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- (IFN-) for up to 24hrs. Lucigenin-enhanced chemiluminescence, real-time PCR, Western blot, promoter-luciferase reporter analysis and chromatin immunoprecipitation assays were employed to investigate Nox regulation. IFN- dose-dependently induced Nox activity and expression, nuclear translocation and up-regulation of C/EBP, C/EBP and C/EBP protein expression levels. Silencing of C/EBP, C/EBP or C/EBP reduced significantly but differentially the IFN--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/EBP, C/EBP or C/EBP enhanced the luciferase level directed by the promoters of the Nox subtypes. Chromatin immunoprecipitation demonstrated the physical interaction of C/EBP, C/EBP and C/EBP proteins with the Nox1/4/5 promoters. C/EBP transcription factors are important regulators of Nox enzymes in IFN--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.