Epithelial cells lining the upper airways are characterized by low sodium absorption and elevated chloride secretion. Together, the movement of these ions creates the osmotic drive to hydrate the airways. Recent studies indicate that influenza is capable of directly modulating the vectorial transport of sodium and chloride ions. However, the direct impact of influenza has not been studied with respect to potassium channels. This is significant because potassium conductance creates the driving force for chloride secretion. Disruptions to this process leads to edema formation in the lungs and can subsequently cause Acute Respiratory Distress Syndrome. Additionally, it has been demonstrated that the induction of pro-inflammatory cytokines in infected cells may contribute to altered ion channel function, further exacerbating edema formation. The purpose of this study was to assess the direct and indirect effects of influenza virus infection on potassium and chloride ion channel function in a secretory epithelial cell model. In order to assess the direct effects we exposed polarized epithelial cell monolayers to varying doses of H1N1 virus. Potassium and chloride channel function was measured by means of short-circuit current in an Ussing chamber. The immune response to viral infection was determined by RT-qPCR and Bioplex suspension array. Virus conditioned media (CM), and IL-8 were used to characterize the indirect effects on non-infected cells. We observed an increase in chloride secretion, consistent with edema formation, when 60% of the epithelium was infected, and after CM treatment. This observation correlated with increased potassium channel conductance through the calcium-activated (KCNN4) and cAMP-activated potassium channels (KCNQ1), which was ameliorated upon specific inhibition of these channels. The data suggest that the mixture of pro-inflammatory cytokines induced by viral infection directly up-regulate these potassium channels. However, treatment with IL-8 also appears to increase chloride secretion, although the underlying mechanism remains to be determined, as it is not mediated through KCNN4 and KCNQ1. We conclude that the strong induction of cytokines in infected cells act in a paracrine manner on non-infected cells to increase potassium channel conductance. This up-regulation of potassium channels subsequently drives an increase in chloride secretion, leading to fluid build-up in the lungs and edema formation.

Cystic Fibrosis (CF) is caused by mutations in CFTR, a protein for chloride efflux in epithelial cells. VIP is a peptide that activates CFTR and improves membrane stability; VIP has 3 receptors VPAC1, VPAC2 and PAC1 that can cause CFTR phosphorylation. VIP-knockout (VIPKO) mice experience inflammation and reduced CFTR membrane localization comparable to CF phenotypes, that’s reversible after 3 weeks of VIP treatment (VIPKOT). In this thesis western blotting showed VPAC1 and VPAC2 expression increased in VIPKO and VIPKOT lung and duodenum tissues. The expression and maturation of CFTR was unchanged in both VIPKO and VIPKOT tissues. The results showed absence of VIP caused increased receptor expression in VIPKO mice, after VIP treatment VIPKO mice maintained increased receptor expression. VIP treatment reduces inflammation and restores existing CFTR membrane localization in VIPKO mice. VIP receptor expression may be important for future treatment of CF for CFTR localization and reducing tissue inflammation.

Misfolding of membrane proteins as a result of mutations that disrupt their functions in substrate transport across the membrane or signal transduction is the cause of many significant human diseases. Yet, we still have a limited understanding of the direct consequences of these mutations on folding and function - a necessary step toward the rational design of corrective therapeutics. This thesis addresses the gap in understanding the residue-specific implications for folding through a series of experiments that utilize the cystic fibrosis transmembrane conductance regulator (CFTR) as a model in various contexts. We first examined the thermodynamic implications of mutations in the soluble nucleotide binding domain 1 (NBD1) of CFTR. We found that mutations can have a significant effect on thermodynamic stability that is masked in non-physiological conditions. Our studies were then focussed on a membrane-embedded hairpin CFTR fragment comprised of transmembrane segments 3 (TM3) and 4 (TM4) to evaluate the direct effects of mutations on folding in a systematic manner. It was found that the translocon-mediated membrane insertion of helices closely parallels a basic hydrophobic-aqueous partitioning event. This study was then extended to determine residue-specific effects on helix-helix association. We found that this process is not solely dependent on hydropathy, but there is a context dependence of these results with regard to residue position within the helix. Overall, these findings constitute a key step in relating mutation-derived effects on membrane protein folding to the underlying basis of human disease such as cystic fibrosis.

Les canaux TRP (Transient Receptor Potential) sont des acteurs clés de l'homéostasie calcique. Plusieurs de ces canaux interviennent dans l'influx calcique des cellules épithéliales bronchiques, notamment TRPC6, qui est impliqué dans un couplage fonctionnel avec le canal Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Les mutations du CFTR (F508del et G551D) sont à l'origine de la mucoviscidose (Cystic Fibrosis (CF)), qui aboutit à l'augmentation de l'influx calcique dans les cellules CF. L'objectif de ce travail a été d'étudier l'implication des canaux TRP dans la dérégulation de l'influx calcique des cellules épithéliales bronchiques CF. Nous avons mis en évidence que CFTR régulait négativement l'activité de TRPC6, tandis que l'influx calcique via TRPC6 permettait de potentialiser l'activité du canal muté CFTR-G551D, activé au préalable par le VX-770. Nous proposons donc une nouvelle stratégie thérapeutique, combinant un potentiateur de CFTR et un activateur spécifique de TRPC6. Nous nous sommes ensuite intéressés au rôle des canaux TRPV, en particulier TRPV5 et TRPV6, dans l'influx calcique des cellules épithéliales bronchiques. Nous avons observé que l'influx Ca2+ constitutif, attribuable à ces deux canaux, était doublé dans les cellules CF, dû à une augmentation de l'activité de TRPV6. En effet, l'expression de la PLC-δ1, une enzyme régulant négativement TRPV6, est dramatiquement réduite dans les cellules CF. La correction de l'adressage du F508del-CFTR a permis de normaliser l'activité de TRPV6 sans restaurer l'expression de la PLC-δ1 dans les cellules CF, suggérant un contrôle plus complexe de TRPV6 dans les cellules épithéliales bronchiques. / TRP (Transient Receptor Potential) channels are keys actors of Ca2+ homeostasis. Several of these channels are involved in the Ca2+ influx of bronchial epithelial cells, including TRPC6 which is implicated in a functional coupling with the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) channel. CFTR mutation leads Cystic Fibrosis (CF) disease and causes abnormal Ca2+ homeostasis trought an increased of Ca2+ influx in CF bronchial epithelial cells. Our objective is to investigate the implication of TRP channels in abnormal Ca2+ influx of CF bronchial epithelial cells.We showed that CFTR down regulates TRPC6 activity whereas Ca2+ influx through TRPC6 potentiates G551D-CFTR, activated by VX-770. We propose a new therapeutic strategy that combines a CFTR potentiator and a specific activator of TRPC6. Then, we focused on the role of TRPV channels, particularly TRPV5 and TRPV6, in Ca2+ influx of bronchial epithelial cells. We observed that constitutive Ca2+ influx, related to TRPV5/TRPV6 activity, was twice higher in CF cells due to the increase of TRPV6 activity. The expression of PLC-δ1, an enzyme that negatively regulates TRPV6 activity, is dramatically decreased in CF cells. The correction of F508del-CFTR trafficking allows TRPV6 activity normalization but do not restore PLC-δ1 expression level in CF cells, suggesting a more complex control of TRPV6 in bronchial epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that when mutated causes the disease cystic fibrosis (CF). Many obstacles hinder the understanding of CF disease pathogenesis, impeding advancements in understanding how mutations cause disease, and slowing the progress towards new treatments. To this end, we have profiled the transcriptome (mRNA and microRNA) of human and newborn pig CF and non-CF airway epithelia. We show that the use of cross-species transcriptomics allows the identification of genes differentially expressed owing to the loss of CFTR, and not due to confounding environmental or secondary disease progression influences. The identification of reduced OAS1 expression in CF samples is a case in point. We also demonstrate the utility of transcriptome profiling and longitudinal studies in pigs, providing greater understanding of the molecular mechanisms underlying CF disease progression. MicroRNAs (miRNAs) comprise a large family of ~21-nt long non-coding RNAs that function as key post-transcriptional regulators of gene expression. Very little is known of how CFTR is regulated in the cell, both transcriptionally and post-transcriptionally. We discovered three miRNAs: miR-509-3p, miR-494 and miR-138 with possible CFTR regulatory functions. miR-509-3p or -494 directly target the CFTR mRNA, and decrease CFTR levels when over expressed; while inhibiting them had the opposite effect. Upon stimulating human airway epithelial cells with TNFα or IL-1β, we observed an increase in expression of both miRNAs mediated in part by the NF-κB transcription factor complex, with a concurrent decrease in CFTR expression. Gene ontology classification of predicted targets of miR-509-3p and/or miR-494 expressed in the airway epithelium revealed enrichment for genes in ion transport pathways. To our knowledge, this is the first suggestion of a possible role for miRNAs regulating a broad range of important epithelial electrolyte and fluid transport proteins. The study of miR-138 mediated regulation of CFTR expression has led to novel discoveries in the field of CFTR transcriptional control. We discovered SIN3A to be a novel transcriptional repressor of CFTR, interacting with CTCF on the CFTR promoter at the -20.9 kb DHS. By validating SIN3A as a conserved target of miR-138, we also discovered miR-138 to be a novel transcriptional regulator/activator of CFTR. The most common CFTR mutation, ΔF508, causes protein misfolding, degradation, and CF. Manipulating the miR-138/SIN3A regulatory network improved the biosynthesis of CFTR-ΔF508, restoring Cl- transport to human CF airway epithelia. To our knowledge, this is the first example of an individual miRNA having such broad regulatory functions. This discovery also provided novel targets for restoring CFTR function in cells affected by the most common CF mutation. To this end, we are utilizing the molecular signatures of miR-138 over-expression and SIN3A knockdown to identify candidate genes for RNA interference screens, and to identify candidate small molecule drugs that might mimic the effects of these two interventions. The goal of this approach is to develop a new therapeutic agent that restores anion transport to airway epithelia and other cell types and tissues affected by CF.

The human colonic cell line T84, a model for studying epithelial chloride secretion and cystic fibrosis chloride channel (CFTR) function, was used to investigate the regulatory role of estrogens in transepithelial ion transport. Estrogens and other steroid hormones do not stimulate chloride secretion by themselves. However, 17 β-estradiol (17β-E2) rapidly (within seconds to minutes) potentiates carbachol- and thapsigargin-stimulated chloride secretion measured as short circuit current in voltage-clamped T84 monolayer cultures. The cholinergic agonist carbachol and the SR Ca2+ ATPase inhibitor thapsigargin stimulate chloride secretion by elevating intracellular calcium. 17α-estradiol, a stereoisomer that does not activate nuclear estrogen receptors, is equipotent with 17β-E2. Other non-estrogen steroids produce much less, if any, potentiation of calcium-stimulated chloride secretion. The estrogen receptor antagonist tamoxifen does not block 17β-E2 potentiation of calcium-stimulated chloride secretion, indicating that the classical estrogen receptors are not involved. Potentiation is greater when 17β-E2 is applied to the apical membrane than to the basolateral membrane. 17β-E2 effects on chloride secretion coincide with an increase in monolayer electrical conductance, which is consistent with activation of one or more ion channel species. Potentiation is not blocked by the chloride channel blockers DIDS and NPPB but is abolished by the PKA inhibitor H89, suggesting that 17β-E2 potentiation depends on the activity of CFTR but not other types of apical membrane chloride channels. 17β-E2 does not increase the activity of calcium-activated potassium channels in the basolateral membrane as measured in nystatin-permeabilized monolayers. 17β-E2 effects are not blocked by the MAP kinase kinase inhibitor PD 98059, or by the PKC inhibitor bisindoylmaleamide, suggesting that these signal transduction pathways are not involved. 17β-E2 potentiation requires extracellular Ca2+. Paradoxically, 17β-E2 reduces the rise in intracellular free Ca2+ levels in thapsigargin-stimulated T84 cells, as measured by fura-2 fluorescence. From my studies I conclude that 17β-E2 causes an increase in the sensitivity of T84 cells to calcium-elevating secretagogues. This effect may be due to nongenomic actions of 17β-E2 on CFTR function and/or the activity of store-operated calcium channels, which leads to a change in CFTR functional regulation.

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