in a primary T cell response. 


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Reversible differentiation of human monoblastic leukemia U937 cells by ML-9, an inhibitor of myosin light chain kinase. 
Human monoblastic leukemia U937 cells are induced to differentiate into monocytes and macrophages by various agents. We have shown that 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9), an inhibitor of myosin light chain kinase, induces differentiation of monocytoid leukemia cell lines U937 and THP-1 but not of myeloblastic leukemic ML-1 cell or erythroleukemia K562 cells. In the present study, we further analyzed the effect of ML-9 in comparison with that of 1 alpha, 25-dihydroxyvitamin D3 (VD3) a typical inducer of monocytic differentiation. ML-9 induced nitroblue tetrazolium (NBT)-reducing activity of U937 cell more rapidly than VD3: This differentiation marker was induced significantly after incubation with ML-9 and VD3 for 4 hours and 1 day, respectively. ML-9 also induced alpha-naphthyl acetate esterase (ANAE) activity, another monocytic differentiation marker, more rapidly than VD3. The maximum levels of these markers induced by ML-9 were comparable to those induced by VD3, but after removal of ML-9 from the medium by washing the cells, the expressions of theses markers decreased within 4 hours and reached basal levels in 1 day, indicating that ML-9's induction of expression of differentiation-associated phenotypes was reversible. The growth inhibition of U937 cells by ML-9 was also reversible. Similar effects were observed in another line of human monoblastic cells, THP-1. ML-9 had little or no effect on the morphology of U937 cells but increased the expression of monocyte-macrophage lineage-associated surface antigen, CD14, to some extent. Irreversible terminal differentiation induced by VD3 is associated with down regulation of the expression of c-myc and upregulation of the expression of c-fos and c-jun, but ML-9 did not affect the expression of these oncogenes appreciably. ML-9-induced differentiation was also reversible when the cells were cultured with cultured with ML-9 plus an anti-cancer drug such as 1-beta-D-arabino-furanosylcytosine or daunomycin. it became irreversible, however, upon simultaneous treatment with dexamethasone and transforming growth factor-beta 1 (TGF-beta 1), which did not induce differentiation of U937 cells but caused growth arrest of the cells in the G0/G1 phase of the cell cycle. These results suggest that ML-9 should be useful for studying the mechanisms of monocytic differentiation. 


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GATA-1 and erythropoietin cooperate to promote erythroid cell survival by regulating bcl-xL expression. 
The transcription factor GATA-1 is essential for normal erythropoiesis. By examining in vitro-differentiated embryonic stem cells, we showed previously that in the absence of GATA-1, committed erythroid precursors fail to complete maturation and instead undergo apoptosis. The mechanisms by which GATA-1 controls cell survival are unknown. Here we report that in erythroid cells, GATA-1 strongly induces the expression of the anti-apoptotic protein bcl-xL, but not the related proteins bcl-2 and mcl-1. Consistent with a role for bcl-xL in mediating GATA-1-induced erythroid cell survival, in vitro-differentiated bcl-xL-/- embryonic stem cells fail to generate viable mature definitive erythroid cells, a phenotype resembling that of GATA-1 gene disruption. In addition, we show that erythropoietin, which is also required for erythroid cell survival, cooperates with GATA-1 to stimulate bcl-xL gene expression and to maintain erythroid cell viability during terminal maturation. Together, our data show that bcl-xL is essential for normal erythroid development and suggest a regulatory hierarchy in which bcl-xL is a critical downstream effector of GATA-1 and erythropoietin-mediated signals. 


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Estrogen receptor diminishes DNA-binding activities of chicken GATA-1 and CACCC-binding proteins. 
The estrogen receptor (ER) repressed erythroid differentiation and erythroid-specific gene expression. In this study, we investigated the effect of ER alpha (referred to throughout as ER) on DNA-binding activities of transcription factors involved in regulating the expression of erythroid-specific genes, and, in particular, the histone H5 gene. Using electrophoretic mobility shift assays, we found that in the presence of rabbit reticulocyte lysate, human ER reduced the binding activities of chicken immature erythrocyte nuclear extracted proteins to GATA and CACCC sites in the H5 promoter and enhancer. In contrast, the binding activities of NF1 and Sp1 were not affected by ER. Binding of ER to an estrogen response element was enhanced by addition of rabbit reticulocyte lysate. This lysate was also necessary for ER to diminish the DNA-binding activity of GATA-1. These results suggest that additional factor(s) are necessary for full ER function. Both GATA-1 and CACCC-binding proteins are critical for the developmentally regulated expression of erythroid-specific genes. We hypothesize that interference in DNA-binding activities of GATA-1 and CACCC-binding proteins is the mechanism by which the ER inhibits regulation of these genes. 


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Transcription factor requirements for U2 snRNA-encoding gene activation in B lymphoid cells. 
Transcription of a human U2 small nuclear RNA(snRNA)-encoding gene in HeLa cells requires a distal enhancer element, which is composed of one octamer motif (Oct) and three Sp 1-binding sites. To study the transcription factor requirement in B-cells, different U2 enhancer constructions were transfected into the lymphoid cell line, BJA-B. The results showed that the activation of U2 snRNA transcription in B-cells also requires an enhancer comprising both the Oct and at least one Sp 1-binding site. Deletion of all the Sp 1-binding sites from the enhancer reduces transcription by 80-90% in HeLa, as well as in BJA-B cells, whereas the removal of the octamer-binding site reduces transcription to levels below detection in both cell types. Enhancers containing a single Oct have, nevertheless, the capacity to partially activate U2 snRNA transcription in both HeLa cells, in which only OTF-1 is expressed, and in BJA-B cells in which OTF-2 is the predominantly expressed octamer-binding factor. The most likely interpretation of our results is that both the ubiquitous transcription factor, OTF-1, and the B-cell-specific transcription factor, OTF-2, can activate U2 snRNA transcription. The results also revealed a similar functional cooperation between the transcription factors which bind to the Oct and the adjacent Sp 1-binding site in BJA-B cells, as has been observed in HeLa cells, since a template which contains a weak binding site for OTFs expresses wild-type levels of U2 snRNA in both cell types when the weak octamer-binding site is combined with a Sp 1-binding site. 


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Unexpected and coordinated expression of Spi-1, Fli-1, and megakaryocytic genes in four Epo-dependent cell lines established from transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen. 
Most erythroleukemic cell lines established in vitro coexpress erythrocytic and megakaryocytic markers that often are associated with expression of Spi-1 and/or Fli-1 transcription factors known as transactivators of megakaryocyte-specific promoters. In the present study, we examined the possibility of establishing new cell lines keeping strictly erythroid-specific properties in vitro through the targeted and conditional immortalization of erythrocytic progenitors. For that purpose, we established several lines of transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen. As expected, these transgenic mice developed splenomegaly due to the massive amplification of Ter 119 positive erythroid nucleated cells expressing T antigen. Despite this drastic effect in vivo, the in vitro immortalization of erythropoietin-dependent erythroid progenitors unexpectedly occurred at low frequency, and all four cell lines established expressed both erythrocytic (globins) and megakaryocytic markers (glycoprotein IIb, platelet factor 4) as well as Spi-1 and Fli-1 transcripts at permissive temperature. Switching the cells to the nonpermissive temperature led to a marked increase in globin gene expression and concomitant decrease in expression of Spi-1, Fli-1, and megakaryocytic genes in an erythropoietin-dependent manner. Interestingly, enhanced expression of Spi-1 and Fli-1 genes already was detected in the Ter 119 positive cell population of transgenic mice spleen in vivo. However, like normal Ter 119 erythroid cells, these Ter 119 positive cells from transgenic mice still expressed high levels of beta-globin and very low or undetectable glycoprotein IIb and platelet factor 4 megakaryocytic transcripts. Taken together, these data indicate that the unexpected expression of megakaryocytic genes is a specific property of immortalized cells that cannot be explained only by enhanced expression of Spi-1 and/or Fli-1 genes. 


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EBF and E47 collaborate to induce expression of the endogenous immunoglobulin surrogate light chain genes. 
Early B cell factor (EBF) and E47 participate in the transcriptional control of early B lymphocyte differentiation. With the aim of identifying genetic targets for these transcription factors, we stably transfected cDNAs encoding EBF or a covalent homodimer of E47, individually or together, into immature hematopoietic Ba/F3 cells, which lack both factors. In combination, EBF and E47 induce efficient expression of the endogenous immunoglobulin surrogate light chain genes, lambda5 and VpreB, whereas other pre-B cell-specific genes remain silent. Multiple functionally important EBF and E47 binding sites were identified in the lambda5 promoter/enhancer region, indicating that lambda5 is a direct genetic target for these transcription factors. Taken together, these data suggest that EBF and E47 synergize to activate expression of a subset of genes that define an early stage of the B cell lineage. 


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Characterization of the human platelet/endothelial cell adhesion molecule-1 promoter: identification of a GATA-2 binding element required for optimal transcriptional activity. 
Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on platelets, endothelial cells, and certain leukocyte subsets. To examine the factors controlling vascular-specific expression of PECAM-1, we cloned the 5'-flanking region of the PECAM-1 gene and analyzed its transcriptional activity. 5'-Rapid amplification of cDNA ends (5'-RACE) analysis showed that transcription initiation occurred at several closely spaced nearby sites originating approximately 204 bp upstream from the translation start site. Analysis of the sequence immediately upstream from the transcription initiation site (TIS) showed no canonical TATA or CAAT elements, however an initiator element commonly found in TATA-less promoters encompassed the TIS. 5'-serially truncated PECAM-1 promoter segments cloned in front of a luciferase reporter drove transcription in both a lineage- and orientation-specific manner. Putative cis-acting control elements present within a 300-bp core promoter included two ets sites, an Sp1 site, tandem E-box domains, two GATA-associated sites (CACCC), an AP-2 binding site, and a GATA element at -24. Mutational analysis showed that optimal transcriptional activity required the GATA sequence at position -24, and gel-shift assays further showed that the GATA-2 transcription factor, but not GATA-1, bound to this region of the PECAM-1 promoter. Understanding the cis- and transacting factors that regulate the tissue-specific expression of PECAM-1 should increase our understanding of the mechanisms by which vascular-specific gene expression is achieved. 


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Ectopic expression of a conditional GATA-2/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner. 
The GATA factors are a family of transcriptional regulatory proteins in eukaryotes that share extensive homology in their DNA-binding domains. One enigmatic aspect of GATA factor expression is that several GATA proteins, which ostensibly share the same DNA-binding site specificity, are coexpressed in erythroid cells. To elucidate the roles of individual GATA factors in erythropoiesis, conditional alleles of GATA-1, GATA-2, and GATA-3 were prepared by fusing each of the factors to the hormone-binding domain of the human estrogen receptor (ER). These GATA/ER chimeric factors were shown to be hormone-inducible trans-activating proteins in transient transfection assays. When stably introduced into primary erythroblasts or conditionally transformed erythroid progenitors cells, exogenous GATA-2/ER promoted proliferation and inhibited terminal differentiation in an estrogen-dependent manner. These phenotypic effects are specifically attributable to the action of ectopically expressed GATA-2/ER because erythroblasts expressing exogenous GATA-2 are constitutively arrested in differentiation and because erythroid progenitors expressing either Gal/ER or GATA-3/ER do not display a hormone-responsive block in differentiation. Thus, the GATA-2 transcription factor appears to play a role in regulating the self-renewal capacity of early erythroid progenitor cells. 


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Phorbol ester reduces constitutive nuclear NF kappa B and inhibits HIV-1 production in mature human monocytic cells. 
NF kappa B is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocytic leukemias. There is little information available on the response of NF kappa B to cytokines in normal human monocytes. We have used a 32P-labeled oligonucleotide derived from human immunodeficiency virus (HIV-1) long terminal repeat, which contains a tandem repeat of the NF kappa B binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NF kappa B in extracts of nuclei from normal human monocytes. Treatment of normal monocytes with 12-0-tetradecanoyl phorbol-13-acetate (TPA) for 4-24 h caused the complete disappearance of NF kappa B from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP-1. The constitutive transcription factor SP1 was unaffected by addition of TPA. The disappearance of NF kappa B from the nucleus was concentration dependent between 10 and 50 ng/ml of phorbol ester. In THP-1 cells, TPA also induced a new, faster-migrating NF kappa B species not induced in monocytes. Protein kinase C inhibitor staurosporine, but not cyclic nucleotide-dependent protein kinase inhibitor HA-1004, also dramatically reduced constitutive levels of nuclear NF kappa B. Finally, TPA addition to monocytes infected with HIV-1 inhibited HIV-1 replication, as determined by reverse transcriptase assays, in a concentration-dependent manner. These results are in striking contrast to the increase in nuclear NF kappa B and HIV-1 replication induced by phorbol esters in promonocytic leukemia cells U937 and HL-60, and emphasize the importance of studying cytokine regulation of HIV-1 in normal monocytes. 


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Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity. 
We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells. 


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Expression of mRNA for the GATA-binding proteins in human eosinophils and basophils: potential role in gene transcription. 
The expression of the hematopoietic transcription factors GATA-1, GATA-2, and GATA-3 was studied in eosinophils and basophils. Eosinophils express mRNA for GATA-1, GATA-2, and GATA-3. Basophils express GATA-2 and GATA-3. Treatment of HL-60 eosinophilic sublines with either interleukin-5 or butyric acid increased the expression of GATA-1 mRNA concomitant with the expression of eosinophil-specific genes, whereas levels of GATA-2 mRNA remained relatively constant. The presence of mRNA for these proteins in eosinophils and basophils suggests that gene transcription in these lineages may be regulated by GATA-binding proteins. 


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Human monocyte binding to fibronectin enhances IFN-gamma-induced early signaling events. 
Leukocyte integrins are fundamentally important in modulating adhesion to extracellular matrix components and to other cells. This integrin-mediated adhesion controls leukocyte arrest and extravasation during the onset of inflammatory responses. Moreover, integrin-ligand interactions trigger signaling pathways that may influence leukocyte phenotype and function at sites of inflammation. In the current studies, we evaluated the combinatorial effects of monocyte adhesion and IFN-gamma on intracellular signaling pathways. IFN-gamma triggers a well-defined signal transduction pathway, which although not directly stimulated by monocyte adherence to fibronectin or arginine-glycine-aspartate (RGD)-coated substrata, was enhanced significantly in these matrix-adherent cells. Compared with monocytes in suspension or adherent on plastic surfaces, monocytes adherent to fibronectin or RGD exhibited a greater than threefold increase in steady state levels of IFN-gamma-induced mRNA for the high affinity Fc gammaRI receptor. By electrophoretic mobility shift assays, this increase in mRNA was associated with a 5- to 10-fold increase in the STAT1-containing DNA-binding complex that binds to Fc gammaRI promoter elements. Furthermore, the tyrosine phosphorylation of STAT1 and the tyrosine kinases JAK1 and JAK2 was enhanced significantly in RGD-adherent monocytes compared with control cells. These results suggest a novel mechanism by which integrin-mediated cell adhesion can modulate the magnitude of cytokine-induced signal transduction pathways, thereby amplifying cellular events leading to monocyte activation and inflammation. 


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Repression by Ikaros and Aiolos is mediated through histone deacetylase complexes. 
Here we show that the lymphoid lineage-determining factors Ikaros and Aiolos can function as strong transcriptional repressors. This function is mediated through two repression domains and is dependent upon the promoter context and cell type. Repression by Ikaros proteins correlates with hypo-acetylation of core histones at promoter sites and is relieved by histone deacetylase inhibitors. Consistent with these findings, Ikaros and its repression domains can interact in vivo and in vitro with the mSin3 family of co-repressors which bind to histone deacetylases. Based on these and our recent findings of associations between Ikaros and Mi-2-HDAC, we propose that Ikaros family members modulate gene expression during lymphocyte development by recruiting distinct histone deacetylase complexes to specific promoters. 


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Bcl-2-mediated drug resistance: inhibition of apoptosis by blocking nuclear factor of activated T lymphocytes (NFAT)-induced Fas ligand transcription. 
Bcl-2 inhibits apoptosis induced by a variety of stimuli, including chemotherapy drugs and glucocorticoids. It is generally accepted that Bcl-2 exerts its antiapoptotic effects mainly by dimerizing with proapoptotic members of the Bcl-2 family such as Bax and Bad. However, the mechanism of the antiapoptotic effects is unclear. Paclitaxel and other drugs that disturb microtubule dynamics kill cells in a Fas/Fas ligand (FasL)-dependent manner; antibody to FasL inhibits paclitaxel-induced apoptosis. We have found that Bcl-2 overexpression leads to the prevention of chemotherapy (paclitaxel)-induced expression of FasL and blocks paclitaxel-induced apoptosis. The mechanism of this effect is that Bcl-2 prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes, a transcription factor activated by microtubule damage) by binding and sequestering calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT to move to the nucleus. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, it appears that paclitaxel and other drugs that disturb microtubule function kill cells at least in part through the induction of FasL. Furthermore, Bcl-2 antagonizes drug-induced apoptosis by inhibiting calcineurin activation, blocking NFAT nuclear translocation, and preventing FasL expression. The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2. Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation. 


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NF-kappaB activation is a critical regulator of human granulocyte apoptosis in vitro. 
During beneficial inflammation, potentially tissue-damaging granulocytes undergo apoptosis before being cleared by phagocytes in a non-phlogistic manner. Here we show that the rate of constitutive apoptosis in human neutrophils and eosinophils is greatly accelerated in both a rapid and concentration-dependent manner by the fungal metabolite gliotoxin, but not by its inactive analog methylthiogliotoxin. This induction of apoptosis was abolished by the caspase inhibitor zVAD-fmk, correlated with the inhibition of nuclear factor-kappa B (NF-kappaB), and was mimicked by a cell permeable inhibitory peptide of NF-kappaB, SN-50; other NF-kappaB inhibitors, curcumin and pyrrolidine dithiocarbamate; and the proteasome inhibitor, MG-132. Gliotoxin also augmented dramatically the early (2-6 h) pro-apoptotic effects of tumor necrosis factor-alpha (TNF-alpha) in neutrophils and unmasked the ability of TNF-alpha to induce eosinophil apoptosis. In neutrophils, TNF-alpha caused a gliotoxin-inhibitable activation of an inducible form of NF-kappaB, a response that may underlie the ability of TNF-alpha to delay apoptosis at later times (12-24 h) and limit its early killing effect. Furthermore, cycloheximide displayed a similar capacity to enhance TNF-alpha induced neutrophil apoptosis even at time points when cycloheximide alone had no pro-apoptotic effect, suggesting that NF-kappaB may regulate the production of protein(s) which protect neutrophils from the cytotoxic effects of TNF-alpha. These data shed light on the biochemical and molecular mechanisms regulating human granulocyte apoptosis and, in particular, indicate that the transcription factor NF-kappaB plays a crucial role in regulating the physiological cell death pathway in granulocytes. 


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Tissue and cell-type specific expression of the tuberous sclerosis gene, TSC2, in human tissues. 
TSC2 is a gene on chromosome 16p13.3 associated with the autosomal dominant neurocutaneous disorder, tuberous sclerosis complex (TSC). By using a partial nucleotide sequence from the cloned TSC2 and polymerase chain reaction methodology, we constructed a digoxigenin-labeled complementary DNA probe to examine TSC2 gene expression in autopsy- or biopsy-derived human tissues by in situ hybridization. TSC2 messenger RNA was widely expressed in various cell types throughout the body, including epithelia, lymphocytes, and cells with endocrine functions, e.g., adrenal cortex and anterior pituitary. It was prominently and selectively (within the central nervous system) expressed in pyramidal cells of the cerebral cortex and other motor neurons, e.g., in spinal cord and brainstem nuclei. Visceral TSC2 expression was comparable in autopsy tissues from patients with and without TSC; TSC2 messenger RNA expression was most prominent in cells with a rapid mitotic rate and turnover, e.g., epithelia and lymphocytes, with central nervous system pyramidal cells and other neurons being an obvious exception, and/or in cells with important secretory/transport functions. This widespread expression of the TSC2 gene supports the view that it encodes a protein vital to cell growth and metabolism or one that functions as a tumor/growth suppressor. 


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Retinoid X receptor and c-cerbA/thyroid hormone receptor regulate erythroid cell growth and differentiation. 
Nuclear receptors are important regulators of erythroid cell development. Here we investigated the impact of retinoid X receptor (RXR), retinoic acid receptor (RAR), and of the c-erbA/thyroid hormone (T3) receptor (c-erbA/TR) on growth and differentiation of erythroid cells using an in vitro culture system of stem cell factor-dependent erythroid progenitors. RXR, RAR, and c-erbA/TR-specific ligands were found to induce erythroid-specific gene expression and to accelerate erythroid differentiation in culture, with T3 being most effective. Furthermore, while ligand-activated c-erbA/TR accelerated differentiation, unliganded c-erbA/TR effectively blocked differentiation and supported sustained progenitor growth in culture. Thus, c-erbA/TR appears to act as a binary switch affecting erythroid cell fate: unliganded c-erbA/TR supports growth while ligand-activated c-erbA/TR induces differentiation. Additionally, to determine the impact of RXR for erythroid cell development, dominant interfering mutant RXRs, lacking the transcriptional activator functions AF-1 and AF-2, or AF-2 only, or the entire DNA-binding domain, were introduced into erythroid progenitor cells via recombinant retrovirus vectors and analyzed for RXR-specific effects. It was found that expression of wild-type RXR and of the RXR mutants devoid of AF-1 and/or AF-2 supported a transient outgrowth of erythroid cells. In marked contrast, expression of the dominant interfering deltaDNA-binding domain RXR, containing a deletion of the entire DNA-binding domain, was incompatible with erythroid cell growth in vitro, suggesting a pivotal role of RXR for erythroid cell development. 


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Protease inhibitors block lipopolysaccharide induction of tissue factor gene expression in human monocytic cells by preventing activation of c-Rel/p65 heterodimers. 
Tissue factor (TF) is expressed rapidly by human monocytes exposed to bacterial endotoxin (lipopolysaccharide, or LPS). Transcriptional regulation is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site in the TF promoter. Nuclear translocation of cytosolic c-Rel/p65 heterodimers and other members of the NF-kappa B/Rel family requires dissociation and proteolytic degradation of the inhibitor protein, I kappa B alpha. The protease inhibitors N alpha-tosylphenylalanyl chloromethyl ketone (TPCK) and N alpha-tosyl-L-lysine chloromethyl ketone (TLCK) block activation of NF-kappa B/Rel proteins by preventing degradation of I kappa B alpha. To determine if TPCK and TLCK inhibited LPS induction of TF expression, freshly isolated human monocytes and monocytic THP-1 cells were pretreated with these inhibitors for 30 min before LPS stimulation. Both TPCK and TLCK inhibited LPS induction of TF protein, TF mRNA and TF promoter activity in a dose-dependent manner. These inhibitors specifically prevented degradation of I kappa B alpha and nuclear translocation of c-Rel/p65 heterodimers. In contrast, TPCK and TLCK did not block induction of an immediate-early gene encoding the transcription factor, Egr-1. Taken together, these data indicated that inhibiting nuclear translocation of c-Rel/p65 heterodimers prevented LPS induction of TF gene transcription in monocytic cells. 


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Increased interleukin 2 transcription in murine lymphocytes by ciprofloxacin. 
The fluoroquinolone antibiotic, ciprofloxacin (cipro), induces hyperproduction of interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) in stimulated human peripheral blood lymphocytes. In this investigation an enhanced and prolonged IL-2 and IL-2 mRNA response was also detected in both stimulated (T cell mitogens or alloantigens) murine splenocytes and in the stimulated murine T cell line EL-4 in the presence of ciprofloxacin (5-80 micrograms/ml) as compared to control cells without antibiotics. However, in contrast to human lymphocytes, IFN-gamma production was inhibited and IFN-gamma mRNA levels were unaffected at 24 h and only slightly upregulated at 48 and 72 h of culture in murine splenocytes incubated with cipro (20 micrograms/ml). EL-4 cells were transfected with a plasmid containing the IL-2 promoter and enhancer region linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Analysis of CAT activity revealed that cipro enhanced IL-2 gene induction. In addition, EL-4 cells incubated with ciprofloxacin showed an early peak and more activated nuclear factor of activated T cells (NFAT-1) as compared to control cells without antibiotics. Cipro did not affect the nuclear transcription factors AP-1 or NFIL-2A. Taken together, cipro inhibited IFN-gamma synthesis, but enhanced IL-2 production in murine lymphocytes by means of influencing NFAT-1 and causing an increased IL-2 transcription. 


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Interleukin-12 expression in B cells by transformation with Epstein-Barr virus. 
Although interleukin (IL)-12 was originally purified from an Epstein-Barr (EBV)-transformed B cell line and the high correlation of EBV infection and IL-12 expression has been suggested, no study has reported whether EBV infection is directly linked to IL-12 expression. To address this issue, we have investigated IL-12 expression in B cells during in vitro transformation with EBV. Human peripheral B cells became capable of constitutively producing p40 by in vitro transformation with EBV, coincident with the expression of latent membrane protein 1 (LMP1) of EBV. These B cells expressed p40 and p35 mRNA, and phorbol myristate acetate (PMA) stimulation strongly enhanced p40 and p70 production. Furthermore, transfection with LMP1 expression vector into a human B lymphoma cell line, Daudi, led to p40 production with nuclear factor (NF)-kappaB activation. These results suggest that transformation of primary B cells with EBV induces IL-12 expression potentially through LMP1 expression. Copyright 1998 Academic Press. 


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Downstream activation of a TATA-less promoter by Oct-2, Bob1, and NF-kappaB directs expression of the homing receptor BLR1 to mature B cells. 
The chemokine receptor, BLR1, is a major regulator of the microenvironmental homing of B cells in lymphoid organs. In vitro studies identify three essential elements of the TATA-less blr1 core promoter that confer cell type- and differentiation-specific expression in the B cells of both humans and mice, a functional promoter region (-36 with respect to the transcription start site), a NF-kappaB motif (+44), and a noncanonical octamer motif (+157). The importance of these sites was confirmed by in vivo studies in gene-targeted mice deficient of either Oct-2, Bob1, or both NF-kappaB subunits p50 and p52. In all of these animals, the expression of BLR1 was reduced or absent. In mice deficient only of p52/NF-kappaB, BLR1 expression was unaffected. Thus our data demonstrate that BLR1 is a target gene for Oct-2, Bob1, and members of the NF-kappaB/Rel family and provides a link to the impaired B cell functions in mice deficient for these factors. 


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Defective transcription of the IL-2 gene is associated with impaired expression of c-Fos, FosB, and JunB in anergic T helper 1 cells. 
Anergic CD4+ Th cells do not produce IL-2 when challenged with Ag-pulsed accessory cells because of a transcriptional defect. In this work, we report that these anergic T cells are defective in their ability to up-regulate protein binding and transactivation at two critical IL-2 DNA enhancer elements: NF-AT (nuclear factor of activated T cells; a sequence that binds a heterotrimeric NFATp, Fos, and Jun protein complex) and Activator Protein-1 (AP-1) (that binds Fos and Jun heterodimers). Western blot analysis of nuclear extracts showed that the impaired DNA-protein interactions in anergic T cells were associated with poor expression of the inducible AP-1 family members c-Fos, FosB, and JunB. However, the reduced expression of these proteins was not the result of a global TCR/CD3-signaling defect because CD3 cross-linking induced an equivalent increase in intracellular-free calcium ions, as well as NFATp dephosphorylation, translocation to the nucleus, and DNA binding in both normal and anergic T cells. Thus, defective IL-2 gene transcription appears to be due, at least in part, to a selective block in the expression of the AP-1 Fos and Jun family members in anergic T cells. 


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Structure and expression of the human GATA3 gene. 
GATA3, a member of the GATA family that is abundantly expressed in the T-lymphocyte lineage, is thought to participate in T-cell receptor gene activation through binding to enhancers. To understand GATA3 gene regulation, we cloned the human gene and the 5' end of the mouse GATA3 gene. We show that the human GATA3 gene contains six exons distributed over 17 kb of DNA. The two human GATA3 zinc fingers are encoded by two separate exons highly conserved with those of GATA1, but no other structural homologies between these two genes can be found. The human and mouse GATA3 transcription units start at a major initiation site. The promoter sequence analysis of these two genes revealed that they are embedded within a CpG island and share structural features often found in the promoters of housekeeping genes. Finally, we show that a DNA fragment containing the human GATA3 transcription unit, 3 kb upstream from the initiation site and 4 kb downstream from the polyadenylation site, displays T-cell specificity. 


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Agonistic activity of a CD40-specific single-chain Fv constructed from the variable regions of mAb G28-5. 
A single-chain Fv (sFv) was expressed from the variable regions of the CD40-specific mAb G28-5. The molecule bound CD40 with a high affinity (2.2 nM) and was a monomer in solution. Surprisingly, G28-5 sFv was a potent CD40 agonist that rapidly crosslinked CD40 on the cell surface but did not crosslink CD40-Ig in solution. G28-5 sFv was a more potent agonist than G28-5 IgG and was able to stimulate CD40 responses by B cells and monocytes. G28-5 IgG partially blocked, whereas G28-5 sFv augmented CD40 responses during stimulation with natural ligand (gp39-CD8 fusion protein). These results indicate that the functional activity of ligands built from the binding site of G28-5 is highly dependent upon the size and physical properties of the molecule both in solution and on the cell surfaces. 


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Stimulation of a human T-cell clone with anti-CD3 or tumor necrosis factor induces NF-kappa B translocation but not human immunodeficiency virus 1 enhancer-dependent transcription. 
The expression of transiently transfected expression vectors under the control of the long terminal repeat (LTR) of the human immunodeficiency virus (HIV) or its enhancer sequence and the translocation of the HIV enhancer-binding protein NF-kappa B were analyzed in two human T-cell clones stimulated through their T-cell receptor complex or by tumor necrosis factor or phorbol 12-myristate 13-acetate. We found a dissociation of NF-kappa B translocation from transactivation of either the HIV LTR or the HIV enhancer. Interleukin 2 induced proliferation but not NF-kappa B translocation or LTR transactivation. Phorbol ester or specific antigen recognition induced HIV LTR transactivation, whereas stimulation with tumor necrosis factor or antibody to CD3 did not. The two latter signals were nevertheless able to induce NF-kappa B translocation with a pattern in the band-shift assay indistinguishable from that observed using phorbol ester. Our finding that induction of NF-kappa B by tumor necrosis factor or antibody to CD3 is not sufficient to induce HIV enhancer-dependent transcription in cloned T cells contrasts with results obtained in most lymphoblastoid T-cell lines and indicates that normal T lymphocytes differ from tumoral T cells in terms of requirements for HIV LTR activation. Furthermore, our results suggest that events linked to T-cell activation, in addition to NF-kappa B translocation per se, induce functional interactions of the NF-kappa B complex with the HIV enhancer. 


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Human T-cell leukemia virus type 1 tax protein abrogates interleukin-2 dependence in a mouse T-cell line. 
Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia. Tax, the viral protein, is thought to be crucial in the development of the disease, since it transforms healthy T cells in vitro and induces tumors in transgenic animals. We examined the effect of Tax activity on the growth of the interleukin-2 (IL-2)-dependent T-cell line CTLL-2. Stable expression of Tax in CTLL-2 transformed cell growth from being IL-2 dependent to IL-2 independent. Tax stimulated transcription through NF-kappaB and the cyclic AMP-responsive element-like sequence in the HTLV-1 promoter. The finding of Tax mutants segregating these two pathways suggested that the NF-kappaB pathway was essential for IL-2-independent growth of CTLL-2 cells while the CRE pathway was unnecessary. However, both pathways were necessary for another transformation-related activity (colony formation in soft agar) of CTLL-2/Tax. Our results show that Tax has at least two distinct activities on T cells, and suggest that Tax plays a crucial role in IL-2-independent T-cell transformation induced by HTLV-1, in addition to its well-known IL-2-dependent cell transformation. 


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Lipopolysaccharide-induced E-selectin expression requires continuous presence of LPS and is inhibited by bactericidal/permeability-increasing protein. 
Endothelial cells stimulated by LPS express E-selectin, which plays an important role in mediating neutrophil adhesion during inflammation. E-selectin is induced within 1-2 h, peaks at 4-6 h, and gradually returns to basal level by 24 h. rBPI21, a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), inhibited LPS-induced E-selectin expression when added at the same time as, and up to 6 h after, LPS. Delayed administration of rBPI21 also affected LPS-mediated activation of the nuclear factor, NF-kappa B. Two to 4 h following LPS addition to endothelial cells, when NF-kappa B was already activated, addition of rBPI21 resulted in marked reduction of NF-kappa B detectable at 4 or 6 h. These results indicate that endothelial activation requires continuous presence of LPS, and rBPI21 acts to reverse LPS-mediated endothelial activation by interrupting the on-going LPS signal. 


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Soluble tumor necrosis factor receptors inhibit phorbol myristate acetate and cytokine-induced HIV-1 expression chronically infected U1 cells. 
Recombinant human tumor necrosis factor (TNF) binding protein-1 (r-h TBP-1) and recombinant human soluble dimeric TNF receptor (rhu TNFR:Fc) were used to determine the relative contributions of TNF to phorbol myristate acetate (PMA) and cytokine-induced human immunodeficiency virus type 1 (HIV-1) replication in chronically infected cell lines. Treatment of HIV-1-infected promonocytic U1 cells with r-h-TBP-1 or rhu TNFR:Fc reduced PMA-induced HIV-1 p24 antigen production in a concentration-dependent manner, with a maximal inhibition of approximately 90%. Maximal inhibition of p24 antigen production in T-lymphocytic ACH-2 cells was 47% with r-hTBP-1 and 42% with rhu TNFR:Fc. r-hTBP-1 and rhu TNFR:Fc also decreased p24 antigen synthesized by U1 cells in response to other stimuli, including phytohemagglutinin (PHA)-induced supernatant, granulocyte-macrophage colony-stimulating factor, interleukin-6, and TNF. Addition of r-hTBP-1 to U1 cells during the last 4 h of a 24 h incubation with PMA still inhibited p24 antigen production by 15%. U1 cells stimulated with 10(-7) M PMA released approximately 1 ng/ml endogenous TBP-1 with an initial peak observed at 1 h and a second peak at 24 h after PMA stimulation. r-hTBP-1 also partially reversed inhibition of U1 cellular proliferation caused by PMA. Both r-hTBP-1 and rhu TNFR:Fc blocked PMA induction of nuclear factor (NK)- kappa B DNA-binding activity in U1 cells in association with decreases in HIV-1 replication. We conclude that soluble TNF receptors can inhibit stimuli-induced HIV-1 expression and NK- kappa B DNA-binding activity in chronically infected U1 cells. 


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Transcriptional basis for hyporesponsiveness of the human inducible nitric oxide synthase gene to lipopolysaccharide/interferon-gamma. 
The work reported here resolves, at the level of gene regulation, the controversy as to whether or not human monocytes/macrophages can produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS), with or without co-stimulation by interferon-gamma (IFN-gamma). Studies included structural comparison of the promoters for human and mouse inducible NO synthase (iNOS) genes, transfection and assay of human and mouse iNOS promoter regions in response to LPS +/- IFN-gamma, and electrophoretic mobility shift assays of kappa B response elements. Two explanations for hyporesponsiveness of the human iNOS promoter to LPS +/- IFN-gamma were found: (1) multiple inactivating nucleotide substitutions in the human counterpart of the enhancer element that has been shown to regulate LPS/IFN-gamma induced expression of the mouse iNOS gene; and (2) and absence of one or more nuclear factors in human macrophages (e.g., an LPS-inducible nuclear factor-kappa B/Rel complex), that is (are) required for maximal expression of the gene. The importance of resolution of this controversy is that future research in this area should be directed toward the understanding of alternative mechanisms that can result in the successful production of NO. 


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SRC-related proto-oncogenes and transcription factors in primary human T cells: modulation by cyclosporin A and FK506. 
Activation of T lymphocytes induces transcription of genes encoding for lymphokines. Interleukin-2 (IL-2) gene expression is controlled transcriptionally by the cooperative activity of specific trans-activating factors that bind to the IL-2 enhancer. Cyclosporin A (CsA) and FK506 inhibit the production of IL-2 in T lymphocytes at the level of gene transcription. A member of the src gene family, the lymphocyte-specific protein tyrosine kinase, p56lck, has been implicated in IL-2 production. CsA was found not to inhibit lck gene expression, nor the activity of the lck gene product. However, CsA and FK506 inhibit the appearance of DNA binding activity of factors that bind to the NF-AT and AP-1 sites in the IL-2 enhancer. Since the induction of NF-AT and AP-1 is induced by the same stimuli that stimulate IL-2 production, these results indicate that the immunosuppressant action of CsA and FK506 is exerted at the level of these trans-activating factors. 


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Transcriptional regulation during T-cell development: the alpha TCR gene as a molecular model. 
The regulation of gene expression during lymphocyte differentiation is a complex process involving interactions between multiple positive and negative transcriptional regulatory elements. In this article, transcriptional regulation of the archetypal T-cell-specific gene, alpha TCR, is discussed. Major recent developments, including the identification of novel families of transcription factors that regulate multiple T-cell genes during thymocyte ontogeny and T-cell activation, are described. 


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Regulation of GM-CSF gene transcription by core-binding factor. 
GM-CSF gene activation in T cells is known to involve the transcription factors nuclear factor-kappa B, AP-1, NFAT, and Sp1. Here we demonstrate that the human GM-CSF promoter and enhancer also encompass binding sites for core-binding factor (CBF). Significantly, the CBF sites are in each case contained within the minimum essential core regions required for inducible activation of transcription. Furthermore, these core regions of the enhancer and promoter each encompass closely linked binding sites for CBF, AP-1, and NFATp. The GM-CSF promoter CBF site TGTGGTCA is located 51 bp upstream of the transcription start site and also overlaps a YY-1 binding site. A 2-bp mutation within the CBF site resulted in a 2-3-fold decrease in the activities of both a 69-bp proximal promoter fragment and a 627-bp full-length promoter fragment. Stepwise deletions into the proximal promoter also revealed that the CBF site, but not the YY-1 site, was required for efficient induction of transcriptional activation. The AML1 and CBF beta genes that encode CBF each have the ability to influence cell growth and differentiation and have been implicated as proto-oncogenes in acute myeloid leukemia. This study adds GM-CSF to a growing list of cytokines and receptors that are regulated by CBF and which control the growth, differentiation, and activation of hemopoietic cells. The GM-CSF locus may represent one of several target genes that are dysregulated in acute myeloid leukemia. 


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HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state. 
This study demonstrates that human immunodeficiency virus type 1 (HIV-1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HIV-1 replication through activation of NF-kappa B. In HeLa cells stably transfected with the HIV-1 tat gene (HeLa-tat cells), expression of the Tat protein enhanced both TNF-induced activation of NF-kappa B and TNF-mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein. TNF-mediated activation of NF-kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat-mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV-1 Tat suppressed the expression of Mn-dependent superoxide dismutase (Mn-SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn-SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn-SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat1-72), known to transactivate the HIV-1 long terminal repeat (LTR), no longer affected Mn-SOD expression, the cellular redox state or TNF-mediated cytotoxicity. Thus, our experiments demonstrate that the C-terminal region of HIV-1 Tat is required to suppress Mn-SOD expression and to induce pro-oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio. (ABSTRACT TRUNCATED AT 250 WORDS) 


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E2F-1 and a cyclin-like DNA repair enzyme, uracil-DNA glycosylase, provide evidence for an autoregulatory mechanism for transcription. 
The cell cycle-dependent transcription factor, E2F-1, regulates the cyclin-like species of the DNA repair enzyme uracil-DNA glycosylase (UDG) gene in human osteosarcoma (Saos-2) cells. We demonstrate, through the deletion of the human UDG promoter sequences, that expression of E2F-1 activates the UDG promoter through several E2F sites. The major putative downstream site for E2F, located in the first exon, serves as a target for E2F-1/DP1 complex binding in vitro. We also provide evidence for the functional relationship between the cyclin-like UDG gene product and E2F. High levels of UDG expression in a transient transfection assay result in the down-regulation of transcriptional activity through elements specific for E2F-mediated transcription. Overexpression of UDG in Saos 2 cells was observed to delay growth late in G1 phase and transiently arrest these cells from progressing into the S phase. This hypothetical model integrates one mechanism of DNA repair with the cell cycle control of gene transcription, likely through E2F. This implicates E2F as a multifunctional target for proteins and enzymes, possibly, responsive to DNA damage through the negative effect of UDG on E2F-mediated transcriptional activity. 


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A novel T-cell trans-activator that recognizes a phorbol ester-inducible element of the interleukin-2 promoter. 
The interleukin 2 (IL-2) gene promoter is recognized by several cell-type-specific and ubiquitous transcriptional regulators that integrate information transmitted by various signaling systems leading to IL-2 production and T-cell activation. Using a combination of transfection, protein-DNA binding, and in vitro transcription methods, we have discovered the novel T-cell-specific transcriptional activator TCF-1 (for T-Cell Factor-1), which recognizes a T-cell-specific response element (TCE) located within the IL-2 promoter. Although the TCE is similar in sequence to a consensus NF kappa B site, several criteria indicate that TCF-1 is distinct from NF kappa B. However, like NF kappa B, TCF-1 activity is induced by phorbol esters and other T-cell activators. 


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Isolation of cDNA clones for 42 different Kruppel-related zinc finger proteins expressed in the human monoblast cell line U-937. 
To study the complexity and structural characteristics of zinc finger proteins expressed during human hematopoiesis and to isolate novel regulators of blood cell development, a degenerate oligonucleotide probe specific for a consensus zinc finger peptide domain was used to isolate 63 cDNA clones for Kruppel-related zinc finger genes from the human monoblast cell line U-937. By extensive nucleotide sequence and Northern blot analysis, these cDNA clones were found to originate from approximately 42 different genes (HZF 1-42) of which only 8 have previously been described. Northern blot analysis showed that a majority of these genes were expressed at comparable levels in U-937 and HeLa cells. The large number of individual genes represented among the 63 clones and their apparent non-cell-type-specific expression suggest that the majority of the Kruppel-related zinc finger genes are likely to be expressed in most human tissues. In contrast, some of the genes displayed a restricted expression pattern, indicating that they represent potential regulators of monocyte differentiation or proliferation. Detailed structural analysis of the first 12 cDNAs (HZF 1-10) and a partial characterization of HZF 11-42 revealed that a common feature of human Kruppel-related zinc finger proteins is the presence of tandem arrays of zinc fingers ranging in number from 3 to over 20 that are preferentially located in the carboxy-terminal regions of the proteins. In addition, several novel KRAB-containing zinc finger genes and a novel conserved sequence element were identified. 


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Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell types. 
Starting with a replication-incompetent molecular clone of human immunodeficiency virus type 1, lacking all the NF-kappa B and Sp1 binding sites present in the native long terminal repeat (LTR), proviruses containing reconstructed LTRs with individual or combinations of NF-kappa B and Sp1 elements were generated and evaluated for their capacity to produce virus progeny following transfection-cocultivation. Virus stocks obtained from these experiments exhibited a continuum of replicative capacities in different human T-cell types depending on which element(s) was present in the LTR. For example, in experiments involving proviral clones with LTRs containing one or two NF-kappa B elements (and no Sp1 binding sites), a hierarchy of cellular permissivity to virus replication (peripheral blood lymphocytes = MT4 greater than H9 greater than CEM greater than Jurkat) was observed. Of note was the associated emergence of second-site LTR revertants which involved an alteration of the TATA box. These results suggest that the human immunodeficiency virus type 1 LTR possesses functional redundancy which ensures virus replication in different T-cell types and is capable of changing depending on the particular combination of transcriptional factors present. 


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Oxidant-regulation of gene expression in the chronically inflamed intestine. 
It is becoming increasingly apparent that the chronic gut inflammation observed in the idiopathic inflammatory bowel diseases (e.g. ulcerative colitis, Crohn's disease) is associated with enhanced production of leukocyte-derived oxidants. Oxidants such as hydrogen peroxide are known to activate certain transcription factors such as nuclear transcription factor kappa beta. Nuclear transcription factor kB (NF-kappa B) is a ubiquitous transcription factor and pleiotropic regulator of numerous genes involved in the immune and inflammatory responses. This transcription factor is activated via the selective phosphorylation, ubiquination and degradation of its inhibitor protein I-kB thereby allowing translocation of NF-kappa B into the nucleus where it upregulates the transcription of a variety of adhesion molecules (e.g. ICAM-1, VCAM-1), cytokines (TNF, IL-1, IL-6) and enzymes (iNOS). The proteolytic degradation of the post-translationally modified I-kappa B is known to be mediated by the 26S proteasome complex. Based upon work from our laboratory, we propose that inhibition of NF-kappa B activation produces significant anti inflammatory activity which may be mediated by the inhibition of transcription of certain pro-inflammatory mediators and adhesion molecules. 


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Coexpression of the interleukin-13 and interleukin-4 genes correlates with their physical linkage in the cytokine gene cluster on human chromosome 5q23-31. 
Interleukin-13 (IL-13) and IL-4 are cytokines produced by T cells that are encoded by the q23-31 region of human chromosome 5. To investigate the regulation of IL-13 gene expression by T cells, we isolated and sequenced the human IL-13 gene, analyzed its 5'-flanking region for potential transcriptional activation elements, and examined its expression in nontransformed T-lineage cell populations. The human IL-13 gene was located 12.5-kb upstream of the IL-4 gene and 2-kb downstream of a CpG island. The IL-13 gene 5' flank region included a segment with sequence homology to P elements of the IL-4 promoter involved in transcriptional activation in T cells. Mutation of the IL-13 P element site significantly reduced IL-13 promoter activity in response to T-cell activation. Oligonucleotides containing the IL-13 or IL-4 P element sites specifically bound the transcriptional activator protein, nuclear factor-activated T cells, preformed (NF-ATp), when incubated with nuclear protein extracts from activated T cells. Similar to IL-4, IL-13 mRNA expression was highest in T-cell populations enriched for cells that had previously been primed in vivo or in vitro, indicating that priming increases the expression of the IL-13 and IL-4 genes in a coordinate manner. Because the primed T cells contain higher levels of nuclear NF-ATp, capable of binding to P elements of the IL-4 and IL-13 promoters, than do freshly-isolated T cells, the NF-AT-binding P elements are attractive candidates to mediate the coordinate expression of these two cytokine genes. 


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Gene transcription through activation of G-protein-coupled chemoattractant receptors. 
Receptors for leukocyte chemoattractants, including chemokines, are traditionally considered to be responsible for the activation of special leukocyte functions such as chemotaxis, degranulation, and the release of superoxide anions. Recently, these G-protein-coupled serpentine receptors have been found to transduce signals leading to gene transcription and translation in leukocytes. Transcription factors, such as NF kappa B and AP-1, are activated upon stimulation of the cells with several chemoattractants at physiologically relevant concentrations. Activation of transcription factors through these receptors involves G-protein coupling and the activation of protein kinases. The underlying signaling pathways appear to be different from those utilized by TNF-alpha, a better characterized cytokine that induces the transcription of immediate-early genes. Chemoattractants stimulate the expression of several inflammatory cytokines and chemokines, which in turn may activate their respective receptors and initiate an autocrine regulatory mechanism for persistent cytokine and chemokine gene expression. 


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AML and Ets proteins regulate the I alpha1 germ-line promoter. 
The immunoglobulin heavy chain (IgH) class switch recombination of B lymphocytes preferentially targets unrearranged IgH genes that have already been rendered transcriptionally active. Transcription of the germ-line IgH genes is controlled by intervening (I) regions upstream of their switch regions. The I alpha1 promoter activates transcription of the human germ-line C alpha1 gene for IgA1 and mediates the transforming growth factor (TGF)-beta1 responsiveness of this locus. Here we show that the I alpha1 promoter contains several binding sites for the AML/PEBP2/CBF family of transcription factors and that AML and Ets proteins are major regulators of the basal and TGF-beta-inducible promoter activity. Our data constitute a starting point for studies to elucidate the molecular mechanism by which TGF-beta regulates IgA production. 


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CD30 is a CD40-inducible molecule that negatively regulates CD40-mediated immunoglobulin class switching in non-antigen-selected human B cells. 
We used our monoclonal model of germinal center maturation, CL-01 B cells, to investigate the role of CD30 in human B cell differentiation. CL-01 cells are IgM+ IgD+ CD30+ and switch to IgG, IgA, and IgE when exposed to CD40L and IL-4. Switching is hampered by CD30 coengagement, possibly through interference with the CD40-mediated NF-kappaB-dependent transcriptional activation of downstream C(H) genes. The physiological relevance of this phenomenon is emphasized by similar CD30-mediated effects in naive B cells. Expression of CD30 by these cells is induced by CD40L but is inhibited by B cell receptor coengagement and/or exposure to IL-6 and IL-12. Our data suggest that CD30 critically regulates the CD40-mediated differentiation of non-antigen-selected human B cells. 


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Regulation of NF-kappa B activity by I kappa B alpha and I kappa B beta stability. 
Transcription factor NF-kappa B must be released from cytoplasmic inhibitory molecules (I kappa Bs) in order to move to the nucleus and to activate its target genes. Little is known about the mechanisms regulating the maintenance of constitutive nuclear NF-kappa B in some cell-types and of sustained nuclear NF-kappa B activity after stimulation. Increased turnover has been implicated in the regulation of constitutive NF-kappa B activity in mature B cells. We therefore compared the turnover of I kappa B alpha and I kappa B beta in mature B cells and HeLa cells. Both proteins display a high turnover in B cells although I kappa B beta is considerably more stable than I kappa B alpha. The half-life of both inhibitors is increased in HeLa cells. In contrast, all other NF-kappa B/I kappa B molecules tested are relatively stable in both cell-types. The elevated turnover of endogenous I kappa B alpha in Namalwa cells is inhibited by a proteasome inhibitor and thus seems to be driven by the same degradation machinery as the slower turnover in non-B cells. Furthermore, we investigated the processes involved in persistent activation of NF-kappa B. TNF-alpha signaling leads to a rapid depletion of cellular I kappa B beta pools. I kappa B alpha is efficiently resynthesized whereas I kappa B beta levels stay low for a prolonged time. NF-kappa B binding activity can be detected for several hours after stimulation. We found that removal of the TNF-alpha containing medium causes a rapid decrease in nuclear NF-kappa B. A phosphoform of newly synthesized I kappa B alpha is visible when degradation by the proteasome is inhibited and new I kappa B alpha displays the same properties regarding phosphorylation and degradation in response to a second inducer. There is no significant difference in the turnover of pre- and post-inductive I kappa B alpha. These observations suggest that resynthesis of I kappa B alpha and removal of the stimulus are obligatory steps for the inactivation of nuclear NF kappa B. 


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HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor kappa B [see comments] 
The HIV-1 accessory gene product Vpr can influence viral pathogenesis by affecting viral replication as well as host cell transcription and proliferation. We have investigated the effects of Vpr on host cell activation and confirm that it influences cellular proliferation. However, we have also found that Vpr modulates T-cell receptor (TCR)-triggered apoptosis in a manner similar to that of glucocorticoids. In the absence of TCR-mediated activation, Vpr induces apoptosis whereas in its presence, Vpr interrupts the expected induction of apoptosis. This regulation of apoptosis is linked to Vpr suppression of NF-kappa B activity via the induction of I kappa B, an inhibitor of NF-kappa B. Further, Vpr suppresses expression of IL-2, IL-10, IL-12, TNF alpha and IL-4, all of which are NF-kappa B-dependent. The effects of Vpr could be reversed by RU486. Our finding that Vpr can regulate NF-kappa B supports the hypothesis that some aspects of viral pathogenesis are the consequence of cell dysregulation by Vpr. 


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Differential interaction of nuclear factors with the leukocyte-specific pp52 promoter in B and T cells. 
The leukocyte-specific, cytoskeleton-binding pp52 (LSP-1, WP-34) protein is widely expressed in multiple leukocyte lineages, including B and T lymphocytes, granulocytes, and macrophages. We previously detected a tissue-specific promoter preceding the exon encoding the N terminus of the pp52 leukocyte protein. Here we describe the functional characterization of this promoter and identification of the factors in B and T cells that regulate its activity. The pp52 promoter contains an initiator specifying the unique 5' terminus of pp52 mRNA, tandem pairs of Ets and SP1 motifs, and a lone C/EBP motif. All these motifs are essential and collectively control transcriptional activity. DNA binding studies and Ab supershift assays revealed that different combinations of factors interact with these motifs in B cells vs T cells. The Ets motifs are preferentially bound by PU-1 in B cell extracts from all stages of development, whereas a different Ets family member reacts with these motifs in T cell extracts. The C/EBP motif is bound by Ig/EBP-1 in pre-B cell and T cell extracts, but is replaced by nuclear factor-IL-6beta or a nuclear factor-IL-6beta-Ig/EBP-1 heterodimer in plasmacytoma cell extracts. Despite its reported role as a negative regulator of transcription, Ig/EBP-1 appears to exert a stimulatory effect on this promoter. These findings reveal the features controlling the pp52 promoter in B and T cells and provide the foundation for determining the regulation of this promoter in other leukocyte lineages. 


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Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B [published erratum appears in Science 1991 Oct 4;254(5028):11] 
A DNA probe that spanned a domain conserved among the proto-oncogene c-rel, the Drosophila morphogen dorsal, and the p50 DNA binding subunit of NF-kappa B was generated from Jurkat T cell complementary DNA with the polymerase chain reaction (PCR) and degenerate oligonucleotides. This probe was used to identify a rel-related complementary DNA that hybridized to a 2.6-kilobase messenger RNA present in human T and B lymphocytes. In vitro transcription and translation of the complementary DNA resulted in the synthesis of a protein with an apparent molecular size of 65 kilodaltons (kD). The translated protein showed weak DNA binding with a specificity for the kappa B binding motif. This protein-DNA complex comigrated with the complex obtained with the purified human p65 NF-kappa B subunit and binding was inhibited by I kappa B-alpha and -beta proteins. In addition, the 65-kD protein associated with the p50 subunit of NF-kappa B and the kappa B probe to form a complex with the same electrophoretic mobility as the NF-kappa B-DNA complex. Therefore the rel-related 65-kD protein may represent the p65 subunit of the active NF-kappa B transcription factor complex. 


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Inhibition of phorbol ester-induced monocytic differentiation by dexamethasone is associated with down-regulation of c-fos and c-jun (AP-1). 
Previous studies have shown that treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with induction of monocytic differentiation and expression of the c-jun and c-fos early response genes. The present work demonstrates that the glucocorticoid dexamethasone inhibits TPA-induced increases in c-jun and c-fos mRNA levels in U-937 leukemia cells. These findings were associated with a block in appearance of the monocytic phenotype, including inhibition of TPA-induced increases in lamin A, lamin C, and vimentin transcripts. Other studies have demonstrated that TPA-induced monocytic differentiation and expression of the c-jun and c-fos genes in myeloid leukemia cells are regulated by protein kinase C (PKC). The finding that dexamethasone has no effect on TPA-induced activation of PKC suggests that this glucocorticoid inhibits signals downstream or parallel to this enzyme. Nuclear run-on assays demonstrate that: (1) induction of c-jun and c-fos expression by TPA is regulated by transcriptional mechanisms, (2) TPA-induced expression of c-jun and c-fos does not require protein synthesis, and (3) TPA-induced expression of both genes is inhibited at the transcriptional level by dexamethasone. To further define the effects of dexamethasone at the molecular level, we prepared a series of deleted c-jun promoter fragments linked to the chloramphenicol acetyltransferase (CAT) gene. Increases in CAT activity during transient expression of these constructs in TPA-treated U-937 cells could be assigned to the region (-97 to -20) of the promoter that contains the AP-1 binding site. This induction of CAT activity was sensitive to dexamethasone. These findings suggest that dexamethasone down-regulates TPA-induced transcription of the c-jun gene during monocytic differentiation by inhibiting activation of the AP-1 site. 


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Induction of interleukin-12 p40 transcript by CD40 ligation via activation of nuclear factor-kappaB. 
Interleukin-12 is produced in response to infection with bacteria or parasites or to bacterial constituents such as LPS in monocytes/macrophages and dendritic cells, and also generated by the interaction between activated T cells and antigen-presenting cells via CD40-CD40 ligand (CD40L). So far, transcriptional analyses of p40 have been carried out only using bacterial constituents such as LPS as stimuli. In the present study, we have characterized the transcriptional induction of p40 by CD40 ligation in a human B lymphoblastoid cell line, Daudi, and a human acute monocytic leukemia cell line, THP-1. These cells, stimulated by an agonistic monoclonal antibody against CD40 or by transfection with a CD40L expression vector, secreted p40 and showed enhanced p40 mRNA expression. Sequence analysis of the p40 promoter region identified two potential nuclear factor (NF)-kappaB binding sites conserved between mouse and human. Electrophoretic mobility shift assay revealed that the potential NF-kappaB binding sequence which is located around 120 bp upstream of the transcription initiation site in murine and human p40 genes formed an NF-kappaB complex with nuclear extract from Daudi cells stimulated by CD40 ligation. Moreover, transfection of Daudi cells with the polymerized NF-kappaB binding sequence ligated to a thymidine kinase/chloramphenicol acetyltransferase (CAT) reporter plasmid greatly induced CAT activity, but transfection with the polymerized mutated NF-kappaB binding sequence did not. These results suggest that the NF-kappaB binding site located around 120 bp upstream of the transcription initiation site in murine and human p40 promoter regions could be important for the p40 induction by CD40 ligation via activation of NF-kappaB. 


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Surfactant protein A activates NF-kappa B in the THP-1 monocytic cell line. 
The expression of many genes for which products are involved in inflammation is controlled by the transcriptional regulator nuclear factor (NF)-kappa B. Because surfactant protein (SP) A is involved in local host defense in the lung and alters immune cell function by modulating the expression of proinflammatory cytokines as well as surface proteins involved in inflammation, we hypothesized that SP-A exerts its action, at least in part, via activation of NF-kappa B. We used gel shift assays to determine whether SP-A activated NF-kappa B in the THP-1 cell line, a human monocytic cell line. Activation of NF-kappa B in THP-1 cells by SP-A doses as low as 1 microgram/ml occurred within 30 min of SP-A treatment, peaked at 60 min, and then declined. This activation is inhibited by known inhibitors of NF-kappa B or by simultaneous treatment of the cells with surfactant lipids. Moreover, the NF-kappa B inhibitors blocked SP-A-dependent increases in tumor necrosis factor-alpha mRNA levels. These observations suggest a mechanism by which SP-A plays a role in the pathogenesis of some lung conditions and point to potential therapeutic measures that could be used to prevent SP-A induced inflammation in the lung. 


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Inactivation of IkappaBbeta by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-kappaB. 
In resting T lymphocytes, the transcription factor NF-kappaB is sequestered in the cytoplasm via interactions with members of the I kappa B family of inhibitors, including IkappaBalpha and IkappaBbeta. During normal T-cell activation, IkappaBalpha is rapidly phosphorylated, ubiquitinated, and degraded by the 26S proteasome, thus permitting the release of functional NF-kappaB. In contrast to its transient pattern of nuclear induction during an immune response, NF-kappaB is constitutively activated in cells expressing the Tax transforming protein of human T-cell leukemia virus type I (HTLV-1). Recent studies indicate that HTLV-1 Tax targets IkappaBalpha to the ubiquitin-proteasome pathway. However, it remains unclear how this viral protein induces a persistent rather than transient NF-kappaB response. In this report, we provide evidence that in addition to acting on IkappaBalpha, Tax stimulates the turnover Of IkappaBbeta via a related targeting mechanism. Like IkappaBalpha, Tax-mediated breakdown of IkappaBbeta in transfected T lymphocytes is blocked either by cell-permeable proteasome inhibitors or by mutation Of IkappaBbeta at two serine residues present within its N-terminal region. Despite the dual specificity of HTLV-1 Tax for IkappaBalpha and IkappaBbeta at the protein level, Tax selectively stimulates NF-kappaB-directed transcription of the IkappaBalpha gene. Consequently, IkappaBbeta protein expression is chronically downregulated in HTLV-1-infected T lymphocytes. These findings with IkappaBbeta provide a potential mechanism for the constitutive activation of NF-kappaB in Tax-expressing cells. 


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The role of nuclear factor-kappa B in cytokine gene regulation. 
Transcription factors are DNA-binding proteins that regulate gene expression. Nuclear factor-kappa B (NF-kappa B) is a critical transcription factor for maximal expression of many cytokines that are involved in the pathogenesis of inflammatory diseases, such as adult respiratory distress syndrome (ARDS) and sepsis syndrome. Activation and regulation of NF-kappa B are tightly controlled by a group of inhibitory proteins (I kappa B) that sequester NF-kappa B in the cytoplasm of immune/inflammatory effector cells. NF-kappa B activation involves signaled phosphorylation, ubiquitination, and proteolysis of I kappa B. Liberated NF-kappa B migrates to the nucleus, where it binds to specific promoter sites and activates gene transcription. The activation of NF-kappa B initiates both extracellular and intracellular regulatory events that result in autoregulation of the inflammatory cascade through modulation of NF-kappa B activation. Recently, activation of NF-kappa B has been linked to ARDS and has been shown to be a critical proximal step in the initiation of neutrophilic inflammation in animal models. Activation of NF-kappa B can be inhibited in vivo by treatment with antioxidants, corticosteroids, and the induction of endotoxin tolerance. Identification of more specific and efficacious inhibitors of NF-kappa B activation might prove beneficial for the treatment of cytokine-mediated inflammatory diseases. 


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Superantigens activate HIV-1 gene expression in monocytic cells. 
Binding of superantigens to MHC class II molecules results in transduction of biochemical signals leading to cellular activation and gene expression. We demonstrate that the staphylococcal superantigens toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA) activate HIV-1-LTR-driven transcription of chloramphenicol acetyl transferase in the human monocytic cell line THP-1. Induction of HIV-1-LTR-driven transcription in THP-1 cells by superantigens was associated with the induction of nuclear factor-kappa B DNA-binding activity. Superantigens also increased viral protein secretion from the granulocyte-macrophage colony-stimulating factor-pretreated chronically infected human monocytic cell line U1. Induction of HIV-1 gene expression in monocytic cells by superantigens occurred via tumor necrosis factor-alpha-dependent and -independent mechanisms. Our results suggest that superantigens and other MHC class II ligands may activate HIV-1 gene expression in monocytes/macrophages. 


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Glucocorticoid-induced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor. 
Induction of apoptosis in lymphocytes, which may account for the therapeutic effects of glucocorticoids in various diseases including leukemia, depends on the glucocorticoid receptor. However, the events leading from the activated receptor to cell lysis are not understood. A prevailing hypothesis postulates induction of so-called 'lysis genes' by the activated receptor. In this study, we show that an activation-deficient glucocorticoid receptor mutant is as effective as the wild-type receptor in repression of AP-1 activity, inhibition of interleukin-2 production, inhibition of c-myc expression and induction of apoptosis. Furthermore, we show that retinoic acid can also induce apoptosis in these cells through the retinoic acid receptor, whose repressive functions but not target site specificity, are similar to those of the glucocorticoid receptor. Therefore, the primary effect of the receptor in glucocorticoid-mediated apoptosis correlates with transcriptional repression rather than activation and could be mediated by interference with other transcription factors required for cell survival. 


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OBF-1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins. 
Recent biochemical and genetic studies indicate that in addition to the octamer-binding proteins Oct-1 and Oct-2, other B cell components are required for lymphoid-restricted, octamer site-mediated immunoglobulin gene promoter activity. Using a genetic screen in yeast, we have isolated B cell-derived cDNAs encoding Oct-binding factor 1 (OBF-1), a novel protein that specifically associates with Oct-1 and Oct-2. Biochemical studies demonstrate that OBF-1 has no intrinsic DNA-binding activity and recognizes the POU domains of Oct-1 and Oct-2, but not those of Oct-4 and Oct-6. The OBF-1 mRNA is expressed in a highly cell-specific manner, being most abundant in B cells and essentially absent in most of the other cells or tissues tested. Furthermore, expression of OBF-1 in HeLa cells selectively stimulates the activity of a natural immunoglobulin promoter in an octamer site-dependent manner. Thus, OBF-1 has all the properties expected for a B cell-specific transcriptional coactivator protein. 


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T cells from renal cell carcinoma patients exhibit an abnormal pattern of kappa B-specific DNA-binding activity: a preliminary report. 
Recent data suggest that the poor induction of a T-cell response to human renal cell carcinoma (RCC) may be related to alterations in signal transduction pathways. We report that T cells from RCC patients have two alterations in kappa B motif-specific DNA-binding activity. The first alteration involves the constitutive expression of substantial kappa B-binding activity in nuclear extracts, which was observed in the electrophoretic mobility shift assay. The magnitude of kappa B activity in unstimulated patient T cells was similar to that observed in T cells from normal individuals that had been activated in vitro. On the basis of Western blotting experiments using antibodies to kappa B/Rel family proteins, the kappa B-binding activity constitutively expressed in T cells from RCC patients is composed mostly of the NF-kappa B1 (p50) subunit. The second abnormality in kappa B-binding activity in T cells from these patients is that RelA, a member of the Rel homology family which is part of the normal NF-kappa B complex, was not induced in the nucleus following activation. Western blotting analysis did not detect any RelA in nuclear extracts either before or after stimulation of T cells. The altered kappa B-binding activity in T cells from RCC patients may impair their capacity to respond normally to various stimuli. 


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Resistance to tumor necrosis factor induced apoptosis in vitro correlates with high metastatic capacity of cells in vivo. 
TNF is one of the cytokines secreted by the cells of the immune system. Our data demonstrate that those cell lines lacking capability to form metastatic tumors in vivo are susceptible to TNF induced apoptosis in vitro. However, cell lines with high metastatic potential are resistant to TNF in vitro. Furthermore, the same cell lines were resistant to cytolytic action of other cytotoxic proteins secreted by LAK cells. Our data showed that TNF resistance in vitro correlates with the increased level of transcription factor NF-kappaB. This finding may provide a tool to improve current protocols of immunotherapy and insights to how tumor cells are or are not killed by LAK cells. 


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Evidence for a trans-acting activator function regulating the expression of the human CD5 antigen. 
Interspecies somatic cell hybrids were generated by fusing the mouse T-lymphoma cell line, BW5147, with normal human T lymphocytes at different stages of differentiation. Thymocytes, activated peripheral T lymphocytes, or an activated T-cell clone were used as human partners, respectively, in three independent fusions. Irrespective of the human cell partner used for fusion, a certain number of hybrids lost CD5 surface expression over a period of time in culture. Analysis at the phenotype and genetic level showed that lack of CD5 expression was due neither to segregation of human autosome 11, on which the CD5 gene has been mapped, nor to deletion of the CD5 structural gene. Furthermore, loss of CD5 surface expression correlated with the absence of specific mRNA. Since these hybrids preferentially segregate human chromosomes, these results indicate the existence of a non-syntenic trans-active locus, or loci, positively controlling the expression of the human CD5 gene. 


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Suppression by azelastine hydrochloride of NF-kappa B activation involved in generation of cytokines and nitric oxide. 
The influence of the anti-allergy agent azelastine hydrochloride (Azeptin) on NF-kappa B activation associated with the generation of cytokines and nitric oxide (NO) was investigated in various kinds of human and mouse cells. Azeptin dose-dependently suppressed both DNA and protein synthesis in human gingival fibroblasts (HF) and also suppressed blastogenesis of human peripheral blood lymphocytes (PBL). Generation of tumor necrosis factor-alpha, interleukin 1-beta, granulocyte-macrophage colony-stimulating factor and interleukin-6 from 10(-5) M Azeptin-treated PBL and human monocytes (HM) was decreased to approximately 1/3 to 2/3 of the control levels. In parallel with the decreased cytokine generation, each cytokine mRNA was less expressed in the presence of 10(-5) M Azeptin. In addition, both inducible nitric oxide synthase-mRNA level and NO generation in mouse peritoneal macrophages were suppressed by 10(-5) M Azeptin. Being compatible with those results, Azeptin (10(-5) M) suppressed activation of NF-kappa B in PBL, HM and HF. These results appear to indicate that suppression of cytokine and NO generation by Azeptin results at least partially from the inhibition of NF-kappa B activation. 


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Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A [see comments] 
The flow of information from calcium-mobilizing receptors to nuclear factor of activated T cells (NFAT)-dependent genes is critically dependent on interaction between the phosphatase calcineurin and the transcription factor NFAT. A high-affinity calcineurin-binding peptide was selected from combinatorial peptide libraries based on the calcineurin docking motif of NFAT. This peptide potently inhibited NFAT activation and NFAT-dependent expression of endogenous cytokine genes in T cells, without affecting the expression of other cytokines that require calcineurin but not NFAT. Substitution of the optimized peptide sequence into the natural calcineurin docking site increased the calcineurin responsiveness of NFAT. Compounds that interfere selectively with the calcineurin-NFAT interaction without affecting calcineurin phosphatase activity may be useful as therapeutic agents that are less toxic than current drugs. 


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Effects of prostaglandin E2 on Th0-type human T cell clones: modulation of functions of nuclear proteins involved in cytokine production. 
The effects of prostaglandin E2 (PGE2) on cytokine production and proliferation of the CD4+ human helper T cell clone SP-B21 were investigated. In cells stimulated with anti-CD3 mAb, PGE2 inhibited cell proliferation and the production of all the cytokines examined. Addition of rIL-2 fully restored the proliferative response and partially restored the production of IL-4 and IL-5, but not that of other cytokines. In contrast, in cells stimulated with phorbol myristate acetate (PMA)/A23187, PGE2 enhanced the production of IL-4 and IL-5, and only partially inhibited the production of other cytokines. Therefore, the effects of PGE2 vary depending on the mode of T cell activation, and the IL-4 and IL-5 are regulated differently from other cytokines. In a mobility shift assay, only the NF-kappa B (p50/p50) homodimer was observed in a complex formed with the kappa B sequence in unstimulated SP-B21 cells. When cells were stimulated with anti-CD3 mAb or PMA/A23187, a complex formation of NF-kappa B (p50/p65) heterodimer with the kappa B sequence was induced. Interestingly, PGE2 or di-butyryl (Bt2)cAMP abolished the binding of NF-kappa B (p50/p65) heterodimer to the kappa B sequence in cells stimulated with anti-CD3 mAb but not with PMA/A23187. Our results suggest that the target of PGE2 action is a component in the signal transduction pathway leading to the activation of protein kinase C. However, the inhibition of the T cell activation signals by PGE2 is selective. PGE2 enhanced the complex formation with NF-AT, AP-1 and CLE0 sequences when the cells were activated by either anti-CD3 mAb or PMA/A23187 stimulation. It seems therefore that PGE2, by elevating cAMP levels, interferes with the activation pathway for NF-kappa B but not for NF-AT, AP-1 or CLE0 binding protein. 


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Cell-to-cell contact activates the long terminal repeat of human immunodeficiency virus 1 through its kappaB motif. 
Cell-to-cell contact between peripheral blood lymphocytes and transfected human colonic carcinoma cell line HT29 activates transcription of the long terminal repeats (LTR) of human immunodeficiency virus. HIV-1 LTR transcription is controlled by a complex array of virus-encoded and cellular proteins. Using various constructs expressing a lacZ reporter gene under the control of the intact or three deleted forms of HIV-1 LTR, we obtained evidence that the kappaB regulatory elements located in the U3 region are involved in cell-to-cell activation of HIV-1 LTR. Cell-to-cell contact activates in vitro binding of the nuclear factor kappaB (NF-kappaB) p50/p65 heterodimer to an HIV-1 kappaB oligonucleotide. Cell-to-cell contact activation of NF-kappaB was only partially inhibited by 100 microM pyrrolidine dithiocarbamate and was not correlated with a significant decrease of cellular inhibitor kappaB alpha. NF-kappaB nuclear activation was not detectable before 1 h after cell contact and was dependent on protein synthesis. 


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