Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro

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Abstract
Connective tissue growth factor/hypertrophic chondrocyte‐specific gene product Hcs24 (CTGF/Hcs24) promotes the proliferation and differentiation of chondrocytes and endothelial cells which are involved in endochondral ossification (Shimo et al., 1998, J Biochem 124:130–140; Shimo et al., 1999, J Biochem 126:137–145; Nakanishi et al., 2000, Endocrinology 141:264–273). To further clarify the role of CTGF/Hcs24 in endochondral ossification, here we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of osteoblastic cell lines in vitro. A binding study using 125I‐labeled recombinant CTGF/Hcs24 (rCTGF/Hcs24) disclosed two classes of specific binding sites on a human osteosarcoma cell line, Saos‐2. The apparent dissociation constant (Kd) value of each binding site was 17.2 and 391 nM, respectively. A cross‐linking study revealed the formation of 125I‐rCTGF/Hcs24‐receptor complex with an apparent molecular weight of 280 kDa. The intensity of 125I‐rCTGF/Hcs24‐receptor complex decreased on the addition of increasing concentrations of unlabeled rCTGF/Hcs24, but not platelet‐derived growth factor‐BB homodimer or basic fibroblast growth factor. These findings suggest that osteoblastic cells have specific receptor molecules for CTGF/Hcs24. rCTGF/Hcs24 promoted the proliferation of Saos‐2 cells and a mouse osteoblast cell line MC3T3‐E1 in a dose‐ and time‐dependent manner. rCTGF/Hcs24 also increased mRNA expression of type I collagen, alkaline phosphatase, osteopontin, and osteocalcin in both Saos‐2 cells and MC3T3‐E1 cells. Moreover, rCTGF/Hcs24 increased alkaline phosphatase activity in both cells. It also stimulated collagen synthesis in MC3T3‐E1 cells. Furthermore, rCTGF/Hcs24 stimulated the matrix mineralization on MC3T3‐E1 cells and its stimulatory effect was comparable to that of bone morphogenetic protein‐2. These findings indicate that CTGF/Hcs24 is a novel, potent stimulator for the proliferation and differentiation of osteoblasts in addition to chondrocytes and endothelial cells. Because of these functions, we are re‐defining CTGF/Hcs24 as a major factor to promote endochondral ossification to be called “ecogenin: endochondral ossification genetic factor.” J. Cell. Physiol. 184:197–206, 2000.