Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor κB-binding sites
Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor κB-binding sites
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DOI：
10.1074/jbc.273.35.22201
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发表时间：
1998-08-28
影响因子：
4.8
通讯作者：
Moss, J
中科院分区：
文献类型：
--
作者：
Marks-Konczalik, J;Chu, SC;Moss, J
检测AP-1和NF-kappaB转录因子在细胞因子诱导的人诱导型一氧化氮合酶(HiNOS)启动子活性中的作用。含有AP-1和NF-kappa B位点突变的荧光素酶报告质粒在A549细胞中瞬时表达，并在含有IL-1-β、干扰素-γ和肿瘤坏死因子-α的细胞因子混合物(CM)处理后，检测启动子的活性。在-8.3kb启动子和-5.574 kb短启动子中，AP-1七肽上游-5301b碱基对的突变使基因激活降低了90%。AP-1(At-5115)或NF-kappa B(At-115和-8283)位点的破坏分别使启动子活性降低45%、67%和52%。在这两个核因子-kappaB位点突变的结构中，对CM的反应性降低了85%。凝胶滞留分析显示，CM增加了AP-1和NF-kappaB的结合。超位移分析确定Jun D和Fra-2为AP-1复合体的组成成分。每个kappa B结合不同互补的核因子-kappaB/Rel家族成员(下游位点，Rel A/p50；上游位点，Rel A/Rel A)。在CM处理1h后，Rel A的表达最强，而I kappa B-α的表达最低，与核转录因子kappaB结合活性的峰值相对应。因此，AP-1和NF-kappaB是诱导hiNOS基因转录的重要顺式元件。
The involvement of AP-1 and NF-kappa B transcription factors in cytokine-mediated induction of human inducible nitric oxide synthase (hiNOS) promoter activity was examined. Luciferase reporter plasmids, containing mutations in AP-1 and NF-kappa B sites, in a hiNOS promoter extending from -8.3 kilobase pairs (kb) to +168, were transiently expressed in A549 cells, and promoter activity was determined after treatment with a cytokine mixture (CM) containing interleukin 1-beta, interferon-gamma, and tumor necrosis factor-alpha. Mutation of the AP-1 heptad located -5301 base pairs upstream decreased gene activation by 90% in a -8.3-kb promoter and a shorter -5.574-kb promoter. Disruption of AP-1 (at -5115) or NF-kappa B (at -115 and -8283) sites reduced promoter activity by 45, 67, and 52%, respectively. Responsiveness to CM was decreased by 85% in constructs mutated in both NF-kappa B sites. By gel retardation analyses, CM increased AP-1- and NF-kappa B binding. Supershift analysis identified Jun D and Fra-2 as components of AP-1 complexes. Each kappa B Site bound different complements of NF-kappa B/Rel family members (downstream site, Rel A/p50; upstream site, Rel A/Rel A). Rel A was maximally, whereas I kappa B-alpha was minimally, expressed in nuclei after 1 h of CM treatment, corresponding with the peak in NF-kappa B inding activity. Thus, AP-1 and NF-kappa B are important cis-elements for induction of hiNOS gene transcription.