Purpose. Vascular endothelial growth factor (VEGF) mitogenic action is mediated through a cell surface receptor (KDR/flk-1) whose expression is restricted primarily to endothelial cells in vivo. We determined DNA elements in the upstream untranslated region (UTR) of the human KDR gene which confer endothelial-specific expression. Methods. A 100kb genomic fragment encompassing the KDR gene was obtained by PI phage cloning and 4 KB of the KDR UTR was subcloned into a luciferase reporter gene expression plasmid. Deletion and site specific mutagenesis was performed. The gene constructs were transiently transfected into bovine retinal endotheiial cells (BREC), pericytes (RFC), and pigment epithelial cells (RPE) using lipofectamine. Promoter activity was determined by luciferase assay. DNA binding proteins were identified in nuclear extracts using gel mobility shift assays. Results. Transfection of BREC with wild-type KDR promoter resulted in a 407fold increase in activity over promoterless control, an effect 5 & 14 times greater than in RFC & RPE, respectively. Stimulation as a percent of maximally driven promoter expression in each cell type was 46% for BRE but only 6% for RPC and 3% for RPE. Gel mobility shift assays identified a 50 bp element which bound transcription factor(s) produced by BREC but not RPC or RPE cells. Site-specific mutagenesis of this region localized specific DNA binding to a 12 base pair region, containing 3 essential residues. Mutations in this region reduced luciferase expression over 65% in BRE, with little effect in either RPC or RPE. Conclusions. These data identify a 12 base pair DNA element which binds a nuclear factor preferentially produced in endothelial cells that confers endothelial-selective expression.
|Original language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience