stimulated. (E) FP (10 nM) reduces the ability of anti-CD3/CD28-stimulated GATA-3 to associate with the native IL-5 promoter 60 min after stimulation. Data are also shown graphically as mean±SEM of three independent experiments. All data were analysed by ANOVA followed by Newman-Keuls post-test.

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.

Effect on MKP-1
Dexamethasone inhibits p38 MAPK function in a cell type–specific manner through the rapid induction of the dual kinase phosphatase MKP-1 (MAPK phosphatase-1), and this effect lasts for up to 24 h [28]. FP (10−8 M) treatment of HuT-78 cells activated by anti-CD3/CD28 in vitro significantly decreased p38 MAPK phosphorylation (Figure 3A) and activity measured by phosphorylation of the downstream target ATF-2 (Figure 3B). This effect was detected at 30 min and lasted for at least 14 h (Figure 3B). FP (10−8 M) also significantly reduced GATA-3 serine phosphorylation induced by anti-CD3/CD28 stimulation in both a time- and concentration-dependent manner (Figure 3C). This reduction in GATA-3 phosphorylation was also seen with lower concentrations of FP. We found that FP significantly induced MKP-1 mRNA in both a time- and concentration-dependent manner, reaching a plateau at 10−8 M after 10 min (Figure 3D and 3E). However, the effects of FP on GATA-3 nuclear import, importin-α association and IL-4 mRNA expression are seen at 10,000-fold lower concentrations (10−12 M, see Figure 2).
10.1371/journal.pmed.1000076.g003 Figure 3  Fluticasone propionate–mediated inhibition of p38 MAP kinase phosphorylation and activation is associated with a marked down-regulation of GATA-3 serine phosphorylation.
(A) Western blot analysis shows that FP (10−8 M, 30 min) treatment reduced dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK in anti-CD3/CD28–co-stimulated HuT-78 cells. (B) Time course of the effect of FP (10−8 M) on phosphorylation of activated transcription factor 2 (ATF-2), a measure of p38 MAPK activity. (C) FP-induced inhibition of p38 MAPK activity is associated with the decrease of anti-CD3/CD28 co-stimulation–induced serine phosphorylation (P-Ser) of GATA-3. For (A–C), quantification of the densitometry data is also shown. 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) FP induced MKP-1 mRNA in a concentration-dependent manner. All results are representative of at least three independent experiments and where appropriate expressed as means±SEM, *p<0.05. (E) FP induces MKP-1 mRNA in a time-dependent manner. Results are representative of two independent experiments. All data except (E) were analysed by ANOVA followed by Newman-Keuls post-test.   Using an in vitro competition assay (Figure 4A) utilizing purified activated GATA-3, importin-α, and activated GR, we demonstrated that activated GR significantly increased GR-importin-α association in the presence and absence of activated GATA-3 (Figure 4B). This effect is not mutual, since activated GATA-3 did not block GR–importin-α association (Figure 4C). These data also suggest that both activated GR and phospho-GATA-3 can directly associate with importin-α (Figure 4D) and that activated GR attenuates the phospho-GATA-3/importin-α interaction in a concentration-dependent manner (Figure 4E). Together, this suggests that ligand-activated GR may compete with phospho-GATA-3 for importin-α and thereby limit GATA-3 nuclear import.
10.1371/journal.pmed.1000076.g004 Figure 4  Fluticasone propionate competes with phospho-GATA-3 for importin-α.
(A) schematic representation of the in vitro binding competition assay. (B) GR isolated from FP (10−8 M) stimulated cells enhances GR–importin-α binding in the presence (•) and absence (▪) of activated GATA-3. * p<0.05 compared to no activated GR. (C) GATA-3 isolated from anti-CD3/CD28–stimulated cells does not attenuate GR–importin-α association. *p<0.05 compared to control. (D) Activated GR blocks the ability of purified phospho-GATA-3 isolated from anti-CD3/CD28–stimulated cells interacting with immobilised importin-α in an in vitro binding assay. *p<0.05 compared to GATA-3 isolated from unstimulated cells. # p<0.05 compared to stimulated GATA-3-importin binding. (E) The effect of activated (•) versus unstimulated (○) GR on attenuation of GATA-3–importin-α association was concentration-dependent. *p<0.05, **p<0.01 between groups. All results are expressed as mean±SEM of three independent experiments and analysed by ANOVA followed by Newman-Keuls post-test.   Other possible interpretations of our results could include an effect of FP on GATA-3 nuclear export and/or degradation. Leptomycin B, which inhibits nuclear export, did not affect the ability of FP to block GATA-3 nuclear localization (Figure 5A). Additionally, FP had no effect on whole cell GATA-3 expression during the time course of these experiments (Figure 5B). Nor did addition of FP subsequent to anti-CD3/CD28 nuclear translocation affect GATA-3 nuclear residency (Figure 5C), suggesting that activated GR does not enhance GATA-3 nuclear export. Finally, the effect of FP on GATA-3 nuclear import was not nonspecific, since FP (10−8 M) had no effect on p65 nuclear translocation measured at 60 min (Figure 5D).
10.1371/journal.pmed.1000076.g005 Figure 5  Fluticasone propionate does not affect GATA-3 nuclear export.
(A) Western blot analysis showing that the nuclear export inhibitor leptomycin B (2 nM) does not affect the ability of FP (10−8 M) to prevent anti-CD3/CD28–stimulated GATA-3 nuclear localization measured at 60 min. ***p<0.001 compared to unstimulated cells, ### p<0.001 compared to anti-CD3/CD28–stimulated cells. (B