GATA-3 nuclear import plays a critical role in regulating Th2 cytokine gene expression: Effects of fluticasone

Abstract

Rationale The subcellular localization of transcription factors is critical for correct cellular function. The transcription factor GATA-3 is crucial for the activation of T-helper type 2 (Th2) gene expression which orchestrates inflammation in asthma and contains a nuclear import signal. Methods Hut-78 cells and PBLs were stimulated with CD3/CD28 cross-linking and protein-protein and protein-DNA interactions detected using co-immunoprecipitations and chromatin immunoprecipitation. IL-4 and IL-5 expression was measured by RT-PCR. Results We have demonstrated in Hut-78 and in T-lymphocytes ex vivo and in vivo that the nuclear import of the GATA-3 protein is regulated by its direct interaction with the nuclear import protein importin-α after stimulation by CD3/CD28. The imported GATA-3 binds to and activates IL-4 and IL-5 cytokine promoters. SB203850, a selective p38 MAPK inhibitor, caused a decrease in GATA-3 serine phosphorylation induced by CD3/CD28 engagement and subsequent impairment of GATA-3-importin-α interaction and GATA-3 nuclear localization. Fluticasone prevented GATA-3 nuclear import both in vivo and in vitro. This was due to a combination of mutual competition for importin-α at early time points (<30 minutes) and a longer term effect of inhibition of p38 MAPK activation leading to reduced GATA-3 phosphorylation. Conclusions These result suggest that GATA-3 nuclear import is one of the most important steps in the regulation of Th2 cytokine gene expression and is directly regulated by corticosteroids.