Expression and Regulation of Microsomal Prostaglandin E Synthase-1 in Human Osteoarthritic Cartilage and Chondrocytes XINFANG LI, HASSAN AFIF, SARANETTE CHENG, JOHANNE MARTEL-PELLETIER, JEAN-PIERRE PELLETIER, PIERRE RANGER, and HASSAN FAHMI ABSTRACT. Objective. Elevated production of prostaglandin E2 (PGE2) plays an important role in the pathogen- esis of arthritis. Recently, an inducible microsomal prostaglandin E synthase-1 (mPGES-1) was identified. This enzyme is functionally coupled with cyclooxygenase-2 (COX-2) and converts the COX product PGH2 to PGE2. We analyzed expression of mPGES-1 in human normal and osteoarthritic (OA) cartilage and determined the effect of different inflammatory agonists on the expression of mPGES-1 in OA chondrocytes. Methods. Expression of mPGES-1 mRNA and protein in cartilage was determined by quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. OA chondrocytes were treated with different inflammatory agents, and mPGES-1 protein expression was evaluated by Western blot. Activation of the mPGES-1 promoter was assessed in transient trans- fection experiments. Results. Levels of mPGES-1 mRNA and protein were markedly elevated in OA versus normal car- tilage. Treatment of chondrocytes with interleukin 1ß (IL-1ß) induced expression of mPGES-1 pro- tein in a dose- and time-dependent manner. This appears to occur at the transcriptional level, as IL- 1ß induced expression of mPGES-1 mRNA and the activity of this gene promoter. Tumor necrosis factor-α (TNF-α) and IL-17 also upregulated expression of mPGES-1 protein and displayed a syn- ergistic effect with IL-1ß. Peroxisome proliferator-activated receptor-γ ligands, 15-deoxy-∆12,14- prostaglandin J2 and troglitazone, inhibited IL-1ß-induced mPGES-1 protein expression, an effect that was reversed by exogenous PGE2. Conclusion. Our study shows that mPGES-1 expression is upregulated in OA versus normal carti- lage and that proinflammatory cytokines increased mPGES-1 expression in chondrocytes. These data suggest that mPGES-1 may prove to be an interesting therapeutic target for controlling PGE2 synthesis. (J Rheumatol 2005;32:887–95) Key Indexing Terms: MICROSOMAL PROSTAGLANDIN E SYNTHASE-1 CARTILAGE CHONDROCYTES OSTEOARTHRITIS 1 Biochemical, genetic, and clinical evidence indicates that of PGE2 . Treatment with neutralizing anti-PGE2 antibodies prostaglandin E2 (PGE2) plays a critical role in inflamma- prevents acute and chronic inflammation in a rat adjuvant 2 tion and in the pathophysiology of articular joint diseases, arthritis model . More direct evidence for the role of PGE2 such as rheumatoid arthritis (RA) and osteoarthritis (OA). in arthritis has been provided by gene targeting studies. 3 For example, arthritic joint tissues produce large quantities Genetic disruption of either the PGE2 receptor EP4 or cyclooxygenase-2 (COX-2)4, one of the key enzymes in From the Osteoarthritis Research Unit, Centre hospitalier de l’Université PGE2 biosynthesis, reduced incidence and severity of colla- de Montréal, Hôpital Notre-Dame, Montréal, Québec, Canada. gen-induced arthritis in mice. These animals showed Supported by the Canadian Institutes of Health Research (CIHR), Grant IMH-63168, and the Fonds de Recherche en santé du Québec (FRSQ) reduced inflammation and less cartilage and bone destruc- Subvention d’Établissement de Jeune Chercheur JC2836. S. Cheng is tion. The role of PGE2 in arthritis is also supported by effec- supported by a fellowship from the CIHR Training on Mobility and Posture tive suppression of pain and inflammatory responses in Deficiencies (MENTOR). H. Fahmi is a Research Scholar of FRSQ. arthritis by nonsteroidal antiinflammatory drugs (NSAID) X. Li, MSc; H. Afif, PhD; S. Cheng, MSc; J. Martel-Pelletier, PhD; 5,6 J-P Pelletier, MD; H. Fahmi, PhD, Osteoarthritis Research Unit, Centre that reduce PGE2 biosynthesis . hospitalier de l’Université de Montréal; P. Ranger, MD, Hopital Sacré- Chondrocytes are a major source of PGE2 in the joint; the Coeur. production of this prostanoid can be induced by proinflam- Address reprint requests to H. Fahmi, Osteoarthritis Research Unit, matory cytokines, mitogens, mechanical stress, and trau- Centre hospitalier de l’Université de Montréal, Hôpital Notre-Dame, 5,7,8 1560 rue Sherbrooke est, Montréal, Québec H2L 4M1, Canada. ma . The synthesis of PGE2 from arachidonic acid (AA) E-mail: [email protected] requires 2 enzymes acting sequentially. Cyclooxygenases Accepted for publication December 29, 2004. catalyze the conversion of AA to the intermediate prostanoid Personal non-commercial use only. The Journal of Rheumatology Copyright © 2005. All rights reserved. Li, et al: PGE synthase in OA cartilage 887 Downloaded on September 27, 2021 from www.jrheum.org 16 PGH2. Two isoforms of the COX enzyme have been identi- digestion as described . Briefly, this consisted of 2 mg/ml pronase for 1 h fied: COX-1 is constitutively expressed in most tissues, followed by 1 mg/ml collagenase for 6 h (type IV; Sigma-Aldrich) at 37°C whereas COX-2 is induced by various stimuli including in DMEM and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). The digested tissue was briefly centrifuged and the pellet was washed. The lipopolysaccharides, growth factors, and proinflammatory isolated chondrocytes were seeded at high density in tissue culture flasks 5,9 cytokines (reviewed in ). Subsequently, PGE synthase and cultured in DMEM supplemented with 10% heat-inactivated FCS. At (PGES) converts COX-derived PGH2 into PGE2. At least 3 confluence, the chondrocytes were detached, seeded at high density, and distinct PGES isoforms have been identified10. Cytosolic allowed to grow in DMEM, supplemented as above. The culture medium PGES (cPGES), which is identical to the heat shock protein was changed every second day, and 24 h before the experiment the cells were incubated in fresh medium containing 0.5% FCS. Only first-passage 90-associated protein p23, is ubiquitously and constitutive- chondrocytes were used. ly expressed, and displays functional coupling with COX-1. Immunohistochemistry. Cartilage specimens were processed for immuno- In contrast, microsomal PGES-1 (mPGES-1), originally histochemistry as described16. Specimens were fixed in 4% paraformalde- designated microsomal glutathione S-transferase 1-like 1, is hyde and embedded in paraffin. Sections (5 µm) of paraffin-embedded an inducible enzyme that exhibits preferential functional specimens were deparaffinized in toluene, and dehydrated in a graded coupling with COX-2. The most recently identified isoform, series of ethanol. The specimens were then preincubated with chondroiti- nase ABC (0.25 U/ml in phosphate buffered saline, PBS, pH 8.0) for 60 mPGES-2, is ubiquitously expressed in diverse tissues, but min at 37°C, followed by 30 min incubation with Triton X-100 (0.3%) at 10 its function and regulation remain obscure . room temperature. Slides were then washed in PBS followed by 2% hydro- The upregulation of mPGES-1 expression has been gen peroxide/methanol for 15 min. They were further incubated for 60 min with 2% normal serum (Vector Laboratories, Burlingame, CA, USA) and reported in conditions in which PGE2 has been implicated, such as arthritis11, and studies with mPGES-1-deficient overlaid with primary antibody for 18 h at 4°C in a humidified chamber. The antibody was a rabbit polyclonal anti-human mPGES-1 (Cayman), mice have shown that induced PGE2 synthesis is largely used at 10 µg/ml. Each slide was washed 3 times in PBS pH 7.4 and stained 12,13 dependent on this enzyme . Proinflammatory cytokines using the avidin-biotin complex method (Vectastain ABC kit; Vector). The interleukin 1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) color was developed with 3,3’-diaminobenzidine (DAB; Vector) containing have been shown to induce mPGES-1 expression in several hydrogen peroxide. Slides were counterstained with eosin. The specificity tissues and cell types, including synovial fibroblasts and of staining was evaluated by using antibody that had been preadsorbed (1 14,15 h, 37°C) with a 20-fold molar excess of the specific corresponding peptide, osteoblasts . However, little is known about the expres- and by substituting the primary antibody with non-immune rabbit IgG sion and regulation of mPGES-1 in cartilage. (Chemicon, Temecula, CA, USA; used at the same concentration as the To understand the regulation of PGE2 production in joint primary antibodies). The evaluation of positive-staining chondrocytes was tissues, we analyzed mPGES-1 expression in normal and performed using our published method16. For each specimen, 6 microscop- OA cartilage. Further, we explored the effect of different ic fields were examined under 40× magnification. The total number of chondrocytes and the number of chondrocytes staining positive were eval- inflammatory agonists on the expression of mPGES-1 in uated and results were expressed as the percentage of chondrocytes stain- OA chondrocytes. ing positive (cell score). RNA extraction and reverse transcriptase-polymerase chain reaction (RT- MATERIALS AND METHODS PCR). Total RNA from homogenized cartilage or stimulated chondrocytes Reagents. Recombinant human (rh) IL-1ß was obtained from Genzyme was isolated using the TRIzol® reagent (Invitrogen) according to the man- (Cambridge, MA, USA), rhTNF-α and rhIL-17 were from R&D Systems ufacturer’s instructions. To remove contaminating DNA, isolated RNA was ∆12,14 (Minneapolis, MN, USA). 15-deoxy- -prostaglandin J2 (15d-PGJ2),