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

描述（由申请人提供）：我们的长期目标是确定破骨细胞（OCs）中Ca2+振荡促进骨重塑的分子机制，并为指导更有效的治疗方法提供基础，以促进骨骼健康。最近的突破性研究表明，rankl诱发的Ca2+振荡在诱导NFATc1激活和OC分化中起着开关作用。然而，RANKL诱导的启动和维持钙离子振荡以促进OC分化的因素，以及钙离子振荡如何在分子水平上诱导破骨细胞发生，在很大程度上仍然未知。我们最近的研究表明，调节剂g蛋白信号12 （RGS12）是RGS家族中一个多结构域和最大的蛋白，在Ca2+振荡和OC分化中起重要作用。我们使用可诱导的Mx1-cre删除造血系细胞中的RGS12，发现突变小鼠（RGS12 /cre）呈骨硬化，OC数量和骨吸收显著减少。此外，我们发现RGS12直接与n型Ca2+通道和钙敏感受体（CaRs）相互作用，可能是通过磷酸酪氨酸结合（PTB）和RGS结构域，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。众所周知，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Ca2+浓度方面起着关键作用，RGS蛋白在调节CaRs和刺激PLC？通过抑制G？我的信号。此外，胞内Ca2+振荡主要是由胞外Ca2+通过L型和n型钙通道内流产生的，RGS12能够通过其PTB结构域与n型Ca2+通道直接相互作用并调节通道活性。基于这些发现，我们的中心假设是RGS12是OC分化和功能中Ca2+振荡所必需的，并且RGS12与G？i蛋白、CaRs、Ca2+通道和其他异源二聚化伙伴在OC分化过程中调节rankl诱导的Ca2+振荡- nfatc1途径。我们将通过两个具体目标来验证这一假设：目标1，我们将通过使用RGS12flox/flox/Mx1-Cre、RGS12flox/flox/LysM-Cre和RGS12flox/flox/ cathepink - cre分析RGS12缺失的RGS12突变小鼠在OC分化早期和晚期的骨骼表型，确定在OC谱系中RGS12诱导的Ca2+振荡对OC分化和骨发育和重塑的功能是必需的。我们将进一步确定RGS12在OVX后骨吸收和骨形成增强中的作用。然后，我们将描述RGS12在体外OC分化早期和晚期调节Ca2+振荡、OC分化和功能中的作用。目的2，我们将研究影响RGS12 / /cre小鼠OC分化的信号通路。我们将通过鉴定和表征RGS12及其异源二聚化伙伴参与RGS12诱导的OCs Ca2+振荡的功能域来进一步阐明其机制。

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.