Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta

Vascular endothelial growth factor (VEGF) mediates angiogenic activity in a variety of estrogen target tissues. To determine whether estrogen has a direct transcriptional effect on VEGF gene expression, we developed a model system by transiently transfecting human VEGF promoter-luciferase reporter constructs into primary human endometrial cells and into Ishikawa cells, derived from a well-differentiated human endometrial adenocarcinoma. In primary endometrial epithelial cells, treatment with 17beta-estradiol (E(2)) resulted in a 3.8-fold increase in luciferase activity, whereas a 3. 2-fold induction was demonstrated for stromal cells. Our Ishikawa cells had less than 100 functional estrogen receptors (ER)/cell and were therefore cotransfected with expression vectors encoding either the alpha- or the beta-form of the human ER. In cells cotransfected with ERalpha, E(2) induced 3.2-fold induction in VEGF-promoter luciferase activity. A 2.3-fold increase was observed in cells cotransfected with ERbeta. Through specific deletions, the E(2) response was restricted to a single 385-bp PvuII-SstI fragment in the 5' flanking DNA. Cotransfection of this upstream region with a DNA binding domain ER mutant, or site-directed mutagenesis of a variant ERE within this fragment, resulted in the loss of the E(2) response. Electromobility shift assays demonstrated that this same ERE sequence specifically binds estradiol-ER complexes. These studies demonstrate that E(2)-regulated VEGF gene transcription requires a variant ERE located 1.5 kb upstream from the transcriptional start site. Site-directed mutagenesis of this ERE abrogated E(2)-induced VEGF gene expression.