Syndecan-4 Regulates Activation of WNT Signaling in Chondrocytes.

Bertrand J, Stange R, Nalesso G, Sherwood J, Godmann L, Echtermeyer F, Dell'Accio F, Pap T

Abstract

Background/Purpose: Blockade of syndecan-4 (Sdc4) signaling protectsmice from cartilage degradation in experimentally induced osteoarthritis(OA). Cartilage damage results in changes of chondrocyte phenotype inducedby various signaling pathways including the activation of WNT signaling.Here, we hypothesized that Sdc4 modulates chondrocyte phenotype by thespecific modulation of WNT signaling.Methods: In vitro analyses were performed using neonatal wild type (wt)and Sdc4/ chondrocytes, or a blocking Sdc4 antibody on wt chondrocytes.The influence of 100 ng/ ml WNT3a on glycosaminoglycan (GAG)production was analyzed using alcian blue staining of micromass cultures.The expression of chondrocyte marker genes (aggrecan, collagen 2, MMP13)was measured by quantitative RT-PCR. The effects of WNT3a on canonical and noncanonical WNT signaling were analyzed using Western Blot detectionof -catenin, pLRP-6, pCamKII and pJNK and a TCF/LEF reporterassay. To investigate the in vivo relevance of the investigated pathwaysinduction of OA in wt and Sdc4/ mice was performed using the DMMmodel and stainings for -catenin and pCamKII were performed.Results: Micromass cultures revealed a higher basal GAG production bySdc4/ than wt chondrocytes. Furthermore, WNT3a stimulation led to adecrease in GAG in wt, which was not observed in Sdc4/ chondrocytes.These findings were confirmed by a 10 higher basal production of aggrecanand collagen 2 in Sdc4/ compared to wt chondrocytes. The expression ofboth genes was 10 fold increased by stimulation with WNT3a, whereasWNT3a led to a decrease in the expression of both genes in wt chondrocytes.Inverse results were found for MMP13, which was significantly less expressedin Sdc4/ chondrocytes and was not upregulated upon WNT3astimulation. Western blot analyses of canonical WNT signaling showed that catenin is strongly reduced and not upregulated upon stimulation withWNT3a in Sdc4/ chondrocytes. Confirming these findings we also foundless phosphorylation of LRP6 and less activation of the TCF/Lef reporterupon WNT3a stimulation in Sdc4/ chondrocytes. Noncanonical WNTsignaling was increased in Sdc4/ under basal conditions, but decreasedupon WNT3a stimulation in Sdc4/and increased in wt chondrocytes. Thesame blockade of canonical and downregulation of noncanonical WNTsignaling upon WNT stimulation were obtained by using a blocking Sdc-4antibody. In vivo analyses of canonical WNT signaling confirmed thedecreased basal activation and the missing increase after induction of OA inSdc4/ mice.Conclusion: We conclude from our data that Sdc4 is a major regulator ofcellular response to WNT through the prevention of the induction ofcanonical WNT signaling. Therefore, we suggest that the blockade of Sdc-4protects from OA induced changes in chondrocyte phenotype by inhibitingWNT induced differentiation of chondrocytes.