CFTR regulation of airway epithelial cell migration

描述(申请人提供)：囊性纤维化(CF)是高加索人最常见的缩短寿命的疾病(12，63)。尽管疾病的严重程度各不相同，但很大一部分受影响的人会发展为阻塞性肺疾病，并逐渐丧失肺功能。粘液堵塞、睫毛功能不良、慢性炎症和呼吸道感染是该病的常见表现(63)。最近观察到CF患者的呼吸道上皮细胞表现出异常再生，感染和炎症加剧了这种再生[28]。囊性纤维化跨膜电导调节因子(CFTR)在这一过程中的作用还知之甚少。在这项应用中提出的初步实验表明，CFTR功能是有效的呼吸道上皮细胞迁移和伤口修复所必需的。通过RNA干扰阻断通道活动或沉默表达，通过阻止片状脂体突起和降低细胞向伤口的迁移速度，显着延迟伤口愈合。此外，我们最新的初步数据显示，与正常受试者的细胞相比，表达最常见的(F508)突变的CF患者的支气管上皮细胞也显示出明显的伤口愈合延迟。因此，这项建议的第一个目标是了解CFTR参与呼吸道上皮细胞迁移和修复的机制。我们的初步实验提出的假设是，CFTR通过调节细胞外pH促进迁移，特别是在细胞的前沿，减少通道活动增强粘附性，减少板脂蛋白突起和细胞迁移速度。第二个目的是研究原代培养的人支气管上皮细胞中，CFTR和阴离子交换器的功能偶联所介导的细胞迁移依赖于氯离子和HCO3的分子机制。我们认为，氯-HCO3交换器与CFTR并行地发挥作用，以介导氯-的摄取，以换取HCO3流出到细胞外基质(ECM)。CFTR为氯循环提供了一条关键途径，它维持了氯-HCO3交换器的活性，从而维持了ECM内的缓冲能力。为了解决这些假设，我们计划使用比率成像方法来测量膜-ECM界面上细胞外pH(Phe)的变化，并应用自参考离子选择微电极技术来研究CFTR在膜-ECM微环境中调节Phe的作用。我们还将确定阴离子交换器的分子同一性，并研究其在ECM pH调节中的作用。了解CFTR促进呼吸道上皮细胞修复和重建的细胞机制可能对开发新的药物治疗具有重要影响，这些药物治疗可能会减少感染、慢性炎症和重塑的加重效应，这些因素导致了CF肺功能的进行性丧失。 公共卫生相关性：尽管我们对CF的遗传和分子基础的了解有了很大的进步，但我们对涉及肺功能障碍的致病事件的理解仍然存在重大差距。我们的初步发现强调了CFTR在伤口愈合过程中以前未被描述的角色，这可能会增强我们对CF病理生理学的理解。从拟议的研究中获得的见解可能会导致确定新的药物靶点或治疗策略，以限制肺功能的进行性恶化。

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common life-shortening disease in Caucasians (12,63). Although severity of the disease is variable, a large proportion of affected individuals develop obstructive lung disease with progressive loss of pulmonary function. Mucus plugging, poor ciliary function, chronic inflammation and infection of the airways are common manifestations of the disease (63). Recently it was observed that airway epithelia from CF patients exhibit abnormal regeneration which is exacerbated by infection and inflammation (28). The role of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in this process is poorly understood. Preliminary experiments presented in this application show that CFTR function is required for efficient airway epithelial cell migration and wound repair. Blocking channel activity or silencing expression by RNA interference produced a significant delay in wound healing by impeding lamellipodia protrusion and reducing the rate of cell migration into the wound. Moreover, our most recent preliminary data using bronchial epithelial cells obtained from CF patients expressing the most common (?F508) mutation also exhibited significantly delayed wound healing compared to cells from normal subjects. Thus the first objective of this proposal is to understand the mechanism by which CFTR participates in airway epithelial cell migration and restitution. The hypothesis suggested by our preliminary experiments is that CFTR promotes migration through regulation of extracellular pH, particularly at the leading edge of the cell and that reduced channel activity enhances adhesion and decreases lamellipodia protrusion and the rate of cell migration. The second objective is to investigate the molecular mechanisms responsible for Cl- and HCO3- dependency of cell migration mediated by the putative functional coupling of CFTR and anion exchangers in primary human bronchial epithelial cells. We propose that a Cl-HCO3 exchanger functions in parallel with CFTR to mediate Cl- uptake in exchange for HCO3 efflux into the extracellular matrix (ECM). CFTR provides a critical pathway for Cl- recycling which sustains the activity of the Cl-HCO3 exchanger so that buffering capacity within the ECM is maintained. To address these hypotheses, we plan to measure changes in extracellular pH (pHe) at the membrane-ECM interface using a ratio imaging approach and to apply self-referencing ion-selective microelectrode technology to study the role of CFTR in regulating pHe within the membrane-ECM microenvironment. We will also determine the molecular identity of the anion exchanger and examine its role in regulation of ECM pH. Understanding the cellular mechanisms involved in the contribution of CFTR to airway epithelial cell repair and restitution may have significant impact on the development of new pharmacotherapies that could potentially reduce the exacerbating effects of infection, chronic inflammation and remodeling that contribute to the progressive loss of pulmonary function observed in CF. PUBLIC HEALTH RELEVANCE: Despite dramatic advances in our knowledge of the genetic and molecular basis for CF there are still significant gaps in our understanding of the pathogenic events involved in pulmonary dysfunction. Our preliminary findings highlight a previously uncharacterized role for CFTR in the process of wound healing that could enhance our understanding of CF pathophysiology. Insights gained from the proposed studies may lead to identification of new drug targets or therapeutic strategies to limit the progressive deterioration of lung function.