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Adult Cartilage-Specific Peroxisome Proliferator–Activated Receptor Gamma Knockout Mice Exhibit the Spontaneous Osteoarthritis Phenotype

2013; Elsevier BV; Volume: 182; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2012.12.012

1525-2191

Faezeh Vasheghani, Roxana Monemdjou, Hassan Fahmi, Yue Zhang, Gemma Pérez, Meryem Blati, René St‐Arnaud, Jean‐Pierre Pelletier, Frank Beier, Johanne Martel‐Pelletier, Mohit Kapoor,

Bone and Joint Diseases

Osteoarthritis (OA) is an age-related progressive degenerative joint disease. Peroxisome proliferator–activated receptor gamma (PPARγ), a transcription factor, is suggested as an attractive therapeutic target to counteract degradative mechanisms associated with OA. Studies suggest that activation of PPARγ by its agonists can reduce the synthesis of OA catabolic and inflammatory factors and the development of cartilage lesions in OA animal models. Because these agonists impart several PPARγ-independent effects, the specific in vivo function of PPARγ in cartilage homeostasis and OA remains largely unknown. Herein, we describe the in vivo role of PPARγ in OA using cartilage-specific PPARγ knockout (KO) mice generated using the Cre-lox system. Adult PPARγ KO mice exhibited a spontaneous OA phenotype associated with enhanced cartilage degradation, hypocellularity, synovial and cartilage fibrosis, synovial inflammation, mononuclear cell influx in the synovium, and increased expression of catabolic factors, including matrix metalloproteinase-13, accompanied by an increase in staining for matrix metalloproteinase–generated aggrecan and type II collagen neoepitopes (VDIPEN and C1-2C). We demonstrate that PPARγ-deficient articular cartilage exhibits elevated expression of the additional catabolic factors hypoxia-inducible factor-2α, syndecan-4, and a disintegrin and metalloproteinase with thrombospondin motifs 5 and of the inflammatory factors cyclooxygenase-2 and inducible nitric oxide synthase. In conclusion, PPARγ is a critical regulator of cartilage health, the lack of which leads to an accelerated spontaneous OA phenotype. Osteoarthritis (OA) is an age-related progressive degenerative joint disease. Peroxisome proliferator–activated receptor gamma (PPARγ), a transcription factor, is suggested as an attractive therapeutic target to counteract degradative mechanisms associated with OA. Studies suggest that activation of PPARγ by its agonists can reduce the synthesis of OA catabolic and inflammatory factors and the development of cartilage lesions in OA animal models. Because these agonists impart several PPARγ-independent effects, the specific in vivo function of PPARγ in cartilage homeostasis and OA remains largely unknown. Herein, we describe the in vivo role of PPARγ in OA using cartilage-specific PPARγ knockout (KO) mice generated using the Cre-lox system. Adult PPARγ KO mice exhibited a spontaneous OA phenotype associated with enhanced cartilage degradation, hypocellularity, synovial and cartilage fibrosis, synovial inflammation, mononuclear cell influx in the synovium, and increased expression of catabolic factors, including matrix metalloproteinase-13, accompanied by an increase in staining for matrix metalloproteinase–generated aggrecan and type II collagen neoepitopes (VDIPEN and C1-2C). We demonstrate that PPARγ-deficient articular cartilage exhibits elevated expression of the additional catabolic factors hypoxia-inducible factor-2α, syndecan-4, and a disintegrin and metalloproteinase with thrombospondin motifs 5 and of the inflammatory factors cyclooxygenase-2 and inducible nitric oxide synthase. In conclusion, PPARγ is a critical regulator of cartilage health, the lack of which leads to an accelerated spontaneous OA phenotype. Osteoarthritis (OA) is a progressive degenerative joint disease and the most common form of arthritis. It is an age-related disease in which the effects of aging contribute to disease progression.1Goldring M.B. Goldring S.R. Osteoarthritis.J Cell Physiol. 2007; 213: 626-634Crossref PubMed Scopus (1002) Google Scholar, 2Tanaka S. Hamanishi C. Kikuchi H. Fukuda K. Factors related to degradation of articular cartilage in osteoarthritis: a review.Semin Arthritis Rheum. 1998; 27: 392-399Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 3Attur M. Samuels J. Krasnokutsky S. Abramson S.B. Targeting the synovial tissue for treating osteoarthritis (OA): where is the evidence?.Best Pract Res Clin Rheumatol. 2010; 24: 71-79Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar OA is characterized by cartilage deterioration, hypocellularity, synovitis, and remodeling of the subchondral bone. Symptoms usually appear in middle age, and >50% of Americans 65 years and older show radiologic signs of the disease.4Lawrence R.C. Felson D.T. Helmick C.G. Arnold L.M. Choi H. Deyo R.A. Gabriel S. Hirsch R. Hochberg M.C. Hunder G.G. Jordan J.M. Katz J.N. Kremers H.M. Wolfe F. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States, part II.Arthritis Rheum. 2008; 58: 26-35Crossref PubMed Scopus (3022) Google Scholar As age is a major contributor to the disease and individuals are living longer owing to increased life expectancy, OA is a growing socioeconomic and clinical concern. Peroxisome proliferator–activated receptor gamma (PPARγ) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. PPARγ was originally identified as a key regulator of adipocyte differentiation and lipid metabolism.5Chawla A. Schwarz E.J. Dimaculangan D.D. Lazar M.A. Peroxisome proliferator-activated receptor (PPAR) gamma: adipose-predominant expression and induction early in adipocyte differentiation.Endocrinology. 1994; 135: 798-800Crossref PubMed Scopus (617) Google Scholar Synthetic agonists of PPARγ, such as thiazolidinediones, are clinically used for the treatment of type 2 diabetes mellitus. Apart from its role in adipogenesis and insulin sensitization, it is now known that PPARγ plays a key role in a variety of pathophysiologic processes. Mouse studies using agonists of PPARγ and targeted deletion of PPARγ in specific cells/tissues have been useful in dissecting the key pathophysiologic role of PPARγ in a variety of disorders, including wound healing/fibrosis,6Sha W. Thompson K. South J. Baron M. Leask A. Loss of PPARgamma expression by fibroblasts enhances dermal wound closure.Fibrogenesis Tissue Repair. 2012; 5: 5Crossref PubMed Scopus (15) Google Scholar, 7Kapoor M. McCann M. Liu S. Huh K. Denton C.P. Abraham D.J. Leask A. Loss of peroxisome proliferator-activated receptor gamma in mouse fibroblasts results in increased susceptibility to bleomycin-induced skin fibrosis.Arthritis Rheum. 2009; 60: 2822-2829Crossref PubMed Scopus (64) Google Scholar, 8Wu M. Melichian D.S. Chang E. Warner-Blankenship M. Ghosh A.K. Varga J. Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma.Am J Pathol. 2009; 174: 519-533Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar tumorigenesis in the breast,9Skelhorne-Gross G. Reid A.L. Apostoli A.J. Di Lena M.A. Rubino R.E. Peterson N.T. Schneider M. Sengupta S.K. Gonzalez F.J. Nicol C.J. Stromal adipocyte PPARgamma protects against breast tumorigenesis.Carcinogenesis. 2012; 33: 1412-1420Crossref PubMed Scopus (20) Google Scholar oxidative damage to the myocardium,10Ding G. Fu M. Qin Q. Lewis W. Kim H.W. Fukai T. Bacanamwo M. Chen Y.E. Schneider M.D. Mangelsdorf D.J. Evans R.M. Yang Q. Cardiac peroxisome proliferator-activated receptor gamma is essential in protecting cardiomyocytes from oxidative damage.Cardiovasc Res. 2007; 76: 269-279Crossref PubMed Scopus (116) Google Scholar intraepithelial neoplasia in the prostate,11Jiang M. Fernandez S. Jerome W.G. He Y. Yu X. Cai H. Boone B. Yi Y. Magnuson M.A. Roy-Burman P. Matusik R.J. Shappell S.B. Hayward S.W. Disruption of PPARgamma signaling results in mouse prostatic intraepithelial neoplasia involving active autophagy.Cell Death Differ. 2010; 17: 469-481Crossref PubMed Scopus (46) Google Scholar pancreatic inflammation,12Ivashchenko C.Y. Duan S.Z. Usher M.G. Mortensen R.M. PPAR-gamma knockout in pancreatic epithelial cells abolishes the inhibitory effect of rosiglitazone on caerulein-induced acute pancreatitis.Am J Physiol Gastrointest Liver Physiol. 2007; 293: G319-G326Crossref PubMed Scopus (27) Google Scholar and T-cell–mediated gut inflammation.13Guri A.J. Mohapatra S.K. Horne II, W.T. Hontecillas R. Bassaganya-Riera J. The role of T cell PPAR gamma in mice with experimental inflammatory bowel disease.BMC Gastroenterol. 2010; 10: 60Crossref PubMed Scopus (61) Google Scholar PPARγ is also believed to be a potential therapeutic target for the treatment of OA based on the fact that PPARγ agonists can reduce the synthesis of various catabolic and inflammatory factors involved in the pathogenesis of OA. For example, it can reduce inflammatory cytokines such as IL-1β, tumor necrosis factor-α, prostaglandin E2, IL-6, nitric oxide (NO), and matrix metalloproteinases (MMPs) such as MMP-1 and MMP-13 (reviewed in the study by Fahmi et al14Fahmi H. Martel-Pelletier J. Pelletier J.P. Kapoor M. Peroxisome proliferator-activated receptor gamma in osteoarthritis.Mod Rheumatol. 2011; 21: 1-9Crossref PubMed Scopus (54) Google Scholar). Moreover, PPARγ agonists can reduce the development of cartilage lesions in vivo in experimental dog15Boileau C. Martel-Pelletier J. Fahmi H. Mineau F. Boily M. Pelletier J.P. The peroxisome proliferator-activated receptor gamma agonist pioglitazone reduces the development of cartilage lesions in an experimental dog model of osteoarthritis: in vivo protective effects mediated through the inhibition of key signaling and catabolic pathways.Arthritis Rheum. 2007; 56: 2288-2298Crossref PubMed Scopus (60) Google Scholar and guinea pig16Kobayashi T. Notoya K. Naito T. Unno S. Nakamura A. Martel-Pelletier J. Pelletier J.P. Pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, reduces the progression of experimental osteoarthritis in guinea pigs.Arthritis Rheum. 2005; 52: 479-487Crossref PubMed Scopus (86) Google Scholar models of OA and have also been shown to inhibit OA-involved signaling pathways, such as the mitogen-activated protein kinases extracellular signal–regulated kinase 1/2 and p38 and NF-κB. Apart from its anticatabolic and anti-inflammatory effects, we and others also showed that PPARγ exhibits antifibrotic properties.6Sha W. Thompson K. South J. Baron M. Leask A. Loss of PPARgamma expression by fibroblasts enhances dermal wound closure.Fibrogenesis Tissue Repair. 2012; 5: 5Crossref PubMed Scopus (15) Google Scholar, 7Kapoor M. McCann M. Liu S. Huh K. Denton C.P. Abraham D.J. Leask A. Loss of peroxisome proliferator-activated receptor gamma in mouse fibroblasts results in increased susceptibility to bleomycin-induced skin fibrosis.Arthritis Rheum. 2009; 60: 2822-2829Crossref PubMed Scopus (64) Google Scholar Loss of PPARγ in fibroblasts results in excessive inflammation and fibrosis in a mouse model,7Kapoor M. McCann M. Liu S. Huh K. Denton C.P. Abraham D.J. Leask A. Loss of peroxisome proliferator-activated receptor gamma in mouse fibroblasts results in increased susceptibility to bleomycin-induced skin fibrosis.Arthritis Rheum. 2009; 60: 2822-2829Crossref PubMed Scopus (64) Google Scholar and PPARγ attenuates fibrosis by blocking activation of the transforming growth factor β/Smad signaling pathway.8Wu M. Melichian D.S. Chang E. Warner-Blankenship M. Ghosh A.K. Varga J. Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma.Am J Pathol. 2009; 174: 519-533Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 17Gong K. Xing D. Li P. Aksut B. Ambalavanan N. Yang Q. Nozell S.E. Oparil S. Chen Y.F. Hypoxia induces downregulation of PPAR-gamma in isolated pulmonary arterial smooth muscle cells and in rat lung via transforming growth factor-beta signaling.Am J Physiol Lung Cell Mol Physiol. 2011; 301: L899-L907Crossref PubMed Scopus (51) Google Scholar, 18Zhang G.Y. Cheng T. Zheng M.H. Yi C.G. Pan H. Li Z.J. Chen X.L. Yu Q. Jiang L.F. Zhou F.Y. Li X.Y. Yang J.Q. Chu T.G. Gao W.Y. Activation of peroxisome proliferator-activated receptor-gamma inhibits transforming growth factor-beta1 induction of connective tissue growth factor and extracellular matrix in hypertrophic scar fibroblasts in vitro.Arch Dermatol Res. 2009; 301: 515-522Crossref PubMed Scopus (25) Google Scholar, 19Zhang G.Y. Yi C.G. Li X. Ma B. Li Z.J. Chen X.L. Guo S.Z. Gao W.Y. Troglitazone suppresses transforming growth factor-beta1-induced collagen type I expression in keloid fibroblasts.Br J Dermatol. 2009; 160: 762-770Crossref PubMed Scopus (36) Google Scholar, 20Ghosh A.K. Bhattacharyya S. Wei J. Kim S. Barak Y. Mori Y. Varga J. Peroxisome proliferator-activated receptor-gamma abrogates Smad-dependent collagen stimulation by targeting the p300 transcriptional coactivator.FASEB J. 2009; 23: 2968-2977Crossref PubMed Scopus (103) Google Scholar, 21Fan F. Li Y. Duan X. Zhao T. Pan D. Chen H. Rosiglitazone attenuates activation of human Tenon's fibroblasts induced by transforming growth factor-beta1.Graefes Arch Clin Exp Ophthalmol. 2012; 250: 1213-1220Crossref PubMed Scopus (13) Google Scholar In vitro and in vivo studies using agonists of PPARγ have been helpful yet unable to define the specific in vivo role of PPARγ in the pathogenesis of OA. In fact, it is now understood that some of these agonists may also act through mechanisms independent of PPARγ activation. To devise OA therapeutic strategies targeting PPARγ, it is essential to first determine the role of this complex transcription factor in cartilage health and physiologic function in vivo. PPARγ knockout (KO) mice exhibit embryonic lethality due to placental defects.22Barak Y. Nelson M.C. Ong E.S. Jones Y.Z. Ruiz-Lozano P. Chien K.R. Koder A. Evans R.M. PPAR gamma is required for placental, cardiac, and adipose tissue development.Mol Cell. 1999; 4: 585-595Abstract Full Text Full Text PDF PubMed Scopus (1613) Google Scholar Therefore, to specifically elucidate the in vivo role of PPARγ in cartilage health and OA, we generated cartilage-specific PPARγ KO mice using the Cre-lox system. Using these mice, we previously showed that cartilage-specific deletion of PPARγ results in delayed endochondral ossification and cartilage growth and developmental defects.23Monemdjou R. Vasheghani F. Fahmi H. Perez G. Blati M. Taniguchi N. Lotz M. St-Arnaud R. Pelletier J.P. Martel-Pelletier J. Beier F. Kapoor M. Association of cartilage-specific deletion of peroxisome proliferator-activated receptor gamma with abnormal endochondral ossification and impaired cartilage growth and development in a murine model.Arthritis Rheum. 2012; 64: 1551-1561Crossref PubMed Scopus (43) Google Scholar In the present study, for the first time, to our knowledge, we show that loss of PPARγ in the cartilage results in the OA phenotype in adult PPARγ KO mice. Genetically modified mice harboring a cartilage-specific deletion of PPARγ were generated using the Cre-lox method in which mice carrying Cre recombinase under the control of the collagen type II promoter were used to induce specific recombination in chondrocytes as previously established.24Terpstra L. Prud'homme J. Arabian A. Takeda S. Karsenty G. Dedhar S. St-Arnaud R. Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes.J Cell Biol. 2003; 162: 139-148Crossref PubMed Scopus (184) Google Scholar Briefly, mice containing a PPARγ gene flanked by loxP sites (C57BL/6-PPARγfl/fl; The Jackson Laboratory, Bar Harbor, ME) were mated with C57BL/6 Col2-Cre transgenic mice to generate mice bearing Col2-Cre and a floxed allele in their germline (genotype: PPARγfl/+; Cre). These mice were backcrossed to homozygote floxed mice in the following cross: PPARγfl/+; Cre × PPARγfl/fl to generate mice with both alleles inactivated in chondrocytes (genotype: PPARγfl/fl;Cre). PPARγfl/fl;Cre mice are referred to as homozygote PPARγ KO mice, PPARγfl/+;Cre mice are referred to as heterozygote PPARγ KO mice, and PPARγfl/fl mice without Cre transgene are referred to as control mice. All procedures involving animals were performed according to the approved protocol by the Comité Institutionnel de protection des animaux (Institutional Animal Protection Committee) of the University of Montreal Hospital Research Centre, Montreal, QC, Canada. All the mice were kept in a 12-hour light/dark cycle. Food and water were available ad libitum. Freshly dissected mouse knee joints from 6- and 14-month-old mice were fixed overnight in TissuFix (Chaptec, Montreal, QC, Canada), decalcified for 1.5 hours in RDO rapid decalcifier (Apex Engineering Products Corp., Plainfield, IL), and further fixed in TissuFix overnight, followed by embedding in paraffin and sectioning, which was performed at the Centre for Bone and Periodontal Research at McGill University (Montreal, QC, Canada). Sections (5 μm) were deparaffinized in xylene, followed by a graded series of alcohol washes. Sections were stained with safranin O/fast green (Sigma-Aldrich, Oakville, ON, Canada) according to the manufacturer's recommendations. Slides were evaluated by two independent readers in a blinded manner. To determine the extent of cartilage deterioration, the medial tibial plateau and medial femoral condyle were histologically scored using the method issued by the Osteoarthritis Research Society International as we have previously described.25Glasson S.S. Chambers M.G. Van Den Berg W.B. Little C.B. The OARSI histopathology initiative: recommendations for histological assessments of osteoarthritis in the mouse.Osteoarthritis Cartilage. 2010; 18: S17-S23Abstract Full Text Full Text PDF PubMed Scopus (818) Google Scholar To evaluate the degree of inflammation in the synovium, sections were stained with H&E (Sigma-Aldrich) according to the manufacturer's recommendations. Stained sections were blindly scored for number of mononuclear cells on a scale from 0 to 3: 0, no mononuclear cells; 1, few mononuclear cells; 2, a moderate number of mononuclear cells; and 3, a high number of mononuclear cells. To evaluate fibrosis in the articular cartilage and in the synovium, sections were stained with Masson's trichrome stain (Sigma-Aldrich) according to the manufacturer's recommendations. Immunohistochemical (IHC) analysis and immunofluorescence were performed on the sections obtained from 14-month-old mouse knee joints using the following antibodies: a rat anti-mouse macrophage/monocyte monoclonal antibody (MAB1852; Millipore, Billerica, MA) that recognizes an intracellular antigen of mouse macrophages and monocytes, a rabbit polyclonal anti–C-terminal peptide of aggrecan G1 domain (VDIPEN, a gift from Dr. John S. Mort, Shriners Hospital for Children, Montreal, QC, Canada), a rabbit polyclonal that represents a type II collagen primary cleavage site [COLII-3/4C short peptide (C1-2C), a gift from Dr. Robin Poole, McGill University, Montreal, QC, Canada], a rabbit anti–MMP-13 antibody (Sigma-Aldrich), and a rabbit anti–hypoxia-inducing factor-2 α (HIF-2α; AbCam Inc., Cambridge, MA). IHC analysis was performed using the LSAB+ system horseradish peroxidase kit (Dako, Burlington, ON, Canada) following the manufacturer-recommended protocol as previously reported.23Monemdjou R. Vasheghani F. Fahmi H. Perez G. Blati M. Taniguchi N. Lotz M. St-Arnaud R. Pelletier J.P. Martel-Pelletier J. Beier F. Kapoor M. Association of cartilage-specific deletion of peroxisome proliferator-activated receptor gamma with abnormal endochondral ossification and impaired cartilage growth and development in a murine model.Arthritis Rheum. 2012; 64: 1551-1561Crossref PubMed Scopus (43) Google Scholar Articular cartilage was microdissected from the knee joints of 6-month-old PPARγ-deficient and control mice. Microdissection of articular cartilage was performed under a high-power surgical microscope (Motic SMZ-168; Fisher Scientific Canada, Ottawa, ON, Canada) to carefully dissect only articular cartilage and avoid subchondral bone. Total RNA was then isolated from dissected cartilage using TRIzol (Invitrogen, Burlington, ON, Canada) and RNeasy (Qiagen, Toronto, ON, Canada) reagents according to the manufacturers' recommendations, reverse transcribed, and amplified using the TaqMan Assays-on-Demand (Applied Biosystems, Streetsville, ON, Canada) in a reaction solution containing two unlabeled primers and a 6-carboxyfluorescein–labeled TaqMan MGB probe. Samples were combined with one-step master mix (Eurogentec, San Diego, CA). Amplified sequences were detected using the ABI Prism 7900HT sequence detector (Applied Biosystems) according to the manufacturer's instructions. The expression values were standardized to values obtained with control glyceraldehyde-3-phosphate dehydrogenase RNA primers using the ΔCT method. All the primers and probe sets were obtained from Applied Biosystems, and the data were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels and represent averages and SEM from direct comparison of KO and control littermates. The statistical significance of real-time PCR results was determined by two-way analysis of variance with the Bonferroni posttest using GraphPad Prism, version 3.00 for Windows, software (GraphPad Software Inc., San Diego, CA). Statistical analysis, unless stated otherwise, was performed using the two-tailed Student's t-test. P < 0.05 was considered statistically significant. The generation of cartilage-specific PPARγ conditional KO mice was determined by tail DNA genotyping, which confirmed the presence of the Cre transgene in heterozygote (PPARγ F/W Cre) and homozygote (PPARγ F/F Cre) PPARγ KO mice and its absence in wild-type (control) mice (Figure 1A), as previously reported.23Monemdjou R. Vasheghani F. Fahmi H. Perez G. Blati M. Taniguchi N. Lotz M. St-Arnaud R. Pelletier J.P. Martel-Pelletier J. Beier F. Kapoor M. Association of cartilage-specific deletion of peroxisome proliferator-activated receptor gamma with abnormal endochondral ossification and impaired cartilage growth and development in a murine model.Arthritis Rheum. 2012; 64: 1551-1561Crossref PubMed Scopus (43) Google Scholar We examined the effects of cartilage-specific ablation of PPARγ on the integrity of knee joints in 6- and 14-month-old mice (Figure 1B). Safranin O/fast green staining demonstrated that the articular cartilage (medial femoral condyle and medial tibial plateau) of 6-month-old control mouse knee joints was intact, with no signs of cartilage deterioration. Similarly, in 14-month-old control mouse knee joints, no cartilage degradation was observed; however, as expected, some loss of articular chondrocyte cellularity and proteoglycan loss (loss of safranin O/fast green staining) was observed. In 6-month-old homozygote PPARγ KO mice, we observed early signs of cartilage deterioration in terms of roughening of the articular surface, reduced cellularity, and increased proteoglycan loss (loss of safranin O staining). In 14-month-old homozygote PPARγ KO mice, we observed more profound spontaneous OA phenotypic changes, such as accelerated cartilage degradation, loss of chondrocytes, and calcified cartilage fibrillation on ≥50% of the articular surface. The average Osteoarthritis Research Society International score, to account for OA severity,25Glasson S.S. Chambers M.G. Van Den Berg W.B. Little C.B. The OARSI histopathology initiative: recommendations for histological assessments of osteoarthritis in the mouse.Osteoarthritis Cartilage. 2010; 18: S17-S23Abstract Full Text Full Text PDF PubMed Scopus (818) Google Scholar was significantly higher in the medial tibial plateaus and medial femoral condyles of 14-month-old homozygote PPARγ KO mice compared with control mice (Figure 1C). The OA phenotype was observed in both sexes in PPARγ KO mice. Loss of proteoglycans (loss of safranin O/fast green staining) in control wild-type mice (14 months old) and PPARγ KO mice (6 months old) was mostly observed in the deep zone/calcified cartilage zone rather than in the superficial zone. This is an indication that proteoglycan loss in spontaneous OA models may not necessarily begin from the superficial layer as opposed to injury-induced OA mouse models, such as the destabilization of medial meniscus model, where we usually observe loss of proteoglycans beginning from the superficial layer.26Valverde-Franco G. Pelletier J.P. Fahmi H. Hum D. Matsuo K. Lussier B. Kapoor M. Martel-Pelletier J. In vivo bone-specific EphB4 overexpression in mice protects both subchondral bone and cartilage during osteoarthritis.Arthritis Rheum. 2012; 64: 3614-3625Crossref PubMed Scopus (33) Google Scholar Note that a previous study has shown that C57Bl/6 mice exhibit spontaneous OA characteristics during aging.27van der Kraan P.M. Stoop R. Meijers T.H. Poole A.R. van den Berg W.B. Expression of type X collagen in young and old C57Bl/6 and Balb/c mice: relation with articular cartilage degeneration.Osteoarthritis Cartilage. 2001; 9: 92-100Abstract Full Text PDF PubMed Scopus (24) Google Scholar In this study, lateral and medial compartments of joints were analyzed, and OA lesions were observed mainly on the lateral side of the tibiofemoral joint. In the present study, control wild-type and PPARγ KO mice were also developed on the C57Bl/6 background. Histopathologic scoring, which was performed on the medial compartment, also showed no substantial cartilage degradation apart from some degree of hypocellularity and proteoglycan loss (loss of safranin O/fast green staining) in the articular cartilage of 14-month-old control mice. To examine synovial inflammation, we performed H&E staining on 14-month-old mouse knee joints, which revealed an increased amount of mononuclear cell infiltration in homozygote PPARγ KO mice compared with control mice (Figure 2A). Quantification of the degree of inflammation (amount of mononuclear cells) by two blinded observers further showed significantly increased inflammatory scores in homozygote PPARγ KO mice versus control mice (Figure 2B). In addition, immunofluorescence using antibody to detect mouse monocytes/macrophages showed an increased number of monocytes/macrophages in the synovium of homozygote PPARγ KO mice compared with the synovium of control mice (Figure 2C). Fourteen-month-old mouse knee joints were also stained with trichrome stain to assess joint fibrosis. Trichrome staining clearly showed increased synovial fibrosis in homozygote PPARγ KO mice compared with the synovium of control mice (Figure 2D). Furthermore, fibrotic deposits were also observed in the superficial zone of the articular cartilage and in some regions of the menisci (meniscal attachments) in homozygote PPARγ KO mice compared with control mice. To further assess the extent of cartilage degradation in PPARγ KO mice versus control mice, we performed IHC studies for VDIPEN and C1-2C. IHC studies for VDIPEN, an MMP-generated neoepitope on aggrecan, demonstrated that tibial plateaus of 14-month-old homozygote PPARγ KO mice exhibited enhanced VDIPEN staining compared with control mice (Figure 3A). IHC analysis for the type II collagen breakdown product C1-2C demonstrated that tibial plateaus of 14-month-old homozygote PPARγ KO mice exhibited increased C1-2C staining compared with control mice. Because MMP-13 is one of the major catabolic enzymes associated with the pathogenesis of OA, we performed IHC studies for MMP-13. Tibial plateaus of 14-month-old homozygote PPARγ KO mice exhibited greater MMP-13 expression than those of age-matched control mice. In addition, we determined the expression of HIF-2α, a recently identified catabolic factor in OA.28Yang S. Kim J. Ryu J.H. Oh H. Chun C.H. Kim B.J. Min B.H. Chun J.S. Hypoxia-inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction.Nat Med. 2010; 16: 687-693Crossref PubMed Scopus (371) Google Scholar HIF-2α expression was higher in tibial plateaus of 14-month-old homozygote PPARγ KO mice compared with those of control mice. We next microdissected cartilage from knee joints of 6-month-old control and homozygote PPARγ KO mice and determined the expression of key markers implicated in mediating destructive mechanisms associated with the pathogenesis of OA. By quantitative PCR, we determined the expression of MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4, ADAMTS-5, HIF-2α, and syndecan-4. PPARγ-deficient cartilage exhibited significantly increased expression of ADAMTS-5, MMP-13, syndecan-4, and HIF-2α but not ADAMTS-4 compared with cartilage isolated from age-matched control littermates (Figure 3B). In addition to catabolic markers, we observed significantly elevated expression of key inducible inflammatory enzymes implicated in OA, including cyclooxygenase (COX)-2 and inducible NO synthase, in PPARγ-deficient cartilage compared with control mouse cartilage (Figure 3B). We recently reported that cartilage-specific PPARγ KO mice exhibit cartilage/bone growth and developmental defects associated with delayed endochondral ossification process, growth plate abnormalities, and reduced length of long bones.23Monemdjou R. Vasheghani F. Fahmi H. Perez G. Blati M. Taniguchi N. Lotz M. St-Arnaud R. Pelletier J.P. Martel-Pelletier J. Beier F. Kapoor M. Association of cartilage-specific deletion of peroxisome proliferator-activated receptor gamma with abnormal endochondral ossification and impaired cartilage growth and development in a murine model.Arthritis Rheum. 2012; 64: 1551-1561Crossref PubMed Scopus (43) Google Scholar In the present study, we further demonstrate, for the first time, an important in vivo role of PPARγ in OA through the use of cartilage-specific PPARγ KO mice. We show that adult cartilage-specific PPARγ KO mice develop a spontaneous OA phenotype associated with enhanced cartilage degradation, synovial inflammation, synovial and cartilage fibrosis, increased expression of MMP-generated neoepitopes (VDIPEN and C1-2C), increased expression of catabolic markers (including MMP-13, ADAMTS-5, HIF-2α, and syndecan-4), and increased expression of inflammatory enzymes (including COX-2 and inducible NO synthase). Recent studies have shown that HIF-2α expression is markedly increased in human and mouse OA cartilage and that it causes cartilage destruction by regulating crucial catabolic genes, including MMP-1, MMP-3, MMP-9, MMP-12, MMP-13, ADAMTS-4, NO synthase 2, and COX-2.28Yang S. Kim J. Ryu J.H. Oh H. Chun C.H. Kim B.J. Min B.H. Chun J.S. Hypoxia-inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction.Nat Med. 2010; 16: 687-693Crossref PubMed Scopus (371) Google Scholar In addition to HIF-2α, other key catabolic factors involved in cartilage degradation are ADAMTS-5 and syndecan-4. Syndecan-4 has been shown to regulate ADAMTS-5 activation and cartilage breakdown in OA.29Echtermeyer F. Bertrand J. Dreier R. Meinecke I. Neugebauer K. Fuerst M. Lee Y.J. Song Y.W. Herzog C. Theilmeier G. Pap T. Syndecan-4 regulates ADAMTS-5 activation and cartilage breakdown in osteoarthritis.Nat Med. 2009; 15: 1072-1076Crossref PubMed Scopus (260) Google Scholar The present study shows that PPARγ-deficient cartilage exhibits elevated expression of HIF-2α, which may, in part, be responsible for the increased expression of MMP-13, inducible NO synthase, and COX-2 observed. In addition, increased expression of syndecan-4 in PPARγ-deficient cartilage could contribute toward the increased expression of ADAMTS-5 observed. The up-regulation of these catabolic mediators in the articular cartilage of PPARγ-deficient mice could be a contributing factor resulting in cartilage destruction and the OA phenotype observed. Note that PPARγ expression is reduced in human OA cartilage compared with normal cartilage.30Afif H. Benderdour M. Mfuna-Endam L. Martel-Pelletier J. Pelletier J.P. Duval N. Fahmi H. Peroxisome proliferator-activated receptor gamma1 expression is diminished in human osteoarthritic cartilage and is downregulated by interleukin-1beta in articular chondrocytes.Arthritis Res Ther. 2007; 9: R31Crossref PubMed Scopus (72) Google Scholar This finding suggests that reduced PPARγ expression in OA cartilage may reflect increased expression of inflammatory and catabolic factors. Indeed, treatment of human OA chondrocytes with pro-inflammatory factors, including IL-1β, tumor necrosis factor-α, IL-17, and prostaglandin E2, suppresses PPARγ expression.30Afif H. Benderdour M. Mfuna-Endam L. Martel-Pelletier J. Pelletier J.P. Duval N. Fahmi H. Peroxisome proliferator-activated receptor gamma1 expression is diminished in human osteoarthritic cartilage and is downregulated by interleukin-1beta in articular chondrocytes.Arthritis Res Ther. 2007; 9: R31Crossref PubMed Scopus (72) Google Scholar Also, PPARγ agonists can reduce the expression of inflammatory/catabolic mediators and protect cartilage from degradation.15Boileau C. Martel-Pelletier J. Fahmi H. Mineau F. Boily M. Pelletier J.P. The peroxisome proliferator-activated receptor gamma agonist pioglitazone reduces the development of cartilage lesions in an experimental dog model of osteoarthritis: in vivo protective effects mediated through the inhibition of key signaling and catabolic pathways.Arthritis Rheum. 2007; 56: 2288-2298Crossref PubMed Scopus (60) Google Scholar, 16Kobayashi T. Notoya K. Naito T. Unno S. Nakamura A. Martel-Pelletier J. Pelletier J.P. Pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, reduces the progression of experimental osteoarthritis in guinea pigs.Arthritis Rheum. 2005; 52: 479-487Crossref PubMed Scopus (86) Google Scholar In normal articular cartilage, there seems to be an optimum equilibrium between catabolic/inflammatory factors and PPARγ. In a catabolic and inflammatory event in the cartilage, PPARγ expression is down-regulated and, as a result, cartilage destruction is promoted. Consequently, increased PPARγ can reduce the expression of critical catabolic/inflammatory mediators, thus preventing cartilage damage. One can also speculate that early developmental defects observed in PPARγ KO mice23Monemdjou R. Vasheghani F. Fahmi H. Perez G. Blati M. Taniguchi N. Lotz M. St-Arnaud R. Pelletier J.P. Martel-Pelletier J. Beier F. Kapoor M. 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TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage.J Cell Biol. 2001; 153: 35-46Crossref PubMed Scopus (521) Google Scholar It is hypothesized that one of the reasons for spontaneous OA-like characteristics during aging is that defects in early cartilage development cause skeletal malformations that affect joint geometry and biochemical properties predisposing to OA. As a result, cartilage degradation occurs at a higher rate, as does the incidence of OA. Stattin et al33Stattin E.L. Tegner Y. Domellof M. Dahl N. Familial osteochondritis dissecans associated with early osteoarthritis and disproportionate short stature.Osteoarthritis Cartilage. 2008; 16: 890-896Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar supported this finding by demonstrating that patients with familial dysplasia develop joint malformations and, subsequently, secondary OA. The present data clearly demonstrate that disruption of PPARγ function leads to earlier OA. However, at this moment, we cannot determine whether accelerated OA in the present mice is secondary to the observed developmental defects or whether it is caused by the independent roles of PPARγ in articular cartilage. Specific inactivation of the PPARγ gene in adult articular cartilage (using an inducible Cre system) will be required to resolve this question. In addition, future studies should also be directed toward understanding the role of PPARγ in chondrocyte hypertrophy as studies show that chondrocyte hypertrophy can play a vital role in the development of OA.34van der Kraan P.M. van den Berg W.B. Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration?.Osteoarthritis Cartilage. 2012; 20: 223-232Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar Apart from cartilage destruction, another key phenotypic characteristic associated with the development of OA is synovial inflammation and fibrosis.35Plaas A. Velasco J. Gorski D.J. Li J. Cole A. Christopherson K. Sandy J.D. The relationship between fibrogenic TGFbeta1 signaling in the joint and cartilage degradation in post-injury osteoarthritis.Osteoarthritis Cartilage. 2011; 19: 1081-1090Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar In cartilage-specific PPARγ KO mice, we also observed increased synovial inflammation (increased presence of monocytes/macrophages) and increased synovial and cartilage fibrosis. This is probably due to the fact that PPARγ is a key anti-inflammatory and antifibrotic agent. We previously showed that loss of PPARγ in fibroblasts results in excessive skin inflammation and fibrosis in a bleomycin-induced fibrosis model.7Kapoor M. McCann M. Liu S. Huh K. Denton C.P. Abraham D.J. Leask A. Loss of peroxisome proliferator-activated receptor gamma in mouse fibroblasts results in increased susceptibility to bleomycin-induced skin fibrosis.Arthritis Rheum. 2009; 60: 2822-2829Crossref PubMed Scopus (64) Google Scholar Similarly, agonists of PPARγ attenuate bleomycin-induced skin inflammation and dermal fibrosis and block the activation of transforming growth factor β/Smad signaling.8Wu M. Melichian D.S. Chang E. Warner-Blankenship M. Ghosh A.K. Varga J. Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma.Am J Pathol. 2009; 174: 519-533Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar In addition, agonism of PPARγ has been shown to attenuate cardiac fibrosis by inhibiting myocardial macrophage infiltration.36Caglayan E. Stauber B. Collins A.R. Lyon C.J. Yin F. Liu J. Rosenkranz S. Erdmann E. Peterson L.E. Ross R.S. Tangirala R.K. Hsueh W.A. Differential roles of cardiomyocyte and macrophage peroxisome proliferator-activated receptor gamma in cardiac fibrosis.Diabetes. 2008; 57: 2470-2479Crossref PubMed Scopus (66) Google Scholar Thus, in the present study, it seems that loss of PPARγ results in not only loss of chondroprotective effects but also loss of anti-inflammatory and antifibrogenic effects, resulting in increased synovial inflammation (accumulation of macrophages) and increased synovial and cartilage fibrosis. In closing, these results clearly demonstrate, for the first time, to our knowledge, that PPARγ is a critical regulator of cartilage health and integrity. Cartilage-specific PPARγ KO mice, which exhibit a slow-progressing spontaneous form of OA, provide further insights into endogenous mechanisms associated with the pathology of OA. For example, PPARγ KO mice provide evidence that loss of proteoglycans in spontaneous OA as opposed to injury-induced OA can begin from the deep zone of the articular cartilage and not necessarily from the superficial zone. This model also clearly defines synovial and cartilage fibrosis as key spontaneous OA characteristics in addition to cartilage degradation. Furthermore, this study further supports the role of PPARγ as an anticatabolic, anti-inflammatory, and antifibrotic factor. We thank Stéphane Tremblay and Frédéric Paré for their assistance with histologic and histomorphometric analyses; François Mineau, François-Cyril Jolicoeur, David Hum, and Changshan Geng for their technical assistance; and Virginia Wallis for her help with manuscript preparation (all from the University of Montreal Hospital Research Centre, Montreal, QC, Canada).