Structural, Molecular and Functional Specialization in Osteocyte Mechanosensing

摘要 骨使其结构适应机械负荷。这种适应对于发展正确的 骨骼和维持其完整性贯穿一生。骨细胞是负责 感测和协调对机械负载的响应。我们最近报告的关键发现 在过去的几年里，一个小组确定骨细胞的细胞过程功能是独特的， 机械感觉元件过程对机械刺激的敏感度是 骨细胞胞体此外，这种从细胞过程触发Ca 2+信号传导的情况发生在 通过aVb 3整合素、膜通道和受体的独特复合物， 附着点在小管壁上，我们称之为“骨细胞机械体”。 这一建议是基于一个全球性的假设，即一个新的结构定位于骨细胞 骨细胞机械体处理、检测和传递机械信号。到目前为止， 我们已经鉴定了四种关键的骨细胞机械体成分：αVβ3整联蛋白，泛连接蛋白1， P2 X7受体（P2 X7 R）和CaV3.2 T型钙通道。我们的多学科团队将 通过三个目标中的每一个目标的多种方法来测试这个假设。在目标1中，我们将联合收割机 生物化学技术（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 这种迄今未知的转导复合物的结构和动力学性质， 体外骨细胞中的骨细胞机械小体。在目标2中，我们测试了药理学和 单个机械体组分的遗传改变改变上游（Ca 2+）， 体外骨细胞中的下游（至骨）信号传导。在目标3中，我们将联合收割机 OtGP 3骨细胞Ca 2+报告基因小鼠体内载药/成像系统 操作，以确认关键机械体组分的作用（如目的1和 2)对骨细胞Ca 2+反应和下游信号传导的影响。我们还将使用这种方法来 回答了骨细胞钙离子是否对机械负荷有反应的基本问题 因组成性性激素（雌激素/雄激素）丢失或合成代谢PTH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.