The phosphorylation of endogenous Nedd4-2 In Na(+)-absorbing human airway epithelial cells

Abstract

Neural precursor cell expressed, developmentally down-regulated protein 4-2 (Nedd4-2) mediates the internalisation / degradation of epithelial Na(+) channel subunits (α-, β- and γ-ENaC). Serum / glucocorticoid inducible kinase 1 (SGK1) and protein kinase A (PKA) both appear to inhibit this process by phosphorylating Nedd4-2-Ser(221), -Ser(327) and -Thr(246). This Nedd4-2 inactivation process is thought to be central to the hormonal control of Na(+) absorption. The present study of H441 human airway epithelial cells therefore explores the effects of SGK1 and / or PKA upon the phosphorylation / abundance of endogenous Nedd4-2; the surface expression of ENaC subunits, and electrogenic Na(+) transport. Effects on Nedd4-2 phosphorylation/abundance and the surface expression of ENaC were monitored by western analysis, whilst Na(+) absorption was quantified electrometrically. Acutely (20min) activating PKA in glucocorticoid-deprived (24h) cells increased the abundance of Ser(221)-phosphorylated, Ser(327)-phosphorylated and total Nedd4-2 without altering the abundance of Thr(246)-phosphorylated Nedd4-2. Activating PKA under these conditions did not cause a co-ordinated increase in the surface abundance of α-, β- and γ-ENaC and had only a very small effect upon electrogenic Na(+) absorption. Activating PKA (20min) in glucocorticoid-treated (0.2µM dexamethasone, 24h) cells, on the other hand, increased the abundance of Ser(221)-, Ser(327)- and Thr(246)-phosphorylated and total Nedd4-2; increased the surface abundance of α-, β- and γ-ENaC and evoked a clear stimulation of Na(+) transport. Chronic glucocorticoid stimulation therefore appears to allow cAMP-dependent control of Na(+) absorption by facilitating the effects of PKA upon the Nedd4-2 and ENaC subunits.

Keywords: Cellular signalling; Epithelial Na(+) channel; Protein kinase A; Pulmonary Na(+) absorption; Serum and glucocorticoid regulated kinase 1.

Fig. 1

Effects of dexamethasone upon PKA and SGK1. Control cells were maintained in hormone-free medium for 24 h whilst dexamethasone-stimulated cells were exposed to this synthetic glucocorticoid (0.2 µM) for 24 h or 3 h. All cells were then lysed and aliquots (40 µg) of extracted protein subject to Western analysis. (A) Typical blots showing the effects of dexamethasone upon the abundance of Thr^346/356/366-phosphorylated (upper panel) and total (lower panel) NDRG1. (B) Densitometric analysis showing the pooled data (n=5). (C) Typical Western blots showing the effects of dexamethasone upon the abundance of Ser¹³³-phosphorylated (upper panel) and total (lower panel) CREB. (D) Densitometric analysis showing the pooled data (n=5). Asterixes denote statistically significant difference from the control values measured in hormone-deprived cells (^⁎⁎P<0.02, one- way ANOVA/Bonferroni post-hoc test). All data are mean±S.E.M.

Fig. 2

Activation of PKA and SGK1 by cAMP agonists. Glucocorticoid-deprived cells were exposed (0–24 h) to a cocktail of compounds that promote activation of cAMP-dependent signalling pathways and 40 µg aliquots of extracted protein then subject to Western analysis. (A) Typical Western blots showing the effects of cAMP agonists upon the abundance of Ser¹³³-phosphorylated (upper panel) and total (lower panel) CREB. (B) Densitometric analysis showing the pooled data from 5 such experiments. (C) Western blots showing the effects of cAMP-agonists upon the abundance of Thr^346/356/366-phosphorylated (upper panel) and total (lower panel) NDRG1. (D) Densitometric analysis showing the pooled data (n=5). Asterixes denote statistically significant deviations from control (^⁎, P<0.05; ^⁎⁎⁎P<0.01, one-way ANOVA/Bonferroni post-hoc test).

Fig. 3

Effects of dexamethasone upon the phosphorylation/abundance of Nedd4-2. Nedd4-2 was immunopurified from glucocorticoid-deprived cells and from cells exposed to 0.2 µM dexamethasone for 3 h or 24 h. (A) Typical Western blots showing the effects of dexamethasone stimulation (3 h and 24 h) upon the abundance of the Ser²²¹-phosphorylated, Ser³²⁷-phosphorylated, Thr²⁴⁶-phosphorylated and total Nedd4-2. (B–E) Densitometric analysis showing the pooled data from the entire series of experiments (n=8). Asterixes denote statistically significant differences from the values measured in hormone-deprived cells (^⁎P<0.05, one-way ANOVA/Bonferroni post-hoc test). All data are mean±S.E.M.

Fig. 4

Effects of GSK650394 upon the dexamethasone-induced changes to the phosphorylation / abundance of Nedd4-2. Nedd4-2 was immunopurified from control and GSK650394-treatred (10 µM, 3 h) cells that were either maintained in hormone-free medium or exposed to 0.2 µM dexamethasone for 3 h. (A) Typical Western blots showing the effects of dexamethasone and/or GSK650394 upon the cellular abundance of Ser²²¹-phosphorylated, Ser³²⁷-phosphorylated, Thr²⁴⁶-phosphorylated and total Nedd4-2. (B–E) Densitometric analysis showing the pooled results of the entire series of experiments (n=4). Asterixes denote statistically significant effects of dexamethasone (^⁎P<0.05, ^⁎⁎⁎P<0.01) whilst daggers († P<0.05, ‡ P<0.01) indicate significant effects of GSK650394 (one-way ANOVA / Bonferroni test). All data are mean±S.E.M.

Fig. 5

Effects of dexamethasone upon the surface abundance of α-, β- and γ-ENaC. Surface-exposed proteins from glucocorticoid-deprived cells and from cells exposed to 0.2 µM dexamethasone for 3 h or 24 h were subject to Western analysis using antibodies against α-, β- or γ-ENaC. (A) Typical Western blots showing the effects of dexamethasone (3 h and 24 h) upon the surface abundance of these channel subunits. (B) Densitometric analysis showing the results of the entire series of experiments (n=7). Asterixes denote statistically significant differences from the values measured in hormone-deprived cells (^⁎P<0.05, ^⁎⁎⁎P<0.001one-way ANOVA / Bonferroni post-hoc test). All data are mean±S.E.M.

Fig. 6

Effects of 0.2 µM dexamethasone (3 h) and / or cAMP agonists (20 min) and upon the phosphorylation / abundance of Nedd4-2. Nedd4-2 was immunopurified from glucocorticoid-deprived and dexamethasone-stimulated (0.2 µM, 3 h) cells that had been either maintained under control conditions or exposed to cAMP agonists for the final 20 min of this incubation period. (A) Typical Western blots showing the effects of cAMP and dexamethasone upon the cellular abundance of Ser²²¹-phosphorylated, Ser³²⁷-phosphorylated, Thr²⁴⁶-phosphorylated and total Nedd4-2. (B–E) Densitometric analysis showing the pooled data (n=3) from the entire series of experiments. Asterixes denote statistically significant effects of dexamethasone (^⁎P<0.05, ^⁎⁎⁎ P<0.01) whilst daggers show significant († P<0.05) effects of cAMP agonists (one-way ANOVA / Bonferroni test). All data are mean±S.E.M.

Fig. 7

Effects of 0.2 µM dexamethasone (3 h) and/or cAMP agonists (20 min) upon the surface abundance of α-, β- and γ-ENaC. Surface-exposed proteins were isolated from glucocorticoid-deprived and dexamethasone-stimulated cells that were either maintained under control conditions or exposed to the cAMP agonists for the final 20 min of this incubation period. (A) Typical Western blots showing the effects of dexamethasone and/or cAMP agonists upon the surface abundance of α-, β- and γ-ENaC. (B–E) Densitometric analysis showing the pooled data (mean±S.E.M.) from the entire series of experiments. Asterixes denote statistically significant effects of dexamethasone (^⁎P<0.05, ^⁎⁎⁎ P<0.01) whilst daggers show significant († P<0.05) effects of cAMP agonists (one-way ANOVA/Bonferroni post-hoc test). All data are mean±S.E.M.

Fig. 8

Effects of GSK650394 and/or cAMP agonists upon PKA and SGK1 in cells chronically (24 h) exposed to 0.2 µM dexamethasone. Dexamethasone-treated (0.2 µM, 24 h) cells (n=3) were exposed to cAMP agonists 20 min under standard conditions and in the presence of GSK650394 (10 µM, 3 h) and aliquots (40 µg) of extracted protein then subject to Western analysis. (A) Typical Western blots showing the effects of GSK650394 and/or cAMP agonists upon the abundance of Ser¹³³-phosphorylated and total CREB. (B) Densitometric analysis showing pooled data (n=3) from the entire series of experiments. (C) Typical Western blots showing the effects of GSK650394 and/or cAMP agonists upon the abundance of Thr^346/356/366-phosphorylated (upper panels) and total NDRG1 (lower panels). (D) Densitometric analysis showing pooled data (n=3) from the entire series of experiments. Asterixes denote statistically significant effects of cAMP agonists (^⁎⁎⁎P<0.01) whilst daggers (†, P<0.05) denote statistically significant effects of GSK650394 (one-way ANOVA / Bonferroni post-hoc test). All data are mean±S.E.M.

Fig. 9

Effect of cAMP agonists and / or GSK650394 upon the phosphorylation /abundance of Nedd4-2 in dexamethasone-treated (0.2 µM, 24 h) cells. Proteins immunopurified from dexamethasone-treated (0.2 µM, 24 h) cells either maintained under standard conditions or exposed to cAMP agonists for 20 min (n=3). Experiments were undertaken using both control and GSK650394-treated (10 µM, 3 h) cells. (A) Typical Western blots showing the effects of cAMP and / or GSK650394 upon the cellular abundance of Ser²²¹-phosphorylated, Ser³²⁷-phosphorylated, Thr²⁴⁶-phosphorylated and total Nedd4-2. (B–E) Densitometric analysis showing the pooled data (n=3) from the entire series of experiments. Asterixes denote statistically significant effects of cAMP agonists (^⁎⁎⁎P<0.01) whilst daggers (†, P<0.05) denote statistically significant effects of GSK650394 (one-way ANOVA/Bonferroni post-hoc test). All data are mean±S.E.M.

Fig. 10

Effect of cAMP agonists and / or GSK650394 upon the surface abundance of α-, β- and γ-ENaC in dexamethasone-treated (0.2 µM, 24 h) cells. Surface-exposed proteins were isolated from dexamethasone-treated (0.2 µM, 24 h) cells that had been maintained under standard conditions or exposed to cAMP agonists for 20 min. Experiments were undertaken both under control conditions and in the presence of GSK650394 (10 µM, 3 h). (A) Typical Western blots showing the effects of cAMP and/or GSK650394 upon the surface abundance of α-, β- and γ-ENaC. (B–E) Densitometric analysis showing the pooled data (n=3) from the entire series of experiments. Asterixes denote statistically significant effects of cAMP agonists (^⁎⁎⁎P<0.01) whilst daggers (†, P<0.05) denote statistically significant effects of GSK650394 (one-way ANOVA / Bonferroni post-hoc test). All data are mean±S.E.M.

Fig. 11

Effects of cAMP agonists / GSK650394 on electrogenic Na⁺ absorption in glucocorticoid-deprived and dexamethasone-treated cells. (A) Cells grown to confluence on permeable supports were either deprived of glucocorticoids for 24 h, or exposed to 0.2 µM dexamethasone for 3 h or 24 h. Control/GSK650394-treated (10 µM, 3 h) cells were then mounted in Ussing chambers and amiloride-sensitive short circuit current (I_Amil) quantified as an indicator of the electrogenic Na⁺ transport rate. (B) Cyclic AMP agonist-induced increases in I_Amil above the levels quantified in parallel studies of unstimulated cells that were age matched, at identical passage number and had been maintained in Ussing chambers for identical time periods. Presented values of P denote statistically significant effects of GSK650394 whilst asterixes denote significant effects of dexamethasone (P<0.05, one-way ANOVA/Bonferroni post-hoc test). All data are mean±S.E.M.