OSTEOBLAST MEDIATED TURNOVER OF COLLAGENASE IN BONE

在一些遗传性骨骼疾病中，包括骨硬化症和 骨质疏松症，过量的骨积聚，而在骨质疏松症骨丢失 明显超过骨增量。 从历史上看，骨形成是 与成骨细胞相关，与破骨细胞相关。 然而，积累的数据表明，成骨细胞需要 在再吸收过程中发挥积极作用。 这个细胞已经被证明是 各种吸收过程的主要目标。 这间牢房 显示为多种再吸收剂的主要目标（例如， 甲状旁腺激素、PTH和1，25（OH）2D 3），并且能够产生 中性蛋白酶，如纤溶酶原激活剂和胶原酶（C 'ase）。 我们最近发现，甲状旁腺素刺激分泌后一种酶 大鼠成骨细胞系UMR 106-01，最大浓度 在加入以下物质后12-24小时， 荷尔蒙 这些随后下降到几乎无法检测到的96 H. 进一步的实验表明， 分泌的酶是细胞介导的，并建议膜的作用 C 'ase受体。 我们现在已经证明了这样一种 UMR细胞上的受体，可能类似于丝氨酸的那些报道 蛋白酶 这些似乎是负责激活和/或 分泌酶的内吞作用。 这促使我们制定了 假设成骨细胞上的C 'ase受体参与了 调节细胞外骨钙蛋白酶的数量， 调节这样的部分（例如，通过用PTH处理细胞）可以 决定了胞外酶的丰度和功能， 环境。 因此，本提案的目的是进一步说明 受体，并测试这一假设。 这些目标将会实现 通过：1）描述结合反应的特征，包括 确定PTH后受体数量或亲和力是否发生变化 治疗，2）评估受体在细胞介导的免疫应答中的作用， 转换的配体，C 'ase，以及是否在受体的改变 改变了这一过程，3）确定的生化特性的 通过将125 I标记的C '酶与受体交联， 125 I标记受体，最后，4）使用该信息， 纯化受体和获得肽序列数据的技术。 的 这项工作的结果应有助于确定多个网站的 调节成骨细胞的阐述C 'ase，并揭示了一些光 这种细胞在重塑过程中的综合作用（包括基质 合成和降解）。 在这样做的时候，这些数据也应该提供一些 深入了解许多骨骼疾病，其中骨重塑（？ 异常的C 'ase表达/摄取）已经出错。

In some inherited disorders of the skeleton, including osteopetrosis and osteosclerosis, excess bone accumulates whereas in osteoporosis bone loss significantly exceeds bone gain. Historically, bone formation was associated with the osteoblast and resorption with the osteoclast. However, data has accumulated which indicates that the osteoblast takes an active role in the resorption process. This cell has been shown to be the primary target for a variety of resorption process. This cell has been shown to be the primary target for a variety of resorption agents (e.g., parathyroid hormone, PTH, and 1, 25(OH)2D3) and to be capable of producing neutral proteases, such as plasminogen activator and collagenase (C'ase). We have recently shown that PTH stimulates secretion of the latter enzyme by the rat osteoblastic cell line, UMR 106-01, with maximal concentrations of C'ase appearing in the extracellular medium 12-24 h after addition of the hormone. These subsequently decline becoming almost undetectable by 96 h. Further experiments revealed that the disappearance of previously secreted enzyme was cell-mediated and suggested the action of a membrane receptor for C'ase. We have now demonstrated the existence of such a receptor on UMR cells which may be analogous to those reported for serine proteases. These appear to be responsible for either activation and/or endocytosis of the secreted enzyme. This has led us to formulate the hypothesis that the C'ase receptor on osteoblastic cells is involved in the regulation of the amounts of extracellular bone C'ase and that down- regulation of such a moiety (e.g., by treatment of cells with PTH) may dictate the abundance and function of the enzyme in the extracellular milieu. Thus, the aims of the present proposal are to further characterize the receptor and to test this hypothesis. These aims will be accomplished by: 1) delineating the characteristics of the binding reaction, including determining whether receptor number or affinity changes after PTH treatment, 2) assessing the role of the receptor in the cell-mediated turnover of the ligand, C'ase, and whether an alteration in the receptor modifies this process, 3) determining the biochemical properties of the receptor, both by cross-linking 125I-labelled C'ase to the receptor and by 125I-labelling the receptor, and finally, 4) using this information and techniques to purify the receptor and obtain peptide sequence data. The results of this work should aid in ascertaining the multiple sites of regulation in the osteoblast of elaborated C'ase and shed some light on the comprehensive role of this cell in the remodelling process (both matrix synthesis and degradation). In so doing, the data should also provide some insight into those many skeletal disorders where bone remodelling (? aberrant C'ase expression/uptake) has gone awry.