Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor alpha and SP proteins.
Vascular endothelial growth factor (VEGF) is expressed in multiple hormone-dependent cancer cells/tumors. Treatment of ZR-75 breast cancer cells with 17beta-estradiol (E2) induced a greater than fourfold increase of VEGF mRNA levels. ZR-75 breast cancer cells were transfected with pVEGF1, a construct containing a -2018 to +50 VEGF promoter insert, and E2 induced reporter gene (luciferase) activity. Deletion and mutation analysis of the VEGF gene promoter identified a GC-rich region (-66 to -47) which was required for E2-induced transactivation of pVEGF5, a construct containing the minimal promoter (-66 to +54) that exhibited E2-responsiveness. Interactions of nuclear proteins from ZR-75 cells with the proximal GC-rich region of the VEGF gene promoter were investigated by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results demonstrate that both Sp1 and Sp3 proteins bound the GC-rich motif (-66 to -47), and estrogen receptor alpha (ERalpha) interactions were confirmed by chromatin immunoprecipitation. Moreover, E2-dependent activation of constructs containing proximal and distal GC/GT-rich regions of the VEGF promoter was inhibited in ZR-75 cells transfected with small inhibitory RNAs for Sp1 and Sp3. These results were consistent with a mechanism of hormone activation of VEGF through ERalpha/Sp1 and ERalpha/Sp3 interactions with GC-rich motifs.
Stoner
Matthew
M
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466, USA.
Wormke
Mark
M
Saville
Brad
B
Samudio
Ismael
I
Qin
Chunhua
C
Abdelrahim
Maen
M
Safe
Stephen
S
D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000494:pharmacology*; D020862:GC Rich Sequence; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D009376:Neoplasms, Hormone-Dependent / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D047628:Estrogen Receptor alpha; D004967:Estrogens; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011960:Receptors, Estrogen; D016329:Sp1 Transcription Factor; D042461:Vascular Endothelial Growth Factor A; D004958:Estradiol