Open AccessC/ EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells
Author(s) -
Manea SimonaAdriana,
Todirita Andra,
Raicu Monica,
Manea Adrian
Publication year - 2014
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/jcmm.12289
Subject(s) - nox1 , nadph oxidase , nox4 , ccaat enhancer binding proteins , chromatin immunoprecipitation , promoter , transcription factor , microbiology and biotechnology , luciferase , gene silencing , biology , chemistry , gene expression , reactive oxygen species , biochemistry , nuclear protein , gene , transfection
In atherosclerosis, oxidative stress‐induced vascular smooth muscle cells ( SMC s) 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 SMC s exposed to pro‐inflammatory conditions. Human aortic SMC s 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/ EBP α, C/ EBP β and C/ EBP δ protein expression levels. Silencing of C/ EBP α, C/ EBP β or C/ EBP δ 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/ EBP α, C/ EBP β or C/ EBP δ enhanced the luciferase level directed by the promoters of the Nox subtypes. Chromatin immunoprecipitation demonstrated the physical interaction of C/ EBP α, C/ EBP β and C/ EBP δ proteins with the Nox1/4/5 promoters. C/ EBP transcription factors are important regulators of Nox enzymes in IFN ‐γ‐exposed SMC s. Activation of C/ EBP may induce excessive Nox‐derived reactive oxygen species formation, further contributing to SMC s dysfunction and atherosclerotic plaque development. Pharmacological targeting of C/ EBP ‐related signalling pathways may be used to counteract the adverse effects of oxidative stress.