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.
Highlights
Compelling evidence indicates that the reactive oxygen species (ROS) produced by members of the NADPH oxidase (Nox) family are important regulators of key biological activities such as cell growth, proliferation, differentiation, migration and apoptosis [1,2,3]
We have demonstrated that the Nox activity and expression are up-regulated by interferon c (IFN-c) in human aortic smooth muscle cells (SMCs) by direct and indirect mechanisms involving signal transducer and activator of transcription (STAT), nuclear factor-kB (NF-kB) and activator protein-1 (AP-1) transcription factors [14, 15]
Quiescent SMCs were exposed to 5–100 ng/ ml IFN-c for 24 hrs and the NADPH-dependent O2À production was determined by lucigenin-enhanced chemiluminescence
Summary
Compelling evidence indicates that the reactive oxygen species (ROS) produced by members of the NADPH oxidase (Nox) family are important regulators of key biological activities such as cell growth, proliferation, differentiation, migration and apoptosis [1,2,3]. The uncover of the stream of signalling molecules responsible for Nox up-regulation and the ensuing ROS production may be used to define strategies to counteract the adverse effects of oxidative stress. Oxidative stress has been linked to the dysfunction of vascular smooth muscle cells (SMCs), which further promotes and accelerates the development of atherosclerotic lesions. Phenotypic alterations of SMCs represent a maladaptive response to vascular insults and are considered to be partially mediated by Nox-derived ROS [11, 12]. Smooth muscle cells express typically Nox, Nox and Nox subtypes that are differentially distributed within the cellular compartments and control important redox-sensitive signalling pathways [13]
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