Ubiquitination-driven down-regulation of the cell adhesion molecule Na,K-ATPase β-subunit by hypercapnia in alveolar epithelial cells

Abstract

Acute lung injury (ALI) is associated with alveolo-capillary barrier dysfunction leading to impaired gas exchange and thus elevated CO2 levels (hypercapnia). In addition to the well-studied role of the α-subunit of the Na,K-ATPase in alveolar edema fluid clearance during ALI, the Na,K-ATPase β-subunit located at the sites of cell-cell junction acts as a cell adhesion molecule and therefore might be an important player in the alveolar-capillary barrier repair. Here, we studied the effects of hypercapnia on the stability of Na,K-ATPase β-subunit and on cell junction formation. Exposing human alveolar epithelial A549 cells to elevated CO2 concentrations led to rapid ubiquitination of the Na,K-ATPase β-subunit in the plasma membrane and protein internalization, as assessed by cell-surface biotinylation. Both ubiquitination and endocytosis of the Na,K-ATPase β-subunit were prevented when Lys 5 and 7 were mutated to Arg, highlighting the critical role of these residues in targeting the Na,K-ATPase β-subunit by ubiquitin. After endocytosis, the Na,K-ATPase β-subunit underwent degradation, as evaluated by pulse-chase experiments and by blocking protein synthesis with cycloheximide and evaluating protein levels. The degradation of Na,K-ATPase β-subunit was prevented by inhibition of the proteasome system by MG-132, but not by inhibition of the lysosome by E-64 or chloroquine, suggesting that hypercapnia induces proteasome-mediated degradation of the Na,K-ATPase β-subunit. Similarly, the formation of cell-cell junctions, assessed by the hanging-drop technique, was markedly inhibited by elevated CO2 levels, which was completely prevented when the proteasome system was inhibited. Accordingly, we provide evidence that hypercapnia promotes ubiquitination, endocytosis and proteasomal degradation of the Na,K-ATPase β-subunit in alveolar epithelial cells. Additionally, we show that elevated CO2 concentrations inhibit alveolar epithelial cell junction formation, which may be mediated by surface instability of Na,K-ATPase β-subunit and may lead to persistent alveolar epithelial barrier dysfunction during ALI.