Inhibition of IFN-γ-Mediated Inducible Nitric Oxide Synthase Induction by the Peroxisome Proliferator-Activated Receptor γ Agonist, 15-Deoxy-Δ12,14-Prostaglandin J2, Involves Inhibition of the Upstream Janus Kinase/STAT1 Signaling Pathway

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Abstract
Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been reported to exert anti-inflammatory activities in macrophages by competition for transcriptional coactivators with some transcriptional factors, including NF-κB. In the present study the influence of PPARγ activators on IFN-γ-elicited macrophage stimulation and signaling cascades was investigated. The results show that IFN-γ-induced inducible NO synthase (iNOS) gene transcription, iNOS protein induction, and NO production are more sensitive to inhibition by 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) than by the other two PPARγ agonists, GW1929 and ciglitazone. Delayed addition of 15dPGJ2 for 2 h resulted in reduced inhibition, suggesting action by 15dPGJ2 on the upstream signaling cascades. Immunoblotting, DNA binding, and reporter gene assays consistently revealed the inhibitory ability of 15dPGJ2, but not GW1929 or ciglitazone, on IFN-γ-elicited signaling cascades, including tyrosine phosphorylation of Janus tyrosine protein kinase 2 and STAT1, DNA binding, and IFN regulatory factor-1 trans-activation of STAT1. These effects of 15dPGJ2 were not abrogated by the PPARγ antagonist, bisphenol A diglycidyl ether, indicating the PPARγ-independent actions. 15dPGJ2 also attenuated IL-6-induced tyrosine phosphorylation of STAT1 and STAT3 in Hep3B hepatoma cells. Consistent with the inhibitory effect of reactive oxygen species on STAT1 signaling, STAT1 inhibition by 15dPGJ2 was abrogated by N-acetylcysteine, glutathione, superoxide dismutase, and catalase. Furthermore, 15dPGJ2-induced inhibition of STAT1 phosphorylation and NO production still occurred in the presence of peroxovanadate, ruling out the action mechanism of 15dPGJ2 on tyrosine phosphatase. Taken together, for the first time in this study we demonstrate that 15dPGJ2 can inhibit cytokine-stimulated Janus kinase 2-STAT signaling through a PPARγ-independent, reactive oxygen species-dependent mechanism. These data provide a novel molecular mechanism of iNOS inhibition by 15dPGJ2 and confirm its physiological role in anti-inflammation.