Structural, Molecular and Functional Specialization in Osteocyte Mechanosensing

摘要 骨骼使其结构适应机械负荷。这种适应对于发展右翼是必不可少的 骨骼，并在一生中保持其完整性。骨细胞是负责 感知和协调对机械载荷的响应。我们最近报告的主要发现 在过去的几年中，一个小组确定了骨细胞过程的独特功能 机械传感元件。过程对机械刺激的敏感度是机械刺激的10倍 骨细胞胞体。此外，这种从细胞过程中触发钙信号的过程发生 通过aVb3整合素、膜通道和受体的独特复合体，发生在 附着点指向管壁，我们称之为“骨细胞机械体”。 这一建议是基于一种全球假设，即一种位于骨细胞上的新结构 骨细胞机械小体负责处理、检测和传递机械信号。到目前为止， 我们已经确定了四个关键的骨细胞机械小体成分：αVβ3整合素，pAnnexin1， P2X7受体(P2X7R)和CaV3.2T型钙通道。我们的多学科团队将 在三个目标中的每一个中，通过多种方法来检验这一假设。在目标1中，我们将结合 生化技术(免疫共沉淀、表面等离子体共振)和成像 模式(FRAP、FRET和STORM超分辨率显微镜)以全面定义 这一迄今未知的转导复合体的结构和动力学性质 体外培养骨细胞中的骨细胞机械体。在目标2中，我们测试了药理和 单个机械体组件的基因改变改变上游(钙离子)和 体外培养的骨细胞中的下游(到骨)信号。在《目标3》中，我们将结合我们的小说 OtGP3骨细胞钙离子报告小鼠--体内药物负荷/成像系统 确认关键机械体部件的效果的操作(如目标1和 2)骨细胞钙反应和下游信号转导。我们还将使用此方法来 回答骨细胞钙离子是否对机械负荷作出反应这一根本问题 因失去构成性激素(雌激素/雄激素)或合成代谢的甲状旁腺激素而改变。

ABSTRACT Bone adapts its structure to mechanical loading. This adaption is essential for growing the right skeleton and maintaining its integrity throughout life. Osteocytes are the cells responsible for sensing and coordinating response to mechanical load. Key recent discoveries reported by our group during the last several years established that osteocyte cell processes function as unique mechanosensory elements. Processes are >10-fold more sensitive to mechanical stimuli than osteocyte cell bodies. Moreover, this triggering of Ca2+ signaling from cell processes occurs through a unique complex of aVb3 integrins, membrane channels and receptors, that occur at attachment points to the canalicular walls, and which we call the “Osteocyte mechanosome.” This proposal is based on the global hypothesis that a novel structure localized on osteocyte processes, the osteocyte mechanosome, detects and transduces mechanical signals. To date, we have identified four key osteocyte mechanosome components: αVβ3 integrin, pannexin1, P2X7 receptor (P2X7R) and the CaV3.2 T-type calcium channel. Our multidisciplinary team will test this hypothesis by multiple approaches in each of three aims. In Aim 1 we will combine biochemical techniques (co-immunoprecipitation, surface plasmon resonance) and imaging modalities (FRAP, FRET and STORM super-resolution microscopy) to define comprehensively the structural and dynamic properties of this heretofore unknown transduction complex, the osteocyte mechanosome in osteocytic cells in vitro. In Aim 2 we test how pharmacological and genetic alteration of individual mechanosome components alters upstream (Ca2+) and downstream (to bone) signaling in osteocytic cells in vitro. In Aim 3, we will combine our novel OtGP3 osteocyte Ca2+ reporter mice-in vivo loading/imaging system with pharmacological manipulations to confirm effects of key mechanosome components (as identified in Aims 1 and 2) on osteocyte Ca2+ response and on downstream signaling. We will also use this approach to answer the fundamental question of whether osteocyte Ca2+ responses to mechanical loading altered by loss of constitutive sex hormones (estrogen/androgen) or by anabolic PTH.