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. MCP-1 secretion and mRNA expression and transcription activity stimulated by TNF-alpha or IL-1beta 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-kappaB binding site resulted in a complete loss of MCP-1 stimulation. Deletion of -2585 to -74 decreased reporter activity by approximately 50%, and DMSO inhibited this induction. Deletion of -2614 to -74 containing the A2 NF-kappaB binding site completely abolished responses to stimulation. Mutations of either of the NF-kappaB binding sites decreased promoter activity, which could still be inhibited by DMSO, whereas deletion of both NF-kappaB binding sites abolished induced transcriptional activity. Mutation or deletion of the NF-kappaB 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-kappaB binding sites located far upstream of the 5'-flanking region of the MCP-1 promoter.
Xing
Liyu
L
Department of Pathology University of Michigan, Medical School, Ann Arbor, Michigan 48109, USA.
Remick
Daniel G
DG
D000975:Antioxidants / Q000494:pharmacology*; D002460:Cell Line; D018932:Chemokine CCL2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D015850:Interleukin-6 / Q000032:analysis / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D008156:Luciferases / Q000378:metabolism; D008168:Lung / Q000166:cytology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000975:Antioxidants; D018932:Chemokine CCL2; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; D004121:Dimethyl Sulfoxide