OSTEOCLASTIC PHOSPHOTYROSYL PHOSPHATASE AND RESORPTION

破骨细胞磷酸酪酰磷酸酶和吸收

• 批准号: 6379908
• 负责人: Kin-Hing William Lau
• 金额: $ 26.9万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6379908
• 关键词: SDS polyacrylamide gel electrophoresis; antisense nucleic acid; autoradiography; bone development; computer data analysis; densitometry; enzyme activity; gene expression; human tissue; immunoprecipitation; laboratory rabbit; oligonucleotides; osteoclasts; phosphorylation; physiologic bone resorption; polymerase chain reaction; protein structure function; protein tyrosine phosphatase; scintillation spectrometry; statistics /biometry; western blottings

Bone resorption, mediated by osteoclasts, is essential for bone growth and repair. Past c-src knockout mice studies have indicated that the protein tyrosine kinase (PTK) activity of c-src is essential for mature osteoclastic activity. The c-src PTK activity is regulated predominately by the phosphorylation status of a C-terminal tyr-527 residue: phosphorylation inactivates, and dephosphorylation activates, c-src PTK activity. The phosphorylation is catalyzed by a cytosolic PTK, Csk, but the identity of the phosphotyrosine phosphatase (PTP) activity that mediates the dephosphorylation is unknown. We have recently cloned a structurally unique PTP, termed PTP-oc, from rabbit osteoclast cDNA library. It is expressed preferentially in osteoclasts and precursors. We have a large body of preliminary data to support that PTP-oc may dephosphorylate c-src and may regulate c-src PTK activity in osteoclasts. These findings led us to formulate a working model, in which PTP-oc acts to dephosphorylate tyr-527 of c-src in osteoclasts, resulting in activation of its PTK activity, which in turn leads to activation of osteoclast activity and resorption. We further speculate that some bone resorption activators may stimulate osteoclastic resorption through an increase in the expression and/or the activity of PTP-oc in osteoclasts; whereas some inhibitors may reduce osteoclastic resorption by reducing the PTP-oc expression and/or activity. This application will test three specific hypotheses: 1) suppression of PTP- oc expression in osteoclasts increases c-src tyr-527 phosphorylation, reduces c-src PTK activity, decreases their resorptive activity, and impairs actions of some resorption activators on their c-src PTK and resorptive activities; 2) overexpression of PTP-oc in osteoclasts reduces tyr-527 phosphorylation of c-src, stimulates c-src PTK activity, increases their resorptive activity, and reduces actions of some resorption inhibibitors on their c-src PTK and resorptive activities; 3) overexpression of an inactive PTP-oc mutant in osteoclasts reduces c-src tyr-527 dephosphorylation, leading to a reduction in c-src PTK activity and a decrease in their osteoclast activity; and it also suppresses the actions of some resorption activators on their c-src PTK and resorptive activities. The first hypothesis will be tested in primary rabbit osteoclasts by using specific PTP-oc antisense oligodeoxynucleotides to suppress PTP-oc expression. The other two hypotheses will be evaluated by stably transfecting either a wild type or a "phosphatase-dead" PTP-oc inducible vector in a human U937/ osteoclast cell model. The stable clones will be induced to overexpress either the wild-type or mutant PTP-oc for evaluation. If our hypotheses are correct, this work would provide insights into a) molecular mechanism of osteoclastic resorption, b) regulation of osteoclast activity, and c) the role of PTP-oc in bone resorption. PTP-oc may also be used as a screening target to identify new agents that alter bone resorption, and as such this study may identify a potential therapeutic target for development of more specific and safer modulators of bone resorption.

破骨细胞介导的骨吸收是骨生长所必需的 并进行修复。过去对c-src基因敲除小鼠的研究表明 C-src的蛋白酪氨酸激酶活性是成熟所必需的。 破骨活性。C-src ptk活性受调控 主要由C-末端Tyr-527的磷酸化状态决定 残基：磷酸化失活，去磷酸化激活， C-src PTK活性。磷酸化是由胞浆中的PTK催化的， CSK，但磷酸酪氨酸磷酸酶(PTP)活性相同 目前还不清楚是什么参与了去磷酸化过程。我们最近克隆了 兔破骨细胞中一种结构独特的PTP，命名为PTP-oc 图书馆。它在破骨细胞和前体细胞中优先表达。 我们有大量的初步数据支持PTP-oc可能 去磷酸化c-src并可能调节c-src PTK活性 破骨细胞。这些发现使我们制定了一个工作模型，在 其中PTP-oc作用于去磷酸化破骨细胞中c-src的Tyr-527， 导致其PTK活性激活，进而导致 激活破骨细胞活性和吸收。我们进一步推测 一些骨吸收激活剂可能刺激破骨细胞 通过增加表达和/或活性而发生的吸收 破骨细胞中的PTP-oc；而一些抑制剂可能会减少破骨细胞 通过减少PTP-oc的表达和/或活性来吸收。这 应用程序将检验三个具体的假设：1)抑制PTP- 破骨细胞中OC的表达增加了c-src Tyr-527的磷酸化， 降低c-src ptk活性，降低其吸收活性，以及 削弱某些吸收激活剂对其c-src PTK和 2)破骨细胞中PTP-oc的过表达 减少c-src的Tyr-527磷酸化，刺激c-src的PTK活性， 增加他们的吸收活动，并减少一些人的行为 吸收抑制剂对其c-src PTK和吸收活性的抑制作用； 3)在破骨细胞中过表达失活的ptp-oc突变体可减少 C-src Tyr-527去磷酸化导致c-src PTK减少 活性和破骨细胞活性下降；它还 抑制某些吸收激活剂对其c-src ptk的作用。 和再吸收活动。第一个假设将在#年检验。 特异性PTP-oc反义寡核苷酸原代培养兔破骨细胞 抑制PTP-oc表达的寡核苷酸。另外两个 假说将通过稳定地感染野生型进行评估。 或人U937/pTP-oc诱导载体中的“磷酸酶死亡” 破骨细胞模型。稳定克隆将被诱导过度表达 野生型或突变型ptp-oc用于评估。如果我们的假设 是正确的，这项工作将提供对)分子的洞察 破骨细胞吸收的机制，b)破骨细胞的调节 活性，以及c)PTP-oc在骨吸收中的作用。PTP-OC还可以 被用作筛选目标，以确定改变骨骼的新制剂 吸收，因此这项研究可能确定一种潜在的治疗方法 开发更特异和更安全的骨调节剂的目标 再吸收。