Hydrocortisone Affects the Transport Phenotype of Differentiated Normal Human Bronchial Epithelial Cells

Abstract

Glucocorticoids play a crucial role in transitioning the developing lung epithelium from a secretory to an absorptive tissue. The objective of this study was to evaluate the effects of hydrocortisone (HC) on the transport phenotype of the bronchial epithelium during differentiation. Normal human bronchial epithelial cells (NHBE) were differentiated on polyester membranes and harvested at four time points over a 24 day time period. NHBE cells differentiated in the presence and absence of HC (HC0) along with cells withdrawn from HC after 8 days (HC8) all formed pseudostratified layers, as confirmed by confocal microscopy, and developed transepithelial resistances (TER) greater than 1000 Ωcm2 by day 8. mRNAs associated with bronchial basal cells (p63, NKX2.1) were detected at days 0, 4, 8 and 24 while airway surface markers (FOXJ1, MUC5ac, MUC5b) were not detected until day 4. HC0 and HC8 NHBE cells expressed lower levels of mucin mRNA than control cells at day 24. At day 8, Ussing chamber experiments showed that the total and benzamil sensitive currents were significantly decreased under HC0 conditions (4.0±0.13, 0.46±0.14, respectively) but not under HC8 conditions (8.8±1.2, 5.7±1.3) compared to control (7.1±0.44, 3.9±0.53). At day 24, total and benzamil sensitive currents were significantly decreased under HC0 and HC8 conditions. HC had no effect on CFTRinh‐172 sensitive current, indicating that there was no detectable effect on CFTR‐dependent anion secretion. ENaCα and γ subunits as well as CFTR co‐localized at the apical membrane in control and HC8 cells at day 24 as observed by immunofluorescence. HC0 cells had noticeably less apical fluorescence at day 24 corresponding to reduced ENaCα and γ expression compared to control cells. Interestingly, ENaC subunits and CFTR appeared to localize within the cilia. Control and HC0 NHBE cells stimulated with the selective β2‐AR agonist salbutamol produced outward currents which resulted in increased benzamil sensitive current in control but not HC0 cells. Additionally, NHBE cells stimulated with UTP initially exhibitedan inward, paxilline‐sensitive current characteristic of KCa1.1 channel activation, followed by an outward, DIDS‐sensitive current (TMEM16a/ANO1) in control and HC8 cells at days 8 and 24. HC0 cells stimulated with UTP at day 8 produced only outward currents consistent with reduced KCNMA1 mRNA expression and K+ channel activity. There was a significantly reduced UTP‐induced inward current at day 24 for both HC0 and HC8 cells. Expression of TMEM16a and P2RY2 receptor mRNA was not affected by HC, whereas P2RY4 receptor expression was not detectable at days 8 and 24 in HC0 cells. Immunofluorescence indicated co‐localization of TMEM16a, KCa1.1, and P2Y2 at the apical membrane in control cells suggesting that these proteins form a signaling complex at the apical membrane that may also include P2Y4 receptors. These results demonstrate an important role for glucocorticoids in development of the transport phenotype of bronchial epithelial cells during differentiation.Support or Funding InformationF31EB018707