Nishida T, Nakanishi T, Asano M, Shimo T, Takigawa M
Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Okayama, Japan.
J Cell Physiol. 2000 Aug;184(2):197-206. doi: 10.1002/1097-4652(200008)184:2<197::AID-JCP7>3.0.CO;2-R.
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."
结缔组织生长因子/肥大软骨细胞特异性基因产物Hcs24（CTGF/Hcs24）可促进参与软骨内骨化的软骨细胞和内皮细胞的增殖与分化（下茂等人，1998年，《生物化学杂志》124卷：130 - 140页；下茂等人，1999年，《生物化学杂志》126卷：137 - 145页；中岸等人，2000年，《内分泌学》141卷：264 - 273页）。为了进一步阐明CTGF/Hcs24在软骨内骨化中的作用，我们在此研究了CTGF/Hcs24对体外成骨细胞系增殖和分化的影响。使用（125）I标记的重组CTGF/Hcs24（rCTGF/Hcs24）进行的结合研究揭示了人骨肉瘤细胞系Saos - 2上存在两类特异性结合位点。每个结合位点的表观解离常数（Kd）值分别为17.2和391 nM。交联研究显示形成了表观分子量为280 kDa的（125）I - rCTGF/Hcs24 - 受体复合物。随着未标记的rCTGF/Hcs24浓度增加，（125）I - rCTGF/Hcs24 - 受体复合物的强度降低，但血小板衍生生长因子 - BB同二聚体或碱性成纤维细胞生长因子则无此作用。这些发现表明成骨细胞具有CTGF/Hcs24的特异性受体分子。rCTGF/Hcs24以剂量和时间依赖性方式促进Saos - 2细胞和小鼠成骨细胞系MC3T3 - E1的增殖。rCTGF/Hcs24还增加了Saos - 2细胞和MC3T3 - E1细胞中I型胶原蛋白、碱性磷酸酶、骨桥蛋白和骨钙素的mRNA表达。此外，rCTGF/Hcs24增加了两种细胞中的碱性磷酸酶活性。它还刺激了MC3T3 - E1细胞中的胶原蛋白合成。此外，rCTGF/Hcs24刺激了MC3T3 - E1细胞上的基质矿化，其刺激作用与骨形态发生蛋白 - 2相当。这些发现表明，CTGF/Hcs24除了对软骨细胞和内皮细胞外，还是一种新型的、强效的成骨细胞增殖和分化刺激因子。由于这些功能，我们将CTGF/Hcs24重新定义为促进软骨内骨化的主要因子，称为“生态生成素：软骨内骨化遗传因子”。
