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

RGS12（G 蛋白信号传导调节剂）在骨重塑中的作用

• 批准号: 8842593
• 负责人: SHUYING YANG
• 金额: $ 34.48万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8842593
• 关键词: Adverse effects; Affect; Arthritis; Binding; Biological Assay; Bone Development; Bone Diseases; Bone Marrow Cells; Bone Resorption; Bone remodeling; Boxing; Calcium Channel; Calcium-Sensing Receptors; Calvaria; Caring; Cell Lineage; Cells; Coculture Techniques; Coupled; Disease; Dominant-Negative Mutation; Ectopic Expression; FOS gene; Family; Fc Receptor; Fracture; G Protein Gene; G-substrate; GTP-Binding Protein Regulators; GTP-Binding Proteins; Genes; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Hematopoietic; Heterodimerization; Heterogeneity; Hormones; Immunoprecipitation; In Vitro; Investigation; MMP9 gene; Mass Spectrum Analysis; Mediating; Metabolism; Microarray Analysis; Molecular; Molecular Genetics; Morphology; Mus; Mutant Strains Mice; N-Type Calcium Channels; NF-kappa B; Osteoclasts; Osteogenesis; Osteoporosis; Ovariectomy; PTB Domain; Parathyroid gland; Pathway interactions; Patients; Phenotype; Phosphorylation; Phosphotyrosine; Play; Proteins; Public Health; RGS Domain; RGS Proteins; RNA Interference; Role; Selective Estrogen Receptor Modulators; Signal Pathway; Signal Transduction; Signaling Molecule; Skeleton; Staging; TRANCE protein; TYROBP gene; Testing; Therapeutic Intervention; Tissue-Specific Gene Expression; Western Blotting; Woman; aged; base; bisphosphonate; bone; bone turnover; cathepsin K; cost; effective therapy; extracellular; gain of function; insight; loss of function; men; nuclear factors of activated T-cells; osteoclastogenesis; overexpression; phospholipase C gamma; response; skeletal; transcription factor; vector

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.

描述（由申请人提供）：我们的长期目标是确定破骨细胞（OC）中Ca2+振荡促进骨重塑的分子机制，并为指导更有效的治疗促进骨骼健康提供基础。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 分化中发挥着开关作用。然而，RANKL诱导的启动和维持OC分化的Ca2+振荡的因素以及Ca2+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未知。我们最近的研究表明，G 蛋白信号传导调节器 12 (RGS12) 是 RGS 家族中最大的多结构域蛋白，在 Ca2+ 振荡和 OC 分化中发挥着重要作用。我们使用诱导型 Mx1-cre 删除了造血谱系细胞中的 RGS12，发现突变小鼠 (RGS12¿/¿/cre) 具有骨质疏松性，OC 数量和骨吸收显着减少。此外，我们发现RGS12可能通过磷酸酪氨酸结合（PTB）和RGS结构域直接与N型Ca2+通道和钙敏感受体（CaR）相互作用，并且RGS12及其PTB结构域的异位表达分别增加OC分化期间的RANKL敏感性。众所周知，G 蛋白偶联的 CaR 在控制 OC 分化和存活的信号通路以及维持细胞外 Ca2+ 浓度方面发挥着关键作用，而 RGS 蛋白在调节 CaR 和刺激 PLC? 方面发挥着重要作用。通过抑制 G?i 信号传导来发挥活性。此外，胞质Ca2+振荡主要是由细胞外Ca2+通过L型和N型钙通道流入产生的，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、CaR、Ca2+ 通道和其他异二聚化伙伴相互作用，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检验这一假设：目标 1，我们将通过使用 RGS12flox/flox/Mx1-Cre 分析在 OC 分化早期和晚期删除 RGS12 的 RGS12 突变小鼠的骨表型，确定 OC 谱系中 RGS12 诱导的 Ca2+ 振荡在骨发育和重塑中的 OC 分化和功能所必需的位置， RGS12flox/flox/LysM-Cre 和 RGS12flox/flox/CathepsinK-Cre。我们将进一步确定 RGS12 在 OVX 后增强的骨吸收和形成中的作用。然后我们将描述 RGS12 在调节 Ca2+ 振荡、OC 分化以及体外 OC 分化早期和晚期阶段的功能中的作用。目标 2，我们将研究损害 RGS12¿/¿/cre 小鼠 OC 分化的信号通路。我们将通过识别和表征 RGS12 的功能域及其参与 RGS12 诱导 OC 中 Ca2+ 振荡的异二聚化伙伴，进一步阐明该机制。