Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro.
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 (125)I-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 (125)I-rCTGF/Hcs24-receptor complex with an apparent molecular weight of 280 kDa. The intensity of (125)I-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."
Nishida
T
T
Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Okayama, Japan.
Nakanishi
T
T
Asano
M
M
Shimo
T
T
Takigawa
M
M
D016475:3T3 Cells / Q000166:cytology / Q000201:enzymology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D015152:Blotting, Northern; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003094:Collagen / Q000096:biosynthesis; D055513:Connective Tissue Growth Factor; D006133:Growth Substances / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism* / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008934:Mitogens / Q000378:metabolism* / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011506:Proteins / Q000378:metabolism* / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; D006133:Growth Substances; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008934:Mitogens; D011506:Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D055513:Connective Tissue Growth Factor; D003094:Collagen; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases