Mechanisms of Cytoskeletal and Lysosomal Protein Regulation in Osteoclasts

描述（由申请方提供）：细胞骨架组织和溶酶体分泌对于破骨细胞活化和功能至关重要。然而，调控这些过程的分子机制知之甚少。本基金申请的目的是阐明细胞骨架和溶酶体蛋白调节破骨细胞的机制。PLEKHM 1是一种新发现的在人和大鼠石骨症中发生突变的蛋白质，它与破骨细胞溶酶体的运输和分泌密切相关。此外，Plekhm 1被发现与LIS 1结合，LIS 1是真核细胞中微管动力学的关键调节因子。LIS 1与动力蛋白/动力蛋白相互作用，动力蛋白/动力蛋白是一种调节微管动力学和运输的运动复合物。LIS 1还结合PAF-AH（血小板活化因子（PAF）乙酰水解酶）1b复合物的催化1-亚基，其使PAF失活，PAF是一种对破骨细胞存活和活动具有重要功能的脂质信使。LIS 1还显示出通过调节M-CSF和RANKL信号传导来调节cdc 42，cdc 42是小鼠中骨稳态所需的小GT3。更重要的是，LIS 1-flox;LysM-Cre小鼠，其中LIS 1在骨髓细胞中特异性缺失，具有增加的骨量和受损的破骨细胞形成和骨吸收。这些数据导致的假设，Plekhm 1是必不可少的骨骼稳态和Plekhm 1/LIS 1相互作用是至关重要的溶酶体分泌和骨吸收。LIS 1通过PAF和/或cdc 42调节动力蛋白功能和M-CSF/RANKL信号通路来调节破骨细胞的形成和功能。为了检验这些假设，将使用遗传修饰小鼠和来自这些小鼠的破骨细胞：a）确定Plekhm 1在破骨细胞功能中的作用，并鉴定介导Plekhm 1/LIS 1相互作用的机制（目的1）。B）确定LIS 1是否调节破骨细胞功能，并剖析LIS 1调节破骨细胞中微管组织和组织蛋白酶K分泌的机制（目的2）。c）确定LIS 1是否调节破骨细胞生成，并确定LIS 1调节M-CSF和RANKL信号通路的分子机制（目的3）。

DESCRIPTION (provided by applicant): Cytoskeleton organization and lysosome secretion are critical for osteoclast activation and function. However, the molecular mechanisms regulating these processes are poorly understood. The goal of this grant application is to elucidate the mechanisms by which the cytoskeletal and lysosomal proteins regulate osteoclasts. PLEKHM1, a newly identified protein mutated in human and rat osteopetrosis, is critically involved in osteoclast lysosome trafficking and secretion. Furthermore, Plekhm1 was found to bind to LIS1, a key regulator of microtubule dynamic in eukaryotic cells. LIS1 interacts with dynein/dynactin, a motor complex that regulates microtubule dynamic and transportation. LIS1 also binds to the catalytic 1-subunit of PAF-AH (platelet-activating factor (PAF) acetylhydrolase) 1b complex, which inactivates PAF, a lipid messenger functional important for osteoclast survival and activities. LIS1 has also been shown to regulate cdc42, a small GTPase that is required for bone homeostasis in mice by modulating M-CSF and RANKL signaling. More importantly, LIS1-flox;LysM-Cre mice, in which LIS1 is specifically deleted in myeloid cells, have increased bone mass and impaired osteoclast formation and bone resorption. These data led to the hypothesis that Plekhm1 is essential for skeleton homeostasis and Plekhm1/LIS1 interaction is critical for lysosome secretion and bone resorption. LIS1 regulates osteoclast formation and function through its modulation of dynein function and M-CSF/RANKL signaling pathways via PAF and/or cdc42. To test these hypotheses, genetically modified mice and osteoclasts derived from these mice will be used to: a) determine the role of Plekhm1 in osteoclast function and identify the mechanisms mediating Plekhm1/LIS1 interaction (Aim 1). b) determine whether LIS1 regulates osteoclast function and dissect the mechanisms by which LIS1 regulates microtubule organization and Cathepsin K secretion in osteoclasts (Aim 2). c) determine whether LIS1 regulates osteoclastogenesis and define the molecular mechanisms by which LIS1 modulates M-CSF and RANKL signaling pathways (Aim 3).