95369245>IL-2 gene expression and <n>NF-kappa B</n> activation through <n>CD28</n> requires reactive oxygen production by <n>5-lipoxygenase</n>. Activation of the <n>CD28</n> surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of <n>interleukin-2</n> (<n>IL-2</n>) and cell proliferation. In primary T lymphocytes we show that <n>CD28</n> ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for <n>CD28</n>- mediated activation of the <n>NF-kappa B</n>/<n>CD28</n>-responsive complex and <n>IL-2</n> expression. Delineation of the <n>CD28</n> signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of <n>phospholipase A2</n> and <n>5-lipoxygenase</n>. Our data suggest that <n>lipoxygenase</n> metabolites activate ROI formation which then induce <n>IL-2</n> expression via <n>NF-kappa B</n> activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the <n>CD28</n> costimulatory pathway.
95333264>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 <n>NF-kappa B</n> 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 <n>peri-kappa B factor</n> may ultimately provide insight into the different role monocytes and T cells play in HIV pathogenesis.
95343554>E1A gene expression induces susceptibility to killing by NK cells following immortalization but not adenovirus infection of human cells. Adenovirus (Ad) infection and <n>E1A</n> transfection were used to model changes in susceptibility to NK cell killing caused by transient vs stable <n>E1A</n> 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 <n>E1A</n> 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.
95347379>Distinct signaling properties identify functionally different CD4 epitopes. The <n>CD4 coreceptor</n> 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 <n>CD4</n> triggering on T cell activating signals in a lymphoma model using monoclonal antibodies (mAb) which recognize different CD4 epitopes. We demonstrate that <n>CD4</n> triggering delivers signals capable of activating the <n>NF-AT transcription factor</n> which is required for <n>interleukin-2</n> gene expression. Whereas different anti-CD4 mAb or <n>HIV-1 gp120</n> could all trigger activation of the protein tyrosine kinases <n>p56lck</n> and <n>p59fyn</n> and phosphorylation of the <n>Shc adaptor protein</n>, which mediates signals to Ras, they differed significantly in their ability to activate <n>NF-AT</n>. Lack of full activation of <n>NF-AT</n> 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 <n>CD4 coreceptor</n> involved in activation of the <n>Ras</n>/<n>protein kinase C</n> and calcium pathways.
95280913>Ligand-dependent repression of the erythroid transcription factor <n>GATA- 1</n> 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 <n>erythroid cell-specific histone</n> <n>H5</n>. 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 <n>GATA-1</n>, 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 <n>GATA-1</n> 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 <n>GATA-1</n> 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 <n>GATA-1</n>. <n>GATA-1</n> and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, <n>GATA-1</n> and ER associate in a ligand-dependent manner. Mapping experiments indicate that <n>GATA-1</n> and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of <n>GATA-1</n>. We speculate that estrogens exert effects on erythropoiesis by modulating <n>GATA-1</n> activity through protein-protein interaction with the ER. (ABSTRACT TRUNCATED AT 250 WORDS)
95256242>Mouse <n>interleukin-2</n> receptor alpha gene expression. <n>Interleukin-1</n> and <n>interleukin-2</n> control transcription via distinct cis-acting elements. We have shown that <n>interleukin-1</n> (<n>IL-1</n>) and <n>IL-2</n> control <n>IL-2 receptor alpha</n> (<n>IL-2R alpha</n>) gene transcription in CD4-CD8- murine T lymphocyte precursors. Here we map the cis-acting elements that mediate interleukin responsiveness of the <n>mouse IL-2R alpha</n> gene using a thymic lymphoma-derived hybridoma (PC60). The transcriptional response of the IL-2R alpha gene to stimulation by <n>IL-1</n> + <n>IL-2</n> is biphasic. <n>IL-1</n> induces a rapid, protein synthesis-independent appearance of <n>IL-2R alpha</n> mRNA that is blocked by inhibitors of <n>NF-kappa B</n> activation. It also primes cells to become <n>IL-2</n> responsive and thereby prepares the second phase, in which <n>IL-2</n> induces a 100-fold further increase in <n>IL- 2R alpha</n> transcripts. Transient transfection experiments show that several elements in the promoter-proximal region of the <n>IL-2R alpha</n> gene contribute to <n>IL-1</n> responsiveness, most importantly an <n>NF-kappa B</n> site conserved in the human and mouse gene. <n>IL-2</n> responsiveness, on the other hand, depends on a 78-nucleotide segment 1.3 kilobases upstream of the major transcription start site. This segment functions as an IL- 2-inducible enhancer and lies within a region that becomes <n>DNase I</n> hypersensitive in normal T cells in which <n>IL-2R alpha</n> expression has been induced. <n>IL-2</n> responsiveness requires three distinct elements within the enhancer. Two of these are potential binding sites for STAT proteins.
95338146>Hematopoietic lineage commitment: role of transcription factors. This review focuses on the roles of transcription factors in hematopoietic lineage commitment. A brief introduction to lineage commitment and asymmetric cell division is followed by a discussion of several methods used to identify transcription factors important in specifying hematopoietic cell types. Next is presented a discussion of the use of embryonic stem cells in the analysis of hematopoietic gene expression and the use of targeted gene disruption to analyze the role of transcription factors in hematopoiesis. Finally, the status of our current knowledge concerning the roles of transcription factors in the commitment to erythroid, myeloid and lymphoid cell types is summarized.
95266275>Epstein-Barr virus replicative gene transcription during de novo infection of human thymocytes: simultaneous early expression of <n>BZLF-1</n> and its repressor <n>RAZ</n>. 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 <n>BamHI ZLF-1 gene product</n>, <n>ZEBRA</n>, by RT-PCR, was observed within 8 hr of infection. The appearance of a novel fusion transcript (<n>RAZ</n>), which comprised regions of the <n>BZLF-1</n> locus and the adjacent BRLF-1 locus, was detected by RT-PCR. <n>ZEBRA</n> protein was also identified in infected thymocytes by immunoprecipitation. In addition, we demonstrated that the <n>EBNA-1</n> 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 <n>gp350/220</n>, 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.
95236292>Identification and purification of human Stat proteins activated in response to <n>interleukin-2</n>. A key cytokine induced during the immune response is <n>IL-2</n>. Following T cell activation, the genes encoding <n>IL-2</n> and the various chains of its receptor are transcriptionally induced. In turn, secreted <n>IL-2</n> serves to stimulate the proliferation and differentiation of T lymphocytes. Several recent studies have implicated Jak kinases in the signaling pathway induced by <n>IL-2</n>. Following this lead, we set out to identify transcription factors induced in response to <n>IL-2</n>. Human peripheral blood lymphocytes were observed to contain several <n>IL-2</n>-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 <n>IL-2</n>-inducible component bears significant relatedness to a <n>prolactin-induced transcription factor</n> first identified in sheep mammary gland tissue. We hypothesize that activation of this protein, designated <n>hStat5</n>, helps govern the biological effects of <n>IL-2</n> during the immune response.
95197524><n>E2F-1</n> and a cyclin-like DNA repair enzyme, <n>uracil-DNA glycosylase</n>, provide evidence for an autoregulatory mechanism for transcription. The cell cycle-dependent transcription factor, <n>E2F-1</n>, regulates the cyclin-like species of the DNA repair enzyme <n>uracil-DNA glycosylase</n> (<n>UDG</n>) gene in human osteosarcoma (Saos-2) cells. We demonstrate, through the deletion of the human <n>UDG</n> promoter sequences, that expression of <n>E2F-1</n> activates the <n>UDG</n> 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 <n>UDG</n> gene product and E2F. High levels of <n>UDG</n> expression in a transient transfection assay result in the down-regulation of transcriptional activity through elements specific for E2F-mediated transcription. Overexpression of <n>UDG</n> 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 <n>UDG</n> on E2F-mediated transcriptional activity
95370702>Cellular and molecular mechanisms of <n>IFN-gamma</n> production induced by <n>IL- 2</n> and <n>IL-12</n> in a human NK cell line. <n>Interferon-gamma</n> (<n>IFN-gamma</n>) is an important <n>immunoregulatory protein</n> produced predominantly by T cells and large granular lymphocytes (LGL) in response to different extracellular signals. In particular, two interleukins (ILs), <n>IL-2</n> and <n>IL-12</n>, have been shown to be potent inducers of <n>IFN-gamma</n> gene expression in both T cells and LGL. Although it has been reported that there are some T cell lines that produce <n>IFN- gamma</n> 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 <n>IL-2</n> and <n>IL-12</n>, as measured by increases in <n>IFN-gamma</n> and <n>granulocyte-macrophage colony- stimulating factor</n> (<n>GM-CSF</n>) cytoplasmic mRNA and protein expression. In addition, when used together <n>IL-2</n> and <n>IL-12</n> synergized in the induction of <n>IFN-gamma</n> and <n>GM-CSF</n> and this synergy was attributed to an increased accumulation and stability of the <n>IFN-gamma</n> and <n>GM-CSF</n> mRNAs. To investigate the signaling pathways involved in the gene induction, five inhibitors, cyclosporin A (CsA), <n>transforming growth factor-beta</n>, cycloheximide, genistein, and staurosporine A, were used in analyzing the effects of <n>IL-2</n> and <n>IL-12</n> on NK3.3 cells. The results suggest that activation of <n>protein kinase C</n>, but not new protein synthesis, is required for <n>IL-2</n> induction of IFN-gamma and GM-CSF cytoplasmic mRNA. In contrast, <n>IL-12</n> induction of <n>IFN-gamma</n> cytoplasmic mRNA appears to only partially depend on activation of <n>protein kinase C</n>. Furthermore, both <n>transforming growth factor-beta</n> and genistein, a tyrosine kinase inhibitor, could suppress <n>IL-2</n> and <n>IL-12</n> 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, <n>IL-2</n> but not <n>IL-12</n> induced nuclear factors <n>NF-kappa B</n> and <n>AP1</n>, 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 <n>IL-2</n> and <n>IL-12</n> may have distinct signaling pathways leading to the induction of <n>IFN-gamma</n> and <n>GM-CSF</n> 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.
95349600>A functional T-cell receptor signaling pathway is required for <n>p95vav</n> activity. Stimulation of the <n>T-cell antigen receptor</n> (<n>TCR</n>) induces activation of multiple <n>tyrosine kinases</n>, resulting in phosphorylation of numerous intracellular substrates. One substrate is <n>p95vav</n>, 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 <n>p95vav</n> in <n>TCR</n>-mediated signaling processes is unclear. Here, we show that overexpression of <n>p95vav</n> alone in Jurkat T cells leads to activation of the nuclear factors, including NFAT, involved in <n>interleukin-2</n> expression. Furthermore, <n>p95vav</n> synergizes with <n>TCR</n> stimulation in inducing NFAT- and interleukin-2-dependent transcription. In contrast, NFAT activation by a G-protein-coupled receptor is not modulated by <n>p95vav</n> overexpression, suggesting that the effect is specific to the <n>TCR</n> signaling pathways. Although removal of the first 67 amino acids of <n>p95vav</n> 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 <n>p95vav</n>-induced NFAT activation is not mimicked by Ras activation, though its function is dependent upon Ras and Raf. Furthermore, the activating function of <n>p95vav</n> is blocked by FK506, suggesting that its activity also depends on <n>calcineurin</n>. To further dissect <n>p95vav</n> involvement in <n>TCR</n> signaling, we analyzed various Jurkat mutants deficient in <n>TCR</n> signaling function or <n>TCR</n> expression and showed that an intact <n>TCR</n> signaling pathway is required for <n>p95vav</n> to function. However, overexpression of <n>p95vav</n> does not appear to influence <n>TCR</n>-induced protein tyrosine phosphorylation or increases in cytoplasmic free calcium. Taken together, our data suggest that <n>p95vav</n> plays an important role at an yet unidentified proximal position in the <n>TCR</n> signaling cascade.
95329717>Positive and negative regulation of <n>granulocyte-macrophage colony- stimulating factor</n> promoter activity by <n>AML1-related transcription factor</n>, <n>PEBP2</n>. The <n>granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) gene promoter contains a consensus sequence for the <n>polyomavirus enhancer binding-protein 2</n> (<n>PEBP2</n>) 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. <n>PEBP2</n> alpha A1, alpha B1, and alpha B2 proteins bound the <n>PEBP2</n> site within the mouse <n>GM-CSF</n> promoter. <n>PEBP2</n> alpha A1 and alpha B1 enhanced the expression of the <n>GM-CSF</n> 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 <n>PEBP2</n> site-binding protein(s) that cross-reacted with antimouse alpha A1 antibodies. Northern blot analysis indicated the expression of human <n>PEBP2</n> alpha A, alpha B (AML1), and beta genes in Jurkat cells. Although further studies are required to determine the precise role of <n>PEBP2</n> in the <n>GM-CSF</n> promoter activity, the present findings suggested the importance of the relative ratio of different <n>PEBP2</n> isoforms in regulating the levels of the promoter activity.
96075792><n>IFN-gamma</n> 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 <n>interferon gamma</n> (<n>IFN-gamma</n>), 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 <n>IFN-gamma</n> was primarily manifested at the level of TNF mRNA accumulation. <n>IFN-gamma</n> 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 <n>IFN-gamma</n> or <n>GM-CSF</n>, but not <n>M- CSF</n>. 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 <n>nuclear factor-kappa B</n> activity in these cells as determined by electrophoretic mobility shift assay using a consensus <n>NF-kappa B</n> 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)
95280917>A Myc-associated zinc finger protein binding site is one of four important functional regions in the <n>CD4</n> promoter. The <n>CD4</n> promoter plays an important role in the developmental control of <n>CD4</n> transcription. In this report, we show that the minimal <n>CD4</n> promoter has four factor binding sites, each of which is required for full function. Using biochemical and mutagenesis analyses, we determined that multiple nuclear factors bind to these independent sites. We determined that an initiator-like sequence present at the cap site and an Ets consensus sequence are required for full promoter function. We also demonstrate that the <n>Myc-associated zinc finger protein</n> (<n>MAZ</n>) appears to be the predominant factor binding to one of these sites. This last site closely resembles the ME1a1 G3AG4AG3 motif previously shown to be a critical element in the P2 promoter of the c- myc gene. We therefore believe that the <n>MAZ</n> transcription factor is also likely to play an important role in the control of developmental expression of the <n>CD4</n> gene.
95263486><n>Erythropoietin</n> 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. <n>TAL1</n> 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 <n>glycoprotein hormone erythropoietin</n> (<n>Epo</n>), we investigated whether the expression or activity of the <n>TAL1</n> gene and its protein products were affected by <n>Epo</n> in splenic erythroblasts from mice infected with an anemia-inducing strain of Friend virus (FVA cells). <n>Epo</n> elicited a rapid, dose-related increase in <n>TAL1</n> mRNA by increasing transcription of the gene and stabilizing one of its mRNAs. An <n>Epo</n>-inducible <n>TAL1</n> 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 <n>Epo</n>-induced phosphorylation of nuclear <n>TAL1</n> protein. These results indicate that <n>Epo</n> acts at both transcriptional and posttranscriptional levels on the <n>TAL1</n> locus in Friend virus-induced erythroblasts and establish a link between <n>Epo</n> signaling mechanisms and a member of a family of transcription factors involved in the differentiation of diverse cell lineages.
95397944>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.
95266282>Induction of <n>Sp1</n> phosphorylation and <n>NF-kappa B</n>-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 <n>NF-kappa B</n>, and the promoter domain of the LTR. The inducibility of HIV LTR-driven <n>luciferase</n> expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional <n>Sp1</n> binding elements and the ability of the TATA box to bind the protein <n>TBP</n>. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed <n>Sp1</n> protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or <n>PHA</n> and <n>interleukin 2</n>. Responsiveness of LTR constructs deleted of kappa B elements to <n>HIV Tat</n> expression was increased upon OKA but not TNF stimulation. Our results suggest that <n>SP1</n> phosphorylation induced by OKA, a selective inhibitor of the serine- threonine phosphatase <n>PP2A</n>, facilitates the formation of a transcription complex involving general transcription factors, <n>HIV Tat</n>, and <n>Sp1</n> proteins. The formation of this complex would increase, independently of an in synergy with <n>NF-kappa B</n>, the low basal activity of the HIV LTR observed in normal T lymphocytes.
95236293>The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by <n>IL-2</n>, <n>IL-4</n>, <n>IL-7</n>, <n>IL-13</n>, and <n>IL-15</n>. 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. <n>Interleukin-2</n> (<n>IL-2</n>) rapidly activated <n>Stat5</n> in fresh PBL, and <n>Stat3</n> and <n>Stat5</n> in preactivated PBL. <n>IL-7</n> and <n>IL-15</n> induced the same complexes as <n>IL-2</n>, a feature explained by the existence of similar tyrosine-phosphorylated motifs in the cytoplasmic domains of <n>IL-2R beta</n> and <n>IL-7R</n> that can serve as docking sites for Stat proteins. <n>IL-13</n> Induced the same complexes as <n>IL-4</n>, a finding explained by our studies implicating <n>IL-4R</n> 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.
95184007>Control of <n>I kappa B-alpha</n> proteolysis by site-specific, signal-induced phosphorylation. <n>I kappa B-alpha</n> inhibits transcription factor <n>NF-kappa B</n> by retaining it in the cytoplasm. Various stimuli, typically those associated with stress or pathogens, rapidly inactivate <n>I kappa B-alpha</n>. This liberates <n>NF-kappa B</n> to translocate to the nucleus and initiate transcription of genes important for the defense of the organism. Activation of <n>NF-kappa B</n> correlates with phosphorylation of <n>I kappa B-alpha</n> and requires the proteolysis of this inhibitor. When either serine-32 or serine-36 of <n>I kappa B-alpha</n> was mutated, the protein did not undergo signal-induced phosphorylation or degradation, and <n>NF-kappa B</n> could not be activated. These results suggest that phosphorylation at one or both of these residues is critical for activation of <n>NF-kappa B</n>
95388524>Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-1 and Sp1 motifs. <n>Erythropoietin</n> (<n>Epo</n>), the primary regulator of the production of erythroid cells, acts by binding to a cell surface receptor (<n>EpoR</n>) 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 <n>EpoR</n> gene. In erythroid cells most of the transcription activity was contained in a 150 bp promoter fragment with binding sites for transcription factors <n>AP2</n>, <n>Sp1</n> and the <n>erythroid-specific GATA-1</n>. 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 h<n>EpoR</n> 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 <n>Sp1</n> and two other CCGCCC binding proteins from erythroid and non-erythroid cells could bind to the <n>Sp1</n> binding motif. By increasing <n>GATA-1</n> 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 <n>GATA-1</n> mRNA levels were comparable in OCIM1 and K562. Interestingly, when we mutated the <n>Sp1</n> site, resulting in a marked decrease in hEpoR promoter activity, we could restore transactivation by increasing <n>GATA-1</n> levels in OCIM1 cells. These data suggest that while <n>GATA-1</n> can transactivate the <n>EpoR</n> promoter, the level of hEpoR gene expression does not depend on <n>GATA-1</n> alone. Rather, <n>hEpoR</n> transcription activity depends on coordination between <n>Sp1</n> and <n>GATA-1</n> with other cell-specific factors, including possibly other <n>Sp1</n>-like binding proteins, to provide high level, tissue-specific expression.
95365382>Overexpression of <n>DR-nm23</n>, 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, <n>DR-nm23</n>, 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 <n>nm23</n> function. <n>DR-nm23</n> 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 <n>granulocyte colony-stimulating factor</n> and causes apoptotic cell death. These results are consistent with a role for <n>DR-nm23</n> in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia.
96032864>An interferon-gamma activation sequence mediates the transcriptional regulation of the IgG Fc receptor type IC gene by <n>interferon-gamma</n>. Expression of the <n>IgG Fc receptor type I</n> (<n>Fc gamma RI</n>) on myeloid cells is dramatically increased by treatment with <n>interferon-gamma</n> (<n>IFN- gamma</n>). We observed that <n>Fc gamma RI</n> transcript levels in monoblast- like U937 cells were elevated within 3 hr and peaked 12 hr after exposure to <n>IFN-gamma</n>. Treatment of U937 with <n>IFN-gamma</n> for 9 hr in the presence of cycloheximide led to super-induction of <n>Fc gamma RI</n> expression. Nuclear run-on analysis revealed that the rate of <n>Fc gamma RI</n> transcription was increased by <n>IFN-gamma</n>. 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 <n>IFN-gamma</n> responsiveness on a heterologous promoter. Double-stranded GIRE sequence, but not a scrambled sequence, was specifically bound by nuclear proteins from <n>IFN- gamma</n> treated U937 cells. Gel shift experiments further showed that the <n>STAT1 alpha protein</n> bound to the Fc gamma RIC GIRE in response to <n>IFN- gamma</n> treatment of U937 cells. The Fc gamma RIC GIRE is homologous to the <n>IFN-gamma</n> activation sequence (GAS) of the <n>guanylate binding protein</n> and to X box elements of class II MHC genes. Our results demonstrate that transcriptional regulation of the Fc gamma RIC gene by <n>IFN-gamma</n> involves the binding of <n>STAT1 alpha</n> to a 17-bp GAS homology in the proximal promoter.
95327953>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 <n>interleukin-2</n> (<n>IL-2</n>)-dependent growth; over time, by an unknown mechanism, the cells become <n>IL-2</n>-independent. Whereas the Jak kinases <n>Jak1</n> and <n>Jak3</n> and the signal transducer and activator of transcription proteins <n>Stat3</n> and <n>Stat5</n> are activated in normal T cells in response to <n>IL-2</n>, this signaling pathway was constitutively activated in HTLV-I-transformed cells. In HTLV-I- infected cord blood lymphocytes, the transition from <n>IL-2</n>-dependent to <n>IL-2</n>-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.
95340873>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 <n>vascular cell adhesion molecule-1</n> (<n>VCAM-1</n>). In a concentration-dependent manner, NO inhibited <n>interleukin (IL)-1</n> alpha-stimulated <n>VCAM-1</n> 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, <n>IL-1 beta</n>, <n>IL-4</n>, tumor necrosis factor (<n>TNF alpha</n>), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (<n>E-selectin</n> and to a lesser extent, <n>intercellular adhesion molecule-1</n>) and secretable cytokines (<n>IL-6</n> and <n>IL-8</n>). Inhibition of endogenous NO production by L-N-monomethyl- arginine also induced the expression of <n>VCAM-1</n>, but did not augment cytokine-induced <n>VCAM-1</n> expression. Nuclear run-on assays, transfection studies using various <n>VCAM-1</n> promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses <n>VCAM- 1</n> gene transcription, in part, by inhibiting <n>NF-kappa B</n>. 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.
95280909><n>MIP1 alpha nuclear protein</n> (<n>MNP</n>), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 alpha gene. <n>Murine macrophage inflammatory protein 1 alpha</n> (<n>MIP-1 alpha</n>) and its human equivalent (<n>GOS19</n>, <n>LD78</n>, or <n>AT464</n>) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived <n>MIP-1 alpha</n>/<n>GOS19</n> 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 <n>MIP-1 alpha</n>/<n>GOS19</n>. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the <n>GOS19</n> promoter. One member, <n>ICK-1A</n>, 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 <n>MIP-1 alpha</n> 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.
96055286>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.
95337737>The interleukin-5/receptor interaction activates Lyn and Jak2 tyrosine kinases and propagates signals via the <n>Ras-Raf-1-MAP kinase</n> and the Jak- STAT pathways in eosinophils. We have shown that the interaction of <n>interleukin (IL)-5</n> with the receptor activates <n>Lyn tyrosine kinase</n> within 1 min and <n>Jak2 tyrosine kinase</n> within 1-3 min. <n>IL-5</n> also stimulates GTP binding to <n>p21ras</n>. The signal is subsequently propagated through the activation of <n>Raf-1</n>, MEK, and MAP kinases as shown by their increased autophosphorylation in vitro and phosphorylation in situ. <n>Jak2 kinase</n> 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 <n>IL-5</n> induces two GAS- binding proteins in eosinophils, one of which is <n>STAT1</n>. We conclude that <n>IL-5</n> induced signals are propagated through two distinct pathways: (1) Lyn--&gt;Ras--&gt;<n>Raf-1</n>--&gt;MEK--&gt;MAP kinase and (2) <n>Jak2</n>--&gt;<n>STAT1</n>.
95266250>The <n>retinoblastoma gene product</n> negatively regulates transcriptional activation mediated by the <n>human cytomegalovirus IE2 protein</n>. The <n>IE2 gene product</n> 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 <n>retinoblastoma susceptibility gene product</n> (<n>Rb</n>) dramatically suppressed this <n>IE2</n> transactivation of various promoters. However, unlike another tumor suppressor protein, <n>p53</n>, <n>Rb</n> did not have any significant effect on basal levels of transcription, suggesting that <n>Rb</n> specifically interacts with <n>IE2</n> rather than other cellular factors involved in the general transcription machinery. We found by protein-affinity chromatography that <n>Rb</n> in nuclear extracts or produced by in vitro translation directly bound to <n>IE2</n>. Our results suggest that <n>Rb</n> 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.
96082382>Expression of the <n>nucleoside diphosphate kinase</n> in human skin cancers: an immunohistochemical study. Expression of <n>nucleoside diphosphate(NDP) kinase</n>, which is homologous to the <n>nm23 gene product</n> 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 <n>NDP kinase</n> expression serves as a marker for metastatic potential in human skin cancer, we assessed the levels of <n>NDP kinase</n> expression in 9 keratoacanthomas (KAs), 26 squamous cell carcinomas (SCCs), and 25 basal cell carcinomas (BCCs) using immunohistochemistry. The expression of <n>NDP kinase</n> was intense in KA and SCC compared with BCC. However, the difference of <n>NDP kinase</n> expression between KA and SCC was not statistically significant. And there was no statistically significant difference in <n>NDP kinase</n> 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
95362365><n>RB</n> and a novel E2F-1 binding protein in MHC class II deficient B-cell lines and normal <n>IFN-gamma</n> induction of the class IL transactivator CIITA in class II non-inducible <n>RB</n>-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 <n>IFN-gamma</n> on a wide variety of cell types. Retinoblastoma protein (<n>RB</n>) is a tumor suppressor and functions as a transcriptional repressor by binding and inactivating the transactivator E2F-I. <n>RB</n>-defective tumor lines are non-inducible for MHC class II by <n>IFN-gamma</n>, or very weakly inducible, but transfection of 2 different lines with <n>RB</n> expression vectors re- establishes or substantially enhances class II inducibility. Therefore, we examined the <n>RB</n> 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 <n>RB</n> was detectable in all cases, indicating that loss of <n>RB</n> 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 <n>IFN-gamma</n> induction of CIITA in <n>RB</n>-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 <n>IFN-gamma</n>. CIITA mRNA is normally inducible by <n>IFN-gamma</n> in class II non-inducible, <n>RB</n>-defective lines, and in one line, re-expression of <n>RB</n> has no effect on CIITA mRNA induction levels. Thus, the block in MHC class II inducibility in <n>RB</n>- defective cells is not due to a block in CIITA inducibility.
95365357><n>Interleukin 12</n> induces tyrosine phosphorylation and activation of <n>STAT4</n> in human lymphocytes. <n>Interleukin 12</n> (<n>IL-12</n>) 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 <n>IL-12</n> induces tyrosine phosphorylation of the Janus family tyrosine kinase <n>JAK2</n> and <n>Tyk2</n>, implicating these kinases in the immediate biochemical response to <n>IL-12</n>. 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 <n>IL-12</n> induces tyrosine phosphorylation of a recently identified STAT family member, <n>STAT4</n>, and show that <n>STAT4</n> expression is regulated by T- cell activation. Furthermore, we show that <n>IL-12</n> stimulates formation of a DNA-binding complex that recognizes a DNA sequence previously shown to bind STAT proteins and that this complex contains <n>STAT4</n>. These data, and the recent demonstration of JAK phosphorylation by <n>IL-12</n>, identify a rapid signal-transduction pathway likely to mediate <n>IL-12</n>- induced gene expression.
96007705>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.
95332324>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 <n>cytokine interleukin (IL)-1 beta</n>. The tyrosine kinase inhibitors genistein and herbimycin A block both integrin-mediated tyrosine phosphorylation and increases in <n>IL-1 beta</n> message levels, indicating a causal relationship between the two events. The components tyrosine phosphorylated subsequent to cell adhesion include <n>paxillin</n>, <n>pp125FAK</n>, and the <n>SH2 domain containing tyrosine kinase Syk</n>. In contrast, integrin ligation with antibodies induces tyrosine phosphorylation of <n>Syk</n> but not of <n>FAK</n> or <n>paxillin</n>. In adhering cells, pre-treatment with cytochalasin D suppresses tyrosine phosphorylation of <n>FAK</n> and <n>paxillin</n> but not of <n>Syk</n>, while <n>IL-1 beta</n> message induction is unaffected. These observations indicate that the <n>Syk</n> tyrosine kinase may be an important component of an integrin signaling pathway in monocytic cells, leading to activation of <n>NF-kappa B</n> and to increased levels of cytokine messages.
96065933>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 &lt; 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 <n>trypsin</n> 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.
95392577>Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals. The mRNA for the <n>Duffy blood group antigen</n>, 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 <n>Duffy antigen/chemokine receptor</n> 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.
95287633>Activation of <n>pp90rsk</n> and early growth response-1 gene expression by <n>pokeweed mitogen</n> in human B cells. The present studies have examined the effects of <n>pokeweed mitogen</n> (<n>PWM</n>) on the induction of early growth response-1 gene (EGR-1) in normal human B cells. <n>PWM</n> regulates EGR-1 gene expression by both transcriptional and post-transcriptional mechanisms. Transient transfection assays with EGR-1 promoter fragments linked to the <n>chloramphenicol acetyltransferase</n> (<n>CAT</n>) gene demonstrated that <n>PWM</n> 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 <n>S6 kinase</n> (<n>pp90rsk</n>), evidenced by phosphorylation of <n>S6</n> and serum response factor (SRF) peptides, in <n>PWM</n> treated B cells. Taken together, these findings suggest that <n>PWM</n> is able to initiate an intracytoplasmic signalling cascade and EGR-1 induction in normal human B cells.
95337734>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 (<n>GM-CSF</n>)]. 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 <n>GM-CSF</n> 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.
95221892>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 <n>NGFI-B/nur77</n>. We also propose that the NGFI-B/nur77 family can be classified into two subtypes.
95202809>Aspirin inhibits <n>nuclear factor-kappa B</n> mobilization and monocyte adhesion in stimulated human endothelial cells. BACKGROUND: The induction of <n>vascular cell adhesion molecule-1</n> (<n>VCAM-1</n>) and <n>E-selectin</n> by <n>tumor necrosis factor-alpha</n> (<n>TNF</n>) is mediated by mobilization of the transcription factor <n>nuclear factor-kappa B</n> (<n>NF- kappa B</n>). Since salicylates have been reported to inhibit <n>NF-kappa B</n> activation by preventing the degradation of its inhibitor <n>I kappa B</n>, 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 <n>TNF</n>-induced <n>NF-kappa B</n> mobilization by aspirin at concentrations ranging from 1 to 10 mmol/L. Induction of <n>VCAM-1</n> and <n>E-selectin</n> surface expression by <n>TNF</n> was dose-dependently reduced by aspirin over the same range, while induction of <n>intercellular adhesion molecule-1</n> (<n>ICAM-1</n>) was hardly affected. Aspirin appeared to prevent <n>VCAM-1</n> transcription, since it dose-dependently inhibited induction of <n>VCAM-1</n> mRNA by <n>TNF</n>. As a functional consequence, adhesion of U937 monocytes to <n>TNF</n>-stimulated HUVECs was markedly reduced by aspirin due to suppression of <n>VCAM-1</n> and <n>E-selectin</n> upregulation. These effects of aspirin were not related to the inhibition of <n>cyclooxygenase</n> activity, since indomethacin was ineffective. CONCLUSIONS: Our data suggest that aspirin inhibits <n>NF- kappa B</n> mobilization, induction of <n>VCAM-1</n> and <n>E-selectin</n>, and subsequent monocyte adhesion in endothelial cells stimulated by <n>TNF</n>, thereby providing an additional mechanism for therapeutic effects of aspirin
95355456>Activation of <n>NF-kappa B</n> by phosphatase inhibitors involves the phosphorylation of <n>I kappa B alpha</n> at phosphatase 2A-sensitive sites. Activation of <n>NF-kappa B</n> by various cellular stimuli involves the phosphorylation and subsequent degradation of its inhibitor, <n>I kappa B alpha</n>, although the underlying mechanism remains unclear. In the present study, the role of <n>serine/threonine phosphatases</n> in the regulation of <n>I kappa B alpha</n> 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 <n>I kappa B alpha</n> even in the absence of any cellular stimulus. This action of the phosphatase inhibitors, which is associated with the activation of the RelA.p50 <n>NF-kappa B</n> heterodimer, is not affected by agents that block the induction of <n>I kappa B alpha</n> phosphorylation by <n>tumor necrosis factor alpha</n> (<n>TNF- alpha</n>). Furthermore, the phosphorylated <n>I kappa B alpha</n> from calyculin A-treated cells, but not that from <n>TNF-alpha</n>-stimulated cells, is sensitive to PP-2A in vitro, suggesting the existence of fundamental differences in the phosphorylation of <n>I kappa B alpha</n> induced by the two different <n>NF-kappa B</n> inducers. However, induction of <n>I kappa B alpha</n> phosphorylation by both <n>TNF-alpha</n> and the phosphatase inhibitors is associated with the subsequent degradation of <n>I kappa B alpha</n>. 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 <n>I kappa B alpha</n>, mediated through both the <n>TNF-alpha</n>- inducible and the PP-2A-opposing kinases, may serve to target <n>I kappa B alpha</n> for proteasome-mediated degradation.
95365343><n>Sp1</n> 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 <n>Sp1</n> 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 <n>Sp1</n>.
95363078>Costimulation of human CD4+ T cells with <n>LFA-3</n> and <n>B7</n> induce distinct effects on AP-1 and <n>NF-kappa B</n> 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 <n>B7</n> or <n>LFA-3</n> resulted in distinct cytokine profiles. We now demonstrate that <n>B7</n>, but not <n>LFA-3</n>, strongly costimulated <n>IL-2</n> transcription and mRNA expression in CD4+ T cells. Maximal increase in <n>IL-2</n> transcription was recorded with CHO-DR/<n>B7</n>/<n>LFA-3</n>, suggesting a cooperative effect of <n>B7</n> and <n>LFA-3</n> at the transcriptional level. Gel- shift analysis demonstrated that stimulation of CD4+ T cells with <n>CHO- DR</n> and staphylococcal enterotoxin A was sufficient to induce significant amounts of <n>NF-kappa B</n> binding proteins, whereas induction of AP-1 binding proteins required costimulation. <n>LFA-3</n> induced moderate levels of <n>AP-1</n>, but did not influence the levels of <n>NF-kappa B</n>, while <n>B7</n> costimulation strongly induced both <n>AP-1</n> and substantially enhanced <n>NF-kappa B</n> binding proteins. The CHO-DR/<n>B7</n>/<n>LFA-3</n> triple transfectant induced a further increase in <n>AP-1</n> and <n>NF-kappa B</n> 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 <n>NF-kappa B</n> complex of costimulated CD4+ T cells contained large amounts of p50, substantial amounts of p65, and marginal levels of c-Rel proteins. The <n>AP-1</n> binding proteins contained <n>c-Jun</n>, <n>Jun-D</n>, and <n>Fra-1</n>, but marginal amounts of <n>Jun-B</n> and <n>c-Fos</n>. Our results indicate distinct effects of <n>B7</n> and <n>LFA-3</n> costimulation on the activity of <n>AP-1</n> and <n>NF-kappa B</n>. These may partly account for the differential effects of <n>B7</n> and <n>LFA-3</n> costimulation on <n>IL-2</n> expression.
95336453>The <n>Ah receptor</n> recognizes DNA binding sites for the B cell transcription factor, <n>BSAP</n>: 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 <n>Ah receptor</n>. In addition, the <n>Ah receptor</n> complex recognized a DNA binding site for B cell lineage-specific activator protein (<n>BSAP</n>) in the promoter region of the human CD19 gene which is similar to the consensus <n>Ah receptor</n> DNA binding site. These results suggest that the <n>AhR</n> could interfere with <n>BSAP</n>-stimulated CD19 gene transcription by competition for a common DNA binding site.
95337142>Inhibitory action of nm23 proteins on induction of erythroid differentiation of human leukemia cells. We recently identified a differentiation inhibitory factor (I-factor) in mouse myeloid leukemia M1 cells as a murine homolog of the <n>human nm23-H2 gene product</n>. nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although their mechanisms of action are still unknown. Although all nm23 proteins contain nucleoside diphosphate (NDP) kinase activity, it has not been established that the enzyme activity mediated the various functions of nm23 proteins. In the present experiment, we examined the effect of nm23 proteins on various differentiation induction systems of human leukemic cells including HL-60, U937, HEL/S, KU812F, K562, and HEL cells. Native human erythrocyte NDP kinase protein inhibited the induction of erythroid differentiation of HEL, KU812 and K562 cells, but not the induction of monocytic or granulocytic differentiation of HL-60, U937 and HEL/S cells. The erythroid differentiation of HEL cells was inhibited by recombinant human nm23-H1, -H2, mouse nm23-M1, and -M2 proteins. Moreover, both the <n>mutant nm23-H2His protein</n> and <n>truncated nm23-H2 protein</n> containing N- terminal (1-60) peptide, which do not have <n>NDP kinase</n> activity, also inhibited erythroid differentiation of HEL cells. These results suggest that (1) the differentiation inhibitory activity of <n>I-factor/nm23 protein</n> is not restricted to monocytic differentiation of M1 cells, (2) the inhibitory activity is exhibited without species specificity, and (3) the differentiation inhibitory activity of the <n>nm23/NDP kinase protein</n> is independent of its enzyme activity and requires the presence of N-terminal peptides.
95355137>Lipopolysaccharide-induced <n>E-selectin</n> expression requires continuous presence of LPS and is inhibited by bactericidal/permeability- increasing protein. Endothelial cells stimulated by LPS express <n>E-selectin</n>, which plays an important role in mediating neutrophil adhesion during inflammation. <n>E- selectin</n> is induced within 1-2 h, peaks at 4-6 h, and gradually returns to basal level by 24 h. <n>rBPI21</n>, a recombinant N-terminal fragment of <n>human bactericidal/permeability-increasing protein</n> (<n>BPI</n>), inhibited LPS- induced <n>E-selectin</n> expression when added at the same time as, and up to 6 h after, LPS. Delayed administration of <n>rBPI21</n> also affected LPS- mediated activation of the nuclear factor, <n>NF-kappa B</n>. Two to 4 h following LPS addition to endothelial cells, when <n>NF-kappa B</n> was already activated, addition of <n>rBPI21</n> resulted in marked reduction of <n>NF-kappa B</n> detectable at 4 or 6 h. These results indicate that endothelial activation requires continuous presence of LPS, and <n>rBPI21</n> acts to reverse LPS-mediated endothelial activation by interrupting the on-going LPS signal.
95279882><n>GM-CSF</n> and <n>IL-2</n> 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 <n>granulocyte-macrophage colony- stimulating factor</n> 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 (<n>IL-2</n>) and IL-4 gene transcription in T cells. Costimulatory signals, such as activation of the <n>CD28 cell surface molecule</n> on T cells, lead to activation through a distinct region of the <n>granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) promoter. This region is termed the CK-1 or CD28RE and appears to bind specific members of the <n>NF-kappa B</n> family of transcription factors. Human T leukemia virus type 1 (HTLV-1) infects T cells and can lead to increase <n>GM-CSF</n> expression. We have found that the HTLV-1 transactivator protein, <n>tax</n>, 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 <n>GM-CSF</n> promoter through the CK-1/CD28RE region and also activates <n>nuclear factor-kappa B</n> binding to this region. However, other transcription factors or coactivators of <n>NF-kappa B</n> are required for <n>tax</n> activation but these remain to be identified. The CK-1/CD28RE of <n>GM-CSF</n> shows a high degree of similarity to the <n>IL-2</n> CD28RE and the IL-3 gene also contains a related region. This observation, together with the fact that both <n>GM- CSF</n> and <n>IL-2</n> respond to TCR signals via NFAT, implies a high degree of conservation in the regulation of cytokine gene expression in T cells.
95309632>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.
95221870>Functional characterization of novel <n>IL-2</n> transcriptional inhibitors. <n>IL-2</n>-mediated T cell proliferation is a critical early event in the inflammatory process. Formation of the NFAT-1 transcriptional complex on the <n>IL-2</n> promoter is essential for <n>IL-2</n> 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 <n>beta-galactosidase</n> activity. WIN 61058 and WIN 53071 were identified as microM inhibitors. These compounds also inhibited <n>beta-galactosidase</n> 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 <n>IL-2</n>. WIN 53071 inhibited <n>IL-2</n> production induced in the calcium-dependent PMA and ionomycin pathway. Conversely, <n>calcium-independent anti-CD28 Ab</n> and PMA-induced <n>IL-2</n> 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.
95221386>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 <n>human vacuolar H(+)-ATPase</n> (<n>V-ATPase</n>). We examined mRNA levels of various <n>V-ATPase</n> 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 <n>V-ATPase</n> 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 <n>ribonuclease</n> 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. <n>DNase I</n> footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5'-untranslated and proximal coding regions
96089572>Synergy between signal transduction pathways is obligatory for expression of c-fos in B and T cell lines: implication for c-fos control via surface immunoglobulin and T cell antigen receptors. Expression of the protooncogene c-fos is controlled by three main regulatory pathways involving <n>kinase C</n>, cAMP, and calcium. Kinase C mediates its effects via phosphorylation of <n>serum response factor</n> (<n>SRF</n>) which interacts with the serum response element (SRE); cAMP and calcium mediate their effects via phosphorylation of <n>CREB</n> (<n>cAMP regulatory element binding protein</n>) presumably by activation of a <n>protein kinase A</n> or calmodulin-regulated kinase. We have examined the function of these elements in Burkitt's lymphoma cells (Ramos and Daudi) as well as a T lymphocytic cell line (Jurkat). We have found that stimulation of any one of these pathways alone has little or no effect on c-fos induction. However, <n>kinase C</n> activation (PMA stimulation) combined with either cAMP (forskolin plus MIX) or calcium stimulation (ionophore) leads to greatly enhanced c-fos induction. By contrast, cAMP in the presence of calcium shows no synergy in c-fos induction. Okadaic acid augments PMA- as well as calcium-mediated activation of c-fos, and has little or no effect when combined with cAMP. The main difference between Ramos (B cells) and Jurkat (T cells) in the regulation of c-fos is that cAMP plus calcium is strongly synergistic in Jurkat and is without effect in Ramos. Analysis of <n>AP-1</n> activity using gel mobility shift assays confirms that the requirements for synergy in c-fos mRNA induction are paralleled by requirements for synergy in induction of <n>AP-1</n> activity. Signaling in B cells due to anti-Ig stimulation involves both <n>kinase C</n> activation and release of intracellular calcium, and results in c-fos mRNA induction. Our results indicate that synergy between the <n>kinase C</n> activation and calcium is needed for efficient c-fos induction since neither of these two alone induces c-fos well. That synergy of signaling pathways is relevant for the anti-Ig induction of c-fos is supported by the fact that cAMP-inducing agents and okadaic acid further enhance anti-Ig induction of c-fos. These results suggest that cell-specific patterns of synergy are an essential feature for c-fos induction and may be relevant for c-fos control through B and T cell antigen receptors.
95363089>Multiple signals are required for function of the <n>human granulocyte- macrophage colony-stimulating factor</n> gene promoter in T cells. The <n>human granulocyte-macrophage CSF</n> (<n>GM-CSF</n>) gene is expressed in T cells in response to TCR activation that can be mimicked by treatment of the cells with PMA and Ca2+ ionophore. The gene contains a proximal functional promoter region (-620 to +34), as well as a powerful enhancer located 3 kb upstream, both of which are involved in the response of the gene to TCR activation. The proximal promoter contains a region termed CLEO (-54 to -31) that consists of a purine-rich element abutting an <n>activator protein-1</n> (<n>AP-1</n>)-like site, as well as an upstream <n>nuclear factor-kappa B</n> (<n>NF-kappa B</n>) site (-85 to -76) and a CK- 1 element (-101 to -92). We show in this work that mutations in either the purine-rich region of the CLEO element or the <n>NF-kappa B</n> site result in reduced PMA/Ca2+ activation of a 620-bp human <n>GM-CSF</n> promoter- <n>luciferase</n> reporter construct in Jurkat T cells by 65% and 50%, respectively. The major inducible protein complex that binds to the human CLEO (hCLEO) element is an <n>AP-1</n>-like complex that is inducible by PMA alone, but shows increased binding in response to PMA together with Ca2+ ionophore. Although the binding of this complex is not cyclosporin- sensitive, promoter induction is inhibited by cyclosporin treatment. A second weak inducible complex resembling nuclear factor of activated T cells (NF-AT) was also observed binding to the hCLEO region. By using recombinant proteins, we confirmed that <n>AP-1</n>, <n>NF-ATp</n>, and a higher order <n>NF-ATp</n>/<n>AP-1</n> complex could all form with the hCLEO element, and we have also defined the sequence requirements for binding of each of these complexes. We found that expression of a constitutively active form of <n>calcineurin</n> could substitute for Ca2+ ionophore and synergize with PMA to activate the <n>GM-CSF</n> promoter, and conversely that <n>mutant- activated Ras</n> could substitute for PMA and cooperate with Ca2+ ionophore. Co-expression of <n>Ras</n> and <n>calcineurin</n>, however, did not activate the <n>GM-CSF</n> promoter, but required the additional expression of <n>NF-kappa B</n> p65. These results imply that at least three signals are required to activate the <n>GM-CSF</n> proximal promoter, and that the signals impinge on distinct transcription factors that bind to the hCLEO and <n>NF- kappa B</n> regions of the promoter.
95363073>IL-10 induces the tyrosine phosphorylation of <n>tyk2</n> and <n>Jak1</n> and the differential assembly of <n>STAT1 alpha</n> and STAT3 complexes in human T cells and monocytes. <n>IL-10</n> 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 <n>IL- 10</n> stimulates tyrosine phosphorylation of the signal transducers and activators of transcription, <n>STAT1 alpha</n> and <n>STAT3</n>, in a differential manner such that the relative formation of homo- and heterodimers varies between the two cell types. Moreover, monocytes express a novel <n>IL-10</n>-stimulated STAT protein with an M(r) of <n>70 kDa</n> that is recognized by the anti-<n>STAT3</n> Ab but is not observed in T cells. <n>IL-10</n> treatment of both T cells and monocytes results in the ligand-induced tyrosine phosphorylation of <n>tyk2</n> and <n>Jak1</n>, but not <n>Jak2</n> or <n>Jak3</n>. Selective modulation of immune responsiveness by <n>IL-10</n> in cells such as monocytes and T cells may result in part from the differential activation of STAT protein pairs.
97253017>Use of new biologic markers in the ovulation induction. Biological markers of ovulation, after a great in the past, have been fallen into disuse for the large diffusion of biochemical and biophysical ones. However, the real effect of hormones involved in ovulation is expressed by biological modifications on target tissues. To explore the modifications of not reproductive target tissues as ovulation markers we studied the behaviour of Albuminemia, <n>Platelet Factor IV</n> (as indicator of Platelet Aggregation), Type II estrogenic receptors in 42 ovulation induced women, undergoing our observation. 33 of them had ovulation and 9 developed a LUF syndrome, constituting two biological models of an opposite situation for the three markers observed. All the markers considered were sufficiently sensitive, but among them, <n>Platelet Factor IV</n> was the most reliable to the hormonal ovulatory situation.
95279790>Abnormal regulation of the <n>IL-2</n> 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 <n>IL- 2</n> 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 <n>p59fyn</n> and <n>CD3 zeta</n> tyrosine phosphorylation. The generation of second messengers in T cells normally leads to downstream signaling that results in transcriptional activation of the <n>IL-2</n> gene. We, therefore, compared the activation state of the <n>IL-2</n> gene promoter region in freshly isolated and stimulated Ipr CD4-8- T cells with that of normal T lymphocytes. Levels of the octamer, <n>NF-kappa B</n> (<n>p50-p65 heterodimer</n>), and <n>AP-1</n> 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 <n>IL-2</n> production from Ipr CD4-8- T cells.
95316714>Cross-linking of <n>CD30</n> induces HIV expression in chronically infected T cells. <n>CD30</n>, 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 <n>CD30</n> with an anti-<n>CD30</n>- specific monoclonal antibody, which mimics the described biological activities of the <n>CD30 ligand</n> (<n>CD30L</n>), results in HIV expression. <n>CD30</n> cross-linking does not alter proliferation of ACH-2 cells and the induction of HIV expression is not mediated by endogenous <n>TNF alpha/beta</n>. Furthermore, cross-linking of <n>CD30</n> leads to <n>NF-kappa B</n> activation and enhanced HIV transcription. Thus, <n>CD30</n>-<n>CD30L</n> 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.
95269137>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 <n>CIITA</n> 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 <n>CIITA</n> dependent. Since <n>CIITA</n> overexpression in normal cells did not increase class II expression, we propose that initiation of <n>CIITA</n> expression serves as the on-off switch, while availability of downstream interactor(s) limits transcription.
95256455>Monocyte tethering by <n>P-selectin</n> regulates <n>monocyte chemotactic protein- 1</n> and <n>tumor necrosis factor-alpha</n> secretion. Signal integration and <n>NF- kappa B</n> 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 <n>P-selectin</n>, the most rapidly expressed <n>endothelial tethering factor</n>, increased the secretion of <n>monocyte chemotactic protein-1</n> (<n>MCP-1</n>) and <n>tumor necrosis factor-alpha</n> (<n>TNF-alpha</n>) by the leukocytes when they were stimulated with <n>platelet- activating factor</n>. Increased cytokine secretion was specifically inhibited by <n>G1</n>, an anti-<n>P-selectin</n> mAb that prevents <n>P-selectin</n> from binding to its ligand (<n>P-selectin</n> glycoprotein ligand-1) on myeloid cells. Moreover, tethering by <n>P-selectin</n> specifically enhanced nuclear translocation of <n>nuclear factor-kappa B</n> (<n>NF-kappa B</n>), a transcription factor required for expression of <n>MCP-1</n>, <n>TNF-alpha</n>, and other immediate- early genes. These results demonstrate that <n>P-selectin</n>, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues.
95238333>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 <n>erythroid- enriched NF-E2 factor</n>. 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.
95221342>Nitric oxide-stimulated guanine nucleotide exchange on <n>p21ras</n>. 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 <n>GTP-bound p21ras</n>. In vitro studies using <n>pure recombinant p21ras</n> 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 <n>p21ras</n> is essential for NO-induced downstream signaling, such as <n>NF-kappa B</n> activation, and that endogenous NO can activate <n>p21ras</n> in the same cell. These studies identify <n>p21ras</n> as a target of the same cell. These studies identify <n>p21ras</n> as a target of NO in T cells and suggest that NO activates <n>p21ras</n> by an action which mimics that of guanine nucleotide exchange factors
95394020>Interleukin-2 promoter activity in Epstein-Barr virus-transformed B lymphocytes is controlled by <n>nuclear factor-chi B</n>. The regulation of interleukin (IL)-2 gene expression has been investigated mainly in T lymphocytes, the predominant producers of <n>IL- 2</n>. However, B cells can also synthesize <n>IL-2</n>. In the present study we analyzed the control of <n>IL-2</n> promoter activity in Epstein-Barr virus (EBV)-transformed B cell clones which are capable of secreting <n>IL-2</n> 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 <n>IL-2</n> promoter [<n>distal nuclear factor (NF)-AT</n>, <n>proximal NF-AT</n>, <n>AP- 1/Octamer</n> (<n>UPS</n>) or <n>NF-chi B</n> (<n>TCEd</n>) 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 <n>IL-2</n> promoter bearing a defective <n>NF-chi B</n> 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 <n>IL-2</n>, the activity of the <n>IL-2</n> promoter correlated well with the status of <n>IL-2</n> secretion. Similarly, a human immunodeficiency virus promoter, whose activity is controlled through chi B factors, was found to be active in the <n>IL-2</n> producing EBV-B cells, but inactive in the non-<n>IL-2</n>-producing cells. Electrophoretic mobility shift assays using protein extracts from EBV-B cells and the <n>IL-2</n> <n>NF-chi B</n> probe revealed the constitutive generation of chi B complexes in <n>IL-2</n>-secreting cells consisting mainly of heterodimeric p50/p65 complexes. A weaker chi B complex formation and faster-migrating complexes were detected in non-<n>IL-2</n>-secreting cells. These results demonstrate that the <n>IL-2</n> <n>NF-chi B</n> site is indispensable for the activity of the <n>IL-2</n> promoter in EBV-transformed B cells, whereas other transcription factors appear to be less important for <n>IL-2</n> expression in these cells.
95363069><n>Latent membrane protein-1</n> induces <n>cyclin D2</n> expression, <n>pRb</n> hyperphosphorylation, and loss of <n>TGF-beta 1</n>-mediated growth inhibition in EBV-positive B cells. The normal cell cycle is regulated by several molecules, such as the tumor-suppressor protein <n>pRb</n>, the G1 cyclins, the cyclin-dependent kinases, and their inhibitors. These regulators are targeted by negative growth regulatory signals, such as that provided by <n>TGF-beta</n>. Here, we show that the presence of either wild-type EBV or its <n>transforming latent membrane protein-1</n> (<n>LMP-1</n>) results in the loss of <n>TGF-beta</n> 1-mediated growth inhibition in human B cells. Chemical cross- linking with 125I-labeled <n>TGF-beta</n> 1 showed an essentially normal <n>TGF- beta receptor</n> 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 <n>TGF-beta 1</n>-mediated modulation of junB gene expression. However, <n>TGF-beta 1</n> did not induce dephosphorylation of <n>pRb</n> in EBV (or <n>LMP-1</n>)-positive cells as opposed to EBV-negative cells, suggesting a dichotomy in the <n>TGF-beta 1</n> signaling pathway leading to separable gene regulatory and growth inhibitory responses. Furthermore, <n>LMP-1</n> was found to induce the expression of <n>cyclin D2</n>; 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 <n>LMP-1</n> can lead to <n>pRb</n> hyperphosphorylation and uncontrolled cell proliferation.
95355533>Does thyroidectomy, radioactive iodine therapy, or antithyroid drug treatment alter reactivity of patients' T cells to epitopes of thyrotropin receptor in autoimmune thyroid diseases? The effect of treatment on thyroid antibody production and T cell reactivity to thyroid antigens was studied in 15 patients with Graves' disease (GD) before and after thyroidectomy, 19 patients with GD before and after radioactive iodine (RAI) therapy, and 9 patients maintained euthyroid on antithyroid drugs (ATD). Twenty subjects matched for age and sex without known thyroid disease served as controls. In GD patients, the responses of peripheral blood mononuclear cells (PBMC) and TSH receptor (TSHR)-specific T cell lines to recombinant human TSHR extracellular domain, <n>thyroglobulin</n>, and TSHR peptides were examined on the day of surgery or RAI therapy (day 0) and also 6-8 weeks and 3-6 months thereafter. Reactivity to TSHR peptides before surgery was heterogeneous and spanned the entire extracellular domain. Six to 8 weeks after subtotal thyroidectomy, the number of patients' PBMC responding to any peptide and the average number of recognized peptides decreased. A further decrease in the T cell reactivity to TSHR peptides was observed 3-6 months after surgery. The responses of PBMC from Graves' patients before RAI therapy were less than those in the presurgical group. Six to 8 weeks after RAI therapy, the number of patients responding to any peptide and the average number of recognized peptides increased. Three to 6 months after RAI, T cell responses to TSHR peptides were less than those 6-8 weeks after RAI therapy, but still higher than the values on day 0. Responses of PBMC from patients with GD, maintained euthyroid on ATD, were lower than those before surgery or RAI therapy. The reactivity of T cell lines in different groups reflected a pattern similar to PBMC after treatment. TSHR antibody and microsomal antibody levels decreased after surgery, but increased after RAI therapy. The difference in the number of recognized peptides by patients' PBMC before RAI and surgery may reflect the effect of long term therapy with ATD in the patients before RAI vs. the shorter period in patients before surgery. The decreased T cell reactivity to thyroid antigens after thyroidectomy could be the result of removal of a major part of the thyroid gland or redistribution of suppressor-inducer T cells. The increased T cell response after RAI therapy is probably epitope specific, rather than a response to the whole TSHR molecule. Synchronous recognition of peptides 158-176 and 248-263 is important for the development of GD, and the loss of recognition of one of these epitopes may be an early sign of immune remission and a predictor of euthyroidism.
96030053>Circumvention of tolerance for the nuclear T cell protein <n>TCF-1</n> by immunization of <n>TCF-1</n> knock-out mice. Molecular events that underlie the well-defined phenotypic changes of the differentiating thymocyte are poorly understood. A candidate gene to control thymocyte differentiation, <n>T cell factor-1</n> (<n>TCF-1</n>)* encodes a DNA-binding protein. Its mRNA expression pattern is complex during embryogenesis, yet restricted to lymphocytes postnatally. Expression studies on <n>TCF-1</n> protein have been hampered by the difficulty to raise antibodies due to extreme evolutionary conservation. <n>TCF-1</n> knock-out mice, generated recently in our laboratory, have strongly decreased numbers of thymocytes, but are otherwise normal. We have used these mice to generate anti-<n>TCF-1</n> antibodies. By immunization with a recombinant fusion protein, we show that <n>TCF-1</n> knock-out mice readily yield antiserum titers against human and mouse TCF-1 protein. Wild-type littermates remain unresponsive to <n>TCF-1</n> while they mount a high-titer antibody response to the fusion protein, <n>Maltose Binding Protein</n> (<n>MBP</n>). Subsequently, <n>TCF-1</n>-specific hybridomas could be prepared from the spleens of immunized knock-out mice. This study illustrates the almost complete tolerance of mice for human <n>TCF-1</n> and demonstrates that this tolerance is readily broken by gene knock-out. Furthermore, the usefulness of knock-out mice for the generation of monoclonal antibodies against the gene product of interest is underscored.
95286632>The transcription factor, <n>Nm23H2</n>, binds to and activates the translocated c-myc allele in Burkitt's lymphoma. We have identified an in vivo footprint over the PuF site on the translocated c-myc allele in Burkitt's lymphoma cells. The PuF site on the silent normal c-myc allele was unoccupied. We demonstrated by electrophoretic mobility shift assay, electrophoretic mobility shift assay with antibody, UV cross-linking followed by SDS-gel electrophoresis, and Western analysis that <n>Nm23H2</n> in B cell nuclear extracts bound to the c-myc PuF site. Transfection experiments with c- myc promoter constructs in both DHL-9 and Raji cells revealed that the PuF site functioned as a positive regulatory element in B cells with a drop in activity with mutation of this site. Access to this site is blocked in the normal silent c-myc allele; these data suggest that the <n>Nm23H2</n> protein is involved in deregulation of the translocated c-myc allele in Burkitt's lymphoma cells.
95279435>Identification of two novel regulatory elements within the 5'- untranslated region of the human A gamma-globin gene. Interaction between the stage selector element (SSE) in the proximal gamma-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the beta-globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5'-untranslated region of the gamma-gene also contributes to this competitive silencing of the beta- gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal gamma-promoter in the absence of the SSE, completely silencing a linked beta-promoter. Mutation of a 20-base pair sequence of the gamma-gene 5'- untranslated region (UTR) led to derepression of beta-promoter activity. A marked activation of gamma-promoter activity was also observed with this mutation, suggesting the presence of a repressor. Fine mutagenesis dissected these activities to different regions of the 5'-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5'-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor <n>GATA-1</n>. These results indicate that the 5'-UTR of the gamma-gene contains sequences that may be important for its transcriptional and developmental regulation.
95257963>Coupling of a signal response domain in <n>I kappa B alpha</n> to multiple pathways for <n>NF-kappa B</n> activation. The eukaryotic transcription factor <n>NF-kappa B</n> 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 <n>NF-kappa B</n> 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 <n>NF-kappa B</n>-inducing agents, including T-cell mitogens, proinflammatory cytokines, and viral transactivators such as the <n>Tax protein</n> of human T-cell leukemia virus type 1. To explore these I kappa B alpha-dependent mechanisms for <n>NF- kappa B</n> induction, we identified novel mutants of <n>I kappa B alpha</n> that uncouple its inhibitory and signal-transducing functions in human T lymphocytes. Specifically, removal of the N-terminal 36 amino acids of <n>I kappa B alpha</n> failed to disrupt its ability to form latent complexes with <n>NF-kappa B</n> in the cytoplasm. However, this deletion mutation prevented the induced phosphorylation, degradative loss, and functional release of <n>I kappa B alpha</n> from <n>NF-kappa B</n> in <n>Tax</n>-expressing cells. Alanine substitutions introduced at two serine residues positioned within this N-terminal regulatory region of <n>I kappa B alpha</n> also yielded constitutive repressors that escaped from <n>Tax</n>-induced turnover and that potently inhibited immune activation pathways for <n>NF-kappa B</n> 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 <n>I kappa B alpha</n> that coordinately controls the biologic activities of <n>I kappa B alpha</n> and <n>NF-kappa B</n> in response to viral and immune stimuli.
95255485>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 <n>phytohemagglutinin</n> (<n>PHA</n>)-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 <n>prostate-specific antigen</n> (<n>PSA</n>) 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 <n>vimentin</n>, correlating to cell shape changes, cytokeratins 8 and 18, associated with differentiated cell types of prostate epithelia, and <n>neuron-specific enolase</n> 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.
95229576>Platelet-activating factor stimulates transcription of the <n>heparin- binding epidermal growth factor-like growth factor</n> in monocytes. Correlation with an increased kappa B binding activity. Human peripheral blood monocytes responded to stimulation of <n>platelet- activating factor</n> (<n>PAF</n>) with up-regulation of the transcript for <n>heparin-binding epidermal growth factor-like growth factor</n> (<n>HB-EGF</n>), a potent mitogen for vascular smooth muscle cells. This function of <n>PAF</n> was observed at nanomolar concentrations of the ligand, starting at 30 min after stimulation. The <n>PAF</n>-induced up-regulation of <n>HB-EGF</n> mRNA was accompanied by an increase in kappa B binding activity. These functions of <n>PAF</n> appeared to be mediated through the cell surface <n>PAF</n> receptors, as two <n>PAF</n> receptor antagonists, WEB 2086 and L-659,989, blocked both the up-regulation of <n>HB-EGF</n> mRNA and kappa B binding activity induced by <n>PAF</n>. The antagonists, however, had no effect on phorbol ester- induced up-regulation of <n>HB-EGF</n> mRNA and kappa B binding activity. Pretreatment of monocytes with pertussis toxin inhibited these functions of <n>PAF</n>, whereas cholera toxin had no inhibitory effect. Pyrrolidine dithiocarbamate, an inhibitor for <n>NF-kappa B</n> activation, markedly reduced <n>PAF</n>-stimulated kappa B binding activity as well as up- regulation of <n>HB-EGF</n> mRNA. These results suggest a potential role of <n>PAF</n> in <n>HB-EGF</n> expression and provide evidence that this stimulation may occur through increased kappa B binding activity.
95197681>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 <n>HLA-DQ</n> 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
95373165>Restoration of the <n>Epstein-Barr virus ZEBRA protein</n>'s capacity to disrupt latency by the addition of heterologous activation regions. The <n>ZEBRA protein</n> has a unique biological function among herpesviral proteins. It is responsible for the disruption of Epstein-Barr virus (EBV) latency and the induction of the lytic cycle. <n>ZEBRA</n> is a bZIP transcriptional activator which binds as a dimer to 7-bp response elements within EBV promoters and is directly involved in the stimulation of virus replication at the EBV lytic origin. We have employed the <n>ZEBRA</n>/EBV biological system to test whether a heterologous activation domain can substitute for another activation domain (the <n>ZEBRA</n> domain). The <n>ZEBRA</n> activation region was replaced with the potent acid activation region from the <n>herpes simplex virus VP16 protein</n> or with the activation region of the <n>EBV R protein</n>. Both chimeras were found to transactivate model and native promoters at equivalent or better levels than <n>ZEBRA</n> itself. Activation was not target- or cell- type dependent, nor was it dependent on the presence of virus. These activation domains restored <n>ZEBRA</n>'s ability to induce early antigen and to stimulate origin replication to levels that were equal to or greater than those of wild type. These studies suggest that the specificities of some of the known biological functions of <n>ZEBRA</n> are not dependent upon the nature of the activation domain present within <n>ZEBRA</n>.
95362854><n>Interleukin 4</n> 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 <n>Janus kinase 3</n> and <n>interleukin-4 Stat</n>. Germ line C transcripts can be induced by <n>IL-4</n> 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 <n>IL-4</n> induced a binding activity in the cytosol and nucleus of BL-2 cells. This factor was designated <n>IL-4</n> NAF (<n>IL-4</n>-induced nuclear-activating factors) and was identified as a tyrosine phosphoprotein, which translocates from the cytosol to the nucleus upon <n>IL-4</n> 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 <n>IL-4</n> Stat, also known as <n>Stat6</n>, but not antibodies to other Stat proteins, interfere with the formation of the <n>IL-4</n> NAF complex. Congruous with the involvement of a Stat protein, <n>IL-4</n> induced robust <n>Janus kinase 3</n> (<n>JAK3</n>) activity in BL- 2 cells. Cotransfection of <n>JAK3</n> with <n>IL-4</n> Stat into COS-7 cells produced an intracellular activity which bound the same IFN-gamma activation site-like sequence and comigrated with <n>IL-4</n> NAF in electrophoretic mobility shift assay. These results show that <n>IL-4</n> NAF is <n>IL-4</n> Stat, which is activated by <n>JAK3</n> in response to <n>IL-4</n> receptor engagement.
95329724>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 <n>TAL1</n> 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 <n>TAL1</n> promotes the development of T-ALL. In this report, we show that <n>TAL1</n> is expressed in the leukemic cells of most patients with T-ALL, including many that do not display an apparent <n>TAL1</n> gene alteration. A polymorphic dinucleotide repeat in the transcribed sequences of <n>TAL1</n> was used to determine the allele specificity of <n>TAL1</n> transcription in primary T-ALL cells. Monoallelic expression of <n>TAL1</n> was observed in the leukemic cells of all patients (8 of 8) bearing a <n>TAL1</n> gene rearrangement. In the leukemic cells of patients without detectable <n>TAL1</n> rearrangements, <n>TAL1</n> transcription occurred in either a monoallelic (3 of 7 patients) or a biallelic (4 of 7 patients) fashion. Thus, <n>TAL1</n> activation in these patients may result from subtle alterations in cis-acting regulatory sequences (affecting expression of a single <n>TAL1</n> allele) or changes in trans-acting factors that control <n>TAL1</n> transcription (affecting expression of both <n>TAL1</n> alleles).
95374911>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 <n>erythroid delta-aminolevulinate synthase</n> 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 <n>GATA-1</n> and <n>GATA-2</n> are expressed in all these cases. These results suggest that M7 blasts and TMD blasts correspond to the erythroid/megakaryocytic bipotential progenitor cells.
95308691>Expression of <n>Ah receptor</n> (<n>TCDD receptor</n>) during human monocytic differentiation. We have previously found a high expression of human <n>Ah receptor</n> (<n>TCDD receptor</n>) 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 <n>Ah receptor</n> gene in the monocyte fraction. Then the expression levels of AhR mRNA in various hematopoietic cell lines were examined together with those of <n>Arnt</n> and <n>P450IA1</n>. <n>AhR</n> 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 <n>AhR</n> 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 <n>AhR</n> mRNA. The incubation with <n>transforming growth factor beta 1</n> and 1 alpha,25-dihydroxyvitamin D3 resulted in a 5- to 7-fold increase of <n>AhR</n> mRNA. The HEL cells also exhibited a similar elevation of <n>AhR</n> 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 <n>AhR</n>, induced the expression of the <n>P450IA1</n> gene. These results indicated that expression of <n>AhR</n> mRNA was significantly induced during monocytic differentiation and that the differentiated cells were responsive to xenobiotics. Our results suggest that <n>AhR</n> may play an important role in the function of monocytes and also in the eventual activation of environmental carcinogens.
95261059>Neutrophils and monocytes express high levels of <n>PU.1</n> (<n>Spi-1</n>) but not <n>Spi-B</n>. <n>PU.1</n> (the Spi-1 oncogene) and <n>Spi-B</n> are closely related members of the ets transcription factor family, sharing similar DNA binding specificities mediated by similar DNA binding domains. <n>PU.1</n> and <n>Spi-B</n> have been previously described as being predominantly expressed coordinately in macrophages and B cells, but their expression in early hematopoietic stages and during the course of myeloid differentiation to monocytes and macrophages or to neutrophils has not been extensively investigated. Here, we report that <n>PU.1</n> mRNA is upregulated during myeloid differentiation of human purified CD34+ cells and murine multipotential FDCP-mix A4 cells, suggesting that <n>PU.1</n> is upregulated as an early event during differentiation of multipotential progenitor cells. <n>PU.1</n> expression is maintained at stable levels during differentiation of myeloid cell lines U937 and HL-60 to monocytic and neutrophilic cells. <n>PU.1</n> is expressed at highest levels in mature human monocytes and human peripheral blood neutrophils. In contrast to <n>PU.1</n>, significant levels of <n>Spi-B</n> mRNA and protein are found only in some B- cell lines and spleen but are not found in myeloid cell lines, neutrophils, or macrophages. In vitro translated <n>Spi-B</n> protein can bind to <n>PU.1</n> binding sites in myeloid promoters and transactivate these promoters in nonmyeloid cells. Therefore, although <n>PU.1</n> and <n>Spi-B</n> may bind to similar DNA control elements and have redundancy of transactivation function in vitro, the lack of significant levels of <n>Spi-B</n> in myeloid cells makes it unlikely that <n>Spi-B</n> plays a significant role in myeloid lineage development and gene expression. In contrast, <n>PU.1</n> is expressed at high levels not only in monocytes and macrophages but also in neutrophils, indicating that <n>PU.1</n> can activate gene expression in both major myeloid lineages.
95244883>Identification of a major positive regulatory element located 5' to the human zeta-globin gene. The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive <n>luciferase</n> expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta- globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta- <n>luciferase</n> constructs and the alpha-<n>luciferase</n> constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta- globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5' to the zeta- globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-<n>luciferase</n> constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a <n>GATA-1</n> site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 <n>GATA-1</n> site has a relatively low affinity for <n>GATA-1</n>. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5' to the zeta-globin mRNA cap site, is necessary for high- level promoter activity in K562 cells. This element requires <n>GATA-1</n> and additional unknown factors for maximal activity.
95247773>Analysis of the role of protein kinase C-alpha, -epsilon, and -zeta in T cell activation. T cells express multiple isotypes of <n>protein kinase C</n> (<n>PKC</n>) and although it is well accepted that <n>PKCs</n> have an important role in T cell activation, little is known about the function of individual PKC isotypes. To address this issue, mutationally active PKC-alpha, - epsilon, or -zeta have been transfected into T cells and the consequences for T cell activation determined. <n>p21ras</n> plays an essential role in T cell activation. Accordingly, the effects of the constitutively active PKCs were compared to the effects of mutationally activated <n>p21ras</n>. The data indicate that <n>PKC-epsilon</n> and, to a lesser extent <n>PKC-alpha</n> but not <n>-zeta</n>, can regulate the transcription factors <n>AP-1</n> and nuclear factor of activated T cells (<n>NF-AT-1</n>). The ability of <n>PKC-epsilon</n> to induce transactivation of <n>NF-AT-1</n> and <n>AP-1</n> was similar to the stimulatory effect of a constitutively activated <n>p21ras</n>. <n>PKC- epsilon</n>, but not <n>PKC-alpha</n> nor activated <n>p21ras</n>, was able to induce <n>NF- KB</n> activity. Phorbol esters induce expression of <n>CD69</n> whereas none of the activated PKC isotypes tested were able to have this effect. Activated Src and <n>p21ras</n> were able to induce <n>CD69</n> expression. These results indicate selective functions for different PKC isotypes in T cells. Moreover, the data comparing the effects of activated Ras and PKC mutants suggest that <n>PKC-alpha</n>, <n>p21ras</n>, and <n>PKC-epsilon</n> are not positioned linearly on a single signal transduction pathway.
95204946>Differences in binding of glucocorticoid receptor to DNA in steroid- resistant asthma. Although glucocorticosteroids are a very effective treatment for asthma and other chronic inflammatory diseases, a small proportion of patients are resistant to their therapeutic effects. The molecular mechanism for this steroid resistance is unclear. Steroid resistance cannot be explained by pharmacokinetic mechanisms, by a defect in the binding of steroids to glucocorticoid receptors, nor by defective nuclear translocation of this receptor, thereby suggesting that the molecular abnormality lies distal to nuclear translocation. We examined the ability of nuclear translocated glucocorticoid receptors to bind to their DNA binding sites (GRE) using electrophoretic mobility shift assays in PBMC from patients with steroid-sensitive and steroid- resistant asthma. The binding of the glucocorticoid receptor to DNA in these patients was also studied using Scatchard analysis. Dexamethasone induced a significant rapid and sustained twofold increase in GRE binding in PBMCs from steroid-sensitive asthmatic patients and nonasthmatic individuals, but this was markedly reduced in steroid- resistant asthmatic patients. Scatchard analysis of glucocorticoid receptor-GRE binding showed no change in binding affinity but did show a reduced number of receptors available for DNA binding in the steroid- resistant patients. These results suggest that the ability of the glucocorticoid receptor to bind to GRE is impaired in steroid-resistant patients because of a reduced number of receptors available for binding to DNA.
95195208><n>Interleukin-5</n> signaling in human eosinophils involves <n>JAK2</n> tyrosine kinase and <n>Stat1 alpha</n>. Signaling by a wide variety of cytokines, including interferons, interleukins, and growth factors, involves activation of JAK kinases and Stat (Signal transducers and activators of transcription) proteins. At present, not much is known about the molecular mechanisms by which <n>interleukin-5</n> (<n>IL-5</n>) exerts its diverse biologic effects. Human eosinophils are one of the most important target cells for <n>IL-5</n> and were used here to study <n>IL-5</n> signaling in a primary human cell. <n>IL-5</n> induced rapid and transient tyrosine phosphorylation of <n>JAK2</n>. Moreover, <n>IL-5</n> induced at least two DNA-binding complexes, using nuclear extracts from normal human eosinophils and the IL-6/interferon-gamma response element of the ICAM-1 promoter (ICAM-1 pIRE) in an electromobility shift assay. From supershift experiments it was concluded that one DNA- binding complex contained <n>Stat1 alpha</n>, probably as a homodimer. Both DNA-binding complexes were inhibited by a phosphotyrosine antibody (<n>4G10</n>), suggesting that tyrosine phosphorylation is required for complex formation. IL-3 and <n>granulocyte-macrophage colony-stimulating factor</n> induced, similar to <n>IL-5</n>, two DNA-binding complexes in human eosinophils, including <n>Stat1 alpha</n>. These data show for the first time that molecular mechanisms of <n>IL-5</n> signaling in human eosinophils involve members of the JAK kinase family as well as members of the Stat family
95373132>Infection and replication of Tat- human immunodeficiency viruses: genetic analyses of LTR and tat mutations in primary and long-term human lymphoid cells. <n>Tat</n> 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 <n>Tat</n> 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 <n>Tat</n> is absent. It is possible that <n>Tat</n> 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 <n>Tat</n> mutants containing wild-type LTRs was less efficient than that of the LTR-modified <n>Tat</n> 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, <n>Tat</n> 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 <n>Tat</n>- virus. The implications for in vivo HIV-1 replication and potential application of this system to the study of alternative <n>Tat</n> function are discussed.
95355848>Functional roles of the transcription factor <n>Oct-2A</n> and the high mobility group protein <n>I/Y</n> 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 <n>interferon-gamma</n> (<n>IFN-gamma</n>). Here we show that the lymphoid- specific transcription factor <n>Oct-2A</n> plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) <n>I/Y</n> binds to multiple sites within the DRA promoter, including the <n>Oct-2A</n> binding site. Coexpression of HMG <n>I/Y</n> 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 <n>I/Y</n> stimulates <n>Oct-2A</n> binding to the HLA-DRA promoter. Thus, <n>Oct-2A</n> and HMG <n>I/Y</n> may synergize to activate HLA-DRA expression in B cells. By contrast, <n>Oct-2A</n> is not involved in the <n>IFN-gamma</n> induction of the HLA- DRA gene in HeLa cells, but antisense HMG <n>I/Y</n> 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 <n>HMG I/Y</n> may be important for B cell-specific expression, and is essential for <n>IFN-gamma</n> induction.
95329723>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 <n>EBV-encoded nuclear antigens</n> (EBNA1, 2, 3A, 3B, 3C, -LP), three latent