Inhibition of Wnt/B-catenin pathway by sclerostin promotes cartilage maintenance

Abstract

Purpose: Cartilage homeostasis is regulated by several mechanisms that influence both anabolic and catabolic balance. Among them, the local activation of Wnt pathways plays a major role in chondrocyte activity which might be regulated by Wnt inhibitors. Sclerostin, a Wnt inhibitor mainly produced by osteocytes and hypertrophic chondrocytes, might participate to the crosstalk between bone and cartilage. Therefore, the aim of this study is to determine the effect of Sclerostin in chondrocyte function. Methods: Primary murine chondrocytes, isolated from long bone epiphysis of 6 day-old mice, were cultured with or without Wnt3a conditioned media (Wnt3aCM) and in the presence or absence of mouse recombinant Sclerostin. Proteoglycan release induced by Wnt was quantified in the supernatant of chondrocytes by a colorimetric assay. Activation and inhibition of Wnt pathway was shown by the translocation of β-catenin (IF, TOP-GAL activity). BrdU and Tunel assays were performed to test the effect of Wnt and Sclerostin on cell proliferation and apoptosis. Chondrocyte differentiation induced by Sclerostin was investigated by RT-qPCR through the relative expression of type II and X collagen, Sox9, aggrecan, MMP-3, MMP-13 ADAMTS-4 & 5 genes. Results: Wnt3aCM reduced proteoglycan release in a dose-dependent manner and was rescued by Sclerostin (20ng/ml). This dose was chosen for the following experiments. Wnt inhibited the gene expression of collagen type II (X18-fold), Sox9 (X140 fold) and Aggrecan (X90-fold) and increases the gene expression of metalloproteinases such as Adamts-4 (X5-fold), Adamts-5 (X5.5-fold), MMP3 (X7-fold) and MMP13 (X6.6-fold). Sclerostin was able to inhibit the nuclear translocation of β-catenin induced by Wnt, but failed to exert any effect in the proliferation or apoptosis in primary chondrocytes regardless of Wnt. However, Wnt3a increased the gene expression of collagen type X (X3-fold) which was abolished by Sclerostin (X1.6-fold). Sclerostin significantly inhibited the Wnt-induced increase of catabolic genes (X1.7-fold Adamts-4, X1.6-fold Adamts-5, X3.4-fold MMP3 and X4-fold MMP13) and rescues partially the expression of anabolic genes (X5- fold collagen type II, X6-fold Sox-9 and X11-fold Aggrecan). In addition, Wnt3a induced the expression of Sost mRNA suggesting that Sclerostin might be regulated by Wnt. We further observed an enhanced expression of Sclerostin in osteoarthritic cartilage compared to cartilage of control mice. Conclusions: We here show that Wnt3a increases the catabolic activity and inhibits the anabolic activity in murine chondrocytes. Sclerostin alleviates the expression of catabolic genes induced by Wnt and rescued proteoglycan production. Moreover, Sclerostin promotes the cartilage maintenance through the inhibition of chondrocyte hypertrophy. Finally, the expression of Sclerostin expression in osteoarthritic mice suggests that Sclerostin might be involved in the pathophysiology in cartilage damage and might constitute a target for the prevention of osteoarthritis.