ESTRADIOL REGULATION OF THE HUMAN RETINOIC ACID RECEPTOR CW 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 line s 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 2-13-fold, which in turn results in increased sensitivity of ER-positive HBCs to the growth inhibitory effects of retinoic acid. To investigate the re gulatory mechanisms of estradiol-mediated enhancement of RAR alpha mRN A 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 transientl y transfected ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231 ) HBCs. Deletional promoter constructs were obtained to further deline ate the promoter region responsible for estradiol-mediated enhancement of promoter activity. Here, we present evidence that approximately 13 0 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 bin ding. Further deletional analysis showed that a promoter sequence of 4 2 base pairs, located approximately 100 bases upstream of the transcri ptional start site, contains elements for estradiol-mediated enhanceme nt. Specific deletion of either the Sp1 motif or mutations in the impe rfect half-palindromic estrogen response element motif of this fragmen t abolish its estradiol responsiveness in transient transfections.