Jessica Bertrand

Syndecan-4 regulates ADAMTS-5 activation and cartilage breakdown in osteoarthritis

Aggrecan cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 5 (ADAMTS-5) is crucial for the breakdown of cartilage matrix during osteoarthritis, a degenerative joint disease that leads to the progressive destruction of articular structures. The mechanisms of ADAMTS-5 activation and their links to the pathogenesis of osteoarthritis remain poorly understood, but syndecans have been shown to be involved in the activation of ADAMTS-4 (ref. 3). Here we show that syndecan-4 is specifically induced in type X collagen–producing chondrocytes both in human osteoarthritis and in murine models of the disease. The loss of syndecan-4 in genetically modified mice and intra-articular injections of syndecan-4–specific antibodies into wild-type mice protect from proteoglycan loss and thereby prevent osteoarthritic cartilage damage in a surgically induced model of osteoarthritis. The occurrence of less severe osteoarthritis-like cartilage destruction in both syndecan-4–deficient mice and syndecan-4–specific antibody–treated wild-type mice results from a marked decrease in ADAMTS-5 activity. Syndecan-4 controls the activation of ADAMTS-5 through direct interaction with the protease and through regulating mitogen-activated protein kinase (MAPK)-dependent synthesis of matrix metalloproteinase-3 (MMP-3). Our data suggest that strategies aimed at the inhibition of syndecan-4 will be of great value for the treatment of cartilage damage in osteoarthritis.

Calcification of articular cartilage in human osteoarthritis

OBJECTIVE Hypertrophic chondrocyte differentiation is a key step in endochondral ossification that produces basic calcium phosphates (BCPs). Although chondrocyte hypertrophy has been associated with osteoarthritis (OA), chondrocalcinosis has been considered an irregular event and linked mainly to calcium pyrophosphate dihydrate (CPPD) deposition. The aim of this study was to determine the prevalence and composition of calcium crystals in human OA and analyze their relationship to disease severity and markers of chondrocyte hypertrophy. METHODS One hundred twenty patients with end-stage OA undergoing total knee replacement were prospectively evaluated. Cartilage calcification was studied by conventional x-ray radiography, digital-contact radiography (DCR), field-emission scanning electron microscopy (FE-SEM), and synovial fluid analysis. Cartilage calcification findings were correlated with scores of knee function as well as histologic changes and chondrocyte hypertrophy as analyzed in vitro. RESULTS DCR revealed mineralization in all cartilage specimens. Its extent correlated significantly with the Hospital for Special Surgery knee score but not with age. FE-SEM analysis showed that BCPs, rather than CPPD, were the prominent minerals. On histologic analysis, it was observed that mineralization correlated with the expression of type X collagen, a marker of chondrocyte hypertrophy. Moreover, there was a strong correlation between the extent of mineralization in vivo and the ability of chondrocytes to produce BCPs in vitro. The induction of hypertrophy in healthy human chondrocytes resulted in a prominent mineralization of the extracellular matrix. CONCLUSION These results indicate that mineralization of articular cartilage by BCP is an indissociable process of OA and does not characterize a specific subset of the disease, which has important consequences in the development of therapeutic strategies for patients with OA.

WNT-3A modulates articular chondrocyte phenotype by activating both canonical and noncanonical pathways

A single Wnt can simultaneously activate different pathways with distinct and independent outcomes and reciprocal regulation in human articular chondrocytes.

Cartilage biology, pathology, and repair

Osteoarthritis is one of the most common forms of musculoskeletal disease and the most prominent type of arthritis encountered in all countries. Although great efforts have been made to investigate cartilage biology and osteoarthritis pathology, the treatment has lagged behind that of other arthritides, as there is a lack of effective disease-modifying therapies. Numerous approaches for dealing with cartilage degradation have been tried, but enjoyed very little success to develop approved OA treatments with not only symptomatic improvement but also structure-modifying effect. In this review we discuss the most recent findings regarding the regulation of cartilage biology and pathology and highlight their potential therapeutic values.

Myostatin is a direct regulator of osteoclast differentiation and its inhibition reduces inflammatory joint destruction in mice

Myostatin (also known as growth and differentiation factor 8) is a secreted member of the transforming growth factor-β (TGF-β) family that is mainly expressed in skeletal muscle, which is also its primary target tissue. Deletion of the myostatin gene (Mstn) in mice leads to muscle hypertrophy, and animal studies support the concept that myostatin is a negative regulator of muscle growth and regeneration. However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone. Here we report a previously unknown direct role for myostatin in osteoclastogenesis and in the progressive loss of articular bone in rheumatoid arthritis (RA). We demonstrate that myostatin is highly expressed in the synovial tissues of RA subjects and of human tumor necrosis factor (TNF)-α transgenic (hTNFtg) mice, a model for human RA. Myostatin strongly accelerates receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast formation in vitro through transcription factor SMAD2-dependent regulation of nuclear factor of activated T-cells (NFATC1). Myostatin deficiency or antibody-mediated inhibition leads to an amelioration of arthritis severity in hTNFtg mice, chiefly reflected by less bone destruction. Consistent with these effects in hTNFtg mice, the lack of myostatin leads to increased grip strength and less bone erosion in the K/BxN serum-induced arthritis model in mice. The results strongly suggest that myostatin is a potent therapeutic target for interfering with osteoclast formation and joint destruction in RA.

Syndecans in cartilage breakdown and synovial inflammation

Syndecans are transmembrane heparan sulphate proteoglycans (HSPGs) that have gained increasing interest as regulators of a variety of tissue responses, including cartilage development and remodelling. These proteoglycans are composed of a core protein to which extracellular glycosaminoglycan (GAG) chains are attached. Through these GAG chains, syndecans can interact with a variety of extracellular matrix molecules and bind to a number of soluble mediators including morphogens, growth factors, chemokines and cytokines. The structure and post-translational modification of syndecan GAG chains seem to differ not only from cell to cell, but also during different stages of cellular differentiation, leading to a complexity of syndecan function that is unique among membrane-bound HSPGs. Unlike other membrane-bound HSPGs, syndecans contain intracellular signalling motifs that can initiate signalling mainly through protein kinase C. This Review summarizes our knowledge of the biology of syndecans and the mechanisms by which binding of molecules to syndecans exert different biological effects, particularly in the joints. On the basis of the structural and functional peculiarities of syndecans, we discuss the regulation of syndecans and their roles in the developing joint as well as during degenerative and inflammatory cartilage remodelling as understood from expression studies and functional analyses involving syndecan-deficient mice.

Molecular mechanisms of cartilage remodelling in osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease that is characterized primarily by progressive breakdown of articular cartilage. The loss of proteoglycans, the mineralization of the extracellular matrix (ECM) and the hypertrophic differentiation of the chondrocytes constitute hallmarks of the disease. The pathogenesis of OA includes several pathways, which in single are very well investigated and partly understood, but in their complex interplay remain mainly unclear. This review summarises recent data on the underlying mechanisms, specifically with respect to cell-matrix interactions and cartilage mineralization. It points out why these findings are of importance for future OA research and for the development of novel therapeutic strategies to treat OA.

A homeostatic function of CXCR2 signalling in articular cartilage

Objective ELR+ CXC chemokines are heparin-binding cytokines signalling through the CXCR1 and CXCR2 receptors. ELR+ CXC chemokines have been associated with inflammatory arthritis due to their capacity to attract inflammatory cells. Here, we describe an unsuspected physiological function of these molecules in articular cartilage homeostasis. Methods Chemokine receptors and ligands were detected by immunohistochemistry, western blotting and RT-PCR. Osteoarthritis was induced in wild-type and CXCR2−/− mice by destabilisation of the medial meniscus (DMM). CXCR1/2 signalling was inhibited in vitro using blocking antibodies or siRNA. Chondrocyte phenotype was analysed using Alcian blue staining, RT-PCR and western blotting. AKT phosphorylation and SOX9 expression were upregulated using constitutively active AKT or SOX9 plasmids. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Results CXCL6 was expressed in healthy cartilage and was retained through binding to heparan sulfate proteoglycans. CXCR2−/− mice developed more severe osteoarthritis than wild types following DMM, with increased chondrocyte apoptosis. Disruption of CXCR1/2 in human and CXCR2 signalling in mouse chondrocytes led to a decrease in extracellular matrix production, reduced expression of chondrocyte differentiation markers and increased chondrocyte apoptosis. CXCR2-dependent chondrocyte homeostasis was mediated by AKT signalling since forced expression of constitutively active AKT rescued the expression of phenotypic markers and the apoptosis induced by CXCR2 blockade. Conclusions Our study demonstrates an important physiological role for CXCR1/2 signalling in maintaining cartilage homeostasis and suggests that the loss of ELR+ CXC chemokines during cartilage breakdown in osteoarthritis contributes to the characteristic loss of chondrocyte phenotypic stability.

TRPC6 Regulates CXCR2-Mediated Chemotaxis of Murine Neutrophils

Unraveling the mechanisms involved in chemotactic navigation of immune cells is of particular interest for the development of new immunoregulatory therapies. It is generally agreed upon that members of the classical transient receptor potential channel family (TRPC) are involved in chemotaxis. However, the regulatory role of TRPC channels in chemoattractant receptor-mediated signaling has not yet been clarified in detail. In this study, we demonstrate that the TRPC6 channels play a pronounced role in CXCR2-mediated intermediary chemotaxis, whereas N-formyl-methionine-leucine-phenylalanine receptor–mediated end-target chemotaxis is TRPC6 independent. The knockout of TRPC6 channels in murine neutrophils led to a strongly impaired intermediary chemotaxis after CXCR2 activation which is not further reinforced by CXCR2, PI3K, or p38 MAPK inhibition. Furthermore, CXCR2-mediated Ca2+ influx but not Ca2+ store release was attenuated in TRPC6−/− neutrophils. We demonstrate that the TRPC6 deficiency affected phosphorylation of AKT and MAPK downstream of CXCR2 receptor activation and led to altered remodeling of actin. The relevance of this TRPC6-depending defect in neutrophil chemotaxis is underscored by our in vivo findings. A nonseptic peritoneal inflammation revealed an attenuated recruitment of neutrophils in the peritoneal cavity of TRPC6−/− mice. In summary, this paper defines a specific role of TRPC6 channels in CXCR2-induced intermediary chemotaxis. In particular, TRPC6-mediated supply of calcium appears to be critical for activation of downstream signaling components.

Syndecan 4 supports bone fracture repair, but not fetal skeletal development, in mice

OBJECTIVE Syndecan 4, a heparan sulfate proteoglycan, has been associated with osteoarthritis. The present study was undertaken to analyze the functional role of syndecan 4 in endochondral ossification of mouse embryos and in adult fracture repair, which, like osteoarthritis, involves an inflammatory component. METHODS Sdc4 promoter activity was analyzed in Sdc4(-/-) lacZ-knockin mice, using β-galactosidase staining. Endochondral ossification in embryos from embryonic day 16.5 was assessed by histologic and immunohistologic staining. Bone fracture repair was analyzed in femora of adult mice on days 7 and 14 postfracture. To evaluate Sdc2 and Sdc4 gene expression with and without tumor necrosis factor α (TNFα) and Wnt-3a stimulation, quantitative real-time polymerase chain reaction was performed. RESULTS In Sdc4(-/-) lacZ-knockin animals, syndecan 4 promoter activity was detectable at all stages of chondrocyte differentiation, and Sdc4 deficiency inhibited chondrocyte proliferation. Aggrecan turnover in the uncalcified cartilage of the epiphysis was decreased transiently in vivo, but this did not lead to a growth phenotype at birth. In contrast, among adult mice, fracture healing was markedly delayed in Sdc4(-/-) animals and was accompanied by increased callus formation. Blocking of inflammation via anti-TNFα treatment during fracture healing reduced these changes in Sdc4(-/-) mice to levels observed in wild-type controls. We analyzed the differences between the mild embryonic and the severe adult phenotype, and found a compensatory up-regulation of syndecan 2 in the developing cartilage of Sdc4(-/-) mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt-3a in vitro led to increased expression of syndecan 2, while stimulation with TNFα resulted in up-regulation of syndecan 4 but decreased expression of syndecan 2. TNFα stimulation reduced syndecan 2 expression and increased syndecan 4 expression even in the presence of Wnt-3a, suggesting that inflammation has a strong effect on the regulation of syndecan expression. CONCLUSION Our results demonstrate that syndecan 4 is functionally involved in endochondral ossification and that its loss impairs fracture healing, due to inhibition of compensatory mechanisms under inflammatory conditions.