RGD-CAP (βig-h3) is expressed in precartilage condensation and in prehypertrophic chondrocytes during cartilage development

Abstract RGD-CAP (βig-h3), isolated from cartilage as a collagen-associated protein, was demonstrated to have a binding ability to collagen and to enhance the adhesion of chondrocytes via integrin α 1 β 1 . However, the role of this protein in cartilage development remains unclear. In this study, we investigated the expression of RGD-CAP (βig-h3) in chick embryos and cultured mesenchymal stem cells (MSCs) during the differentiation to chondrocytes. The effects of recombinant RGD-CAP on adhesion and DNA synthesis of MSCs and mineralization were also examined. Tissue sections from chick embryos at Hamburger–Hamilton (HH) stages 19–37 were immunostained with anti-chick RGD-CAP antibodies. The expression of RGD-CAP was slightest in chick embryos at HH stage 19, whereas a considerable expression of RGD-CAP was observed in the developing vertebrae and precartilage aggregate in the limb bud of chick embryos at HH stage 26. The expression of RGD-CAP was significantly reduced in vertebrae of chick embryo at HH stage 32. Reverse transcriptional polymerase chain reaction (RT-PCR) analysis showed that RGD-CAP was highly expressed in cultured MSCs and decreased by 4-day treatment with 10 –8 M dexamethasone when MSCs proliferated to adipocyte-like cells, whereas it was recovered by co-treatment with 3 ng/ml TGF-β for 8-12 days when MSCs proliferated to hypertrophic chondrocyte-like cells. The adhesion and DNA synthesis of MSCs cultured on RGD-CAP-coated dishes increased significantly compared with the controls. RGD-CAP was distributed in the prehypertrophic zone in matured cartilage of the vertebrae of chick embryos at HH stage 37. Recombinant RGD-CAP inhibited the mineralization of hypertrophic chondrocytes. These results suggest that RGD-CAP (βig-h3) exerts an essential role in the early cartilage development by enhancing the adhesion and growth of the pre-chondrogenic cells, and functions as a negative regulator for mineralization at the terminal stage of the chondrogenic differentiation.