Thrombin stimulation of the vascular cell adhesion molecule-1 promoter in endothelial cells is mediated by tandem nuclear factor-kappa B and GATA motifs.
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.
Minami
T
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Department of Molecular Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
Aird
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WC
D020816:Amino Acid Motifs; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000378:metabolism*; D050989:GATA2 Transcription Factor; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; C494711:GATA2 protein, human; D016328:NF-kappa B; D012333:RNA, Messenger; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D012313:RNA; D008156:Luciferases; D012260:Ribonucleases; D013917:Thrombin