Gap Junction and Bone Cell Response to Physical Signals

描述（由申请方提供）：新出现的证据表明，连接蛋白和间隙连接细胞间通讯（GJIC）在骨转换中起关键作用，尤其是在对机械负荷的反应中。体外研究表明，GJIC对于骨细胞网络对机械负荷的最大反应至关重要，并预测在没有GJIC的情况下，骨对机械负荷的反应较低。然而，我们实验室以及其他实验室最近的体内数据表明，非常矛盾的是，骨细胞连接蛋白43（Cx43）（骨中主要的间隙连接蛋白）的缺乏实际上会增加骨对机械负荷的合成代谢反应。此外，我们的实验室以及另一个实验室最近已经证明，Cx43缺乏使骨对机械卸载的分解代谢作用脱敏。这些最新的发展表明，在我们的理解骨连接蛋白，GJIC和mechanotransduction的范式转变。也就是说，抑制骨细胞Cx43表达或GJIC对骨对其机械环境的响应具有有益作用。然而，这种令人惊讶的发展背后的机制尚不清楚。为了解决这一问题，我们将研究抑制骨细胞Gja 1表达增加骨对机械负荷的合成代谢反应，并通过涉及细胞凋亡、RANKL/OPG和WNT/β-连环蛋白信号通路的机制减少对卸载的分解代谢反应的假设。在这个为期5年的项目中，我们将使用体外细胞培养和良好建立的以及新开发的基因工程体内小鼠模型来检查3个特定目标：1）检查机械负荷对骨细胞选择性Cx43缺陷小鼠和骨细胞同时缺乏Cx43和β-连环蛋白的小鼠的骨的影响; 2）检查通过后肢悬吊（HLS）的机械卸载对来自目的1中检查的相同基因型的骨的影响;和3）检查流体流动对具有和不具有Cx43的骨细胞中的WNT/β-连环蛋白和RANKL/OPG信号传导的影响。我们的研究的预期成果将有助于骨适应其力学环境的分子水平的知识，为代谢性骨病提供开创性的机制见解，并促进新的骨骼再生策略的发展，从而为我们的研究重点提供广泛的转化基础。

DESCRIPTION (provided by applicant): Emerging evidence suggests that connexins and gap junctional intercellular communication (GJIC) play a critical role in bone turnover especially in response to mechanical load. In vitro studies suggest that GJIC is critical for maximal bone cell network response to mechanical load and predict that, in the absence of GJIC, bone would be less responsive to mechanical load. However, recent in vivo data from our laboratory, as well as others, suggest that, quite paradoxically, deficiency in bone cell connexin 43 (Cx43), the predominant gap junction protein in bone, actually increases bone's anabolic response to mechanical load. Furthermore, our laboratory as well as one other has recently demonstrated that Cx43 deficiency desensitize bone to the catabolic effect of mechanical unloading. These recent developments suggest a paradigm shift in our understanding of connexins, GJIC and mechanotransduction in bone. That is, inhibiting bone cell Cx43 expression or GJIC has a beneficial effect on bone's response to its mechanical environment. However, the mechanism underlying this surprising development is unknown. To address this we will examine the hypothesis that inhibiting osteocytic Gja1 expression increases the anabolic response of bone to mechanical load and decreases the catabolic response to unloading through a mechanism involving apoptosis, RANKL/OPG and WNT/ß-catenin signaling pathways. During this 5 year project we will use in vitro cell culture and well established, as well as newly developed, genetically engineered in vivo mouse models, to examine 3 specific aims: 1) Examine the effect of mechanical loading on bone from osteocyte selective Cx43-deficient mice and mice with osteocytes deficient in both Cx43 and ß-catenin; 2) Examine the effect of mechanical unloading via hind limb suspension (HLS) on bone from the same genotypes examined in aim 1; and 3) Examine the effect of fluid flow on WNT/ß-catenin and RANKL/OPG signaling in osteocytic cells with and without Cx43. The expected outcomes of our research will contribute molecular-level knowledge of bone adaptation to its mechanical environment, provide seminal mechanistic insights into metabolic bone disease and facilitate development of new skeletal regeneration strategies, thereby providing a broad translational basis for our research focus.