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Purification of Receptors to Parathyroid Hormone from Cultured Chondrocytes and the Mechanism of its Signal Transduction

培养软骨细胞甲状旁腺激素受体的纯化及其信号转导机制

基本信息

批准号：
01571016
负责人：
ASADA Akira
金额：
$ 1.34万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

1) Parathyroid Hormone (PTH) receptors on cultured rabbit costal chondrocytes were demonstrated using HPLC-purified, radio-iodinated [Nle^<8,>-Nle^<18>, Tyr^<34>]bovine PTH- (1-34) amide. Both Growth Cartilage (GC) cells and resting cartilage cells were shown to have a single class of saturable, high affinity PTH binding sites with a dissociation constant of 0.6-0.7 nM. The number of receptors per cell was approximately 40,000-90,000 on GC cells. After crosslinking the receptors on these cells with the radioligand, One, major^<125>l-labeled band of 76 kDa was separated by SDS-PAGE.2) Treatment with retinoic acid, epidermal growth factor and fibroblast growth factor of rabbit costal chondrocytes decreased both proteoglycan synthesis and the number PTH receptors while treatment with insulin-like growth factor-I, dibutyryl cyclic AMP and transforming growth factor-beta resulted in increases in proteoglycan synthesis as well as in the number of PTH receptors. However, their affinity did no … More t change by these treatments. The PTH-stimulated cyclic AMP level in chondrocytes pretreated with retinoic acid, epidermal growth factor and fibroblast growth factor was lower than that in control cells while the PTH-stimulated cvclic AMP level in chondrocytes pretreated with insulin-like growth factor-I and transforming growth factor-beta was higher tnan that in control cells. These observations suggest that the increase in the number of PTH receptors on chondrocytes is closely related with expression of the differentiated phenotype of chondrocytes and that the number of the receptors is a good marker of the differentiated phenotype of chondrocytes.3) Three clonal cell lines with responsiveness to PTH during more than 3 years, more than 58 passages in culture were established from growth cartilage of mouse ribs. Among the three clonal cell lines, a cell line, named MGC/T1.18, showed the highest responsiveness to PTH : The hormone increased intacellular cAMP level in the cells to 200 times that of control. Less

1)使用HPLC纯化的放射性碘标记的[Nle^<8，>-Nle^，Tyr^ ]牛PTH-（1-34）酰胺证明培养的兔肋软骨细胞上的甲状旁腺激素（PTH）受体<18><34>。生长软骨（GC）细胞和静息软骨细胞都显示出具有一类可饱和的高亲和力PTH结合位点，解离常数为0.6-0.7 nM。GC细胞上每个细胞的受体数量约为40，000 - 90，000。2<125>）用维甲酸、表皮生长因子和成纤维细胞生长因子处理兔肋软骨细胞后，蛋白多糖合成减少，PTH受体数量减少，而用胰岛素样生长因子-I、二丁酰环腺苷酸和转化生长因子-β处理后，蛋白多糖合成增加，PTH受体数量增加。然而，他们的亲和力并没有 ...更多信息这些治疗不会改变。经维甲酸、表皮生长因子和成纤维细胞生长因子预处理的软骨细胞中PTH刺激的cAMP水平低于对照细胞，而经胰岛素样生长因子-I和转化生长因子-β预处理的软骨细胞中PTH刺激的cAMP水平高于对照细胞。这些结果表明，软骨细胞表面甲状旁腺素受体数量的增加与软骨细胞分化表型的表达密切相关，甲状旁腺素受体的数量是软骨细胞分化表型的一个很好的标志。3）从小鼠肋骨生长软骨中建立了3株对甲状旁腺素有反应的克隆细胞系。在三个克隆细胞系中，命名为MGC/T1.18的细胞系显示出对PTH的最高反应性：该激素使细胞内cAMP水平增加至对照的200倍。少