Enhanced AP-1 and NF-κB activities and stability of interleukin 8 (IL-8) transcripts are implicated in IL-8 mRNA superinduction in lung epithelial H292 cells
Enhanced AP-1 and NF-κB activities and stability of interleukin 8 (IL-8) transcripts are implicated in IL-8 mRNA superinduction in lung epithelial H292 cells
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DOI：
10.1042/bj3300429
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发表时间：
1998-02-15
影响因子：
4.1
通讯作者：
Lutter, R
中科院分区：
文献类型：
--
作者：
Roger, T;Out, TA;Lutter, R
抑制蛋白质合成可能会导致短寿命转录物的超诱导，并可归因于转录物的稳定和/或基因转录的增加。对这些过程的动力学知之甚少，相关的转录元件尚未确定。在这项研究中，我们描述了白细胞介素8 (IL-8) mRNA的超诱导，一种重要的炎症介质，在肺上皮样H292细胞中，并确定了潜在的分子机制和动力学。环己亚胺（CHI, 10 μ g/ml）是一种蛋白质合成抑制剂，可使H292细胞中IL-8 mRNA水平升高30倍。肿瘤坏死因子α (tnf - α)诱导IL-8 mRNA 3倍，与CHI协同作用，使IL-8 mRNA在6小时内增加150倍。CHI早期增加了IL-8 mRNA的稳定性（从培养液培养40分钟到CHI培养4小时以上）。通过转染IL-8启动子表明，CHI也增加了转录。截断和突变的构建体鉴定出NF-kappa B和AP-1结合位点是IL-8基因转录和IL-8 mRNA超诱导的主要顺式作用元件。电泳迁移率转移实验表明，CHI增加了NF-kappa B并延长了AP-1 dna结合活性，tnf - α和CHI对IL-8 mRNA表达的协同作用与AP-1 dna结合活性的进一步增加是平行的。在加入CHI和tnf - α之间的4小时后，这种协同作用仍然被注意到。综上所述，我们的研究结果表明CHI干扰了IL-8 mRNA表达的转录后和转录抑制机制。
Inhibition of protein synthesis may result in superinduction of short-lived transcripts and has been attributed variably to stabilization of transcripts and/or increased gene transcription. Little is known about the kinetics of these processes and relevant transcriptional elements have not been identified. In this study, we describe superinduction of interleukin 8 (IL-8) mRNA, an important inflammatory mediator, in lung epithelial-like H292 cells and identify the underlying molecular mechanisms and their kinetics. Cycloheximide (CHI, 10 mu g/ml), an inhibitor of protein synthesis, maximally increased IL-8 mRNA levels 30-fold in H292 cells. Tumour necrosis factor alpha (TNF-alpha), which induced IL-8 mRNA 3-fold, synergized with CHI causing a 150-fold increase at 6 h. CHI early on increased the stability of IL-8 mRNA (from 40 min in cells cultured with medium to more than 4h with CHI). CHI also increased transcription as shown by transfection with IL-8 promoter constructs. Truncated and mutated constructs identified NF-kappa B and AP-1 binding sites as primary cis-acting elements in IL-8 gene transcription and IL-8 mRNA superinduction. Electrophoretic mobility shift assays indicated that CHI increased NF-kappa B and prolonged AP-1 DNA-binding activities and that the synergism of TNF-alpha and CHI on IL-8 mRNA expression was paralleled by a further increase of AP-1 DNA-binding activity. This synergism was still noticed when 4h elapsed between the addition of CHI and that of TNF-alpha. Taken together, our results indicate that CHI interferes with both post-transcriptional and transcriptional repressive mechanisms of IL-8 mRNA expression.