Connexin 43 Hemichannels and Signaling In Bone

描述（由申请人提供）：连接蛋白（Cx）是细胞间通讯以及细胞与其环境通讯所必需的蛋白质家族。Cx43是骨细胞中表达最丰富的连接蛋白。Cx43表达在骨骼中的重要性已经通过Cx43缺失小鼠胚胎中成骨细胞功能障碍和延迟骨化的证明而确立。然而，Cx43的作用，特别是Cx43半通道在非对立的细胞膜中表达，是远远没有被完全理解。在导致该应用的研究中，证明了双膦酸盐除了阻止破骨细胞介导的再吸收之外，还可以通过促进成骨细胞和骨细胞活力至少部分地保持骨强度，并且这种存活效应需要Cx43表达。因此，通过二膦酸盐抑制细胞凋亡需要Cx43半通道的打开、细胞外信号调节激酶（ERK）的激活以及由于它们与支架蛋白（β-arrestin）的相互作用而导致的激酶的核外保留。Cx43也需要在体外通过甲状旁腺激素（PTH）预防成骨细胞凋亡;在Cx43缺陷小鼠中，对间歇性给予激素的合成代谢反应减弱。此外，Cx43还可能参与骨骼中的机械传导。因此，机械刺激增加成骨细胞和骨细胞中的Cx43表达、间隙连接通讯和半通道开放。基于这一证据，假设Cx43是成骨细胞和骨细胞中由药理学、激素和机械刺激激活的信号通路的关键组分，通过其参与半通道或间隙连接或其支架特性。该假设将通过体外和体内方法的组合进行检验。目的1中的研究将使用选择性激活该途径的双膦酸盐类似物来检查Cx43/β-arrestin/ERK途径对于双膦酸盐对骨的作用的体内相关性;并且它们将描述Cx43、ERK和β-arrestin的结合导致ERK保留在核外和Cx43内化的机制。目的2中的研究将通过检查对Cx43缺陷的小鼠间歇性施用PTH缺乏合成代谢作用是否是由于PTH不能防止成骨细胞凋亡，来确定Cx43在PTH体内抗凋亡作用中的作用。目的3明确Cx43半通道在体内外机械力信号转导中的作用。这将通过研究Cx43缺陷小鼠中基因表达的变化和对机械负荷的合成代谢反应是否改变来实现;以及Cx43是否是体外成骨细胞和骨细胞中机械刺激作用所必需的。我们希望这项工作将为改善骨脆性增加疾病的治疗提供机会。公共卫生相关性：这些研究将促进我们对Cx43在骨骼对药物、激素和机械刺激的反应中的作用的理解。我们希望这项工作将为改善骨脆性增加疾病的治疗提供机会。

DESCRIPTION (provided by applicant): Connexins (Cx) are a family of proteins essential for cell-to-cell communication and for the communication of cells with their environment. Cx43 is the most abundant connexin expressed in bone cells. The importance of Cx43 expression in the skeleton has been established by the demonstration of the osteoblast dysfunction and delay ossification in embryos of Cx43 null mice. However, the role of Cx43 and, in particular Cx43 hemichannels expressed in unopposed cell membranes, is far from being completely understood. In studies leading to this application it was demonstrated that bisphosphonates, besides stopping osteoclast-mediated resorption, might preserve bone strength at least in part by promoting osteoblast and osteocyte viability and that Cx43 expression is required for this survival effect. Thus, inhibition of apoptosis by bisphosphonates requires opening of Cx43 hemichannels, activation of the extracellular signal-regulated kinases (ERKs) and extra-nuclear retention of the kinases due to their interaction with the scaffolding protein (-arrestin. Cx43 is also required for prevention of osteoblast apoptosis by parathyroid hormone (PTH) in vitro; and the anabolic response to intermittent administration of the hormone is blunted in mice deficient mice in Cx43. In addition, Cx43 might be also involved in mechanotransduction in the skeleton. Thus, mechanical stimulation increases Cx43 expression, gap junction communication and hemichannel opening in osteoblasts and osteocytes. Based on this evidence, it is hypothesized that Cx43 is a crucial component of signaling pathways activated by pharmacologic, hormonal and mechanical stimuli in osteoblasts and osteocytes, via its involvement in hemichannels or gap junctions or its scaffolding properties. This hypothesis will be tested by a combination of in vitro and in vivo approaches. Studies in Aim 1 will examine the in vivo relevance of the Cx43/(-arrestin/ERK pathway for bisphosphonate actions on bone using bisphosphonate analogs that selectively activate this pathway; and they will delineate the mechanism by which association of Cx43, ERKs and (-arrestin leads to retention of ERKs outside the nucleus and the internalization of Cx43. Studies in Aim 2 will determine the role of Cx43 in the anti-apoptotic effect of PTH in vivo by examining whether the lack of anabolic effect of intermittent PTH administration to mice deficient in Cx43 is due to the inability of PTH to prevent osteoblast apoptosis. Aim 3 will define the role of Cx43 hemichannels in mechanotransduction in vivo and in vitro. This will be accomplished by investigating whether changes in gene expression and the anabolic response to mechanical loading are altered in Cx43 deficient mice; and whether Cx43 is required for the effects of mechanical stimulation in osteoblasts and osteocytes in vitro. We expect that this work will provide opportunities to improve the treatment of diseases with increased bone fragility. PUBLIC HEALTH RELEVANCE: These studies will advance our understanding of the role of Cx43 in the response of the skeleton to pharmacotherapeutic, hormonal and mechanical stimuli. We expect that this work will provide opportunities to improve the treatment of diseases with increased bone fragility.