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

干扰素（IFN）由于其抗生长、抗病毒和免疫调节特性而在癌症和病毒性疾病的治疗中成为极其有用的细胞因子。IFN的生物学作用由早期应答基因的表达介导，所述早期应答基因部分地由Jak/Stat信号传导途径的激活调节。的抗增殖 I型IFN在Jurkat细胞（一种人类T细胞系）中的作用需要 一组T细胞受体（TCR）信号传导组分的活化：蛋白质 酪氨酸激酶和蛋白酪氨酸磷酸酶。刺激Jurkat 细胞与IFN-β诱导核转位的NFAT，转录 因子激活响应TCR刺激。为了探索NFAT在I型IFN信号传导中的作用，用NFAT驱动的荧光素酶报告基因转染Jurkat细胞。用IFN-β孵育细胞抑制PMA和离子霉素诱导的NFAT依赖性转录。这种负调控作用被显性负Stat 1突变体的表达所逆转。相反，用离子霉素处理Jurkat细胞抑制IFN-β诱导的ISRE驱动的荧光素酶报告基因的转录活性，这是通过用环孢菌素A（CsA）（一种钙调磷酸酶的特异性抑制剂，NFAT的上游激活剂）预处理细胞可逆的。孵育正常外周血T细胞与PMA加离子霉素抑制特异性IFN诱导基因的表达和CsA恢复这些基因的表达。此外，Stat 1和NFAT蛋白在T细胞中相关。这些结果为NFAT参与T细胞中I型IFN调节的信号级联提供了第一个证据。我们将通过执行以下目标来检验NFAT和Stat 1相互抑制转录活性的假设：（1）确定T细胞与IFN-β和T细胞受体激动剂的组合孵育是否改变NFAT或Stat 1活化、与DNA的结合或核定位。(2)确定这些蛋白质相互作用所需的Stat 1和NFAT结构域，以及这些因子调节基因表达是否需要这种相互作用。

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.