Molecular mechanisms of inducible nitric oxide synthase induction by interferon-γ in rat aortic smooth muscle cells

Abstract

The aim of this study is to investigate the molecular mechanisms of inducible nitric oxide synthase (NOS) induction by interferon-γ (IFN-γ) in rat aortic smooth muscle cells (RASMC) and the interactions between transcription factors and the rat NOS promoter that are responsible for the IFN-γ-induced NOS expression. Measurements of NOS activity levels (nitrite accumulation), NOS protein levels and NOS promoter activity demonstrated that IFN-γ enhanced the interleukin-1β, (IL-1β)-induced NOS expression in RASMC, but that IFN-γ alone had no effect. By electrophoretic mobility shift assay (EMSA), IFN-γ was shown to activate interferon regulatory factor I (IRF-1) and signal transducers and activators of transcription 1 (Stat1). However, it had no effect on nuclear factor kappa B (NF-κB) or CCAAT box/enhancer binding protein (C/EBP) activation. Conversely, IL-1β activated NF-κB and C/EBP, but had no effect on IRF-1 or Stat1 activation. Deletion of a gamma interferon activated site (GAS site, −936 to −928 bp) increased the IL-1β-induced promoter activity, but did not significantly change the iNOS promoter induction by IFN-γ+IL-1β. The IFN-γ enhancement was partially abolished by the GAS site deletion, suggesting that the GAS site participates in the IFN-γ-enhanced NOS expression. Mutations of a reverse IRF site (−918 to −907 bp) completely abolished the NOS induction by IFN-γ, but it did not decrease the IL-1β-induced NOS promoter activity. These results suggest that the reverse IRF site is responsible for at least part of NOS induction by IFN-γ in RASMC. Deletion mutations that disrupted both the IRF site and the overlapping C/EBP site (−910 to −902 bp) abolished both the IL-1β-induced promoter activity and the IFN-γ enhancement, suggesting that the C/EBP site participates in the IL-1β-induced NOS expression. Mutations of a reverse NF-κB site (−901 to −892 bp) abolished the IL-1β-induced promoter activity, but not the IFN-γ enhancement, suggesting that the NF-κB binding mediates the IL-1β-induced, but not the IFN-γ-enhanced, NOS expression in RASMC. In summary, this study demonstrates that, in RASMC, IRF-1 and Stat1 activation mediates NOS induction by IFN-γ, and that the NF-κB and C/EBP activation mediates NOS induction by IL-1β, but not by IFN-γ.