Determining the mechanism and clinical relevance of sphingomyelinase-mediated decreases in transepithelial CFTR current in primary bronchial epithelial cells Pubblico

Abstract

The Cystic Fibrosis transmembrane conductance regulator (CFTR) is an anion channel whose dysfunction causes Cystic Fibrosis (CF). Loss of CFTR function in pulmonary epithelial cells causes surface dehydration, mucus accumulation, inflammation, and bacterial infections resulting in lung failure. Little has been done to evaluate the effects of lipid perturbation on CFTR activity, despite CFTR residing in the plasma membrane. This work focuses on the acute effects of sphingomyelinase (SMase), a bacterial virulence factor secreted by CF-relevant airway bacteria which degrades sphingomyelin into ceramide, on the electrical circuitry of pulmonary epithelial monolayers. We report that basolateral SMase decreases CFTR-mediated transepithelial anion secretion in primary airway epithelial cells from explant tissue, with current CFTR modulators unable to rescue this effect. Using a holistic ion homeostasis approach, we determined that basolateral SMase inhibits apical and basolateral conductance in non-CF primary cells without affecting paracellular permeability. In CF cells (ΔF508/ΔF508), SMase was found to inhibit only apical conductance. We then explored the mechanisms underlying this effect. SMase increased the abundance of dihydroceramides, a result mimicked by blockade of ceramidase enzyme using ceranib-1, which also decreased CFTR currents. The SMase-mediated inhibitory mechanism did not involve removal of CFTR from the apical surface relative to total CFTR, nor did it involve activation of 5' adenosine monophosphate-activated protein kinase. To determine the pathological relevance of these sphingolipid imbalances, we evaluated the sphingolipid profiles of CF cells as compared to non-CF controls. Sphingomyelins, ceramides, and dihydroceramides largely were increased in CF cells. Correction of ΔF508-CFTR trafficking with VX445+VX661 decreased some sphingomyelins and all ceramides, but exacerbated increases in dihydroceramides. Additional treatment with the CFTR potentiator VX770 did not affect these changes, suggesting rescue of misfolded CFTR was sufficient. We furthermore determined that CF cells express more acid-SMase protein than non-CF cells. Lastly, we determined that airway-like neutrophils, which are increased in the CF lung, secrete acid-SMase. Identifying the complete mechanism of SMase-mediated inhibition of CFTR will be important, given the imbalance of sphingolipids in CF cells and the secretion of acid-SMase from cell types relevant to CF pathophysiology.

Table of Contents

CHAPTER 1 – INTRODUCTION 1

1.1 Cystic Fibrosis: an overview of the disease 1

1.1.1 European folklore and salty sweat 1

1.1.2 Dr. Dorothy Anderson and cystic fibrosis of the pancreas 4

1.1.3 The issue today – pulmonary manifestations of CF 5

1.1.4 Other organs affected by CF 9

1.2 CFTR: an overview of the protein 10

1.2.1 Protein structure and activity 10

1.2.2 CF-causing mutations 14

1.3 Highly effective modulator therapies and other developing therapies 20

1.3.1 The potentiator – VX770 20

1.3.2 The correctors –VX809, VX661, and VX445 22

1.3.3 Other emerging therapies 24

1.4 CF animal and cell models 25

1.4.1 CF mouse models 25

1.4.2 CF ferret models 28

1.4.3 CF pig models 29

1.4.4 CF cell models 31

1.5 Lipid imbalances in CF epithelia 33

1.5.1 Fatty acids are imbalanced in CF epithelia 35

1.5.2 Cholesterol is increased in CF epithelia 37

1.5.3 Ceramides are imbalanced in CF epithelia 40

1.5.4 Gangliosides are imbalanced in CF epithelia 47

1.5.5 Sphingosine and sphingosine-1-phosphate are imbalanced in CF epithelia 47

1.6 How lipids generally affect membrane protein activity 49

1.6.1 Lipid-dependent membrane mechanics affect membrane protein activity 50

1.6.2 Direct allosteric lipid interactions affect membrane protein activity 52

1.6.3 Lipid-mediated membrane surface localization affects membrane protein activity 53

1.6.4 Lipid-mediated signaling cascades affect membrane protein activity 54

1.6.5 How lipids interact with ABC transporters 54

1.7 How lipids can affect CFTR 57

1.7.1 Detergents and membrane lipids affect CFTR stability and ATPase activity 57

1.7.2 Arachidonic acid affects CFTR activity 61

1.7.3 Ceramide and its derivatives affect CFTR activity 62

1.7.4 Lipid rafts affect CFTR activity 65

1.8 Summary 67

CHAPTER 2 – SPHINGOMYELINASE DECREASES TRANSEPITHELIAL ANION SECRETION IN AIRWAY EPITHELIAL CELLS IN PART BY INHIBITING CFTR-MEDIATED APICAL CONDUCTANCE 68

2.1 Introduction 69

2.2 Materials and methods 70

2.2.1 Airway epithelial cells from human donors 70

2.2.2 Calu-3 and 16HBE bronchial epithelial cell culture 72

2.2.3 Purification of bacterial SMase 73

2.2.4 Lipidomics mass spectrometry 73

2.2.5 Short-circuit current measurements 75

2.2.6 Impedance measurements 77

2.2.7 Calcein flux assay 79

2.2.8 Statistical analyses 80

2.3 Results 80

2.3.1 WT SMase affects the sphingolipid profile in nHBE cells 80

2.3.2 WT SMase decreases short-circuit CFTR current in nHBE cells and nHTE cells 87

2.3.3 Differential effect of WT SMase on immortalized Calu-3 and 16HBE cells 91

2.3.4 WT SMase affects impedance-derived apical and basolateral conductance in nHBE cells 93

2.3.5 WT SMase does not affect paracellular permeability in nHBE cells 96

2.3.6 WT SMase inhibits CFTR current and conductance in cfHBE cells 102

2.4 Discussion 108

CHAPTER 3 – MECHANISTIC ANALYSIS AND SIGNIFICANCE OF SMASE-MEDIATED DECREASES IN TRANSEPITHELIAL CFTR CURRENTS IN HBE CELLS 113

3.1 Introduction 113

3.2 Materials and methods 115

3.2.1 Airway epithelial cells from human donors 115

3.2.2 Purification of bacterial SMase 115

3.2.3 Lipidomics mass spectrometry 116

3.2.4 Short-circuit current measurements 116

3.2.5 Analysis of surface expression of CFTR in nHBE cells 116

3.2.6 Neutrophil transmigration 117

3.2.7 Western blot analysis 118

3.2.8 Statistical analyses 119

3.3 Results 119

3.3.1 Inhibiting CDases with ceranib-1 affects CFTR currents similarly to WT SMase 119

3.3.2 Ceranib-1 affects total cellular dihydroceramides, as does SMase 123

3.3.3 No decrease in relative CFTR surface expression was detected following SMase treatment 123

3.3.4 AMPK is not involved in the WT SMase-mediated decrease in transepithelial CFTR currents 126

3.3.5 Ceramide and dihydroceramide levels in cfHBE compared to nHBE cells 130

3.3.6 Effects of modulator therapy on sphingolipids in cfHBE cells 135

3.3.7 Imbalance of acid-SMase in cfHBE cells and the effects of modulators 138

3.3.8 LTB4-transmigrated neutrophils secrete acid-SMase 138

3.4 Discussion 140

3.4.1 Limitations 145

CHAPTER 4 – DETERMINING THE EFFECTS OF CHOLESTEROL EXTRACTION ON CFTR CHANNEL ACTIVITY 147

4.1 Introduction 147

4.2 Methods 148

4.2.1 FRT cells 148

4.2.2 Primary bronchial epithelial cells 149

4.2.3 Transepithelial short-circuit current analysis 149

4.2.4 Cholesterol staining with filipin III 149

4.2.5 Mass spectrometry analysis of cholesterol 150

4.3 Results 151

4.3.1 MβCD extracts cholesterol from FRT and nHBE cells 151

4.3.2 MβCD shifts the forskolin dose-response curve of CFTR to the right in FRT cells 151

4.3.3 MβCD shifts the forskolin dose-response curve of CFTR to the right in nHBE cells 155

4.4 Discussion 158

CHAPTER 5 – UTILIZATION OF THE PLANAR LIPID BILAYER TECHNIQUE TO STUDY SINGLE-CHANNEL CFTR ACTIVITY 160

5.1 Introduction 161

5.2 Methods 161

5.2.1 Growth and maintenance of BHK cells stably expressing His-tagged CFTR 161

5.2.2 Generation of His-tagged CFTR microsomes from BHK cells 162

5.2.3 Purification of His-tagged CFTR from BHK cells and generation of proteoliposomes 162

5.2.4 Planar Lipid Bilayer 164

5.3 Results 168

5.3.1 A CFTR-like current can be detected in His-CFTR-expressing BHK microsomes 168

5.3.2 The CFTR current from His-CFTR-expressing BHK Microsomes is sensitive to INH172 171

5.3.3 Purified CFTR proteoliposomes can be analyzed by PLB but are not as sensitive to INH172 171

5.4 Discussion 174

CHAPTER 6 – THE EFFECTS OF LONG-TERM HYPERGLYCEMIA ON TIGHT JUNCTION FIDELITY IN NON-CF AND CF 16HBE CELLS 178

6.1 Introduction 178

6.2 Methods 180

6.2.1 16HBE cells 180

6.2.2 Transepithelial short-circuit current analysis 180

6.2.3 Transcriptional expression analysis 181

6.2.4 Immunofluorescence staining 183

6.3 Results 184

6.3.1 CFTR mutation and long-term glucose growth affect TER in 16HBE cells 184

6.3.2 CFTR mutation and chronic hyperglycemia affect mRNA expression of tight junction proteins 184

6.3.3 CFTR mutation and long-term hyperglycemia affect CLDN1 protein expression 186

6.3.4 CFTR mutation and long-term glucose growth affect CLDN3 protein expression and localization 189

6.3.5 CFTR mutation and long-term glucose growth affect CLDN4 protein expression and localization 194

6.3.6 CFTR mutation and long-term glucose growth affect CLDN7 protein expression and localization 197

6.4 Discussion 197

CHAPTER 7 – CONCLUSIONS 202

CHAPTER 8 – APPENDIX OF ADDITIONAL PROJECTS 207

8.1 Identification of CFTR in lamprey intestine 207

8.2 Development of the Programable Automated Cell Culture System (PACCS) 207

8.3 Expanding the applicability of a new impedance analysis technology 209

8.4 Determining the impacts of β2AR agonists on VX770-mediated potentiation of CFTR 210

8.5 Evaluating the importance of cholesterol on CLC channels 211

CHAPTER 9 – APPENDIX OF DETAILED ELECTROPHYSIOLOGY METHODS 213

9.1 Ussing chamber analysis 213

9.1.1 Recording solutions 213

9.1.2 Sources of reagents and equipment 214

9.1.3 Setting up and running the experiment 214

9.1.4 Application notes 217

9.2 PLB analysis 217

9.2.1 Sources of reagents and equipment 217

9.2.2 Electrophysiology Physical Setup 218

9.2.3 Running an Experiment 219

REFERENCES 224

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Physiology Biophysics, General Biology, Cell
Parola chiave	Ussing chamber lipidomics primary bronchial epithelial cells CFTR sphingomyelinase impedance analysis
Committee Chair / Thesis Advisor	Nael McCarty, Emory University
Committee Members	Andrew Jenkins, Emory University Michael Koval, Emory University Eric Ortlund, Emory University Joshua Chandler, Emory University

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