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Lack of cystic fibrosis transmembrane conductance regulator disrupts fetal airway development in pigs.

ABSTRACT: Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing the lungs to chronic infection and inflammation. In young infants with CF, structural airway defects are increasingly recognized before the onset of significant lung disease, which suggests a developmental origin and a possible role in lung disease pathogenesis. The role(s) of CFTR in lung development is unclear and developmental studies in humans with CF are not feasible. Young CF pigs have structural airway changes and develop spontaneous postnatal lung disease similar to humans; therefore, we studied lung development in the pig model (non-CF and CF). CF trachea and proximal airways had structural lesions detectable as early as pseudoglandular development. At this early developmental stage, budding CF airways had smaller, hypo-distended lumens compared to non-CF airways. Non-CF lung explants exhibited airway lumen distension in response to forskolin/IBMX as well as to fibroblast growth factor (FGF)-10, consistent with CFTR-dependent anion transport/secretion, but this was lacking in CF airways. We studied primary pig airway epithelial cell cultures and found that FGF10 increased cellular proliferation (non-CF and CF) and CFTR expression/function (in non-CF only). In pseudoglandular stage lung tissue, CFTR protein was exclusively localized to the leading edges of budding airways in non-CF (but not CF) lungs. This discreet microanatomic localization of CFTR is consistent with the site, during branching morphogenesis, where airway epithelia are responsive to FGF10 regulation. In summary, our results suggest that the CF proximal airway defects originate during branching morphogenesis and that the lack of CFTR-dependent anion transport/liquid secretion likely contributes to these hypo-distended airways.

SUBMITTER: Meyerholz DK

PROVIDER: S-EPMC6019641 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Lack of cystic fibrosis transmembrane conductance regulator disrupts fetal airway development in pigs.

Meyerholz David K DK Stoltz David A DA Gansemer Nick D ND Ernst Sarah E SE Cook Daniel P DP Strub Matthew D MD LeClair Erica N EN Barker Carrie K CK Adam Ryan J RJ Leidinger Mariah R MR Gibson-Corley Katherine N KN Karp Philip H PH Welsh Michael J MJ McCray Paul B PB

Laboratory investigation; a journal of technical methods and pathology 20180221 6

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing the lungs to chronic infection and inflammation. In young infants with CF, structural airway defects are increasingly recognized before the onset of significant lung disease, which suggests a developmental origin and a possible role in lung disease pathogenesis. The role(s) of CFTR in lung development is unclear and developmental studies in humans with CF are not feasible. Young ...[more]

PMID: 29467455

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Project description:Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin–proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B. Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio. Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level. Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

Project description:Excessive consumption of ethanol is one of the most common causes of acute and chronic pancreatitis. Alterations to the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) also cause pancreatitis. However, little is known about the role of CFTR in the pathogenesis of alcohol-induced pancreatitis.We measured CFTR activity based on chloride concentrations in sweat from patients with cystic fibrosis, patients admitted to the emergency department because of excessive alcohol consumption, and healthy volunteers. We measured CFTR levels and localization in pancreatic tissues and in patients with acute or chronic pancreatitis induced by alcohol. We studied the effects of ethanol, fatty acids, and fatty acid ethyl esters on secretion of pancreatic fluid and HCO3(-), levels and function of CFTR, and exchange of Cl(-) for HCO3(-) in pancreatic cell lines as well as in tissues from guinea pigs and CFTR knockout mice after administration of alcohol.Chloride concentrations increased in sweat samples from patients who acutely abused alcohol but not in samples from healthy volunteers, indicating that alcohol affects CFTR function. Pancreatic tissues from patients with acute or chronic pancreatitis had lower levels of CFTR than tissues from healthy volunteers. Alcohol and fatty acids inhibited secretion of fluid and HCO3(-), as well as CFTR activity, in pancreatic ductal epithelial cells. These effects were mediated by sustained increases in concentrations of intracellular calcium and adenosine 3',5'-cyclic monophosphate, depletion of adenosine triphosphate, and depolarization of mitochondrial membranes. In pancreatic cell lines and pancreatic tissues of mice and guinea pigs, administration of ethanol reduced expression of CFTR messenger RNA, reduced the stability of CFTR at the cell surface, and disrupted folding of CFTR at the endoplasmic reticulum. CFTR knockout mice given ethanol or fatty acids developed more severe pancreatitis than mice not given ethanol or fatty acids.Based on studies of human, mouse, and guinea pig pancreata, alcohol disrupts expression and localization of the CFTR. This appears to contribute to development of pancreatitis. Strategies to increase CFTR levels or function might be used to treat alcohol-associated pancreatitis.

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Lack of cystic fibrosis transmembrane conductance regulator disrupts fetal airway development in pigs.

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Lack of cystic fibrosis transmembrane conductance regulator disrupts fetal airway development in pigs.

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