Enhanced BMP-2/BMP-4 ratio in patients with peripheral spondyloarthritis and in cytokine- and stretch-stimulated mouse chondrocytes.

BACKGROUND: Excessive bone formation in the entheses is one of the features of peripheral spondyloarthritis (SpA). Complex pathological mechanisms connecting inflammation, mechanical stress, and ossification are probably involved. We focused on bone morphogenetic protein (BMP)-2, -4, and -7 as possible mediators of this process. METHODS: BMP-2, -4, and -7 concentration was measured by ELISA in synovial fluids (SFs) of SpA (n = 56) and osteoarthritic (n = 21) patients. Mouse organotypic ankle cultures were challenged by a pro-inflammatory cocktail. Mouse primary chondrocytes, osteoblasts, or tenocytes were treated with TNF-α, interleukin (IL)-17, or IL-22 and/or subjected to cyclic stretch, or with recombinant BMP-2 or -4. RESULTS: In SpA SFs, if BMP-7 was barely detectable, BMP-2 concentration was higher and BMP-4 was lower than in osteoarthritic samples, so that BMP-2/BMP-4 ratio augmented 6.5 folds (p < 0.001). In SpA patients, TNF-α, IL-6, and IL-17 levels correlated this ratio (n = 21). Bmp-2/Bmp-4 ratio was similarly enhanced by cytokine treatment in explant and cell cultures, at mRNA level. In particular, simultaneous application of TNF-α and cyclical stretch induced a 30-fold increase of the Bmp-2/Bmp-4 ratio in chondrocytes (p = 0.027). Blockade of prostaglandin E2 and IL-6 production had almost no effect on the stretch-induced regulation of Bmp-2 or -4. Osteoinductive effects of BMP-4, and to a lesser extend BMP-2, were identified on cultured chondrocytes and tenocytes. CONCLUSIONS: Our results first settle that BMP factors are locally deregulated in the SpA joint. An unexpected decrease in BMP-4 could be associated to an increase in BMP-2, possibly in response to mechanical and/or cytokine stimulations.

The role of sphingosine 1-phosphate metabolism in bone and joint pathologies and ectopic calcification.

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 ¼, 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.

Cytokine-Induced and Stretch-Induced Sphingosine 1-Phosphate Production by Enthesis Cells Could Favor Abnormal Ossification in Spondyloarthritis.

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.

Design, synthesis and biological evaluation of inhibitors of cathepsin K on dedifferentiated chondrocytes.

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.

Effects of phospholipase D during cultured osteoblast mineralization and bone formation.

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.

Involvement of sphingosine kinase/sphingosine 1-phosphate metabolic pathway in spondyloarthritis.

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 sphingosine 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±4.2µM, n=21) compared to healthy donors (1.6±0.9µM, 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.

TNAP stimulates vascular smooth muscle cell trans-differentiation into chondrocytes through calcium deposition and BMP-2 activation: Possible implication in atherosclerotic plaque stability.

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.

Wnt5a is expressed in spondyloarthritis and exerts opposite effects on enthesis and bone in murine organ and cell cultures.

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.

Protective role of frizzled-related protein B on matrix metalloproteinase induction in mouse chondrocytes.

INTRODUCTION: Our 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. METHODS: Cartilage 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-1ß (IL-1ß) (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 ß-catenin was assessed by RT-PCR and western blot. RESULTS: Cartilage 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-1ß dose-dependently induced Mmp-13 and -3 gene expression and protein release by cultured chondrocytes. IL-1ß-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 ß-catenin accumulation at protein level suggests that the enhanced catabolic response of FrzB-/- chondrocytes to IL-1ß and load may be associated with an over-stimulation of the canonical Wnt/ß-catenin pathway. CONCLUSIONS: Our 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/ß-catenin pathway.

Expression and function of visfatin (Nampt), an adipokine-enzyme involved in inflammatory pathways of osteoarthritis.

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 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 <0.0005). Nampt activity was identified ex vivo in synovium. In vitro, visfatin/Nampt significantly induced the expression of interleukin 6, keratinocyte chemoattractant and monocyte chemoattractant protein 1 in chondrocytes and osteoblasts. APO866 decreased the mRNA and protein levels of these pro-inflammatory cytokines in the two cell types (up to 94% and 63% inhibition, respectively). Levels of growth factors (vascular endothelial growth factor, transforming growth factor ß) and hypertrophic genes were unchanged with treatment. CONCLUSION: Visfatin/Nampt is released by all human OA tissues in a dimeric enzymatically active conformation and mostly by the synovium, which displays Nampt activity. The Nampt activity of visfatin is involved in chondrocyte and osteoblast activation, so targeting this enzymatic activity to disrupt joint tissue interactions may be novel in OA therapy.

Alteration of cartilage mechanical properties in absence of ß1 integrins revealed by rheometry and FRAP analyses.

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 ß1 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 ß1 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 ß1 integrins on the surface of chondrocytes increases the stiffness and modifies the diffusion properties of costal cartilage. Our data imply that ß1 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 ß1 integrins in the cartilage function.

Identification of soluble 14-3-3∊ as a novel subchondral bone mediator involved in cartilage degradation in osteoarthritis.

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.

Stress-induced cartilage degradation does not depend on the NLRP3 inflammasome in human osteoarthritis and mouse models.

OBJECTIVE: The main feature of osteoarthritis (OA) is degradation and loss of articular cartilage. Interleukin-1ß (IL-1ß) is thought to have a prominent role in shifting the metabolic balance toward degradation. IL-1ß 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-1ß and the inflammasome in cartilage breakdown. METHODS: We assessed IL-1ß 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-1ß 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-1ß. 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-1ß. 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-1ß inhibitors observed in previous trials.

Dynamic compression of chondrocyte-agarose constructs reveals new candidate mechanosensitive genes.

Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-ß pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-ß pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond to mechanical forces.

Induction of the chemokine IL-8/Kc by the articular cartilage: possible influence on osteoarthritis.

PURPOSE: IL-8 and its murine equivalent keratinocyte chemoattractant (Kc), chemokines produced by chondrocytes, contribute to the pathophysiology of osteoarthritis. However, the mechanisms leading to their production are poorly known. We aimed to investigate whether mechanical (compression), inflammatory (IL-1ß) and metabolic (visfatin) stresses may induce the release of Kc when applied on murine cartilage. METHODS: Mouse cartilage explants were subjected to intermittent compression for 4, 6 and 24h. Primary cultures of immature murine articular chondrocytes were obtained by enzymatic digestion of articular cartilage from 6-days-old newborns mice. The effect of compression, IL-1ß (10, 50, 100pg/mL) and of visfatin (5µg/mL) on the release of Kc was assessed by ELISA. IL-8 levels in conditioned media from human OA joint tissues (cartilage or synovium) were also assessed. RESULTS: In comparison with non-compressed explants, loading increased Kc release of 3.2-, 1.9- and 2.0-fold at 4, 6 and 24h respectively (P<0.004, n=9). IL-1ß triggered an increase of Kc release by primary cultured chondrocytes of 4.1-, 15.5- and 35.2-fold at 10, 50 and 100pg/mL of IL-1ß respectively (P<0.05, n=4). Likewise, visfatin (5µg/mL) induced an increase in Kc release of 56.5±25.2 fold (P=0.002, n=6). IL-8 was released in conditioned media by synovium as well as by cartilage. CONCLUSION: We show for the first time that IL-8/Kc is highly responsive to mechanical, inflammatory and metabolic stresses, strengthening the hypothesis that IL-8/Kc could be added to the cytokines which may have a deleterious impact in osteoarthritis.

BMP-2 and TGF-beta1 differentially control expression of type II procollagen and alpha 10 and alpha 11 integrins in mouse chondrocytes.

Bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1 are multifunctional cytokines both proposed as stimulants for cartilage repair. Thus it is crucial to closely examine and compare their effects on the expression of key markers of the chondrocyte phenotype, at the gene and protein level. In this study, the expression of alpha 10 and alpha 11 integrin subunits and the IIA/IIB spliced forms of type II procollagen have been monitored for the first time in parallel in the same in vitro model of mouse chondrocyte dedifferentiation/redifferentiation. We demonstrated that TGF-beta1 stimulates the expression of the non-chondrogenic form of type II procollagen, IIA isoform, and of a marker of mesenchymal tissues, i.e. the alpha 11 integrin subunit. On the contrary, BMP-2 stimulates the cartilage-specific form of type II procollagen, IIB isoform, and a specific marker of chondrocytes, i.e. the alpha 10 integrin subunit. Collectively, our results demonstrate that BMP-2 has a better capability than TGF-beta1 to stimulate chondrocyte redifferentiation and reveal that the relative expressions of type IIB to type IIA procollagens and alpha 10 to alpha 11 integrin subunits are good markers to define the differentiation state of chondrocytes. In addition, adenoviral expression of Smad6, an inhibitor of BMP canonical Smad signaling, did not affect expression of total type II procollagen or the ratio of type IIA and type IIB isoforms in mouse chondrocytes exposed to BMP-2. This result strongly suggests that signaling pathways other than Smad proteins are involved in the effect of BMP-2 on type II procollagen expression.

Investigating conversion of mechanical force into biochemical signaling in three-dimensional chondrocyte cultures.

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.

Molecular analysis of chondrocytes cultured in agarose in response to dynamic compression.

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 protein-2 stimulates chondrogenic expression in human nasal chondrocytes expanded in vitro.

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.

Knockdown of the intraflagellar transport protein IFT46 stimulates selective gene expression in mouse chondrocytes and affects early development in zebrafish.

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.