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

Abstract

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 (I(sc)) 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 I(sc) 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 I(sc) 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 I(sc), 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.

Fig. 1.

Dexamethasone (Dex) induces endogenous amiloride-sensitive short-circuit current (I_sc) in the CFBE41o⁻ parental cell model of cystic fibrosis bronchial epithelia. CFBE41o⁻ parental cells were grown in the absence or presence of 1 μM Dex for 24 h as described in materials and methods. A: representative I_sc profile for control (●) and Dex-treated (○) cells. Addition of 10 μM amiloride, addition of 10 μM forskolin and 100 μM IBMX, imposition of a basolateral-to-apical Cl⁻ gradient (Apical Low Cl⁻), and addition of 10 μM CFTR_inh-172 (Inh-172) inhibitor are indicated. B: summary data for amiloride-sensitive I_sc. P value was determined by 2-tailed t-test. C: summary data for cystic fibrosis transmembrane conductance regulator (CFTR) functional expression, defined in materials and methods. Solid bar, No Dex; open bar, + Dex. Means ± SE for n individual experiments are shown.

Fig. 2.

Dex induces endogenous amiloride-sensitive I_sc in CFBE41o⁻ ΔF508-CFTR-overexpressing cells but not in CFBE41o⁻ wild-type (wt)-expressing cells. CFBE41o⁻ ΔF508-CFTR-overexpressing and wt-expressing cells were grown in the absence or presence of 1 μM Dex for 24 h as described in materials and methods. Summary data for amiloride-sensitive I_sc as determined in Ussing chambers are presented. Solid bar, No Dex; open bar, + Dex. Means ± SE for n individual experiments are shown. P value for the comparison of Dex-treated ΔF508-CFTR-overexpressing and wt-expressing cells was determined by 2-tailed t-test.

Fig. 3.

Hydrocortisone mimics the effect of Dex, which modestly induces serum- and glucocorticoid-induced kinase 1 (SGK1) expression. CFBE41o⁻ parental (A) or ΔF508-CFTR-overexpressing (B) cells were grown under control conditions or exposed to 1 μM hydrocortisone, aldosterone, or Dex for 24 h before determination of amiloride-sensitive I_sc. Data are presented as amiloride-sensitive I_sc relative to the mean amiloride-sensitive I_sc of Dex-treated cells (means ± SE for n replicates). For C–E, CFBE41o⁻ parental, ΔF508-CFTR-overexpressing, and wt-overexpressing cells were grown in the absence or presence of 1 μM Dex for 24 h as described in materials and methods. C: whole cell lysates were prepared, and immunoblot detection of SGK1, and of GAPDH as a loading control, was performed as described in materials and methods. Representative immunoblots are shown. Molecular mass is shown in kilodaltons. D: densitometry was performed as described in materials and methods. Data are presented as the expression of SGK1 in the presence of Dex relative to that in the absence of Dex (means ± SE for the indicated number of independent experiments). Statistical significance was determined by t-test for each cell line, and all had statistically significant increases in SGK1 expression relative to non-Dex-treated cells (P < 0.05). There was no statistically significant difference in the Dex-induced increase in SGK1 expression among the 3 cell lines [P = not significant (ns) by ANOVA]. E: whole cell lysates were subject to immunoprecipitation (IP) using anti-SGK1 (Parental) or anti-phosphorylated (phospho-, P-) Thr256-SGK1 (ΔF508, WT-CFTR). Precipitated proteins were resolved by SDS-PAGE, and phospho-Thr256-SGK1 (Parental) or total SGK1 (ΔF508 and WT-CFTR) in the precipitates was revealed by immunoblot. Similar results were obtained when the antibodies were used in reverse sequence, and data are representative of 3 experiments for each cell line.

Fig. 4.

CFBE41o⁻ wt CFTR-expressing cells have reduced whole cell abundance of α- and γ-epithelial sodium channel (ENaC). CFBE41o⁻ parental, wt-overexpressing, and ΔF508-CFTR-overexpressing cells were grown in the absence or presence of 1 μM Dex for 24 h as described in materials and methods. Whole cell lysates were prepared, and immunoblot detection of CFTR (representative of 3 independent immunoblots), of α- and γ-ENaC, and of GAPDH as a loading control was performed as described in materials and methods. A: representative immunoblots. There was no other immunoreactivity noted outside of the area of these blots presented. B: densitometry of the α- and γ-ENaC immunoblots was performed as described in materials and methods. Data are presented as means ± SE, with statistical significance determined by ANOVA as indicated. Number of independent experiments analyzed were: α-ENaC, n = 4; γ-ENaC, n = 4.

Fig. 5.

Amiloride-sensitive I_sc in CFBE41o⁻ parental, CFBE41o⁻ ΔF508, and CFBE41o⁻ wt cells before and after trypsin treatment of the apical surface. CFBE41o⁻ parental, ΔF508-CFTR-overexpressing, and wt-expressing cells were grown in the presence of 1 μM Dex for 24 h, and amiloride-sensitive I_sc was determined in Ussing chambers before (gray bars) and after (open bars) 5 min of exposure to 10 μg/ml trypsin in the apical bath. Data are presented as means ± SE for n individual experiments, with P value determined by ANOVA.

Fig. 6.

Influence of 4-phenylbutyrate (4PBA) on amiloride-sensitive I_sc and ΔF508 in CFBE41o⁻ parental and ΔF508-overexpressing cells. A: CFBE41o⁻ parental or ΔF508-overexpressing cells were incubated for 24 h in the presence of 1 mM 4PBA and then for a 2nd 24 h with 1 mM 4PBA and 1 μM Dex. Amiloride-sensitive I_sc was determined before and after addition of 10 μg/ml trypsin to the apical surface. Data are presented as means ± SE for n experiments. P values were determined by paired t-test. B: CFTR-mediated I_sc was determined for CFBE41o⁻ ΔF508-overexpressing cells after incubation with 1 mM 4PBA for 48 h either in the absence (closed bar) or presence (open bar) of 1 μM Dex for the latter 24 h of incubation with 4PBA. Data are presented as means ± SE for n experiments with a 2-tailed t-test used for comparison.

Fig. 7.

Influence of 4PBA on ENaC retrieval from the apical membrane in CFBE41o⁻ parental and ΔF508-overexpressing cells. CFBE41o⁻ parental (A) or ΔF508-overexpressing (B) cells were incubated for 24 h in the absence (●) or presence (○) of 1 mM 4PBA for 48 h. In all cases, 1 μM Dex was added to the media for the latter 24 h of this incubation. Amiloride-sensitive I_sc was determined before and at the indicated times after addition of 100 μM cycloheximide to both apical and basolateral baths and are expressed relative to the amiloride-sensitive current before incubation with cycloheximide (means ± SE). Error bars contained within the symbols are not apparent. Apparent first-order rate constants (k_app) were obtained using the Regression Wizard of SigmaPlot 2000 (means ± SE) and compared by a 2-tailed t-test.

Fig. 8.

Low temperature inhibits Dex-induced expression of endogenous amiloride-sensitive I_sc. CFBE41o⁻ parental or ΔF508-overexpressing cells were incubated for 24 h at 27°C and then for a 2nd 24 h at 27°C in either the absence (closed bars) or presence (open bars) of 1 μM Dex. Amiloride-sensitive I_sc (A) and CFTR (B) functional expression were determined in Ussing chambers as described in materials and methods. Data are presented as means ± SE for n individual experiments. A 2-tailed t-test was used to determine statistical significance.