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

Noor A.S Ismail,Deborah L Baines,Stuart M Wilson

doi:10.1016/j.ejphar.2014.03.005

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-Ser221, -Ser327 and -Thr246. 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 Ser221-phosphorylated, Ser327-phosphorylated and total Nedd4-2 without altering the abundance of Thr246-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 Ser221-, Ser327- and Thr246-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.

Highlights

The controlled absorption of Naþ from the liquid film covering the lung / airway epithelia is vital to the integrated functioning of Abbreviations: cyclic adenosine monophosphate (cAMP), cyclic 3050 adenosine monophosphate; CREB, cyclic AMP response element binding protein; EDTA, ethylene diamine tetra acetic acid; epithelial Naþ channels (ENaC), epithelial sodium channel, IAmil, amiloride-sensitive component of the transepithelial current; IBMX, isomethyl butyl xanthine; Nedd4-2, neural precursor cell expressed, developmentally down-regulated protein 4-2; Nmyc-downstream regulated gene 1 (NDRG1), protein encoded by n-myc downstream regulated gene 1; PAGE, polyacylamide gel electrophoresis; protein kinase A (PKA), cyclic adenine nucleotide-dependent protein kinase; SDS, sodium dodecyl sulphate; serum / glucocorticoid-inducible kinase 1 (SGK1), serum and glucocortcoid-inducible kinase 1; s.e.m., standard error of the mean n Corresponding author at: Wolfson Research Institute, Durham University School of Medicine, Pharmacy and Health Queen's Campus Stockton on Tees TS17 6BH
Minor adjustments were made to the contrast / brightness of presented images but these changes were applied this synthetic glucocorticoid for 24 h showed that this response was not sustained and these data confirm that glucocorticoids evoke transient activation of SGK1 in H441 cells
The present studies of endogenously expressed proteins show that dexamethasone activates SGK1, induces phosphorylation of Nedd4-2 and increases the surface abundance of ENaC subunits

Summary

Introduction

The controlled absorption of Naþ from the liquid film covering the lung / airway epithelia is vital to the integrated functioning of Abbreviations: cAMP, cyclic 3050 adenosine monophosphate; CREB, cyclic AMP response element binding protein; EDTA, ethylene diamine tetra acetic acid; ENaC, epithelial sodium channel, IAmil, amiloride-sensitive component of the transepithelial current; IBMX, isomethyl butyl xanthine; Nedd, neural precursor cell expressed, developmentally down-regulated protein 4-2; NDRG1, protein encoded by n-myc downstream regulated gene 1; PAGE, polyacylamide gel electrophoresis; PKA, cyclic adenine nucleotide-dependent protein kinase; SDS, sodium dodecyl sulphate; SGK1, serum and glucocortcoid-inducible kinase 1; s.e.m., standard error of the mean n Corresponding author at: Wolfson Research Institute, Durham University School of Medicine, Pharmacy and Health Queen's Campus Stockton on Tees TS17 6BH. Glucocorticoids are clearly important to the induction and maintenance of this Naþ absorbing phenotype and synthetic glucocorticoids are used in the clinical management of conditions, such as respiratory distress and pulmonary oedema, that involve dysfunction of pulmonary Naþ absorption (Barker and Olver, 2002; Boucher, 2004; 2007; Matthay et al, 2002; Olver et al, 2004; Wilson et al, 2007) Despite their clear importance, the mechanisms that allow these hormones to control pulmonary Naþ transport are not understood fully (Barnes, 2011; Matthay et al., 2002; Olver et al, 2004).

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