CFTR regulation of airway epithelial cell migration

CFTR对气道上皮细胞迁移的调节

• 批准号: 8089552
• 负责人: SCOTT M. O'GRADY
• 金额: $ 17.9万
• 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 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8089552
• 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（由申请人提供）：囊性纤维化 (CF) 是白种人中最常见的缩短寿命的疾病 (12,63)。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患上阻塞性肺病，并伴有肺功能进行性丧失。粘液堵塞、纤毛功能差、气道慢性炎症和感染是该疾病的常见表现（63）。最近观察到，CF 患者的气道上皮表现出异常再生，感染和炎症会加剧这种异常再生 (28)。囊性纤维化跨膜电导调节器 (CFTR) 在此过程中的作用尚不清楚。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，会阻碍片状伪足突出并降低细胞迁移到伤口的速率，从而显着延迟伤口愈合。此外，我们最新的初步数据显示，与来自正常受试者的细胞相比，从表达最常见 (?F508) 突变的 CF 患者中获得的支气管上皮细胞也显示出伤口愈合明显延迟。因此，该提案的首要目标是了解 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（特别是在细胞的前缘）促进迁移，并且通道活性降低可增强粘附并减少片状伪足突出和细胞迁移速率。第二个目标是研究原代人支气管上皮细胞中 CFTR 和阴离子交换剂的假定功能耦合介导的细胞迁移的 Cl- 和 HCO3- 依赖性的分子机制。我们提出 Cl-HCO3 交换器与 CFTR 并行发挥作用，介导 Cl- 吸收，以换取 HCO3 流出到细胞外基质 (ECM)。 CFTR 为 Cl 循环提供了一条关键途径，可维持 Cl-HCO3 交换器的活性，从而维持 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测量膜-ECM界面处的细胞外pH（pHe）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微环境中pHe的作用。我们还将确定阴离子交换剂的分子特性并检查其在 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会对新药物疗法的开发产生重大影响，这些新药物疗法可能会减少感染、慢性炎症和重塑的加剧效应，这些效应会导致 CF 中观察到的肺功能进行性丧失。 公共卫生相关性：尽管我们对 CF 遗传和分子基础的了解取得了巨大进展，但我们对肺功能障碍致病事件的理解仍然存在重大差距。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确定新的药物靶点或治疗策略，以限制肺功能的逐渐恶化。