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A3829 - Na,K-ATPase αβ Heterodimers and FXYD5 Compete for the Regulation of the Alveolar Epithelial Barrier
Author Block: O. Vagin1, E. Tokhtaeva1, J. I. Sznajder2, L. A. Dada2; 1Physiology, UCLA and VAGLAHS, Los Angeles, CA, United States, 2Northwestern University, Chicago, IL, United States.
Rationale: FXYD5, a regulatory Na,K-ATPase subunit, has been identified as an anti-adhesive and pro-inflammatory contributor to acute lung injury in a mice model. FXYD5 levels are elevated in mice acutely exposed to lipopolysaccharide (LPS), and FXYD5 silencing ameliorates LPS-induced impairment of the alveolar barrier. FXYD5 levels are also increased in the lungs of ARDS patients. In contrast, the αβ Na,K-ATPase plays a critical role in maintaining the alveolar epithelial barrier, both by pumping ions and by acting as a cell adhesion molecule. The Na,K-ATPase abundance in alveolar epithelial cells is decreased in ARDS patients, and over-expression of either α or β subunit in mice improves the barrier function. Here, we determined whether the roles αβ and FXYD5 in the regulation of the alveolar barrier permeability are interdependent.
Methods: LPS was instilled intratracheally into C57BL/6 mice (3 mg/kg) or added to cultured primary mouse alveolar type 2 (AT2) cells and an AT2 cell line (100 ng/ml) for 2-24h. In cells, Na,K-ATPase and FXYD5 were over-expressed using adenovirus and silenced using siRNA. Abundance and ubiquitylation of the subunits were determined by surface biotinylation/immunoblotting and immunoprecipitation/immunoblotting, respectively.
Results: LPS treatment of mice or AT2 cells increased the levels of FXYD5 and decreased the levels of α and β subunits at the plasma membrane. In cultured cells, FXYD5 silencing prevented the decrease in α and β subunits in response to LPS. FXYD5 over-expression induced ubiquitylation of α and β subunits and decreased their surface and total amount. Bafilomycin increased vesicular accumulation of α and β subunits and partially prevented FXYD5-induced decrease in their levels independently of new protein synthesis, suggesting that the mature subunits are internalized and degraded through the endosomal/lysosomal pathway. Conversely, the over-expression of α or β subunit induced FXYD5 ubiquitylation and degradation through the endosomal/lysosomal pathway.
Conclusions The decrease in αβ levels in response to LPS in cultured cells is mediated through the increase in FXYD5. FXYD5 and αβ Na,K-ATPase induce mutual degradation, suggesting that LPS-induced increase in FXYD5 promotes αβ degradation, which, in turn, increases FXYD5, resulting in self-amplifying loop and escalating the impairment of epithelial junctions. Further, the results suggest that the αβ Na,K-ATPase in normal epithelia preserves the integrity of the barrier not only by pumping ions and forming intercellular bridges but also by maintaining low levels of FXYD5. Antagonistic relationship between FXYD5 and αβ Na,K-ATPase might contribute to the regulation of the alveolar barrier permeability in vivo.