Mechanotransduction of shear stress: from ATP release to CFTR regulation

描述(由申请人提供)： 在这项应用中，Brian Button博士提出了一项原创性研究，以探索剪切力介导的ATP释放的调节机制。ATP的释放和对嘌呤能受体的自分泌/旁分泌刺激参与了多种不同细胞类型的多种细胞功能的调节。嘌呤能信号的一个关键作用是调节各种离子转运过程，包括CFTR氯通道。外部机械应力，包括剪切、压缩、拉伸和细胞膨胀，是一种普遍存在的刺激ATP释放的机制。然而，外界压力对ATP释放的机械转导机制尚不清楚。最近，PI和他的合作者发现，压力的振荡性质，如在正常呼吸中所经历的，对于刺激ATP的释放是必不可少的。此外，他们发现，在正常呼吸的生理范围内，振荡应激的大小和ATP释放速率之间的关系最陡峭，而更强的力产生的反应更弱。这些结果导致PI假设细胞在机械刺激过程中可以主动调节ATP释放的速度，从而保护自己免受不受调节的ATP释放和对嘌呤受体过度刺激的潜在有害影响。初步结果表明，振荡应激介导的ATP释放是通过细胞骨架和胞吐依赖的分泌途径传递外部和纤毛搏动介导力的机制发生的。该项目旨在系统地研究参与ATP释放的机械转导途径的几个组成部分，并确定其在调节上皮功能中的生理作用。为了实现这些目标，应聘者将使用归类为三个具体目标的各种技术。目的1将重点研究应激刺激的ATP释放的动力学特性，并确定参与囊泡介导的过程的细胞骨架元件。目的2将验证这样的假设，即高于生理范围的幅度的振荡切应力通过改变细胞膜的属性来减少ATP的释放。最后，目标3将测试呼吸道上皮是否通过纤毛跳动传递到细胞骨架产生的内应力来感知并响应覆盖粘液的水合状态的变化。总之，这些研究旨在为调节ATP释放以响应外部和内部力量的机制提供宝贵的见解，这可能会导致发现调节ATP释放的新的治疗方法，这在囊性纤维化等疾病中非常重要，在囊性纤维化中，ATP的释放已被证明能刺激粘液清除。这一K01奖项将为巴顿博士追求他的职业目标--成为一名独立研究员并建立科学资助机会--奠定基础。

DESCRIPTION (provided by applicant): In this application, Dr. Brian Button is proposing original research to investigate the mechanisms regulating shear stress-mediated ATP release. ATP release and autocrine/paracrine stimulation of purinergic receptors has been implicated in the regulation of a wide array of cell functions in numerous diverse cell types. A key role of purinergic signaling is the regulation of various ion transport processes, including the CFTR chloride channel. External mechanical stresses, including shear, compression, stretch, and cell swelling represent a ubiquitous mechanism to stimulate ATP release. However, the mechanisms responsible for mechanotransduction of external stresses to ATP release are unknown. Recently, the PI and collaborators discovered that the oscillatory nature of stress, such as experienced during normal breathing, is essential to stimulate ATP release. Furthermore, they found that the relationship between the magnitude of oscillatory stress and the rate of ATP release was steepest within the physiological range of normal breathing, whereas stronger forces generated weaker responses. These results lead the PI to hypothesize that cells can actively regulate the rate of ATP release during mechanical stimulation, thus protecting themselves from the potentially detrimental effect of unregulated ATP release and over-stimulation of purinoceptors. Preliminary results suggest that oscillatory stress-mediated ATP release occurs by a mechanism involving transmission of external and cilia beating-mediated forces through the cytoskeleton and exocytosis-dependent secretory pathways. The work outlined in this project is designed to systematically address several components of the mechanotransduction pathway involved in ATP release and establish its physiological role in the regulation of epithelial function. To achieve these objectives, the candidate will employ a variety of techniques grouped into three Specific Aims. Aim 1 will focus on the kinetic properties of stress-stimulated ATP release and identify the cytoskeletal elements involved in the vesicular-mediated process. Aim 2 will test the hypothesis that oscillatory shear stress of magnitude above physiological ranges reduces ATP release by altering the properties of the cell membrane. Finally, Aim 3 will test whether airway epithelia sense and respond to changes in the hydration status of the overlying mucus by internal stresses generated by cilia beating transmitted to the cytoskeleton. Together, these studies are designed to provide invaluable insights into the mechanism regulating ATP release in response to external and internal forces, which may potentially lead to the discovery of novel therapeutic approaches to modulate ATP release, important in such diseases as cystic fibrosis, where ATP release has been shown to stimulate mucus clearance. This K01 award will provide the foundation for Dr. Button to pursue his career goals of becoming an independent investigator and establishing scientific funding opportunities.