Fechir Marcel, Linker Katrin, Pautz Andrea, Hubrich Thomas, Förstermann Ulrich, Rodriguez-Pascual Fernando, Kleinert Hartmut
Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, 55101 Mainz, Germany.
Mol Pharmacol. 2005 Jun;67(6):2148-61. doi: 10.1124/mol.104.008763. Epub 2005 Mar 18.
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 MAPKalpha 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.
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的稳定性通常取决于p38丝裂原活化蛋白激酶(p38 MAPK)的激活。在人DLD-1细胞中,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(SB203580)或显性负性p38 MAPKα蛋白的过表达抑制p38 MAPK,导致人iNOS mRNA和蛋白表达降低,而人iNOS启动子活性未受影响。受p38 MAPK途径调控并参与多种mRNA稳定性调节的一种重要RNA结合蛋白是锌指蛋白TTP。核糖核酸酶保护、定量实时聚合酶链反应和蛋白质印迹实验表明,用于诱导DLD-1细胞中iNOS表达的细胞因子也增强了锌指蛋白TTP的表达。SB203580孵育降低了细胞因子介导的锌指蛋白TTP表达增强。在稳定转染的DLD-1或A549/8细胞中过表达或下调锌指蛋白TTP,通过调节iNOS-mRNA稳定性,始终导致iNOS表达增强或降低。在紫外线交联实验中,重组锌指蛋白TTP不与人iNOS mRNA相互作用。然而,免疫共沉淀实验表明锌指蛋白TTP与KH型剪接调节蛋白(KSRP)相互作用,已知KSRP可将含有富含AU元件的mRNA募集到外泌体进行降解。这种锌指蛋白TTP-KSRP相互作用在细胞因子作用下增强,在SB203580处理下减弱。我们得出结论,锌指蛋白TTP通过增强人iNOS mRNA的稳定性来正向调节人iNOS的表达。由于锌指蛋白TTP不直接与人iNOS mRNA结合,而是与KSRP相互作用,锌指蛋白TTP可能通过捕获KSRP-外泌体复合物来稳定iNOS mRNA。