PTH Signaling through LRP6

描述（由申请人提供）：PTH是钙稳态和骨代谢的主要调节剂，其信号传导系统已成为开发骨质疏松症新型合成代谢治疗方法的主要靶点。然而，PTH在骨中发挥作用的确切机制尚未完全了解。尽管在确定成骨细胞中PTH下游信号方面取得了进展，但可能存在新的信号传导组分，以弥合我们对PTH骨合成代谢作用的理解中的一些主要差距。我们的特点是一个新的PTH信号通路在成骨细胞。我们的初步数据表明，PTH与其受体PTH 1 R的结合诱导了Wnt的辅助受体LRP 6与PTH 1 R的结合。含有PTH、PTH 1 R和LRP 6的三元复合物的形成促进了LRP 6的快速磷酸化，这导致轴蛋白向LRP 6的募集和2-连环蛋白的稳定。PKA的激活对于PTH诱导的2-catenin稳定是必需的，但对于Wnt信号传导不是必需的。我们证明LRP 6辅助受体介导成骨细胞中PTH激活的2-连环蛋白信号转导，这首次表明LRP 6是PTH/PTH 1 R复合物的组分。重要的是，体内研究证实，PTH治疗导致成骨细胞中LRP 6的磷酸化和2-连环蛋白的量增加，同时增加大鼠的骨形成。因此，我们假设PTH诱导的LRP 6磷酸化，这是通过PKA的活化诱导，是PTH对骨的合成代谢作用所必需的。本提案的目的是表征成骨细胞中响应于PTH刺激的LRP 6激活的机制，并检查LRP 6在体内PTH骨合成代谢作用中的作用。该提案分为三个目标。在目的I中，将表征LRP 6内的PKA磷酸化位点，并将确定PKA磷酸化的LRP 6在成骨细胞中激活LRP 6 -2-连环蛋白信号传导中的作用。在目的II中，将定义LRP 6在原代颅骨前成骨细胞和骨髓基质细胞中介导PTH刺激的成骨细胞活性的需要。在目的III中，将使用OC-Cre介导的LRP 6条件性缺失的小鼠模型来检查体内PTH骨合成代谢作用中LRP 6的需求。公共卫生相关性：负责甲状旁腺素对骨的合成代谢作用的机制尚未完全了解。该提案将描述LRP 6，Wnt的辅助受体，如何在成骨细胞中激活PTH刺激和LRP 6在PTH骨合成代谢作用中的作用。

DESCRIPTION (provided by applicant): PTH is a primary regulator of calcium homeostasis and bone metabolism, and its signaling system has served as a major target for the development of novel anabolic therapeutic approaches for osteoporosis. However, the exact mechanisms by which PTH exerts its actions in bone are not fully understood. Despite the progress having been made in determining the PTH downstream signals in osteoblasts, novel signaling components may exist to bridge some major gaps in our understanding of the bone anabolic effects of PTH. We have characterized a novel PTH signaling pathway in osteoblasts. Our preliminary data show that binding of PTH to its receptor PTH1R induced association of LRP6, a coreceptor of Wnt, with PTH1R. The formation of the ternary complex containing PTH, PTH1R and LRP6 promoted rapid LRP6 phosphorylation, which resulted in the recruitment of axin to LRP6, and stabilization of 2-catenin. Activation of PKA is essential for PTH-induced 2-catenin stabilization, but not for Wnt signaling. Our demonstration of that the LRP6 coreceptor mediates PTH- activated 2-catenin signaling in osteoblasts suggests for the first time that LRP6 is the component of PTH/PTH1R complex. Importantly, in vivo studies confirmed that PTH treatment led to phosphorylation of LRP6 and an increase in amount of 2-catenin in osteoblasts with a concurrent increase in bone formation in rat. Therefore, we hypothesize that PTH-induced phosphorylation of LRP6, which is induced through activation of PKA, is required for PTH anabolic effect on bone. The objective of this proposal is to characterize the mechanisms of the LRP6 activation in response to PTH stimulation in osteoblasts and examine the role of LRP6 in PTH bone anabolic action in vivo. The proposal is organized into three aims. In aim I, the PKA phosphorylation sites within LRP6 will be characterized, and the role of PKA-phosphorylated LRP6 in activation of LRP6-2-catenin signaling in osteoblasts will be determined. In aim II, the requirement of LRP6 in mediating PTH-stimulated osteoblast activities in primary calvarial preosteoblasts and bone marrow stromal cells will be defined. In aim III, the requirement of LRP6 in PTH bone anabolic action in vivo will be examined using a mouse model of OC- Cre-mediated conditional deletion of LRP6. PUBLIC HEALTH RELEVANCE: The mechanisms responsible for anabolic actions of PTH on bone are not completely understood. This proposal will characterize how LRP6, a coreceptor for Wnt, is activated in osteoblasts upon PTH stimulation and the role of LRP6 in PTH bone anabolic action.