Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been reported to exert anti-inflammatory activities in macrophages by competition for transcriptional coactivators with some transcriptional factors, including NF-kappaB. In the present study the influence of PPARgamma activators on IFN-gamma-elicited macrophage stimulation and signaling cascades was investigated. The results show that IFN-gamma-induced inducible NO synthase (iNOS) gene transcription, iNOS protein induction, and NO production are more sensitive to inhibition by 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) than by the other two PPARgamma agonists, GW1929 and ciglitazone. Delayed addition of 15dPGJ(2) for 2 h resulted in reduced inhibition, suggesting action by 15dPGJ(2) on the upstream signaling cascades. Immunoblotting, DNA binding, and reporter gene assays consistently revealed the inhibitory ability of 15dPGJ(2), but not GW1929 or ciglitazone, on IFN-gamma-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 15dPGJ(2) were not abrogated by the PPARgamma antagonist, bisphenol A diglycidyl ether, indicating the PPARgamma-independent actions. 15dPGJ(2) 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 15dPGJ(2) was abrogated by N-acetylcysteine, glutathione, superoxide dismutase, and catalase. Furthermore, 15dPGJ(2)-induced inhibition of STAT1 phosphorylation and NO production still occurred in the presence of peroxovanadate, ruling out the action mechanism of 15dPGJ(2) on tyrosine phosphatase. Taken together, for the first time in this study we demonstrate that 15dPGJ(2) can inhibit cytokine-stimulated Janus kinase 2-STAT signaling through a PPARgamma-independent, reactive oxygen species-dependent mechanism. These data provide a novel molecular mechanism of iNOS inhibition by 15dPGJ(2) and confirm its physiological role in anti-inflammation.