Molecular mechanisms of lysosome secretion in osteoclasts and bone Homeostasis

破骨细胞溶酶体分泌与骨稳态的分子机制

基本信息

批准号：
10449976
负责人：
HAIBO ZHAO
金额：
$ 29.91万
依托单位：
SOUTHERN CALIFORNIA INST FOR RES/EDUC
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10449976
关键词：
3-Dimensional Actins Adaptor Signaling Protein Affect American Amino Acids Arthritis Attenuated Automobile Driving Binding Biology Bone Diseases Bone Matrix Bone Resorption Bone remodeling Cell physiology Cells Complex Cytokine Receptors Cytoskeleton Defect Docking Dynein ATPase Event Fracture Goals Hand Homeostasis Human Hydrochloric Acid In Vitro Integrin Signaling Pathway Integrins Kinesin Light Link Lysosomal Storage Diseases Lysosomes MAP3K7 gene Macrophage Colony-Stimulating Factor Mammalian Cell Mediating Metabolic Bone Diseases Microscopic Microscopy Microtubule-Associated Proteins Microtubules Molecular Molecular Motors Monitor Mouse Strains Mus Mutation Organ Organelles Osteoclasts Osteoporosis Pathway interactions Phosphorylation Play Positioning Attribute Process Proteins Public Health Rattus Resolution Role SRC gene Seminal Serine Signal Transduction TNFSF11 gene Techniques Testing Transportation Tubulin Tyrosine Work base bone bone loss bone mass bone resorbing activity cathepsin K cell type cytokine in vivo knock-down novel novel therapeutics phosphoproteomics protein function real-time images seal substantia spongiosa therapeutic candidate therapeutic target trafficking two-photon

项目摘要

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.

项目概要破骨细胞酸化再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）包围的褶皱边界。 RANKL 是一个开创性的破骨细胞分化的细胞因子。此外，RANKL 直接刺激成熟骨的骨吸收活性。破骨细胞与免疫受体和整合素信号通路协同作用。虽然分子机制调节破骨细胞的细胞骨架和肌动蛋白环的形成已被充分了解，破骨细胞如何激活信号在骨吸收过程中刺激溶酶体分泌以及溶酶体如何与骨结合并沿骨运输微管是完全未知的。我们和其他人的工作表明 PLEKHM1 作为破骨细胞细胞内溶酶体运输和分泌的不可或缺的调节剂。通过磷酸化蛋白质组学，我们发现 PLEKHM1 S491 和 Y991 可能被 RANKL 或免疫受体信号传导磷酸化，并且 c-Src 分别对破骨细胞骨吸收至关重要。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 在溶酶体中与 DEF8 和 RAB7 形成三元复合物。 PLEKHM1 还与 FAM98A 和 NDEL1 相互作用在破骨细胞中。 FAM98A 具有保守的微管蛋白结合域，可介导转运蛋白的对接将货物或细胞器装载到微管上； NDEL1 与细胞质动力蛋白和/或驱动蛋白相互作用促进细胞器沿微管运输。基于这些前提，我们假设来自 RANKL、M-CSF、免疫受体和/或整合素的破骨细胞激活信号刺激溶酶体分泌，独立于其在肌动蛋白环形成中的作用，通过磷酸化和激活普莱克姆1。活性 PLEKHM1 与微管相关蛋白 FAM98A 和 NDEL1 相互作用，从而链接溶酶体形成微管并促进溶酶体运输至褶皱边界。 FAM98A 和 NDEL1 在体内骨吸收和骨重塑中发挥重要作用。为了检验这些相互关联的假设，我们将 (a) 定义磷酸化和激活 PLEKHM1 的信号级联以及它们如何刺激破骨细胞中的溶酶体分泌（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。（目标2）。顺利完成拟议的工作应该增进我们对破骨细胞生物学关键方面的理解，即 RANKL、M-CSF、免疫受体和/或整合素信号在溶酶体分泌中的特定作用以及破骨细胞中微管依赖性溶酶体运输的分子基础。鉴于有证据表明 PLEKHM1 对于破骨细胞中的溶酶体运输是不可或缺的，但在其他细胞类型中则不然。 PLEKHM1 及其相互作用蛋白调节溶酶体分泌的分子机制可能确定代谢性骨疾病的破骨细胞特异性候选治疗靶点。