Hypercapnia Impairs ENaC Cell Surface Stability by Promoting Phosphorylation, Polyubiquitination and Endocytosis of β-ENaC in a Human Alveolar Epithelial Cell Line.

Paulina Gwoździńska,Werner Seeger,Susanne Herold,Rory E Morty,István Vadász,Benno A Buchbinder,Konstantin Mayer

doi:10.3389/fimmu.2017.00591

Abstract

Acute lung injury is associated with formation of pulmonary edema leading to impaired gas exchange. Patients with acute respiratory distress syndrome (ARDS) require mechanical ventilation to improve oxygenation; however, the use of relatively low tidal volumes (to minimize further injury of the lung) often leads to further accumulation of carbon dioxide (hypercapnia). Hypercapnia has been shown to impair alveolar fluid clearance (AFC), thereby causing retention of pulmonary edema, and may lead to worse outcomes; however, the underlying molecular mechanisms remain incompletely understood. AFC is critically dependent on the epithelial sodium channel (ENaC), which drives the vectorial transport of Na+ across the alveolar epithelium. Thus, in the current study, we investigated the mechanisms by which hypercapnia effects ENaC cell surface stability in alveolar epithelial cells (AECs). Elevated CO2 levels led to polyubiquitination of β-ENaC and subsequent endocytosis of the α/β-ENaC complex in AECs, which were prevented by silencing the E3 ubiquitin ligase, Nedd4-2. Hypercapnia-induced ubiquitination and cell surface retrieval of ENaC were critically dependent on phosphorylation of the Thr615 residue of β-ENaC, which was mediated by the extracellular signal-regulated kinase (ERK)1/2. Furthermore, activation of ERK1/2 led to subsequent activation of AMP-activated protein kinase (AMPK) and c-Jun N-terminal kinase (JNK)1/2 that in turn phosphorylated Nedd4-2 at the Thr899 residue. Importantly, mutation of Thr899 to Ala markedly inhibited the CO2-induced polyubiquitination of β-ENaC and restored cell surface stability of the ENaC complex, highlighting the critical role of Nedd4-2 phosphorylation status in targeting ENaC. Collectively, our data suggest that elevated CO2 levels promote activation of the ERK/AMPK/JNK axis in a human AEC line, in which ERK1/2 phosphorylates β-ENaC whereas JNK mediates phosphorylation of Nedd4-2, thereby facilitating the channel–ligase interaction. The hypercapnia-induced ENaC dysfunction may contribute to impaired alveolar edema clearance and thus, interfering with these molecular mechanisms may improve alveolar fluid balance and lead to better outcomes in patients with ARDS.

Highlights

Carbon dioxide (CO2) is formed as a by-product of cellular respiration and is eliminated from the body during breathing [1]
To test whether high CO2 levels promote endocytosis of ENaC, A549 cells were co-transfected with plasmids encoding the human α- and β-subunit of ENaC and the plasma membrane (PM) abundance of these proteins was measured after exposure of cells to physiological or elevated CO2 concentrations at a pHe of 7.4 for 30 min
We show that elevated CO2 levels initiate a specific signaling pattern leading to ubiquitination-mediated retrieval of ENaC from the PM, thereby reducing cell surface abundance of the channel in a human alveolar epithelial cells (AECs) line

Summary

Introduction

Carbon dioxide (CO2) is formed as a by-product of cellular respiration and is eliminated from the body during breathing [1]. It is increasingly evident that the alveolar epithelium is capable of sensing of elevated CO2 levels, which initiate specific signaling signatures and alter the function of alveolar epithelial and other cells [2, 4] While some of these effects are anti-inflammatory, which may be beneficial in the context of excessive inflammation, others impair innate immunity, mitochondrial function, cellular repair, and alveolar epithelial barrier function, which are clearly detrimental in the setting of ARDS [5–10]. In order to keep the alveolar space “dry,” excess alveolar liquid is reabsorbed from the air space into the interstitium by a well-characterized active sodium transport process in which Na+ enters the alveolar epithelial type I and type II cells through the apically located epithelial sodium channel (ENaC) and is subsequently pumped out basolaterally by the Na,K-ATPase This creates a Na+ gradient, which drives paracellular movement of water leading to its clearance from the alveolar space [11, 12]. It has been clearly demonstrated that in most patients with ARDS alveolar fluid clearance (AFC) is impaired and that those patients with ARDS and impaired AFC the mortality is significantly higher than in ARDS patients with normal AFC [13]

Methods

Results

Conclusion