Importance of Beta-Catenin Signaling in Osteocytes Associated with Anabolic Load

描述(申请人提供)：骨质疏松症是一种衰弱的疾病，影响着全球7500万人，估计医疗费用为480亿美元。骨量的一个主要决定因素是骨骼在日常活动中所承受的机械载荷，这刺激了适应性建模。骨细胞是嵌入在骨基质中的细胞，被认为负责感知和协调骨骼中的适应性反应。作为对机械负荷的反应，在经历高机械刺激的部位的骨细胞似乎是施加负荷的初始感知者，并将这种力转化为导致骨形成的早期生化信号。然而，目前对这些细胞如何单独和共同感知和整合整个骨骼上感知的应变以启动特定部位的适应性建模反应知之甚少。硬化素和b-连环蛋白介导的WNT信号转导通路对Wnt/b-连环蛋白通路产生正反两方面的影响，在骨形成过程中起着重要作用。我们的初步数据表明，在机械负荷后，骨细胞中的b-连环蛋白被迅速激活。我们的数据还表明，负载信号被传播到邻近的细胞，到硬化素蛋白表达下调的位置。基于此，我们的总体假设是，当SOST下调时，b-catenin信号受一组骨细胞中机械刺激的大小控制，该刺激激活该通路，随后将其传播到邻近区域。在这一应用中，我们建议使用b-连环蛋白信号和SOST表达作为读数，以确定骨形成的驱动因素(b-连环蛋白)及其负调控因子(骨细胞特异性分子SOST)是如何在三维机械响应性骨模型中被骨细胞中的机械载荷控制的。这个体内模型将应变或流体流动的剪应力大小作为机械刺激(一个原因)与报告活动或基因表达(一个机制)与骨形成(一个生物反应(一个效应))相结合。提出了以下具体目标，目标1)。确定b-连环蛋白途径对合成代谢负荷的反应的时间和空间激活，目标2)。确定SOST表达在时间和空间上的下调，以响应合成代谢负荷，并目的3)。确定增加或减少b-catenin信号对SOST/skerostin表达和诱导骨形成所需的机械刺激阈值的影响。我们认为，增加基础水平的b-连环蛋白活性降低了机械刺激的阈值，以诱导骨形成对负荷的反应，反之亦然。β-连环蛋白活性和Sost/skerostin表达的变化将与应变或流体流动剪切应力大小在三维上相关。在三维而不是二维上思考和可视化骨骼对机械载荷的响应将为了解骨骼对载荷的响应提供新的见解，并有助于我们理解应变和骨细胞响应之间的基本关系。与公共卫生相关。骨质疏松症是一种骨量低的疾病，是一种使人衰弱的疾病，全球有7500万人受到困扰，医疗费用估计为480亿美元。骨量的一个主要决定因素是骨骼在日常活动中所承受的机械载荷，这刺激了适应性建模。骨细胞是嵌入在骨基质中的细胞，被认为负责感知和协调骨骼中的适应性反应。在这一应用中，我们评估了一种药理学试剂(氯化锂)的剂量，它可以与机械载荷协同作用，激活骨细胞中一条强大的途径(Wnt/b-catenin信号)，从而导致在机械应力峰值位置发生骨形成。值得注意的是，未能促进这些部位的骨形成可导致骨折。我们还评估了参与该途径的分子的激活，并显示了这些分子的激活和骨形成与三维机械刺激峰值位置的关系。因此，这项研究对于通过机械负荷调节骨量和评估降低骨折风险的分子途径具有重要意义。

DESCRIPTION (provided by applicant): Osteoporosis is a debilitating disease that affects 75 million people worldwide with an estimated $48 billion in healthcare costs. A major determinant of bone mass is the mechanical loading to which the skeleton is subjected during daily activity, which stimulates adaptive modeling. Osteocytes, cells embedded in the bone matrix, are thought to be responsible for sensing and coordinating adaptive responses in the skeleton. In response to mechanical loading, osteocytes at sites experiencing high mechanical stimulus appear to be the initial perceivers of applied load and to translate this force into early biochemical signals that lead to bone formation. However, little is currently known about how these cells individually and collectively sense and integrate strains perceived over the entire bone in order to initiate site-specific adaptive modeling responses. Wnt signaling mediated by sclerostin and b-catenin, molecules that negatively and positively influence the Wnt/b-catenin pathway, clearly plays an important role in bone formation. Our preliminary data suggests that b-catenin is rapidly activated in osteocytes following mechanical loading. Our data also suggests that the load signal is propagated to adjacent cells, to sites where sclerostin protein expression is down regulated. Based on this, our overall hypothesis is that b-catenin signaling is controlled by magnitude of mechanical stimulus in a subset of osteocytes that activate the pathway followed by its subsequent propagation to adjacent regions when Sost becomes down regulated. In this application, we propose to use b-catenin signaling and Sost expression as readouts to determine how a driver of bone formation (b-catenin) and its negative regulator (Sost, an osteocyte specific molecule) are controlled by mechanical loading in osteocytes within a three dimensional mechanically responsive bone model. This in vivo model integrates strain or fluid flow shear stress magnitudes as the mechanical stimulus (a cause) with reporter activity or gene expression (a mechanism) with bone formation, a biological response (an effect). The following specific aims are proposed, Aim 1). Determine the temporal and spatial activation of the b-catenin pathway in response to anabolic load, Aim 2). Determine the temporal and spatial down regulation of Sost expression in response to anabolic load, and Aim 3). Determine the effects of increasing or decreasing b-catenin signaling on Sost/sclerostin expression and mechanical stimulus thresholds required to induce bone formation. We propose that increasing the basal level of b-catenin activity decreases the threshold of mechanical stimulus required to elicit bone formation in response to load and vice versa. Changes in b-catenin activity and Sost/sclerostin expression will be correlated with strain or fluid flow shear stress magnitudes in 3 dimensions. Thinking and visualizing bone responses to mechanical load in 3 dimensions instead of 2 dimensions will provide novel insights into how bone responds to load and will contribute to our understanding of the fundamental relationship between strain and bone cell responses. PUBLIC HEALTH RELEVANCE. Osteoporosis, a disease of low bone mass, is a debilitating disease that afflicts 75 million people worldwide with an estimated $48 billion in healthcare costs. A major determinant of bone mass is the mechanical loading to which the skeleton is subjected during daily activity, which stimulates adaptive modeling. Osteocytes, cells embedded in the bone matrix, are thought to be responsible for sensing and coordinating adaptive responses in the skeleton. In this application, we evaluate the dosage of a pharmacological agent (lithium chloride) that can synergize with mechanical loading to activate a powerful pathway (the Wnt/b-catenin signaling) in osteocytes such that it leads to bone formation occurring at sites of peak mechanical stresses. It is noted that failure to enhance bone formation at these sites can result in bone fractures. We also evaluate the activation of molecules that are involved in the pathway and show the activation of these molecules and bone formation in relationship to sites of peak mechanical stimuli in three dimensions. Therefore, this research is important in building an insightful foundation for regulation of bone mass by mechanical loading and in evaluating molecular pathways to reduce fracture risk.