title: Vascular endothelial growth factor receptor-2 expression is down-regulated by 17beta-estradiol in MCF-7 breast cancer cells by estrogen receptor alpha/Sp proteins.
abstract: 17beta-Estradiol (E2) induces and represses gene expression in breast cancer cells; however, the mechanisms of gene repression are not well understood. In this study, we show that E2 decreases vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in MCF-7 cells, and this gene was used as a model for investigating pathways associated with E2-dependent gene repression. Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich motifs at -58 and -44 are critical for the E2-dependent decreased response in MCF-7 cells. Mutation or deletion of these GC-rich elements results in loss of hormone responsiveness and shows that the -60 to -37 region of the VEGFR2 promoter is critical for both basal and hormone-dependent decreased VEGFR2 expression in MCF-7 cells. Western blot, immunofluorescent staining, RNA interference, and EMSAs support a role for Sp proteins in hormone-dependent down-regulation of VEGFR2 in MCF-7 cells, primarily through estrogen receptor (ER)alpha/Sp1 and ERalpha/Sp3 interactions with the VEGFR2 promoter. Using chromatin immuno-precipitation and transient transfection/RNA interference assays we show that the ERalpha/Sp protein-promoter interactions are accompanied by recruitment of the co-repressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) to the promoter and that SMRT and NCoR knockdown reverse E2-mediated down-regulation of VEGFR2 expression in MCF-7 cells. This study illustrates that both SMRT and NCoR are involved in E2-dependent repression of VEGFR2 in MCF-7 cells.
authors.affiliation: Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, College Station, TX 77843-4466, USA.
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Higgins
authors.forename: Shengxi
authors.initials: S
authors.lastname: Liu
authors.forename: Maen
authors.initials: M
authors.lastname: Abdelrahim
authors.forename: Kathryn
authors.initials: K
authors.lastname: Vanderlaag
authors.forename: Xinyi
authors.initials: X
authors.lastname: Liu
authors.forename: Weston
authors.initials: W
authors.lastname: Porter
authors.forename: Richard
authors.initials: R
authors.lastname: Metz
authors.forename: Stephen
authors.initials: S
authors.lastname: Safe
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008957:Models, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051703:Sp Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D014162:Transfection; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics*
chemical_list: D047628:Estrogen Receptor alpha; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051703:Sp Transcription Factors; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D004958:Estradiol; D040301:Vascular Endothelial Growth Factor Receptor-2