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

项目总结 骨量是由成骨细胞的骨形成和骨吸收之间的平衡决定的。 破骨细胞。骨细胞，即嵌入骨骼中的有丝分裂后细胞，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子。因此，以骨细胞为靶点的疗法 治疗绝经后骨质疏松症的新策略前景看好。骨细胞对外部信号做出反应，如 作为甲状旁腺激素，来协调骨骼重建。信号级联过程中的关键一步 骨细胞对甲状旁腺激素的反应是抑制激酶盐诱导的蛋白激酶2(SIK2)。小分子SIK2 抑制剂，如YKL-05-099，模拟甲状旁腺素的作用。尽管取得了这些进展，但目前主要的知识差距 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 将确定盐诱导蛋白在体内骨细胞中的作用。同时缺乏SIK2和SIK3的小鼠 表达DMP1-Cre的细胞已经产生，并显示出非常令人想起的骨骼表型 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 甲状旁腺功能亢进性骨病。在骨细胞中表达的SIK亚型中，SIK2是 独特的甲状旁腺素反应，并作为一个开关负责甲状旁腺素介导的底物去磷酸化和 靶基因表达。SIK2条件性基因敲除小鼠将接受间歇性甲状旁腺素治疗，每天一次，共4次 通过先进的放射学和组织学技术评估骨表型。目标2 这一建议将确定不同的SIK底物在骨细胞甲状旁腺激素反应中的作用。我们 目前已知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的上调。然而，甲状旁腺素调节了大量的 骨细胞中的靶基因，以及这些事件在甲状旁腺整体反应中的相对贡献 荷尔蒙仍不清楚。此外，甲状旁腺激素是否调节额外的SIK底物的磷酸化 是未知的。我们已经进行了磷蛋白组学分析来鉴定新的磷蛋白，其 甲状旁腺素和小分子SIK抑制剂均可降低其丰度。在这样做的过程中，我们发现FOX03是一种 新型SIK基板。在这里，甲状旁腺激素调节FOXO_3磷酸化的机制，亚细胞 本地化，以及目标基因的表达将被探索。此外，我们还将使用功能损失 CRTC2和FOXO_3在甲状旁腺激素调控靶基因表达中相对贡献的研究方法 在体外和体内。综上所述，这些研究将大大提高我们对PTH/SIK如何 信号轴控制着骨细胞的生物学。详细了解将SIK抑制和SIK抑制 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，并显著促进 Sik抑制剂作为骨合成代谢治疗药物的研究进展。

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.