C/EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells

In atherosclerosis,oxidative stress-induced vascular smooth muscle cells (smcs) dysfunction is partially mediated by up-regulated nadph oxidase (nox); the mechanisms of enzyme regulation are not entirely defined. ccaat/enhancer-binding proteins (c/ebp) regulate cellular proliferation and differentiation,and the expression of many inflammatory and immune genes. we aimed at elucidating the role of c/ebp in the regulation of nox in smcs exposed to pro-inflammatory conditions. human aortic smcs were treated with interferon-γ (ifn-γ) for up to 24 hrs. lucigenin-enhanced chemiluminescence,real-time pcr,western blot,promoter-luciferase reporter analysis and chromatin immunoprecipitation assays were employed to investigate nox regulation. ifn-γ dose-dependently induced nox activity and expression,nuclear translocation and up-regulation of c/ebpα,c/ebpβ and c/ebpδ protein expression levels. silencing of c/ebpα,c/ebpβ or c/ebpδ reduced significantly but differentially the ifn-γ-induced up-regulation of nox activity,gene and protein expression. in silico analysis indicated the existence of typical c/ebp sites within nox1,nox4 and nox5 promoters. transient overexpression of c/ebpα,c/ebpβ or c/ebpδ enhanced the luciferase level directed by the promoters of the nox subtypes. chromatin immunoprecipitation demonstrated the physical interaction of c/ebpα,c/ebpβ and c/ebpδ proteins with the nox1/4/5 promoters. c/ebp transcription factors are important regulators of nox enzymes in ifn-γ-exposed smcs. activation of c/ebp may induce excessive nox-derived reactive oxygen species formation,further contributing to smcs dysfunction and atherosclerotic plaque development. pharmacological targeting of c/ebp-related signalling pathways may be used to counteract the adverse effects of oxidative stress. © 2014 the authors.