Estradiol regulation of the human retinoic acid receptor alpha gene in human breast carcinoma cells is mediated via an imperfect half-palindromic estrogen response element and Sp1 motifs.
Estrogen receptor (ER)-positive human breast carcinoma (HBC) cell lines express significantly higher levels of retinoic acid receptor alpha (RAR alpha) (isoform 1) mRNA than ER-negative HBCs. Estradiol enhances RAR alpha mRNA expression in different ER-positive HBCs by 2-3-fold, which in turn results in increased sensitivity of ER-positive HBCs to the growth inhibitory effects of retinoic acid. To investigate the regulatory mechanisms of estradiol-mediated enhancement of RAR alpha mRNA expression, the functional promoter for the human RAR alpha isoform 1 was cloned and used to assess estradiol-mediated promoter-dependent enhancement of firefly luciferase reporter gene activity in transiently transfected ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231) HBCs. Deletional promoter constructs were obtained to further delineate the promoter region responsible for estradiol-mediated enhancement of promoter activity. Here, we present evidence that approximately 130 bp of the promoter fragment preceding the transcriptional start site are responsible for estradiol-mediated enhancement of hRAR alpha gene expression. The estradiol-mediated enhancement is dependent on ER binding. Further deletional analysis showed that a promoter sequence of 42 base pairs, located approximately 100 bases upstream of the transcriptional start site, contains elements for estradiol-mediated enhancement. Specific deletion of either the Sp1 motif or mutations in the imperfect half-palindromic estrogen response element motif of this fragment abolish its estradiol responsiveness in transient transfections.
Rishi
A K
AK
Department of Medicine, University of Maryland School of Medicine, Baltimore, USA.
Shao
Z M
ZM
Baumann
R G
RG
Li
X S
XS
Sheikh
M S
MS
Kimura
S
S
Bashirelahi
N
N
Fontana
J A
JA
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics* / Q000648:ultrastructure; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004958:Estradiol / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D000072482:Retinoic Acid Receptor alpha; D016329:Sp1 Transcription Factor / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; C000607071:RARA protein, human; D011960:Receptors, Estrogen; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D016329:Sp1 Transcription Factor; D004958:Estradiol; D008156:Luciferases