Vascular endothelial growth factor receptor-2 expression is induced by 17β-estradiol in ZR-75 breast cancer cells by estrogen receptor α/Sp proteins

Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17 beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ER alpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ER alpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ER alpha/Sp3 and ER alpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ER alpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ER alpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ER alpha/Sp1 in breast cancer cell lines.