Gap Junction and Bone Cell Responses to Physical Signals

描述(申请人提供)：骨细胞网络对负荷诱导的机械信号作出反应的机制尚不清楚。该项目的长期目标是更好地了解由多种细胞类型组成的骨细胞网络中的机械转导，以及在体内干扰这种机械转导如何影响负荷诱导的成骨。我们认为，细胞间的缝隙连接通讯和从成骨细胞或成骨细胞释放的核苷酸，特别是三磷酸腺苷(ATP)，对于最大化骨细胞对物理环境的反应都是必不可少的。我们的中心假设是，生物物理信号，如液体流动，通过一种机制刺激成骨细胞的增殖和分化，该机制涉及细胞内钙离子的动员，GJIC的激活和通过缝隙连接半通道释放ATP。我们将通过完成四个具体目标来验证这一假说：1)在存在和不存在抑制胞浆钙动员的药物的情况下，量化液体流动对GJIC、GJ半通道的激活和骨细胞释放ATP的影响；2)研究液体流动对骨细胞增殖的影响；3)研究液体流动对骨细胞分化的影响；4)研究连接蛋白缺乏小鼠骨骼中负荷诱导的成骨作用。在这个为期五年的项目中，我们将利用一种新颖的共培养流体流动装置、shRNA策略、定点突变一种创新的蛋白质组学方法、一种具有良好特性的在体骨骼加载装置和转基因小鼠模型来检查暴露于机械信号下的骨细胞是否向成骨细胞传递增殖和分化诱导信号，这是骨细胞生物学的教条，实验支持令人惊讶地很少，如果是的话，这对体内机械转导的重要性。了解力学信号是如何被骨细胞检测并在整个骨细胞网络中传递的，对于了解骨骼如何适应其物理环境非常重要。这将反过来为许多肌肉骨骼病理的新治疗靶点提供洞察力。此外，了解力学信号如何调控骨细胞的增殖和分化将有助于设计新的骨组织工程方案的体外环境，即生物反应器。

DESCRIPTION (provided by applicant): The mechanism by which bone cell networks respond to load-induced mechanical signals is poorly understood. The long term goals of this project are to gain a better understanding of mechanotransduction in bone cell networks, composed of multiple cell types, and how disrupting this mechanotransduction in vivo affects load-induced osteogenesis. We propose that gap junctional intercellular communication and release of nucleotides, specifically adenosine triphosphate (ATP), from osteocytic or osteoblastic cells, are both essential to maximize bone cell response to the physical environment. Our central hypothesis is that biophysical signals, such as fluid flow, stimulate osteoblast proliferation and differentiation via a mechanism involving mobilization of cytosolic Ca2+, activation of GJIC and release of ATP through gap junction hemichannels. We will examine this hypothesis through the completion of four specific aims: 1) quantify the effect of fluid flow, in the presence and absence of agents that inhibit cytosolic Ca2+ mobilization, on GJIC, activation of GJ hemichannels and release of ATP by bone cells; 2) examine the effect of fluid flow on bone cell proliferation; 3) examine the effect of fluid flow on bone cell differentiation and 4) examine load-induced osteogenesis in bones isolated from connexin deficient mice. During this five-year project we will utilize a novel co-culture fluid flow apparatus, shRNA strategies, site-directed mutagenesis an innovative proteomics approach, a well characterized in vivo bone loading apparatus and transgenic murine models to examine whether osteocytic cells exposed to mechanical signals communicate proliferation and differentiation inducing signals to osteoblastic cells, a dogma of bone cell biology with surprisingly little experimental support, and if so the importance of this to in vivo mechanotransduction. An understanding of how mechanical signals are detected by bone cells and communicated throughout the bone cell network is important to understanding how bone adapts to its physical environment. This will in turn provide insights as to novel therapeutic targets for many musculoskeletal pathologies. Additionally, an understanding of how mechanical signals regulate bone cell proliferation and differentiation would be beneficial in designing in vitro environments, i.e. bioreactors, for novel bone tissue engineering protocols.