Promoter Elements Responsible for Antioxidant Regulation of MCP-1 Gene Expression

Abstract

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. MCP-1 secretion and mRNA expression and transcription activity stimulated by TNF-α or IL-1β 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 A1 NF-κB binding site resulted in a complete loss of MCP-1 stimulation. Deletion of −2585 to −74 decreased reporter activity by ∼50%, and DMSO inhibited this induction. Deletion of −2614 to −74 containing the A2 NF-κB binding site completely abolished responses to stimulation. Mutations of either of the NF-κB binding sites decreased promoter activity, which could still be inhibited by DMSO, whereas deletion of both NF-κB binding sites abolished induced transcriptional activity. Mutation or deletion of the NF-κB 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-κB binding sites located far upstream of the 5′-flanking region of the MCP-1 promoter.