Trafficking of immature ΔF508-CFTR to the plasma membrane and its detection by biotinylation

2009 ◽  
Vol 419 (1) ◽  
pp. 211-221 ◽  
Author(s):  
Yishan Luo ◽  
Ken McDonald ◽  
John W. Hanrahan
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Molecular Mass ◽  
Protein A ◽  
Airway Epithelial Cells ◽  
Mutant Form ◽  
Western Blots ◽  
Δf508 Cftr ◽  
R Domain

Recent studies suggest that immature, core-glycosylated ΔF508-CFTR [the predominant mutant form of the CFTR (cystic fibrosis transmembrane conductance regulator)] can reach the plasma membrane under some conditions. In the present study we investigated this possibility since it has implications for understanding how therapeutics rescue the trafficking of mutant CFTR and perhaps other misfolded proteins. Core-glycosylated CFTR was labelled and pulled down on streptavidin beads after exposure to sulfo-NHS-SS-biotin [biotin attached to a reactive NHS (N-hydroxysuccinimide) ester with a disulfide spacer; molecular mass=606.7 Da]; however, intracellular proteins were also detected in the precipitates. When the R domain of CFTR was expressed in the cytosol of BHK (baby-hamster kidney) cells as a soluble polypeptide it was also labelled after surface biotinylation and pulled down on streptavidin beads. Intracellular biotinylation was reduced when cells were treated with sulfo-NHS-LC-biotin (biotin attached to a reactive NHS ester with an aminocaproic acid spacer) or sulfo-NHS-PEO12-biotin [biotin attached to a reactive NHS ester with a poly(ethylene glycol) spacer], but the reduction could be explained by the lower reactivity of these reagents. The R domain was detected on Western blots after loading <0.25% of the pulldown sample (∼0.01% of total lysate protein), a fraction that could be ascribed to cells that were permeable to ethidium homodimer-1 (molecular mass=856.8 Da) and propidium iodide (molecular mass=668.6 Da). When BHK cells were incubated at 29 °C to rescue ΔF508-CFTR trafficking, and then biotinylated and sorted to remove permeable cells, labelling of core-glycosylated ΔF508-CFTR was no longer detected although a weak signal was still observed using CFBE (cystic fibrosis bronchial epithelial) cells. These results suggest that there is weak surface expression of immature ΔF508-CFTR on airway epithelial cells and demonstrate the need to remove permeable cells when studying CFTR glycoforms by surface biotinylation.

scholarly journals Nedd4–2 does not regulate wt-CFTR in human airway epithelial cells

2012 ◽  
Vol 303 (8) ◽  
pp. L720-L727 ◽  
Author(s):  
Katja Koeppen ◽  
Chris Chapline ◽  
J. Denry Sato ◽  
Bruce A. Stanton
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Apical Plasma Membrane ◽  
Respiratory Pathogens ◽  
Airway Epithelial ◽  
Human Airway ◽  
Δf508 Cftr ◽  
Sirna Knockdown ◽  
Human Airway Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− channel in airway epithelial cells, plays an important role in maintaining the volume of the airway surface liquid and therefore mucociliary clearance of respiratory pathogens. A recent study has shown that the E3 ubiquitin ligase Neural precursor cells expressed developmentally downregulated (Nedd4–2) ubiquitinates ΔF508-CFTR in pancreatic epithelial cells and that siRNA-mediated silencing of Nedd4–2 increases plasma membrane ΔF508-CFTR. Because the role of Nedd4–2 in regulating wild-type (wt)-CFTR in airway epithelial cells is unknown, studies were conducted to test the hypothesis that Nedd4–2 also ubiquitinates wt-CFTR and regulates its plasma membrane abundance. We found that Nedd4–2 did not affect wt-CFTR Cl− currents in Xenopus oocytes. Likewise, overexpression of Nedd4–2 in human airway epithelial cells did not alter the amount of ubiquitinated wt-CFTR. siRNA knockdown of Nedd4–2 in human airway epithelial cells had no effect on ubiquitination or apical plasma membrane abundance of wt-CFTR. Thus Nedd4–2 does not ubiquitinate and thereby regulate wt-CFTR in human airway epithelial cells.

scholarly journals Exocytosis is not involved in activation of Cl− secretion via CFTR in Calu-3 airway epithelial cells

1998 ◽  
Vol 275 (4) ◽  
pp. C913-C920 ◽  
Author(s):  
Johannes Loffing ◽  
Bryan D. Moyer ◽  
David McCoy ◽  
Bruce A. Stanton
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Critical Role ◽  
Airway Epithelial Cells ◽  
Cell Phenotype ◽  
Apical Plasma Membrane ◽  
Airway Epithelial ◽  
Intracellular Pool ◽  
Airway Function

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channel, which mediates transepithelial Cl− transport in a variety of epithelia, including airway, intestine, pancreas, and sweat duct. In some but not all epithelial cells, cAMP stimulates Cl− secretion in part by increasing the number of CFTR Cl− channels in the apical plasma membrane. Because the mechanism whereby cAMP stimulates CFTR Cl− secretion is cell-type specific, our goal was to determine whether cAMP elevates CFTR-mediated Cl− secretion across serous airway epithelial cells by stimulating the insertion of CFTR Cl− channels from an intracellular pool into the apical plasma membrane. To this end we studied Calu-3 cells, a human airway cell line with a serous cell phenotype. Serous cells in human airways, such as Calu-3 cells, express high levels of CFTR, secrete antibiotic-rich fluid, and play a critical role in airway function. Moreover, dysregulation of CFTR-mediated Cl− secretion in serous cells is thought to contribute to the pathophysiology of cystic fibrosis lung disease. We report that cAMP activation of CFTR-mediated Cl− secretion across human serous cells involves stimulation of CFTR channels present in the apical plasma membrane and does not involve the recruitment of CFTR from an intracellular pool to the apical plasma membrane.

Pseudomonas aeruginosainhibits endocytic recycling of CFTR in polarized human airway epithelial cells

2006 ◽  
Vol 290 (3) ◽  
pp. C862-C872 ◽  
Author(s):  
Agnieszka Swiatecka-Urban ◽  
Sophie Moreau-Marquis ◽  
Daniel P. MacEachran ◽  
John P. Connolly ◽  
Caitlin R. Stanton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Membrane Expression ◽  
Endocytic Recycling ◽  
Human Airway ◽  
Δf508 Cftr ◽  
Human Airway Epithelial Cells

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), ΔF508, leads to the absence of CFTR Cl−channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of ΔF508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl−channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl−secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl−secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and ΔF508-CFTR Cl−channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of ΔF508-CFTR.

scholarly journals Three-Dimensional Airway Spheroids and Organoids for Cystic Fibrosis Research

2021 ◽  
Vol 1 (4) ◽  
pp. 229-247
Author(s):  
Onofrio Laselva ◽  
Massimo Conese
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Fluid Transport ◽  
Protein A ◽  
Three Dimensional ◽  
Airway Epithelial Cells ◽  
Culture Conditions ◽  
Airway Epithelial ◽  
Cellular Models ◽  
Apical Side

Cystic fibrosis (CF) is an autosomal recessive multi-organ disease caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, with morbidity and mortality primacy related to the lung disease. The CFTR protein, a chloride/bicarbonate channel, is expressed at the apical side of airway epithelial cells and is mainly involved in appropriate ion and fluid transport across the epithelium. Although many animal and cellular models have been developed to study the pathophysiological consequences of the lack/dysfunction of CFTR, only the three-dimensional (3D) structures termed “spheroids” and “organoids” can enable the reconstruction of airway mucosa to model organ development, disease pathophysiology, and drug screening. Airway spheroids and organoids can be derived from different sources, including adult lungs and induced pluripotent stem cells (iPSCs), each with its advantages and limits. Here, we review the major features of airway spheroids and organoids, anticipating that their potential in the CF field has not been fully shown. Further work is mandatory to understand whether they can accomplish better outcomes than other culture conditions of airway epithelial cells for CF personalized therapies and tissue engineering aims.

Terminal sialylation is altered in airway cells with impaired CFTR-mediated chloride transport

2001 ◽  
Vol 280 (3) ◽  
pp. L482-L492 ◽  
Author(s):  
Dianne Kube ◽  
Lynn Adams ◽  
Aura Perez ◽  
Pamela B. Davis
Keyword(s):  
Cystic Fibrosis ◽  
Sialic Acid ◽  
Cell Lines ◽  
Chloride Transport ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Wild Type ◽  
Δf508 Cftr ◽  
R Domain

Reduced terminal sialylation at the surface of airway epithelial cells from patients with cystic fibrosis may predispose them to bacterial infection. To determine whether a lack of chloride transport or misprocessing of mutant cystic fibrosis transmembrane conductance regulator (CFTR) is critical for the alterations in glycosylation, we studied a normal human tracheal epithelial cell line (9/HTEo−) transfected with the regulatory (R) domain of CFTR, which blocks CFTR-mediated chloride transport; ΔF508 CFTR, which is misprocessed, wild-type CFTR; or empty vector. Reduced cAMP-stimulated chloride transport is seen in the R domain and ΔF508 transfectants. These two cell lines had consistent, significantly reduced binding of elderberry bark lectin, which recognizes terminal sialic acid in the α-2,6 configuration. Binding of other lectins, including Maakia amurensis lectin, which recognizes sialic acid in the α-2,3 configuration, was comparable in all cell lines. Because the cell surface change occurred in R domain-transfected cells, which continue to express wild-type CFTR, it cannot be related entirely to misprocessed or overexpressed CFTR. It is associated most closely with reduced CFTR activity.

scholarly journals Regulation of endogenous ENaC functional expression by CFTR and ΔF508-CFTR in airway epithelial cells

2011 ◽  
Vol 300 (1) ◽  
pp. L88-L101 ◽  
Author(s):  
Ronald C. Rubenstein ◽  
Shannon R. Lockwood ◽  
Ellen Lide ◽  
Rebecca Bauer ◽  
Laurence Suaud ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Functional Expression ◽  
Surface Expression ◽  
Airway Epithelial Cells ◽  
Cell Surface Expression ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Δf508 Cftr

The functional expression of the epithelial sodium channel (ENaC) appears elevated in cystic fibrosis (CF) airway epithelia, but the mechanism by which this occurs is not clear. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) alters the trafficking of endogenously expressed human ENaC in the CFBE41o− model of CF bronchial epithelia. Functional expression of ENaC, as defined by amiloride-inhibited short-circuit current ( Isc) in Ussing chambers, was absent under control conditions but present in CFBE41o− parental and ΔF508-CFTR-overexpressing cells after treatment with 1 μM dexamethasone (Dex) for 24 h. The effect of Dex was mimicked by incubation with the glucocorticoid hydrocortisone but not with the mineralocorticoid aldosterone. Application of trypsin to the apical surface to activate uncleaved, “near-silent” ENaC caused an additional increase in amiloride-sensitive Isc in the Dex-treated cells and was without effect in the control cells, suggesting that Dex increased ENaC cell surface expression. In contrast, Dex treatment did not stimulate amiloride-sensitive Isc in CFBE41o− cells that stably express wild-type (wt) CFTR. CFBE41o− wt cells also had reduced expression of α- and γ-ENaC compared with parental and ΔF508-CFTR-overexpressing cells. Furthermore, application of trypsin to the apical surface of Dex-treated CFBE41o− wt cells did not stimulate amiloride-sensitive Isc, suggesting that ENaC remained absent from the surface of these cells even after Dex treatment. We also tested the effect of trafficking-corrected ΔF508-CFTR on ENaC functional expression. Incubation with 1 mM 4-phenylbutyrate synergistically increased Dex-induced ENaC functional expression in ΔF508-CFTR-overexpressing cells. These data support the hypothesis that wt CFTR can regulate the whole cell, functional, and surface expression of endogenous ENaC in airway epithelial cells and that absence of this regulation may foster ENaC hyperactivity in CF airway epithelia.

scholarly journals Deleterious impact of Pseudomonas aeruginosa on cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells

2015 ◽  
Vol 45 (6) ◽  
pp. 1590-1602 ◽  
Author(s):  
Nguyen Thu Ngan Trinh ◽  
Claudia Bilodeau ◽  
Émilie Maillé ◽  
Manon Ruffin ◽  
Marie-Claude Quintal ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Short Circuit ◽  
Airway Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Cystic Fibrosis Transmembrane

The epithelial response to bacterial airway infection, a common feature of lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis, has been extensively studied. However, its impact on cystic fibrosis transmembrane conductance regulator (CFTR) channel function is not clearly defined. Our aims were, therefore, to evaluate the effect of Pseudomonas aeruginosa on CFTR function and expression in non-cystic fibrosis airway epithelial cells, and to investigate its impact on ΔF508-CFTR rescue by the VRT-325 corrector in cystic fibrosis cells.CFTR expression/maturation was evaluated by immunoblotting and its function by short-circuit current measurements.A 24-h exposure to P. aeruginosa diffusible material (PsaDM) reduced CFTR currents as well as total and membrane protein expression of the wildtype (wt) CFTR protein in CFBE-wt cells. In CFBE-ΔF508 cells, PsaDM severely reduced CFTR maturation and current rescue induced by VRT-325. We also confirmed a deleterious impact of PsaDM on wt-CFTR currents in non-cystic fibrosis primary airway cells as well as on the rescue of ΔF508-CFTR function induced by VRT-325 in primary cystic fibrosis cells.These findings show that CFTR function could be impaired in non-cystic fibrosis patients infected by P. aeruginosa. Our data also suggest that CFTR corrector efficiency may be affected by infectious components, which should be taken into account in screening assays of correctors.

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