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: 7158599
• 负责人: ABDUL B ABOU-SAMRA
• 金额: $ 13.05万
• 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 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7158599
• 关键词: Achievement; Adult; Affect; Biology; Bone Density; Bone Development; Bone Diseases; Bone Growth; Bone Matrix; Bone Resorption; Bone and Cartilage Funding; Calcium; Cells; Complex; Cyclic AMP; Development; ES Cell Line; Epiphysial cartilage; Exhibits; Fetus; Goals; Grant; Homeostasis; Injection of therapeutic agent; Intracellular Second Messenger; Ions; Knock-in Mouse; Ligands; Maps; Mediating; Minerals; Modeling; Mus; Newborn Infant; Osteogenesis; Parathyroid Hormone Receptor; Parathyroid Hormone Receptors; Parathyroid Hormones; Peptide Receptor; Phenotype; Phosphorylation; Phosphorylation Site; Physiological; Play; Prevention; Property; Regulation; Role; Second Messenger Systems; Signal Transduction; Techniques; Therapeutic Agents; bone; bone cell; cartilage development; desensitization; design; homologous recombination; human PTH protein; in vivo; inorganic phosphate; mature animal; mouse model; mutant; novel therapeutics; parallel processing; parathyroid hormone-related protein; receptor; receptor internalization; response

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）：甲状旁腺激素（PTH)/PTH相关肽（PTHrP）受体（PTH /PTHrP受体或PTH1R）通过其同源配体PTH或PTHrP激活，启动两个平行过程：1）刺激细胞内第二信使，导致矿物离子稳态和骨细胞发育、分化和成熟的生物学效应；2) PTH1R的磷酸化，受体-配体复合物的内化以及由该受体介导的生物反应的脱敏。PTH1R磷酸化、内化和脱敏的生理作用尚不清楚。我们最近绘制了PTH1R的磷酸化位点，并发现了一个磷酸化缺陷（pd） PTH1R，它在配体刺激的内化中存在缺陷。利用同源重组技术，我们“敲入”了突变的pdPTH1R，以取代ES细胞系中的正常PTH1R，并已经开发出了纯合敲入（pd/pd）小鼠。pd/pd小鼠是可育的、可存活的、正常血钙的。然而，这些小鼠的甲状旁腺激素和磷酸盐水平都很低。此外，这些小鼠对PTH注射表现出延长和夸大的cAMP反应。甲状旁腺激素生物学中一个有趣的谜题是了解间歇给药甲状旁腺激素如何促进骨形成，而连续给药甲状旁腺激素如何促进骨吸收。在Specific Aim 1中，我们建议建立表达pdPTH1R的敲入小鼠模型，以了解PTH1R磷酸化、内化和脱敏在骨发育、分化和成熟中的作用，以及PTH间歇性给药在体内的合成代谢作用。在Specific Aim 2中，我们将检查小鼠pdPTH1R敲除的骨和软骨表型，以评估PTH1R的磷酸化和内化对骨和软骨细胞发育、分化和成熟的调节很重要的假设。在Specific Aim 3中，我们将使用该模型来检验PTH1R磷酸化和内化对间歇性PTH给药的合成代谢作用的表达起重要作用的假设。实现这个项目的目标将增加我们对PTH/PTHrP受体的磷酸化和内化如何影响骨中PTH行为的表达（形成和吸收）的理解；这对于了解骨病和设计新的治疗药物以预防和逆转骨病是至关重要的。