MECHANOTRANSDUCTION IN BONE VIA OSCILLATING FLUID FLOW

描述(申请人提供)：在之前的资助期间，我们获得了强有力的结果，表明负载引起的液体振荡流动对成骨细胞和骨细胞都是一个重要的细胞物理信号。利用我们定制的动态流动系统，我们能够证明，在没有其他物理或生化信号的情况下，振荡流动可以通过细胞内钙动员、前列腺素E2释放和MAP激酶活性来调节细胞代谢。然而，我们还没有发现由振荡流体流动激活的分子力学转导机制。候选人可以预期经历由于流动而产生的负荷，并具有生化信号潜力。这一概念模型得到了我们在前一个资金阶段所做的观察的支持，即延伸到流场中的膜蛋白多糖的降解对流动的响应有很大的影响。此外，我们有初步迹象表明，肌动蛋白和粘着斑激酶(FAK)参与了液体流诱导的信号转导。因此，这个五年项目的中心假设是，振荡流体通过一种分子机制调节骨细胞的新陈代谢，该机制涉及细胞骨架所受的力，并通过局部黏附部位传递到整合素。为了验证这一假设，我们将对细胞结构蛋白进行系统的多水平评估，包括肌动蛋白、整合素和连接蛋白，这两个方面都是根据振荡流对这些蛋白的影响(目标1)以及每个蛋白在传导对流的反应中的作用(目标2)。此外，来自我们实验室和其他实验室的有力证据表明，粘着斑激酶(FAK)酪氨酸磷酸化是一种特殊的参与。这与最近开发的针对FAK磷酸化的分子工具相结合，促使我们对两个特定的FAK信号通路进行更深入的研究(目标3)。最后，利用一种新的微型流动室，我们将确定骨细胞突起是否是一种对流体剪切力具有增强敏感性的特殊结构(目标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).