PTH receptor Signaling and Diabetes-induced Bone Disease

骨细胞通过调节成骨细胞的产生和活性， 破骨细胞;但对骨细胞在骨病理生理学中的功能知之甚少。PI在 这项优异奖的上一个资助期显示，甲状旁腺激素1受体（PTH 1 R）的激活 骨细胞中的信号传导抑制骨细胞衍生的骨抑制剂Sost/sclerostin的表达 形成，并增加骨细胞中RANKL的表达，RANKL是破骨细胞分化的主要诱导剂。 此外，RANKL启动子中PTH调节结构域的缺失可消除PTH 1 R驱动的再吸收 激活骨细胞。此外，PTH 1 R骨细胞缺失的小鼠表现出降低的再吸收， 对甲状旁腺素的合成代谢反应缺陷。这项应用的研究还证实，患有糖尿病的小鼠 链脲佐菌素（S-DM）诱导的骨质疏松症表现为低骨量和较差的力学和材料性能; 吸收增加、形成减少和骨髓脂肪细胞（BMAT）增加;以及 骨细胞凋亡增加和Sost/sclerostin的高表达。此外，用抗糖尿病药物治疗S-DM小鼠， 通过PTH 1 R起作用的PTH相关蛋白（PTHrP）衍生肽（1-37）纠正了这些变化， 并激活存活信号，防止骨细胞凋亡。这项研究的长期目标是 确定靶向骨细胞及其产物治疗骨病的潜力。具体目标 该提案的目的是揭示PTH 1 R信号传导保护骨骼退化的潜在机制 在DM。我们假设，PTH或阿巴帕林（FDA批准）激活的骨细胞中的PTH 1 R信号传导 骨合成代谢剂）通过调节骨细胞衍生因子抵消DM在骨中的破坏作用， 从而维持骨质量和强度、保持骨细胞活力并降低BMAT。这一假设 将使用已建立的1型和2型DM、S-DM和高脂饮食（HFD-DM）的两种鼠模型进行测试， 分别与低胰岛素血症和高胰岛素血症相关，并使用药理学和遗传学工具激活 或抑制PTH 1 R信号传导，并干扰骨细胞基因产物。我们将努力实现以下目标： 目的1将检测PTH或abaloparavine对PTH 1 R信号传导的药理学激活是否恢复 S-DM或HFD-DM小鼠（近交系C57 BL/6和远交系Swiss韦伯斯特品系）的骨量和强度;以及 揭示潜在的细胞和分子机制。目的2将检查骨细胞对PTH 1 R的贡献 通过研究PTH或abaloparylate在S-DM中的作用， 骨细胞中PTH 1 R缺失的HFD-DM和对照小鼠（DMP 1 - 8 kb-Cre）。目标3将检查 骨细胞来源的Wnt/βcatenin拮抗剂在S-DM或HFD诱导的骨骼退化中的作用。 DM，通过研究骨细胞中缺乏Sost、Dkk 1或两者的小鼠（Sostf/f; Dkk 1f/f; DMP 1 - 8 kb-Cre）或 表达LRP 5高骨量突变pG 171 V的小鼠（对Sost和Dkk 1介导的抑制具有抗性）， Wnt-β连环蛋白信号传导）免受S-DM和HFD-DM诱导的对骨的影响。

Osteocytes play a critical role in bone homeostasis by regulating the production and activity of osteoblasts and osteoclasts; but less is known about osteocyte function in bone pathophysiology. Work by the PI during the previous funding period of this Merit award showed that activation of parathyroid hormone 1 receptor (PTH1R) signaling in osteocytes inhibits the expression of Sost/sclerostin, the osteocyte-derived inhibitor of bone formation, and increases in osteocytes the expression of RANKL, the master inducer of osteoclast differentiation. Moreover, deletion of PTH-regulated domains in the RANKL promoter abolished resorption driven by PTH1R activation in osteocytes. Further, mice with osteocytic deletion of PTH1R exhibit decreased resorption and defective anabolic response to PTH. Work leading to this application also established that mice with diabetes mellitus induced by streptozotocin (S-DM) exhibit low bone mass and inferior mechanical and material properties; increased resorption, decreased formation, and increased bone marrow adipocytes (BMAT); as well as increased osteocyte apoptosis and high expression of Sost/sclerostin. Further, treatment of S-DM mice with a PTH related protein (PTHrP)-derived peptide (1-37), which acts through the PTH1R, corrected these changes, and activated survival signaling preventing osteocyte apoptosis. The long term goal of this research is to determine the potential of targeting osteocytes and their products for treating bone maladies. The specific goal of this proposal is to unveil the mechanisms underlying protection of skeletal deterioration by PTH1R signaling in DM. We hypothesize that PTH1R signaling in osteocytes activated by PTH or abaloparatide (FDA-approved bone anabolic agents) counteracts the damaging actions of DM in bone by regulating osteocyte-derived factors, thus maintaining bone mass and strength, preserving osteocyte viability, and reducing BMAT. This hypothesis will be tested using two murine models of established type 1 and type 2 DM, S-DM and high fat diet (HFD-DM), associated with low versus high insulinemia, respectively, and using pharmacologic and genetic tools to activate or inhibit PTH1R signaling, and to interfere with osteocytic gene products. We will pursue the following aims: Aim 1 will examine whether pharmacologic activation of PTH1R signaling with PTH or abaloparatide restores bone mass and strength in S-DM or HFD-DM mice (in inbred C57BL/6 and outbred Swiss Webster strains); and reveal underlying cellular and molecular mechanisms. Aim 2 will examine osteocyte contribution to PTH1R signaling protective action on DM bone disease, by investigating the effect of PTH or abaloparatide in S-DM, HFD-DM and control mice with deletion of the PTH1R in osteocytes (DMP1-8kb-Cre). And Aim 3 will examine the role of osteocyte-derived Wnt/βcatenin antagonists on the skeletal deterioration induced by S-DM or HFD- DM, by investigating whether mice lacking Sost, Dkk1, or both in osteocytes (Sostf/f; Dkk1f/f; DMP1-8kb-Cre) or mice expressing the LRP5 high bone mass mutation pG171V (resistant to Sost- and Dkk1-mediated inhibition of Wnt-βcatenin signaling) are protected from S-DM and HFD-DM induced effects on bone.