La mucoviscidose est la maladie autosomique récessive grave la plus fréquente dans la population d'origine caucasienne. Elle est due a des anomalies du gène CFTR, dont les multiples mutations décrites rendent compte en partie de la grande variabilité phénotypique. A l'heure du développement de thérapies ciblées selon les mutations portées par les patients, mieux comprendre les mécanismes sous-jacents des relations génotype-phénotype semble de première importance. La première partie de ce travail est focalisée sur la relation génotype-phénotype. Par une étude rétrospective de 694 demandes de diagnostic prénatal de la mucoviscidose sur signes d'appel échographique, nous définissons les profils d'anomalies digestives les plus discriminants, et proposons en conséquence une révision de la stratégie d'analyse moléculaire du gène CFTR. La deuxième partie concerne la mise en place d’outils nécessaires à l’exploration fonctionnelle du promoteur CFTR. En effet, dans les formes atypiques de la maladie, la fonction résiduelle de CFTR peut expliquer le phénotype. Des anomalies de régulation de la transcription peuvent parfois être à l’origine de telles formes modérées. La mise en place des outils d’analyse des variants du promoteur permettra de mieux interpréter leur pathogénicité et d’ouvrir de nouvelles pistes pour la compréhension de la régulation de ce gène. La troisième partie s'intéresse a l'inflammation pulmonaire anormalement régulée qui est une caractéristique phénotypique et le premier facteur de morbidité et de mortalité de la mucoviscidose. La protéine COMMD1 est une protéine pleiotrope participant a de nombreux processus cellulaires, principalement par un mécanisme de stabilisation d'interactions protéiques. Elle est impliquée dans les trois voies thérapeutiques : modulation de CFTR, restauration du liquide de surface des voies aériennes et inhibition de l'inflammation. Notre étude a permis d'observer l'activité anti-inflammatoire de COMMD1 dans le contexte d'inflammation exacerbée décrite chez les patients atteints de mucoviscidose. La réduction de cette réaction exacerbée fait partie des enjeux thérapeutiques actuels et nous montrons ici que la protéine COMMD1 est un bon candidat comme modérateur de l'inflammation mediee par NF-kB dans cette pathologie. / Cystic fibrosis (CF) is the most common severe autosomal recessive disorder in the Caucasian population. Apart from classical CF, there is a broad range of phenotypes associated with a huge genotypic variability concerning the mutations in the CFTR gene. In order to develop a mutation specific therapeutic approach, a better understanding of the phenotype]genotype correlation and its underlying mechanism is primordial. In the first part of our work, we focused on genotype‐phenotype correlation. With a retrospective study on 694 cases of prenatal diagnosis of CF for fetal bowel anomalies, we report on the most evocative digestive abnormal patterns and propose to revise current strategies for the CFTR gene analysis. The second part concerns the CFTR promoter functional analysis. Mutations which conserve a residual CFTR channel function, such as mutations affecting the gene regulation, can be involved in atypical phenotypes. However, knowledge about the CFTR promoter reminds poor and the clinical significance of new variants identified in this region is difficult to evaluate. Our implementation of functional analysis tools will improve the appreciation of such new variants in the CFTR promoter and open new insights for the gene regulation study. In the third part, we contributed to study the inappropriate pulmonary inflammation which characterizes CF, the respiratory affection being the major factor of morbidity and mortality in the disease. COMMD1 is a pleiotropic protein involved in CFTR trafficking, ionic exchanges in the airways surface liquid and inflammation inhibition. In our study, we show the anti]inflammatory role of COMMD1 in the context of cystic fibrosis. Modulation of the exaggerated inflammation belongs to currently therapeutic challenges, and we show the ability of COMMD1, a protein partner of CFTR, to buffer the NF-kB pathway activation

La mutation F508del dans la protéine CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) est considérée comme un facteur de risque indépendant de maladie osseuse liée à la mucoviscidose. Nous avons évalué la densité minérale osseuse et les paramètres histomorphométriques de la formation osseuse et la masse osseuse chez des souris homozygotes F508del-CFTR (F508del) et contrôle(WT), de la même portée, âgées de 6 semaines (pré-pubères), 10 semaines (pubères) et 14 semaines (jeunes adultes), dans deux sexes. L'architecture osseuse des souris F508del et WT a été évaluée par densitométrie osseuse, micro-CT et l'analyse des paramètres dynamiques de la formation osseuse a été réalisée par double marquage, in vivo. Les niveaux de sérum de l'insuline-like growth factor 1 (IGF-1) et l'ostéocalcine ont également été déterminés. Une diminution de la densité minérale osseuse, de la masse osseuse et une altération de l'architecture osseuse trabéculaire fémorale ont été observées chez les souris F508del par rapport aux témoins à 6, 10 et 14 semaines. Une diminution du taux de formation osseuse dans F508del été mise en évidence, par rapport aux souris témoins, indépendamment de l'âge et du sexe. En outre, nous avons observé un taux sérique d'IGF-1 plus faibles chez la souris F508del par rapport aux souris WT tandis que le taux sérique d'ostéocalcine est normal. Nos résultats démontrent que la mutation F508del-CFTR affecte la masse osseuse trabéculaire en réduisant la formation osseuse.Le N-butyldeoxynojyrimicin (NB-DNJ, le miglustat [Zavesca], les Laboratoires Actelion, Suisse) est un médicament approuvé pour le traitement de la pathologie osseuse de la maladie de Gaucher de type I. Il a été montré que le miglustat normalise le transport des ions sodiques et chlorures dépendant de la protéine CFTR au niveau de la muqueuse nasale des souris F508del. La microarchitecture osseuse de la souris F508del par rapport à son contrôle a été évaluée après l'administration du miglustat à une dose de 120 mg / kg / jour par gavage pendant 28 jours. Les niveaux de sérum IGF-1 et 17β-estradiol (E2) ont également été déterminés. Le traitement, de 4 semaines, avec le miglustat, normalise le volume osseux trabéculaire des vertèbres lombaires de la souris F508del. Cette augmentation du volume osseux est associée à une augmentation du taux de formation osseuse et une augmentation sérique de E2, mais pas au taux sérique d'IGF-1 chez la souris F508del traitées par rapport aux souris non traitées F508del.Nos données montrent clairement que l'administration orale de miglustat normalise la masse osseuse par une augmentation de la formation osseuse chez des souris F508del. Ces résultats appuient fortement le potentiel thérapeutique du miglustat chez les patients atteints de la maladie osseuse associée à la mucoviscidose. / The F508del mutation in the cystic fibrosis transmembrane conductance regulator (Cftr) gene is believed to be an independent risk factor for cystic fibrosis-related bone disease.We evaluated the bone mineral density and histomorphometric parameters of bone formation and bone mass in F508del-CFTR homozygous mice (F508del) and littermate (WT) controls at 6 (prepubertal), 10 (pubertal) and 14 (young adult) weeks of age in two genders. The bone architecture of F508del and WT mice was evaluated by bone densitometry, micro-CT and analysis of dynamic parameters of bone formation. Levels of serum insulin-like growth factor 1 (IGF-1) and osteocalcin were also determined. Reduced bone mineral density, lower femoral bone mass and altered trabecular bone architecture were observed in F508del compared to controls at 6, 10, and 14 weeks of age. A decrease in bone formation rate in F508del was evidenced compared to control mice, independently of age and sex. Additionally, we found lower IGF-1 levels in F508del mice compared to WT mice whereas osteocalcin level was normal. Our findings demonstrate that the F508del mutation in CFTR impacts trabecular bone mass by reducing bone formation.N-butyldeoxynojyrimicin (NB-DNJ, miglustat [Zavesca], Laboratoires Actelion, Suisse) an approved drug for treating bone pathology in type I Gaucher disease, was reported to normalizes sodium and Cftr-dependent chloride transport in nasal mucosa of F508del cystic fibrosis mice. The bone microarchitecture of F508del mice relative to WT littermates was evaluated after an administration of 120 mg/kg/day miglustat by oral gavage for 28 days. Levels of serum IGF-1 and 17β-estradiol (E2) were also determined. Once-day treatments with miglustat, over 4 weeks, normalized trabecular bone volume of the lumbar spine in F508del mice. This increase of bone volume was related to both an increased rate of bone formation and increased serum E2 level but not IGF-1 level in miglustat-treated F508del mice compared to untreated F508del mice.Our data provides clear evidence that oral administration of miglustat normalizes bone mass by increasing bone formation in F508del mice; these findings strongly support the therapeutic potential of miglustat in patients with cystic fibrosis related bone disease.Key words : cystic fibrosis, bone disease, miglustat

The intrinsically disordered nonphosphorylated and phosphorylated R region of CFTR and its interactions with NBD1 and SLC26A3 STAS have been characterized at residue-specific resolution, primarily using NMR. Limited chemical shift dispersion indicates that the R region is intrinsically disordered in solution and that no global folding event occurs upon phosphorylation. Chemical shifts of backbone nuclei and sidechain carbons were assigned. SSP values indicate that phosphorylation acts as a structural switch, with a reduction in helical propensity in multiple nonphosphorylated R region segments. Free nonphosphorylated and phosphorylated R region were characterized using a variety of structural probes. Fast timescale motion indicates the presence of structural contacts in many R region segments. Hydrodynamic radii are intermediate to those expected for fully folded or denatured proteins, with the phosphorylated R region being slightly more compact. The nonphosphorylated R region was further characterized, including measurements of molecular dimensions, N-H bond vector orientation and inter-residue distances from 6 spin label sites. Using these parameters as input to the program ENSEMBLE enabled calculation of a representative pool of nonphosphorylated R region conformations, indicating the presence of transient contacts that could not be directly discerned from the input data. Examining labeled R region with the addition of unlabeled NBD1 provided evidence that multiple segments of nonphosphorylated R region bind and are released from NBD1 with varying affinities in a highly dynamic equilibrium. Phosphorylation relieves these interactions, with the exception of limited R region interactions near S768 when NBD1 is ATP-bound. Largely similar nonphosphorylated R region residues bind both ATP-bound ΔF508 and wild-type NBD1. Addition of unlabeled R region to labeled ATP-bound NBD1 caused spectral changes indicative of a direct interaction with more than one surface or conformational changes within NBD1 that are transmitted from one binding surface to other surface(s). Binding of unlabeled SLC26A3 STAS domain to labeled phosphorylated R region was also monitored and indicated that similar R region segments bind NBD1 and STAS, suggesting a direct competition between these two domains for binding. A model is proposed where the R region acts as a regulatory hub, integrating interactions with a variety of partners to regulate channel function.

The intrinsically disordered nonphosphorylated and phosphorylated R region of CFTR and its interactions with NBD1 and SLC26A3 STAS have been characterized at residue-specific resolution, primarily using NMR. Limited chemical shift dispersion indicates that the R region is intrinsically disordered in solution and that no global folding event occurs upon phosphorylation. Chemical shifts of backbone nuclei and sidechain carbons were assigned. SSP values indicate that phosphorylation acts as a structural switch, with a reduction in helical propensity in multiple nonphosphorylated R region segments. Free nonphosphorylated and phosphorylated R region were characterized using a variety of structural probes. Fast timescale motion indicates the presence of structural contacts in many R region segments. Hydrodynamic radii are intermediate to those expected for fully folded or denatured proteins, with the phosphorylated R region being slightly more compact. The nonphosphorylated R region was further characterized, including measurements of molecular dimensions, N-H bond vector orientation and inter-residue distances from 6 spin label sites. Using these parameters as input to the program ENSEMBLE enabled calculation of a representative pool of nonphosphorylated R region conformations, indicating the presence of transient contacts that could not be directly discerned from the input data. Examining labeled R region with the addition of unlabeled NBD1 provided evidence that multiple segments of nonphosphorylated R region bind and are released from NBD1 with varying affinities in a highly dynamic equilibrium. Phosphorylation relieves these interactions, with the exception of limited R region interactions near S768 when NBD1 is ATP-bound. Largely similar nonphosphorylated R region residues bind both ATP-bound ΔF508 and wild-type NBD1. Addition of unlabeled R region to labeled ATP-bound NBD1 caused spectral changes indicative of a direct interaction with more than one surface or conformational changes within NBD1 that are transmitted from one binding surface to other surface(s). Binding of unlabeled SLC26A3 STAS domain to labeled phosphorylated R region was also monitored and indicated that similar R region segments bind NBD1 and STAS, suggesting a direct competition between these two domains for binding. A model is proposed where the R region acts as a regulatory hub, integrating interactions with a variety of partners to regulate channel function.

During fetal development, the lung is filled with fluid that is secreted by an active Cltransport promoting lung growth. The basolateral Na+,K+,2Cl- cotransporter (NKCC1) participates in Cl- secretion. The apical Cl- channels responsible for secretion are unknown but studies suggest an involvement of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is developmentally regulated with a high expression in early fetal development and a decline in late gestation. Perinatal lung transition is triggered by hormones that stimulate alveolar Na+ channels resulting in fluid absorption. Little is known on how hormones affect pulmonary Cl- channels. Since the rise of fetal cortisol levels correlates with the decrease in fetal CFTR expression, a causal relation may be assumed. The aim of this study was to analyze the influence of glucocorticoids on pulmonary Cl- channels. Alveolar cells from fetal and adult rats, A549 cells, bronchial Calu-3 and 16HBE14o- cells, and primary rat airway cells were studied with real-time quantitative PCR and Ussing chambers. In fetal and adult alveolar cells, glucocorticoids strongly reduced Cftr expression and channel activity, which was prevented by mifepristone. In bronchial and primary airway cells CFTR mRNA expression was also reduced, whereas channel activity was increased which was prevented by LY-294002 in Calu-3 cells. Therefore, glucocorticoids strongly reduce CFTR expression while their effect on CFTR activity depends on the physiological function of the cells. Another apical Cl- channel, anoctamin 1 showed a glucocorticoid-induced reduction of mRNA expression in alveolar cells and an increase in bronchial cells. Furthermore, voltage-gated chloride channel 5 and anoctamine 6 mRNA expression were increased in alveolar cells. NKCC1 expression was reduced by glucocorticoids in alveolar and bronchial cells alike. The results demonstrate that glucocorticoids differentially modulate pulmonary Clchannels and are likely causing the decline of CFTR during late gestation in preparation for perinatal lung transition.

Contexte. Les protéines transmembranaires ont une importance considérable tant au niveau de la survie d'une cellule qu'au niveau de ces interactions avec les autres cellules. En raison de contraintes techniques, la cristallisation de ce type de protéine demeure très complexe, ce qui limite grandement l’exploration de leur structure. Pour contourner ces difficultés, différents outils de prédiction ont été développés,en se fondant originellement sur l'hydrophobicité des régions enfouies dans la membrane. Méthode. L'outil développé repose sur une dérivation de la moyenne d'hydrophobicité calculée sur deux ensembles de taille de fenêtres. Le premier ensemble (G1) contient des petites tailles de fenêtres ce qui correspond à des événements locaux, tandis que le second (G2) correspond à des tailles de fenêtres plus larges, adaptées à la taille des hélices formant certaines protéines transmembranaires. La variation d'hydrophobicité est obtenue en dérivant les moyennes d'hydrophobicité. Un consensus est établi pour chaque groupe, et les résultats sont comparés à un ensemble de protéines transmembranaires cristallisées. Résultats. Les variations d'hydrophobicité G2 sont liées aux extrémités des hélices transmembranaires,tandis que les variations G1 sont en relation avec la limites des structures et certaines irrégularités structurelles.Ces résultats nous ont amené à introduire une nouvelle notion : les unités transmembranaires(TMU). Les TMU consistent en un ensemble de sous-structures qui composent les structures transmembranaires. / Background. Few high-resolution structures of integral membranes proteins are available, as crystallization of such proteins needs yet to overcome too many technical limitations. Nevertheless, prediction oftheir transmembrane (TM) structure by bioinformatics tools provides interesting insights on the topology of these proteins.Method. We describe here how to extract new information from the analysis of hydrophobicity variations or hydrophobic pulses (HPulses) in the sequence of integral membrane proteins using the Hydrophobic Pulse Predictor, a new tool we developed for this purpose. To analyze the primary sequence of 70 integralmembrane proteins we defined two levels of analysis : G1-HPulses for sliding windows of n=2 to 6 andG2-HPulses for sliding windows of n=12 to 16.Results. The G2-HPulse analysis of 541 transmembrane helices allowed the definition of the new conceptof transmembrane unit (TMU) that groups together transmembrane helices and segments with potentialadjacent structures. In addition, the G1-HPulse analysis identified helix irregularities that correspondedto kinks, partial helices or unannotated structural events. These irregularities could represent key dynamicelements that are alternatively activated depending on the channel status as illustrated by the crystalstructures of the lactose permease in different conformations. Our results open a new way in the understanding of transmembrane secondary structures : hydrophobicity through hydrophobic pulses stronglyimpacts on such embedded structures and is not confined to define the transmembrane status of aminoacids.

Ausgangslage: Die genetischen Grundlagen der chronischen Pankreatitis sind zum heutigen Zeitpunkt nur unzureichend erforscht. Mutationen im Gen des Serinprotease-Inhibitors Kazal Type 1 (SPINK1) und heterozygote Mutationen im CFTR-Gen wurden in zahlreichen Untersuchungen gehäuft bei Patienten mit chronischer Pankreatitis nachgewiesen. Methodik: Es wurden retrospektiv anhand der Daten der Pankcourse Studie (2004-2007) Untersuchungen bei Patienten mit chronischer Pankreatitis zur Häufigkeit von SPINK1- und CFTR-Mutationen sowie zum Manifestationszeitpunkt der Erkrankung durchgeführt. In Fall-Kontroll-Analysen wurde untersucht, ob sich Unterschiede in den jeweiligen Krankheitsverläufen nachweisen lassen. Ergebnisse: Eine heterozygote SPINK1-Mutation (N34S) konnte bei 11,5% und eine CFTR-Mutationen bei 24% der untersuchten Patienten nachgewiesen werden. Bei Patienten mit SPINK1-Mutation fand sich im Gegensatz zu Patienten mit CFTR-Mutation eine signifikant frühere Krankheitsmanifestation als bei Patienten ohne Mutationsnachweis. Patienten mit SPINK1-Mutation mussten zudem seltener und später operiert werden als Patienten ohne Mutation. Bei Patienten mit CFTR-Mutation zeigte sich ein signifikant früheres Auftreten von Stenosierungen und Konkrementen des D. pancreaticus im Vergleich zur Kontrollgruppe. Schlussfolgerung: Die ätiologische Bedeutung von SPINK1- und CFTR-Mutationen konnte bestätigt werden. Es fanden sich einzelne Hinweise auf einen durch die jeweilige Mutation verursachten charakteristischen Krankheitsverlauf, was durch weitergehende Untersuchungen bestätigt werden muss.

La mucoviscidose (CF, Cystic Fibrosis) est la maladie génétique la plus fréquente dans les populations d’origine caucasienne. Les malades présentent une symptomatologie variée, dominée par une bronchopneumopathie chronique obstructive due à des sécrétions de mucus abondantes et anormalement épaisses et une réponse inflammatoire chronique excessive. La mucoviscidose résulte de mutations dans le gène codant la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator), dont la plus fréquente est la délétion d’une phénylalanine en position 508 (F508del) qui est à l’origine d’un adressage défectueux et d’une fonction altérée de la protéine. Afin d’identifier différents partenaires moléculaires de CFTR qui participent à son processus de maturation, à son trafic à la membrane plasmique ou à sa fonction, un criblage double hybride de levure a été effectué en utilisant la troisième boucle intra-cytoplasmique de CFTR (ICL3) comme appât. A l’issue de ce criblage, 14 clones indépendants ont pu être identifiés dont la protéine COMMD1 qui a initialement été décrite comme un régulateur de l’homéostasie du cuivre, de l’absorption sodique et de la voie de signalisation NF-?B. L’objectif principal de ce travail a été de déterminer quel pouvait être l’impact de la protéine COMMD1 sur la maturation et le trafic intracellulaire de CFTR afin de déterminer le rôle de cette protéine dans la physiopathologie de la mucoviscidose. Nous avons montré que la protéine COMMD1 est un nouveau partenaire cytoplasmique du canal CFTR, qui régule le trafic intracellulaire de ce canal par inhibition de l’ubiquitinylation probablement au niveau des endosomes d’endocytose et de recyclage. Notre étude permet de proposer une nouvelle voie de trafic de CFTR via un modèle d’ubiquitinylation régulé par COMMD1. Au cours de cette étude, nous avons également identifié le récepteur 1 de la transferrine (TFR1) comme un nouveau partenaire de COMMD1 dont le mécanisme de régulation semble similaire à celui proposé pour CFTR. Dans un second temps, nous nous sommes intéressés aux propriétés inhibitrices de COMMD1 dans la réaction inflammatoire. COMMD1 a été décrit comme le prototype d’une nouvelle famille de protéines jouant un rôle dans l’inhibition de la voie de signalisation NF-kB. Nous avons observé que la distribution subcellulaire de COMMD1 est différente dans les cellules CF et non-CF. Nous avons mis en évidence que la surexpression de COMMD1 dans les cellules épithéliales bronchiques CF, qui présentent une inflammation excessive, pouvait restaurer un niveau d’inflammation comparable aux cellules non-CF. COMMD1 est impliquée dans plusieurs processus cellulaires altérés dans la physiopathologie de la mucoviscidose, affectant le trafic du canal CFTR, l’absorption de sodium et la réponse inflammatoire. Comprendre comment moduler d’une part le processus d’absorption/sécrétion des ions par le trafic de canaux ioniques et d’autre part, l’inflammation des cellules CF par rapport aux non-CF, devrait permettre d’identifier de nouvelles pistes thérapeutiques. Ces traitements permettraient à la fois de réduire la réaction inflammatoire exacerbée, sans nuire à l’activité essentielle de défense contre les pathogènes, et d’améliorer la sécrétion de fluide chez les patients atteints de mucoviscidose / Cystic fibrosis is mainly caused by mutations interfering with the biosynthetic folding of the CFTR protein. The aim of this study was to find proteins able to interact with CFTR and modify its processing. We have identified COMMD1 as a new CFTR partner. COMMD1 is a regulator of copper homeostasis and sodium uptake through interaction with ENaC, as well as the prototype of a new protein family that plays a role in inhibiting NF-?B signalling Co-immunoprecipitation experiments showed that COMMD1 associates with endogenous CFTR in HT29 cells and with F508del-CFTR in heterologously expressing epithelial cells. COMMD1 sub-cellular distribution is both nuclear and cytoplasmic, and more precisely in vesicular cytoplasmic compartments, as assessed by immunocytochemical microscopy. Further studies showed COMMD1 partial codistribution with an early endosomal compartments (TfR). COMMD1 is not involved in CFTR processing (C band) but wt-CFTR cell surface expression was halfreduced when COMMD1 expression was silenced. Unlike F508del-CFTR in temperature rescue, COMMD1 over-expression increased 15% wt-CFTR cell surface expression. Assessment of CFTR ubiquitination showed that COMMD1 over-expression strongly decreased CFTR ubiquitination therefore increasing CFTR cell surface expression. Finally, these data indicate that COMMD1 vesicular compartment is involved in CFTR trafficking through inhibition of CFTR ubiquitination. Understanding how COMMD1 modulation modifies transepithelial transport and inflammation in CF versus non CF cells should give new therapeutic clues to reduce exacerbated inflammation and improve fluid secretion in CF patients

Background: Excess plugging of small airways is associated with premature death in chronic obstructive pulmonary disease (COPD). Over-expression of beta-epithelial sodium channel (β-ENaC) in airway epithelia in mice resulted in plugging of small airways while cystic fibrosis transmembrane regulator (CFTR) negatively regulated ENaC activity in cell models. Purpose: To test the hypothesis that accumulation of mucus exudates observed with the progression of COPD is related to excess airway epithelial sodium re-absorption as a result of over-expression of β-ENaC and reduced expression of CFTR by small airway epithelia. Methods: Small airway epithelial samples from frozen lungs from patients at different levels of COPD severity were isolated by laser capture microdissection (LCM). β-ENaC, CFTR, and β-actin (control) gene expression was determined by qRT-PCR and compared to expression in entire airways and lung parenchyma surrounding these airways. β-ENaC protein as well as epithelial mucin expression and mucus plugging were localized and quantified after immunohistochemical and periodic acid Schiff staining, respectively. Results: β-ENaC mRNA expression had a strong positive correlation with that of CFTR (p<O.0001) in airway epithelia and surrounding lung parenchyma (p=O.Ol) but not whole airways. β-ENaC mRNA and protein expression were positively correlated (p=O.4O, p=O.O5) and protein expression significantly increased with GOLD stage of COPD severity. Epithelial mucin expression positively correlated with β-ENaC (p=O.38, p=O.O5) and CFTR (p=OAO, p=O.O4.) mRNA and with mucus plugging (p=O. 43 , ptO.OOO2). CFTR mRNA also correlated positively with mucus plugging (p=O. 48 , p=O.O2). Conclusions: Strong positive correlations between β-ENaC and CFTR mRNA expression that are limited to the lung parenchyma and epithelium suggest a novel mechanism of mRNA regulation. This differs from their functional relationship where an inverse relationship between CFTR expression and β-ENaC activity has been reported. Positive correlations of epithelial mucin or mucus plugging with CFTR mRNA but not β-ENaC protein expression in the small airway epithelium suggest that CFTR may regulate mucin at this site independently of β-ENaC protein. The relationship between β-ENaC mRNA andepithelial mucin expression could be due to strong correlations between β-ENaC and CFTR mRNA expression but β-ENaC’s relationship with COPD GOLD stage suggests it may nevertheless play a role in COPD.

Background: Excess plugging of small airways is associated with premature death in chronic obstructive pulmonary disease (COPD). Over-expression of beta-epithelial sodium channel (β-ENaC) in airway epithelia in mice resulted in plugging of small airways while cystic fibrosis transmembrane regulator (CFTR) negatively regulated ENaC activity in cell models. Purpose: To test the hypothesis that accumulation of mucus exudates observed with the progression of COPD is related to excess airway epithelial sodium re-absorption as a result of over-expression of β-ENaC and reduced expression of CFTR by small airway epithelia. Methods: Small airway epithelial samples from frozen lungs from patients at different levels of COPD severity were isolated by laser capture microdissection (LCM). β-ENaC, CFTR, and β-actin (control) gene expression was determined by qRT-PCR and compared to expression in entire airways and lung parenchyma surrounding these airways. β-ENaC protein as well as epithelial mucin expression and mucus plugging were localized and quantified after immunohistochemical and periodic acid Schiff staining, respectively. Results: β-ENaC mRNA expression had a strong positive correlation with that of CFTR (p<O.0001) in airway epithelia and surrounding lung parenchyma (p=O.Ol) but not whole airways. β-ENaC mRNA and protein expression were positively correlated (p=O.4O, p=O.O5) and protein expression significantly increased with GOLD stage of COPD severity. Epithelial mucin expression positively correlated with β-ENaC (p=O.38, p=O.O5) and CFTR (p=OAO, p=O.O4.) mRNA and with mucus plugging (p=O. 43 , ptO.OOO2). CFTR mRNA also correlated positively with mucus plugging (p=O. 48 , p=O.O2). Conclusions: Strong positive correlations between β-ENaC and CFTR mRNA expression that are limited to the lung parenchyma and epithelium suggest a novel mechanism of mRNA regulation. This differs from their functional relationship where an inverse relationship between CFTR expression and β-ENaC activity has been reported. Positive correlations of epithelial mucin or mucus plugging with CFTR mRNA but not β-ENaC protein expression in the small airway epithelium suggest that CFTR may regulate mucin at this site independently of β-ENaC protein. The relationship between β-ENaC mRNA andepithelial mucin expression could be due to strong correlations between β-ENaC and CFTR mRNA expression but β-ENaC’s relationship with COPD GOLD stage suggests it may nevertheless play a role in COPD.