METHODS: THP-1 cells were cultured with dexamethasone overnight prior to IFN-γ exposure. RNA was isolated for quantitative RT-PCR (qPCR) and chromatin-immunoprecipitation (CHIP) assay was performed for binding of STAT1 and GR to CXCL10 gene regulatory sequences. Similarly treated human PBMCs underwent imaging cytometry (AMNIS Imagestream) for GR nuclear translocation.
RESULTS: In THP-1 cells, dexamethasone induction of the GR transactivation target Dual Specificity Phosphatase-1 (DUSP1) was not impaired by IFN-γ. ChIP analysis revealed association of both STAT1 and GR with the key CXCL10 promoter regulatory sequence ISRE in the presence of IFN-γ alone; this association was further increased with dexamethasone pretreatment. Imaging cytometry showed marked nuclear GR translocation with dexamethasone. However, IFN-γ alone also induced nuclear GR translocation.
CONCLUSIONS: We have shown that IFN-γ causes un-liganded GR nuclear translocation without inducing GR-mediated transactivation but promotes binding to a key regulatory sequence, ISRE, in the CXCL10 promoter. Combined exposure of cells to IFN-γ and dexamethasone induces increased association of STAT1 and GR with the ISRE. This suggests that GR may stabilize IFN-γ-activated STAT1 on target CXCL10 promoter sequences. Ultimately, IFN-γ and CS cooperation could foster a feed forward loop, as CXCL10 recruits IFN-γ producing Th1 cells to sites of inflammation.