A +220 GATA motif mediates basal but not endotoxin-repressible expression of the von Willebrand factor promoter in Hprt-targeted transgenic mice.

Abstract

The von Willebrand factor (VWF) gene is a marker for spatial and temporal heterogeneity of the endothelium. A GATA motif at +220 has been implicated in basal VWF expression in vitro. Other studies have shown that GATA3 and VWF are transcriptionally downregulated in response to inflammatory mediators. Our goal was to determine the importance of the +220 GATA motif in mediating expression of VWF promoter in vivo, and to elucidate whether the GATA element plays a role in spatial and/or temporal regulation of VWF expression. ChIP and electrophoretic mobility shift assays were carried out in human umbilical vein endothelial cells (HUVEC). Reporter gene constructs containing 3.6 kb of the human VWF promoter with and without a mutation of the +220 GATA element were transfected into cultured endothelial cells or targeted to the Hprt locus of mice. The Hprt-targeted mice were subjected to endotoxemia. In protein-DNA binding assays, the +220 GATA motif bound GATA-2, -3 and -6. Mutation of the GATA site resulted in reduced basal promoter activity in HUVEC. When targeted to the Hprt locus of mice, the GATA mutation resulted in a significant, proportionate reduction of promoter activity in LacZ expressing vascular beds. Systemic administration of lipopolysaccharide (LPS) resulted in a widespread reduction in VWF mRNA expression and promoter activity. LPS-mediated repression of the VWF promoter was unaffected by the GATA mutation. A region of the VWF promoter between -2182 and the end of the first intron contains information for LPS-mediated gene repression. The +220 GATA motif is important for basal, but not LPS-repressible expression of the VWF gene.