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Interactions of PTH and Wnt signaling in bone formation

PTH 和 Wnt 信号在骨形成中的相互作用

基本信息

批准号：
10395962
负责人：
JOY Y WU
金额：
$ 34.42万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10395962
关键词：
Adult Aging Antibodies Biology Cells Combined Modality Therapy Cytometry Data Development Fluorescence Gene Expression Genes Genetic Growth Hip Fractures Histology Impairment Individual Injections Investigation Knock-out Lead Ligands Measures Mesenchymal Stem Cells Methods Mus Osteoblasts Osteogenesis Osteoporosis PTH gene Parathyroid Hormone Receptor Pathway interactions Pharmaceutical Preparations Pharmacology Population Positioning Attribute Proteins Publishing Receptor Signaling Recombinants Role Signal Pathway Signal Transduction Techniques Testing WNT Signaling Pathway Walking Water age related base beta catenin bone bone cell bone mass cohort fracture risk gene network hormonal signals inhibitor innovation microCT multidisciplinary novel osteoporosis with pathological fracture osteoprogenitor cell prevent programs regenerative approach response single-cell RNA sequencing transcriptome sequencing

项目摘要

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.

项目摘要骨质疏松症是常见的和毁灭性的，因为20%的成年人髋部骨折在1年内死亡，另外50% 再也不能独立行走了促进骨形成的药物可能是一种上级治疗方法。骨质疏松重组甲状旁腺激素（PTH）和抗sclerostin抗体增加骨形成，分别靶向PTH受体（PTH 1 R）和Wnt信号通路。然而，这些药物需要注射，只能使用两年。在小鼠中，PTH和抗硬化素联合治疗抗体比单独使用任何一种药物都更有效。了解结合的机制 PTH 1 R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。我们将测试我们的中心假设，即PTH 1 R是Wnt完全刺激骨形成所必需的。从机制上讲，我们认为PTH 1 R是稳定Wnt效应子β-连环蛋白所必需的。骨祖细胞，这反过来又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来激活骨骼中缺乏PTH 1 R的小鼠的Wnt信号传导：水溶性Wnt替代物，并通过基因敲除Wnt抑制剂sclerostin。我们建议使用两个创新的方法，以克服目前的障碍，了解PTH和Wnt信号如何在骨骼中相互作用。首先，我们将使用质谱细胞术（CyTOF）分析>40个参数的表达，使我们能够区分间充质干细胞、骨祖细胞和成骨细胞，并同时检查 PTH 1 R和Wnt信号在这些人群中。其次，我们将使用单细胞RNA测序来评估骨祖细胞中的PTH 1 R和Wnt信号传导。我们有初步的数据表明，在没有PTH 1 R信号的情况下，在骨祖细胞中，增加的Wnt信号传导不能增加骨。在具体目标1中，我们将确定完整的PTH 1 R信号传导是通过药理学和遗传学激活的Wnt依赖性骨形成所必需的 1月龄对照（PTH 1 ROsxWT）和PTH 1 ROsxKO小鼠中Wnt信号传导的变化。我们将评估骨形成，组织学、定量组织形态学和显微计算机断层扫描（µCT）。具体目标2 确定PTH 1 R是否是Wnt信号传导所必需的以增加骨祖细胞数量，对来自Aim 1的小鼠的骨细胞进行质量细胞计数，使用抗体组区分间充质干细胞，骨祖细胞和成骨细胞。我们将同时评估每个人群中的PTH 1 R和Wnt信号。我们将使用单细胞RNA测序（scRNA-seq）来确定PTH 1 R是否是Wnt信号传导所必需的。增加骨祖细胞中的成骨细胞基因程序。在目标3中，我们将在成人中验证我们的发现使用µCT、组织形态计量学、质量流式细胞术和scRNA-seq.这些目标的成功完成将使人们更详细地了解 PTH 1 R和Wnt合作增加骨形成的机制。