Nuclear Factor-κB p65 Mediates the Assembly and Activation of the TNF-Responsive Element of the Murine Monocyte Chemoattractant-1 Gene

Abstract

Abstract TNF-α 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 κB sites, a novel dimethylsulfate-hypersensitive sequence, and a promoter proximal SP-1 site were required for TNF induction. Although the κB sites and the hypersensitive sequence function as a NF-κB-mediated enhancer, regulating induction by TNF, stereospecific alignment of the κB 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-κB. Examination of MCP-1 induction in NF-κB-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-κB activation, pyrrolidine dithiocarbamate, in wild-type and NF-κB mutant cells was examined. The results suggested that TNF activates NF-κB through both pyrrolidine dithiocarbamate-sensitive and -insensitive mechanisms. This study illustrates the crucial role for NF-κB p65 in the induction of the MCP-1 gene by TNF and in the assembly of a NF-κB dependent enhancer in vivo.