Mechanisms of cytoskeletal and lysosomal protein regulation in osteoclasts

描述(申请人提供)：细胞骨架组织和溶酶体分泌对破骨细胞的激活和功能至关重要。然而，调控这些过程的分子机制却知之甚少。这项拨款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。PLEKHM1是最近在人和大鼠骨化症中发现的一种突变蛋白，它与破骨细胞溶酶体的运输和分泌密切相关。此外，Plekhm1被发现与真核细胞中微管动力学的关键调节因子Lis1结合。Lis1与dynein/dynactin相互作用，dynein/dynactin是一种调节微管动态和运输的运动复合体。Lis1还与PAF-AH(血小板激活因子(PAF)乙酰水解酶)1b复合体的催化1亚基结合，从而使PAF失活，PAF是破骨细胞生存和活动的重要脂质信使功能。Lis1还被证明通过调节M-CSF和RANKL信号来调节CDC42，这是一种小的GTP酶，是小鼠骨骼稳态所必需的。更重要的是，Lis1-Flox；LysM-Cre小鼠，其中Lis1在髓系细胞中特异性缺失，骨量增加，破骨细胞形成和骨吸收受损。这些数据导致假设，Plekhm1对骨骼动态平衡是必不可少的，Plekhm1/Lis1相互作用对溶酶体分泌和骨吸收至关重要。Lis1通过PAF和/或CDC42调节动力蛋白功能和M-CSF/RANKL信号通路，从而调节破骨细胞的形成和功能。为了验证这些假说，转基因小鼠和来自这些小鼠的破骨细胞将被用于：a)确定Plekhm1在破骨细胞功能中的作用，并确定介导Plekhm1/Lis1相互作用的机制(目标1)。B)确定Lis1是否调节破骨细胞的功能，并剖析Lis1调节破骨细胞微管组织和组织蛋白酶K分泌的机制(目标2)。C)确定Lis1是否调节破骨细胞的形成，并确定Lis1调节M-CSF和RANKL信号通路的分子机制(目标3)。 公共卫生相关性：破骨细胞活动增强会导致骨质流失，导致重大公共卫生问题。我们已经在破骨细胞中发现了有助于其活性的新蛋白质。我们计划确定这些蛋白质是如何调节破骨细胞的，这增加了我们能够为代谢性骨疾病的治疗提供新的治疗靶点的可能性。

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). PUBLIC HEALTH RELEVANCE: Enhanced osteoclast activity causes bone loss, leading to major public health problems. We have identified novel proteins in osteoclasts which contribute to their activity. We plan to determine how these proteins regulate the osteoclast, raising the possibility that we will be able to provide new therapeutic targets for the treatment of metabolic bone diseases.