Inhibition of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor alpha and Sp3proteins

Treatment of HEC1A endometrial cancer cells with 10 nM 17 beta-estradiol (E 2) resulted in decreased vascular endothelial growth factor (VEGF) mRNA exp ression, and a similar response was observed using a construct, pVEGF1, con taining a VEGF gene promoter insert from -2018 to +50, In HEC1A cells trans iently transfected with pVEGF1 and a series of deletion plasmids, it was sh own that E2-dependent down-regulation was dependent on wild-type estrogen r eceptor alpha (ER alpha) and reversed by the anti-estrogen ICI 182,780, and this response was not affected by progestins, Deletion analysis of the VEG F gene promoter identified an overlapping G/GC-rich site between -66 to -47 that was required for decreased transactivation by E2. Protein-DNA binding studies using electrophoretic mobility shift and DNA footprinting assays s howed that both Sp1 and Sp3 proteins bound this region of the VEGF promoter . Coimmunoprecipitation and pull-down assays demonstrated that Sp3 and ER a lpha proteins physically interact, and the interacting domains of both prot eins are different from those previously observed for interactions between Sp1 and ER alpha proteins, Using a dominant negative form of Sp3 and transc riptional activation assays in Schneider SL-2 insect cells, it was confirme d that ER alpha-Sp3 interactions define a pathway for E2-mediated inhibitio n of gene expression, and this represents a new mechanism for decreased gen e expression by E2.