MECHANOTRANSDUCTION IN BONE VIA OSCILLATING FLUID FLOW

描述（由申请人提供）：在之前的资助期内，我们获得的结果强烈表明，由于载荷引起的振荡流体流动是成骨细胞和骨细胞的重要细胞物理信号。利用我们定制的动态流动系统，我们能够表明，振荡流体流动可以通过细胞内钙动员，前列腺素E2释放和MAP激酶活性在没有其他物理或生化信号的情况下调节细胞代谢。然而，我们还没有发现的分子mechanotransduction机制激活振荡流体流动。可以预期候选人会因流动而经历负荷，并具有生化信号传导潜力。这个概念模型是支持我们的观察，在以前的资金期间，膜蛋白聚糖的降解延伸到流场有一个显着的效果对流量的响应。此外，我们有初步的迹象表明，肌动蛋白和粘着斑激酶（FAK）参与流体流动诱导的信号。因此，这个为期五年的项目的中心假设是，振荡流体流动调节骨细胞代谢的分子机制，涉及力所经历的细胞骨架和通过粘着斑网站传递到整合素。为了验证这一假设，我们将进行一个系统的多层次的评价细胞结构蛋白，包括肌动蛋白，整合素，连接蛋白在振荡流对这些蛋白质的影响（目的1）和每个转导的响应流（目的2）的作用。此外，我们实验室和其他实验室的强有力证据表明粘着斑激酶（FAK）酪氨酸磷酸化特异性参与。这与最近开发的靶向FAK磷酸化的分子工具相结合，促使我们对两种特异性FAK信号通路进行更深入的研究（目的3）。最后，利用一种新型的微加工流动室，我们将确定骨细胞过程是否是一种对流体剪切力具有增强敏感性的特殊结构（目的4）。

DESCRIPTION (provided by applicant): In the prior funding period we obtained results strongly suggesting that oscillatory fluid flow due to loading is an important cellular physical signal for both osteoblasts and osteocytes. Utilizing our custom built dynamic flow system, we were able to show that oscillatory fluid flow can regulate cell metabolism via intracellular calcium mobilization, prostaglandin E2 release, and MAP kinase activity in the absence of other physical or biochemical signals. However, we have not uncovered the molecular mechanotransduction mechanism activated by oscillatory fluid flow. Candidates can be expected to experience load due to flow and have biochemical signaling potential. This conceptual model is supported by our observation made in the prior funding period that degradation of membrane proteoglycans extending into the flow field has a dramatic effect on the response to flow. Also, we have preliminary indications that actin and focal adhesion kinase (FAK) are involved fluid flow induced signaling. Thus, the central hypothesis of this five year project is that oscillatory fluid flow regulates bone cell metabolism via a molecular mechanism involving forces experienced by the cytoskeleton and transmitted through focal adhesion sites to integrins. To test this hypothesis we will undertake a systematic multilevel evaluation of cell structural proteins to include actin, integrins, and linker proteins both in terms of the effect of oscillatory flow on these proteins (aim 1) and the role of each in transducing the response to flow (aim 2). Additionally, strong evidence from our laboratory and others suggests specific involvement of focal adhesion kinase (FAK) tyrosine phosphorylation. This combined with recently developed molecular tools targeting FAK phosphorylation motivate us to perform a more in-depth investigation of two specific FAK signal pathways (aim 3). Finally, utilizing a novel microfabricated flow chamber, we will determine if the osteocyte process is a specialized structure with enhanced sensitivity to fluid shear forces (aim 4).