Role of interferon regulatory factor 1 in induction of nitric oxide synthase.
Role of interferon regulatory factor 1 in induction of nitric oxide synthase.
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DOI：
10.1084/jem.180.3.977
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发表时间：
1994-09-01
期刊：
影响因子：
--
通讯作者：
Xie QW
中科院分区：
文献类型：
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作者：
Martin E;Nathan C;Xie QW
干扰素 γ (IFN-gamma) 与细菌脂多糖 (LPS) 协同相互作用，诱导 iNOS 转录，iNOS 是一氧化氮合酶的亚型，其活性不依赖于 Ca2+ 升高和外源性钙调蛋白。为了定义介导 IFN-γ 依赖性协同作用的顺式作用元件，我们在与报告基因融合的 iNOS 启动子构建体中进行了删除，转染 RAW 264.7 巨噬细胞，并用 IFN-γ 和/或 LPS 处理细胞。该分析涉及位置-951至-911的区域，这是已知结合IFN-γ响应转录因子的四个增强子元件的簇，包括位于核苷酸-913至-923处的干扰素调节因子结合位点(IRF-E)。在全长iNOS启动子的背景下，IRF-E内两个保守核苷酸的位点特异性取代消除了IFN-γ对转录协同增强的贡献。使用含有 IRF-E 的探针进行的电迁移率测定揭示了 RAW 264.7 巨噬细胞核中存在复合物，该复合物仅在用 IFN-γ 处理后才存在，该复合物与抗 IRF-1 免疫球蛋白 G 发生特异性反应，并且其中包括以 40-45 kD 迁移的物种，与鼠 IRF-1 的表观分子量一致。因此，IFN-γ对RAW 264.7巨噬细胞中iNOS转录的协同贡献需要IRF-1与iNOS启动子中的IRF-E结合。结合 Kamijo 等人的工作。 (Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, K. S. Im, T. Kimura, S. Green et al. 1994. Science [Wash. DC]. 263:1612)，这些发现将 iNOS 确定为第一个需要 IRF-1 进行 IFN-γ 依赖性转录调节的基因。
Interferon gamma (IFN-gamma) interacts synergistically with bacterial lipopolysaccharide (LPS) to induce transcription of iNOS, the isoform of nitric oxide synthase whose activity is independent of elevated Ca2+ and exogenous calmodulin. To define a cis-acting element mediating IFN- gamma-dependent synergy, we made deletions in iNOS promoter constructs fused to reporter genes, transfected RAW 264.7 macrophages, and treated the cells with IFN-gamma and/or LPS. This analysis implicated the region from positions -951 to -911, a cluster of four enhancer elements known to bind IFN-gamma-responsive transcription factors, including an interferon regulatory factor binding site (IRF-E) at nucleotides -913 to -923. Site-specific substitution of two conserved nucleotides within IRF-E in the context of the full-length iNOS promoter ablated IFN- gamma's contribution to synergistic enhancement of transcription. Electromobility shift assays performed with a probe containing IRF-E revealed the existence of a complex in nuclei of RAW 264.7 macrophages that was present only after treatment with IFN-gamma, which reacted specifically with anti-IRF-1 immunoglobulin G and which included a species migrating at 40-45 kD, consistent with the apparent molecular weight of murine IRF-1. Thus, the synergistic contribution of IFN-gamma to transcription of iNOS in RAW 264.7 macrophages requires that IRF-1 bind to IRF-E in the iNOS promoter. In conjunction with the work of Kamijo et al. (Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, K. S. Im, T. Kimura, S. Green et al. 1994. Science [Wash. DC]. 263:1612), these findings identify iNOS as the first gene that requires IRF-1 for IFN-gamma-dependent transcriptional regulation.