Pmepa1调控破骨细胞溶酶体运输对骨吸收的作用及机制研究

Increased bone resorption is one cause of osteoporosis. Bone resorption is performed by osteoclasts which transport lysosome to secret acid enzyme like cathepsin K (Catk) and hydrochloride acid on bone surface. However, the mechanism of lysosome transport in osteoclasts is still unclear. Previously, we found Prostate transmembrane protein androgen induced 1(Pmepa1) was expressed in osteoclasts and its protein was localized to lysosome. Pmepa1 was also found to regulate bone resorption via secretion of Catk. In further analysis, we found inhibition of Pmepa1 expression reduced the secretion hydrochloride in osteoclasts, and impaired transport of lysosome in osteoclasts. We also found that Pmepa1 was co-localized with the dynactin 5 and dynactin 6 from dynein-dynactin complex which are the main regulator of lysosome transport. From these analyses, we hypothesize that Pmepa1 would bind to dynactin 5 and dynactin 6 to modulate transport of lysosome to bone surface to perform bone resorption. This project will show the mechanism of Pmepa1 regulating transport of lysosome and the role in bone resorption in osteoclasts via western blot, immunoprecipitation，osteoporosis animal model and using lenti-virus. This project will provide the mechanism of Pmepa1 modulating lysosome transport and the role of Pmepa1 in osteoporosis. And this project would also provide a new way and scientific evidence to treat osteoporosis.

骨吸收增强是骨质疏松症(OP)发病的重要原因之一。破骨细胞(OC)通过运输溶酶体至骨组织表面后分泌组织蛋白酶K(Catk)和盐酸进行骨吸收，而调控溶酶体运输的机制尚未明确。我们已证实，跨膜前列腺雄激素诱导蛋白1(Pmepa1)在OC中高表达，且主要定位于溶酶体，通过调控Catk的分泌影响骨吸收活动。我们进一步研究发现：下调Pmepa1的表达抑制了盐酸的释放及溶酶体的运输，促使骨吸收减少；Pmepa1与调控溶酶体运输的关键调节因子-动力蛋白复合物中的dynactin5和6亚基共定位。由此，我们提出假设：OC中Pmepa1通过与动力蛋白复合物的dynactin5和6亚基结合，调控溶酶体的运输，促进骨吸收。本项目拟在前期研究基础上，通过免疫印迹、慢病毒感染、免疫沉淀、动物实验等探讨Pmepa1调控溶酶体运输的具体分子机制及对骨吸收的作用，旨在为OP的防治提供新的思路和科学依据。

破骨细胞的骨吸收活动增强是导致骨质疏松症的重要原因之一。溶酶体是参与骨吸收活动的重要细胞器。然而，溶酶体在骨吸收互动过程中的发挥的相关作用及其机制尚不明确。我们在项目前期发现，跨膜前列腺雄激素诱导蛋白1(Pmepa1)在破骨细胞中高表达，且主要定位于溶酶体；在破骨细胞中下调Pmepa1的表达抑制了骨吸收互动，并影响了溶酶体的运输。我们在本项目中拟探讨Pmepa1调控溶酶体运输的具体分子机制及对骨吸收的作用，并揭示相关机制。我们在本项目中发现，与对照组相比，Pmepa1基因敲除小鼠的骨量明显增加；在体外破骨细胞中敲除Pmepa1基因，明显抑制了骨吸收活动，减少了氢离子的分泌，并影响了溶酶体在破骨细胞中的分布。以上结果提示，Pmepa1通过调控溶酶体的移动，参与骨吸收活动。为了进一步探究Pmepa1调控溶酶体移动的机制，我们探讨了Rab相互作用的溶酶体蛋白（RILP）的功能。我们发现在破骨细胞中，我们发现在破骨细胞中，Pmepa1与RILP相互作用，并推测Pmepa1可能与RILP相互作用调控溶酶体在破骨细胞中的分布。为了证实推测，我们首先验证了RILP在破骨细胞中的功能。我们发现在破骨细胞中，抑制RILP基因的表达减少了破骨细胞的形成，抑制了骨吸收活动。而且，在破骨细胞中抑制RILP的表达，溶酶体的分布也发生了变化。此外，在体内抑制RILP的表达，减少了骨吸收的面积。以上结果提示，在破骨细胞中Pmepa1是调控溶酶体分布的关键蛋白，并且通过调控溶酶体分布参与骨吸收活动。而且，Pmepa1可能是与RILP相互作用调控溶酶体的移动。本项目进一步揭示了破骨细胞骨吸收活动的相关机制，并且为下一步探讨Pmepa1与RILP调控溶酶体分布的研究，奠定了坚实的基础。