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

Authors

  • Simona-Adriana Manea,

    1. Molecular and Cellular Pharmacology – Functional Genomics Laboratory, Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Bucharest, Romania
    Search for more papers by this author
  • Andra Todirita,

    1. Molecular and Cellular Pharmacology – Functional Genomics Laboratory, Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Bucharest, Romania
    Search for more papers by this author
  • Monica Raicu,

    1. Molecular and Cellular Pharmacology – Functional Genomics Laboratory, Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Bucharest, Romania
    Search for more papers by this author
  • Adrian Manea

    Corresponding author
    1. Molecular and Cellular Pharmacology – Functional Genomics Laboratory, Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Bucharest, Romania
    • Correspondence to: Adrian MANEA, Ph.D.,

      Institute of Cellular Biology and Pathology “Nicolae Simionescu”, Molecular and Cellular Pharmacology – Functional Genomics Laboratory 8, B.P. Hasdeu Street, Bucharest 050568, Romania.

      Tel.: (+4021) 319 27 37

      Fax: (+4021) 319 45 19

      E-mail: adrian.manea@icbp.ro

    Search for more papers by this author

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.

Ancillary