Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin E(2 )synthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δ(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(1α )and PGE(2 )in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)γ agonists. Real-time PCR analysis showed that IL-1β 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(1α )and PGE(2 )peaked 24 hours after stimulation with IL-1β; the induction of PGE(2 )was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Δ(12,14)prostaglandin J(2 )(15d-PGJ(2)) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 μM), with more potency on PGE(2 )level than on 6-keto-PGF(1α )level (-90% versus -66% at 10 μM). 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 μM. Inhibitory effects of 10 μM 15d-PGJ(2 )were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent mechanism. EMSA and TransAM(® )analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression and PGE(2 )production, whereas 15d-PGJ(2 )inhibited NF-κB 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-κB activation; third, 15d-PGJ(2 )strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ(2 )occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ(2).