Ping D, Boekhoudt G H, Rogers E M, Boss J M
Department of Microbiology and Immunology, Emory University School of Medicine, Emory University, Atlanta, GA 30322, USA.
J Immunol. 1999 Jan 15;162(2):727-34.
TNF-alpha transcriptionally regulates murine monocyte chemoattractant protein-1 (MCP-1) expression. Three approaches were used to determine the mechanism by which TNF regulates MCP-1. Mutation analysis showed that two distal kappa B sites, a novel dimethylsulfate-hypersensitive sequence, and a promoter proximal SP-1 site were required for TNF induction. Although the kappa B sites and the hypersensitive sequence function as a NF-kappa B-mediated enhancer, regulating induction by TNF, stereospecific alignment of the kappa B sites was not critical. Trans-activation studies conducted by cotransfection of p50 and/or p65 expression vectors with MCP-1 constructions showed that TNF regulates MCP-1 through NF-kappa B. Examination of MCP-1 induction in NF-kappa B-disrupted embryonic fibroblasts showed that p65 was necessary for both the induction and the TNF-induced protein occupancy of the enhancer in vivo. The action of the antioxidant inhibitor of NF-kappa B activation, pyrrolidine dithiocarbamate, in wild-type and NF-kappa B mutant cells was examined. The results suggested that TNF activates NF-kappa B through both pyrrolidine dithiocarbamate-sensitive and -insensitive mechanisms. This study illustrates the crucial role for NF-kappa B p65 in the induction of the MCP-1 gene by TNF and in the assembly of a NF-kappa B dependent enhancer in vivo.
肿瘤坏死因子-α（TNF-α）通过转录调控小鼠单核细胞趋化蛋白-1（MCP-1）的表达。本研究采用了三种方法来确定TNF调节MCP-1的机制。突变分析表明，TNF诱导需要两个远端κB位点、一个新的硫酸二甲酯超敏序列和一个启动子近端SP-1位点。尽管κB位点和超敏序列作为NF-κB介导的增强子发挥作用，调节TNF的诱导，但κB位点的立体特异性排列并不关键。通过将p50和/或p65表达载体与MCP-1构建体共转染进行的反式激活研究表明，TNF通过NF-κB调节MCP-1。在NF-κB缺失的胚胎成纤维细胞中检测MCP-1的诱导情况，结果显示p65对于体内增强子的诱导和TNF诱导的蛋白占据都是必需的。研究了NF-κB激活的抗氧化剂抑制剂吡咯烷二硫代氨基甲酸盐在野生型和NF-κB突变细胞中的作用。结果表明，TNF通过吡咯烷二硫代氨基甲酸盐敏感和不敏感的机制激活NF-κB。本研究阐明了NF-κB p65在TNF诱导MCP-1基因以及体内组装NF-κB依赖性增强子过程中的关键作用。
