Connexin 43 Hemichannels and Signaling In Bone

描述（由申请人提供）：连接蛋白（Cx）是细胞间通信和细胞与环境通信所必需的蛋白质家族。Cx43是骨细胞中表达量最多的连接蛋白。Cx43缺失小鼠胚胎成骨细胞功能障碍和骨化延迟的研究证实了骨骼中Cx43表达的重要性。然而，Cx43的作用，特别是在非对抗细胞膜中表达的Cx43半通道，还远未完全了解。在导致这种应用的研究中，已证明双膦酸盐除了阻止破骨细胞介导的再吸收外，还可能至少部分地通过促进成骨细胞和骨细胞的活力来保持骨强度，并且这种存活效果需要Cx43的表达。因此，双膦酸盐抑制细胞凋亡需要打开Cx43半通道，激活细胞外信号调节激酶（ERKs），以及由于这些激酶与支架蛋白（-阻滞蛋白）相互作用而使这些激酶在核外保留。体外实验还需要Cx43来预防甲状旁腺激素（PTH）诱导的成骨细胞凋亡；在缺乏Cx43的小鼠中，间歇性给药的合成代谢反应变得迟钝。此外，Cx43也可能参与骨骼的机械传导。因此，机械刺激增加了成骨细胞和骨细胞中Cx43的表达、间隙连接通信和半通道打开。基于这一证据，我们假设Cx43是成骨细胞和骨细胞中被药理、激素和机械刺激激活的信号通路的关键组成部分，通过参与半通道或间隙连接或其支架特性。这一假设将通过体外和体内方法的结合来验证。Aim 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/ -抑制蛋白/ERK途径与双膦酸盐对骨作用的体内相关性；他们将描述Cx43、ERKs和-抑制蛋白的关联导致ERKs在细胞核外保留和Cx43内化的机制。Aim 2中的研究将通过检验间歇性PTH给药对Cx43缺乏的小鼠缺乏合成代谢作用是否是由于PTH无法阻止成骨细胞凋亡，来确定Cx43在体内PTH抗凋亡作用中的作用。目的3将定义Cx43半通道在体内和体外机械转导中的作用。这将通过研究Cx43缺陷小鼠的基因表达变化和对机械负荷的合成代谢反应是否改变来实现；体外成骨细胞和骨细胞的机械刺激作用是否需要Cx43。我们期望这项工作将提供机会，以改善疾病的治疗增加骨脆性。公共卫生相关性：这些研究将促进我们对Cx43在骨骼对药物治疗、激素和机械刺激的反应中的作用的理解。我们期望这项工作将提供机会，以改善疾病的治疗增加骨脆性。

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.