Molecular cloning of the receptors for chondrosarcoma-derived chondrocyte growth factor Ecogenin/CTGF

(1)用差异显示-聚合酶链式反应(Differential Display-PCR)从软骨细胞系HCS-2/8中克隆出结缔组织生长因子(CTGF)，重组CTGF(RCTGF)可刺激软骨细胞的增殖、成熟和分化，(3)软骨细胞系HCS-2/8上存在两种CTGF受体，其高亲和力可能是包括整合素在内的细胞黏附分子。低亲和力的受体可能是包括蛋白多糖在内的胞外基质化合物。(4)信号抑制剂抑制实验表明，CTGF刺激的HCS-2/8细胞通过MEK和ERK等MAP激酶途径介导细胞间信号转导。(5)CTGF偶联亲和层析从HCS-2/8细胞膜和胞浆部分纯化CTGF结合蛋白。结果从HCS-2/8细胞中分离纯化出4种结合蛋白，分别为膜结合蛋白34、44、66 kDa，胞质结合蛋白50 kDa。这四种蛋白的表达受CTGF刺激的调控，提示它们在功能上与CTGF相关。(6)根据酪氨酸激酶区之间的共同序列，用简并引物从HCS-2/8细胞中克隆了酪氨酸激酶型受体的cDNA。序列分析表明，在HCS-2/8细胞中存在多种类型的酪氨酸激酶受体，其中包括新的受体。这些受体的表达受CTGF刺激的调节。此外，CTGF产生细胞高水平表达这些受体。这些结果表明，它们在功能上与CTGF相关。

(1) Connective tissue growth factor (CTGF) was cloned from a chondrocyte-derived chondrocytic cell line, HCS-2/8 by differential display-PCR.(2) Recombinant CTGF (rCTGF) stimulated the proliferation, maturation and differentiation of chondrocytes.(3) Two types of receptors for CTGF were found on a chondrocyte-derived chondrocytic cell line, HCS-2/8. The receptor with high affinity was supposed to be cell adhesion molecules including integrins. The receptor with low affinity was supposed to be extracellular matrix compounds including proteoglycans.(4) The inhibitory experiments using signal inhibitors showed that intercellular signal transduction in HCS-2/8 cells caused by the stimulation of CTGF was mediated by MAP kinase-pathways including MEK and ERK.(5) CTGF-binding proteins were purified from membrane fractions and cytoplasmic fractions of HCS-2/8 cells with CTGF-conjugated affinity chromatography. As a result, four binding proteins (34, 44, 66 kDa from membrane fractions 50 kDa from cytoplasmic fractions) were purified from HCS-2/8 cells. The expression of these four proteins were regulated by the stimulation of CTGF, suggesting that they were functionally associated with CTGF.(6) The cDNA of tyrosine kinase-type receptors were cloned from HCS-2/8 cells using degenerate primers corresponding to the consensus sequences between tyrosine kinase-domains. The sequence analysis of these cDNA revealed that there were several types of tyrosine kinase-receptors including novel receptors in HCS-2/8 cells. The expression of these receptors were regulated by the stimulation of CTGF. In addition, CTGF-producing cells showed high level of expression of these receptors. These results suggest that they were functionally associated with CTGF.