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型的细胞因子和细胞外刺激之间的串扰进行的。干扰素γ (IFNγ)和肿瘤坏死因子α (TNFα)或脂多糖（LPS）经常协同调节许多炎症基因的表达。在本研究中，我们探索了协同激活因子creb结合蛋白（CBP）通过整合IFNγ/STATI和TNFα/NF-κB信号通路之间的串导，共同诱导CXCL9基因的转录激活的机制。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多余的，但n端和c端区域对于转录协同作用是必要的。共免疫沉淀实验显示STAT1和NF-κB RelA（p65）在体内同时与CBP相关。此外，染色质免疫沉淀显示，IFNγ和TNFα的共刺激导致cxcl9基因启动子区域STAT1、CBP和RNA聚合酶II的募集增加。这些结果表明，STAT1/NF-κB依赖的转录协同作用是通过CBP与STAT1和NF-κB同时相互作用，增强RNA聚合酶II复合物向启动子的募集。在这项研究中，我们还研究了丝裂原活化蛋白（MAP）激酶在LPS和ifn γ刺激的巨噬细胞中STAT1和NE-κ b介导的转录协同作用中的作用。P38 MAP激酶抑制剂SB203580显著抑制LPS和IFNγ刺激的巨噬细胞样RAW264.7细胞中CXCL9 mRNA的表达。由于使用CXCL9启动子构建的报告基因分析也表明，SB203580抑制了IFNγ和lps诱导的协同启动子活性，因此抑制作用在转录水平上。这些结果表明p38 MAP激酶参与了ifn γ和lps诱导的转录协同作用。最后，我们评估了IFNγ诱导的CXCL9和CXCL10基因在几种人肿瘤细胞系中的表达，发现这些趋化因子基因在人鳞癌Ca9-22和胶质瘤细胞系A172中不被IFNγ诱导。然而，表达的损害不是由于ifn γ激活的STAT1的任何缺陷。相反，在表达ifn γ诱导的CXCL9和CXCL10基因的细胞中可见的构成性NF-κB活性在Ca9-22和A172细胞中不存在。我们的数据表明，组成性NF-κB活性是ifn γ诱导的抗肿瘤趋化因子CXCL9和CXCL10表达的关键决定因素，而组成性NF-κB活性的下调可能会抑制这些ifn γ诱导的趋化因子在人肿瘤细胞中的表达。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