PTH Signaling through LRP6

描述(申请人提供)：甲状旁腺素是钙稳态和骨代谢的主要调节器，其信号系统已成为开发新的骨质疏松症合成代谢治疗方法的主要靶点。然而，甲状旁腺激素在骨骼中发挥作用的确切机制还不完全清楚。尽管在确定成骨细胞的甲状旁腺素下游信号方面已经取得了进展，但可能存在新的信号成分来弥合我们对甲状旁腺素的骨合成代谢作用的一些主要认识上的差距。我们在成骨细胞中研究了一种新的甲状旁腺素信号通路。我们的初步数据表明，PTH与其受体PTH1R的结合诱导了Wnt的辅助受体LRP6与PTH1R的结合。PTH、PTH1R和LRP6三元复合体的形成促进了LRP6的快速磷酸化，导致Axin重新聚集到LRP6，并稳定了2-catenin。PKA的激活是PTH诱导的2-连环蛋白稳定所必需的，但对Wnt信号转导不是必需的。在成骨细胞中，LRP6辅受体介导了PTH激活的2-连环蛋白信号转导，这首次表明LRP6是PTH/PTH1R复合体的组成部分。重要的是，体内研究证实，甲状旁腺素治疗导致LRP6的磷酸化和成骨细胞中2-连环素的数量增加，同时增加了大鼠骨形成。因此，我们推测PTH诱导的LRP6的磷酸化是PTH对骨的合成作用所必需的，这种磷酸化是通过激活PKA来诱导的。本研究的目的是研究成骨细胞中LRP6在甲状旁腺激素(PTH)刺激下活化的机制，并探讨LRP6在体内甲状旁腺激素骨合成代谢中的作用。该提案分为三个目标。在AIM I中，将鉴定LRP6中的PKA磷酸化位点，并将确定PKA磷酸化的LRP6在成骨细胞中激活LRP6-2-catenin信号中的作用。在AIM II中，将确定LRP6在原代颅骨前成骨细胞和骨髓基质细胞中介导甲状旁腺素刺激的成骨细胞活动的需求。在目标III中，将使用OC-Cre介导的条件删除LRP6的小鼠模型来研究体内甲状旁腺激素骨合成作用中LRP6的需求。公共卫生相关性：甲状旁腺素对骨骼的合成代谢作用的机制还不完全清楚。本研究将描述成骨细胞中Wnt的辅助受体LRP6如何在甲状旁腺激素刺激下被激活，以及LRP6在甲状旁腺激素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.