G PROTEIN SIGNALING IN OSTEOBLASTS

描述（由申请人提供）：激素、生长因子和机械应变等外在因素通过对成骨细胞的作用对骨骼生长和重塑产生影响。因此，在定义特定的细胞内信号通路如何介导这些因子在控制成骨细胞功能中的作用方面有很大的兴趣。G蛋白信号在对许多骨骼刺激的反应中发生，这种信号的结果取决于G蛋白的性质；信号的时间传递；以及成骨细胞的表型状态。要更清楚地了解促钙剂如甲状旁腺激素如何在骨骼中引发其复杂作用，需要在体内环境中直接解决这些问题的研究。在目前的建议中，我们将评估成骨细胞中Gs和Gi信号在介导合成代谢骨骼反应中的作用。在一种方法中，称为rassl的新型G蛋白偶联受体将靶向转基因小鼠的成骨细胞。rassl激活特定的G蛋白通路，以响应合成激动剂的管理。Gs-和Gi rassl的激活将使我们能够解剖这些途径在骨骼反应中的作用。在第二种方法中，Gs和Gi的功能将分别通过cr介导的功能性Gs- α等位基因的切除和百日咳毒素催化亚基的靶向表达在体内成骨细胞中消融。我们将确定消融这些信号通路对正常骨骼稳态和甲状旁腺激素合成代谢反应的影响。机制研究将评估G蛋白信号对体内和从转基因小鼠分离的骨髓基质细胞（BMSCs）中成骨细胞增殖和凋亡的影响。G蛋白信号与最近被证明对骨形成至关重要的两条通路（LRP/典型Wnt通路和最近发现的RSK2/ATF4通路）的趋同将在骨髓间充质干细胞中进行探索。我们建议：1)评估成骨细胞Gs和Gi信号在成熟小鼠骨骼稳态调节中的作用。我们将确定成骨细胞在不同分化阶段受调控的、间歇性的Gs和Gi信号的影响；确定条件敲除Gs和Gi信号的骨骼效应；并评估操纵Gs和Gi信号引发这些影响的机制；2)确定Gs和Gi信号在PTH合成代谢反应中的作用及其作用机制。这些研究将为研究成骨细胞不同分化阶段G蛋白信号对骨形成和骨吸收的调控提供新的思路。它们还可能提供G蛋白信号与骨合成代谢作用之间的新联系，从而确定治疗骨质疏松症的新靶点。

DESCRIPTION (provided by applicant): Extrinsic factors such as hormones, growth factors, and mechanical strain produce their effects on skeletal growth and remodeling via actions on osteoblasts. Accordingly, there is great interest in defining how specific intracellular signaling pathways mediate the effects of these factors in controlling osteoblast function. G protein signaling occurs in response to many skeletal stimuli, and the consequence of this signaling depends on the nature of the G protein; the temporal delivery of the signal; and the phenotypic state of the osteoblast. A clearer understanding of how calciotropic agents such as PTH elicit their complex effects in bone requires studies that address these issues directly in an in vivo context. In the present proposal, we will assess the role of Gs and Gi signaling in osteoblasts in mediating anabolic skeletal responses. In one approach, novel G protein-coupled receptors termed RASSLs will be targeted to osteoblasts in transgenic mice. RASSLs activate specific G protein pathways in response to administration of synthetic agonists. Activation of Gs- and Gi RASSLs will allow us to dissect the role of these pathways in skeletal responses. In a second approach, Gs and Gi function will be ablated in osteoblasts in vivo by cre-mediated excision of functional Gs-alpha alleles and by targeted expression of the catalytic subunit of pertussis toxin, respectively. We will determine the effects of ablating these signaling pathways on normal skeletal homeostasis and on the anabolic response to PTH. Mechanistic studies will be carried out to assess the effects of G protein signals on osteoblast proliferation and apoptosis in vivo and in bone marrow stromal cells (BMSCs) isolated from the transgenic mice. Convergence of G protein signals with two pathways recently shown to be essential for bone formation - the LRP/canonical Wnt pathway and the recently identified RSK2/ATF4 pathway- will be explored in BMSCs. We propose to: 1) assess the role of osteoblast Gs and Gi signaling in the regulation of skeletal homeostasis in mature mice. We will determine the effects of regulated, intermittent Gs and Gi signaling in osteoblasts at different stages of differentiation; determine the skeletal effect of conditional knockout of Gs and Gi signaling; and assess the mechanisms by which manipulation of Gs and Gi signaling elicits these effects; and 2) determine the contribution of Gs and Gi signaling to the anabolic response to PTH and the mechanisms of these effects. These studies will shed new light on the control of bone formation and bone resorption by G protein signaling in osteoblasts at different stages of differentiation. They may also provide new links between G protein signals and anabolic effects in bone, thereby identifying new therapeutic targets for the treatment of osteoporosis. LAY DESCRIPTION: We will explore how activation of specific signals in bone-forming cells can lead to increases in bone mass. The results may lead to new approaches to the treatment of osteoporosis.