PTH Signaling through LRP6

描述（由申请人提供）：甲状旁腺激素是钙稳态和骨代谢的主要调节因子，其信号系统已成为开发新型骨质疏松症合成代谢治疗方法的主要靶点。然而，PTH在骨骼中发挥作用的确切机制尚不完全清楚。尽管在确定成骨细胞中甲状旁腺激素下游信号方面取得了进展，但可能存在新的信号成分来弥补我们对甲状旁腺激素骨合成代谢作用的理解中的一些主要空白。我们在成骨细胞中发现了一种新的PTH信号通路。我们的初步数据表明，PTH与其受体PTH1R结合可诱导Wnt的一种辅助受体LRP6与PTH1R结合。含有PTH、PTH1R和LRP6的三元配合物的形成促进了LRP6的快速磷酸化，导致轴素向LRP6募集，2-catenin稳定。PKA的激活对pth诱导的2-连环蛋白稳定至关重要，但对Wnt信号传导却不是如此。我们对LRP6辅助受体在成骨细胞中介导PTH激活的2-catenin信号传导的研究首次表明LRP6是PTH/PTH1R复合物的组成部分。重要的是，体内研究证实，PTH治疗导致大鼠成骨细胞中LRP6磷酸化和2-catenin数量增加，同时骨形成增加。因此，我们假设PTH诱导的LRP6磷酸化（通过激活PKA诱导）是PTH对骨的合成代谢作用所必需的。本研究的目的是表征LRP6在成骨细胞中对甲状旁腺激素刺激的激活机制，并研究LRP6在体内甲状旁腺激素骨合成代谢中的作用。该建议分为三个目标。在目的1中，将对LRP6内的PKA磷酸化位点进行表征，并确定PKA磷酸化的LRP6在成骨细胞中激活LRP6-2-catenin信号通路中的作用。在aim II中，我们将定义LRP6在原发性颅骨前成骨细胞和骨髓基质细胞中介导甲状旁腺激素刺激的成骨细胞活性的需要。在aim III中，我们将使用OC- cre介导的LRP6条件缺失小鼠模型来研究体内PTH骨合成代谢对LRP6的需求。公共卫生相关性：骨促甲状旁腺激素合成代谢作用的机制尚不完全清楚。本研究将描述Wnt的一种辅助受体LRP6是如何在PTH刺激下在成骨细胞中被激活的，以及LRP6在PTH骨合成代谢中的作用。

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.