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

表征小鼠模型生理和病理条件下破骨细胞内源性负调节因子控制骨稳态的机制

• 批准号: 10402666
• 负责人: Wei Chen
• 金额: $ 32.44万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10402666
• 关键词: AKT Signaling Pathway; Aging; Animal Model; Attenuated; Autoimmunity; Biological Assay; Biology; Bone Development; Bone Diseases; Bone Resorption; Bone remodeling; Cartilage; Cell Lineage; Co-Immunoprecipitations; Collagen Arthritis; Coupled; Data; Dendritic Cells; Disease; Down-Regulation; Exhibits; G-Protein-Coupled Receptors; Generations; Genes; Goals; Guanine; Homeostasis; Immunologist; Inflammation; Interdisciplinary Study; Investigation; Knowledge; Ligands; Microarray Analysis; Mus; Nuclear; Osteoclasts; Osteolytic; Osteoporosis; Ovariectomy; Pathologic; Phenotype; Physiological; Proteins; Regulation; Research; Rheumatoid Arthritis; Role; Signal Pathway; Signal Transduction; System; TNF Receptor-Associated Factors; TRAF6 gene; Testing; Therapeutic; Tissues; Transgenic Mice; base; bone; bone loss; conditional knockout; design; effective therapy; gain of function; human disease; human model; improved; in vivo; inhibitor/antagonist; innovation; insight; loss of function; monocyte; mouse model; new therapeutic target; novel; novel therapeutic intervention; osteoclastogenesis; overexpression; receptor; repaired; side effect; therapeutic target

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.

该应用程序的直接目标是了解破骨细胞 (OC) 分化是如何通过以下方式调节的： 负内源性调节因子，可能为骨疾病提供新的治疗靶点，例如 骨质疏松症和类风湿性关节炎（RA）。虽然 OC 分化的正调节剂通过 核因子kB受体激活剂(RANK)配体(RANKL)-RANK信号轴已被广泛研究 研究表明，对 OC 分化的负调节因子的理解是难以捉摸的。确定关键的负面监管因素 为了研究 OC 分化，我们利用了微阵列、基因下调和破骨细胞生成检测，从而 鸟嘌呤蛋白 α 亚基 13（Gα13，由 Gna13 基因编码）的分离作为潜在的 OC 阴性 调节器。为了能够体内研究 Gα13 在 OC 分化中的作用，我们生成了 OC 谱系 通过将 Gna13f/f 小鼠与 LysM-Cre (OC 前体特异性）小鼠。我们的初步数据显示 Gna13f/fLysM-Cre 小鼠表现出严重的 OC 分化急剧增加导致骨质疏松。我们还注意到 Gα13 缺乏会减弱 RhoA 活性并促进 OC 中的 Akt 活性。始终如一地，我们表明，作为原则策略的证明，本地 持续活跃的 Gα13 过度表达可以防止骨和软骨损失，同时还可以减弱 RA 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 负调节因子，控制 OC 细胞谱系定型和骨稳态分化 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，我们将描述以下骨骼的表型： Gnα13f/fLysM-Cre 小鼠并阐明 Gα13 如何激活 RhoA 信号传导的机制 减弱 Akt 信号传导以负向调节 OC 细胞谱系定型和分化。在目标 2 中，我们将 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 结合。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 Gna13 CKO 和 分别在功能丧失和功能获得分析中过度表达转基因小鼠。这项研究 可能为了解 OC 分化负调节因子在骨稳态中的作用提供重要见解 以及OC过度分化的溶骨性骨病。从这项研究中获得的知识可能会产生 通过表征 OC 中可能靶向治疗的 Gα13 信号传导，确定潜在的治疗靶点 溶骨性骨疾病通过模仿正常的OC抑制信号通路来控制OC的形成。一个 多学科研究团队，包括一位在 OC 生物学和动物模型方面具有专业知识的骨生物学家 骨骼疾病；骨生物学家，具有 OC 生物学和细胞信号传导方面的专业知识；和一位免疫学家 为了实现研究目标，已经建立了包括 RA 在内的自身免疫性疾病方面的专业知识。