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

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.

项目概要 骨量由成骨细胞的骨形成与骨吸收之间的平衡决定 破骨细胞。骨细胞，即嵌入骨骼内的有丝分裂后细胞，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，针对骨细胞的治疗代表 有前景的治疗绝经后骨质疏松症的新策略。骨细胞对外部信号做出反应，例如 作为甲状旁腺激素，协调骨骼重塑。信号级联中的关键一步 骨细胞对 PTH 的反应是抑制激酶盐诱导激酶 2 (SIK2)。小分子SIK2 抑制剂，例如 YKL-05-099，模仿 PTH 作用。尽管取得了这些进步，但目前仍存在重大知识差距 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 将确定盐诱导激酶在体内骨细胞中的作用。同时缺乏 SIK2 和 SIK3 的小鼠 表达 DMP1-Cre 的细胞已生成，并显示出与 甲状旁腺功能亢进症。将对这些小鼠进行详细的骨表型分析，重点是 甲状旁腺功能亢进性骨病之间的相似之处。在骨细胞中表达的 SIK 亚型中，SIK2 是 具有独特的 PTH 响应性，并充当负责 PTH 介导的底物去磷酸化和 目标基因表达。 SIK2条件性敲除小鼠将接受间歇性PTH治疗，每日一次，持续4次 周，并通过先进的放射学和组织学技术评估由此产生的骨表型。目标 2 的 该提案将定义不同 SIK 底物在骨细胞 PTH 反应中的贡献。我们 目前已知 PTH 介导的 HDAC4/5 去磷酸化调节硬化蛋白表达，并且 PTH- 介导的 CRTC2 去磷酸化控制 RANKL 上调。然而，PTH 调节大量 骨细胞中的靶基因，以及这些事件对甲状旁腺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 尚不清楚。我们进行了磷酸蛋白质组分析来鉴定新的磷蛋白，其 PTH 和小分子 SIK 抑制剂都会降低丰度。在此过程中，我们确定 FOXO3 是 新型SIK基板。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，我们将采用功能丧失 研究 CRTC2 和 FOXO3 在 PTH 调节的靶基因表达中相对贡献的方法 体外和体内。总而言之，这些研究将显着提高我们对 PTH/SIK 如何进行治疗的认识。 信号轴控制骨细胞生物学。详细了解 SIK 抑制和连接的步骤 基因表达的调控将阐明甲状旁腺激素作用的新机制 骨。此外，这项工作将确定新的骨质疏松药物靶点，并显着促进 开发 SIK 抑制剂作为骨合成代谢治疗剂。