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FXYD5 Modulates Na,K-Atpase Activity and Is Increased in Cystic Fibrosis Airway Epithelia

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FXYD5 Modulates Na,K-Atpase Activity and Is Increased in Cystic Fibrosis Airway Epithelia FXYD5 MODULATES NA,K-ATPASE ACTIVITY AND IS INCREASED IN CYSTIC FIBROSIS AIRWAY EPITHELIA By TIMOTHY J. MILLER Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Dissertation Advisor: Dr. Pamela B. Davis Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May, 2008 Case Western Reserve University School of Graduate Studies We hereby approve the dissertation of Timothy J. Miller candidate for the Ph.D degree *. (signed) Michael Maguire (chair of the committee) Pamela Davis Mitch Drumm Ruth E. Siegel ___________________________ Date March 21, 2008 * We also certify that written approval has been obtained for any proprietary material contained therein. ii DEDICATION The achievement of any worthwhile goal is often accomplished through hard work and commitment. It is often easier to succor such accolades with the inspiration of a beautiful wife and son. For her love, patience and guidance throughout our journey, her supremely generous and kind nature, and the joy of my life, I dedicate this thesis to my wife, Molly Megan Gallogly, and my son, Gavin Bryce Miller. iii TABLE OF CONTENTS LIST OF TABLES……………………………………………………………………. vii LIST OF FIGURES………………………………………………………………….. viii LIST OF ABBREVIATIONS…………………………………………………………. ix ACKNOWLEDGEMENTS……………………………………………………...……. xi ABSTRACT………………………………………………………………………….. xiv CHAPTER 1: BACKGROUND…………………………………………………….….1 Cystic Fibrosis is caused by defective ion transport……………………..….1 Na+ hyperabsorption and airway surface liquid dehydration…………….....3 The contribution of the Na,K-ATPase………………………………………...6 The FXYD family: modulators of Na,K-ATPase activity………………….....8 FXYD5…………………………………………………………………………..11 CHAPTER 2: CHARACTERIZATION OF HUMAN AND MOUSE FXYD5….….18 Abstract………………………………………………………………………....18 Introduction……………………………………………………………………..19 Methods…………………………………………………………………………21 Results………………………………………………………………………….30 A comparison of human and mouse expression profiles………….30 Analysis of human and mouse FXYD5……………………………...33 Discussion………………………………………………………………………36 CHAPTER 3: FXYD5 MODULATES NA,K-ATPASE ACTIVITY AND IS INCREASED IN CF AIRWAY EPITHELIA…………………………………………58 Abstract……………………………………………………………………..…..58 Introduction………………………………………………………………….….60 Methods…………………………………………………………………………62 Results………………………………………………………………………….73 Flag-FXYD5 is expressed at the cell membrane…………………...73 iv FXYD5 alters Na,K-ATPase pump kinetics…………………………74 FXYD5 is downregulated after ENaC activation……………………75 FXYD5 is decreased in the lungs of Scnn1b transgenic mice……76 FXYD5 is upregulated in the nasal epithelia of CF mice………….76 FXYD5 is increased in lungs of CF mice……………………………77 CFTR inhibition upregulates FXYD5 in human epithelia…………..78 Discussion………………………………………………………………………80 CHAPTER 3 ADDENDUM: FXYD5 IS INCREASED IN THE LUNGS OF CF MICE AFTER INFECTION WITH P. AERUGINOSA………………..102 Introduction……………………………………………………………………102 Methods……………………………………………………………………….105 Results and Discussion……………………………………………………...109 CHAPTER 4: S163 IS CRITICAL FOR FXYD5 MODULATED WOUND HEALING IN AIRWAY EPITHELIA………………………………………………..113 Abstract………………………………………………………………………..113 Introduction……………………………………………………………………114 Methods……………………………………………………………………….116 Results………………………………………………………………………...121 Mutations in Ser163 alter FXYD5 cellular localization……………121 S163 mutations alter FXYD5/Na,K-ATPase interaction………….122 S163 modulates wound healing in murine airway epithelia……..123 Discussion…………………………………………………………………….124 CHAPTER 5: SUMMARY, CONCLUSIONS AND FUTURE DIRECTIONS…..135 Summary………………………………………………………………………135 FXYD5 modulates Na,K-ATPase pump affinity for Na+ and K+....136 Consequences of increased FXYD5 expression in the airway….139 Secondary effects of FXYD5/Na,K-ATPase interaction………….143 Pro-inflammatory signals upregulate FXYD5 expression………..148 Future directions……………………………………………………………...151 v APPENDICES………………………………………………………………………..161 Neighbor joining alignment of known FXYD5 sequences………..……...161 Human and mouse primer sequences……………………………………..162 Future directions materials and methods………………………………….163 REFERENCE LIST…………………………………………………………………..169 vi LIST OF TABLES Table 2-1. Comparison of human and mouse FXYD5………………………….…41 Table 3-1. Comparison of intracellular Na+ and extracellular K+ activation of Na,K-ATPase pump activity in FXYD5 transfected MDCK cells..........................89 LIST OF FIGURES Figure 1-1. Post-Albers Na,K-ATPase transport scheme…………………………16 Figure 1-2. Human FXYD5 cDNA and protein sequence…………………………17 Figure 2-1. Unigene profile of human FXYD5 tissue expression………………...43 Figure 2-2. Unigene profile of murine Fxyd5 tissue expression………………….46 Figure 2-3. Tissue distribution of murine Fxyd5 expression….…………………..48 Figure 2-4. Fxyd5 is highly expressed in murine lung tissue……………………..49 Figure 2-5. FXYD5 is expressed is multiple cell lines……………………………..50 Figure 2-6. Antibody 562 is specific for murine FXYD5 …………..………………51 Figure 2-7. FXYD5 is strongly expressed in murine epidermis …..……………...53 Figure 2-8. Murine FXYD5 tissue expression ………..…………………………….54 Figure 2-9. Alignment of human and mouse FXYD5………………………………55 Figure 2-10. Comparison of human and murine FXYD5 chimeras……….……...57 Figure 3-1. Flag-FXYD5 is membrane localized in MDCK cells..........................86 Figure 3-2. FXYD5 modulates Na,K-ATPase activity..........................................88 Figure 3-3. ENaC activation increases Na,K-ATPase but decreases FXYD5.....91 Figure 3-4. FXYD5 is decreased in lungs from Scnn1b overexpressing mice.....93 Figure 3-5. FXYD5 is upregulated in the nasal epithelia of S489X-/- CF mice…95 Figure 3-6. FXYD5 is increased in airway epithelia of CF mice…………………..97 Figure 3-7. CFTR inhibition upregulates FXYD5 in human airway epithelia…….99 Figure 3-8. Model of FXYD5 role in Na+ absorption in CF airway epithelia……101 Figure 3-9. Fxyd5 is increased in CF mouse lungs after bacterial infection…...112 Figure 4-1. Immunoblot of FXYD5-Flag in HEK 293 and LA4 cells…………….128 Figure 4-2. S163 mutations alter FXYD5 membrane localization………………130 Figure 4-3. Ser163 modulates FXYD5 interaction with Na,K-ATPase……..…..132 Figure 4-4. FXYD5 modulates wound repair in murine airway epithelial cells...134 Figure 5-1. Regulation and role of FXYD5 in ASL dehydration.........................142 Figure 5-2. Helical wheel plot of human FXYD5 transmembrane domain……..146 Figure 5-3. FXYD5 S170 is an SGK1 consensus phosphorylation site………..158 Figure 5-4. SGK1 phosphorylates FXYD5 S170………………………………….159 Figure 5-5. FXYD5 S170 mutations affect cell localization in MDCK cells…….160 viii LIST OF ABBREVIATIONS a.a.: amino acid ASL: airway surface liquid ATP: adenosine triphosphate BALF: broncheoalveolar lavage fluid Bp: base pair Ca+2: calcium CCL2: chemokine (C-C motif) ligand 2 CCR2: chemokine (C-C motif) receptor 2 CF: cystic fibrosis CFTR: cystic fibrosis transmembrane conductance regulator CHIF: corticosteroid hormone-induced factor COPD: chronic obstructive pulmonary disorder DDD: digital differential display ENaC: epithelial sodium channel EST: expressed sequence tag FXYD: phenylalanine-X-tyrosine-aspartic acid HEK293: human embryonic kidney cells 293 Kb: kilobase kDa: kilodalton MDCK: madin-darby kidney cells MCC: mucociliary clearance Na,K-ATPase: sodium, potassium adenosine triphosphatase ix Nasal PD: nasal potential difference NF-κB: nuclear factor kappa-B PA: Pseudomonas aeruginosa PCL: pericilliary layer RAA: renin-angiontensin-aldosterone axis RIC: Related to Ion Channel RT: room temperature SDS: sodium dodecyl sulfate SGK1: serum/glucocorticoid induced kinase siRNA: silenced RNA qRT-PCR: quantitative reverse-transcription polymerase chain reaction x Acknowledgments The decision to go back to graduate school wasn’t easy, and wouldn’t have been possible without help from my father, Peter V. Miller. The “Miller Foundation” is gratefully acknowledged for emotional, financial and humoral support over the past 7 years. Similarly, my mother, Wendy E. Murray, was a source of strength and determination for me and I’m proud to have had her support. I left a wonderful workplace to join the ranks of graduate students, but it was due to the research I performed at Copernicus Therapeutics, Inc. that convinced me I wanted to work on cystic fibrosis for my thesis project. It was quite a boon to have a general field of work already chosen, and it helped propel my initial studies. I’ll always value the time I spent at Copernicus, and I thank them and Dr. Mark Cooper, M.D. for supporting my transition from employee to student. While I may have had a general interest in CF research, this project originated from an observation made by Dr. Aura Perez, M.D., Ph.D. At the time, the FXYD family had just been identified, and FXYD5 came up on a short list of genes altered on a CF microarray. She generously allowed me to make this my project. The rest is history. No student can perform their work alone. I was able to successfully publish my work only after collaboration with the members of the Davis lab. In particular, Yongyi Qian was a source of information for experimental design as well as a contributor to the FXYD5 project. She is a valuable asset to any lab, xi and will be missed. Similarly, Xuguang Chen was helpful in troubleshooting a variety of experiments and was always willing to listen about my next “big experiment”. He helped provide a fun and interactive environment as well as graduate student solidarity. Much of my thesis work was made possible as a result of the hard working
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