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Ca 2+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诱发的Ca 2+振荡在诱导NFATc 1激活和OC分化中起着开关作用。然而，由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是未知的。我们最近的研究表明，G蛋白信号转导调节子12（RGS 12），一个多结构域和最大的蛋白质在RGS家族，在钙振荡和OC分化中起着至关重要的作用。我们使用诱导型Mx 1-cre删除造血谱系细胞中的RGS 12，发现突变小鼠（RGS 12 <$/<$/cre）患有骨硬化症，OC数量和骨吸收显著减少。此外，我们发现RGS 12可能通过磷酸酪氨酸结合（PTB）和RGS结构域与N型Ca 2+通道和钙敏感受体（汽车）直接相互作用，并且RGS 12及其PTB结构域的异位表达分别增加了OC分化期间RANKL的敏感性。已知G蛋白偶联的汽车在控制参与OC分化和存活的信号通路以及维持细胞外Ca 2+浓度中起关键作用，而RGS蛋白在调节汽车和刺激PLC中起重要作用。通过抑制G？i信令。此外，胞质Ca 2+振荡主要由细胞外Ca 2+通过L-和N-型钙通道的内流产生，并且RGS 12能够通过其PTB结构域与N-型Ca 2+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，RGS 12是必需的钙振荡OC的分化和功能，RGS 12与G？i蛋白、汽车、Ca 2+通道和其他异源二聚化伴侣，以调节OC分化过程中RANKL诱导的Ca 2+振荡-NFATc 1通路。我们将通过两个具体目标来检验这一假设：目的1，我们将通过使用RGS 12 flox/flox/Mx 1-Cre分析在OC分化的早期和晚期阶段具有RGS 12缺失的RGS 12突变小鼠的骨表型来确定在OC谱系中RGS 12诱导的Ca 2+振荡对于OC分化和在骨发育和重塑中的功能是必需的，RGS12flox/flox/LysM-Cre和RGS12flox/flox/CathepsinK-Cre。我们将进一步确定RGS 12在OVX后骨吸收和骨形成中的作用。然后，我们将在体外研究RGS 12在调节Ca 2+振荡、OC分化和OC分化早期和晚期功能中的作用。目的2，我们将研究在RGS 12 <$/<$/cre小鼠中损害OC分化的信号通路。我们将通过鉴定和表征RGS 12的功能结构域及其参与RGS 12诱导的OC中Ca 2+振荡的异源二聚化伙伴来进一步阐明机制。