STAT1 pathway is involved in activation of caprine arthritis-encephalitis virus long terminal repeat in monocytes.

The caprine arthritis-encephalitis virus (CAEV) long terminal repeat (LTR) is activated by gamma interferon (IFN-gamma) in promonocytic cells. We have previously shown that a 70-bp element is necessary and sufficient for the response of the CAEV LTR to this cytokine. At the 5' end, this 70-bp IFN-gamma response element contains sequence similarity to the gamma activated site (GAS). Here we demonstrate that the putative GAS element in the CAEV LTR binds specifically to a cellular factor induced by IFN-gamma in promonocytic cells. Substitution mutations in this consensus sequence eliminate binding of the inducible factor. The GAS element from the 70-bp motif is sufficient to confer responsiveness to IFN-gamma using a heterologous minimal promoter. Consistent with the binding data, the same mutations in the GAS element eliminate responsiveness to IFN-gamma in the context of both a functional CAEV LTR and a heterologous promoter. The cellular factor that binds to the GAS element is present from 5 min to 14 h after stimulation with IFN-gamma. Binding of the nuclear factor to the GAS element in the CAEV LTR is inhibited by antibody directed against STAT1 (p91/84). Thus, the GAS sequence in the CAEV LTR is essential for the response to IFN-gamma and a STAT1-like factor binds to this site. The STAT-1 signaling pathway provides at least one mechanism for activation of the CAEV LTR by IFN-gamma in monocytes. These data are the first demonstration of a role for a STAT family member in the regulation of a viral promoter.     

信号转导和转录活化因子3通过趋化因子CX3C配体1促进血管内皮细胞增殖迁移

信号转导和转录活化因子3 (STAT3)与趋化因子CX3C配体1 (Fractalkine/CX3CL1)在血管炎症和损伤中起重要作用,为了探讨STAT3是否通过CX3CL1促进血管内皮细胞增殖和迁移,在血管内皮细胞(HUVEC)中过表达或敲降STAT3,通过quantitative real-time PCR、Western blotting实验确定STAT3对CX3CL1表达的影响。构建含有STAT3结合位点及突变STAT3结合位点的CX3CL1启动子荧光素酶报告基因质粒,利用荧光素酶活性分析实验研究STAT3对CX3CL1启动子转录活性的作用。利用MTT实验检测过表达或敲降STAT3或CX3CL1对血管内皮细胞增殖率的影响。利用划痕实验检测过表达或敲降STAT3或CX3CL1对血管内皮细胞迁移率的影响。结果显示,过表达STAT3可以促进CX3CL1表达,敲降STAT3可以使CX3CL1表达下调。STAT3可以直接结合到CX3CL1的启动子促进其转录激活,其促进作用依赖于CX3CL1启动子上的GAS位点。敲降STAT3可以抑制血管内皮细胞的迁移,过表达CX3CL1拮抗该抑制作用。总结得出,STAT3通过结合到CXCL1启动子促进CX3CL1转录与表达进而促进血管内皮的增殖与迁移。     

Enhancer role of STAT5 in CD2 activation of IFN-gamma gene expression

IFN-gamma is an important immunoregulatory protein with tightly controlled expression in activated T and NK cells. Three potential STAT binding regions have been recognized within the IFN-gamma promoter: 1) an IL-12-mediated STAT4 binding site at -236 bp; 2) a newly identified IL-2-induced STAT5 binding element at -3.6 kb; and 3) CD2-mediated STAT1 and STAT4 binding to an intronic element in mucosal T cells. However, functional activation of these sites remains unclear. In this study we demonstrate CD2-mediated activation of the newly characterized -3.6-kb IFN-gamma STAT5 binding region. CD2 signaling of human PBMC results in activation of the -3.6-kb IFN-gamma promoter, whereas mutation of the -3.6-kb STAT5 site attenuates promoter activity. Functional activation is accompanied by STAT5A but little STAT5B nucleoprotein binding to the IFN-gamma STAT5 site, as determined by competition and supershift assays. STAT5 activation via CD2 occurs independent of IL-2. Western and FACS analysis shows increased phospho-STAT5 following CD2 signaling. AG490, a tyrosine kinase inhibitor affecting Jak proteins, inhibits CD2-mediated IFN-gamma mRNA expression, secretion, and nucleoprotein binding to the IFN-gamma STAT5 site in a dose-dependent fashion. This report is the first to describe CD2-mediated activation of STAT5 and supports STAT5 involvement in regulation of IFN-gamma expression.     

Interferon-gamma-induced epithelial ICAM-1 expression and monocyte adhesion. Involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway.

Interferon-gamma (IFN-gamma) induced intercellular adhesion molecule-1 (ICAM-1) expression in human NCI-H292 epithelial cells, as shown by enzyme-linked immunosorbent assay and immunofluorescence staining. The enhanced ICAM-1 expression resulted in increased adhesion of U937 cells to NCI-H292 cells. Tyrosine kinase inhibitors (genistein or herbimycin), Src family inhibitor (PP2), or a phosphatidylinositol-phospholipase C inhibitor (U73122) attenuated the IFN-gamma-induced ICAM-1 expression. Protein kinase C (PKC) inhibitors (staurosporine or Ro 31-8220) also inhibited IFN-gamma-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression; this effect was inhibited by tyrosine kinase or Src inhibitor. ICAM-1 promoter activity was enhanced by IFN-gamma and TPA in cells transfected with pIC339-Luc, containing the downstream NF-kappaB and gamma-activated site (GAS) sites, but not in cells transfected with GAS-deletion mutant, pIC135 (DeltaAP2). Electrophoretic gel mobility shift assay demonstrated that GAS-binding complexes in IFN-gamma-stimulated cells contained STAT1alpha. The IFN-gamma-induced ICAM-1 promoter activity was inhibited by tyrosine kinase inhibitors, a phosphatidylinositol-phospholipase C inhibitor, or PKC inhibitors, and the TPA-induced ICAM-1 promoter activity was also inhibited by tyrosine kinase inhibitors. Cotransfection with a PLC-gamma2 mutant inhibited IFN-gamma- but not TPA-induced ICAM-1 promoter activity. However, cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited both IFN-gamma- and TPA-induced ICAM-1 promoter activity. The ICAM-1 promoter activity was stimulated by cotransfection with wild type PLC-gamma2, PKCalpha, c-Src, JAK1, or STAT1. An immunocomplex kinase assay showed that both IFN-gamma and TPA activated c-Src and Lyn activities and that these effects were inhibited by staurosporine and herbimycin. Thus, in NCI-H292 epithelial cells, IFN-gamma activates PLC-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and c-Src or Lyn, resulting in activation of STAT1alpha, and GAS in the ICAM-1 promoter, followed by initiation of ICAM-1 expression and monocyte adhesion.     

Inhibition of IFN-gamma-induced janus kinase-1-STAT1 activation in macrophages by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide

The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two immunomodulatory neuropeptides that affect both innate and acquired immunity, down-regulate IL-12 p40 and inducible NO synthase expression in LPS/IFN-gamma-stimulated macrophages. We showed previously that VIP/PACAP inhibit NF-kappaB nuclear translocation through the stabilization of IkappaB and reduce IFN regulatory factor-1 (IRF-1) binding to the regulatory elements found in the IL-12 p40 and inducible NO synthase promoters. In this paper we studied the molecular mechanisms involved in the VIP/PACAP regulation of IRF-1 transactivating activity. Our studies indicate that the inhibition in IRF-1 binding correlates with a reduction in IRF-1 protein and mRNA in IFN-gamma-treated Raw 264.7 macrophages. In agreement with the described Janus kinase (Jak)1/Jak2/STAT1/IRF-1 activation pathway, VIP/PACAP inhibit Jak1/Jak2, STAT1 phosphorylation, and the binding of STAT1 to the GAS sequence motif in the IRF-1 promoter. The effects of VIP/PACAP are mediated through the specific VIP/PACAP receptor-1 and the cAMP/protein kinase A (PKA) transduction pathway, but not through the induction of suppressor of cytokine signaling-1 or suppressor of cytokine signaling-3. Because IFN-gamma is a major stimulator of innate immune responses in vivo, the down-regulation of IFN-gamma-induced gene expression by VIP and PACAP could represent a significant element in the regulation of the inflammatory response by endogenous neuropeptides.