PTH1R REGULATION IN BONE BIOLOGY USING A MOUSE MODEL

使用小鼠模型研究骨生物学中的 PTH1R 调节

• 批准号: 6736473
• 负责人: ABDUL B ABOU-SAMRA
• 金额: $ 40.34万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-15 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6736473
• 关键词: biological models; bone development; cartilage development; cell differentiation; chondrocytes; enzyme linked immunosorbent assay; genetically modified animals; hormone receptor; in situ hybridization; laboratory mouse; model design /development; osteoblasts; parathyroid hormones; phosphorylation; physiologic bone resorption; receptor expression

DESCRIPTION (provided by applicant): Activation of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (the PTH/PTHrP receptor or PTH1R) by its cognate ligands, PTH or PTHrP, initiates two parallel processes: 1) stimulation of intracellular second messengers leading to biologic effects on mineral ion homeostasis and bone cell development, differentiation, and maturation; and 2) phosphorylation of PTH1R, internalization of the receptor - ligand complexes and desensitization of the biologic responses mediated by this receptor. The physiological role of PTH1R phosphorylation, internalization and desensitization is not known. We have recently mapped the phosphorylation sites on PTH1R and developed a phosphorylation-deficient (pd) PTH1R, which is defective in ligand-stimulated internalization. Using homologous recombination techniques we have "knocked in" the mutant pdPTH1R to replace the normal PTH1R in ES cell lines and have already developed homozygous knock-in (pd/pd) mice. The pd/pd mice are fertile, viable, and normocalcemic. However, these mice have low PTH and low phosphate levels. In addition, these mice showed prolonged and exaggerated cAMP response to PTH injection. One intriguing puzzle in PTH biology is to understand how intermittent PTH administration promotes bone formation whereas continuous PTH administration promotes bone resorption. In Specific Aim 1 we propose to develop a knock in mouse model expressing the pdPTH1R to understand the role of PTH1R phosphorylation, internalization and desensitization in bone development, differentiation and maturation and in the anabolic effects of intermittent PTH administration in vivo. In Specific Aim 2 we shall examine the bone and cartilage phenotype of the pdPTH1R knock in mouse to assess the hypothesis that phosphorylation and internalization of the PTH1R is important for the regulation of bone and chondrocytic cell development, differentiation and maturation. In Specific Aim 3 we shall use this model to examine the hypothesis that PTH1R phosphorylation and internalization play an important role for the expression of the anabolic actions of intermittent PTH administration. Achievement of the goals of this project will increase our understanding of how phosphorylation and internalization of the PTH/PTHrP receptor affects the expression of PTH actions in bone (formation and resorption); this is essential to understand bone diseases and design new therapeutic agents targeted for their prevention and reversal.

描述（由申请人提供）：通过同源配体，PTH或PTHRP激活与PTH或PTH相关的肽（PTH）受体（PTHRP）受体（PTH/PTHRP受体或PTH1R）的激活和成熟； 2）PTH1R的磷酸化，受体配体复合物的内在化以及该受体介导的生物学反应的脱敏。 PTH1R磷酸化，内在化和脱敏的生理作用尚不清楚。我们最近在PTH1R上绘制了磷酸化位点，并开发了磷酸化缺陷型（PD）PTH1R，该磷酸化位点在配体刺激的内在化中有缺陷。使用同源重组技术，我们“在”突变体PDPTH1R中“敲击”以替代ES细胞系中的正常PTH1R，并且已经开发了纯合敲入（PD/PD）小鼠。 PD/PD小鼠是肥沃的，可行的和正常的。但是，这些小鼠的PTH和低磷酸盐水平较低。此外，这些小鼠表现出长时间和夸张的cAMP对PTH注射的反应。 PTH生物学中的一个有趣的难题是了解间歇性PTH给药如何促进骨骼形成，而连续的PTH给药会促进骨吸收。在特定目标1中，我们建议在表达PDPTH1R的小鼠模型中开发敲击，以了解PTH1R磷酸化，内在化和脱敏在骨发育，分化和成熟以及体内间歇性PTH给药的合成代谢作用中的作用。在特定目标2中，我们将检查小鼠PDPTH1R敲击的骨和软骨表型，以评估以下假设：PTH1R的磷酸化和内在化对于调节骨骼和软骨细胞的发育，分化和成熟至关重要。在特定目标3中，我们将使用该模型来检验以下假设：PTH1R磷酸化和内在化对表达间歇性PTH给药的合成代谢作用起着重要作用。实现该项目的目标将增加我们对PTH/PTHRP受体的磷酸化和内在化如何影响骨骼中PTH作用的表达（形成和吸收）的理解；这对于了解骨骼疾病并设计针对预防和逆转的新治疗剂至关重要。