IFN Regulatory Factor-1-Mediated Transcriptional Activation of Mouse STAT-Induced STAT Inhibitor-1 Gene Promoter by IFN-γ

Abstract

STAT-induced STAT inhibitor-1 (SSI-1), also referred to as suppressor of cytokine signaling-1 and JAK-binding protein, is a member of a new family, the members of which are negative regulators of cytokine signals. SSI-1 is induced by various cytokines; however, the transcriptional mechanism of the SSI-1 gene is not fully understood. Here, we showed that transcription of the mouse SSI-1 gene was initiated from six adjoining sites accompanying three GC boxes and a single GC box-like element near them, but not from the TATA box or an initiator sequence. We also showed that IFN-γ induced SSI-1 mRNA more strongly than IL-6 in NIH-3T3 fibroblasts and that this IFN-γ effect was mediated by Stat1. To determine the signal pathway downstream of Stat1, transcriptional activities of several mutant promoters were examined. The region mediating stimulatory effect of IFN-γ to the gene transcription was localized to the −88/−60 region containing three tandem GAAA units, named variant IFN-γ-responsive element (VIRE), while four IFN-γ activation site (GAS)-like elements located far upstream were not related to the IFN-γ response. Gel-shift assays revealed that IFN-γ induced IFN regulatory factor-1 (IRF-1) binding to VIRE, but not that of IRF-2 or three components of ISGF3. Furthermore, forced expression of IRF-1 mimicked and that of IRF-2 inhibited the stimulatory effect of IFN-γ on SSI-1 gene transcription. Finally, mouse embryonal fibroblasts lacking IRF-1 showed impaired SSI-1 mRNA induction by IFN-γ. These results demonstrated that IRF-1, which is induced by activation of Stat1, mediated transcriptional activation of the SSI-1 gene by IFN-γ via VIRE.