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

脂多糖（LPS）和干扰素γ (IFNγ)协同调节巨噬细胞趋化因子等多种基因的表达。虽然这些炎症基因的转录调控受到转录因子NF-κB和STAT1的调控，但LPS和IFNγ诱导的蛋白激酶在转录调控中的作用尚不完全清楚。在本研究中，我们通过一系列实验来确定p38 MAP激酶在巨噬细胞系RAW264.7细胞中对趋化因子CXCL9基因转录调控中的功能作用，得到如下结果。1)为了确定p38 MAP激酶是否调控lps诱导的NF-κB依赖的转录活性，我们构建了NF-κB RelA转激活域融合到GAL4 dna结合域的表达载体，并将含有GAL4 dna结合序列的荧光素酶报告基因构建体共转染RAW264.7细胞，并评估了p38 MAP激酶是否调控lps诱导的NF-κB依赖的转录活性。结果表明，LPS刺激增强了rela依赖性的转录活性，而这种增强的活性被p38MAPK抑制剂SB203580抑制。2)染色质免疫沉淀分析表明，IFNγ和LPS共处理RAW264.7细胞时，在CXCL9基因启动子区域协同募集STAT1、RelA和RNA聚合酶II，这种协同募集被SB203580.3抑制。尽管从LPS和IFNγ诱导的CXCL9基因转录活性中可以看出，诱导的程度并不是协同的。4) Anisomycin是一种p38 MAPK激活剂，能增强Ser727的磷酸化作用，但对NF-κB的激活没有影响，仅能微弱增强ifn γ诱导的CXCL9基因表达，提示仅磷酸化Ser727不足以协同诱导CXCL9基因。这些结果表明，在IFNγ和lps刺激的巨噬细胞中，p38MAPK调节STAT1-和rela介导的CXCL9基因启动子/增强区增强体的形成，从而导致协同转录活性。少

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