PTH actions on early cells of the osteoblast lineage

PTH 对成骨细胞谱系早期细胞的作用

基本信息

批准号：
10207597
负责人：
HENRY M. KRONENBERG
金额：
$ 40.85万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10207597
关键词：
Ablation Address Adipocytes Adult Affect Age Bone Marrow Bone Surface CXCL12 gene Cells Characteristics Chondrocytes Collaborations Complement Cyclic AMP-Dependent Protein Kinases Data Endosteum Epiphysial cartilage Exhibits Fracture Gene Expression Gene Expression Profile Gene Expression Regulation Genes Genetic Hematopoiesis Heterogeneity Heterotrimeric GTP-Binding Proteins Hormonal Hormone Receptor Hormone use Human In Vitro Individual Knock-in Knock-out Life Location Marrow Mediating Metaphysis Methods Molecular Mus Nature Osteoblasts Osteocytes Osteogenesis Osteoporosis PTH gene Paper Parathyroid Hormone Receptor Pathway interactions Periosteum Phosphotransferases Population Proliferating Publishing Reserve Cell Reticular Cell Rodent Role SOX9 protein Signal Pathway Signal Transduction Signaling Molecule Sodium Chloride Stromal Cells Supporting Cell Testing Time Wild Type Mouse base bone bone mass calcium metabolism cell type conditional knockout experimental study genetic makeup hormonal signals hormone analog in vivo kinase inhibitor long bone mutant osteoprogenitor cell parathyroid hormone (1-34)parathyroid hormone-related protein precursor cell progenitor receptor repaired response single cell sequencing single-cell RNA sequencing skeletal skeletal stem cell stem stem cells transcription factor

项目摘要

Parathyroid hormone (PTH), whether administered intermittently to treat osteoporosis, or administered continuously in rodent experiments, increases bone formation rate. While osteoblasts, osteocytes, and bone lining cells contribute to the PTH response, the role of osteoblast precursors in the PTH response is poorly understood. We have used expression of Sox9, a gene expressed in many stem cell types, to mark early cells in the osteoblast lineage and to understand how PTH affects these cells. In Aim 1, we will determine the signaling pathways inside osteoblast precursors that are activated downstream from the PTH receptor in order to increase the number of these precursors. Because Gs, the heterotrimeric G protein, is an important signaling relay downstream of the PTH receptor in mature osteoblasts and osteocytes, we will knockout Gsa in Sox9-expressing osteoblast precursors and their descendants. Because mice with a mutant PTH receptor (so- called DSEL receptor) that cannot activate Gq/11 have abnormal bone, we will determine whether PTH increases the number of osteoblast precursors in DSEL receptor mice in the same way that PTH does in wild type mice. Because salt-induced kinases (SIKs) are prominent regulatory targets of PTH signaling in osteocytes, we will determine whether inhibition of SIKs is an important strategy downstream of activation of Gsa in the PTH-induced increase in the number of osteoblast precursors. In Aim 2, we will address the heterogeneity of the early cells of the osteoblast lineage. Osteoblast precursors can be found in the growth plate, bone marrow, and periosteum. We will use lineage tracing in Aim 2A to determine whether growth plate osteoblast precursors (marked with PTHrPcreERt) become Sox9-expressing cells in the marrow and whether Sox9 marked cells become CXCL12-abundant reticular (CAR) cells in the marrow. Further, we have found that Sox9-marked cells in the metaphysis, the endosteum and periosteum have strikingly different paths to osteogenesis. To determine these paths, and also to compare Sox9-marked paths with those marked by growth plate stem cells and marrow CAR cells, we will use single cell RNA sequencing in collaboration with Dr. Alexandra-Chloé Villani, a leader in this field in Aim 2B. The characterization of the genetic makeup of individual cells will allow us to define the variety of distinct fates of skeletal stem cells and the signaling molecules/transcription factors that regulate these cells. In Aim 3, we will use these same methods to determine how PTH increases the numbers of cells descended from skeletal precursors and how the cellular pathways change when Gs signaling is blocked in these cells. Single cell RNA sequencing will be used to determine the mechanisms downstream of the pathways established in Aim 1 that PTH uses to increase the numbers of osteoblast precursors. Thus, we will clarify the relationships between the varieties of skeletal stem cells and how PTH changes the pathways used by skeletal precursors.

甲状旁腺激素 (PTH)，无论是间歇性施用以治疗骨质疏松症，还是施用在啮齿动物实验中持续增加成骨细胞、骨细胞和骨的形成率。衬里细胞有助于 PTH 反应，成骨细胞前体在 PTH 反应中的作用很差我们已经使用 Sox9（一种在许多干细胞类型中表达的基因）的表达来标记早期细胞。在目标 1 中，我们将确定成骨细胞谱系中的 PTH 如何影响这些细胞。成骨细胞前体内部的信号通路在 PTH 受体下游被激活，以便因为Gs（异源三聚体G蛋白）是一种重要的前体物质。成熟成骨细胞和骨细胞中 PTH 受体下游的信号传递，我们将敲除 Gsa 表达 Sox9 的成骨细胞前体及其后代。因为具有突变 PTH 受体的小鼠（所以- 不能激活Gq/11的人称为DSEL受体）有骨骼异常，我们将确定是否有PTH 增加 DSEL 受体小鼠中成骨细胞前体的数量，其方式与野生 PTH 的作用相同因为盐诱导激酶 (SIK) 是 PTH 信号传导的重要调节靶点。骨细胞，我们将确定 SIK 的抑制是否是骨细胞激活下游的重要策略 PTH 中的 Gsa 诱导的成骨细胞前体数量增加。在目标 2 中，我们将解决以下问题。成骨细胞谱系早期细胞的异质性可以在生长过程中发现。我们将使用 Aim 2A 中的谱系追踪来确定生长板是否存在。成骨细胞前体（用 PTHrPcreERt 标记）成为骨髓中表达 Sox9 的细胞，并且是否 Sox9 标记的细胞在骨髓中变成富含 CXCL12 的网状 (CAR) 细胞。干骺端、内膜和骨膜中的 Sox9 标记细胞具有显着不同的途径确定这些路径，并将 Sox9 标记的路径与标记的路径进行比较。生长板干细胞和骨髓CAR细胞，我们将与Dr.合作使用单细胞RNA测序。 Alexandra-Chloé Villani，Aim 2B 中该领域的领导者。单个细胞将使我们能够定义骨骼干细胞的各种不同命运和信号传导在目标 3 中，我们将使用这些相同的方法来调节这些细胞。确定 PTH 如何增加骨骼前体细胞的数量以及细胞如何当这些细胞中的 Gs 信号传导被阻断时，通路会发生变化。确定目标 1 中建立的途径的下游机制，PTH 用于增加因此，我们将阐明骨骼干品种之间的关系。细胞以及 PTH 如何改变骨骼前体使用的途径。