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

描述（由申请人提供）：骨质疏松症是一种使人衰弱的疾病，影响着全世界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负荷，这刺激了适应性建模。骨细胞，嵌入在骨基质中的细胞，被认为负责感知和协调骨骼的适应性反应。在机械负荷的响应中，处于高机械刺激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集体感知和整合整个骨骼感知的应变，以启动特定部位的适应性建模反应知之甚少。由sclerostin和b-catenin介导的Wnt信号，这些分子对Wnt/b-catenin通路有消极和积极的影响，显然在骨形成中起重要作用。我们的初步数据表明，在机械负荷后，b-连环蛋白在骨细胞中迅速激活。我们的数据还表明，负载信号被传播到邻近的细胞，到硬化蛋白表达下调的位点。基于此，我们的总体假设是，b-catenin信号是由骨细胞亚群中机械刺激的大小控制的，当Sost下调时，骨细胞亚群激活该通路，随后将其传播到邻近区域。在本应用中，我们建议使用b-catenin信号传导和Sost表达作为读数，以确定骨形成的驱动因子（b-catenin）及其负调节因子（Sost，一种骨细胞特异性分子）如何在三维机械响应骨模型中由骨细胞中的机械负荷控制。该体内模型将应变或流体流动剪切应力大小作为机械刺激（原因）与报告活性或基因表达（机制）结合起来，作为骨形成的生物反应（效果）。提出了以下具体目标：目标1)。确定b-catenin通路在响应合成代谢负荷时的时空激活，目的2)。确定Sost表达在合成代谢负荷下的时空下调，以及Aim 3)。确定增加或减少b-catenin信号传导对Sost/sclerostin表达和诱导骨形成所需的机械刺激阈值的影响。我们提出，增加b-连环蛋白活性的基础水平降低了引起骨形成的机械刺激阈值，反之亦然。b-catenin活性和Sost/sclerostin表达的变化与应变或流体流动剪切应力大小在三维上相关。从三维而不是二维的角度来思考和可视化骨骼对机械负荷的反应将为骨骼对负荷的反应提供新的见解，并有助于我们理解应变和骨细胞反应之间的基本关系。公共卫生相关性。骨质疏松症是一种骨量低的疾病，是一种使人衰弱的疾病，全世界有7500万人患有骨质疏松症，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负荷，这刺激了适应性建模。骨细胞，嵌入在骨基质中的细胞，被认为负责感知和协调骨骼的适应性反应。在本应用中，我们评估了一种药理学剂（氯化锂）的剂量，该药理学剂可以与机械负荷协同作用，激活骨细胞中一个强大的途径（Wnt/b-catenin信号），从而导致骨形成发生在机械应力峰值的位置。值得注意的是，未能在这些部位加强骨形成可导致骨折。我们还评估了参与该通路的分子的激活，并在三维空间中显示了这些分子的激活和骨形成与峰值机械刺激位点的关系。因此，该研究对于建立机械载荷调节骨量的深刻基础以及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.