Identification of functional estrogen response elements in the gene codingfor the potent angiogenic factor vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent stimulator of angioge nesis and a prognostic factor for many tumors including those of endocrine- responsive tissues such as the breast and uterus, We and others have previo usly shown that VEGF is regulated by estradiol and tamoxifen in the uterus and by estradiol in human breast cancer cells, and pharmacological evidence has suggested that this regulation was mediated by transcriptional activat ion of the estrogen receptor (ER), This prompted us to investigate whether the VEGF gene contains sequences that bind the ER and confer hormonal induc ibility to reporter constructs in the presence of the two ER subtypes, Thes e studies identified two sequences homologous to the consensus estrogen res ponse element, GGTCAnnnTGACC, which bind both ER-alpha and ER-beta, One of these elements is located in the 5'-untranslated region of the VEGF gene (G GGCAaagTGACT), and the other is located in the 5'-untranslated region (CAGC AcccTGCCC), Competition with excess unlabeled oligonucleotides indicates th at these two elements hind both ERs specifically, mutations in either half- site of the two elements abolish receptor binding, and ER-alpha and ER-beta specific antibodies interact with complexes formed with the corresponding receptor subtypes, In cells containing either ER-alpha or ER-beta, the 3'-e lement behaves as a traditional enhancer that confers hormone inducibility to reporter constructs in an orientation-independent manner, and transcript ional activity is blocked by the pure antiestrogen ICI 182,780, The pattern of transcriptional activity of the element located in the 9'-flanking regi on is more complex, In the orientation found in the endogenous gene, this e lement is nonresponsive to ER-P but confers estrogen-dependent inhibition o f transcription with ER-alpha that is blunted by ICI 182,780, In the opposi te orientation, the 5'-element confers hormone inducibility with either ER- alpha or -beta, and ICI 182,780 blocks activation by ER-alpha but not by ER -beta, These findings support the hypotheses that estrogens directly regula te VEGF transcription in target tissues and tumors, although such regulatio n appears likely to involve a complex interplay of cis- and trans-acting el ements not previously observed for other hormone-responsive genes.