The Role of the Osteoblast Secretome in Bone Formation

保持健康的骨骼需要破骨细胞(OCs)的偶联活动，这些细胞吸收有机的 和骨的无机成分，以及成骨细胞(OBS)，即对产生和钙化有反应的细胞 基质蛋白。这两种类型的细胞都通过调节的胞吐作用来执行它们的功能，在这个过程中 特化的囊泡定位于特定的细胞域，导致极化分泌。因此，我们 假设OB的分泌囊泡蛋白对OB的功能和维持很重要 骨量的比例。与最近对成骨细胞分化的转录调控的见解不同， 成骨细胞分泌基质蛋白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，我们开发了实验工具，使我们能够1 溶酶体，从而通过质谱仪测定其蛋白质含量，2)内源敲除 利用Lenti和逆转录病毒介导的shRNA转导候选分泌基因并用 抗RNAi突变体。因此，我们能够a)在OBS中鉴定新的分泌泡蛋白，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 OBS中的候选调节蛋白之一，骨活素(OA)目前正在研究中。办公自动化以OCS表示 和OBS，在体外定位于这两种细胞的溶酶体中，并调节骨形成和吸收。 此外，已经有了一种小鼠的骨关节炎突变模型。因此，我们的具体目标是A)识别 成骨细胞分泌泡蛋白的质谱学研究及其成骨特性 通过shRNA介导的基因体外敲除功能，以及B)并行，以表征其作用 利用现有的小鼠模型在体内形成骨形成中的候选OB分泌蛋白OA。 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 对于肌肉骨骼生物学和医学，即核心B-肌肉骨骼的结构和强度，以及核心 C-In原位分子分析，在这些研究完成后，我们将能够在体内提出 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。

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.