Hypercapnia impairs cell junction formation by promoting TRAF2 E3 ligase-mediated ubiquitination and endocytosis of the Na,K-ATPase beta-subunit in alveolar epithelial cells
INTRODUCTION: In patients with acute respiratory distress syndrome (ARDS) disruption of the epithelial barrier results in accumulation of edema fluid in the airspaces, impairing gas exchange and leading to elevated CO2 levels (hypercapnia). Moreover, lung-protective mechanical ventilation in patients often results in further hypercapnia. The formation of new cell-cell contacts after ARDS is essential for the re-establishment of an intact and functional alveolar epithelium, capable of clearing lung edema and performing gas exchange. The Na,K-ATPase beta-subunit is a cell-adhesion molecule with a key role in the formation and stability of cell junctions and therefore might be important in the repair of the alveolar epithelium. However, the effects of hypercapnia on the stability of the Na,K-ATPase beta-subunit and on the re-organization of adherens junctions have not been studied before. In the present work we tested the hypothesis that hypercapnia inhibits cell-cell contact formation by promoting the down-regulation of the Na,K-ATPase beta-subunit. We aimed to elucidate the molecular mechanism underlying the (dis)regulation of adherens junction formation by hypercapnia.METHODS AND RESULTS: Exposing alveolar epithelial cells to elevated CO2 at constant extracellular pH of 7.4 led to the ubiquitination of the Na,K-ATPase beta-subunit at the plasma membrane which resulted in a significant reduction of the protein abundance at the cell surface, as determined by cell-surface biotinylation and confocal microscopy. Internalization of the Na,K-ATPase beta-subunit was followed by proteasome-dependent degradation of the protein, as assessed by pulse-chase experiments with impermeable biotin. The simultaneous mutations of lysines 5 and 7 of the Na,K-ATPase beta-subunit to arginine prevented hypercapnia-induced ubiquitination and endocytosis of the protein, demonstrating that ubiquitin chains covalently-attached to the Na,K-ATPase beta-subunit during hypercapnia act as an endocytosis signal. Mutation of serine 11 of the Na,K-ATPase beta-subunit to alanine prevented hypercapnia-induced ubiquitination and degradation of the protein. In contrast, mutation of serine 11 to aspartate which mimics phosphorylation did not prevent hypercapnia-induced effects, suggesting that phosphorylation of the Na,K-ATPase beta-subunit at serine 11 is a prerequisite for the ubiquitination of the protein. Coimmunoprecipitation and in vitro interaction studies showed that the serine/threonine kinase protein kinase C-zeta; (PKC-zeta), known to be activated by hypercapnia, interacted with the Na,K-ATPase beta-subunit and this interaction was dependent on the serine 11 of the Na,K-ATPase beta-subunit. Moreover, PKC-zeta activity was required for the hypercapnia-mediated effects, since the chemical inhibition or knock-down of the kinase prevented the endocytosis of the Na,K-ATPase beta-subunit under hypercapnic conditions. By the use of a protein-interaction microarray we identified the E3 ligase TRAF2 as an interactive partner for Na,K-ATPase beta-subunit. This interaction was further confirmed by coimmunoprecipitation and in vitro interaction studies. TRAF2 led to ubiquitination of the Na,K-ATPase beta-subunit in vitro and in vivo. Moreover, knock-down of TRAF2 prevented the hypercapnia-induced endocytosis of the Na,K-ATPase beta-subunit, demonstrating that TRAF2 is the E3 ligase that mediates the Na,K-ATPase beta-subunit ubiquitination in hypercapnia. Most importantly, by cell aggregation assays we demonstrated that hypercapnia led to impaired cell junction formation, effect that was prevented by the simultaneous mutations of lysines 5 and 7 of the Na,K-ATPase beta-subunit, demonstrating that hypercapnia-mediated down-regulation of the Na,K-ATPase beta-subunit at the plasma membrane is the underlying mechanism by which hypercapnia inhibits cell-cell adhesion.CONCLUSIONS: Here we report a novel mechanism by which hypercapnia affects the function of the alveolar epithelium. We provide evidence that hypercapnia (independently of pH) promotes ubiquitination of the Na,K-ATPase beta-subunit at the plasma membrane leading to the endocytosis of the protein, which results in reduced ability of alveolar cells to form intercellular junctions. We demonstrate that ubiquitination depends on PKC-zeta activity and we identify TRAF2 as the E3 ligase that mediates the hypercapnia-induced ubiquitination of the Na,K-ATPase beta-subunit. Thus, hypercapnia may impair restoration of the alveolo-capillary barrier in patients with ARDS upon hypercapnia by the down-regulation of the Na,K-ATPase beta-subunit.
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