Molecular mechanisms of lysosome secretion in osteoclasts and bone Homeostasis

项目摘要 破骨细胞酸化骨吸收微环境，并通过分泌 溶酶体穿过由紧密密封区（肌动蛋白环）限定的皱褶边界。RANKL是一种开创性的 破骨细胞分化的细胞因子。此外，RANKL直接刺激骨吸收活性的成熟 破骨细胞与免疫受体和整联蛋白信号传导途径一致。虽然分子机制 调节破骨细胞细胞骨架和肌动蛋白环的形成是很好理解的，破骨细胞激活信号如何 刺激骨吸收过程中的溶酶体分泌，以及溶酶体如何与骨吸收蛋白连接并沿骨吸收蛋白沿着运输。 微管是完全未知的。我们和其他人的工作表明PLEKHM 1是一种 破骨细胞内溶酶体运输和分泌的不可缺少的调节剂。通过磷酸化蛋白质组学， 我们发现PLEKHM 1 S491和Y 991通过RANKL或免疫受体信号转导而磷酸化， c-Src分别是破骨细胞骨吸收的关键。此外，PLEKHM 1与TAK 1相互作用， RAP 1B-RANKL和整合素信号通路的两个关键分子。此外，我们发现PLEKHM 1 在溶酶体处与DEF 8和RAB 7形成三元复合物。PLEKHM 1还与FAM 98 A和NDEL 1相互作用， 在破骨细胞中。FAM 98 A具有一个保守的微管蛋白结合结构域，其介导转运蛋白的对接。 NDEL 1与细胞质动力蛋白和/或驱动蛋白相互作用 以促进细胞器沿微管沿着运输。基于这些前提，我们假设， 源自RANKL、M-CSF、免疫受体和/或整联蛋白的破骨细胞活化信号刺激 溶酶体分泌，独立于它们在肌动蛋白环形成中的作用，通过磷酸化和激活 PLEKHM 1.活性PLEKHM 1与微管相关蛋白FAM 98 A和NDEL 1相互作用，从而连接 溶酶体的微管和促进溶酶体运输到皱褶的边界。FAM 98 A和NDEL 1 在体内骨吸收和骨重建中起重要作用。为了验证这些假设，我们 将（a）定义磷酸化和激活PLEKHM 1的信号级联，以及它们如何刺激 破骨细胞中的溶酶体分泌（Aim 1）;（B）确定PLEKHM 1-FAM 98 A和PLEKHM 1-NDEL 1的作用 破骨细胞中溶酶体分泌的相互作用和小鼠骨稳态。（目标2）。成功完成 的建议工作应该推进我们的破骨细胞生物学的关键方面的理解，即 RANKL、M-CSF、免疫受体和/或整联蛋白信号传导在溶酶体分泌中的特异性作用， 破骨细胞中微管依赖性溶酶体转运的分子基础。鉴于有证据表明， PLEKHM 1对于破骨细胞中的溶酶体运输是不可缺少的，但在其他细胞类型中不是，阐明了破骨细胞中的溶酶体运输是不可缺少的。 PLEKHM 1及其相互作用蛋白调节溶酶体分泌的分子机制可以确定 破骨细胞特异性代谢性骨疾病的候选治疗靶点。

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.