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.

To elucidate the mechanisms involved in cell protection by aurintricarboxylic acid (ATA), an endonuclease inhibitor, high nitric oxide (NO)-induced macrophage apoptosis was studied. In RAW 264.7 macrophages, a high level of NO production accompanied by cell apoptosis was apparent with lipopolysaccharide (LPS) treatment. Direct NO donor sodium nitroprusside (SNP) also dramatically induced cell death, with an EC 50 of 1 mM. Coincubation of ATA (1-500 M) in LPS-stimulated RAW 264.7 cells resulted in a striking reduction of NO production and cell apoptosis, whereas only a partial cell protection was achieved in response to SNP. This suggests that abrogation of inducible nitric-oxide synthase (iNOS)-dependent NO production might contribute to ATA protection of LPS-treated cells. Immunoblotting and reverse transcription-polymerase chain reaction analysis revealed that ATA down-regulated iNOS protein through transcriptional inhibition of iNOS gene expression but was unrelated to iNOS protein stability. ATA not only inhibited nuclear factor-B (NF-B) activation through impairment of the targeting and degradation of IBs but also reduced LPS-induced activator protein-1 (AP-1) activation. These actions of ATA were not caused by the influence on LPS binding to macrophage membrane. Kinase assays indicated that ATA inhibited IB kinase (IKK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK) activity both in vivo and in vitro, suggesting a direct interaction between ATA and these signaling molecules. Taken together, these results provide novel action targets of ATA and indicate that ATA protection of macrophages from LPS-mediated cell death is primarily the result of its inhibition of NO production, which closely relates to the inactivation of NF-B and AP-1 and inhibition of IKK, ERK and p38 MAPK.