Celic S, Katayama Y, Chilco P J, Martin T J, Findlay D M
St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
J Endocrinol. 1998 Sep;158(3):377-88. doi: 10.1677/joe.0.1580377.
We have previously shown that an exogenous type I collagen matrix can regulate expression of mRNA for parathyroid hormone (PTH)-related protein (PTHrP) and its receptor, the PTH/PTHrP receptor, in the UMR106-06 osteogenic sarcoma cell line, which is considered to be representative of a relatively mature osteoblast phenotype. Consistent with those data, we show here that growth of UMR106-06 cells on type I collagen increased PTH/PTHrP receptor-binding capacity. Analysis of the binding data showed that the number of PTH/PTHrP receptors expressed by cells cultured on collagen was at least 2-fold greater than that of cells cultured on plastic. Expression of mRNA encoding alkaline phosphatase (ALP) and osteopontin (OP) was also upregulated in cells cultured on collagen, suggesting that interaction with collagen promotes the osteoblast phenotype in this cell line. Retinoic acid (RA), which has also been shown to promote osteoblastic differentiation, synergized with type I collagen to cause super-induction of OP mRNA. In contrast, RA abolished the collagen-induced increase in ALP mRNA and PTH/PTHrP receptor mRNA. The collagen-mediated increase in the expression of OP and PTH/PTHrP receptor mRNA, but not that of ALP, was perturbed by prior covalent modification of the collagen by non-enzymatic glycation. The collagen effects did not occur via interaction with RGD amino acid domains in type I collagen, but evidence was obtained for involvement of the DGEA amino acid cell-binding domain. The mechanism by which plating of UMR106-06 cells on a type I collagen substrate affects PTH/PTHrP receptor mRNA levels was investigated. Inhibition of cytoskeletal organization using cytochalasin D, and inhibitors of protein phosphatases, protein kinase C, phospholipase C and cyclooxygenase, did not abrogate the collagen-mediated effects. In contrast, treatment of cells with the protein tyrosine kinase inhibitor genistein, but not herbimycin A, dose-dependently abolished the collagen effects on the expression of PTH/PTHrP receptor, ALP and OP mRNA. These results show that a type I collagen substrate influences the expression of osteoblast-associated genes in a cell model of mature osteoblasts and suggests that this involves, at least in part, changes in intracellular tyrosine phosphorylation.
我们之前已经表明，外源性I型胶原基质可以调节甲状旁腺激素（PTH）相关蛋白（PTHrP）及其受体（PTH/PTHrP受体）的mRNA表达，在UMR106 - 06骨肉瘤细胞系中，该细胞系被认为是相对成熟的成骨细胞表型的代表。与这些数据一致，我们在此表明，UMR106 - 06细胞在I型胶原上生长增加了PTH/PTHrP受体结合能力。结合数据分析表明，在胶原上培养的细胞表达的PTH/PTHrP受体数量比在塑料上培养的细胞至少多2倍。编码碱性磷酸酶（ALP）和骨桥蛋白（OP）的mRNA表达在胶原上培养的细胞中也上调，这表明与胶原的相互作用促进了该细胞系中的成骨细胞表型。视黄酸（RA）也已被证明可促进成骨细胞分化，它与I型胶原协同作用导致OP mRNA的超诱导。相反，RA消除了胶原诱导的ALP mRNA和PTH/PTHrP受体mRNA的增加。胶原介导的OP和PTH/PTHrP受体mRNA表达的增加，但不是ALP的增加，被胶原预先通过非酶糖基化的共价修饰所干扰。胶原的作用不是通过与I型胶原中的RGD氨基酸结构域相互作用发生的，但有证据表明DGEA氨基酸细胞结合结构域参与其中。研究了UMR106 - 06细胞接种在I型胶原底物上影响PTH/PTHrP受体mRNA水平的机制。使用细胞松弛素D抑制细胞骨架组织，以及蛋白磷酸酶、蛋白激酶C、磷脂酶C和环氧化酶的抑制剂，并没有消除胶原介导的作用。相反，用蛋白酪氨酸激酶抑制剂染料木黄酮处理细胞，但不是赫曲霉素A，剂量依赖性地消除了胶原对PTH/PTHrP受体、ALP和OP mRNA表达的影响。这些结果表明，I型胶原底物在成熟成骨细胞的细胞模型中影响成骨细胞相关基因的表达，并表明这至少部分涉及细胞内酪氨酸磷酸化的变化。
