A4.05 SMOC2, a secreted calcium-binding protein from cartilage extracellular matrix is an inhibitor of cartilage and bone formation

Background and objectives

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. We evaluated the impact of SMOC2 on in vitro chondrogenesis and on in vitro osteogenesis and mineralization.

Materials and methods

Smoc2 was stably overexpressed (Smoc2+) or silenced (Smoc2-). We also stably overexpressed Smoc2 lacking the Calcium-binding domain (ΔCaBD). ATDC5 cells were cultured as micromasses. Gene expression of chondrogenic genes (Acan, Col2a1, Col10a1), Wnt- and BMP genes was analysed by RT-qPCR. Safranin O, Sirius red, Alcian Blue and Alizarin red staining were performed to evaluate collagens, proteoglycans and mineralization. MC3T3 cells were cultured in monolayer. Alkaline phosphatase activity (ALP) was measured and mineralization was analysed by alizarin red staining. Gene expression of extracellular matrix genes, genes linked to mineralization and TGFβ superfamily genes was analysed by RT-qPCR. Intracellular signalling pathways (PKCα, ERK1/2, SMAD1/5/8, p38MAPK) were investigated by Western blot. Data were translationally validated in human periosteal and endothelial cells.

Results

Mineralization and ALP activity was reduced in Smoc2+ MC3T3 cells. Gene expression analysis showed an overall altered differentiation when overexpressing Smoc2. The activation status of PKCα, ERK1/2, p38MAPK and SMAD1/5/8 was significantly modified in Smoc2+ cells. We could not observe an effect on osteogenesis when silencing Smoc2. ΔCaBD cells however exhibited less of the inhibiting effects of Smoc2+ cells. Moreover we could observe partial restoration after addition of extracellular calcium to the culture medium of differentiating Smoc2+ cells. Alizarin red staining and ALP activity was reduced in hPDCs cultured in the presence of Smoc2+ supernatant compared to controls. However, hPDCs stimulated with ΔCaBD supernatants exhibited a lesser decrease of these markers compared to Smoc2+. Alizarin red quantification showed the same effect for HUVECs. In ATDC5 cells, Smoc2 overexpression altered chondrogenic markers and reduced Wnt and BMP signalling. Smoc2 silencing enhanced chondrogenic differentiation by increasing BMP signalling.

Conclusions

SMOC2 can regulate osteogenesis and chondrogenesis. Silencing of Smoc2 did not affect osteogenesis suggesting a limited endogenous role. SMOC2 appears to exert its effects through interaction with calcium and by interfering with BMP and Wnt signalling.