Mechanotransduction of shear stress: from ATP release to CFTR regulation

描述（由申请人提供）： 在这项申请中，Brian Button博士提出了一项原创性研究，旨在研究调节剪切应力介导的ATP释放的机制。ATP释放和嘌呤能受体的自分泌/旁分泌刺激与许多不同细胞类型中多种细胞功能的调节有关。嘌呤能信号传导的一个关键作用是调节各种离子转运过程，包括CFTR氯离子通道。外部机械应力，包括剪切、压缩、拉伸和细胞肿胀代表了刺激ATP释放的普遍机制。然而，负责机械转导的外部应力ATP释放的机制是未知的。最近，PI和合作者发现，压力的振荡性质，如在正常呼吸期间经历的，对刺激ATP释放至关重要。此外，他们发现振荡应力的大小和ATP释放速率之间的关系在正常呼吸的生理范围内是最陡峭的，而更强的力产生更弱的反应。这些结果导致PI假设细胞可以在机械刺激期间主动调节ATP释放速率，从而保护自身免受不受调节的ATP释放和嘌呤受体过度刺激的潜在有害影响。初步结果表明，振荡应力介导的ATP释放发生的机制，涉及通过细胞骨架和胞吐依赖的分泌途径的外部和纤毛跳动介导的力量的传输。本项目概述的工作旨在系统地解决ATP释放所涉及的机械转导途径的几个组成部分，并建立其在上皮功能调节中的生理作用。为了实现这些目标，候选人将采用分为三个具体目标的各种技术。目的1将集中在应激刺激ATP释放的动力学特性，并确定参与囊泡介导的过程中的细胞骨架元素。目的2将测试的假设，振荡剪切应力的幅度以上的生理范围减少ATP的释放，通过改变细胞膜的性质。最后，目标3将测试气道上皮细胞是否通过传递到细胞骨架的纤毛跳动产生的内应力感知和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释放的机制提供宝贵的见解，以响应外部和内部力量，这可能会导致发现新的治疗方法来调节ATP释放，这在囊性纤维化等疾病中很重要，其中ATP释放已被证明可以刺激粘液清除。这个K 01奖项将为巴顿博士追求成为独立研究者和建立科学资助机会的职业目标提供基础。

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.