Osteocyte control of bone formation via Sost

描述（由申请人提供）：最近令人信服的证据表明，硬化蛋白——骨中骨细胞独家表达的Sost基因的产物——拮抗wnt和bmp的促成骨作用；提供一种长期寻找的骨细胞调节骨形成的分子方法。PI及其合作者的这项应用表明，体内和体外，甲状旁腺激素（PTH）的慢性升高可有效降低骨细胞中Sost/sclerostin的表达，这表明由骨细胞介导的PTH依赖性成骨细胞形成的新机制。机械载荷也使成骨细胞数量增加；PTH相关肽（PTHrP）是这种合成代谢作用的潜在介质，因为它的表达在机械刺激下增加。值得注意的是，Sost的表达受到体外机械刺激和体内骨负荷的抑制。此外，仅在骨细胞中过表达组成活性PTH 1受体（PTHR1） （DMP1-caPTHR1）的转基因小鼠表现出Sost表达降低和骨量显著增加。基于这些证据，我们假设骨细胞中PTHR1信号的激活导致Sost基因表达的快速和直接抑制，这反过来又导致PTH全身性升高时骨形成增加，以及通过局部PTHrP增加骨负荷。这一假设将通过使用体外生成的骨细胞和真实骨细胞的体外研究以及使用转基因和敲除小鼠的体内方法来验证。Aim 1的研究将阐明PTH和PTHrP快速抑制Sost表达的信号通路。在Aim 2中，将确定拉伸或振荡流体流动诱导的机械刺激对体外Sost表达的抑制是否需要PTHR1信号和PTHrP；在小鼠尺骨负荷模型中，骨负荷导致的硬化蛋白表达降低是否与PTHrP升高和骨形成增加存在空间关系。在Aim 3中，将通过互补的转基因和敲除方法来确定体内骨细胞中PTHR1激活或缺失的后果。我们还将研究PTH升高或负荷诱导的成骨反应，或DMP1- caPTHRI小鼠的高骨量表型是否可以通过Sost过表达或阻断Wnt信号通路来逆转，或至少改善。此外，将确定PTHR1在骨细胞中特异性敲除的小鼠（DMP1-10kb-Cre/PTHR1小鼠）中Sost表达的减少和对PTH或机械负荷的成骨反应是否被消除。这些研究将促进对骨细胞控制骨形成的理解，并将阐明这些细胞对PTH、PTHrP和机械刺激的成骨作用的贡献。我们期望这项工作将为通过作用于骨细胞导致骨合成代谢的新治疗方法的发展提供机会。

DESCRIPTION (provided by applicant): Recent compelling evidence demonstrates that sclerostin - the product of the Sost gene exclusively expressed by osteocytes in bone - antagonizes the pro-osteoblastogenic actions of Wnts and BMPs; providing a long-sought molecular means by which osteocytes regulate bone formation. Work leading to this application by the PI and collaborators demonstrated that chronic elevation of parathyroid hormone (PTH) potently decreases Sost/sclerostin expression in osteocytes in vivo and in vitro, suggesting a novel mechanism for PTH-dependent osteoblastogenesis mediated by osteocytes. Mechanical loading also increases osteoblast number; and a potential mediator of this anabolic effect is PTH related peptide (PTHrP), as its expression is increased by mechanical stimulation. Notably, Sost expression in inhibited by mechanical stimuli in vitro and by bone loading in vivo. Moreover, transgenic mice overexpressing a constitutively active PTH 1 receptor (PTHR1) exclusively in osteocytes (DMP1-caPTHR1) exhibit decreased Sost expression and a remarkable increase in bone mass. Based on these lines of evidence, it is hypothesized that activation of PTHR1 signaling in osteocytes leads to a rapid and direct inhibition of Sost gene expression, which, in turn, is responsible for increased bone formation in response to systemic elevation of PTH as well as to bone loading through local increase in PTHrP. This hypothesis will be tested by a combination of in vitro studies using osteocytes generated in vitro and authentic osteocytes, and in vivo approaches using transgenic and knock out mice. Studies in Aim 1 will elucidate the signaling pathways responsible by the rapid inhibition of Sost expression by PTH and PTHrP. In Aim 2, it will be determined whether suppression of Sost expression in vitro by mechanical stimulation induced by stretching or oscillating fluid flow requires PTHR1 signaling and PTHrP; and whether the decreased sclerostin expression by bone loading is spatially related to increased PTHrP and increased bone formation using the model of ulna loading in mice. In Aim 3, the consequences of PTHR1 activation or deletion in osteocytes in vivo will be established by complementary transgenic and knock out approaches. It will be also examined whether the osteogenic response induced by PTH elevation or loading, or the high bone mass phenotype of DMP1- caPTHRI mice are reversed, or at least ameliorated, by Sost overexpression or by blocking the Wnt signaling pathway. Furthermore, it will be established whether the reduction in Sost expression and the osteoblastogenic response to PTH or mechanical loading are abrogated in mice in which the PTHR1 is knocked out specifically in osteocytes (DMP1-10kb-Cre/PTHR1 mice). These studies will advance understanding of the control of bone formation by osteocytes and will elucidate the contribution of these cells to the osteoblastogenic actions of PTH, PTHrP, and mechanical stimuli. We expect that this work will provide opportunities for the development of novel therapeutic approaches leading to bone anabolism through actions on osteocytes.