Antagonistic Relationship between Na,K‐ATPase αβ Heterodimers and FXYD5 in the Regulation of the Epithelial Integrity

Abstract

RationaleThe αβ Na,K‐ATPase heterodimer plays a critical role in maintaining epithelial integrity by acting as a cell adhesion molecule. The α and β protein abundance at the plasma membrane of epithelial cells (EC) are decreased in patients with impaired alveolar, prostate or urinary epithelial barrier, and over‐expression of either α or β subunit in mice improves the epithelial barrier function. On the other hand, over‐expression of FXYD5, a regulatory Na,K‐ATPase subunit, impairs epithelial junctions by disrupting the intercellular bridges formed by Na,K‐ATPase αβ heterodimers. FXYD5 levels in EC are increased in patients with impaired epithelial junctions and in mice exposed to lipopolysaccharide (LPS), a bacterial toxin that is implicated in the epithelial barrier disruption. In the current study, we determined whether the decrease in αβ and increase in FXYD5 are interdependent and whether the balance between αβ and FXYD5 contributes to the regulation of the epithelial barrier.Methods and ResultsIntratracheal instillation of LPS into mice increased FXYD5 levels and decreased the Na,K‐ATPase α and β protein abundance at the plasma membrane of alveolar EC in parallel with increased alveolar epithelial barrier permeability as assessed by the increased protein concentration in the broncho‐alveolar lavage fluid (BALF). FXYD5 silencing in mice by intratracheal instillation of shRNA lentivirus decreased protein concentration in BALF after LPS treatment, and FXYD5 silencing in alveolar EC using siRNA prevented the decrease in α and β subunits in response to LPS, indicating that FXYD5 contributes to both the impairment of the barrier and decrease in αβ in response to LPS. FXYD5 adenoviral over‐expression in alveolar, gastric or renal EC induced α ubiquitylation, decreased surface and total levels of both α and β subunits. Conversely, the over‐expression of α and β (individually or together) induced FXYD5 ubiquitylation and decreased its surface and total levels in these cells. Exogenous (under constitutive promoters) α subunit and FXYD5 were affected similarly as their endogenous counterparts by the over‐expression of FXYD5 and α, respectively, suggesting a post‐transcriptional mechanism. The over‐expression of an unrelated protein did not affect the ubiquitylation or abundance of the Na,K‐ATPase subunits.ConclusionsIn normal epithelial cells, the high levels Na,K‐ATPase αβ heterodimers preserve epithelial integrity not only by forming intercellular bridges, but also by promoting FXYD5 degradation. The increase in FXYD5 in response to LPS decreases the α and β protein abundance, which, in turn, increases FXYD5, hence, amplifying the damage to the epithelial barrier.Support or Funding InformationThis work was supported, in part, by National Institutes of Health Grants R37‐HL48129 (J.I.S.), HL071643 (J.I.S., and L.A.D.), HL113350 (L.A.D. and O.V.), USVA 2I01BX001006 (G.S.), DK105156‐01 (G.S.)