Gap Junction and Bone Cell Response to Physical Signals

描述（由申请人提供）：越来越多的证据表明，连接蛋白和间隙连接细胞间通讯（GJIC）在骨转换中起着关键作用，特别是在响应机械负荷时。体外研究表明，GJIC对于骨细胞网络对机械负荷的最大响应至关重要，并预测，在没有GJIC的情况下，骨对机械负荷的响应会降低。然而，最近来自我们实验室以及其他实验室的体内数据表明，相当矛盾的是，骨细胞连接蛋白43 (Cx43)（骨中主要的间隙连接蛋白）的缺乏实际上增加了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证明，Cx43缺乏会使骨骼对机械卸载的分解代谢作用脱敏。这些最近的发展表明，我们对骨连接蛋白、GJIC和机械转导的理解发生了范式转变。也就是说，抑制骨细胞Cx43表达或GJIC对骨对其机械环境的反应有有益的影响。然而，这一惊人发展背后的机制尚不清楚。为了解决这个问题，我们将研究抑制骨细胞Gja1表达的假设，即通过涉及凋亡、RANKL/OPG和WNT/ß-catenin信号通路的机制，增加骨对机械负荷的合成代谢反应，减少对卸载的分解代谢反应。在这个为期5年的项目中，我们将使用体外细胞培养和完善的，以及新开发的，基因工程的体内小鼠模型，来研究3个具体目标：1)研究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ß-catenin的小鼠的骨的影响；2)研究通过后肢悬吊（HLS）进行机械卸载对目的1中检测的相同基因型骨的影响；3)观察液体流动对含和不含Cx43的骨细胞WNT/ß-catenin和RANKL/OPG信号通路的影响。我们研究的预期结果将有助于了解骨对其机械环境的适应分子水平，为代谢性骨疾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.