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: 10415056
• 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/10415056
• 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; 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; loss of function; mimetics; novel; paracrine; phosphoproteomics; protein activation; radiological imaging; response; salt-inducible kinase; skeletal; small molecule; synergism; targeted treatment; transcription factor; 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; 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; loss of function; mimetics; novel; paracrine; phosphoproteomics; protein activation; radiological imaging; 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 骨骼质量取决于成骨细胞之间骨骼形成和骨吸收之间的平衡。 破骨细胞。骨细胞，嵌入骨骼内的有丝分裂细胞，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素。因此，靶向骨细胞的疗法代表 有希望治疗绝经后骨质疏松症的新策略。骨细胞对外部提示做出反应， 作为甲状旁腺激素，可以编排骨骼重塑。信号级联的关键步骤 骨细胞对PTH的反应是激酶盐诱导激酶2（SIK2）的抑制作用。小分子sik2 抑制剂，例如YKL-05-099，模仿PTH作用。尽管有这些进步，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。目的1本提案 将确定盐诱导激酶在体内骨细胞中的作用。小鼠在 已经生成了表达DMP1-Cre-Cre的细胞，并且显示骨骼表型相当让人联想到 甲状旁腺功能亢进。将对这些小鼠进行详细的骨表型分析，重点是 甲状旁腺疾病之间的相似性。在骨细胞中表达的sik同工型中，sik2是 独特的PTH响应性，充当负责PTH介导的底物去磷酸化和 靶基因表达。 SIK2有条件敲除小鼠将每天用间歇性PTH治疗4 通过晚期放射学和组织学技术评估的几周和由此产生的骨表型。目标2 该建议将定义骨细胞中PTH响应中不同SIK底物的贡献。我们 目前知道PTH介导的HDAC4/5去磷酸化调节硬化素的表达，并且PTH- 介导的CRTC2去磷酸化控制RANKL上调。但是，PTH调节了大量 骨细胞中的靶基因，以及这些事件对甲状旁腺的总体反应的相对贡献 激素仍然未知。此外，PTH是否调节其他SIK底物的磷酸化 不知道。我们已经进行了磷酸蛋白质组学分析，以鉴定新的磷蛋白 PTH和小分子SIK抑制剂都会降低丰度。这样，我们确定了foxo3是一个 新颖的Sik底物。在这里，PTH调节FOXO3磷酸化的机制，亚细胞 将探索定位和靶基因表达。此外，我们将运用功能丧失 研究CRTC2和FOXO3在PTH调节的靶基因表达中的相对贡献的方法 体外和体内。综上所述，这些研究将大大提高我们对PTH/SIK的了解 信号轴控制骨细胞生物学。详细了解有关SIK抑制和连接的步骤 基因表达的调节将阐明甲状旁腺激素作用于中的新型机制 骨。此外，这项工作将确定新型的骨质疏松药物靶标，并显着促进 将SIK抑制剂作为骨合成代谢剂的发展。