The role of salt inducible kinases in parathyroid hormone action in bone

盐诱导激酶在骨甲状旁腺激素作用中的作用

• 批准号: 9980386
• 负责人: Marc Nathan Wein
• 金额: $ 40.16万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980386
• 关键词: Address; Anabolism; Binding; Biology; Bone Diseases; Bone Resorption; Bone remodeling; CREB1 gene; Cell Nucleus; Cells; Cellular biology; Cues; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Development; Diagnostic radiologic examination; Drug Targeting; Enhancers; Equilibrium; Event; FOXO3A gene; GTP-Binding Protein alpha Subunits, Gs; Gene Expression; Gene Expression Regulation; Genes; Goals; HDAC4 gene; Histological Techniques; Histone Deacetylase; Hormone Responsive; Hyperparathyroidism; In Vitro; Injections; Innate Bone Remodeling; Insulin-Like Growth Factor I; Knockout Mice; Knowledge; Link; Mediating; Mitotic; Molecular; Mus; Nuclear Translocation; Osteoblasts; Osteoclasts; Osteocytes; Osteogenesis; Osteoporosis; PTH gene; Parathyroid Hormone Receptor; Pathway interactions; Phenotype; Phosphoproteins; Phosphorylation; Phosphotransferases; Play; Postmenopausal Osteoporosis; Production; Protein Dephosphorylation; Protein Isoforms; Proteins; Public Health; Regulation; Resistance; Role; Signal Pathway; Signal Transduction; Stimulus; TNFSF11 gene; Testing; Time; Up-Regulation; Work; aging population; bone; bone cell; bone mass; conditional knockout; cost; defined contribution; effective therapy; fragility fracture; hormonal signals; hormone analog; in vivo; inhibitor/antagonist; loss of function; mimetics; novel; paracrine; phosphoproteomics; protein activation; response; salt-inducible kinase; skeletal; small molecule; synergism; targeted treatment; transcription factor

Project summary Bone mass is determined by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteocytes, post-mitotic cells embedded within bone, control this balance by producing paracrine factors that regulate osteoblast and osteoclast activity. Therefore, therapies that target osteocytes represent promising new strategies to treat post-menopausal osteoporosis. Osteocytes respond to external cues, such as parathyroid hormone, to orchestrate bone remodeling. A crucial step in the signaling cascade through which osteocytes respond to PTH is inhibition of the kinase salt inducible kinase 2 (SIK2). Small molecule SIK2 inhibitors, such as YKL-05-099, mimic PTH action. Despite these advances, major knowledge gaps currently exist in our understanding of how the PTH/SIK signaling axis regulates skeletal biology. Aim 1 of this proposal will determine the role of salt inducible kinases in osteocytes in vivo. Mice lacking both SIK2 and SIK3 in DMP1-Cre-expressing cells have been generated, and display skeletal phenotypes quite reminiscent of hyperparathyroidism. Detailed bone phenotypic analysis of these mice will be performed, focusing on similarities between hyperparathyroid bone disease. Of the SIK isoforms expressed in osteocytes, SIK2 is uniquely PTH-responsive, and acts as a switch responsible for PTH-mediated substrate dephosphorylation and target gene expression. SIK2 conditional knockout mice will be treated with intermittent PTH once daily for 4 weeks, and resultant bone phenotypes assessed by advanced radiographic and histologic techniques. Aim 2 of this proposal will define the contribution of distinct SIK substrates in PTH responses in osteocytes. We currently know that PTH-mediated HDAC4/5 dephosphorylation regulates sclerostin expression, and that PTH- mediated CRTC2 dephosphorylation controls RANKL upregulation. However, PTH regulates a large number of target genes in osteocytes, and the relative contributions of these events to the overall response to parathyroid hormone remains unknown. Furthermore, whether PTH regulates phosphorylation of additional SIK substrates is not known. We have performed phosphoproteomic profiling to identify novel phosphoproteins whose abundance is decreased by both PTH and small molecule SIK inhibitors. In doing so, we identified FOXO3 is a novel SIK substrate. Here, the mechanisms through which PTH regulates FOXO3 phosphorylation, subcellular localization, and target gene expression will be explored. In addition, we will employ loss of function approaches to study the relative contribution of CRTC2 and FOXO3 in PTH-regulated target gene expression in vitro and in vivo. Taken together, these studies will significantly advance our knowledge of how the PTH/SIK signaling axis controls osteocyte biology. A detailed understanding of the steps linking SIK inhibition and regulation of gene expression will illuminate novel mechanisms through which parathyroid hormone acts in bone. Furthermore, this work will identify novel osteoporosis drug targets, and significantly facilitate the development of SIK inhibitors as bone anabolic treatment agents.

项目摘要 骨质量由成骨细胞的骨形成和成骨细胞的骨吸收之间的平衡决定。 破骨细胞骨细胞，有丝分裂后的细胞嵌入骨，通过产生旁分泌来控制这种平衡 调节成骨细胞和破骨细胞活性的因子。因此，靶向骨细胞的疗法代表了 治疗绝经后骨质疏松症的新策略。骨细胞对外界信号有反应， 作为甲状旁腺激素，来协调骨骼重塑。信号级联的关键一步， 骨细胞对PTH的反应是抑制激酶盐诱导激酶2（SIK 2）。小分子SIK 2 抑制剂如YKL-05-099模拟PTH作用。尽管取得了这些进展，但目前的主要知识差距 存在于我们对PTH/SIK信号轴如何调节骨骼生物学的理解中。本提案的目标1 将确定体内骨细胞中盐诱导激酶的作用。小鼠缺乏SIK 2和SIK 3， 已经产生了表达DMP 1-Cre的细胞，并显示出骨骼表型， 甲状旁腺机能亢进将对这些小鼠进行详细的骨表型分析，重点是 甲状旁腺增生性骨病之间的相似之处。在骨细胞中表达的SIK同种型中，SIK 2是 独特的PTH响应，并作为负责PTH介导的底物去磷酸化的开关， 靶基因表达。SIK 2条件性敲除小鼠将用间歇性PTH每天一次治疗4 周，并通过先进的放射学和组织学技术评估所得的骨表型。目标2 该建议将确定骨细胞中不同的SIK底物在PTH应答中的作用。我们 目前已知PTH介导的HDAC 4/5去磷酸化调节硬化蛋白表达，并且PTH- 介导的CRTC 2去磷酸化控制RANKL上调。然而，PTH调节大量的 骨细胞中的靶基因，以及这些事件对甲状旁腺激素总体反应的相对贡献。 激素仍然未知。此外，PTH是否调节其他的SIK底物的磷酸化， 是未知的。我们已经进行了磷酸蛋白质组学分析，以确定新的磷酸蛋白， 丰度被PTH和小分子SIK抑制剂降低。在这样做的过程中，我们确定FOXO 3是一种 新型的SIK衬底。在这里，PTH调节FOXO 3磷酸化、亚细胞 定位和靶基因表达将被探索。另外，我们将采用功能丧失 研究CRTC 2和FOXO 3在PTH调节的靶基因表达中的相对作用的方法 在体外和体内。综上所述，这些研究将显着推进我们的知识如何PTH/SIK 信号轴控制骨细胞生物学。详细了解连接抑制SIK和 基因表达的调节将阐明甲状旁腺激素在 骨头此外，这项工作将确定新的骨质疏松症药物靶点，并显着促进 作为骨合成代谢治疗剂的SIK抑制剂的开发。