Role of RGS12, a Regulator of G protein Signaling, in Bone Remodeling

描述(申请人提供)：我们的长期目标是明确破骨细胞(OCs)中的钙振荡促进骨重建的分子机制，并为指导更有效的治疗以促进骨骼健康提供基础。最近的突破性研究表明，RANKL诱导的钙振荡在诱导NFATc1激活和OC分化中起启动作用。然而，RANKL诱导的启动和维持OC分化的Ca~(2+)振荡的因素以及Ca~(2+)振荡如何在分子水平上诱导破骨细胞生成仍然很大程度上是未知的。我们最近的研究表明，G蛋白信号转导调节因子12(RGS12)是RGS家族中最大的多域蛋白，在钙振荡和OC分化中起着重要作用。我们用可诱导的Mx1-cre基因删除了造血祖细胞中的RGS12，发现突变的小鼠(RGS12�/�/cre)具有成骨作用，OC数量和骨吸收显著减少。此外，我们还发现RGS12可能通过磷酸酪氨酸结合(PTB)和RGS结构域与N型钙通道和钙敏感受体(CARS)直接相互作用，并且RGS12及其PTB结构域的异位表达分别增加了OC分化过程中RANKL的敏感性。已知G蛋白偶联的CARS在控制OC分化和存活的信号通路以及维持细胞外钙离子浓度方面起着关键作用，而RGS蛋白在调节CARS和刺激PLC？抑制G？I信号的活性。此外，细胞内钙振荡主要由细胞外钙离子通过L和N型钙通道内流产生，RGS12能够通过其PTB结构域与N型钙通道直接相互作用并调节通道活动。基于这些发现，我们的中心假设是RGS12是OC分化和功能中钙振荡所必需的，RGS12与G？I蛋白、CARS、钙通道和其他异源二聚体伙伴相互作用，调节RANKL诱导的OC分化过程中的钙振荡-NFATc1通路。我们将通过两个特定的目标来验证这一假说：目的1，我们将通过使用RGS12 FLOX/FLOX/Mx1-Cre、RGS12FLOX/FLOX/LysM-Cre和RGS12FLOX/FLOX/LysM-Cre和RGS12FLOX/FLOX/CathepsinK-Cre来分析在OC分化早期和后期RGS12缺失的RGS12突变小鼠的骨表型，从而确定在OC谱系中RGS12诱导的钙振荡是OC分化所必需的。我们将进一步确定RGS12在去势后促进骨吸收和骨形成中的作用。然后，我们将在体外研究RGS12在调节钙离子振荡、OC分化和OC分化早期和晚期的功能中的作用。目的研究影响�/�/cre小鼠OC分化的信号转导途径。我们将通过鉴定和表征RGS12及其异二聚化伙伴的功能结构域来进一步阐明RGS12诱导OCS钙振荡的机制。

DESCRIPTION (provided by applicant): Our long-term goal is to define the molecular mechanisms by which Ca2+ oscillations in osteoclasts (OCs) contribute to bone remodeling and provide the basis for guiding more effective therapies to promote skeleton health. Recent breakthrough studies have shown RANKL-evoked Ca2+ oscillations play a switch-on role in inducing NFATc1 activation and OC differentiation. However, the factors induced by RANKL that initiate and maintain Ca2+ oscillations for OC differentiation and how Ca2+ oscillations induc osteoclastogenesis at the molecular level are still largely unknown. Our recent investigations suggest that Regulator of G-protein Signaling 12 (RGS12), a multi-domain and the largest protein in the RGS family, plays essential roles in Ca2+ oscillations and OC differentiation. We deleted RGS12 in hematopoietic lineage cells using inducible Mx1-cre and found that the mutant mice (RGS12�/�/cre) are osteopetrotic with significantly reduced OC numbers and bone resorption. Additionally, we found that RGS12 directly interacts with N-type Ca2+ channels and calcium- sensing receptors (CaRs), likely through phosphotyrosine-binding (PTB) and RGS domains, and that ectopic expression of RGS12 and its PTB domain respectively increases RANKL sensitivity during OC differentiation. It is known that the G protein-coupled CaRs play a pivotal role in controlling signaling pathways involved in OC differentiation and survival and maintaining extracellular Ca2+ concentrations, and that RGS proteins play essential roles in modulating CaRs and stimulating PLC? activity by inhibiting G?i signaling. Furthermore, cytosolic Ca2+ oscillations are generated mainly by influx of extracellular Ca2+ through L- and N-type calcium channels, and that RGS12 is capable of direct interaction with N-type Ca2+ channel through its PTB domain and modulates channel activity. Based on these findings, our central hypothesis is that RGS12 is required for Ca2+ oscillations in OC differentiation and function, and that RGS12 interacts with G?i protein, CaRs, Ca2+ channels, and other heterodimerization partners to regulate the RANKL-induced Ca2+ oscillation-NFATc1 pathway during OC differentiation. We will test this hypothesis through two specific aims: Aim 1, we will determine where in the OC lineage RGS12-induced Ca2+ oscillations are required for OC differentiation and function in bone development and remodeling by analyzing the bone phenotype of RGS12 mutant mice with deletion of RGS12 at early and late stages of OC differentiation using RGS12flox/flox/Mx1-Cre, RGS12flox/flox/LysM-Cre and RGS12flox/flox/CathepsinK-Cre. We will further etermine the role of RGS12 in the enhanced bone resorption and formation that occurs after OVX. We will then characterize the role of RGS12 in regulating Ca2+ oscillations, OC differentiation and function at early and late stages of OC differentiation in vitro. Aim 2, we will investigate the signaling pathways that impair OC differentiation in RGS12�/�/cre mice. We will further elucidate the mechanism by identifying and characterizing the functional domains of RGS12 and their heterodimerization partners involved in RGS12-induced Ca2+ oscillation in OCs.