scholarly journals Reduced GM1 ganglioside in CFTR-deficient human airway cells results in decreased β1-integrin signaling and delayed wound repair

2014 ◽  
Vol 306 (9) ◽  
pp. C819-C830 ◽  
Author(s):  
Yutaka Itokazu ◽  
Richard E. Pagano ◽  
Andreas S. Schroeder ◽  
Scott M. O'Grady ◽  
Andrew H. Limper ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Migration ◽  
Epithelial Cells ◽  
Wound Repair ◽  
Airway Epithelial Cells ◽  
Integrin Signaling ◽  
Gm1 Ganglioside ◽  
Airway Epithelial ◽  
Hairpin Rna ◽  
Short Hairpin

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function reduces chloride secretion and increases sodium uptake, but it is not clear why CFTR mutation also results in progressive lung inflammation and infection. We previously demonstrated that CFTR-silenced airway cells migrate more slowly during wound repair than CFTR-expressing controls. In addition, CFTR-deficient cells and mouse models have been reported to have altered sphingolipid levels. Here, we investigated the hypothesis that reduced migration in CFTR-deficient airway epithelial cells results from altered sphingolipid composition. We used cell lines derived from a human airway epithelial cell line (Calu-3) stably transfected with CFTR short hairpin RNA (CFTR-silenced) or nontargeting short hairpin RNA (controls). Cell migration was measured by electric cell substrate impedance sensing (ECIS). Lipid analyses, addition of exogenous glycosphingolipids, and immunoblotting were performed. We found that levels of the glycosphingolipid, GM1 ganglioside, were ∼60% lower in CFTR-silenced cells than in controls. CFTR-silenced cells exhibited reduced levels of activated β1-integrin, phosphorylated tyrosine 576 of focal adhesion kinase (pFAK), and phosphorylation of Crk-associated substrate (pCAS). Addition of GM1 (but not GM3) ganglioside to CFTR-silenced cells restored activated β1-integrin, pFAK, and pCAS to near control levels and partially restored (∼40%) cell migration. Our results suggest that decreased GM1 in CFTR-silenced cells depresses β1-integrin signaling, which contributes to the delayed wound repair observed in these cells. These findings have implications for the pathology of cystic fibrosis, where altered sphingolipid levels in airway epithelial cells could result in a diminished capacity for wound repair after injury.

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

1995 ◽  
Vol 268 (1) ◽  
pp. C243-C251 ◽  
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.

G protein G alpha i-2 inhibits outwardly rectifying chloride channels in human airway epithelial cells

1995 ◽  
Vol 269 (2) ◽  
pp. C451-C456 ◽  
Author(s):  
E. M. Schwiebert ◽  
D. C. Gruenert ◽  
W. B. Guggino ◽  
B. A. Stanton
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
G Protein ◽  
G Proteins ◽  
Airway Epithelial Cells ◽  
Protein G ◽  
Airway Epithelial ◽  
Human Airway ◽  
Cl Channels ◽  
Human Airway Epithelial Cells

Previously we demonstrated that the heterotrimeric G protein, G alpha i-2, inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels in human airway epithelial cells (E. M. Schwiebert, F. Gesek, L. Ercolani, C. Wjasow, D. C. Gruenert, and B. A. Stanton. Am. J. Physiol. 267 (Cell Physiol. 36): C272-C281, 1994, and E. M. Schwiebert, N. L. Kizer, D. C. Gruenert, and B. A. Stanton. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992). The goal of the present study was to determine if G proteins also regulate outwardly rectifying Cl- channels (ORCC), a distinct class of Cl- channels regulated defectively by protein kinase A (PKA) in cystic fibrosis (CF). To this end, we used the patch-clamp technique to study ORCC in a normal human airway epithelial cell line (9HTEo-) that expresses CFTR and ORCC. Stimulation of G proteins with GTP and GTP gamma S decreased the single-channel open probability (Po) of ORCC, whereas inhibition of G proteins by GDP beta S increased the Po. Moreover, pertussis toxin (PTX), an uncoupler of Gi and G(o) subclasses of heterotrimeric G proteins, also increased the Po. Purified G alpha i-2 decreased the Po. In contrast, other PTX-sensitive G proteins, G alpha i-1, G alpha i-3, and G alpha o, had no effect on Po. We propose that G alpha i-2 couples to a receptor whose agonist negatively regulates ORCC in human airway epithelial cells.

scholarly journals Agonist binding to β-adrenergic receptors on human airway epithelial cells inhibits migration and wound repair

2015 ◽  
Vol 309 (12) ◽  
pp. C847-C855 ◽  
Author(s):  
Elizabeth R. Peitzman ◽  
Nathan A. Zaidman ◽  
Peter J. Maniak ◽  
Scott M. O'Grady
Keyword(s):  
Epithelial Cells ◽  
Protein Phosphatase ◽  
Adrenergic Receptors ◽  
Wound Repair ◽  
Wound Closure ◽  
Basal Membrane ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Phosphatase 2A ◽  
Β Adrenergic Receptors

Human airway epithelial cells express β-adrenergic receptors (β-ARs), which regulate mucociliary clearance by stimulating transepithelial anion transport and ciliary beat frequency. Previous studies using airway epithelial cells showed that stimulation with isoproterenol increased cell migration and wound repair by a cAMP-dependent mechanism. In the present study, impedance-sensing arrays were used to measure cell migration and epithelial restitution following wounding of confluent normal human bronchial epithelial (NHBE) and Calu-3 cells by electroporation. Stimulation with epinephrine or the β2-AR-selective agonist salbutamol significantly delayed wound closure and reduced the mean surface area of lamellipodia protruding into the wound. Treatment with the β-AR bias agonist carvedilol or isoetharine also produced a delay in epithelial restitution similar in magnitude to epinephrine and salbutamol. Measurements of extracellular signal-regulated kinase phosphorylation following salbutamol or carvedilol stimulation showed no significant change in the level of phosphorylation compared with untreated control cells. However, inhibition of protein phosphatase 2A activity completely blocked the delay in wound closure produced by β-AR agonists. In Calu-3 cells, where CFTR expression was inhibited by RNAi, salbutamol did not inhibit wound repair, suggesting that β-AR agonist stimulation and loss of CFTR function share a common pathway leading to inhibition of epithelial repair. Confocal images of the basal membrane of Calu-3 cells labeled with anti-β1-integrin (clone HUTS-4) antibody showed that treatment with epinephrine or carvedilol reduced the level of activated integrin in the membrane. These findings suggest that treatment with β-AR agonists delays airway epithelial repair by a G protein- and cAMP-independent mechanism involving protein phosphatase 2A and a reduction in β1-integrin activation in the basal membrane.

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