Promoter elements responsible for antioxidant regulation of MCP-1 gene expression

Monocyte chemoattractant protein-1 (MCP-1) is produced by different cells in response to inflammatory stimulation. In the present study, a series of human MCP-1 promoter reporter genes were constructed to illustrate elements involved in antioxidant dimethyl sulfoxide (DMSO) inhibition of MCP-1 gene expression. MCPI secretion and mRNA expression and transcription activity stimulated by TNF-alpha or IL-1 beta were significantly inhibited by 1% DMSO in alveolar type II epithelial cells (A549). Deletion of - 7537 to -2741 caused a 77 % decrease in reporter activity, but DMSO inhibition was still present. Deletion of -7537 to -2616 containing the Al NF-kappa B binding site resulted in a complete loss of MCP-1 stimulation. Deletion of -2585 to -74 decreased reporter activity by similar to 50%, and DMSO inhibited this induction. Deletion of -2614 to -74 containing the A2 NF-kappa B binding site completely abolished responses to stimulation. Mutations of either of the NF kappa B binding sites decreased promoter activity, which could still be inhibited by DMSO, whereas deletion of both NF-kappa B binding sites abolished induced transcriptional activity. Mutation or deletion of the NF-kappa B binding sites significantly decreased or abolished reporter activity in response to reactive oxygen intermediates (ROI), generated by xanthine plus xanthine oxidase. In conclusion, DMSO inhibits MCP-1 gene expression through both NF-kappa B binding sites located far upstream of the 5'-flanking region of the MCP-1 promoter.