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Role of p38 MAP Kinase in the Regulation of Inflammatory Gene Expression

p38 MAP 激酶在炎症基因表达调节中的作用

基本信息

批准号：
17591949
负责人：
OHMORI Yoshihiro
金额：
$ 2.3万
依托单位：
Meikai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591949/
关键词：
Chemokine Transcription factor Phosphorylation MAP kinase NF-κB STAT LPS Interferon interferon

项目摘要

Lipopolysaccharide (LPS) and interferon gamma (IFNγ) have been show to cooperatively regulate the expression of many macrophage genes such as chemokines. Although the transcriptional regulation of these inflammatory genes has been regulated by transcription factors NF-κB and STAT1, the role of protein kinase induced by LPS and IFNγ in the transcriptional regulation has not been fully understood. In the present study, we performed a series of experiments to determine the functional role of p38 MAP kinase in the transcriptional regulation of the chemokine CXCL9 gene in macrophage cell line RAW264.7 cells, and obtained the following results.1) To determine whether p38 MAP kinase regulates LPS-induced NF-κB-dependent transcriptional activity, we constructed an expression vector in which NF-κB RelA transactivation domain fused to the GAL4 DNA-binding domain, co-transfected with a luciferase reporter construct containing GAL4 DNA-binding sequences into RAW264.7 cells, and assessed for the lu … More ciferase activity. The results demonstrated that stimulation with LPS enhanced the RelA-dependent transcriptional activity and the enhanced activity was suppressed by SB203580, an inhibitor of p38MAPK.2) Chromatin immunoprecipitation analysis demonstrated that co-treatment of RAW264.7 cells with IFNγ and LPS cooperatively recruited STAT1, RelA and RNA polymerase II at the promoter region of the CXCL9 gene and that this cooperative recruitment was suppressed by SB203580.3) Phosphorylation on Ser727 of STAT1 was additively enhanced by co-treatment with LPS and IFNγ, though the magnitude of induction was not synergistic as seen in the transcriptional activity of the CXCL9 gene induced by LPS and IFNγ.4) Anisomycin, a p38 MAPK activator which enhances Ser727 phosphorylation but has no effect on NF-κB activation, only marginally potentiated IFNγinduced CXCL9 gene expression, suggesting that phosphorylation on Ser727 alone is not sufficient for the synergistic induction of CXCL9 gene.These results suggest p38MAPK regulates the STAT1- and RelA-mediated enhanceosome formation at the promoter/enhance region of the CXCL9 gene in IFNγ and LPS-stimulated macrophages, which formation then leads to the synergistic transcriptional activity. Less

脂多糖（Lipopolysaccharide，LPS）和干扰素γ（interferon γ，IFNγ）协同调节巨噬细胞趋化因子等基因的表达。虽然这些炎症基因的转录调控受转录因子NF-κB和STAT 1的调控，但LPS和IFNγ诱导的蛋白激酶在转录调控中的作用尚未完全清楚。在本研究中，我们进行了一系列的实验来确定p38 MAP激酶在巨噬细胞系RAW264.7细胞中趋化因子CXCL 9基因转录调节中的功能作用，并获得了以下结果：1）为了确定p38 MAP激酶是否调节LPS诱导的NF-κ B依赖的转录活性，我们构建了一个表达载体，其中NF-κB RelA反式激活结构域与GAL 4 DNA结合结构域融合，与含有GAL 4 DNA结合序列的荧光素酶报告基因构建体共转染到RAW 264.7细胞中，并评估了lu ...更多信息 ciferase活性结果表明，LPS刺激可增强Rel A依赖的转录活性，而p38 MAPK抑制剂SB 203580可抑制这种活性。2）染色质免疫沉淀分析表明，IFNγ和LPS共同处理RAW 264. 7细胞后，STAT 1协同募集，在CXCL 9基因的启动子区域的RelA和RNA聚合酶II，并且这种合作募集被SB 203580抑制。用LPS和IFNγ处理，尽管诱导的幅度不是协同的，如在LPS和IFNγ诱导的CXCL 9基因的转录活性中所见。4）茴香霉素，一种增强Ser 727磷酸化但对NF-κB活化没有影响的p38 MAPK活化剂，仅略微增强IFNγ诱导的CXCL 9基因表达，这些结果表明p38 MAPK在启动子区调控STAT 1和RelA介导的增强体形成，而在启动子区调控增强体形成。在IFNγ和LPS刺激的巨噬细胞中，CXCL 9基因的增强区，其形成然后导致协同转录活性。少