Osteoarthritis (OA) is a slowly progressing disease, resulting in the degradation of cartilage and the loss of joint functionality. The cartilage extracellular matrix (ECM) is degraded and undergoes remodelling in OA progression. Chondrocytes start to express degrading proteases but are also reactivated and synthesise ECM proteins. The spectrum of these newly synthesised proteins and their involvement in OA specific processes and cartilage repair is hardly investigated. Human articular cartilage obtained from OA patients undergoing knee replacement surgery was evaluated according to the OARSI histopathology grading system. Healthy, non-OA cartilage samples were used as controls. The expression and distribution of thrombospondin-4 (TSP-4) and the closely related COMP were analysed on the gene level by PCR and on the protein level by immunohistology and immunoblot assays. The potential of TSP-4 as a diagnostic marker was evaluated by immunoblot assays, using serum samples from OA patients and healthy individuals. The functional role of both proteins was further investigated in in vitro studies using chondrocytes isolated from femoral condyles of healthy pigs. The effect of COMP and TSP-4 on chondrocyte migration and attachment was investigated via transwell and attachment assays, respectively. Moreover, the potential of COMP and TSP-4 to modulate the chondrocyte phenotype by inducing gene expression, ECM protein synthesis and matrix formation was investigated by immunofluorescence staining and qPCR. The activation of cartilage relevant signalling pathways was investigated by immunoblot assays. These results showed for the first time the presence of TSP-4 in articular cartilage. Its amount dramatically increased in OA compared to healthy cartilage and correlated positively with OA severity. In healthy cartilage TSP-4 was primarily found in the superficial zone while it was wider distributed in the middle and deeper zones of OA cartilage. The amount of specific TSP-4 fragments was increased in sera of OA patients compared to healthy controls, indicating a potential to serve as an OA biomarker. COMP was ubiquitously expressed in healthy cartilage but degraded in early as well as re-expressed in late-stage OA. The overall protein levels between OA severity grades were comparable. Contrary to TSP-4, COMP was localised primarily in the upper zone of OA cartilage, in particular in areas with severe damage. COMP could attract chondrocytes and facilitated their attachment, while TSP-4 did not affect these processes. COMP and TSP 4 were generally weak inducers of gene expression, although both could induce COL2A1 and TSP-4 additionally COL12A1 and ACAN after 6 h. Correlating data were obtained on the protein level: COMP and TSP-4 promoted the synthesis and matrix formation of collagen II, collagen IX, collagen XII and proteoglycans. In parallel, both proteins suppressed chondrocyte hypertrophy and dedifferentiation by reducing collagen X and collagen I. By analysing the effect of COMP and TSP-4 on intracellular signalling, both proteins induced Erk1/2 phosphorylation and TSP-4 could further promote Smad2/3 signalling induced by TGF-β1. None of the two proteins had a direct or modulatory effect on Smad1/5/9 dependent signalling. In summary, COMP and TSP-4 contribute to ECM maintenance and repair by inducing the expression of essential ECM proteins and suppressing chondrocyte dedifferentiation. These effects might be mediated by Erk1/2 phosphorylation. The presented data demonstrate an important functional role of COMP and TSP-4 in both healthy and OA cartilage and provide a basis for further studies on their potential in clinical applications for OA diagnosis and treatment.
Clonal growth of human articular cartilage and the functional role of the periosteum in chondrogenesis
