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（LRP 5），正在成为骨量和强度的关键调节因子。LRP 5的功能丧失突变导致人类疾病骨质疏松-假神经胶质瘤综合征（OPPG），其特征在于骨量和强度严重降低。LRP 5中的其他突变与高骨量（HBM）疾病相关，其特征在于骨量和强度增加。用Lrp 5中的功能缺失突变工程化的小鼠（其模拟OPPG状况）在体内和体外都显著降低了骨骼对机械负荷的响应性，这表明Lrp 5在骨细胞响应负荷的能力中起关键作用（例如，锻炼）。本申请的目标是精确地理解LRP 5如何参与骨骼对机械负荷的响应，这最终可以提出用于预防或治疗常见骨疾病的新方法-NIAMS（NIH）的主要使命。其中解决的问题是1）机械反应性过程中的哪些步骤受到LRP 5功能丧失和导致HBM表型的错义突变的影响？2)机械反应性是由经典Wnt信号介导的吗？3)哪些配体参与了Lrp 5传递机械信息的过程？4)如果有的话，哪些抑制蛋白参与了这一过程？5)成骨细胞系中Wnt/Lrp 5信号是否调节废用或过度使用时的吸收信号？拟议的项目是两个骨骼生物学实验室（印第安纳州大学和凯斯西储大学）之间的合作，这有助于补充专业知识，将有利于阐明Lrp 5的作用，在骨机械反应性在多个层面。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和2种HBM突变菌株杂交的OPPG（Lrp 5-/-）小鼠，以确定Lrp 5对机械响应性的作用机制。为了更全面地剖析Lrp 5在机械信号转导中的作用，将从这些小鼠中收获原代成骨细胞并在体外进行机械刺激。具体而言，我们将研究（目的1）Lrp 5在负荷诱导的成骨细胞生命阶段中的作用（目的2）其中Lrp 5活性发生在机械转导信号级联中，包括鉴定上游调节剂和下游信号转导靶途径;（目的3）Lrp 5在调节机械诱导的促吸收标志物（包括OPG和RANKL）表达中的作用;以及（目的4）两种HBM突变调节机械转导的作用机制。深入了解Lrp 5在机械反应性中的活性机制在公共卫生竞技场中具有很大的潜力，可以了解负荷对骨量、脆性和骨折风险的骨生成作用。

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.