title: Positive regulation of NADPH oxidase 5 by proinflammatory-related mechanisms in human aortic smooth muscle cells.
abstract: NADPH oxidase Nox5 subtype expression is significantly increased in vascular smooth muscle cells (SMCs) underlying fibro-lipid atherosclerotic lesions. The mechanisms that up-regulate Nox5 are not understood. Consequently, we characterized the promoter of the human Nox5 gene and investigated the role of various proinflammatory transcription factors in the regulation of Nox5 in human aortic SMCs. The Nox5 promoter was cloned in the pGL3 basic reporter vector. Functional analysis was done employing 5' deletion mutants to identify the sequences necessary to effect high levels of expression in SMCs. Transcriptional initiation site was detected by rapid amplification of the 5'-cDNA ends. In silico analysis indicated the existence of typical NF-kB, AP-1, and STAT1/STAT3 sites. Transient overexpression of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 increased significantly the Nox5 promoter activity. Chromatin immunoprecipitation demonstrated the physical interaction of c-Jun/AP-1 and STAT1/STAT3 proteins with the Nox5 promoter. Lucigenin-enhanced chemiluminescence, real-time PCR, and Western blot assays showed that pharmacological inhibition and the silencing of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 reduced significantly the interferon γ-induced Ca(2+)-dependent Nox activity and Nox5 expression. Up-regulated Nox5 correlated with increases in intracellular Ca(2+), an essential condition for Nox5 activity. NF-kB, AP-1, and STAT1/STAT3 are important regulators of Nox5 in SMCs by either direct or indirect mechanisms. Overexpressed Nox5 may generate free radicals in excess, further contributing to SMCs dysfunction in atherosclerosis.
authors.affiliation: Petru Poni Institute of Macromolecular Chemistry of the Romanian Academy, 41A, Grigore Ghica Voda Alley, 700487, Iasi, Romania. adrian.manea@icbp.ro
authors.forename: Adrian
authors.initials: A
authors.lastname: Manea
authors.forename: Simona A
authors.initials: SA
authors.lastname: Manea
authors.forename: Irina C
authors.initials: IC
authors.lastname: Florea
authors.forename: Catalina M
authors.initials: CM
authors.lastname: Luca
authors.forename: Monica
authors.initials: M
authors.lastname: Raicu
mesh_terms: D000263:Adenylate Kinase / Q000378:metabolism*; D000818:Animals; D002319:Cardiovascular System / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress
chemical_list: D019255:NADPH Oxidases; D000263:Adenylate Kinase