FXYD5 modulates Na,K-ATPase activity and is increased in cystic fibrosis airway epithelia

Cystic fibrosis (CF) is a common genetic disease among Caucasians, caused by lesions in the cystic fibrosis transmembrane conductance regulator gene, CFTR. This defect leads to the inability to transport chloride through the apical membrane, particularly in epithelia lining the airway. A hallmark of CF is the hyperabsorption of sodium across the airway epithelia, leading to decreased airway surface liquid volume and the inability to clear sticky, mucous plugs from the airway surface. This contributes to the smoldering inflammation and excessive growth of pathogenic bacteria typically seen in the lungs of CF patients and is often the primary cause of mortality.

Although the exact link between CFTR defects and the inability to clear trapped, pathogenic bacteria remains unclear, it has been postulated that increased sodium absorption and decreased airway surface liquid are critical modulators of disease pathogenesis in the lung. Transepithelial sodium absorption begins with entry through the epithelial sodium channel (ENaC) in the apical membrane and exits through the Na,K-ATPase in the basolateral membrane. We identified FXYD5, also known as Dysadherin, as a gene upregulated in CF airway epithelia. FXYD5 belongs to a family of tissue-specific regulators of the Na,K-ATPase, and thus we hypothesized that FXYD5 may modulate Na,K-ATPase activity and contribute to disease pathogenesis in CF.

Therefore, we determined the effects of FXYD5 overexpresison on Na,K-ATPase activity in an epithelial cell model. FXYD5 significantly increased the apparent affinity for Na⁺ 2-fold, and decreased the apparent affinity for K+ by 60% with a 2-fold increase in V_max(K+), a pattern that would increase activity and Na+ removal from the cell. To test the effect of increased sodium uptake on FXYD5 expression, we analyzed MDCK cells stably transfected with an inducible vector expressing all three ENaC subunits. Na,K-ATPase activity increased 6-fold after 48-hour ENaC induction, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that overexpress ENaC, suggesting that chronic Na⁺ absorption by itself downregulates epithelial FXYD5 expression.

However, we counterintuitively found that FXYD5 was significantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry and immunoblot analysis (P<0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared to controls (P<0.02). Treatment of human tracheal epithelial (HTE) cells with a CFTR inhibitor (CTFR_inh-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression (P<0.001), which was abrogated inhibitors of NF-κB. Similarly, stimulation of NF-κB activity with the pro-inflammatory cytokines TNFα/IL-1β upregulated FXYD5 expression and was blocked by a separate chemical inhibitor of NF-κB. We also found that overexpression of FXYD5 increased wound healing in airway epithelial cells, which was modulated by negative charge at S163, suggesting a role for FXYD5 in epithelial motility and regeneration in the airway. Collectively, these data show that FXYD5 is upregulated in CF epithelia and this change may exacerbate the Na⁺ hyperabsorption and surface liquid dehydration observed in CF airway epithelia.