Molecular mechanisms of lysosome secretion in osteoclasts and bone Homeostasis

项目总结 破骨细胞可酸化骨吸收微环境，并通过分泌 溶酶体穿过褶皱的边缘，由紧密的密封区(肌动蛋白环)包围。RANKL是一个开创性的 破骨细胞分化的细胞因子。此外，RANKL还可直接刺激成熟者的骨吸收活动 破骨细胞与免疫受体和整合素信号通路协调。而分子机制 调控破骨细胞细胞骨架和肌动蛋白环的形成是很清楚的，破骨细胞是如何激活信号的 在骨吸收过程中刺激溶酶体的分泌，以及溶酶体是如何连接到骨吸收区域并沿骨吸收区域运输的 微管是完全未知的。我们和其他人的研究表明，PLEKHM1是一种 破骨细胞内溶酶体运输和分泌的不可或缺的调节因子。通过磷酸蛋白质组学， 我们发现PLEKHM1 S491和Y991可能被RANKL或免疫受体信号和 C-Src分别对破骨细胞的骨吸收起关键作用。此外，PLEKHM1与TAK1相互作用， RAP1B-RANKL和整合素信号通路的两个关键分子。此外，我们发现PLEKHM1 在溶酶体上与DEF8和RAB7形成三元络合物。PLEKHM1还与FAM98A和NDEL1相互作用 在破骨细胞中。FAM98A有一个保守的微管蛋白结合域，它介导了运输的 在微管上的货物或细胞器；NDEL1与细胞质动力蛋白和/或动力蛋白相互作用 以促进细胞器沿微管的运输。基于这些前提，我们假设 来自RANKL、M-CSF、免疫受体和/或整合素刺激的破骨细胞激活信号 溶酶体的分泌，与它们在肌动蛋白环形成中的作用无关，通过磷酸化和激活 PLEKHM1。活性PLEKHM1与微管相关蛋白FAM98A和NDEL1相互作用，从而连接 溶酶体到微管，并促进溶酶体运输到褶皱的边界。FAM98A和NDEL1 在体内的骨吸收和骨重建中起重要作用。为了检验这些相互关联的假设，我们 将(A)定义使PLEKHM1磷酸化和激活的信号级联以及它们如何刺激 破骨细胞溶酶体分泌(Aim1)；(B)确定PLEKHM1-FAM98A和PLEKHM1-NDEL1的作用 破骨细胞溶酶体分泌与小鼠骨稳态的相互作用。(AIM2)。成功完成 建议的工作应该促进我们对破骨细胞生物学的关键方面的理解，即 RANKL、M-CSF、免疫受体和/或整合素信号在溶酶体分泌和 破骨细胞微管依赖的溶酶体转运的分子基础。鉴于有证据表明 PLEKHM1对于溶酶体在破骨细胞中的运输是必不可少的，但在其他类型的细胞中不是。 PLEKHM1及其相互作用蛋白调节溶酶体分泌的分子机制可能识别 针对代谢性骨病的破骨细胞特异性候选治疗靶点。

PROJECT SUMMARY Osteoclasts acidify the resorptive microenvironment and digest the organic matrix of bone via secretion of lysosomes through the ruffled border circumscribed by a tight sealing zone (actin ring). RANKL is a seminal cytokine for osteoclast differentiation. In addition, RANKL directly stimulates the bone-resorbing activity of mature osteoclasts in concert with immunoreceptor and integrin signaling pathways. While the molecular mechanisms regulating osteoclast cytoskeleton and actin-ring formation are well understood, how osteoclast activating signals stimulate lysosome secretion during bone resorption and how lysosomes are linked to and transported along the microtubules are completely unknown. The work from us and others has implicated PLEKHM1 as an indispensable regulator of intracellular lysosome trafficking and secretion in osteoclasts. By phospho-proteomic, we found that PLEKHM1 S491 and Y991, putatively phosphorylated by RANKL or immunoreceptor signaling and c-Src, respectively, are critical for osteoclast bone resorption. Moreover, PLEKHM1 interacts with TAK1 and RAP1B - two key molecules of the RANKL and integrin signaling pathways. Additionally, we found that PLEKHM1 forms a ternary complex with DEF8 and RAB7 at lysosomes. PLEKHM1 also interacts with FAM98A, and NDEL1 in osteoclasts. FAM98A has a conservative tubulin-binding domain that mediates the docking of transported cargoes or organelles onto the microtubules; and that NDEL1 interacts with cytoplasmic dynein and/or kinesins to promote organelle transport along the microtubules. Based on these lines of premise, we hypothesize that osteoclast activating signals derived from RANKL, M-CSF, immunoreceptors, and/or integrins stimulate lysosome secretion, independently of their role in actin ring formation, by phosphorylating and activating PLEKHM1. Active PLEKHM1 interacts with microtubule-associate proteins FAM98A and NDEL1, thereby, links lysosomes to microtubules and facilitates lysosome transportation to the ruffled border. FAM98A and NDEL1 play an important role in bone resorption and bone remodeling in vivo. To test these interrelated hypotheses, we will (a) Define the signaling cascades that phosphorylate and activate PLEKHM1 and how they stimulate lysosome secretion in osteoclasts (Aim1); (b) Determine the role of PLEKHM1-FAM98A and PLEKHM1-NDEL1 interactions in lysosome secretion in osteoclasts and bone homeostasis in mice. (Aim2). Successful completion of the proposed work should advance our understanding of critical aspects of osteoclast biology, namely the specific role of RANKL, M-CSF, immunoreceptors, and/or integrin signaling in lysosome secretion and the molecular basis of microtubule-dependent lysosome transportation in osteoclasts. In view of the evidence that PLEKHM1 is indispensable for lysosome trafficking in osteoclasts, but not in other cell types, elucidation of the molecular mechanisms by which PLEKHM1 and its interacting proteins regulate lysosome secretion may identify osteoclast-specific candidate therapeutic targets for metabolic bone diseases.