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

此应用程序的直接目标是了解破骨细胞(OC)分化是如何通过 负性内源性调节因子，可能为骨骼疾病提供新的治疗靶点，如 骨质疏松和类风湿关节炎(RA)。尽管OC分化的正向调节因子通过 核因子kB受体激活剂(RANK)配体(RANKL)-RANK信号轴被广泛研究 研究表明，对于OC分化的负性调节因子的理解是难以捉摸的。找出关键的负面调节因素 对于OC的分化，我们利用微阵列、基因下调和破骨细胞生成分析，导致 潜在OC阴性的鸟嘌呤蛋白α亚基13(Gα13，由GNA13基因编码)的分离 调整器。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 Gna 13f/f小鼠与LysM-Cre(OC)杂交建立特异性Gna 13条件性基因敲除(CKO)小鼠模型 前体特异性)小鼠。我们的初步数据显示，Gna 13f/fLysM-Cre小鼠表现出严重的 由于OC分化急剧增加而导致的骨质疏松症。我们还注意到Gα13缺乏会减弱RhoA 活动并促进OCS中的Akt活动。我们始终表明，作为原则性战略的证明，本地 结构性活性Gα13过表达可以防止骨和软骨的丢失，同时也可以减轻 类风湿关节炎小鼠模型的炎症反应。根据我们的初步数据，我们假设Gα13是一个关键 骨质疏松症的负性调节因子--控制骨质疏松症细胞系定位和分化的成骨细胞 在生理和病理条件下，通过激活RhoA，减弱AKT信号通路。 为了检验我们的假设，本文提出了三个具体目标。在目标1中，我们将描述骨的表型。 GNα13f/fLysM-Cre小鼠，阐明Gα13激活RhoA信号转导途径的机制 减弱Akt信号，以负向调节OC细胞系的承诺和分化。在《目标2》中我们将 通过G蛋白偶联受体的特性确定OCS中Gα13上游信号级联 (GPCRs)与Gα13结合。我们将描述Gα13在骨骼中的作用机制 使用树突状细胞(DC)、单核细胞和OC特异性的GNA13 CKO和 在功能丧失和功能获得分析中分别过表达转基因小鼠。本研究 可能为研究成骨细胞分化的负性调节因子在骨稳态中的作用提供重要的见解 OC分化过度的溶骨性骨病。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Gα13信号在OCS中可能的治疗靶点 溶骨性骨病通过模仿正常的OC抑制信号通路控制OC的形成。一个 多学科研究团队，包括一名在OC生物学和动物模型方面具有专业知识的骨生物学家 一位在OC生物学和细胞信号方面有专长的骨生物学家；以及一位 已经建立了包括RA在内的自身免疫性疾病的专业知识来实现这一研究目标。

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.