A Critical Role for Stat2 in Type 1 Intferon-Induced Apoptosis

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Interferons (IFNs) have become extremely useful cytokines in the treatment of cancer and viral diseases due to their antigrowth, antiviral and immunomodulatory properties. The biological actions of IFNs are mediated by expression of early responsive genes regulated in part by the activation of the Jak/Stat signaling pathway. The antiproliferative actions of type I IFNs in Jurkat cells, a human T cell line, require activation of a set of T cell receptor (TCR) signaling components: protein tyrosine kinases and a protein tyrosine phosphatase. Stimulation of Jurkat cells with IFN-beta induces nuclear translocation of NFAT, a transcription factor activated in response to TCR stimulation. To explore the role of NFAT in type I IFN signaling, Jurkat cells were transfected with a NFAT driven luciferase reporter. Incubation of cells with IFN-beta inhibited PMA and ionomycin induced NFAT dependent transcription. This negative regulatory effect was reversed by expression of a dominant negative Stat1 mutant. Conversely, treatment of Jurkat cells with ionomycin inhibited IFN-beta induced transcriptional activity of an ISRE driven luciferase reporter that is reversible by pretreatment of cells with cyclosporine A (CsA), a specific inhibitor of calcineurin, the upstream activator of NFAT. Incubation of normal peripheral blood T cells with PMA plus ionomycin inhibited the expression of specific IFN inducible genes and CsA restored expression of these genes. Moreover, Stat1 and NFAT proteins are associated in T cells. These results provide first evidence for the involvement of NFAT in type I IFN regulated signaling cascades in T cells. We will test the hypothesis that NFAT and Stat1 reciprocally repress each others’ transcriptional activities by performing the following aims: (1) Determine whether incubation of T cells with IFN-beta in combination with T-cell receptor agonists alters NFAT or Stat1 activation, binding to DNA or nuclear localization. (2) Determine the domains of Stat1 and NFAT that are required for these proteins to interact and whether this interaction is required for these factors to regulate gene expression.