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

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.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00409.2011 ◽

2012 ◽

Vol 303 (8) ◽

pp. L720-L727 ◽

Cited By ~ 14

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.

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Flagellin From Pseudomonas aeruginosa Modulates SARS-CoV-2 Infectivity in Cystic Fibrosis Airway Epithelial Cells by Increasing TMPRSS2 Expression

Frontiers in Immunology ◽

10.3389/fimmu.2021.714027 ◽

2021 ◽

Vol 12 ◽

Author(s):

Manon Ruffin ◽

Jeanne Bigot ◽

Claire Calmel ◽

Julia Mercier ◽

Maëlle Givelet ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Critical Role ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Health Crisis ◽

Chronic Respiratory Diseases ◽

Cf Transmembrane Conductance Regulator ◽

Main Component

In the coronavirus disease 2019 (COVID-19) health crisis, one major challenge is to identify the susceptibility factors of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in order to adapt the recommendations for populations, as well as to reduce the risk of COVID-19 development in the most vulnerable people, especially patients with chronic respiratory diseases such as cystic fibrosis (CF). Airway epithelial cells (AECs) play a critical role in the modulation of both immune responses and COVID-19 severity. SARS-CoV-2 infects the airway through the receptor angiotensin-converting enzyme 2, and a host protease, transmembrane serine protease 2 (TMPRSS2), plays a major role in SARS-CoV-2 infectivity. Here, we show that Pseudomonas aeruginosa increases TMPRSS2 expression, notably in primary AECs with deficiency of the ion channel CF transmembrane conductance regulator (CFTR). Further, we show that the main component of P. aeruginosa flagella, the protein flagellin, increases TMPRSS2 expression in primary AECs and Calu-3 cells, through activation of Toll-like receptor-5 and p38 MAPK. This increase is particularly seen in Calu-3 cells deficient for CFTR and is associated with an intracellular increased level of SARS-CoV-2 infection, however, with no effect on the amount of virus particles released. Considering the urgency of the COVID-19 health crisis, this result may be of clinical significance for CF patients, who are frequently infected with and colonized by P. aeruginosa during the course of CF and might develop COVID-19.

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Faculty Opinions recommendation of Toll-like receptor 4 is not targeted to the lysosome in cystic fibrosis airway epithelial cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717986343.793506134 ◽

2015 ◽

Author(s):

Uta Griesenbach

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Cystic Fibrosis Airway

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Faculty Opinions recommendation of Cystic fibrosis transmembrane conductance regulator is expressed in mucin granules from Calu-3 and primary human airway epithelial cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718017856.793479176 ◽

2013 ◽

Author(s):

Steven M Rowe ◽

Sammeta Vamsee Raju

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Transmembrane Conductance Regulator ◽

Airway Epithelial ◽

Transmembrane Conductance ◽

Human Airway ◽

Cystic Fibrosis Transmembrane ◽

Human Airway Epithelial Cells

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Differential expression of ORCC and CFTR induced by low temperature in CF airway epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.1.c243 ◽

1995 ◽

Vol 268 (1) ◽

pp. C243-C251 ◽

Cited By ~ 37

Author(s):

M. E. Egan ◽

E. M. Schwiebert ◽

W. B. Guggino

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Incubation Temperature ◽

Cell Types ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Channel Activity ◽

Patch Clamp Recording ◽

Cl Channel ◽

Channel Expression

When nonepithelial cell types expressing the delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) mutation are grown at reduced temperatures, the mutant protein can be properly processed. The effect of low temperatures on Cl- channel activity in airway epithelial cells that endogenously express the delta F508-CFTR mutation has not been investigated. Therefore, we examined the effect of incubation temperature on both CFTR and outwardly rectifying Cl- channel (ORCC) activity in normal, in cystic fibrosis (CF)-affected, and in wild-type CFTR-complemented CF airway epithelia with use of a combination of inside-out and whole cell patch-clamp recording, 36Cl- efflux assays, and immunocytochemistry. We report that incubation of CF-affected airway epithelial cells at 25-27 degrees C is associated with the appearance of a protein kinase A-stimulated CFTR-like Cl- conductance. In addition to the appearance of CFTR Cl- channel activity, there is, however, a decrease in the number of active ORCC when cells are grown at 25-27 degrees C, suggesting that the decrease in incubation temperature may be associated with multiple alterations in ion channel expression and/or regulation in airway epithelial cells.

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PBA increases CFTR expression but at high doses inhibits Cl−secretion in Calu-3 airway epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.277.4.l700 ◽

1999 ◽

Vol 277 (4) ◽

pp. L700-L708 ◽

Cited By ~ 11

Author(s):

Johannes Loffing ◽

Bryan D. Moyer ◽

Donna Reynolds ◽

Bruce A. Stanton

Keyword(s):

Protein Expression ◽

Apical Membrane ◽

Basolateral Membrane ◽

Airway Epithelial Cells ◽

Cell Phenotype ◽

Apical Plasma Membrane ◽

Airway Epithelial ◽

High Concentration ◽

Cycle Enzyme ◽

Cf Transmembrane Conductance Regulator

Sodium 4-phenylbutyrate (PBA), a short-chain fatty acid, has been approved to treat patients with urea cycle enzyme deficiencies and is being evaluated in the management of sickle cell disease, thalassemia, cancer, and cystic fibrosis (CF). Because relatively little is known about the effects of PBA on the expression and function of the wild-type CF transmembrane conductance regulator (wt CFTR), the goal of this study was to examine the effects of PBA and related compounds on wt CFTR-mediated Cl−secretion. To this end, we studied Calu-3 cells, a human airway cell line that expresses endogenous wt CFTR and has a serous cell phenotype. We report that chronic treatment of Calu-3 cells with a high concentration (5 mM) of PBA, sodium butyrate, or sodium valproate but not of sodium acetate reduced basal and 8-(4-chlorophenylthio)-cAMP-stimulated Cl−secretion. Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and increased the intensity of CFTR staining in the apical plasma membrane. PBA also increased protein expression of Na+-K+-ATPase. PBA reduced CFTR Cl−currents across the apical membrane but had no effect on Na+-K+-ATPase activity in the basolateral membrane. Thus a high concentration of PBA (5 mM) reduces Cl−secretion by inhibiting CFTR Cl−currents across the apical membrane. In contrast, lower therapeutic concentrations of PBA (0.05–2 mM) had no effect on cAMP-stimulated Cl−secretion across Calu-3 cells. We conclude that PBA concentrations in the therapeutic range are unlikely to have a negative effect on Cl−secretion. However, concentrations >5 mM might reduce transepithelial Cl−secretion by serous cells in submucosal glands in individuals expressing wt CFTR.

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