Background SMOC2, a secreted calcium-binding protein of the BM-40/SPARC family was identified from a chondrogenic extract of articular cartilage and is increased in osteoarthritic cartilage. Previous research demonstrated that Wnt signalling needs to be carefully balanced in cartilage to ensure homeostasis. We evaluated the effect of SMOC2 on in vitro chondrogenesis and its relationship with Wnt signalling.
Materials and methods Smoc2 was stably overexpressed or knocked down in ATDC5 cells cultured as micromasses. Impact of SMOC2 on chondrogenesis was evaluated by gene expression analysis of chondrogenic markers (Acan, Col2a1, Col10a1) and by Sirius red, Alcian Blue and Alizarin red staining to evaluate collagens, proteoglycans and mineralization. Femoral head caps of 6 weeks old mice were isolated and stimulated with IL-1β to induce cartilage breakdown. Influence of SMOC2 on cartilage breakdown was evaluated by co-stimulating femoral head caps with recombinant SMOC2 protein. Glycosaminoglycan release in the culture medium was measured by performing a DMMB assay. The influence of SMOC2 on canonical Wnt signalling was assessed by performing a TOP/FOP-flash reporter assay in ATDC5 cells. Human articular chondrocytes were stimulated with Wnt agonist CHIR99021 and recombinant SMOC2 protein to validate the effect of SMOC2 on Wnt signalling. Gene expression of canonical Wnt markers was analysed.
Results Gene expression of chondrogenic markers and staining intensity of Alcian blue and Alizarin red in ATDC5 micromasses was strongly reduced in Smoc2 overexpressing cells compared to control. Mice femoral head caps stimulated with recombinant SMOC2 protein showed an increased glycosaminoglycan release after IL-1β treatment in culture medium in comparison to IL-1β stimulation alone. ATDC5 cells stimulated with recombinant SMOC2 exhibit decreased luciferase activity in the Wnt reporter assay. In contrast we observed an increased luciferase activity in ATDC5 cells with transient Smoc2 knockdown. This inhibitory effect of Smoc2 on canonical Wnt signalling was also observed in human articular chondrocytes.
Conclusion Smoc2 negatively affects chondrogenic differentiation of precursor cells and increases IL-1β induced breakdown in mice cartilage. Inhibition of canonical Wnt signalling is identified as contributing mechanism.