Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δ12,14prostaglandin J2

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.