Abstract
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.
Highlights
The vascular adhesion molecule-1 (VCAM-1) promoter has been cloned and well characterized [6]
These results suggest that thrombin stimulation of VCAM-1 in endothelial cells is mediated by the coordinate action of nuclear factor B (NF-B) and GATA transcription factors
We show that the effect of thrombin on VCAM-1 expression is mediated by a combination of NF-B and GATA motifs in the upstream promoter region
Summary
The VCAM-1 promoter has been cloned and well characterized [6]. Previous studies have shown that two tandem NF-B elements located at position Ϫ77 and Ϫ63, relative to the transcriptional start site are necessary for mediating response to inflammatory mediators (6 – 8). Thrombin plays a role in inflammation by activating a variety of cell types, including endothelial cells, smooth muscle cells, and leukocytes. Most of these cellular effects are mediated by PAR1, a G-protein-coupled receptor. Given that chronic inflammation is characterized by increased monocyteendothelial cell interactions, an understanding of the molecular mechanisms by which thrombin induces VCAM-1 expression may provide important insight about the link between coagulation and chronic inflammatory disease states. We show that the effect of thrombin on VCAM-1 expression is mediated by a combination of NF-B and GATA motifs in the upstream promoter region These findings are the first to demonstrate a link between thrombin signaling and GATA binding activity
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