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• W2489431013 abstract "Current description of osteoarthritis includes the involvement of synovial inflammation. Studies contributing to understanding the mechanisms of cross-talk and feedback among the joint tissues could be relevant to the development of therapies that block disease progression. During osteoarthritis, synovial fibroblasts exposed to anomalous mechanical forces and an inflammatory microenvironment release factors such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) metalloproteinases that mediate tissue damage and perpetuate inflammation. We therefore studied the production of ADAMTS by synovial fibroblasts and their contribution to cartilage degradation. Moreover, we analyzed the implication of two mediators present in the osteoarthritis joint, IL-1β as proinflammatory cytokine, and 45-kDa fibronectin fragments as products of matrix degradation. We reported that synovial fibroblasts constitutively express and release ADAMTS 4, 5, 7, and 12. Despite the contribution of both mediators to the stimulation of Runx2 and Wnt/β-catenin signaling pathways, as well as to ADAMTS expression, promoting the degradation of aggrecan and cartilage oligomeric matrix protein from cartilage, fibronectin fragments rather than IL-1β played the major pathological role in osteoarthritis, contributing to the maintenance of the disease. Moreover, higher levels of ADAMTS 4 and 7 and a specific regulation of ADAMTS-12 were observed in osteoarthritis, suggesting them as new potential therapeutic targets. Therefore, synovial fibroblasts provide the biochemical tools to the chronicity and destruction of the osteoarthritic joints. Current description of osteoarthritis includes the involvement of synovial inflammation. Studies contributing to understanding the mechanisms of cross-talk and feedback among the joint tissues could be relevant to the development of therapies that block disease progression. During osteoarthritis, synovial fibroblasts exposed to anomalous mechanical forces and an inflammatory microenvironment release factors such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) metalloproteinases that mediate tissue damage and perpetuate inflammation. We therefore studied the production of ADAMTS by synovial fibroblasts and their contribution to cartilage degradation. Moreover, we analyzed the implication of two mediators present in the osteoarthritis joint, IL-1β as proinflammatory cytokine, and 45-kDa fibronectin fragments as products of matrix degradation. We reported that synovial fibroblasts constitutively express and release ADAMTS 4, 5, 7, and 12. Despite the contribution of both mediators to the stimulation of Runx2 and Wnt/β-catenin signaling pathways, as well as to ADAMTS expression, promoting the degradation of aggrecan and cartilage oligomeric matrix protein from cartilage, fibronectin fragments rather than IL-1β played the major pathological role in osteoarthritis, contributing to the maintenance of the disease. Moreover, higher levels of ADAMTS 4 and 7 and a specific regulation of ADAMTS-12 were observed in osteoarthritis, suggesting them as new potential therapeutic targets. Therefore, synovial fibroblasts provide the biochemical tools to the chronicity and destruction of the osteoarthritic joints. Osteoarthritis (OA), one of the leading causes of substantial physical and psychological disability worldwide, is a complex disease with a prevalence of >70% in the population >55 years.1Brooks P. Inflammation as an important feature of osteoarthritis.Bull World Health Organ. 2003; 81: 689-690PubMed Google Scholar, 2Goldring M.B. Goldring S.R. Osteoarthritis.J Cell Physiol. 2007; 213: 626-634Crossref PubMed Scopus (1029) Google Scholar Current description of OA includes not only the remodeling of articular cartilage and adjacent bone, but also the involvement of synovial inflammation, which is characterized by thickening of synovium or, indirectly, by joint effusion. Synovial membrane inflammation and proliferation in OA joints trigger the production of cytokines and proteinases that damage connective tissues, including the cartilage.3Goldring M.B. Marcu K.B. Cartilage homeostasis in health and rheumatic diseases.Arthritis Res Ther. 2009; 11: 224Crossref PubMed Scopus (505) Google Scholar, 4Benito M.J. Veale D.J. FitzGerald O. van den Berg W.B. Bresnihan B. Synovial tissue inflammation in early and late osteoarthritis.Ann Rheum Dis. 2005; 64: 1263-1267Crossref PubMed Scopus (699) Google Scholar In this sense, research on inhibitory mediators of synovial activation could identify ways to avoid the progressive cartilage degradation and functional impairment. The role of synovial fibroblasts (SFs) as active drivers of joint destruction in rheumatoid arthritis is well established,5Lefevre S. Meier F.M. Neumann E. Muller-Ladner U. Role of synovial fibroblasts in rheumatoid arthritis.Curr Pharm Des. 2015; 21: 130-141Crossref PubMed Scopus (80) Google Scholar but their behavior in healthy subjects and OA patients is poorly understood. It has been described that OA-SF exposed to anomalous mechanical forces and to an inflammatory microenvironment, release factors such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), that mediate tissue damage and perpetuate inflammation.2Goldring M.B. Goldring S.R. Osteoarthritis.J Cell Physiol. 2007; 213: 626-634Crossref PubMed Scopus (1029) Google Scholar, 6Bondeson J. Blom A.B. Wainwright S. Hughes C. Caterson B. van den Berg W.B. The role of synovial macrophages and macrophage-produced mediators in driving inflammatory and destructive responses in osteoarthritis.Arthritis Rheum. 2010; 62: 647-657Crossref PubMed Scopus (274) Google Scholar, 7Sutton S. Clutterbuck A. Harris P. Gent T. Freeman S. Foster N. Barrett-Jolley R. Mobasheri A. The contribution of the synovium, synovial derived inflammatory cytokines and neuropeptides to the pathogenesis of osteoarthritis.Vet J. 2009; 179: 10-24Crossref PubMed Scopus (158) Google Scholar, 8Lin E.A. Liu C.J. The role of ADAMTSs in arthritis.Protein Cell. 2010; 1: 33-47Crossref PubMed Scopus (69) Google Scholar, 9Yamanishi Y. Boyle D.L. Clark M. Maki R.A. Tortorella M.D. Arner E.C. Firestein G.S. Expression and regulation of aggrecanase in arthritis: the role of TGF-beta.J Immunol. 2002; 168: 1405-1412Crossref PubMed Scopus (154) Google Scholar Therefore, studies contributing to a better understanding of the cross-talk and feed-back mechanisms among the joint tissues could be relevant to the development of new therapies able to block disease progression. Our aim was to elucidate the role of SF in the cartilage joint degradation in OA patients through the production of ADAMTS and to characterize these metalloproteinases in SF from healthy donors (HD). We have mapped the expression and function of aggrecanases ADAMTS 4 and 5, which degrade aggrecan, one of the main components of the cartilage extracellular matrix (ECM) that facilitates cartilage to resist compression.10Fosang A.J. Rogerson F.M. Identifying the human aggrecanase.Osteoarthritis Cartilage. 2010; 18: 1109-1116Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar We have also characterized ADAMTS 7 and 12, involved in destruction of cartilage oligomeric matrix protein (COMP), a noncollagenous component of the cartilage ECM that contributes to its assembly and to the cartilage integrity.11Acharya C. Yik J.H. Kishore A. Van Dinh V. Di Cesare P.E. Haudenschild D.R. Cartilage oligomeric matrix protein and its binding partners in the cartilage extracellular matrix: interaction, regulation and role in chondrogenesis.Matrix Biol. 2014; 37: 102-111Crossref PubMed Scopus (109) Google Scholar Moreover, we studied the physiopathological effect of two mediators present in OA joint microenvironment: the catabolic cytokine IL-1β and the 45-kDa fibronectin fragments (Fn-fs) as products of cartilage ECM degradation.12Sandy J.D. Chan D.D. Trevino R.L. Wimmer M.A. Plaas A. Human genome-wide expression analysis reorients the study of inflammatory mediators and biomechanics in osteoarthritis.Osteoarthritis Cartilage. 2015; 23: 1939-1945Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 13Ding L. Guo D. Homandberg G.A. Fibronectin fragments mediate matrix metalloproteinase upregulation and cartilage damage through proline rich tyrosine kinase 2, c-src, NF-kappaB and protein kinase Cdelta.Osteoarthritis Cartilage. 2009; 17: 1385-1392Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 14Ding L. Guo D. Homandberg G.A. The cartilage chondrolytic mechanism of fibronectin fragments involves MAP kinases: comparison of three fragments and native fibronectin.Osteoarthritis Cartilage. 2008; 16: 1253-1262Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 15Yasuda T. Cartilage destruction by matrix degradation products.Mod Rheumatol. 2006; 16: 197-205Crossref PubMed Google Scholar This study is the first to report the expression and release of ADAMTS 7 and 12 by SF from HD and OA patients, both constitutively and after IL-1β or Fn-fs stimulation. Besides, the capacity of SF to attach and degrade the cartilage ECM, generating glycosaminoglycans (GAGs) and releasing COMP, is also described. Finally, we study the activation of Runx2 and β-catenin, two signaling pathways related to ADAMTS expression.16Thirunavukkarasu K. Pei Y. Moore T.L. Wang H. Yu X.P. Geiser A.G. Chandrasekhar S. Regulation of the human ADAMTS-4 promoter by transcription factors and cytokines.Biochem Biophys Res Comm. 2006; 345: 197-204Crossref PubMed Scopus (53) Google Scholar, 17Thirunavukkarasu K. Pei Y. Wei T. Characterization of the human ADAMTS-5 (aggrecanase-2) gene promoter.Mol Biol Rep. 2007; 34: 225-231Crossref PubMed Scopus (51) Google Scholar, 18Yuasa T. Otani T. Koike T. Iwamoto M. Enomoto-Iwamoto M. Wnt/β-catenin signaling stimulates matrix catabolic genes and activity in articular chondrocytes: its possible role in joint degeneration.Lab Invest. 2008; 88: 264-274Crossref PubMed Scopus (162) Google Scholar Our study reports that SF activated by mediators present in the joint, such as a proinflammatory cytokines and Fn-fs, release ADAMTS, which contribute to the maintenance of cartilage destruction in osteoarthritic patients. Synovial tissue was obtained from 20 active OA patients (16 women and 4 men) aged between 48 and 87 years, at the time of knee prosthetic replacement surgery. Patients had advanced disease and were diagnosed with primary OA, excluding trauma, inflammatory disease, and other structural causes of secondary OA. Control samples from HD were obtained from four patients (two women and two men) aged between 35 and 72 years, at the time of knee arthroscopic evaluation. These patients were diagnosed with meniscopathy caused by trauma to the knee or sports injury, excluding inflammatory and rheumatic diseases. The study was performed according to the recommendations of the Declaration of Helsinki and approved by the Clinical Research Ethics Committee of the Hospital La Princesa (Madrid, Spain). All biopsy samples were obtained after subjects gave informed consent. SF cultures were established by explant growth of synovial biopsies, cultured in Dulbecco's modified Eagle's medium (DMEM) with 25 mmol/L HEPES and 4.5 g/L glucose, completed with 10% heat-inactivated fetal bovine serum (Lonza Ibérica S.A.U., Barcelona, Spain), 1% l-glutamine, and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA) at 37°C and 5% CO2. After three passages, residual contamination by macrophages was avoided, previously assessed by flow cytometry analysis of SF with a purity of 95%.19Juarranz Y. Gutierrez-Canas I. Santiago B. Carrion M. Pablos J.L. Gomariz R.P. Differential expression of vasoactive intestinal peptide and its functional receptors in human osteoarthritic and rheumatoid synovial fibroblasts.Arthritis Rheum. 2008; 58: 1086-1095Crossref PubMed Scopus (62) Google Scholar Monocultures of SF were used for experiments until passage 8. Despite the use of cells at varying passage numbers, all comparisons within a same experimentation were made on SF at an identical passage number and at 80% to 90% confluence. For treatments, HD- and OA-SF were cultured in serum-free DMEM with 1% l-glutamine and 1% antibiotic-antimycotic, in the absence (untreated) or presence of the following agents: 10 ng/mL IL-1β (ImmunoTools, Friesoythe, Germany) or 10 nmol/L Fn-fs 45 kDa (Sigma-Aldrich, St Louis, MO). SFs were seeded in 100-mm petri dishes (3 × 105cells per dish) and cultured in the absence or presence of 10 ng/mL IL-1β or 10 nmol/L Fn-fs 45 kDa for 24 hours. Total RNA was obtained using TriReagent (Sigma-Aldrich). Two microgram of RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Semiquantitative real-time PCR analysis was performed using a TaqMan Gene Expression Master Mix with manufactured-predesigned primers and probes for β-actin (NM001101.3), ADAMTS-4 (NM005099.4), ADAMTS-5 (NM007038.3), ADAMTS-7 (NM014272.3), and ADAMTS-12 (NM030955.2) (Applied Biosystems). We normalized the target gene expression to the housekeeping gene, β-actin (2−ΔCt). For relative quantification, results were presented as the relative expression with respect to the untreated condition using the formula 2−ΔΔCt, as previously described.19Juarranz Y. Gutierrez-Canas I. Santiago B. Carrion M. Pablos J.L. Gomariz R.P. Differential expression of vasoactive intestinal peptide and its functional receptors in human osteoarthritic and rheumatoid synovial fibroblasts.Arthritis Rheum. 2008; 58: 1086-1095Crossref PubMed Scopus (62) Google Scholar SFs were seeded in 6-well plates (6 × 104 cells per well) and cultured in the absence or presence of 10 ng/mL IL-1β or 10 nmol/L Fn-fs 45 kDa, for 24 hours. Levels of ADAMTS were measured in the culture supernatants using commercial enzyme-linked immunosorbent assay (ELISA) kits for ADAMTS 4 and 5 (Cloud-Clone Corp., Houston, TX), and for ADAMTS 7 and 12 (MyBioSource, San Diego, CA). SFs were seeded in 100-mm petri dishes (3 × 105 cells per dish) and cultured in the absence or presence of 10 ng/mL IL-1β or 10 nmol/L Fn-fs 45 kDa, for 24 hours. Aggrecanase activity was measured in the SF culture supernatants using a Sensitive Aggrecanase Activity ELISA Kit (MD Bioproducts, Zürich, Switzerland), according to the manufacturer's instructions. Briefly, this assay consists of two modules. In the Aggrecanase Module, a modified interglobular domain (aggrecan-IGD-s) is digested with aggrecanases, and its proteolytic cleavage releases an aggrecan peptide (ARGSVIL-peptide-s), which is then quantified with antibodies in the ELISA Module. SFs were seeded on glass coverslips (2.5 × 104 cells per glass), fixed with paraformaldehyde, and permeabilized with Tween-20 in phosphate-buffered saline. Cells were blocked with phosphate-buffered saline containing donkey serum and incubated with rabbit polyclonal anti-human antibodies for ADAMTS 4, 5, 7, or 12 (Sigma-Aldrich). After washing, cells were incubated with AlexaFluor 488 donkey anti-rabbit IgG antibody (Invitrogen). Coverslips were counterstained with Hoechst. Background fluorescence was reduced with Sudan Black in ethanol. Negative controls were performed in the absence of primary antibodies (data not shown). Fluorescence was examined using an Olympus BX51 microscope with DP72 camera model (objective 40×). SFs were seeded in 150-mm petri dishes (8 × 105 cells per dish). A Nuclear Extract Kit (Active Motif, Rixensart, Belgium) was used for nuclear extracts preparation, and the protein content was measured with a QuantiPro BCA Assay Kit (Sigma-Aldrich). Cytoplasmic extracts obtained were stored at −80°C for later use in the Western blots. Nuclear extracts (12 μg per well) were added to a 96-well plate, and Runx2 activity was measured using a TransAM AML-3/Runx2 kit (Active Motif). Time course of Runx2 activation after incubation with 10 ng/mL IL-1β or 10 nmol/L Fn-fs 45 kDa was studied (data not shown), and the experiments were performed at 60 or 30 minutes of treatment, respectively. To detect β-catenin levels, a β-catenin (Total) and a (Phospho) InstantOne ELISA kits were used (eBioscience, San Diego, CA) with SF cellular lysates. Briefly, SFs seeded in 100-mm petri dishes (3 × 105 cells per dish) were scraped into phosphate-buffered saline, centrifuged, and resuspended in the Cell Lysis Buffer Mix (eBioscience). Protein content was measured by QuantiPro BCA Assay Kit. Levels of β-catenin in the cellular lysates were measured after 60 minutes of treatment with 10 ng/mL IL-1β or 10 nmol/L Fn-fs 45 kDa. For the detection of ADAMTS, SFs were seeded in 100-mm dishes and cultured to confluence. Culture supernatants were collected. For protein purification and concentration, Amicon Ultra 0.5 mL centrifugal filters (Merck Millipore, Darmstadt, Germany) were used. For Runx2 and β-catenin, the cytoplasmic extracts previously obtained were used, and protein content was measured by QuantiPro BCA Assay Kit. Cytoplasmic extracts (15 μg/well) and culture supernatants were subjected to SDS-PAGE and blotted on a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked with Tris-buffered saline containing bovine serum albumin and Tween-20, and incubated with mouse monoclonal anti-human ADAMTS-4, ADAMTS-5 (R&D Systems), Runx2, or β-catenin (Santa Cruz Biotechnology) antibodies, or rabbit polyclonal anti-human ADAMTS-7 or ADAMTS-12 antibodies (Abcam, Cambridge, UK). Appropriate horseradish peroxidase–conjugated secondary antibodies were applied and detected by Western blot Luminol Reagent (Santa Cruz Biotechnology). For Runx2 and β-catenin, we used β-actin as a loading control. Protein bands were scanned and quantified with the Bio-Rad Quantity One program. For blockade experiments, HD- and OA-SF were seeded in 100-mm dishes (3 × 105 cells per dish) and cultured in serum-free DMEM with 1% l-glutamine and 1% antibiotic-antimycotic, in the absence or presence of 10 μmol/L of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinases inhibitor, PD98059, consequently implicated in the inhibition of ERK; 10 μmol/L of MAPK) p38 inhibitor, SB203580 (Calbiochem, EMD Biosciences, San Diego, CA), or 200 ng/mL of Wnt inhibitor, DDK-1 (R&D Systems) for 1 hour. These treatments were followed by stimulation with 10 ng/mL IL-1β or 10 nmol/L Fn-fs 45 kDa, for 24 hours. Total RNA was obtained, and quantitative RT-PCR for ADAMTS 4 and 5 was performed as previously described.19Juarranz Y. Gutierrez-Canas I. Santiago B. Carrion M. Pablos J.L. Gomariz R.P. Differential expression of vasoactive intestinal peptide and its functional receptors in human osteoarthritic and rheumatoid synovial fibroblasts.Arthritis Rheum. 2008; 58: 1086-1095Crossref PubMed Scopus (62) Google Scholar Release of GAG and COMP from cartilage was measured in culture supernatants from wells containing co-cultures of SF over cartilage explants.20Pretzel D. Pohlers D. Weinert S. Kinne R.W. In vitro model for the analysis of synovial fibroblast-mediated degradation of intact cartilage.Arthritis Res Ther. 2009; 11: R25Crossref PubMed Scopus (42) Google Scholar OA human cartilages were obtained from three patients undergoing total hip arthroplasty from Hospital del Mar (Barcelona, Spain). Fixed diameter (6 mm) and height (2 mm) sections were collected from cartilage areas without macroscopic signs of OA. Samples were frozen at −80°C and stored until testing. One explant per well was attached to a 24-well plate. HD- or OA-SF were added drop-wise on top of the cartilage surface (2 × 104 SF per explant). After 3 hours of incubation, wells were filled with DMEM in the absence or presence of 10 ng/mL IL-1β or 10 nmol/L Fn-fs 45 kDa, and cultures were continued for 14 days. Culture supernatants were collected for detection of GAG and COMP, using a Blyscan Sulfated Glycosaminoglycan Assay (Biocolor Ltd County Antrim, Ireland, UK), and a Quantikine Human COMP Immunoassay (R&D Systems, Abingdon, OX, UK), respectively. Frozen sections were prepared using a cryostat and stained with Alcian blue and Callejas's tricromic. Sections were observed using an Olympus BX51 microscope with DP72 camera model (objective 20×). Data were analyzed using the GraphPad Prism software version 6 (Graphpad Software Inc., La Jolla, CA). Data were subjected to normality test (Kolmogórov-Smirnov test) and equal variance test (F-test). Statistical differences between sample groups were assessed using Student's two-tailed t-test or unpaired t-test with Welch's correction, in case of groups with different variances. P < 0.05 was considered statistically significant. Results are presented as the means ± SEM. We explored the constitutive expression of ADAMTS in HD- and OA-SF by quantitative RT-PCR (Figure 1). Patterns of constitutive ADAMTS gene expression were similar in untreated HD- and OA-SF. ADAMTS-5 was the most expressed followed by ADAMTS 4, 7, and 12. ADAMTS-5 transcripts were nearly 50-fold higher than ADAMTS-12. Comparing HD- and OA-SF, ADAMTS 4 and 7 transcripts were twofold and fourfold higher in OA- than in HD-SF, respectively. However, ADAMTS 5 and 12 mRNA levels were similar. ADAMTS protein expression was confirmed by immunocytochemistry. Untreated HD- and OA-SF displayed similar morphology and both showed cytoplasmic immunostaining for ADAMTS 4, 5, 7, and 12 (Figure 2). No staining was observed in isotype controls (data not shown). ADAMTS were also evaluated in untreated SF culture supernatants by Western blot (Figure 3). We confirmed that all ADAMTS are released to the medium by HD- and OA-SF. Western blots for ADAMTS 4 and 5 (Figure 3, A and B) revealed bands corresponding to the active forms (between 48 and 74 kDa), and additional bands with a higher molecular weight. ADAMTS-7 and ADAMTS-12 Western blots (Figure 3, C and D) showed bands with the predicted molecular weight of the enzymes (between 114 and 201 kDa), and additional smaller bands. Because SF has been suggested to represent an important source of aggrecanases within the joint mediating cartilage destruction,10Fosang A.J. Rogerson F.M. Identifying the human aggrecanase.Osteoarthritis Cartilage. 2010; 18: 1109-1116Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar we next studied the effect of IL-1β and Fn-fs on their production. IL-1β and Fn-fs increased the transcript and protein of ADAMTS-4 in HD- and OA-SF compared with the untreated cells (Figure 4, A and B). A significant increase in ADAMTS-5 transcript and protein was detected in OA-SF for both stimuli, whereas in HD stimulation was only observed in the protein after treatment with IL-1β (Figure 4, C and D). Altogether, these results show that IL-1β increased ADAMTS 4 and 5 in HD- and OA-SF, whereas Fn-fs showed more specific effects in OA, resulting in a significant augment of ADAMTS-4 in both, and in a restricted stimulation of ADAMTS-5 production in OA-SF. ADAMTS 4 and 5 cleave aggrecan within the interglobular domain at the Glu-373 and Ala-374 bond.21Huang K. Wu L.D. Aggrecanase and aggrecan degradation in osteoarthritis: a review.J Int Med Res. 2008; 36: 1149-1160Crossref PubMed Scopus (141) Google Scholar, 22Kelwick R. Desanlis I. Wheeler G.N. Edwards D.R. The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family.Genome Biol. 2015; 16: 113Crossref PubMed Scopus (389) Google Scholar Thus, we assessed the ability of ADAMTS 4 and 5 produced by cultured SF to this cleavage by measuring aggrecanase activation and the ARGSVL-peptide-s released in culture supernatants by means of ELISA. Both the constitutive aggrecanase activity (Figure 4E) and the derived peptide (data not shown) were significantly greater in OA- compared to HD-SF. Fn-fs significantly increased aggrecanase activity (Figure 4F) and the derived peptide (data not shown) exclusively in OA-SF, whereas IL-1β did not induce any change. Interestingly, these results correlated with the Fn-fs induction of ADAMTS 4 and 5 in OA-SF. The aggrecanase activity yields the generation of GAGs from the aggrecan in cartilage ECM. Hence, we next studied the potential capacity of SF to degrade cartilage by measuring GAG release in supernatants from cartilage-SF co-cultures. After 14 days of in vitro co-cultures, a monolayer of SF was observed exclusively on the cartilage surface (Figure 4G) that did not appear in the control (Figure 4H). The effects of IL-1β and Fn-fs after 14 days of treatment were evaluated. The constitutive release of GAGs to the medium was significantly greater in OA-SF compared to HD-SF. IL-1β induced no change in GAGs levels, whereas Fn-fs enhanced significantly the release of GAGs in both HD- and OA-SF (Figure 4I). Because Runx2 transcription factor and Wnt/β-catenin signaling are involved in aggrecanase gene expression,16Thirunavukkarasu K. Pei Y. Moore T.L. Wang H. Yu X.P. Geiser A.G. Chandrasekhar S. Regulation of the human ADAMTS-4 promoter by transcription factors and cytokines.Biochem Biophys Res Comm. 2006; 345: 197-204Crossref PubMed Scopus (53) Google Scholar, 17Thirunavukkarasu K. Pei Y. Wei T. Characterization of the human ADAMTS-5 (aggrecanase-2) gene promoter.Mol Biol Rep. 2007; 34: 225-231Crossref PubMed Scopus (51) Google Scholar, 18Yuasa T. Otani T. Koike T. Iwamoto M. Enomoto-Iwamoto M. Wnt/β-catenin signaling stimulates matrix catabolic genes and activity in articular chondrocytes: its possible role in joint degeneration.Lab Invest. 2008; 88: 264-274Crossref PubMed Scopus (162) Google Scholar and also seem to be implicated in the OA pathology,23Alcaraz M.J. Megias J. Garcia-Arnandis I. Clerigues V. Guillen M.I. New molecular targets for the treatment of osteoarthritis.Biochem Pharmacol. 2010; 80: 13-21Crossref PubMed Scopus (112) Google Scholar, 24Ji Q. Xu X. Xu Y. Fan Z. Kang L. Li L. Liang Y. Guo J. Hong T. Li Z. Zhang Q. Ye Q. Wang Y. miR-105/Runx2 axis mediates FGF2-induced ADAMTS expression in osteoarthritis cartilage.J Mol Med (Berl). 2016; 94: 681-694Crossref PubMed Scopus (51) Google Scholar we decided to examine whether IL-1β or Fn-fs could alter the ADAMTS 4 and 5 expressions by the modulation of these factors. Indeed, we found that both IL-1β and Fn-fs significantly induced nuclear activation of Runx2 in HD and OA-SF (Figure 5A), consistent with its reduction in the cytoplasm after the stimulation with both mediators (Figure 5C). Nonetheless, the data were consistent with a role for Runx2 in ADAMTS4 transcription because the transcript abundance with IL-1β, on both HD and OA, was approximately twofold greater than in untreated controls. Furthermore, treatment with Fn-fs stimulated ADAMTS4 expression was approximately 2.5-fold in OA cells and approximately 1.3-fold in HD cells. In contrast to ADAMTS-4, ADAMTS-5 expression showed no stimulation of HD-SF by either IL-1β or Fn-fs, but approximately a 1.6-fold increase by both IL-1β and Fn-fs in OA. In the cytoplasm, β-catenin is regulated by interaction with a multiprotein complex that phosphorylates it to be degraded by proteasomes. On activation of Wnt signaling, non-phosphorylated β-catenin is transported to the nucleus, where it couples with the complex T-cell factor/lymphoid-enhancing factor to initiate the transcription of ADAMTS 4 and 5 genes.25Thomas R.S. Clarke A.R. Duance V.C. Blain E.J. Effects of Wnt3A and mechanical load on cartilage chondrocyte homeostasis.Arthritis Res Ther. 2011; 13: R203Crossref PubMed Scopus (39) Google Scholar We measured both β-catenin forms in cellular lysates of SF, where levels of phosphorylated β-catenin were undetectable by ELISA (data not shown). Thus, we measured the total β-catenin that mainly represented the active form. The β-catenin content of whole cell lysates was approximately 1.5-fold higher for untreated OA-SF than untreated HD. Moreover, β-catenin levels increased to approximately twofold after treatment with either IL-1β or Fn-fs exclusively in OA (Figure 5B), which correlated with the reduction observed in cytosplasmic extracts by Western blot (Figure 5D). To better elucidate the implication of Runx2 and β-catenin in the aggrecanases expression, we performed blockade experiments using inhibitors of two MAPK, ERK, and p38-MAPK, implicated in the activation of Runx2, PD98059, and SB203580, respectively. We also used an inhibitor of Wnt/β-catenin signaling, DDK-1. We showed that PD98059 significantly inhibited mRNA expression of ADAMTS-4 before treatment with IL-1β or Fn-fs, in HD- and OA-SF (Figure 6A). Moreover, PD98059 inhibited the expression of ADAMTS-5 in OA-SF after both stimuli, whereas in HD this inhibition was observed only before stimulation with IL-1β (Figure 6B). On the other hand, SB203580 is involved in the decrease of ADAMTS-4 mRNA expression stimulated by Fn-fs in both, HD- and OA-SF (Figure 6A). Regarding ADAMTS-5, SB203580 significantly inhibited its expression only in OA-SF, after both stimuli (Figure 6B). Moreover, the Wnt/β-catenin inhibitor DKK-1 significantly inhibited the expression of both, ADAMTS 4 and 5, before treatment with IL-1β or Fn-fs, exclusively in OA-SF (Figure 6, C and D). We further studied the effects of IL-1β and Fn-fs on the ADAMTS involved in the degradation of COMP. ADAMTS 7 and 12 share a C-terminal COMP/GEP-binding TSP domain. Their effects in OA are because of the association of this domain with COMP and its subsequent degradation.26Lin E.A. Liu C.J. The emerging roles of ADAMTS-7 and ADAMTS-12 matrix metalloproteinases.Rheumatol Res Rev. 2009; 1: 121-131Google Scholar Significant increases in ADAMTS-7 transcript and protein were detected after IL-1β and Fn-fs stimulation (Figure 7, A and B). Regarding ADAMTS-12, we observed an increase of mRNA and protein induced by both stimuli exclusively in OA-SF (Figure 7, C and D). The release of COMP a" @default.
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• W2489431013 date "2016-09-01" @default.
• W2489431013 modified "2023-10-14" @default.
• W2489431013 title "Healthy and Osteoarthritic Synovial Fibroblasts Produce a Disintegrin and Metalloproteinase with Thrombospondin Motifs 4, 5, 7, and 12" @default.
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• W2489431013 doi "https://doi.org/10.1016/j.ajpath.2016.05.017" @default.
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