PTH actions on early cells of the osteoblast lineage

甲状旁腺激素(PTH)，间歇给药治疗骨质疏松症，或给药 持续进行啮齿动物实验，提高骨形成率。而成骨细胞、骨细胞和骨 衬里细胞参与甲状旁腺激素反应，成骨细胞前体在甲状旁腺激素反应中的作用较差 明白了。我们已经用Sox9的表达来标记早期细胞，Sox9是一种在许多干细胞类型中表达的基因 了解甲状旁腺素是如何影响这些细胞的。在目标1中，我们将确定 成骨细胞前体体内甲状旁腺素受体下游依次激活的信号通路 以增加这些前体的数量。因为Gs，即异三聚体G蛋白，是一种重要的 在成熟的成骨细胞和成骨细胞中，PTH受体下游的信号传递，我们将在 表达SOX9的成骨细胞前体细胞及其后代。因为携带甲状旁腺素受体突变的小鼠(所以- 称为DSEL受体)，不能激活GQ/11的人有异常的骨骼，我们将确定是否PTH 增加DSEL受体小鼠的成骨细胞前体的数量，与PTH在野生小鼠中的作用相同 输入老鼠。因为盐诱导蛋白激酶(SIKs)是PTH信号的重要调控靶点。 ，我们将确定抑制siks是否是激活下游的一个重要策略。 GSA在甲状旁腺激素诱导下成骨细胞前体细胞数量增加。在目标2中，我们将解决 成骨细胞系早期细胞的异质性。成骨细胞前体可以在生长过程中找到。 骨板、骨髓和骨膜。我们将在Aim 2A中使用血统追踪来确定生长板 成骨细胞前体(标记为PTHrPcreERt)在骨髓中成为表达Sox9的细胞，以及 SOX9标记的细胞在骨髓中成为CXCL12丰富的网状细胞(CAR)。此外，我们还发现 干骺端、骨内膜和骨膜中SOX9标记的细胞有截然不同的路径到达 成骨作用。以确定这些路径，并将Sox9标记的路径与 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-ChloéVillani，Aim 2B这一领域的领导者。人类基因构成的特征 单个细胞将允许我们定义骨骼干细胞的各种不同的命运和信号 调节这些细胞的分子/转录因子。在目标3中，我们将使用这些相同的方法 确定甲状旁腺素如何增加骨骼前体细胞的数量，以及细胞如何 当Gs信号在这些细胞中被阻断时，通路就会改变。单细胞RNA测序将用于 确定目标1中建立的通路下游的机制，PTH用来增加 成骨细胞前体细胞的数量。因此，我们将阐明各种骨骼干之间的关系 以及甲状旁腺激素如何改变骨骼前体细胞所使用的途径。

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.