In situ calcium signaling of chondrocytes under serum-free and serum culture

Abstract

Sclerostin failed to restore the expression of the anabolic genes, we investigated the effect of Sclerostin on the activation of Wnt non canonical pathways mediated by Ca2+/CaMKII, JNK and PKC proteins. Methods: Primary murine chondrocytes were cultured with or without Wnt3a conditioned media and in the presence or absence of recombinant Sclerostin. Activation of Ca2+/CaMKII, JNK and PKC pathways was analyzed by Western blotting. The role of the activation of JNK and PKC pathways in the chondrocyte phenotype was investigated using SP600125 and Staurosporin inhibitors respectively. Chondrocyte differentiation was investigated by RT-qPCR through the relative expression of type II and X collagen, Sox9, Aggrecan, MMP-3 & 13 and ADAMTS-4&5 genes and by Western blotting through the protein expression of ADAMTS-4 & 5 and type X Collagen Alcian blue staining and spectrophotometric quantification was also used for analyzing the accumulation of highly sulphated GAG.. Results: Wnt3a increased the gene expression of metalloproteinases such as Adamts-4 & 5 MMP3, 13 and type X collagen. This effect was totally abolished in the presence of Sclerostin. In the other hand, Sclerostin restored partially the expression of type II Collagen, Sox9 and Aggrecan inhibited by Wnt3a. Because of the partial effect of Sclerostin on the anabolic genes, we assessed whether Wnt3a activates b-catenin independent pathways such as JNK, PKC and CaMKbII. Wnt3A promoted the phosphorylation of JNK and PKC without affecting CaMKbII phsphorylation. Moreover, Sclerostin inhibited the phosphorylation of JNK but not that of PKC. We further investigated whether Sclerostininduced inhibition of JNK affects chondrocyte function. We found that Wnt3a decreased the accumulation of highly sulphated GAG while Sclerostin failed in rescuing their amount. Interestingly, Sclerostin was able to rescue the accumulation of highly sulphated GAG and the expression of the anabolic genes when JNK pathway is inhibited by SP600125. Conclusions: We here showed that Wnt3a inhibits the expression of the anabolic genes by activating the non-canonical JNK pathways in chondrocytes. Sclerostin reduced the Wnt-induced activation of JNK pathway. These results show that Sclerostin may play a role in the anabolic activity of chondrocytes.