CFTR regulation of airway epithelial cell migration

CFTR对气道上皮细胞迁移的调节

• 批准号: 7988533
• 负责人: SCOTT M. O'GRADY
• 金额: $ 21.7万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7988533
• 关键词: Address; Adhesions; Affect; Anions; Bacterial Infections; Basal Cell; Bicarbonates; Buffers; Caucasians; Caucasoid Race; Cell membrane; Cells; Chloride Ion; Chlorides; Chronic; Coupling; Critical Pathways; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Dependence; Dependency; Deterioration; Development; Digestion; Disease; Drug Delivery Systems; Epithelial; Epithelial Cells; Event; Exhibits; Exposure to; Extracellular Matrix; Functional disorder; Healed; Human; Image; Impaired wound healing; Individual; Infection; Inflammation; Integrins; Ions; Knowledge; Lead; Life; Lung; Measures; Mediating; Membrane; Microelectrodes; Molecular; Molecular Genetics; Mucous body substance; Mutation; Natural regeneration; Normal Cell; Nude Mice; Obstructive Lung Diseases; Pathway interactions; Peptide Hydrolases; Pharmacotherapy; Play; Population; Process; Proliferating; RNA Interference; Recycling; Regulation; Respiratory physiology; Role; Severity of illness; Site; Staging; Surface; Technology; Therapeutic; Time; Tissues; Water; Wound Healing; airway epithelium; base; cell injury; cell motility; cystic fibrosis airway; cystic fibrosis airway epithelia; cystic fibrosis patients; design; driving force; extracellular; healing; inhibitor/antagonist; insight; mRNA Expression; migration; public health relevance; pulmonary function; repaired; research study; uptake; wound

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.

描述（申请人提供）：囊性纤维化（CF）是白种人中最常见的缩短寿命的疾病（12，63）。虽然疾病的严重程度是可变的，但大部分受影响的个体发展为阻塞性肺疾病，并伴有肺功能的进行性丧失。粘液堵塞、纤毛功能差、慢性炎症和气道感染是该疾病的常见表现（63）。最近观察到CF患者的气道上皮细胞表现出异常再生，其因感染和炎症而加剧（28）。囊性纤维化跨膜传导调节因子（CFTR）在这一过程中的作用知之甚少。本申请中提出的初步实验表明，CFTR功能是有效的气道上皮细胞迁移和伤口修复所必需的。通过RNA干扰阻断通道活性或沉默表达通过阻碍板状伪足突出和降低细胞迁移到伤口中的速率而产生伤口愈合的显著延迟。此外，我们最新的初步数据使用支气管上皮细胞从CF患者表达最常见的（？F508）突变的细胞相比于来自正常受试者的细胞也表现出显著延迟的伤口愈合。因此，该建议的第一个目标是了解CFTR参与气道上皮细胞迁移和恢复的机制。我们的初步实验提出的假设是，CFTR通过调节细胞外pH值促进迁移，特别是在细胞的前缘，并且降低的通道活性增强粘附并降低板状伪足突起和细胞迁移速率。第二个目标是调查负责Cl-和HCO3-依赖性的细胞迁移介导的CFTR和阴离子交换剂在原代人支气管上皮细胞的假定功能耦合的分子机制。我们提出，一个Cl-HCO3交换功能与CFTR平行介导的Cl-吸收，以换取HCO3流出到细胞外基质（ECM）。CFTR提供了Cl-再循环的关键途径，其维持Cl-HCO3交换器的活性，使得ECM内的缓冲能力得以维持。 为了解决这些假设，我们计划使用比率成像方法测量膜-ECM界面处的细胞外pH（pHe）的变化，并应用自参考离子选择性微电极技术来研究CFTR在调节膜-ECM微环境内的pHe中的作用。我们还将确定阴离子交换剂的分子身份，并研究其在ECM pH调节中的作用。了解CFTR对气道上皮细胞修复和恢复的贡献所涉及的细胞机制可能对新药物治疗的开发产生重大影响，这些药物治疗可能会减少感染的恶化作用，慢性炎症和重塑，导致CF中观察到的肺功能进行性丧失。 公共卫生关系：尽管我们对CF的遗传和分子基础的认识有了很大的进步，但我们对肺功能障碍中涉及的致病事件的理解仍然存在很大的差距。我们的初步研究结果强调了CFTR在伤口愈合过程中以前未表征的作用，这可以增强我们对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识别新的药物靶点或治疗策略，以限制肺功能的进行性恶化。