Tristetraprolin regulates the expression of the human inducible nitric-oxide synthase gene
Tristetraprolin regulates the expression of the human inducible nitric-oxide synthase gene
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DOI：
10.1124/mol.104.008763
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发表时间：
2005-06-01
影响因子：
3.6
通讯作者：
Kleinert, H
中科院分区：
文献类型：
--
作者：
Fechir, M;Linker, K;Kleinert, H
人诱导型一氧化氮合酶（iNOS）的表达受转录和转录后机制的调控。mRNA的稳定通常依赖于p38丝裂原活化蛋白激酶（p38 MAPK）的活化。在人DLD-1细胞中，通过化合物4-（4-氟苯基）-2-（4-甲基亚磺酰基苯基）-5-（4-吡啶基）1H-咪唑（SB 203580）或通过显性负性p38 MAPK α蛋白的过表达抑制p38 MAPK导致人iNOS mRNA和蛋白表达的减少，而人iNOS启动子活性不受影响。由p38 MAPK途径调节并参与调节几种mRNA稳定性的重要RNA结合蛋白是三曲脯氨酸。RNA酶保护，定量实时聚合酶链反应和Western印迹实验表明，用于诱导DLD-1细胞中iNOS表达的细胞因子也增强了tristetraprolin的表达。SB 203580孵育降低了曲曲脯氨酸介导的三曲脯氨酸表达增强。在稳定转染的DLD-1-或A549/8细胞中，tristetraprolin的过表达或下调通过调节iNOS-mRNA的稳定性一致地导致iNOS表达的增强或减少。在UV交联实验中，重组tristetraprolin不与人iNOS mRNA相互作用。然而，免疫共沉淀实验显示tristetraprolin与KH型剪接调节蛋白（KSRP）的相互作用，已知其将含有富含AU的元件的mRNA募集到外泌体中进行降解。细胞因子增强了这种三曲脯氨酸-KSRP相互作用，SB 203580处理降低了这种相互作用。我们得出结论，tristetraprolin积极调节人iNOS的表达，通过提高稳定性的人iNOS mRNA。由于tristetraprolin不直接结合人iNOS mRNA，但与KSRP相互作用，因此tristetraprolin可能通过捕获KSRP-外泌体复合物来稳定iNOS mRNA。
The expression of human inducible NO synthase ( iNOS) is regulated both by transcriptional and post-transcriptional mechanisms. Stabilization of mRNAs often depends on activation of p38 mitogen-activated protein kinase ( p38 MAPK). In human DLD-1 cells, inhibition of p38 MAPK by the compound 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) or by overexpression of a dominant-negative p38 MAPK alpha protein resulted in a reduction of human iNOS mRNA and protein expression, whereas human iNOS promoter activity was not affected. An important RNA binding protein regulated by the p38 MAPK pathway and involved in the regulation of the stability of several mRNAs is tristetraprolin. RNase protection, quantitative real-time polymerase chain reaction, and Western blot experiments showed that cytokines used to induce iNOS expression in DLD-1 cells also enhanced tristetraprolin expression. SB203580 incubation reduced cytokine-mediated enhancement of tristetraprolin expression. Overexpression or down-regulation of tristetraprolin in stably transfected DLD-1- or A549/8 cells consistently resulted in enhanced or reduced iNOS expression by modulating iNOS-mRNA stability. In UV cross-linking experiments, recombinant tristetraprolin did not interact with the human iNOS mRNA. However, coimmunoprecipitation experiments showed interaction of tristetraprolin with the KH-type splicing regulatory protein ( KSRP), which is known to recruit mRNAs containing AU-rich elements to the exosome for degradation. This tristetraprolin-KSRP interaction was enhanced by cytokines and reduced by SB203580 treatment. We conclude that tristetraprolin positively regulates human iNOS expression by enhancing the stability of human iNOS mRNA. Because tristetraprolin does not directly bind to the human iNOS mRNA but interacts with KSRP, tristetraprolin is likely to stabilize iNOS mRNA by capturing the KSRP-exosome complex.