Beta-Catenin Signaling in Osteocytes Associated with Load Induced Bone Formation

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负荷，这会刺激自适应建模。骨细胞是嵌入骨基质中的细胞，被认为负责感知和协调骨骼中的适应性反应。在响应机械负荷，骨细胞在网站经历高机械刺激似乎是施加负荷的初始感知器，并将这种力量转化为早期的生化信号，导致骨形成。然而，目前很少有人知道这些细胞如何单独和集体的感觉和整合应变感知整个骨，以启动特定位点的自适应建模反应。由sclerostin和b-连环蛋白介导的Wnt信号传导，这些分子对Wnt/b-连环蛋白途径产生负面和正面影响，显然在骨形成中起重要作用。我们的初步数据表明，b-连环蛋白在机械负荷后的骨细胞中被迅速激活。我们的数据还表明，负载信号传播到邻近细胞，到sclerostin蛋白表达下调的位点。基于此，我们的总体假设是，b-连环蛋白信号传导是由骨细胞亚群中的机械刺激的大小控制的，所述骨细胞亚群激活该通路，随后当Sost下调时，该通路传播到相邻区域。在本申请中，我们建议使用β-连环蛋白信号传导和Sost表达作为读数，以确定骨形成的驱动因子（β-连环蛋白）及其负调节因子（Sost，骨细胞特异性分子）如何通过三维机械响应骨模型内骨细胞中的机械负荷来控制。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）与骨形成（生物反应（效应））整合。提出了以下具体目标：目标1）。确定响应合成代谢负荷的b-连环蛋白途径的时间和空间激活，目的2）。确定响应于合成代谢负荷的Sost表达的时间和空间下调，以及目的3）。确定增加或减少β-连环蛋白信号传导对Sost/sclerostin表达和诱导骨形成所需的机械刺激阈值的影响。我们建议，增加的基础水平的b-连环蛋白活性降低的阈值所需的机械刺激引起骨形成的负荷，反之亦然。β-连环蛋白活性和Sost/sclerostin表达的变化将与三维中的应变或流体流动剪切应力大小相关。在三维而不是二维中思考和可视化骨骼对机械载荷的响应将为骨骼如何响应载荷提供新的见解，并将有助于我们理解应变和骨细胞响应之间的基本关系。公共卫生相关性。骨质疏松症是一种低骨量疾病，是一种使人衰弱的疾病，困扰着全世界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，这会刺激自适应建模。骨细胞是嵌入骨基质中的细胞，被认为负责感知和协调骨骼中的适应性反应。在本申请中，我们评估了药理学试剂（氯化锂）的剂量，该剂量可以与机械负荷协同作用，以激活骨细胞中的强大途径（Wnt/b-连环蛋白信号传导），从而导致在峰值机械应力部位发生骨形成。值得注意的是，未能增强这些部位的骨形成可能导致骨折。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峰值机械刺激位点的关系。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.