Molecular Mechanisms of Inflammatory Gene Expression by Thanscriptional factors STAT1 and NF-κB

细胞因子介导的细胞间通讯通常是通过不同类别的细胞因子和细胞外刺激之间的串扰来协调的。干扰素γ和肿瘤坏死因子α或脂多糖经常协同调节多种炎症基因的表达。在本研究中，我们探讨了辅活化子CREB结合蛋白整合干扰素γ/STATI和肿瘤坏死因子α/核因子-κB信号通路之间的串扰，协同诱导γ诱导的趋化因子CXCL9基因转录激活的机制。对CBP突变体的实验表明，尽管CBP的组蛋白乙酰转移酶活性是必不可少的，但N-末端和C-末端区域是转录协同所必需的。免疫共沉淀实验表明，STAT1和NF-κB relA(P65)在体内同时与CBP结合。此外，染色质免疫沉淀显示，共刺激与…更多的干扰素γ和肿瘤坏死因子α导致STAT1、CBP和RNA聚合酶II在CXCL9基因启动子区域的募集增加。本研究还探讨了丝裂原活化蛋白(MAP)在STAT1和NE-κB介导的巨噬细胞转录协同中的作用，以及NE-κB介导的转录协同作用。P38MAPK抑制剂SB203580显著抑制脂多糖和干扰素γ刺激的巨噬细胞样RAW264.7细胞中CXCL9mRNA的表达。用CXCL9启动子构建的报告基因分析也表明，SB203580抑制了干扰素γ和脂多糖诱导的协同启动子活性。这些结果表明p38 MAPK参与了干扰素γ和脂多糖诱导的转录协同作用。最后，我们检测了干扰素γ诱导的几种人肿瘤细胞系中CXCL9和CXCL10基因的表达，发现这些趋化因子基因在人鳞癌细胞株Ca9-22和人脑胶质瘤细胞系A172中不受干扰素γ的诱导。然而，该表达的损害并不是由于干扰素γ激活的STAT1中的任何缺陷。相反，在表达干扰素κ诱导的CXCL9和CXCL10基因的细胞中发现的结构性NF-γB活性在Ca9-22和A172细胞中缺失。我们的结果表明，结构性的NF-κB活性是干扰素γ诱导的抗肿瘤趋化因子CXCL9和CXCL10表达的关键决定因素，下调结构性的NF-κB活性可能会抑制这些干扰素γ诱导的趋化因子在肿瘤细胞中的表达。较少

Cytokine-mediated intercellular communication is often orchestrated through crosstalk between different classes of cytokines and extracellular stimuli. Interferon gamma (IFNγ) and tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS) often cooperatively regulate the expression of many inflammatory gene expressions. In the present study we explored the mechanisms through which coactivator CREB-binding protein (CBP) integrates the crosstalk between IFNγ/STATI and TNFα/NF-κB signaling pathways to cooperatively induce the transcriptional activation of the gene for CXCL9, an IENγ-inducible chemokine. Experiments with CBP mutants indicated that the N-terminal and C-terminal regions were necessary for the transcriptional synergy, although the histone acetyltransferase activity of CBP was dispensable. Co-immunoprecipitation assay demonstrated that STAT1 and NF-κB RelA(p65) simultaneously associated with CBP in vivo. Furthermore, chromatin immunoprecipitation revealed that costimulation wi … More th IFNγ and TNFα resulted in increased recruitment of STAT1, CBP and RNA polymerase II at the promoter region of the CXCL 9 gene. These results demonstrate that the STAT1/NF-κB-dependent transcriptional synergy results from the enhanced recruitment of RNA polymerase II complex to the promoter via simultaneous interaction of CBP with STAT1 and NF-κB.In this study, we also examined the role of mitogen-activated protein (MAP) kinase in STAT1 and NE-κB-mediated transcriptional synergy in LPS and IFNγ-stimulated macrophages. P38 MAP kinase inhibitor SB203580 markedly suppressed expression of CXCL9 mRNA in macrophage-like RAW264.7 cells stimulated with LPS and IFNγ. The inhibitory effect was at transcriptional level because reporter gene analysis using a CXCL9 promoter construct also showed that IFNγ and LPS-induced synergistic promoter activity was suppressed by SB203580. These results indicate that p38 MAP kinase is involved in the IFNγ-and LPS-induced transcriptional synergy.Finally, we evaluated the IFNγ-induced expression of CXCL9 and CXCL10 genes in several human tumor cell lines and found that these chemokine genes were not inducible by IFNγ in human squamous carcinoma line Ca9-22 and human glioma cell line A172. However, the impairment for the expression was not due to any defect in IFNγ-activated STAT1. Instead, constitutive NF-κB activity, which was seen in cells that expressed the IFNγ-induced CXCL9 and CXCL10 genes, was absent in Ca9-22 and A172 cells. Our data indicate that constitutive NF-κB activity is a critical determinant for the expression of the IFNγ-induced anti-tumor chemokines CXCL9 and CXCL10 and that downregulation of constitutive NF-κB activity may inhibit the expression of these IFNγ-inducible chemokines in human tumor cells. Less