Regulation of Airway Epithelial Cell Migration by the Cystic Fibrosis Transmembrane Conductance Regulator
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Regulation of Airway Epithelial Cell Migration by the Cystic Fibrosis Transmembrane Conductance Regulator A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Katherine Rebecca Schiller IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Scott M. O’Grady, Advisor May 2010 © Katherine Rebecca Schiller 2010 Acknowledgments First and foremost I would like to thank the members of my thesis committee, Dr. John Collister, and Dr. Paulo Kofuji for their time and input on my thesis project. I would also especially like to thank Dr. Mathur Kannan for his help and suggestions on my current thesis project as well as my last. I thank my husband for his love and support, and his patience and understanding throughout my graduate training. Finally, I thank my advisor, Dr. Scott O’Grady. His time and dedication to my training has given me valuable skills on which to build for my future endeavors in this field. i Abstract The airway epithelium is a critical barrier between the noxious particles in inspired air and the lung tissue. Mucociliary clearance is an essential function of the airway epithelium to protect against infection and airway damage. By this process, inhaled particles are trapped in the mucus lining the airway and propelled out of the airways by ciliary movement. Transport of Cl- and Na+ ions controls fluid secretion in the airways and the depth and viscosity of the periciliary liquid layer essential for cilia movement. The ion channel cystic fibrosis transmembrane conductance regulator - - (CFTR), transports Cl and HCO3 in the airways and other tissues, and controls the depth of the periciliary liquid layer. Cystic fibrosis is a fatal genetic disease in which CFTR is dysfunctional. As a result, mucociliary clearance is impaired and airways become chronically infected with microorganisms. Microorganism colonization results in recurrent inflammation and cycles of damage to the epithelial barrier followed by wound repair, eventually resulting in airway remodeling and ultimately respiratory failure. It has recently been demonstrated that the loss of CFTR function impairs epithelial restitution in the absence of infection. This suggests the CFTR plays a direct role in airway wound repair. To test this hypothesis, the role of CFTR in the initial step of epithelial wound repair-cell migration- was assessed. An impedance based assay was used to objectively measure cell migration rates of human bronchial epithelial cells. Inhibition of CFTR transport and silencing of CFTR protein expression were used to assess the effect of the loss of functional CFTR on cell migration rate. The migration rate of an airway epithelial ii cell line isolated from a cystic fibrosis patient was also compared to that of an airway epithelial cell line isolated from a healthy lung. A functional effect of CFTR on the process of lamellipodia protrusion during cell migration was also assessed morphometrically. Time lapse video images were also captured during wound closure of airway epithelial cells to assess how cell migration occurs. Additionally, the dependence - - of airway epithelial cell migration on Cl and HCO3 transport was assessed using ion substitution and selective inhibitors of CFTR and other transporters of these ions. Further, the effect of changes in extracellular pH on migration rate was assessed. - - The results of these studies revealed a role for CFTR transport of Cl and HCO3 to regulate airway epithelial cell migration. These studies contribute to the basic understanding of the repair process of the airways following damage and may potentially be important for the design of new treatments to limit airway damage in cystic fibrosis. iii Table of Contents List of Tables _______________________________________________vi List of Figures ______________________________________________vii Chapter 1: Airway Wound Repair and Cystic Fibrosis_____________1 I. Function of the Airway Epithelium_____________________________ 1 II. Cystic Fibrosis_____________________________________________ 5 III. The Role of Airway Wound Repair in CF________________________7 IV. Epithelial Wound Repair and the Role of Ion Channels _____________8 V. Scope, Central Hypothesis and Aims of the Thesis_________________12 Chapter 2: Airway Epithelia Dependence on CFTR for Wound Closure __________________________________________14 I. In Vitro Model of Wound Repair ______________________________14 a. Cell Types Utilized in the Wound Repair Model ________________14 b. In vitro Assay for Measuring Wound Closure Rates ______________19 II. Inhibition of CFTR Channel Activity Slows Wound Closure_________23 III. Silencing CFTR Protein Expression Slows Wound Closure _________26 IV. Time Lapse Imaging of Wound Closure _________________________29 V. Effect of CFTR Inhibition on Lamellipodia Protrusion _____________32 VI. CF Airway Epithelial Cells and Inflammatory Mediators ___________35 VII. Conclusions ______________________________________________39 Chapter 3: Ionic Mechanism of CFTR Regulation of Cell Migration 42 - - I. Role of Cl and HCO3 Transport in Cell Migration ________________42 - - II. Cl and HCO3 Transporters that may Cooperate with CFTR ________49 a. Anion Exchangers ________________________________________51 - - b. Solute carrier 26 Cl /HCO3 Exchangers _______________________52 + - c. Na /HCO3 Cotransporters _________________________________52 d. CFTR __________________________________________________54 III. Measurement of Migration with Inhibition of Multiple Transporters and CFTR _______________________________________________55 IV. Conclusions ______________________________________________61 Chapter 4: Discussion________________________________________66 I. Model of the Role of CFTR in Airway Epithelial Cell Migration_____66 iv II. Future Experiments ________________________________________70 a. Localization of CFTR and other Transporters Involved in Migration ______________________________________________71 b. Measurement and Identification of Ions Transported During Migration ______________________________________________73 c. Migration of CF Airway Epithelial Cells in Vitro and in Vivo _____75 d. Modulation of Airway Epithelial Cell Migration Rates by Cytokines and Infection____________________________________________78 III. Conclusions ______________________________________________82 References _________________________________________________________84 Appendix A: Statistical Analyses ______________________________98 v List of Tables Chapter 2 Table 1: Primer Sets Used for qPCR________________________41 Chapter 3 Table 1: Primers Used for qPCR________________________65 vi List of Figures Chapter 1 1. The Airway Epithelium________________________________1 Chapter 2 1. Expression of Airway Epithelial Cell Type Markers ______________18 2. Proliferation of Airway Epithelial Cells ____________________20 3. Measurement of Wound Closure by CIS ____________________22 4. Migration with CFTR Inhibited ______________________________24 5. Migration Rates with Inhibition of CFTR _______________________25 6. Proliferation with Inhibition of CFTR _________________________26 7. Silencing of CFTR Protein Expression _________________________28 8. CFTR Silencing Effect on Migration and Proliferation ____________29 9. Time Lapse Images of Wound Closure-Calu-3 __________________31 10. Time Lapse Images of Wound Closure-NHBE __________________32 11. Lamellipodia Protrusion ___________________________________ 34 12. Lamellipodia Area ________________________________________35 13. Migration of CF Cells ______________________________________37 14. IFNγ and Migration________________________________________38 Chapter 3 - 1. Migration in Cl Free Media ____________________________44 - 2. Migration in Low HCO3 Media__________________________47 3. Extracellular pH and Migration __________________________48 4. Transporter and Exchanger Expression _____________________50 - - - - 5. Cl and HCO3 Transport in Cl Free and Low HCO3 Media _______55 6. Viability with DIDS Treatment __________________________56 - 7. Inhibition of CFTR in Cl Free Media ______________________57 - 8. Inhibition of CFTR and Low HCO3 ______________________59 9. Migration with DIDS _________________________________60 Chapter 4 1. Putative Model of the Role of CFTR in Airway Epithelial Cell Migration 68 vii Chapter I Airway Wound Repair and Cystic Fibrosis I. Function of the Airway Epithelium Airway surfaces are continuously exposed to noxious inhaled particles, allergens and pathogens (Mall 2008). The airway epithelium is critical for protection from infection and damage by these agents (Sparrow et al. 1995). The cornerstone of airway defense is mucociliary clearance, a process by which inhaled particles are trapped in mucus lining the airway, and this mucus is propelled out of the airways towards the mouth by ciliary Airway Lumen movement. The Mucus Layer mucociliary clearance Periciliary Liquid Cilia mechanism is dependent on three functional elements (Fig. 1) that coordinate b to accomplish Fig. 1 The Airway Epithelium The bronchial airways are lined with a pseudostratified epithelium clearance: (i) ciliated composed of ciliated, mucus secreting (shaded cell) and basal cells (b, arrows). The airway surface liquid epithelial cells that traps inhaled particles has a mucus and periciliary liquid layer. connected by intercellular tight junctions, (ii) airway surface liquid (ASL) which is partitioned into a low viscosity periciliary liquid layer that