The role of Piezo1 in bone homeostasis and mechanotransduction

项目总结/摘要 机械刺激促进骨骼生长，对成年期骨骼的稳态至关重要。损失 机械信号降低了骨量并增加了骨折风险。骨细胞，是埋藏在 骨基质来源于成骨细胞，能够感知机械负荷的变化并协调骨 重塑有几条证据表明，钙通道参与了机械传导的感知。 骨细胞负载。例如，钙内流是骨细胞对机械力的最早反应之一 体外和体内刺激。这与钙信号在机械刺激反应中的功能作用一致。 力，骨细胞对机械刺激的反应可以通过阻断钙通道来抑制， 化学阻滞剂。此外，负荷诱导的骨形成在大鼠尺骨显着钝化钙 通道抑制剂然而，由机械力激活的钙通道的特性及其 在骨中作为机械传感器的功能作用尚不清楚。我们已经发现Piezo 1钙通道是 在骨细胞中高度表达，并且其表达和活性通过机械刺激而增加， 骨细胞此外，成骨细胞和骨细胞中Piezo 1的缺失会降低骨量和骨密度。 在小鼠中，这与骨骼对机械刺激的反应性丧失一致。而且 在成骨细胞和骨细胞中缺乏Piezo 1的小鼠中，骨骼对合成代谢负荷的反应减弱。Wnt 1，a 已知Wnt信号传导的配体被机械信号上调并刺激骨形成， 在Piezo 1条件性敲除小鼠中下调。重要的是，Piezo 1被其化学激动剂激活， Yoda 1，模拟流体流动对骨细胞的影响，并增加小鼠的骨量。基于此 证据，我们假设骨细胞通过Piezo 1感知机械信号的变化， 促进骨形成部分通过激活信号通路，增加Wnt 1的表达。测试 基于这一假设，我们将确定骨细胞表达Piezo 1是否是机械传感所必需的 在老鼠的骨骼中。我们将产生小鼠，其中Piezo 1从骨细胞中删除，但不是成骨细胞， 并将其骨骼表型与成骨细胞和骨细胞中缺乏Piezo 1的小鼠中观察到的骨骼表型进行比较。 我们还将在成年小鼠出生后删除Piezo 1，并研究其对胫骨机械负荷的反应 压缩（目标1）。此外，为了了解Piezo 1如何促进骨形成，我们将确定 使用小鼠遗传方法研究Wnt 1在体内Piezo 1介导的骨形成中的作用（Aim 2）。在目标3中， 将确定Piezo 1是否负责骨骼对使用尾悬架卸载的反应 模型最后，我们将确定Piezo 1的药理学激活是否可以防止与骨丢失相关的骨丢失。 与卸载或增加老年小鼠的骨量有关。这项工作的成功完成应建立一个新的 了解骨骼对合成代谢机械负荷的反应，并可能提出新的策略 开发合成代谢疗法，治疗与废用或衰老有关的骨质流失。

PROJECT SUMMARY/ABSTRACT Mechanical stimuli promote bone growth and are critical for skeletal homeostasis during adulthood. Loss of mechanical signals decreases bone mass and increases fracture risk. Osteocytes, which are cells buried in the bone matrix and derived from osteoblasts, are able to sense changes in mechanical load and orchestrate bone remodeling. Several lines of evidence suggest that calcium channels are involved in the sensing of mechanical load by osteocytes. For example, calcium influx is one of the earliest responses of osteocytes to mechanical stimuli in vitro and in vivo. Consistent with a functional role for calcium signaling in the response to mechanical forces, the response of osteocytes to mechanical stimuli can be inhibited by blocking calcium channels using chemical blockers. Moreover, load-induced bone formation in the rat ulna is significantly blunted by calcium channel inhibitors. However, the identity of the calcium channels activated by mechanical forces and their functional role as mechanosensors in bone remain unclear. We have found that Piezo1 calcium channel is highly expressed in osteocytes, and that its expression and activity are increased by mechanical stimulation in osteocytes. In addition, deletion of Piezo1 in osteoblasts and osteocytes decreases both bone mass and bone strength in mice, consistent with loss of skeletal responsiveness to mechanical stimulation. Moreover, the skeletal response to anabolic loading is blunted in mice lacking Piezo1 in osteoblasts and osteocytes. Wnt1, a ligand for Wnt signaling that is known to be upregulated by mechanical signals and stimulate bone formation, is downregulated in Piezo1 conditional knockout mice. Importantly, activation of Piezo1 by its chemical agonist, Yoda1, mimics the effects of fluid flow on osteocytes and increases bone mass in mice. Based on this evidence, we hypothesize that osteocytes sense changes in mechanical signals through Piezo1 and thereby promote bone formation in part by activating signaling pathways that increase the expression of Wnt1. To test this hypothesis, we will determine whether Piezo1 expression by osteocytes is required for mechanical sensing in the murine skeleton. We will generate mice in which Piezo1 is deleted from osteocytes, but not osteoblasts, and compare their skeletal phenotype to that observed in mice lacking Piezo1 in osteoblasts and osteocytes. We also will delete Piezo1 postnatally in adult mice and investigate their response to mechanical loads by tibia compression (Aim 1). In addition, to understand how Piezo1 promotes bone formation, we will determine the role of Wnt1 in Piezo1-mediated bone formation in vivo using a mouse genetic approach (Aim 2). In Aim 3, we will determine whether Piezo1 is responsible for the skeletal response to unloading using a tail-suspension model. Lastly, we will determine whether pharmacological activation of Piezo1 prevents bone loss associated with unloading or increases bone mass in old mice. Successful completion of this work should establish a new model for understanding the skeletal response to anabolic mechanical loading and may suggest new strategies to develop anabolic therapies for bone loss related to disuse or aging.