Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo, 202-8585, Japan.
Immunol Cell Biol. 2018 Nov;96(10):1095-1103. doi: 10.1111/imcb.12185. Epub 2018 Jun 28.
The signal transducer and activator of transcription 1 (STAT1), a pivotal transcription factor in Janus kinase (JAK)-STAT signaling, regulates the expression of a wide range of immune-related genes, including interferon (IFN) regulatory factor 1 (IRF1). In this study, we found that IRF1 could induce STAT1 phosphorylation and in turn STAT1 activation. When IRF1 was transiently expressed in HEK293 cells, STAT1 phosphorylated at Y701, dimerized and bound to an oligonucleotide containing a gamma-activated sequence (GAS) derived from the IRF1 promoter. IRF1 expression also induced GAS-dependent promoter reporter activity, and phosphorylation of JAK1, a kinase upstream of STAT1. Although no direct interaction between IRF1 and STAT1 was observed, the transactivation domain of IRF1 was required for IRF1-mediated STAT1 activation, indicating the involvement of gene product(s) regulated by IRF1. Moreover, supernatants from cells expressing IRF1 induced phosphorylation of STAT1 and JAK1, and subsequent GAS binding by STAT1 that could not be blocked by treatment with antibodies against IFN-β or IFN-γ. IFN-γ-induced STAT1 phosphorylation persisted for up to 30 h following stimulation of HEK293, but declined in IRF1-deficient HEK293 cells. IRF1-promoter activity induced by IFN-γ was also reduced in IRF1-deficient HEK293 cells, which could be rescued by complementation with IRF1. Together these results indicate that IRF1 promotes DNA binding of STAT1, which can in turn participate in a positive feedback loop of JAK-STAT signaling.
信号转导子和转录激活子 1（STAT1）是 Janus 激酶（JAK）-STAT 信号通路中的关键转录因子，调节广泛的免疫相关基因的表达，包括干扰素（IFN）调节因子 1（IRF1）。在这项研究中，我们发现 IRF1 可以诱导 STAT1 磷酸化，进而激活 STAT1。当 IRF1 在 HEK293 细胞中转瞬时表达时，STAT1 在 Y701 处磷酸化，二聚化并与来自 IRF1 启动子的包含γ-激活序列（GAS）的寡核苷酸结合。IRF1 表达还诱导 GAS 依赖性启动子报告基因活性，以及 STAT1 的上游激酶 JAK1 的磷酸化。虽然没有观察到 IRF1 和 STAT1 之间的直接相互作用，但 IRF1 的转录激活结构域是 IRF1 介导的 STAT1 激活所必需的，表明涉及受 IRF1 调节的基因产物（s）。此外，表达 IRF1 的细胞的上清液诱导 STAT1 和 JAK1 的磷酸化，以及随后的 STAT1 与 GAS 的结合，这不能通过用抗 IFN-β 或 IFN-γ的抗体处理来阻断。IFN-γ刺激 HEK293 后，STAT1 磷酸化持续长达 30 小时，但在 IRF1 缺陷型 HEK293 细胞中下降。IFN-γ诱导的 IRF1 启动子活性在 IRF1 缺陷型 HEK293 细胞中也降低，这可以通过与 IRF1 互补来挽救。这些结果表明，IRF1 促进 STAT1 的 DNA 结合，而 STAT1 反过来又可以参与 JAK-STAT 信号通路的正反馈环。
