Gap Junction and Bone Cell Responses to Physical Signals

描述（由申请人提供）：骨细胞网络响应载荷诱导的机械信号的机制尚不清楚。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，以及在体内破坏这种机械转导如何影响载荷诱导的成骨。我们认为，骨细胞或成骨细胞的间隙连接细胞间通讯和核苷酸的释放，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，如流体流动，通过一种涉及胞质Ca2+动员、GJIC激活和ATP通过间隙连接半通道释放的机制刺激成骨细胞增殖和分化。我们将通过完成四个特定目标来检验这一假设：1)量化流体流动的影响，在存在和不存在抑制胞质Ca2+动员的药物的情况下，GJIC， GJ半通道的激活和骨细胞释放ATP；2)观察体液流动对骨细胞增殖的影响；3)观察液体流动对骨细胞分化的影响；4)观察连接蛋白缺陷小鼠分离骨的负荷诱导成骨。在这个为期五年的项目中，我们将利用一种新型的共培养流体流动装置、shRNA策略、位点定向诱变、一种创新的蛋白质组学方法、一种特性良好的体内骨负荷装置和转基因小鼠模型来研究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一个原则，但实验支持却很少。如果是这样，这对体内机械转导的重要性。了解机械信号如何被骨细胞检测并通过骨细胞网络传递，对于理解骨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疾病的新治疗靶点提供见解。此外，了解机械信号如何调节骨细胞增殖和分化将有助于设计体外环境，即生物反应器，用于新的骨组织工程方案。

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.