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种主要的健康问题，其特征是由于破骨细胞的发生和活动随着年龄的增长而增加，导致骨重塑和代谢失衡。目前的治疗方案受到较低的反应率或副作用的限制，并且不能有效地解决骨质疏松症的负担和异质性。进一步建立成功的治疗迫切需要更清楚地了解破骨细胞（OC）分化和骨重塑随年龄增长的机制。有证据表明，RANKL诱发的Ca2+振荡在PLC -和NFATc1的激活和OC分化中起着开关作用。进一步的研究表明，FcR和DAP 12调节PLC的磷酸化，而PLC是rankl诱导的Ca2+振荡- NFATc1通路的关键成分。尽管有这些新的见解，但仍不清楚RANKL如何唤起触发NFATc1激活和OC分化的必需Ca2+振荡，并且尚不清楚该机制是否以及如何参与与年龄相关的骨质流失。我们最近的研究表明，G蛋白信号蛋白12 （RGS12）的调控因子在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表达下调抑制Ca2+振荡、NFATc1表达和OC分化。从造血/早期OC谱系细胞（RGS12 / /cre）中删除RGS12可导致骨量显著增加，并且RGS12 / /cre小鼠骨量的增加可避免与年龄相关的骨质流失。我们还发现，小鼠中RGS12的缺失会随着年龄的增长减少OC数量和骨髓脂肪生成水平。基于这些结果，我们假设年龄相关的骨重塑和代谢需要RGS12的表达和功能，RGS12是衰老过程中控制Ca2+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。在Aim 1中，我们将通过表征RGS12条件敲除小鼠的表型和病理机制，揭示RGS12在OC分化中的功能和衰老过程中的功能。我们将通过Mx1-cre和Cathepsin K-Cre小鼠分析幼年（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。我们将进一步在体外表征衰老RGS12 / /cre小鼠的OCs，并确定间充质谱系细胞对衰老RGS12 / /cre小鼠OCs和骨骼表型的贡献。在Aim 2中，我们将通过表征病理机制和途径、RGS12结构域功能及其异源二聚化伙伴，阐明RGS12相互作用的机制，这些相互作用赋予Ca2+振荡和OCs与年龄的特异性。本项目旨在发现RGS12在病理年龄状态下OC分化活化中的作用及机制，为年龄相关性骨质疏松等骨病提供新的更有效的治疗靶点。

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 PLC� and NFATc1, and OC differentiation. Further study showed that FcR� and DAP 12 regulate the phosphorylation of PLC�, 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.