Ligand-Activated GR Competes with GATA-3 for Importin-α
We confirmed and extended previous data [20] to show that ligand-activated GR as well as GATA-3 uses importin-α for its nuclear import (Figure 2A and 2B). This interaction between GR and importin-α was significant at concentrations as low as 10−12 M and was maximal with 10−8 M FP. Subsequent GR nuclear translocation was rapid and sustained at significant levels for at least 14 h (Figure 2B). Using IP-Western blotting we showed that FP at 10−12–10−8 M decreased the association between GATA-3 and importin-α induced by anti-CD3/CD28 stimulation in a concentration-dependent manner (Figure 2C). In addition, using GFP-labelled GATA-3 and confocal microscopy we demonstrated that GATA-3 nuclear import following anti-CD3/CD28 stimulation for 30 min was attenuated by pretreatment with FP (10−8 M) (Figure 2D).
10.1371/journal.pmed.1000076.g002 Figure 2  Fluticasone propionate reduces GATA-3 association with importin-α and GATA-3 nuclear import.
(A) Western blot analysis demonstrates a time- (at 10−8 M FP) and concentration- (at 60 min after stimulation) dependent induction of FP-activated GR interaction with importin-α (Imp-α). A positive control for GR association with importin is shown. Quantification of the densitometry data is shown below. Each bar represents mean±SEM of at least three independent experiments. *** p<0.001 compared to control, ### p<0.001. (B) Western blot analysis demonstrated a time- (at 10−8 M FP) and concentration- (at 60 min after stimulation) dependent induction of FP-activated GR nuclear translocation measured by IP. Quantification of the densitometry data is shown below. Each bar represents mean±SEM of at least three independent experiments. ***p<0.001 compared to control. (C) Western blot analysis of HuT-78 cells treated with FP and anti-CD3/CD28 co-stimulation demonstrated a concentration-dependent decrease in GATA-3–importin-α association at 20 min. Quantification of the densitometry data is shown below. Each bar represents mean±SEM of at least three independent experiments. ### p<0.001 compared to control, ***p<0.001 compared to αCD3/CD28-stimulated cells. (D) GFP-tagged GATA-3 was overexpressed and cells stimulated (b, c) or not (a) for 30 min with anti-CD3/CD28. The effect of 30 min pretreatment of cells with FP (10−8 M, c) is also shown. All data were analysed by ANOVA followed by Newman-Keuls post-test.

E