Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-γ-treated RAW264.7 murine macrophages

Background: We hypothesized that acetylation of the Slat1 regulates interferon-gamma (IFN-gamma) mediated macrophage expression of inducible nitric oxide, synthase (iNOS). Methods: RAW 264.7 iNOS expression was induced with IFN-gamma. Deacetylase inhibitors trichostatin A (TSA) or valproic acid (VPA) were added. Stat1 and iNOS mRNA and protein were measured. Acetylated Slat1 was determined by immunoprecipitation. Chromatin immunoprecipitation assessed in vivo binding of Stal1 to the iNOS promoter. Results: IFN-gamma significantly increased nitrite, iNOS protein and iNOS mRNA and iNOS promoter activation. (P < .01 vs control for nitrite, protein, and mRNA). TSA-mediated acetylation decreased these to levels that were not different from controls. IFN-gamma increased acetylated Stat1 by 5-fold (P < .02 vs control); TSA + IFN-gamma caused an additional 4-fold increase in acetylated Slat1 (P < .05 vs IFN alone). Slat1 binding to the iNOS promoter increased 8-fold with IFN-gamma, (P < .01 vs control). In TSA + IFN-gamma, Stall binding was not different from controls. Although less potent than TSA, VPA also significantly decreased nitrite, iNOS protein, iNOS mRNA, Stat1 acetylation, and Slat1 binding. Conclusions: Acetylation of Slat1 protein correlates with decreased Stat1 binding to the iNOS promoter with resultant inhibition of IFN-gamma-mediated iNOS expression. Acetylation of the Slat1 protein may downregulate iNOS expression in Proinflammatory states.