Osteocyte control of bone formation via Sost

描述(申请人提供)：最近令人信服的证据表明，硬化素-骨细胞独有表达的SOST基因的产物-拮抗WNTS和BMPs的成骨作用；提供了骨细胞调节骨形成的长期寻求的分子手段。PI及其合作者在这方面的研究表明，甲状旁腺激素(PTH)的慢性升高有效地降低了体内和体外骨细胞中Sost/skerostin的表达，提示了骨细胞介导的PTH依赖的成骨细胞形成的新机制。机械负荷也会增加成骨细胞的数量；这种合成代谢作用的一个潜在中介是甲状旁腺素相关肽(PTHrP)，因为它的表达通过机械刺激而增加。值得注意的是，SOST的表达在体外受到机械刺激的抑制，在体内受到骨负荷的抑制。此外，在骨细胞(DMP1-caPTHR1)中高表达活性PTH1受体(PTHR1)的转基因小鼠，SOST表达降低，骨量显著增加。根据这些证据推测，骨细胞中PTHR1信号的激活导致SOST基因表达的快速而直接的抑制，而SOST基因的表达反过来又负责响应全身甲状旁腺素的升高而增加骨形成，以及通过局部甲状旁腺素增加来增加骨负荷。这一假说将通过使用体外产生的骨细胞和真实的骨细胞进行的体外研究和使用转基因和敲除小鼠的体内方法相结合的方法来验证。目标1的研究将阐明PTH和PTHrP快速抑制SOST表达的信号通路。在目标2中，将利用小鼠尺骨负荷的模型，确定拉伸或振荡液体流动诱导的机械刺激抑制Sost的表达是否需要PTHR1信号和PTHrP；以及骨负荷导致的硬化素表达降低是否与PTHrP增加和骨形成增加在空间上相关。在目标3中，将通过互补转基因和基因敲除的方法来确定体内骨细胞中PTHR1激活或缺失的后果。此外，还将研究是否通过SOST过表达或通过阻断Wnt信号通路，逆转或至少改善由PTH升高或负荷引起的成骨反应，或DMP1-caPTHRI小鼠的高骨量表型。此外，在骨细胞中特异性敲除PTHR1的小鼠(DMP1-10kb-CRE/PTHR1小鼠)，是否可以取消SOST表达的减少和对PTH或机械负荷的成骨反应。这些研究将促进对骨细胞控制骨形成的理解，并将阐明这些细胞在甲状旁腺素、甲状旁腺素和机械刺激的成骨作用中的作用。我们期望这项工作将为开发通过作用于骨细胞而导致骨合成代谢的新的治疗方法提供机会。

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.