Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)gamma agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1beta-stimulated rat chondrocytes: evidence for PPARgamma-independent inhibition by 15-deoxy-Delta12,14prostaglandin 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.
Bianchi
Arnaud
A
Laboratoire de Physiopathologie et Pharmacologie Articulaires, UMR 7561 CNRS-UHP, 54505 Vandoeuvre-lès-Nancy, France. arnaud.bianchi@medecine.uhp-nancy.fr
Moulin
David
D
Sebillaud
Sylvie
S
Koufany
Meriem
M
Galteau
Marie-Madeleine
MM
Netter
Patrick
P
Terlain
Bernard
B
Jouzeau
Jean-Yves
JY
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects*; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D004338:Drug Combinations; D015870:Gene Expression / Q000187:drug effects; D007155:Immunologic Factors / Q000494:pharmacology*; D007375:Interleukin-1 / Q000494:pharmacology*; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D008297:Male; D047495:PPAR gamma / Q000819:agonists*; D015230:Prostaglandin D2 / Q000031:analogs & derivatives* / Q000494:pharmacology; D000071876:Prostaglandin-E Synthases; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D000077154:Rosiglitazone; D045162:Thiazolidinediones / Q000494:pharmacology; D014162:Transfection
C097240:15-deoxy-delta(12,14)-prostaglandin J2; D004338:Drug Combinations; D007155:Immunologic Factors; D007375:Interleukin-1; D047495:PPAR gamma; D012333:RNA, Messenger; D045162:Thiazolidinediones; D000077154:Rosiglitazone; D051546:Cyclooxygenase 2; C489691:Ptgs2 protein, rat; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; D015230:Prostaglandin D2