Thrombin stimulation of the vascular cell adhesion molecule-1 promoter in endothelial cells is mediated by tandem nuclear factor-κB and GATA motifs
Thrombin stimulation of the vascular cell adhesion molecule-1 promoter in endothelial cells is mediated by tandem nuclear factor-κB and GATA motifs
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DOI：
10.1074/jbc.m108363200
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发表时间：
2001-12-14
影响因子：
4.8
通讯作者：
Aird, WC
中科院分区：
文献类型：
--
作者：
Minami, T;Aird, WC
本研究的目的是阐明凝血酶介导的诱导血管粘附分子-1（VCAM-1）的转录机制。用凝血酶处理人脐静脉内皮细胞导致VCAM-1启动子活性增加3.3倍。VCAM-1的上游启动子区包含凝血酶反应元件、两个核因子κ B（NF-κ B）基序和串联加塔基序。在瞬时转染试验中，凝血酶反应元件的突变对凝血酶诱导没有影响。相反，任一NF-κ B位点的突变导致诱导的完全丧失，而两个加塔基序的突变导致凝血酶刺激的显著减少。在电泳迁移率变动分析中，凝血酶处理的内皮细胞的核提取物显示与串联NF-κ B和加塔基序的结合显著增加。NF-κ B复合物与抗p65抗体超移，但与RelB、c-Rel、p50或p52抗体不超移。加塔复合物被针对加塔-2的抗体超移位，但不被针对加塔-3或加塔-6的抗体超移位。含有串联拷贝的VCAM-1加塔基序连接到一个最小的胸苷激酶启动子的构建体被凝血酶诱导2.4倍。总之，这些结果表明，凝血酶刺激血管内皮细胞中的VCAM-1是由NF-κ B和加塔转录因子的协调作用介导的。
The goal of this study was to delineate the transcriptional mechanisms underlying thrombin-mediated induction of vascular adhesion molecule-1 (VCAM-1). Treatment of human umbilical vein endothelial cells with thrombin resulted in a 3.3-fold increase in VCAM-1 promoter activity. The upstream promoter region of VCAM-1 contains a thrombin response element, two nuclear factor kappaB (NF-kappaB) motifs, and a tandem GATA motif. In transient transfection assays, mutation of the thrombin response element had no effect on thrombin induction. In contrast, mutation of either NF-kappaB site resulted in a complete loss of induction, whereas a mutation of the two GATA motifs resulted in a significant reduction in thrombin stimulation. In electrophoretic mobility shift assays, nuclear extracts from thrombin-treated endothelial cells displayed markedly increased binding to the tandem NF-kappaB and GATA motifs. The NF-kappaB complex was supershifted with anti-p65 antibodies, but not with antibodies to RelB, c-Rel, p50, or p52. The GATA complex was supershifted with antibodies to GATA-2, but not GATA-3 or GATA-6. A construct containing tandem copies of the VCAM-1 GATA motifs linked to a minimal thymidine kinase promoter was induced 2.4-fold by thrombin. Taken together, these results suggest that thrombin stimulation of VCAM-1 in endothelial cells is mediated by the coordinate action of NF-kappaB and GATA transcription factors.