The Role of the Osteoblast Secretome in Bone Formation

健康骨骼的维持需要破骨细胞（OC）的偶联活性，破骨细胞（OC）再吸收有机质， 和骨的无机成分，以及成骨细胞（OBs），对生产和钙化反应的细胞 基质蛋白质。这两种细胞类型都通过调节胞吐作用来执行其功能，在该过程中， 专门的囊泡靶向特定的细胞结构域，导致极化分泌。因此我们 假设OB分泌囊泡蛋白对于OB功能和维持是重要的 的骨量。与最近对成骨细胞分化的转录调控的认识相反， 成骨细胞分泌基质蛋白以形成骨的机制在很大程度上是未知的。作为一名飞行员， 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，我们已经开发了实验工具，使我们能够1）免疫纯化 溶酶体，从而通过质谱测定其蛋白质含量，2）敲低内源性 候选分泌基因使用慢病毒和逆转录病毒介导的shRNA转导，并拯救它与一个 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，和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，其中之一是骨激活素（OA）。OA在OC中表达 和OBs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）识别 成骨细胞分泌囊泡蛋白质的质谱分析，并表征其骨形成 在体外通过shRNA介导的基因敲低发挥作用，和B）平行，以表征 候选OB分泌蛋白OA在使用现有鼠模型的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帮助下完成 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，作为未来的一部分， R 01提案。

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.