Lrp5 Signaling in Bone Mechano-Responsiveness

描述（由申请人提供）：细胞表面受体，低密度脂蛋白受体相关蛋白5 (LRP5)，正在成为骨量和强度的关键调节因子。LRP5的功能缺失突变导致人类疾病骨质疏松-假性胶质瘤综合征（OPPG），其特征是骨量和强度严重减少。LRP5的其他突变与高骨量（HBM）疾病有关，其特征是骨量和强度增加。经过Lrp5功能缺失突变改造的小鼠（OPPG模型）在体内和体外都显著降低了骨骼对机械负荷的反应性，这表明Lrp5在骨细胞对负荷（如运动）的反应能力中起着关键作用。当前应用的目标是准确理解LRP5如何参与骨骼对机械负荷的反应，这最终可能为预防或治疗常见骨骼疾病提供新方法-这是NIAMS （NIH）的主要任务。解决的问题包括：1)机械反应性过程中的哪些步骤受到LRP5功能丧失和导致HBM表型的错义突变的影响？2)机械反应是否由典型Wnt信号介导？3)通过Lrp5传递机械信息涉及哪些配体？4)如果有的话，哪些抑制蛋白参与了这个过程？5)成骨细胞谱系中的Wnt/Lrp5信号是否在废弃或过度使用时调节吸收信号？拟议的项目是两个骨骼生物学实验室（印第安纳大学和凯斯西储大学）之间的合作，这两个实验室提供了互补的专业知识，将有助于阐明Lrp5在多个水平上骨力学反应中的作用。为了确定Lrp5对机械反应性的影响机制，我们将使用几种小鼠模型进行体外机械加载和卸载研究，包括与几种报告菌株杂交的OPPG （Lrp5-/-）小鼠和2种HBM突变菌株。为了更全面地分析Lrp5在机械信号转导中的作用，我们将从这些小鼠身上收获原代成骨细胞，并在体外进行机械刺激。具体来说，我们将研究Lrp5在负荷诱导成骨细胞生命阶段（起源、募集、分化和命运）和负荷诱导的典型Wnt信号传导中的作用（目的1）；（目标2）Lrp5活性发生在机械转导信号级联中，包括识别上游调节剂和下游信号转导目标通路；（目的3）Lrp5在调节机械诱导的促吸收标志物表达中的作用，包括OPG和RANKL；（目标4）两种HBM突变调节机械转导的作用机制。了解Lrp5在机械反应性中的活性机制在公共卫生领域具有巨大的潜力，有助于理解载荷对骨量、脆弱性和骨折风险的骨构建效应。

DESCRIPTION (provided by applicant): The cell surface receptor, low-density lipoprotein receptor-related protein 5 (LRP5), is emerging as a key regulator of bone mass and strength. Loss-of-function mutations in LRP5 cause the human disease osteoporosis-pseudoglioma syndrome (OPPG), characterized by severely reduced bone mass and strength. Other mutations in LRP5 have been associated with high bone mass (HBM) disorders, characterized by increased bone mass and strength. Mice engineered with loss-of-function mutations in Lrp5, which model the OPPG condition, have dramatically reduced skeletal responsiveness to mechanical loading both in vivo and in vitro, suggesting that Lrp5 plays a key role in bone cell's ability to respond to loading (e.g., exercise). The goal of the present application is to understand precisely how LRP5 participates in the skeleton's response to mechanical loading, which ultimately could suggest new approaches for preventing or treating common diseases of bone-a primary mission of NIAMS (NIH). Among the questions addressed are 1) What steps in the process of mechanoresponsiveness are affected by loss of LRP5 function and by missense mutations that cause HBM phenotypes? 2) Is mechano-responsiveness mediated by canonical Wnt signaling? 3) What ligands are involved in transmitting the mechanical messages through Lrp5? 4) Which, if any, inhibitory proteins participate in this process? 5) Does Wnt/Lrp5 signaling in osteoblast-lineage cells modulate resorption signaling during disuse or overuse? The proposed project is a collaboration between two skeletal biology labs (Indiana Univ. and Case Western Reserve Univ.), which contribute complementary expertise that will facilitate the elucidation of Lrp5's role in bone mechano-responsiveness at multiple levels. In vitro mechanical loading and unloading studies will be conducted using several mouse models, including OPPG (Lrp5-/-) mice crossed with several reporter strains, and 2 HBM mutant strains, in order to determine the mechanisms of Lrp5's effect on mechano-responsiveness. To more fully dissect the role of Lrp5 in mechanical signal transduction, primary osteoblasts will be harvested from these mice and mechanically stimulated in vitro. Specifically, we will investigate (Aim 1) the role of Lrp5 in load-induced osteoblast lifestages (origin, recruitment, differentiation, and fate) and in load-induced canonical Wnt signaling; (Aim 2) where Lrp5 activity occurs in the mechanotransduction signaling cascade, including identification of upstream modulators and downstream signal transduction target pathways; (Aim 3) the role of Lrp5 in regulating mechanically-induced expression of pro-resorption markers, including OPG and RANKL; and (Aim 4) the mechanism of action by which two HBM mutations modulate mechanotransduction. Insights into the mechanisms of Lrp5 activity in mechano-responsiveness hold great potential in the public health arena for understanding the bone-building effects of loading on bone mass, fragility, and fracture risk.