Wolf Anna Tjärnlund, Medcalf Robert L, Jern Christina
Institute of Clinical Neuroscience, Sahlgrenska University Hospital/Sahlgrenska, Göteborg University, S-413 45 Göteborg, Sweden.
Blood. 2005 Feb 1;105(3):1060-7. doi: 10.1182/blood-2003-12-4383. Epub 2004 Oct 5.
We have previously identified a common polymorphism at the tissue-type plasminogen activator (t-PA) locus (-7351C>T), located within a GC-box in the retinoic acid (RA) and steroid hormone responsive t-PA enhancer. The aim of the present study was to functionally characterize this t-PA variant. Electrophoretic mobility shift assays (EMSAs) using crude nuclear extracts from human endothelial, HeLa, and NT2 neuronal cells revealed a 10-fold greater protein binding affinity to the wild-type C allele compared with the mutant T allele variant. Sp1 and Sp3 were identified as the GC-box binding proteins. Luciferase reporter assays showed that the C allele generated higher transcriptional activity after induction by RA, compared with the T allele variant. Further EMSAs showed that RA treatment enhanced Sp1/Sp3 binding to the GC-box. Formation of the Sp1/Sp3 containing complex was inhibited by anti-RA receptor (RAR) antibodies, suggesting that Sp1/Sp3 and RAR interact. The t-PA -7351C>T polymorphism is therefore functional at the level of transcription. The reduced binding affinity of Sp1/Sp3 to the T allele could explain our earlier observations of a reduced t-PA release and an increased risk of myocardial infarction in individuals carrying this allele.
我们之前在组织型纤溶酶原激活剂（t-PA）基因座（-7351C>T）处鉴定出一种常见的多态性，该基因座位于视黄酸（RA）和类固醇激素反应性t-PA增强子的一个GC盒内。本研究的目的是对这种t-PA变体进行功能表征。使用来自人内皮细胞、HeLa细胞和NT2神经元细胞的粗核提取物进行的电泳迁移率变动分析（EMSA）显示，与突变型T等位基因变体相比，野生型C等位基因的蛋白结合亲和力高10倍。Sp1和Sp3被鉴定为GC盒结合蛋白。荧光素酶报告基因分析表明，与T等位基因变体相比，C等位基因在RA诱导后产生更高的转录活性。进一步的EMSA表明，RA处理增强了Sp1/Sp3与GC盒的结合。抗RA受体（RAR）抗体抑制了含Sp1/Sp3复合物的形成，表明Sp1/Sp3与RAR相互作用。因此，t-PA -7351C>T多态性在转录水平上具有功能。Sp1/Sp3与T等位基因结合亲和力的降低可以解释我们之前观察到的携带该等位基因的个体t-PA释放减少和心肌梗死风险增加的现象。
