The Role of the Osteoblast Secretome in Bone Formation

成骨细胞分泌组在骨形成中的作用

• 批准号: 7680844
• 负责人: HAIBO ZHAO
• 金额: $ 7.56万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-11 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7680844
• 关键词: Biology; Candidate Disease Gene; Cells; Core Facility; Coupled; Exocytosis; Feasibility Studies; Future; Genes; Goals; Hand; In Vitro; Lysosomes; Maintenance; Mass Spectrum Analysis; Mediating; Medicine; Modeling; Molecular; Molecular Analysis; Mus; Musculoskeletal; Mutation; Osteoblasts; Osteoclasts; Osteogenesis; Positioning Attribute; Process; Production; Proteins; Proteomics; RNA Interference; Research; Resistance; Role; Secretory Vesicles; Structure; Testing; Transcriptional Regulation; Universities; Vesicle; Viral; Washington; bone; bone mass; calcification; cell type; genetic regulatory protein; in vivo; insight; mutant; novel; osteoactivin; osteoblast differentiation; secretory protein; small hairpin RNA; tool

Maintenance of healthy bone requires the coupled activities of osteoclasts (OCs), which resorb the organic and inorganic components of bone, and osteoblasts (OBs), cells responsive for production and calcification of matrix proteins. Both cell types execute their function by regulated exocytosis, a process in which specialized vesicles are targeted to specific cell domains, leading to polarized secretion. Thus, we hypothesize that secretory vesicle proteins of the OB are important for OB function and maintenance of bone mass. In contrast to recent insights into transcriptional regulation of osteoblast differentiation, the mechanisms by which osteoblasts secrete matrix proteins to form bone are largely unknown. As a Pilot and Feasibility study, our goal is to identify the molecular mechanisms by which secretory vesicle proteins regulate osteoblast function. In this regard, we have developed experimental tools enabling us to 1) immunopurify lysosomes and thus determine their protein content by mass spectrometry, 2) knockdown endogenous candidate secretory genes using lenti- and retro- viral-mediated transduction of shRNA and rescue it with an RNAi-resistant mutant. We are thus in a position to a) identify novel secretory vesicle proteins in OBs, and b) test their role in secretion and bone formation in vitro. While these proposed studies will identify a number of candidate regulatory proteins in OBs, one, osteoactivin (OA) is presently in hand. OA is expressed in OCs and OBs, localizes in lysosomes in both cell types and regulates bone formation and resorption, in vitro. Moreover, a murine model of OA mutation is already available. Thus, our specific aim is A) to identify osteoblast secretory vesicle proteins by mass-spectrometry and to characterize their bone forming functions by shRNA-mediated gene knockdown in vitro, and B) in parallel, to characterize the role of the candidate OB secretory protein OA in bone formation in vivo using an existing murine model. The latter study will be done with assistance from two core facilities in the Washington University Core center for Musculoskeletal Biology and Medicine, namely Core B-Musculoskeletal Structure and Strength, and Core C-ln situ Molecular Analysis, Following completion of these studies, we will be in a position to propose in vivo functional studies of additional candidate genes identified by our OB proteomics analysis as part of a future R01 proposal.

维持健康的骨骼需要破骨细胞 (OC) 的联合活动，破骨细胞可吸收有机骨 骨的无机成分，以及成骨细胞（OB），对生产和钙化有反应的细胞 基质蛋白。两种细胞类型都通过调节胞吐作用来执行其功能，该过程中 专门的囊泡针对特定的细胞域，导致极化分泌。因此，我们 假设 OB 的分泌囊泡蛋白对于 OB 功能和维持很重要 骨量。与最近对成骨细胞分化的转录调控的见解相反， 成骨细胞分泌基质蛋白形成骨的机制在很大程度上尚不清楚。作为飞行员和 可行性研究，我们的目标是确定分泌囊泡蛋白的分子机制 调节成骨细胞功能。在这方面，我们开发了实验工具，使我们能够 1) 免疫纯化 溶酶体，从而通过质谱法测定其蛋白质含量，2) 敲低内源性 使用慢病毒和逆转录病毒介导的 shRNA 转导来寻找候选分泌基因，并用 RNAi 抗性突变体。因此，我们能够 a) 鉴定 OB 中的新型分泌囊泡蛋白，以及 b) 测试它们在体外分泌和骨形成中的作用。虽然这些拟议的研究将确定一些 OB 中的候选调节蛋白之一，骨激活素（OA）目前已在手。 OA 用 OC 表示 和 OB，定位于两种细胞类型的溶酶体中，并在体外调节骨形成和吸收。 此外，OA 突变的小鼠模型已经可用。因此，我们的具体目标是 A) 确定 通过质谱分析成骨细胞分泌囊泡蛋白并表征其骨形成 通过 shRNA 介导的体外基因敲低发挥功能，并且 B) 平行地表征 使用现有的小鼠模型，研究了候选 OB 分泌蛋白 OA 在体内骨形成中的作用。 后一项研究将在华盛顿大学核心中心的两个核心设施的协助下完成 肌肉骨骼生物学和医学，即核心 B-肌肉骨骼结构和力量，以及核心 C-原位分子分析，完成这些研究后，我们将能够提出体内 我们的 OB 蛋白质组学分析确定的其他候选基因的功能研究是未来的一部分 R01提案。