Hypercapnia impairs ENaC cell surface expression and function by promoting phosphorylation and polyubiquitination of ENaC beta-subunit in alveolar epithelial cells
An elevation of CO2 concentration in the blood (hypercapnia) may occur as a result of poor alveolar gas exchange and is frequently detected in patients with respiratory diseases including those with acute respiratory distress syndrome (ARDS). Hypercapnia may be further exacerbated as a consequence of lung-protective mechanical ventilation with low ... tidal volumes, which is necessary in patients with ARDS. The vectorial Na+ transport across the alveolar epithelium mediated by the epithelial Na+ channel (ENaC) and the Na,K-ATPase provides the driving force for alveolar fluid clearance (AFC). Persistent alveolar edema is a hallmark of ARDS and can be attributed in part to impaired Na+ and fluid reabsorption, which leads to worse outcomes. Thus, the effects of hypercapnia on ENaC function in the alveolar epithelium and understanding of the signaling pathways that may via downregulation of ENaC impair edema clearance in hypercapnic patients with ARDS are of high clinical relevance.The current work describes the molecular mechanisms by which hypercapnia reduces cell surface expression of ENaC in alveolar epithelial cells (AECs) and thus impairs ENaC-driven transepithelial Na+ transport. We found that acute hypercapnia independently of pH (pCO2 ~120 mmHg, pH 7.4 for 30 min) led to polyubiquitination of ß-ENaC and subsequent endocytosis of the a/ß-ENaC complex from the cell surface of primary rat ATII and human alveolar epithelial A549 cells, as assessed by cell surface biotinylation and fluorescent microscopy. In contrast, hypercapnia altered neither mRNA nor intracellular levels of ENaC proteins, indicating that acutely elevated CO2 levels affect the trafficking of the channel rather than its transcription or degradation. Furthermore, our data established that hypercapnia by decreasing ENaC cell surface expression reduced both total (Isc) and amiloride-sensitive (Iamil-sens) Na+ current in H441 human airway epithelial cells. Moreover, the hypercapnia-induced increase in polyubiquitination of ß-ENaC and endocytosis of the a/ß-ENaC complex were prevented by silencing the E3 ubiquitin ligase, Nedd4-2. Co-immunoprecipitation studies confirmed the direct interaction of Nedd4-2 with the ß-subunit of ENaC. Most importantly, the CO2-induced ß-ENaC ubiquitination and a/ß-ENaC retrieval from the cell surface were strongly dependent on the extracellular signal regulated kinase (ERK)1/2 that directly phosphorylated ß-ENaC at the T615 residue. In line with these findings, transfection of A549 cells with a ß-ENaC mutant lacking T615, normalized cell surface density of a/ß-ENaC upon hypercapnic exposure. Activation of ERK1/2 caused 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 T899 residue. Replacement of the T899 residue by an alanine (T899A), which prevented JNK-mediated phosphorylation of Nedd4-2 significantly inhibited polyubiquitination of ß-ENaC and improved cell surface expression of the a/ß-ENaC complex upon CO2 treatment. Similarly, pharmacological inhibition of JNK rescued the abundance of ENaC at the cell surface upon hypercapnia. Additionally, chemical inhibition or genetic ablation of AMPK prevented the hypercapnia-induced ß-ENaC polyubiquitination and internalization of the a/ß-ENaC complex. A comparable reduction of ENaC endocytosis upon hypercapnia treatment was observed when an upstream kinase of AMPK, the Ca2+/calmodulin-dependent protein kinase kinase-ß (CaMKK-ß) was inhibited, confirming that the activation of AMPK was required for ENaC downregulation during acute hypercapnia. Thus, we describe a novel CO2-induced signaling pattern in alveolar epithelial cells, which by activation of the ERK/AMPK/JNK axis impairs cell surface expression and function of ENaC via promoting ubiquitination of ß-ENaC and driving subsequent internalization of the channel. This signaling cascade may further impair alveolar fluid balance therefore leading to worse outcomes in patients with ARDS and thus also represent a potentially targetable pathway that might lead to discovery of novel therapies against alveolar edema in patients with hypercapnic respiratory failure.