Interaction of interferon regulatory factor-1 and nuclear factor κB during activation of inducible nitric oxide synthase transcription
Interaction of interferon regulatory factor-1 and nuclear factor κB during activation of inducible nitric oxide synthase transcription
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DOI：
10.1006/jmbi.1999.2752
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发表时间：
1999-06-11
影响因子：
5.6
通讯作者：
Lowenstein, CJ
中科院分区：
文献类型：
--
作者：
Saura, M;Zaragoza, C;Lowenstein, CJ
我们研究了TNF-α和IFN-γ协同诱导诱导型一氧化氮合酶转录的分子机制。由于TNF-α和IFN-γ部分通过激活NF-κ B和IRF-1刺激细胞，我们假设这两种转录因子相互作用。IRF-1和NF-κ B共定位于刺激的巨噬细胞的细胞核中。免疫共沉淀实验表明，LRF-1和NF-κ B在刺激的细胞中相互作用，但在静息细胞中不相互作用。超位移实验表明，LRF-1和NF-κ B相互作用，同时结合到各自的DNA结合位点。这些结果证明了体内IRF-1和NF-κ B蛋白之间存在物理相互作用。我们接下来提出IRF-1和NF-κ B之间的这种相互作用使iNOS启动子区域的DNA弯曲。使用环化试验，我们证明，从刺激细胞的核提取物加速线性到环状DNA的转化率，从静息细胞的提取物相比。然而，刺激的核提取物不能影响具有突变IRE或κ B位点的启动子的环化速率。此外，缺乏IRF-1和NF-κ B的刺激的核提取物不能诱导环化。我们的结论是，IRF-1和NF-κ B在体内相互作用，这种相互作用物理弯曲indicible一氧化氮合酶启动子DNA。这种相互作用可以解释IFN-γ协同增强诱导型一氧化氮合酶转录的机制。(C)北京：科学出版社.
We investigated the molecular mechanism for the synergistic induction of inducible nitric oxide synthase transcription by TNF-alpha and IFN-gamma. Since TNF-alpha and IFN-gamma stimulate cells in part by activating NF-kappa B and IRF-1, we hypothesized that these two transcription factors interact with each other. IRF-1 and NF-kappa B co-localize in the nucleus of stimulated macrophages. Co-immunoprecipitation experiments show that LRF-1 and NF-kappa B interact in stimulated but not resting cells. Super-shift experiments show that LRF-1 and NF-kappa B interact while binding to their respective DNA binding sites. These results demonstrate the existence of a physical interaction between IRF-1 and NF-kappa B proteins in vivo. We next suggested that this interaction between IRF-1 and NF-kappa B bends the DNA of the iNOS promoter region. Using a cyclization assay, we demonstrate that nuclear extracts from stimulated cells accelerate the rate of conversion of a linear to circular DNA, compared to extracts from resting cells. However, stimulated nuclear extracts cannot affect the rate of cyclization of a promoter with a mutant IRE or kappa B site. Furthermore, stimulated nuclear extracts depleted of IRF-1 and NF-kappa B cannot induce cyclization. We conclude that IRF-1 and NF-kappa B interact in vivo, and that this interaction physically bends the indicible nitric oxide synthase promoter DNA. This interaction may explain the mechanism by which IFN-gamma synergistically augments inducible nitric oxide synthase transcription. (C) 1999 Academic Press.