Pham Nhat-Long, Franzen Amy, Levin Eugene G
Division of Vascular Biology, La Jolla Institute for Molecular Medicine, San Diego Calif 92121, USA.
Arterioscler Thromb Vasc Biol. 2004 May;24(5):982-7. doi: 10.1161/01.ATV.0000126679.70877.d0. Epub 2004 Mar 25.
Analysis of the distribution of endothelial cell tissue plasminogen activator (tPA) in the vasculature of rodents and primates demonstrated that tPA is constitutively expressed predominantly in small artery endothelial cells of brain and lung. The regulatory elements responsible for the highly selective expression of arterial endothelial cell tissue plasminogen activator were sought.
Transcription factor binding sites were defined by electrophoretic mobility-shift assay (EMSA) analysis using rat lung and brain nuclear extracts and the tPA promoter sequence from -609 to +37 bp. Protein binding to the promoter was found to be mediated by an NF1 site between -158 and -145 bp upstream from the transcriptional start site. Specific binding was confirmed through mutational analysis and competition binding studies. Infection of endothelial cells with a tPA promoter-green fluorescent protein (GFP) (-609 to +37 bp) reporter construct resulted in expression of the GFP, whereas no expression was found in smooth muscle cells. Mutation of the NF1 site increased the GFP expression indicating that the element acts as a repressor.
These results suggest that the 600 bp of the tPA promoter upstream of the transcription start site conveys cell specificity to tPA expression and that an NF1 site within this region acts as a repressor.
对啮齿动物和灵长类动物脉管系统中内皮细胞组织型纤溶酶原激活物（tPA）分布的分析表明，tPA主要在脑和肺的小动脉内皮细胞中组成性表达。研究了负责动脉内皮细胞组织型纤溶酶原激活物高度选择性表达的调控元件。
利用大鼠肺和脑的核提取物以及从-609至+37 bp的tPA启动子序列，通过电泳迁移率变动分析（EMSA）确定转录因子结合位点。发现与启动子的蛋白结合是由转录起始位点上游-158至-145 bp之间的一个NF1位点介导的。通过突变分析和竞争结合研究证实了特异性结合。用tPA启动子-绿色荧光蛋白（GFP）（-609至+37 bp）报告基因构建体感染内皮细胞导致GFP表达，而在平滑肌细胞中未发现表达。NF1位点的突变增加了GFP表达，表明该元件起阻遏作用。
这些结果表明，转录起始位点上游600 bp的tPA启动子赋予tPA表达细胞特异性，并且该区域内的一个NF1位点起阻遏作用。
