Regulation of NADPH oxidase subunit p22phox by NF-kB in human aortic smooth muscle cells

Abstract

Accumulating evidence demonstrates the involvement of oxidative stress in the pathophysiology of cardiovascular diseases. The molecular mechanisms accountable for the increased production of reactive oxygen species remain uncertain. Among others, NADPH oxidase is one of the most important sources of superoxide in vascular cells. Here we investigate the role of NF-kB in the regulation of p22phox subunit and NADPH oxidase activity, in human aortic smooth muscle cells. Overexpression of p65/RelA or IKKβ up-regulated p22phox gene promoter activity. Transcription factor pull-down assays demonstrated the physical interaction of p65/RelA protein with predicted NF-kB binding sites. Real time PCR and Western blotting analysis showed that p22phox mRNA and protein expression are significantly down-regulated by NF-kB decoy oligodeoxynucleotides and N-alpha-tosyl-l-phenylalanine chloromethyl ketone (TPCK). Lucigenin-enhanced chemiluminescence assay revealed that NF-kB inhibitors reduce the NADPH-dependent superoxide production. Regulation of NADPH oxidase by NF-kB may represent a possible mechanism whereby pro-inflammatory factors induce oxidative stress in atherosclerosis, hypertension, diabetes, stroke or heart failure.