title: C/EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells.
abstract: 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.
authors.affiliation: Molecular and Cellular Pharmacology - Functional Genomics Laboratory, Institute of Cellular Biology and Pathology "Nicolae Simionescu" of the Romanian Academy, Bucharest, Romania.
authors.forename: Simona-Adriana
authors.initials: SA
authors.lastname: Manea
authors.forename: Andra
authors.initials: A
authors.lastname: Todirita
authors.forename: Monica
authors.initials: M
authors.lastname: Raicu
authors.forename: Adrian
authors.initials: A
authors.lastname: Manea
mesh_terms: D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D017097:Electric Impedance; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D007371:Interferon-gamma; D019255:NADPH Oxidases
keywords: C/EBP; NADPH oxidase; atherosclerosis; inflammation; oxidative stress