Interactions of PTH and Wnt signaling in bone formation

项目摘要 骨质疏松症是一种常见且具有破坏性的疾病，20%的成年髋部骨折患者在一年内死亡，另有50%的患者在1年内死亡 永远不要再独立行走了。促进骨形成的药物可能是一种更好的治疗方法 骨质疏松。重组甲状旁腺激素(PTH)和抗硬化素抗体通过 分别靶向PTH受体(PTH1R)和Wnt信号通路。然而，这些药物需要 注射，最多只能使用两年。甲状旁腺激素与抗硬化素联合治疗小鼠 抗体比单独用药更有效。了解联合的机制 PTH1R和Wnt信号增加骨量可能导致新的治疗方法来降低骨折风险。我们会 测试我们的中心假设，即WNT需要PTH1R来充分刺激骨形成。 从机制上讲，我们认为PTH1R是稳定Wnt效应分子b-catenin所必需的。 骨祖细胞，这反过来最大限度地刺激成骨细胞基因程序的表达。我们 将使用两种方法激活骨骼中PTH1R缺失的小鼠的Wnt信号：一种新的药理学方法 可溶于水的Wnt替代物，并通过基因敲除Wnt抑制物硬化素。我们建议使用两个 克服目前理解甲状旁腺激素和Wnt信号如何在骨骼中相互作用的障碍的创新方法。 首先，我们将使用质量细胞术(CyTOF)来分析&gt；40参数的表达，从而使我们能够区分 间充质干细胞、成骨祖细胞和成骨细胞，并同时检测 PTH1R和Wnt信号在这些人群中的表达。其次，我们将使用单细胞RNA测序来评估 成骨细胞中的PTH1R和Wnt信号。我们有初步数据表明，在没有PTH1R信号的情况下 在骨祖细胞中，增加的Wnt信号不能增加骨骼。在具体目标1中，我们将确定 完整的PTH1R信号是Wnt依赖的骨形成所必需的药理和遗传激活 1个月龄对照组(PTH1ROsxWT)和PTH1ROsxKO小鼠Wnt信号的表达。我们将通过以下方式评估骨形成 组织学、定量组织形态计量学和微计算机断层扫描(µCT)。在具体目标2中，我们将 通过执行以下操作来确定Wnt信号是否需要PTH1R来增加骨祖细胞数量 对来自AIM 1的小鼠的骨细胞进行质量细胞仪检测，使用抗体板来区分间充质干细胞， 骨祖细胞和成骨细胞。我们将同时评估每个种群中的PTH1R和Wnt信号。 我们将使用单细胞RNA测序(scRNA-seq)来确定Wnt信号是否需要PTH1R 增加成骨祖细胞的成骨基因程序。在目标3中，我们将在成人身上验证我们的发现 经Wnt替代配体处理的PTH1ROsxWT和PTH1ROsxKO小鼠，采用µCT、组织形态计量学、质量 流式细胞术和scRNA-seq.成功完成这些目标将使我们更详细地了解 PTH1R和Wnt协同促进骨形成的机制。

Project Summary Osteoporosis is common and devastating, as 20% of adults with a hip fracture die within 1 year and another 50% never walk independently again. Medications to promote bone formation might be a superior approach to treating osteoporosis. Recombinant parathyroid hormone (PTH) and anti-sclerostin antibody increase bone formation by targeting PTH receptor (PTH1R) and Wnt signaling pathways, respectively. However, these medications require injections and can only be used for up to two years. In mice, combined treatment with PTH and anti-sclerostin antibody is more effective than either medication alone. Understanding the mechanisms by which combined PTH1R and Wnt signaling increase bone mass could lead to novel treatments to decrease fracture risk. We will test our central hypothesis that PTH1R is required for Wnt to fully stimulate bone formation. Mechanistically, we propose that PTH1R is required to stabilize the Wnt effector b-catenin in osteoprogenitors, which in turn maximally stimulates expression of the osteoblast gene program. We will use two approaches to activate Wnt signaling in mice lacking PTH1R in bone: pharmacologically with a novel water soluble Wnt surrogate, and genetically by knocking out the Wnt inhibitor sclerostin. We propose to use two innovative methods to overcome current barriers to understanding how PTH and Wnt signaling interact in bone. First, we will use mass cytometry (CyTOF) to analyze expression of >40 parameters, allowing us to distinguish mesenchymal stem cells, osteoprogenitors and osteoblasts, and to simultaneously examine the effects of PTH1R and Wnt signaling in these populations. Second, we will use single-cell RNA-sequencing to evaluate PTH1R and Wnt signaling in osteoprogenitors. We have preliminary data that in the absence of PTH1R signaling in osteoprogenitors, increased Wnt signaling fails to increase bone. In Specific Aim 1 we will determine whether intact PTH1R signaling is required for Wnt-dependent bone formation by pharmacological and genetic activation of Wnt signaling in 1 month-old control (PTH1ROsxWT) and PTH1ROsxKO mice. We will assess bone formation by histology, quantitative histomorphometry, and micro-computed tomography (µCT). In Specific Aim 2 we will determine whether PTH1R is required for Wnt signaling to increase osteoprogenitor numbers by performing mass cytometry on bone cells of mice from Aim 1, using antibody panels to distinguish mesenchymal stem cells, osteoprogenitors and osteoblasts. We will simultaneously evaluate PTH1R and Wnt signaling in each population. We will use single-cell RNA-sequencing (scRNA-seq) to determine whether PTH1R is required for Wnt signaling to increase osteoblast gene programs in osteoprogenitors. In Aim 3 we will validate our findings in adult PTH1ROsxWT and PTH1ROsxKO mice treated with Wnt surrogate ligand, using µCT, histomorphometry, mass cytometry and scRNA-seq. Successful completion of these aims will provide more detailed understanding of the mechanisms by which PTH1R and Wnt cooperate to increase bone formation.