Function of Regulator of G protein signaling in aging skeleton

描述(申请人提供)：年龄相关性骨质疏松症是一个主要的健康问题，其特征是由于破骨细胞的生成和活动随着年龄的增加而导致骨重建和代谢的失衡。目前的治疗方案受限于较低的应答率或副作用，并且不能有效地应对骨质疏松的负担和异质性。在建立成功的治疗方法方面的进一步进展迫切需要更清楚地了解破骨细胞(OC)分化和随年龄变化的骨重建的机制。有证据表明，RANKL诱导的钙振荡在PLCγ和NFATc1的激活以及OC分化中起着启动作用。进一步的研究表明，FcRγ和DAP12调节γ的磷酸化，这是RANKL诱导的钙振荡-NFATc1途径中的关键成分。尽管有这些新的见解，但RANKL如何引发必要的钙振荡，触发NFATc1激活和OC分化，以及这一机制是否以及如何参与与年龄相关的骨丢失，目前尚不清楚。我们最近的研究表明，G蛋白信号蛋白12调节因子(RGS12)在OC分化过程中显著表达，并且随着年龄的增长，RGS12的表达显著增加。RGS12表达下调可抑制细胞内钙离子振荡、NFATc1表达和OC分化。从造血系/早期OC细胞(RGS12//cre)中缺失RGS12导致骨量显著增加，这种增加的骨量在RGS12//cre小鼠中受到保护，不受年龄相关性骨丢失的影响。我们还发现，随着年龄的增长，RGS12基因的缺失会降低小鼠的OC数量和骨髓脂肪生成水平。基于这些结果，我们假设与年龄相关的骨重建和代谢需要RGS12的表达和功能，RGS12是控制衰老过程中钙振荡和OC分化的关键调节因子。我们将通过以下两个具体目标来检验这一假设。目的1通过对RGS12条件性基因敲除小鼠的表型和发病机制的研究，揭示RGS12在OC分化和衰老过程中的作用。我们将分析年轻(6个月)、中年(14个月)和老年(24个月)小鼠的骨表型，以确定早期和晚期OC谱系细胞中RGS12的缺失如何影响出生后年龄相关的骨代谢，方法是使用Mx1-cre和组织蛋白酶K-Cre小鼠。我们将在体外进一步鉴定老龄RGS12//cre小鼠来源的OCs，并确定间充质系细胞对衰老RGS12//cre小鼠OCS和骨骼表型的贡献。在目标2中，我们将通过研究RGS12的致病机制和途径、RGS12结构域的功能以及它的异二聚体伙伴来阐明RGS12相互作用赋予钙离子振荡和OCS与年龄的特异性的机制。本课题的目的是揭示RGS12在病理性年龄条件下OC分化和激活中的作用和机制，为老年性骨质疏松症等骨病的治疗提供新的、更有效的靶点。

DESCRIPTION (provided by applicant): Age-associated osteoporosis is a major health problem, which is characterized by an imbalance in bone remodeling and metabolism due to the increase of osteoclastogenesis and activity with age. The current treatment options have been constrained with lower response rates or side-effects and ineffectively tackle the burden and heterogeneity of osteoporosis. Further progress in establishing successful treatment urgently requires a clearer understanding of the mechanisms of osteoclast (OC) differentiation and bone remodeling with age. Evidences have documented that RANKL -evoked Ca2+ oscillations play a switch-on role in the activation of PLCγ and NFATc1, and OC differentiation. Further study showed that FcRγ and DAP 12 regulate the phosphorylation of PLCγ, which is the critical component involved in the RANKL-induced Ca2+ oscillations- NFATc1 pathway. Despite these new insights, it remains unclear how RANKL evokes the essential Ca2+ oscillations that triggers NFATc1 activation and OC differentiation, and it is unknown whether and how this mechanism is involved in age-associated bone loss. Our recent research demonstrated that Regulator of G protein signaling protein 12 (RGS12) was prominently expressed during OC differentiation and that RGS12 expression was significantly increased with increasing age. Knockdown of RGS12 expression inhibited Ca2+ oscillations, NFATc1 expression and OC differentiation. Deletion of RGS12 from hematopoietic/early OC lineage cells (RGS12 / /cre) led to a significantly increase of bone mass, and this increase of bone mass was protected from age-associated bone loss in RGS12 / /cre mice. We also found that loss of RGS12 in mice decreased OC number and levels of marrow adipogenesis with age. Based on these results, we hypothesize that age-associated bone remodeling and metabolism requires RGS12 expression and function, and RGS12 is a critical regulator in controlling Ca2+ oscillation and OC differentiation during aging. We will test the hypothesis through the following two specific aims. In Aim 1, we will reveal the function of RGS12 in OC differentiation and function during aging through characterization of the phenotypes and pathomechanism of RGS12 conditional knockout mice. We will analyze bone phenotypes in young (6 months (m)), mid-aged (14m) and aged (24m) mice to determine how deletion of RGS12 in early and late OC lineage cells affect postnatal age-associated bone metabolism by using Mx1-cre and Cathepsin K-Cre mice. We will further characterize in vitro of OCs derived from aging RGS12 / /cre mice and determine the contribution of mesenchymal lineage cells to the OCs and skeletal phenotype in aging RGS12 / /cre mice. In Aim 2, we will elucidate the mechanism of RGS12 interactions that confer specificity of Ca2+ oscillation and OCs with age by characterizing pathomechanism and pathways, RGS12 domain functions and its heterodimerization partners. The goal of this project is to discover the role and mechanism of RGS12 in OC differentiation and activation in pathologic age condition, and provide new and more effective therapeutic targets to age-associated osteoporosis and other bone diseases.