OSTEOBLAST MEDIATED TURNOVER OF COLLAGENASE IN BONE

成骨细胞介导骨中胶原酶的周转

• 批准号: 3161091
• 负责人: Nicola C Partridge
• 金额: $ 14.28万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-01 至 1995-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3161091
• 关键词: collagenase; crosslink; hormone receptor; laboratory rat; ligands; osteoblasts; osteopetrosis; osteosclerosis; parathyroid hormones; pathologic bone resorption; protein sequence; tissue /cell culture

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.

在骨骼的一些遗传性疾病中，包括骨腐症和 骨硬化，过剩的骨积聚，而在骨质疏松症中，骨丢失 大大超过了骨骼的增长。从历史上看，骨形成是 与成骨细胞和破骨细胞的吸收有关。 然而，积累的数据表明，成骨细胞采取了 在吸收过程中发挥积极作用。该单元格已被显示为 主要目标为各种再吸收过程。这间牢房一直是 显示为各种再吸收试剂的主要目标(例如， 甲状旁腺激素、甲状旁腺激素和1，25(OH)2D3)，并能够产生 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，甲状旁腺素能刺激后一种酶的分泌。 最大浓度的大鼠成骨细胞系UMR 106-01 加入12-24小时后C‘酶出现在细胞外培养基中 荷尔蒙。随后，这些下降在96年变得几乎不可察觉 H.进一步的实验表明，之前的消失 分泌酶是由细胞介导的，提示是膜的作用。 C‘ase受体。我们现在已经证明了这样一个 UMR细胞上的受体，可能与已报道的丝氨酸类似 蛋白酶。它们似乎负责激活和/或 分泌酶的内吞作用。这导致我们制定了 假设成骨细胞上的C‘ase受体参与了 调节细胞外骨C‘酶的量，并下调- 对这种部分的调节(例如，通过用PTH处理细胞)可以 决定细胞外酶的丰度和功能 周围的环境。因此，本提案的目的是进一步说明 并检验这一假设。这些目标将会实现 通过：1)描述结合反应的特征，包括 甲状旁腺激素后受体数目或亲和力是否改变 治疗，2)评估受体在细胞介导的 配体、C‘酶的周转，以及受体是否发生变化 修改了这一过程，3)确定了 受体，既通过将~(125)I标记的C‘ase与受体交联，也通过 125I-标记受体，最后，4)使用该信息和 纯化受体和获得多肽序列数据的技术。这个 这项工作的结果应该有助于确定多个地点 精制C‘ase对成骨细胞的调节作用 该细胞在重塑过程中的全面作用(两者都是基质 合成和降解)。在这样做的时候，数据还应该提供一些 洞察许多骨骼疾病，其中骨骼重塑(？ 异常C‘ase表达/摄取)出了问题。