Gap Junction and Bone Cell Response to Physical Signals

描述(申请人提供)：越来越多的证据表明，连接蛋白和缝隙连接细胞间通讯(GJIC)在骨转换中起着关键作用，特别是在对机械负荷的反应中。体外研究表明，GJIC对于最大的骨细胞网络对机械负荷的反应至关重要，并预测在没有GJIC的情况下，骨骼对机械负荷的反应将较小。然而，最近来自我们实验室和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)的缺乏实际上会增加骨骼对机械负荷的合成代谢反应。此外，我们的实验室以及其他实验室最近已经证明，Cx43缺乏会使骨骼对机械卸载的分解代谢效应失去敏感性。这些最新的发展表明，我们对连接蛋白、GJIC和骨骼中的机械转导的理解发生了范式转变。也就是说，抑制骨细胞Cx43的表达或GJIC对骨骼对其力学环境的反应具有有益的影响。然而，这一令人惊讶的发展背后的机制尚不清楚。为了解决这一问题，我们将验证一种假说，即抑制骨细胞Gja1的表达增加了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/B-catenin信号通路的机制降低了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成熟的以及新开发的体内基因工程小鼠模型，以检测3个特定目标：1)检测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ç-catenin的小鼠骨骼的影响；2)检测通过后肢悬吊(HLS)机械卸载对与Aim 1中检测到的相同基因型骨的影响；以及3)检测流体流动对有和没有Cx43的成骨细胞中WNT/ü-catenin和RANKL/OPG信号的影响。我们研究的预期结果将为其力学环境提供分子水平的骨适应知识，为代谢性骨疾病提供开创性的力学见解，并促进新的骨骼再生策略的开发，从而为我们的研究重点提供广泛的翻译基础。

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.