Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)gamma agonists on membrane-associated prostaglandin E-2 synthase-1 in IL-1 beta-stimulated rat chondrocytes: evidence for PPAR gamma-independent inhibition by 15-deoxy-Delta(12,14)prostaglandin J(2)

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