Characterizing the mechanism by which endogenous negative regulators of osteoclasts control bone homeostasis under physiological and pathological conditions in mouse models

本申请的直接目标是了解破骨细胞（OC）分化是如何通过以下途径调节的： 负性内源性调节因子，这可能为骨疾病提供新的治疗靶点， 骨质疏松症和类风湿性关节炎（RA）。虽然OC分化的正调节因子通过 核因子kB受体激活因子（RANK）配体（RANKL）-RANK信号传导轴已被广泛研究， 研究表明，对OC分化的负调节因子的理解是难以捉摸的。确定关键的负调节因子 OC分化，我们利用微阵列，基因下调，破骨细胞生成分析，导致 分离出鸟嘌呤蛋白α亚基13（Gα13，由Gna 13基因编码）作为潜在的OC阴性 调节器为了能够在体内研究Gα13在OC分化中的作用，我们产生了OC谱系。 通过将Gna 13 f/f小鼠与LysM-Cre（OC）杂交建立特异性Gna 13条件性敲除（CKO）小鼠模型 前体特异性）小鼠。我们的初步数据显示，Gna 13 f/fLysM-Cre小鼠表现出严重的 骨质疏松症从OC分化急剧增加。我们还注意到，Gα13缺乏会减弱RhoA， 活性并促进OC中Akt活性。一致地，我们表明，作为原则策略的证明， 组成型活性Gα13过表达可以防止骨和软骨损失，同时也减少了 在RA的小鼠模型中的炎症。根据我们的初步数据，我们假设Gα13是一个关键， OC的负调节因子，控制OC细胞谱系定型和分化以实现骨稳态 在生理和病理条件下通过激活RhoA和减弱AKT信号通路。 提出了三个具体目标来检验我们的假设。在目标1中，我们将描述 Gnα 13 f/fLysM-Cre小鼠，并阐明Gα13如何激活RhoA信号传导和 减弱Akt信号传导以负调节OC细胞谱系定型和分化。在目标2中， 通过G蛋白偶联受体的表征确定OC中的上游Gα13信号级联 （GPCR）与Gα13偶联。我们将描述Gα13在骨中作用的机制 通过使用树突状细胞（DC）-、单核细胞-和OC-特异性Gna 13 CKO和 分别在功能丧失和功能获得分析中对过表达转基因小鼠进行比较。本研究 可能提供了重要的见解的作用，负调控OC分化骨稳态 和OC过度分化的溶骨性骨疾病。从这项研究中获得的知识可能会产生 潜在的治疗靶点，通过表征OC中的Gα13信号传导， 通过模拟正常的OC抑制信号通路来控制OC的形成。一 多学科的研究团队，包括一名骨生物学家，具有OC生物学和动物模型的专业知识， 骨疾病;具有OC生物学和细胞信号传导专业知识的骨生物学家;以及具有 为实现研究目标，建立了包括类风湿性关节炎在内的自身免疫性疾病的专业知识。

The immediate goal of this application is to understand how osteoclast (OC) differentiation is regulated through negative endogenous regulators, which may provide novel therapeutic targets for bone diseases, such as osteoporosis and rheumatoid arthritis (RA). Although positive regulators of OC differentiation through the receptor activator of nuclear factor kB (RANK) ligand (RANKL)-RANK signaling axis have been extensively studied, understanding of negative regulators of OC differentiation is elusive. To identify key negative regulators of OC differentiation, we utilized microarray, gene downregulation, and osteoclastogenesis assays, leading to the isolation of guanine protein alpha subunit 13 (Gα13, encoded by the Gna13 gene) as a potential OC negative regulator. To enable in vivo investigation of the role of Gα13 in OC differentiation, we generated an OC-lineage specific Gna13 conditional knockout (CKO) mouse model by crossing Gna13f/f mice with LysM-Cre (OC precursor specific) mice. Our preliminary data showed that Gna13f/fLysM-Cre mice exhibited a severe osteoporosis from a drastic increase in OC differentiation. We also noted that Gα13 deficiency attenuates RhoA activity and promotes Akt activity in OCs. Consistently, we showed that, as a proof of principle strategy, local constitutively active Gα13 overexpression can protect against bone and cartilage loss while also attenuating inflammation in a mouse model of RA. Based on our preliminary data, we hypothesize that Gα13 is a key negative regulator of OC that controls OC cell lineage commitment and differentiation for bone homeostasis under physiological and pathological conditions through activating RhoA and attenuating AKT signaling pathway. Three specific aims are proposed to test our hypothesis. In Aim 1, we will characterize the bone phenotypes of Gnα13f/fLysM-Cre mice and elucidate the mechanism underlying how Gα13 activates RhoA signaling and attenuates Akt signaling to negatively regulate OC cell lineage commitment and differentiation. In Aim 2 we will determine the upstream Gα13 signaling cascade in OCs through characterization of G protein coupled receptors (GPCRs) coupled with Gα13. We will characterize the mechanism underlying the roles of Gα13 in bone remodeling and pathological bone loss by using dendritic cell (DC)-, monocyte- and OC-specific Gna13 CKO and overexpression transgenic mice in the loss-of-function and gain-of-function analysis, respectively. This study may provide important insights into the roles of negative regulators of OC differentiation in bone homeostasis and osteolytic bone diseases of excessive OC differentiation. Knowledge gained from this study may generate potential therapeutic targets, through characterization of Gα13 signaling in OCs that may be targeted in treating osteolytic bone diseases by mimicking normal OC inhibitory signaling pathway to control OC formation. A multidisciplinary research team, including a bone biologist with expertise in OC biology and animal models of bone diseases; a bone biologist with expertise in OC biology and cell signaling; and an immunologist with expertise in autoimmunity diseases including RA has been established to achieve the research goal.