PTH actions on early cells of the osteoblast lineage
PTH actions on early cells of the osteoblast lineage
批准号:
10207597
负责人:
HENRY M. KRONENBERG
金额:
$40.85万
依托单位国家:
美国
项目类别:
财政年份:
2020
资助国家:
美国
项目状态:
已结题
起止时间:
2020-07-01 至 2024-06-30
关键词:
AblationAddressAdipocytesAdultAffectAgeBone MarrowBone SurfaceCXCL12 geneCellsCharacteristicsChondrocytesCollaborationsComplementCyclic AMP-Dependent Protein KinasesDataEndosteumEpiphysial cartilageExhibitsFractureGene ExpressionGene Expression ProfileGene Expression RegulationGenesGeneticHematopoiesisHeterogeneityHeterotrimeric GTP-Binding ProteinsHormonalHormone ReceptorHormone useHumanIn VitroIndividualKnock-inKnock-outLifeLocationMarrowMediatingMetaphysisMethodsMolecularMusNatureOsteoblastsOsteocytesOsteogenesisOsteoporosisPTH genePaperParathyroid Hormone ReceptorPathway interactionsPeriosteumPhosphotransferasesPopulationProliferatingPublishingReserve CellReticular CellRodentRoleSOX9 proteinSignal PathwaySignal TransductionSignaling MoleculeSodium ChlorideStromal CellsSupporting CellTestingTimeWild Type Mousebasebonebone masscalcium metabolismcell typeconditional knockoutexperimental studygenetic makeuphormonal signalshormone analogin vivokinase inhibitorlong bonemutantosteoprogenitor cellparathyroid hormone (1-34)parathyroid hormone-related proteinprecursor cellprogenitorreceptorrepairedresponsesingle cell sequencingsingle-cell RNA sequencingskeletalskeletal stem cellstemstem cellstranscription factor
中文摘要
甲状旁腺激素(PTH),无论是间歇性给药治疗骨质疏松症,还是
在啮齿类动物实验中,持续增加骨形成率。而成骨细胞骨细胞和骨
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
明白我们使用Sox 9基因的表达来标记早期细胞,Sox 9基因在许多干细胞类型中表达
并了解甲状旁腺素如何影响这些细胞。在目标1中,我们将确定
成骨细胞前体细胞内的信号通路,其在PTH受体下游被激活,
来增加这些前体的数量。因为Gs,异三聚体G蛋白,是一个重要的
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sox 9的成骨细胞前体及其后代。因为具有突变型PTH受体的小鼠(因此-
称为DSEL受体)不能激活Gq/11有异常的骨,我们将确定是否PTH
增加DSEL受体小鼠中成骨细胞前体的数量,与野生型PTH相同。
型小鼠。由于盐诱导的激酶(SIKs)是PTH信号传导的主要调节靶点,
骨细胞,我们将确定是否抑制SIKs是一个重要的策略下游的激活,
Gsa在PTH诱导的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中找到。
骨板骨髓和骨膜我们将在Aim 2A中使用谱系追踪来确定生长板是否
成骨细胞前体(标记为PTHrPcreER t)在骨髓中成为Sox 9表达细胞,
Sox 9标记的细胞在骨髓中变成富含CXCL 12的网状(CAR)细胞。此外,我们还发现
Sox 9标记的细胞在干骺端、骨内膜和骨膜中具有显著不同的途径,
成骨为了确定这些路径,并将Sox 9标记的路径与由
生长板干细胞和骨髓CAR细胞,我们将使用单细胞RNA测序与Dr。
Alexandra-Chloé Villani是Aim 2B这一领域的领导者。基因组成的表征
单个细胞将使我们能够确定骨骼干细胞的不同命运和信号转导的多样性。
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来
确定PTH如何增加骨骼前体细胞的数量,以及细胞如何
当Gs信号传导在这些细胞中被阻断时,通路发生变化。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用于增加
成骨细胞前体的数量。因此,我们将阐明骨骼茎品种之间的关系
细胞以及PTH如何改变骨骼前体细胞使用的途径。
英文摘要
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.
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资助金额:$92.4万
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依托单位:
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资助金额:$30.78万
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依托单位:
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