Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/ TSP5) is a major component of the extracellular matrix of the musculoskeletal system. Although COMP/TSP5 abnormalities are associated with several pathological conditions, its normal function remains unclear. This study was undertaken to delineate the function(s) of COMP/TSP5 in cartilage, especially regarding its interaction with chondrocytes. We show that COMP/TSP5 can support chondrocyte attachment and that the RGD sequence in COMP/ TSP5 and the integrin receptors ␣51 and ␣V3 on the chondrocytes are involved in mediating this attachment. The interactions of COMP/TSP5 with the integrins are dependent on COMP/TSP5 conformation. Chondrocyte attachment to COMP/TSP5 in the calcium-replete conformation was inhibited by function-blocking integrin ␣5 and 1 antibodies, suggesting the involvement of the ␣51 integrin. Under this condition, a function-blocking antibody against ␣V3 did not have any effect on cell attachment. On the other hand, chondrocyte attachment to reduced COMP/TSP5 was instead sensitive to ␣V3 function-blocking antibodies, suggesting that COMP/TSP5 mediates attachment through chondrocyte ␣V3 integrin under this condition. Cell attachment to reduced COMP/TSP5 was not inhibited by 1 antibodies. These data indicate that COMP/TSP5 in different conformations can utilize different integrin receptors. These results are the first to demonstrate that COMP/TSP5 can mediate chondrocyte attachment through interactions with integrins. Through these interactions, COMP/ TSP5 may be able to regulate cellular activities and respond to environment in the surrounding cartilage matrix.Cartilage oligomeric matrix protein (COMP), 2 also known as thrombospondin 5 (TSP5), is an abundant extracellular matrix protein that has been shown to exist in tissues of the musculoskeletal system including cartilage, tendon, ligament, and synovium (1-7). Immunohistochemistry studies of COMP/TSP5 distribution in cartilage have revealed specific temporal and spatial patterns, and COMP/TSP5 can be found in cartilage at sites both in proximity to chondrocytes in the pericellular matrix and away from the cells in the territorial and interterritorial matrix (8 -10). In fetal cartilage, COMP/TSP5 exists in the pericellular matrix, especially in the growth cartilage adjacent to the primary ossification center. In adult articular cartilage, COMP/TSP5 is found at higher levels in the interterritorial matrix, with stronger staining in the deeper zone (8 -10).The importance of COMP/TSP5 is suggested by its association with several pathological conditions. Compared with normal cartilage, the levels and patterns of COMP/TSP5 expression were found to change in cartilage of osteoarthritis and rheumatoid arthritis patients, and COMP/TSP5 in serum and synovial fluid has been suggested as a biomarker for these conditions (11-13). The importance of COMP/ TSP5 to cartilage structure and function is further underscored by findings that COMP/TSP5 mutations lead to human skeletal dysplasias, p...
Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix (ECM) of the musculoskeletal system. Its importance is underscored by its association with several growth disorders. In this report, we investigated its interaction with aggrecan, a major component of cartilage ECM. We also tested a COMP/TSP5 mutant, designated MUT3 that accounts for 30% of human pseudoachondroplasia cases, to determine if the mutation affects function. Using a solid-phase binding assay, we have shown that COMP/ TSP5 can bind aggrecan. This binding was decreased with MUT3, or when COMP/TSP5 was treated with EDTA, indicating the presence of a conformation-dependent aggrecan binding site. Soluble glycosaminoglycans (GAGs) partially inhibited binding, suggesting that the interaction was mediated in part through aggrecan GAG side chains. Using affinity co-electrophoresis, we showed that COMP/TSP5, in its calcium-replete conformation, bound to heparin, chondroitin sulfates, and heparan sulfate; this binding was reduced with EDTA treatment of COMP/TSP5. MUT3 showed weaker binding than calcium-repleted COMP/TSP5. Using recombinant COMP/TSP5 fragments, we found that the "signature domain" could bind to aggrecan, suggesting that this domain can mediate the interaction of COMP/TSP5 and aggrecan. In summary, our data indicate that COMP/TSP5 is an aggrecan-binding protein, and this interaction is regulated by the calcium-sensitive conformation of COMP/TSP5; interaction of COMP with aggrecan can be mediated through the GAG side chains on aggrecan and the "signature domain" of COMP/TSP5. Our results suggest that COMP/TSP5 may function to support matrix interactions in cartilage ECM.
SummaryThe specific aim of this study was to investigate the effect of chondroitinase ABC treatment on the frictional response of bovine articular cartilage against glass, under creep loading. The hypothesis is that chondroitinase ABC treatment increases the friction coefficient of bovine articular cartilage under creep. Articular cartilage samples (n=12) harvested from two bovine knee joints (1-3 monthsold) were divided into a control group (intact specimens) and a treated group (chondroitinase ABC digestion), and tested in unconfined compression with simultaneous continuous sliding (±4 mm at 1 mm/s) under a constant applied stress of 0.5 MPa, for 2,500 s. The time-dependent response of the friction coefficient was measured. With increasing duration of loading, treated samples exhibited a significantly higher friction coefficient than control samples as assessed by the equilibrium value (treated: μ eq = 0.19 ± 0.02; control: μ eq = 0.12 ± 0.03; p=0.002), though the coefficient achieved immediately upon loading did not increase significantly (treated: μ min = 0.0053 ± 0.0025; control: μ min = 0.037 ± 0.0013; p=0.19). Our results demonstrate that removal of the cartilage glycosaminoglycans using chondroitinase ABC significantly increases the overall time-dependent friction coefficient of articular cartilage. These findings strengthen the motivation for developing chondroprotective strategies by increasing cartilage chondroitin sulfate content in osteoarthritic joints.
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