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

干扰素(IFN)具有抗生长、抗病毒和免疫调节等特性，已成为治疗癌症和病毒性疾病的重要细胞因子。IFN的生物学作用是由早期反应基因的表达介导的，部分受JAK/STAT信号通路的激活调节。抗增殖剂 在人T细胞系Jurkat细胞中，I型IFN的作用需要 一组T细胞受体(TCR)信号成分的激活：蛋白质 酪氨酸激酶和一种蛋白酪氨酸磷酸酶。对Jurkat的刺激 含有干扰素-β的细胞诱导转录因子NFAT的核转位 对TCR刺激作出反应而激活的因子。为了探讨NFAT在I型干扰素信号转导中的作用，将NFAT驱动的荧光素酶报告基因导入Jurkat细胞。细胞与干扰素-β共同孵育可抑制PMA和离子霉素诱导的NFAT依赖转录。这种负调控效应被显性负STAT1突变体的表达逆转。相反，用离子霉素处理Jurkat细胞可抑制干扰素-β诱导的ISRE驱动的荧光素酶报告基因的转录活性，这种活性可通过用环孢素A(CsA)预处理细胞而可逆。环孢素A是NFAT上游激活剂钙调神经磷酸酶的特异性抑制剂。用PMA加离子霉素孵育正常外周血T细胞可抑制特异性干扰素诱导基因的表达，而CsA可恢复这些基因的表达。此外，STAT1和NFAT蛋白在T细胞中也有关联。这些结果首次证明NFAT参与了I型干扰素调节的T细胞信号级联反应。我们将通过以下目的验证NFAT和STAT1相互抑制彼此转录活性的假设：(1)确定T细胞与干扰素-β结合T细胞受体激动剂是否改变NFAT或STAT1的激活、与DNA的结合或核定位。(2)确定这些蛋白质相互作用所需的STAT1和NFAT结构域，以及这些因子是否需要这种相互作用来调节基因表达。

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.