The Effect of Corticosteroids on GATA-3 Nuclear Translocation and IL-4 mRNA
Corticosteroids are effective in inhibiting GATA-3-regulated IL-4 gene expression in vitro and in vivo [32]. We therefore investigated whether corticosteroids affect anti-CD3/CD28–stimulated nuclear import of GATA-3. Stimulation of cells with anti-CD3/CD28 resulted in a rapid cytoplasmic/nuclear GATA-3 translocation (Figure 1A), confirming our previous results [12]. We also confirmed a clear separation of nuclear and cytosolic fractions as indicated by histone H1 and MEK-1 markers (Figure 1B). The potent topical corticosteroid FP caused sustained loss of nuclear GATA-3 expression and cytoplasmic retention of GATA-3 at concentrations ranging from 10−12 to 10−8 M, which cover the therapeutic range [37]. This effect was concentration- and time-dependent, with a peak effect of 11.6-fold at 30 min at a concentration of 10−8 M (Figure 1C) and was associated with marked reductions in anti-CD3/CD28–stimulated IL-4 and IL-5 mRNA expression (Figure 1D) and a loss of GATA-3 binding to the native IL-5 promoter (Figure 1E).
10.1371/journal.pmed.1000076.g001 Figure 1  Fluticasone propionate down-regulates Th2 cytokine gene expression and inhibits GATA-3 nuclear import.
(A) Anti-CD3/CD28 treatment of HuT-78 cells results in translocation of GATA-3 from the cytoplasm to the nucleus within 30 min. (B) Histone H1 and MEK-1 were used to confirm distinct separation of cytoplasmic and nuclear extracts in three separate experiments. (C) Western blot analysis of FP-treated HuT-78 cells demonstrated impaired nuclear localization of GATA-3 induced by anti-CD3/CD28 co-stimulation in a time- (at 10−8 M FP) and concentration- (at 60 min after stimulation) dependent manner. Cells were pretreated with FP for 30 min prior to stimulation. MEK1 and histone H1 were used to demonstrate equal cytoplasmic and nuclear loading respectively. Results are presented graphically below as mean±SEM of at least three independent experiments. *** p<0.001 compared to t = 0. (D) RT-PCR showing that FP inhibits IL-4 and IL-5 mRNA expression in CD3/CD28-costimulated cells. GAPDH was used as a loading control. Lower panels show graphical analysis of results presented as mean±SEM of at least three independent experiments. ### p<0.001 compared to control, ***p<0.001 compared to anti-CD3/CD28–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.

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