Inhibition of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor alpha and Sp3 proteins.
Treatment of HEC1A endometrial cancer cells with 10 nm 17beta-estradiol (E2) resulted in decreased vascular endothelial growth factor (VEGF) mRNA expression, and a similar response was observed using a construct, pVEGF1, containing a VEGF gene promoter insert from -2018 to +50. In HEC1A cells transiently transfected with pVEGF1 and a series of deletion plasmids, it was shown that E2-dependent down-regulation was dependent on wild-type estrogen receptor alpha (ERalpha) and reversed by the anti-estrogen ICI 182, 780, and this response was not affected by progestins. Deletion analysis of the VEGF 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 showed that both Sp1 and Sp3 proteins bound this region of the VEGF promoter. Coimmunoprecipitation and pull-down assays demonstrated that Sp3 and ERalpha proteins physically interact, and the interacting domains of both proteins are different from those previously observed for interactions between Sp1 and ERalpha proteins. Using a dominant negative form of Sp3 and transcriptional activation assays in Schneider SL-2 insect cells, it was confirmed that ERalpha-Sp3 interactions define a pathway for E2-mediated inhibition of gene expression, and this represents a new mechanism for decreased gene expression by E2.
Stoner
M
M
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466, USA.
Wang
F
F
Wormke
M
M
Nguyen
T
T
Samudio
I
I
Vyhlidal
C
C
Marme
D
D
Finkenzeller
G
G
Safe
S
S
D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D016889:Endometrial Neoplasms / Q000378:metabolism* / Q000473:pathology; D016228:Endothelial Growth Factors / Q000235:genetics* / Q000378:metabolism; D047628:Estrogen Receptor alpha; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008222:Lymphokines / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D011960:Receptors, Estrogen / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D017931:DNA Primers; D004268:DNA-Binding Proteins; D016228:Endothelial Growth Factors; D047628:Estrogen Receptor alpha; D008222:Lymphokines; D012333:RNA, Messenger; D011960:Receptors, Estrogen; C497121:SP3 protein, human; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D051705:Sp3 Transcription Factor