Connexin channels in transducing mechanical signals in bone

项目摘要 骨骼组织所经历的机械载荷在骨的形成和改建过程中起着重要作用。 骨细胞是最丰富的骨细胞类型，也是骨的主要机械感觉细胞。他们 通过协调成骨细胞的形成，从它们在整个骨基质中的位置协调骨重建 和破骨细胞吸收。骨细胞通过间隙连接到邻近的骨细胞和其他骨细胞。 连接通道，并通过半通道进入细胞外环境。这两种类型的通道都是由 连接蛋白(CX)43.Cx43参与骨组织对机械刺激的反应 在基因敲除模型中显示；然而，缝隙连接通道和半通道在 骨细胞，以及这些通道在机械加工过程中的生理作用机制 装载情况在很大程度上仍不得而知。为了剖析这两种类型的通道的生理作用，我们有 最近建立了两种表达Cx43显性负突变的转基因小鼠模型 在骨细胞中。我们还在建立Cx43突变基因敲击小鼠模型，以补充我们的 转基因模型。这些突变损害了骨细胞缝隙连接通道和/或半槽。此外, 我们已经产生了特异性地抑制Cx43半通道的抗体，但不能抑制缝隙连接通道。 此外，我们开发了新的方法，使我们能够在原位评估骨细胞半通道活动。 初步数据显示，Cx43半通道的损伤减弱了机械刺激的合成代谢效应 在骨头上装货。我们和其他人还表明，骨细胞Cx43半管是由 机械应力，体外释放前列腺素等小骨合成代谢因子。我们的目标是 确定Cx43半通道在机械合成代谢中的特殊机制作用 加载到骨骼组织上。提出了三个具体目标：1)检验骨细胞 Cx43半通道在调节骨骼组织机械负荷的合成代谢功能中起着重要作用。 2)检验成骨细胞Cx43半管介导机械负荷合成代谢功能的假说 通过释放PGE2，激活PGE2和Wnt信号。3)检验激活和激活的假设 Cx43半通道失活受整合素活化/细胞骨架和MAPK的特异性调节 分别发信号。拟议的研究预计将产生重大的积极影响，因为它界定了 机械转导机制及其在骨组织中的调节，构成潜力，意义 对开发治疗骨质疏松症和骨质疏松的新治疗剂的贡献 损失。

Project Summary Mechanical loading experienced by skeletal tissues plays an important role in bone formation and remodeling. Osteocytes are the most abundant bone cell type and the major mechanosensory cells of the bone. They orchestrate bone remodeling from their location throughout bone matrix by coordinating osteoblastic formation and osteoclastic resorption. Osteocytes are connected to neighboring osteocytes and other bone cells via gap junction channels and to extracellular environments via hemichannels. Both types of channels are formed by connexin (Cx) 43. The involvement of Cx43 in response to mechanical stimulation of bone tissue has been shown in gene knockout models; however, the distinct functions of gap junction channels and hemichannels in osteocytes, as well as the mechanism underlying the physiological roles of these channels during mechanical loading remain largely unknown. To dissect the physiological roles of these two types of channels, we have recently developed two transgenic mouse models expressing Cx43 dominant negative mutants predominantly in osteocytes. We are also generating a Cx43 mutant gene knockin mouse model to complement our transgenic models. These mutations impair osteocytic gap junction channels and/or hemichannels. In addition, we have generated antibodies that specifically inhibit Cx43 hemichannels, but not gap junction channels. Moreover, we have developed novel approaches that allow us to assess osteocytic hemichannel activity in situ. Preliminary data show that impairment of Cx43 hemichannels attenuates the anabolic effect of mechanical loading on the bone. We and others have also shown that osteocytic Cx43 hemichannels are opened by mechanical stress, releasing small bone anabolic factors including prostaglandins in vitro. The objective is to determine the specific mechanistic role of Cx43 hemichannels in mediating the anabolic effect of mechanical loading on the skeletal tissues. Three specific aims are proposed: 1) To test the hypothesis that osteocytic Cx43 hemichannels play a crucial role in mediating anabolic function of mechanical loading on skeletal tissue. 2) To test the hypothesis that osteocytic Cx43 hemichannels mediate anabolic function of mechanical loading via PGE2 release, and activation of PGE2 and Wnt signaling. 3) To test the hypothesis that activation and inactivation of Cx43 hemichannels are specifically regulated by integrin activation/cytoskeleton and MAPK signaling, respectively. The proposed studies are expected to have a major positive impact by defining the mechanical transduction mechanism and its regulation in bone tissue, constituting potential, significant contributions toward the development of new therapeutic agents for the treatment of osteoporosis and bone loss.