Cdc2-Like Kinases and DNA Topoisomerase I Regulate Alternative Splicing of Tissue Factor in Human Endothelial Cells
Cdc2-Like Kinases and DNA Topoisomerase I Regulate Alternative Splicing of Tissue Factor in Human Endothelial Cells
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DOI：
10.1161/circresaha.108.183905
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发表时间：
2009-03-13
影响因子：
20.1
通讯作者：
Rauch, Ursula
中科院分区：
文献类型：
--
作者：
Eisenreich, Andreas;Bogdanov, Vladimir Y.;Rauch, Ursula
肿瘤坏死因子(TNF)-α刺激的人脐静脉内皮细胞表达两种天然存在的组织因子(TF)，组织因子是凝血的主要起始物：可溶的选择性剪接异构体和全长的组织因子异构体。促成这一现象的监管途径是完全未知的。Cdc2样激酶和DNA拓扑异构酶I通过富含丝氨酸/精氨酸的蛋白的磷酸化来调节选择性剪接。在这项研究中，我们研究了富含丝氨酸/精氨酸的蛋白激酶在肿瘤坏死因子-α刺激后对转铁蛋白剪接的影响。人内皮细胞在用肿瘤坏死因子-α刺激之前，用针对CDc2样激酶和DNA拓扑异构酶I的特异性抑制剂或小干扰RNA进行预处理。采用半定量逆转录聚合酶链式反应、实时荧光定量聚合酶链式反应和免疫印迹法检测组织因子水平。细胞促凝血活性用发色因子活性测定法分析。已知的4种CDC2样蛋白在人内皮细胞中均有表达。选择性抑制CDc2样激酶和DNA拓扑异构酶I可引起肿瘤坏死因子-α刺激的内皮细胞TF生物合成的明显变化，从而影响内皮细胞促凝血活性。这项研究首次证明富含丝氨酸/精氨酸的蛋白激酶调节人内皮细胞中TF-Pre-mRNA的剪接，从而在炎症条件下调节内皮细胞促凝血活性。(中国保监会决议2009；104：589-599。)
Tumor necrosis factor (TNF)-alpha-stimulated human umbilical vein endothelial cells express 2 naturally occurring forms of tissue factor (TF), the primary initiator of blood coagulation: the soluble alternatively spliced isoform and the full-length TF isoform. The regulatory pathways enabling this phenomenon are completely unknown. Cdc2-like kinases and DNA topoisomerase I regulate alternative splicing via phosphorylation of serine/arginine-rich proteins. In this study, we examined effects of serine/arginine-rich protein kinases on TF splicing following stimulation with TNF-alpha. Human endothelial cells were pretreated with specific inhibitors or small interfering RNAs against Cdc2-like kinases and DNA topoisomerase I before stimulation with TNF-alpha. TF levels were determined by semiquantitative RT-PCR, real-time PCR, and Western blotting. Cellular procoagulant activity was analyzed in a chromogenic TF activity assay. All 4 known Cdc2-like kinases forms were expressed in human endothelial cells. Selective inhibition of Cdc2-like kinases and DNA topoisomerase I elicited distinct changes in TF biosynthesis in TNF-alpha-stimulated endothelial cells, which impacted endothelial procoagulant activity. This study is the first to demonstrate that serine/arginine-rich protein kinases modulate splicing of TF pre-mRNA in human endothelial cells and, consequently, endothelial procoagulant activity under inflammatory conditions. (Circ Res. 2009; 104: 589-599.)