OSTEOCLASTIC PHOSPHOTYROSYL PHOSPHATASE AND RESORPTION

破骨细胞磷酸酪酰磷酸酶和吸收

• 批准号: 2822675
• 负责人: Kin-Hing William Lau
• 金额: $ 26.61万
• 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/2822675
• 关键词: SDS polyacrylamide gel electrophoresis; antisense nucleic acid; autoradiography; bone development; 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 和修复。过去的c-src基因敲除小鼠研究表明， c-src的蛋白酪氨酸激酶（PTK）活性是成熟的 骨细胞活性 c-src PTK活性受到调控 主要通过C-末端tyr-527的磷酸化状态 残基：磷酸化失活，去磷酸化活化， c-src PTK活性。磷酸化由胞质PTK催化， Csk，但磷酸酪氨酸磷酸酶（PTP）活性的同一性 介导去磷酸化的蛋白质是未知的。 我们最近克隆了 一种结构独特的PTP，称为PTP-oc，来自兔破骨细胞cDNA 图书馆 它优先在破骨细胞和前体细胞中表达。 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， 该PTP-a用于使破骨细胞中c-src的tyr-527去磷酸化， 导致其PTK活性的激活，这反过来又导致 破骨细胞活性和再吸收的活化。 我们进一步推测 一些骨吸收激活剂可能刺激骨吸收， 通过增加的表达和/或活性的吸收 破骨细胞中的PTP-oc;而一些抑制剂可能会减少破骨细胞 通过降低PTP-α的表达和/或活性来抑制再吸收。 这 应用程序将测试三个特定的假设：1）抑制PTP- 破骨细胞中的OC表达增加c-Src Tyr-527磷酸化， 降低c-src PTK活性，降低其再吸收活性，和 损害一些再吸收激活剂对其c-src PTK的作用， 骨吸收活性; 2）破骨细胞中PTP-α的过度表达 减少c-src的tyr-527磷酸化，刺激c-src PTK活性， 增加它们的吸收活性，并减少一些 再吸收抑制剂对其c-src PTK和再吸收活性的影响; 3)破骨细胞中失活的PTP-oc突变体的过度表达降低了 c-src tyr-527去磷酸化，导致c-src PTK减少 活性和破骨细胞活性的降低;而且 抑制某些再吸收激活剂对其c-src PTK的作用 和再吸收活动。 第一个假设将在 特异性PTP-oc反义核酸诱导兔原代破骨细胞分化 寡脱氧核苷酸抑制PTP-oc表达。 另外两 假设将通过稳定地检测野生型 或"磷酸酶死亡"的PTP-α诱导型载体在人U937/ 破骨细胞模型。 稳定的克隆将被诱导过表达 用于评估的野生型或突变型PTP-α。 如果我们的假设 是正确的，这项工作将提供深入了解a）分子 骨细胞再吸收的机制，B）破骨细胞的调节 活性，和c）PTP-α在骨吸收中的作用。 PTP-α还可以 用作筛选靶点，以鉴定改变骨的新试剂 因此，这项研究可能会确定一种潜在的治疗方法， 开发更特异和更安全骨调节剂的目标 再吸收