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.
Mueller
M D
MD
Center for Reproductive Sciences, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, CA 94143-0556, USA.
Vigne
J L
JL
Minchenko
A
A
Lebovic
D I
DI
Leitman
D C
DC
Taylor
R N
RN
D002478:Cells, Cultured; D004717:Endometrium / Q000378:metabolism*; D016228:Endothelial Growth Factors / Q000096:biosynthesis* / Q000235:genetics*; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D005260:Female; D018014:Gene Transfer Techniques; D006801:Humans; D008222:Lymphokines / Q000096:biosynthesis* / Q000235:genetics*; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D008222:Lymphokines; D011960:Receptors, Estrogen; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors