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Spondyloarthritis (SpA) is a common rheumatic disease characterized by enthesis inflammation (enthesitis) and ectopic ossification (enthesophytes). The current pathogenesis model suggests that inflammation and mechanical stress are both strongly involved in SpA pathophysiology. We have previously observed that the levels of sphingosine 1-phosphate (S1P), a bone anabolic molecule, were particularly high in SpA patients' serum compared to healthy donors. Therefore, we wondered how this deregulation was related to SpA molecular mechanisms. Mouse primary osteoblasts, chondrocytes, and tenocytes were used as cell culture models. The sphingosine kinase 1 (Sphk1) gene expression and S1P secretion were significantly enhanced by cyclic stretch in osteoblasts and chondrocytes. Further, TNF-α and IL-17, cytokines implicated in enthesitis, increased Sphk1 mRNA in chondrocytes in an additive manner when combined to stretch. The immunochemistry on mouse ankles showed that sphingosine kinase 1 (SK1) was localized in some chondrocytes; the addition of a pro-inflammatory cocktail augmented Sphk1 expression in cultured ankles. Subsequently, fingolimod was used to block S1P metabolism in cell cultures. It inhibited S1P receptors (S1PRs) signaling and SK1 and SK2 activity in both osteoblasts and chondrocytes. Fingolimod also reduced S1PR-induced activation by SpA patients' synovial fluid (SF), demonstrating that the stimulation of chondrocytes by SFs from SpA patients involves S1P. In addition, when the osteogenic culture medium was supplemented with fingolimod, alkaline phosphatase activity, matrix mineralization, and bone formation markers were significantly reduced in osteoblasts and hypertrophic chondrocytes. Osteogenic differentiation was accompanied by an increase in S1prs mRNA, especially S1P1/3 , but their contribution to S1P-impact on mineralization seemed limited. Our results suggest that S1P might be overproduced in SpA enthesis in response to cytokines and mechanical stress, most likely by chondrocytes. Moreover, S1P could locally favor the abnormal ossification of the enthesis; therefore, blocking the S1P metabolic pathway could be a potential therapeutic approach for the treatment of SpA. © 2019 American Society for Bone and Mineral Research.

Atherosclerotic plaque calcification varies from early, diffuse microcalcifications to a bone-like tissue formed by endochondral ossification. Recently, a paradigm has emerged suggesting that if the bone metaplasia stabilizes the plaques, microcalcifications are harmful. Tissue-nonspecific alkaline phosphatase (TNAP), an ectoenzyme necessary for mineralization by its ability to hydrolyze inorganic pyrophosphate (PPi), is stimulated by inflammation in vascular smooth muscle cells (VSMCs). Our objective was to determine the role of TNAP in trans-differentiation of VSMCs and calcification. In rodent MOVAS and A7R5 VSMCs, addition of exogenous alkaline phosphatase (AP) or TNAP overexpression was sufficient to stimulate the expression of several chondrocyte markers and induce mineralization. Addition of exogenous AP to human mesenchymal stem cells cultured in pellets also stimulated chondrogenesis. Moreover, TNAP inhibition with levamisole in mouse primary chondrocytes dropped mineralization as well as the expression of chondrocyte markers. VSMCs trans-differentiated into chondrocyte-like cells, as well as primary chondrocytes, used TNAP to hydrolyze PPi, and PPi provoked the same effects as TNAP inhibition in primary chondrocytes. Interestingly, apatite crystals, associated or not to collagen, mimicked the effects of TNAP on VSMC trans-differentiation. AP and apatite crystals increased the expression of BMP-2 in VSMCs, and TNAP inhibition reduced BMP-2 levels in chondrocytes. Finally, the BMP-2 inhibitor noggin blocked the rise in aggrecan induced by AP in VSMCs, suggesting that TNAP induction in VSMCs triggers calcification, which stimulates chondrogenesis through BMP-2. Endochondral ossification in atherosclerotic plaques may therefore be induced by crystals, probably to confer stability to plaques with microcalcifications.

Sphingolipids display important functions in various pathologies such as cancer, obesity, diabetes, cardiovascular or neurodegenerative diseases. Sphingosine, sphingosine 1-phosphate (S1P), and ceramide are the central molecules of sphingolipid metabolism. Sphingosine kinases 1 and 2 (SK1 and SK2) catalyze the conversion of the sphingolipid metabolite sphingosine into S1P. The balance between the levels of S1P and its metabolic precursors ceramide and sphingosine has been considered as a switch that could determine whether a cell proliferates or dies. This balance, also called « sphingolipid rheostat », is mainly under the control of SKs. Several studies have recently pointed out the contribution of SK/S1P metabolic pathway in skeletal development, mineralization and bone homeostasis. Indeed, SK/S1P metabolism participates in different diseases including rheumatoid arthritis, spondyloarthritis, osteoarthritis, osteoporosis, cancer-derived bone metastasis or calcification disorders as vascular calcification. In this review, we will summarize the most important data regarding the implication of SK/S1P axis in bone and joint diseases and ectopic calcification, and discuss the therapeutic potential of targeting SK/S1P metabolism for the treatment of these pathologies.

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC50 values and inhibition constants (Ki) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with Ki amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.

Background:Excessive bone formation in the entheses is one of the features of peripheral spondyloarthritis. Biomechanical stress is proposed to occupy a central place in spondyloarthritis pathophysiology, but the precise molecular and cellular mechanisms underlying the pathological response of the enthesis are still largely unknown [1]. Besides, physical therapy and exercise are recommended as non-pharmacologic therapies for patients. We focused on the effect of exercising on enthesis ossification.Objectives:We aimed to develop and characterize an in vivo model in mice to study the impact of mechanical stimulation on the enthesis of the Achilles tendon.Methods:DBA/1 mice were subjected to voluntary running exercise by the use of activity wheels for two weeks, and compared to mice housed in standard conditions (n=17 per group). The running performances were recorded. mRNAs were extracted from the long bones (flushed tibia and femur) and the ankles’ entheses for real-time PCR analysis. µCT was performed on the femurs. Alkaline phosphatase activity was detected by histology on the anchorage of the Achilles tendon to the calcaneum, and by enzymatic assay in serum samples. Luminex analysis was also conducted on serum samples for Il-6 and Il-8/Kc detection.Results:Free access to the activity wheel resulted in a running exercise of 5.5±0.8 km/day (approximately 80 km in total) at 14.5±0.5 m/min. No effect was detected on the femur architecture by µCT. Sclerostin (Sost) gene expression was monitored as a mechanosensitive marker. Its expression was expectedly reduced by half in entheseal tissues, but no modulation was observed in long bones (Figure 1). Similarly, exercise-induced regulation of Osterix and Runx2 expressions was observed only in enthesis samples. This tissue-specific pattern was also verified for key genes of the sphingosine-1 phosphate metabolic pathway, which we recently implicated in spondyloarthritis pathophysiology [2]. The in situ staining of alkaline phosphatase activity suggested the presence of more positive cells in the anchorage of Achilles tendon of running mice, compared to control ones. However, alkaline phosphatase activity in serum samples and its gene expression in rough tissue extracts were unchanged. No inflammatory response was detected as Il-8/Kc serum levels were similar in the control and the exercising group (59±14 vs 57±14 pg/mL). In addition, Il-6 was not detected in the serum and its expression was very faint and constant in the tissue extracts.Conclusion:This work is still in progress for a more complete characterization of the model. We believe that this experimental design will be useful to study the role of mechanical stimulation specifically in the enthesis and that it can help to better understand the spondyloarthritis pathophysiology.References:[1]Cambré, et al. Nat Commun, 2018; [2] El Jamal, et al. J Bone Miner Res, 2019Figure 1.Expression level of the mechanosensitive gene Sclerostin (Sost). It dropped in response to exercise in entheseal tissues, but not in long bones, revealing a tissue-specific response to mechanical stimulation.Acknowledgements:Société Arthritis R&D (2020)Disclosure of Interests:None declared

In the context of shoulder surgical replacement, a new generation of spherical interposition implants has been developed, with the implant being a mobile spacer rubbing against the glenoid cartilage and humeral bone cavity. The aim of the present study was to compare pyrocarbon (PyC) versus cobalt-chromium (CoCr) implants, regarding preservation and regeneration of the surrounding tissues. The effect of the biomaterials on chondrocytes was analysed in vitro. Murine primary chondrocytes were grown on discs made of PyC or CoCr using two culture media to mimic either cartilage-like or bone-like conditions (CLC or BLC). Chondrocytes did grow on PyC and CoCr without alteration in cell viability or manifestation of cytotoxicity. The tissue-like cell membranes grown under BLC were examined for the chondrocyte's ability to mineralise (by alizarin red matrix staining, calcium deposit and alkaline phosphatase activity) and for their mechanical properties (by rheological tests). For the chondrocytes grown under CLC and BLC, extracellular matrix components were analysed by histological staining and immunolabelling. Under CLC, PyC promoted type II collagen expression in chondrocytes, suggesting that they may generate a more cartilage-like matrix than samples grown on both CoCr and plastic control. In BLC, the tissue-like cell membranes grown on PyC were more mineralised and homogenous. The mechanical results corroborated the biological data, since the elastic modulus of the tissue-like cell membranes developed on the PyC surface was higher, indicating more stiffness. Overall, the results suggested that PyC might be a suitable biomaterial for spherical interposition implants.

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos-2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos-2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1-selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high-resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.

Objective Mechanical stress plays an important role in cartilage degradation and subchondral bone remodeling in osteoarthritis (OA). The remodeling of the subchondral bone could initiate cartilage loss in OA through the interplay of bone and cartilage. The aim of this study was to identify soluble mediators released by loaded osteoblasts/osteocytes that could induce the release of catabolic factors by chondrocytes. Methods Murine osteoblasts/osteocytes were subjected to cyclic compression, and then conditioned medium from either compressed (CCM) or uncompressed (UCM) cells was used to stimulate mouse chondrocytes. Chondrocyte expression of matrix metalloproteinase 3 (MMP-3), MMP-13, type II collagen, and aggrecan was assessed by reverse transcription–polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. Soluble mediators released by compressed osteoblasts/osteocytes were identified using iTRAQ (isobaric tags for relative and absolute quantification), a differential secretome analysis. Subchondral bone and cartilage samples were isolated from OA patients, and culture medium conditioned with OA subchondral bone or cartilage was used to stimulate human chondrocytes. Results Stimulation of mouse chondrocytes with CCM strongly induced the messenger RNA (mRNA) expression and protein release of MMP-3 and MMP-13 and inhibited the mRNA expression of type II collagen and aggrecan. Differential secretome analysis revealed that 10 proteins were up-regulated in compressed osteoblasts/osteocytes. Among them, soluble 14-3-3∊ (s14-3-3∊) dose-dependently induced the release of catabolic factors by chondrocytes, mimicking the effects of cell compression. Addition of a 14-3-3∊ blocking antibody greatly attenuated the CCM-mediated induction of MMP-3 and MMP-13 expression. Furthermore, in human OA subchondral bone, s14-3-3∊ was strongly released, and in cultures of human OA chondrocytes, s14-3-3∊ stimulated MMP-3 expression. Conclusion The results of this study identify s14-3-3∊ as a novel soluble mediator critical in the communication between subchondral bone and cartilage in OA. Thus, s14-3-3∊ may be a potential target for future therapeutic or prognostic applications in OA.

International audience; Spondyloarthritis (SpA) is a relatively common chronic inflammatory joint disorder, with a prevalence of about 0.2–0.5% worldwide. The primary target of the pathological process is the enthesis, where tendons and ligaments attach to underlying bone. These insertion sites are hotspots of bone formation (enthesophytes), which can lead to ankylosis. Unfortunately, the mechanisms causing the onset and progression of entheseal ossification remain largely unknown. Sphingosine 1-phosphate (S1P), a lipid generated after sphingosine phosphorylation by sphin-gosine kinases 1 and 2 (SK1/2), plays important roles in cell proliferation, differentiation and survival. S1P regulates fundamental biological processes such as cell cycle, inflammatory response or bone homeostasis. Indeed, S1P has been involved in some of most-spread skeletal diseases such as rheumatoid arthritis or osteoarthritis. On the other hand, the implication of S1P in SpA has not been explored yet. In the present work, we observed by ELISA that S1P content was significantly increased in the serum of SpA patients (6.1 ± 4.2 μM, n = 21) compared to healthy donors (1.6 ± 0.9 μM, n = 12). In vitro, gene expression of SK1 and SK2 as well as their activity were increased during differentiation of primary murine chondrocytes and osteoblasts into mineralizing cells. In addition, mRNA of the S1P-specific transporter Spns2 and S1P secretion were augmented. Using the pharmacological drugs SKi (SK pan-inhibitor), PF-543 (SK1 specific inhibitor) or K-145 (SK2 specific inhibitor), we showed that the inhibition of SK1 and/or SK2 decreased matrix mineralization, alkaline phosphatase activity and the mRNA expression of Runx2 and Bglap in chondrocytes and osteoblasts. To our knowledge, this is the first study indicating that S1P levels are significantly increased in serum from SpA patients. Moreover, we showed in vitro that SK activity was involved in the mineralization capacity of osteoblasts and chondrocytes. S1P metabolic pathway may represent an ingenious therapeutic target for SpA in the future.

Objective The main feature of osteoarthritis (OA) is degradation and loss of articular cartilage. Interleukin-1β (IL-1β) is thought to have a prominent role in shifting the metabolic balance toward degradation. IL-1β is first synthesized as an inactive precursor that is cleaved to the secreted active form mainly in the “inflammasome,” a complex of initiators (including NLRP3), adaptor molecule ASC, and caspase 1. The aim of this study was to clarify the roles of IL-1β and the inflammasome in cartilage breakdown. Methods We assessed IL-1β release by cartilage explants from 18 patients with OA. We also evaluated the lipopolysaccharide (LPS)–, IL-1α–, and tumor necrosis factor α (TNFα)–induced activity of matrix metalloproteinase 3 (MMP-3), MMP-9, and MMP-13 in NLRP3-knockout mice and wild-type mice and the inhibition of caspase 1 with Z-YVAD-FMK and the blockade of IL-1β with IL-1 receptor antagonist (IL-1Ra). Cartilage explants from NLRP3-knockout mice and IL-1R type I (IL-1RI)–knockout mice were subjected to excessive dynamic compression (0.5 Hz, 1 MPa) to trigger degradation, followed by assessment of load-induced glycosaminoglycan (GAG) release and MMP enzymatic activity. Results Despite the expression of NLRP3, ASC, and caspase 1, OA cartilage was not able to produce active IL-1β. LPS, IL-1α, and TNFα dose-dependently increased MMP-3, MMP-9, and MMP-13 activity in cultured chondrocytes and in NLRP3−/− chondrocytes, and this effect was not changed by inhibiting caspase 1 or IL-1β. The load-induced increase in GAG release and MMP activity was not affected by knockout of NLRP3 or IL-1RI in cartilage explants. Conclusion OA cartilage may be degraded independently of any inflammasome activity, which may explain, at least in part, the lack of effect of IL-1β inhibitors observed in previous trials.

Resume But de l’etude Le cartilage est un tissu au potentiel de cicatrisation spontane tres limite. La transplantation de chondrocytes autologues (TCA) est une technique mondialement utilisee pour le traitement de lesions limitees de cartilage articulaire. Cependant, cette approche implique au prealable une amplification des chondrocytes sur plastique, ce qui entraine leur dedifferenciation. Ce phenomene se traduit notamment par une perte de l’expression du collagene de type II, proteine majoritaire du cartilage. Le defi actuel de la TCA est de fournir au patient des cellules au phenotype bien differencie. L’objectif de cette etude a ete de tester la capacite de la bone morphogenetic protein (BMP)-2, a redifferencier des chondrocytes humains apres leur amplification in vitro. Materiels et methodes Les chondrocytes ont ete extraits a partir de cartilage de septum nasal obtenu lors de septoplastie. Apres un, deux ou trois passages d’amplification en monocouche sur plastique, la BMP-2 a ete ajoutee au milieu de culture. L’evolution du phenotype a ete suivie en analysant par PCR l’expression de genes codant differentes proteines de reference : procollagene de type II avec rapport des isoformes IIB/IIA, Agrecanne, Sox9, Osteocalcine et procollagene de type I. Resultats Nos resultats montrent que la BMP-2 est capable de stimuler l’expression chondrogenique des chondrocytes amplifies sans stimuler d’expression osteoblastique. Cependant, l’activite stimulatrice de la BMP-2 diminue avec les passages. Conclusion L’efficacite d’un traitement par TCA pourrait etre amelioree en administrant des cellules prealablement cultivees en presence de BMP-2.

The culture of chondrocytes embedded within agarose hydrogels maintains chondrocytic phenotype over extended periods and allows analysis of the chondrocyte response to mechanical forces. The mechanisms involved in the transduction of a mechanical stimulus to a physiological process are not completely deciphered. We present protocols to prepare and characterize constructs of murine chondrocytes and agarose (1 week pre-culture period), to analyze the effect of compression on mRNA level by RT-PCR (2-3 d), gene transcription by gene reporter assay (3 d) and phosphorylation state of signaling molecules by western blotting (3-4 d). The protocols can be carried out with a limited number of mouse embryos or newborns and this point is particularly important regarding genetically modified mice.

Articular cartilage contains an extracellular matrix with characteristic macromolecules such as type II collagen. Because this tissue is avascular and mature chondrocytes do not proliferate, cartilage lesions have a limited capacity for healing after trauma. Autologous chondrocyte implantation (ACI) is widely used for the treatment of patients with focal damage to articular cartilage. However, this method faces a major issue: dedifferentiation of chondrocytes occurs during the long-term culture necessary for mass cell production. The aim of this study was to determine if the step of cell amplification required for ACI could benefit from the use of bone morphogenetic protein (BMP)-2, a potent regulator of chondrogenic expression. Chondrocytes were isolated from human nasal cartilage, a hyaline cartilage like articular cartilage and were serially cultured in monolayers. After one, two or three passages, BMP-2 was used to evaluate the chondrogenic potential of the dedifferentiated chondrocytes, at the gene and protein level. We found that BMP-2 can reactivate the program of chondrogenic expression in dedifferentiated chondrocytes. To gain insight into the molecular mechanisms involved in the responsiveness of chondrocytes to BMP-2, we examined the phosphorylation of Smad proteins and the interaction of the Sry-type high-mobility-group box (Sox) transcription factors with the cartilage-specific enhancer of the type II procollagen gene. Our results show that BMP-2 acts by stimulating Smad phosphorylation and by enhancing DNA-binding of the Sox transcription factors to the specific enhancer of the type II procollagen gene. Thus, this study reveals the potential use of BMP-2 as a stimulatory agent in conventional ACI strategies.

International audience; Spondyloarthritis (SpA) is a chronic inflammatory joint disorder that initiates at the enthesis, where tendons attach to bone through a fibrocartilage zone. At late stages, excessive bone apposition appears within the diseased enthesis. Because Wnt5a participates to normal bone formation and appears related to inflammatory processes, we investigated the role of this Wnt growth factor in inflammation-associated ossification in SpA. The concentration of Wnt5a assessed by enzyme-linked immunosorbent assay in synovial fluids of patients with SpA (2.58 ± 0.98 ng/mL) was higher than in osteoarthritic patients (1.33 ± 0.71 ng/mL). In murine primary cultures of tendon cells, chondrocytes, and osteoblasts and in an organotypic model of mouse ankle, we showed that tumor necrosis factor α reversibly diminished Wnt5a expression and secretion, respectively. Wnt5a decreased gene expression of differentiation markers and mineralization in cultured chondrocytes and reduced alkaline phosphatase activity in Achilles tendon enthesis (-14%) and osteocalcin protein levels released by ankle explants (-36%). On the contrary, Wnt5a stimulated ossification markers' expression in cultured osteoblasts and increased the bone volume of the tibial plateau of the cultured explants (+19%). In conclusion, our results suggest that Wnt5a is expressed locally in the joints of patients with SpA. Wnt5a appears more associated with ossification than with inflammation and tends to inhibit mineralization in chondrocytes and enthesis, whereas it seems to favor the ossification process in osteoblasts and bone. Further studies are needed to decipher the opposing effects observed locally in enthesis and systemically in bone in SpA.

International audience; INTRODUCTION: Visfatin is an adipokine that may be involved in intertissular joint communication in osteoarthritis (OA). With a homodimeric conformation, it exerts nicotinamide phosphoribosyltransferase (Nampt) enzymatic activity, essential for nicotinamide adenine dinucleotide biosynthesis. We examined the tissular origin and conformation of visfatin/Nampt in human OA joints and investigated the role of visfatin/Nampt in OA chondrocytes and osteoblasts by studying Nampt enzymatic activity. METHODS: Synovium, cartilage and subchondral bone from human OA joints were used for protein extraction or incubated for 24 hours in serum-free media (conditioned media), and synovial fluid was obtained from OA patients. Visfatin/Nampt expression in tissular extracts and conditioned media was evaluated by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Nampt activity was assessed in OA synovium by colorimetric assay. Primary cultures of murine chondrocytes and osteoblasts were stimulated with visfatin/Nampt and pretreated or not with APO866, a pharmacologic inhibitor of Nampt activity. The effect on cytokines, chemokines, growth factors and hypertrophic markers expression was examined by quantitative reverse transcriptase polymerase chain reaction and/or ELISA. RESULTS: In tissular explants, conditioned media and synovial fluid, visfatin/Nampt was found as a homodimer, corresponding to the enzymatically active conformation. All human OA joint tissues released visfatin/Nampt (synovium: 628 +/- 106 ng/g tissue; subchondral bone: 195 +/- 26 ng/g tissue; cartilage: 152 +/- 46 ng/g tissue), with significantly higher level for synovium (P

Context Mechanical properties are essential for biological functions of the hyaline cartilage such as energy dissipation and diffusion of solutes. Mechanical properties are primarily dependent on the hierarchical organization of the two major extracellular matrix (ECM) macromolecular components of the cartilage: the fibrillar collagen network and the glycosaminoglycan (GAG)-substituted proteoglycan, mainly aggrecan, aggregates. Interaction of chondrocytes, the only cell type in the tissue, with the ECM through adhesion receptors is involved in establishing mechanical stability via bidirectional transduction of both mechanical forces and chemical signals. In this study, we aimed to determine the role of the transmembrane β1 integrin adhesion receptors in cartilage biomechanical properties by the use of genetic modification in mice. Methods Costal cartilages of wild type and mutant mice lacking β1 integrins in chondrocytes were investigated. Cartilage compressive properties and solute diffusion were characterized by rheometric analysis and Fluorescence Recovery After Photobleaching (FRAP), respectively. Cartilage tissue sections were analyzed by histology, immunohistochemistry and transmission electron microscopy (TEM). Results At the histological level, the mutant costal cartilage was characterized by chondrocyte rounding and loss of tissue polarity. Immunohistochemistry and safranin orange staining demonstrated apparently normal aggrecan and GAG levels, respectively. Antibody staining for collagen II and TEM showed comparable expression and organization of the collagen fibrils between mutant and control cartilages. Despite the lack of gross histological and ultrastructural abnormalities, rheological measurements revealed that the peak elastic modulus in compression of mutant cartilage was 1.6-fold higher than the peak elastic modulus of wild-type sample. Interestingly, the diffusion coefficient within the mutant cartilage tissue was found to be 1.2-fold lower in the extracellular space and 14-fold lower in the pericellular (PCM) space compared to control. Conclusion The results demonstrate that the absence of β1 integrins on the surface of chondrocytes increases the stiffness and modifies the diffusion properties of costal cartilage. Our data imply that β1 integrins-mediated chondrocyte–matrix interactions directly affect cartilage biomechanics probably by modifying physical properties of individual cells. This study thus highlights the crucial role of β1 integrins in the cartilage function.

Mechanical stress plays an important role in cartilage degradation and subchondral bone remodeling in osteoarthritis (OA). The remodeling of the subchondral bone could initiate cartilage loss in OA through the interplay of bone and cartilage. The aim of this study was to identify soluble mediators released by loaded osteoblasts/osteocytes that could induce the release of catabolic factors by chondrocytes.Murine osteoblasts/osteocytes were subjected to cyclic compression, and then conditioned medium from either compressed (CCM) or uncompressed (UCM) cells was used to stimulate mouse chondrocytes. Chondrocyte expression of matrix metalloproteinase 3 (MMP-3), MMP-13, type II collagen, and aggrecan was assessed by reverse transcription-polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. Soluble mediators released by compressed osteoblasts/osteocytes were identified using iTRAQ (isobaric tags for relative and absolute quantification), a differential secretome analysis. Subchondral bone and cartilage samples were isolated from OA patients, and culture medium conditioned with OA subchondral bone or cartilage was used to stimulate human chondrocytes.Stimulation of mouse chondrocytes with CCM strongly induced the messenger RNA (mRNA) expression and protein release of MMP-3 and MMP-13 and inhibited the mRNA expression of type II collagen and aggrecan. Differential secretome analysis revealed that 10 proteins were up-regulated in compressed osteoblasts/osteocytes. Among them, soluble 14-3-3∊ (s14-3-3∊) dose-dependently induced the release of catabolic factors by chondrocytes, mimicking the effects of cell compression. Addition of a 14-3-3∊ blocking antibody greatly attenuated the CCM-mediated induction of MMP-3 and MMP-13 expression. Furthermore, in human OA subchondral bone, s14-3-3∊ was strongly released, and in cultures of human OA chondrocytes, s14-3-3∊ stimulated MMP-3 expression.The results of this study identify s14-3-3∊ as a novel soluble mediator critical in the communication between subchondral bone and cartilage in OA. Thus, s14-3-3∊ may be a potential target for future therapeutic or prognostic applications in OA.

Background Articular cartilage is exposed to high mechanical loads under normal physiological conditions and articular chondrocytes regulate the composition of cartilaginous matrix, in response to mechanical signals. However, the intracellular pathways involved in mechanotransduction are still being defined. Using the well-characterized chondrocyte/agarose model system and dynamic compression, we report protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the effect of compression on steady-state level of mRNA by RT-PCR, gene transcription by gene reporter assay, and phosphorylation state of signalling molecules by Western-blotting. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice. Results Chondrocytes cultured in agarose for one week were surrounded by a newly synthesized pericellular matrix, as revealed by immunohistochemistry prior to compression experiments. This observation indicates that this model system is suitable to study the role of matrix molecules and trans-membrane receptors in cellular responsiveness to mechanical stress. The chondrocyte/agarose constructs were then submitted to dynamic compression with FX-4000C™ Flexercell® Compression Plus™ System (Flexcell). After clearing proteins off agarose, Western-blotting analysis showed transient activation of Mitogen-activated protein kinases (MAPK) in response to dynamic compression. After assessment by capillary electrophoresis of the quality of RNA extracted from agarose, steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as a response to compression, in accordance with the mechanosensitive character observed for these two genes in other studies using cartilage explants submitted to compression. To explore further the biological response of mouse chondrocytes to the dynamic compression at the transcriptional level, we also developed an approach for monitoring changes in gene transcription in agarose culture by using reporter promoter constructs. A decrease in promoter activity of the gene coding for type II procollagen, the most abundant protein in cartilage, was observed in response to dynamic loading. Conclusion The protocols developed here offer the possibility to perform an integrated analysis of the molecular mechanisms of mechanotransduction in chondrocytes, at the gene and protein level.

Bone morphogenetic proteins (BMPs) act as multifunctional regulators in morphogenesis during development. In particular they play a determinant role in the formation of cartilage molds and their replacement by bone during endochondral ossification. In cell culture, BMP-2 favors chondrogenic expression and promotes hypertrophic maturation of chondrocytes. In mouse chondrocytes we have identified a BMP-2-sensitive gene encoding a protein of 301 amino acids. This protein, named mIFT46, is the mouse ortholog of recently identified Caenorhabditis elegans and Chlamydomonas reinhardtii intraflagellar transport (IFT) proteins. After generation of a polyclonal antibody against mIFT46, we showed for the first time that the endogenous protein is located in the primary cilium of chondrocytes. We also found that mIFT46 is preferentially expressed in early hypertrophic chondrocytes located in the growth plate. Additionally, mIFT46 knockdown by small interfering RNA oligonucleotides in cultured chondrocytes specifically stimulated the expression of several genes related to skeletogenesis. Furthermore, Northern blotting analysis indicated that mIFT46 is also expressed before chondrogenesis in embryonic mouse development, suggesting that the role of mIFT46 might not be restricted to cartilage. To explore the role of IFT46 during early development, we injected antisense morpholino oligonucleotides in Danio rerio embryos to reduce zebrafish IFT46 protein (zIFT46) synthesis. Dramatic defects in embryonic development such as a dorsalization and a tail duplication were observed. Thus our results taken together indicate that the ciliary protein IFT46 has a specific function in chondrocytes and is also essential for normal development of vertebrates.Bone morphogenetic proteins (BMPs) act as multifunctional regulators in morphogenesis during development. In particular they play a determinant role in the formation of cartilage molds and their replacement by bone during endochondral ossification. In cell culture, BMP-2 favors chondrogenic expression and promotes hypertrophic maturation of chondrocytes. In mouse chondrocytes we have identified a BMP-2-sensitive gene encoding a protein of 301 amino acids. This protein, named mIFT46, is the mouse ortholog of recently identified Caenorhabditis elegans and Chlamydomonas reinhardtii intraflagellar transport (IFT) proteins. After generation of a polyclonal antibody against mIFT46, we showed for the first time that the endogenous protein is located in the primary cilium of chondrocytes. We also found that mIFT46 is preferentially expressed in early hypertrophic chondrocytes located in the growth plate. Additionally, mIFT46 knockdown by small interfering RNA oligonucleotides in cultured chondrocytes specifically stimulated the expression of several genes related to skeletogenesis. Furthermore, Northern blotting analysis indicated that mIFT46 is also expressed before chondrogenesis in embryonic mouse development, suggesting that the role of mIFT46 might not be restricted to cartilage. To explore the role of IFT46 during early development, we injected antisense morpholino oligonucleotides in Danio rerio embryos to reduce zebrafish IFT46 protein (zIFT46) synthesis. Dramatic defects in embryonic development such as a dorsalization and a tail duplication were observed. Thus our results taken together indicate that the ciliary protein IFT46 has a specific function in chondrocytes and is also essential for normal development of vertebrates.

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S452 MMP1 and ADAMTS4 in FLSs at the gene expression level. Importantly, we also demonstrate that insulin blunts release of MMP1 and MMP13 protein into the culture media, counteracting the effect of TNFa and IL-1b. Thus, insulin appears to play a selective protective role in the diarthrodial joint by suppressing release of catabolic enzymes into the synovial fluid via its effects on FLSs. Further study is needed to determine if insulin resistance in obesity/diabetes impairs this critical role for insulin in protecting cartilage matrix in synovial joints. Although the mechanism of selective suppression of MMP1 and MMP13 by insulin is still unknown, insulin and insulinsensitizing agents could be new disease-modifying interventions in OA treatment. 822 EXPRESSION OF INTERLEUKIN-17 AND INTERLEUKIN-22 IN NON INFLAMMATORY AND INFLAMMATORY SYNOVIAL MEMBRANES FROM OSTEOARTHRITIS PATIENTS C. Deligne y,z, S. Casulli x,z, A. Pigenet y,z, C. Bougault y,z, L. Campillo-Gimenez x,z, F. Berenbaum k,y, C. Elbim x,z, X. Houard y,z. y INSERM UMRS938, Paris, France; zUPMC Univ Paris 06, Paris, France; x INSERM UMRS945, Paris, France; kDept. of Rheumatology, AP-HP SaintAntoine Hosp., Labex Transimmunom, Inflammation-ImmunopathologyBiotherapy Dept. (i2B), Paris, France Purpose: IL-17 and IL-22 are inflammatory cytokines classically involved in chronic inflammation in various diseases. Here, we investigated the expression and the release of IL-17 and IL-22 by OA synovial membranes, in relation to the inflammatory status of synovium. Methods: Synovial membranes from OA knee patients (n 1⁄4 32) were collected at surgery and inflammatory (Infl) and non-inflammatory (NI) areas were separated according to the macroscopic evaluation of inflammation that appeared as hypervascularized areas with hypertrophic and hyperaemic villi. A piece of each tissuewas frozen for mRNA extraction, fixed in paraformaldehyde for histology and incubated in serum free culture medium in order to obtain conditioned media containing soluble factors released by synovial tissues. IL-1b, IL-6, IL-8, IL18, IL-17, IL-22, IL-23, TNF-a, TGF-b1, myeloperoxidase (MPO) andMMP9were analyzed for mRNA expression by quantitative RT-PCR and/or for protein level by ELISA and gelatin zymography. Immunohistochemistry for endothelial cells (CD31) and leukocytes (CD45) among them macrophages (CD68), neutrophils (CD15) and Tand B-lymphocytes (CD3 and CD20, respectively) was performed. Results: Inflammatory areas of OA synovial membranes were characterized by increased CD45þ inflammatory cell infiltration and vessel area/tissue area as compared to non-inflammatory areas (p1⁄4 0.001 and p 1⁄4 0.009, respectively). Macrophages were present in the intimal layer of both NI and Infl OA synovial membranes with higher accumulation in Infl as compared to NI. Only the subintimal layer of Infl areas of OA synovial membranes contained macrophages, Tand B-lymphocytes and some neutrophils. Consistently, the inflammatory markers MMP-9 and MPO were released in significantly higher concentrations by Infl than by NI areas (p 1⁄4 0.026 and p 1⁄4 0.001, respectively). IL-17 and IL-22 were both expressed and released by OA synovial membranes. A stronger mRNA expression was found in Infl for both IL-17 and IL-22. Infl also released significantly higher levels of IL-22 than NI (p 1⁄4 0.046). Strong positive correlations were found between IL-17 and IL-22 at mRNA and protein levels. The expression of IL-17 and IL-22 is controlled by a subset of cytokines, including IL-1b, IL-6, IL-8, IL-18, IL-23, TGFb-1 and TNF-a. With the exception of TNF-a, all were released in significantly higher concentration by Infl as compared to NI areas. IL-12 was not detected in conditioned media of synovial tissues. Conclusions: Our results show an increased infiltration of lymphocytes as well as an increased release of inflammatory cytokines, including ILSynovial gene expression at day 1 after transection (as fold-change compared to healt

International audience; IntrtoductionOur objective was to investigate whether a lack of frizzled-related protein B (FrzB), an extracellular antagonist of the Wnt signaling pathways, could enhance cartilage degradation by facilitating the expression, release and activation of matrix metalloproteinases (MMPs) by chondrocytes in response to tissue-damaging stimuli.MethodsCartilage explants from FrzB−/− and wild-type mice were challenged by excessive dynamic compression (0.5 Hz and 1 MPa for 6 hours). Load-induced glycosaminoglycan (GAG) release and MMP enzymatic activity were assessed. Interleukin-1β (IL-1β) (10, 100 and 1000 pg/mL for 24 hours) was used to stimulate primary cultures of articular chondrocytes from FrzB−/− and wild-type mice. The expression and release of MMP-3 and −13 were determined by RT-PCR, western blot and ELISA. The accumulation of β-catenin was assessed by RT-PCR and western blot.ResultsCartilage degradation, as revealed by a significant increase in GAG release (2.8-fold, P = 0.014) and MMP activity (4.5-fold, P = 0.014) by explants, was induced by an excessive load. Load-induced MMP activity appeared to be enhanced in FrzB−/− cartilage explants compared to wild-type (P = 0.17). IL-1β dose-dependently induced Mmp-13 and −3 gene expression and protein release by cultured chondrocytes. IL-1β-mediated increase in MMP-13 and −3 was slightly enhanced in FrzB−/− chondrocytes compared to wild-type (P = 0.05 and P = 0.10 at gene level, P = 0.17 and P = 0.10 at protein level, respectively). Analysis of Ctnn1b and Lef1 gene expression and β-catenin accumulation at protein level suggests that the enhanced catabolic response of FrzB−/− chondrocytes to IL-1β and load may be associated with an over-stimulation of the canonical Wnt/β-catenin pathway.ConclusionsOur results suggest that FrzB may have a protective role on cartilage degradation and MMP induction in mouse chondrocytes by attenuating deleterious effects of the activation of the canonical Wnt/β-catenin pathway.