A novel estradiol/estrogen receptor α-dependent transcriptional mechanism controls expression of the human prolactin receptor

Abstract

Prolactin exerts diverse functions in target tissues through its membrane receptors, and is a potent mitogen in normal and neoplastic breast cells. Estradiol (E2) induces human prolactin receptor (hPRLR) gene expression through stimulation of its generic promoter (PIII). This study identifies a novel E2-regulated non-estrogen responsive element-dependent transcriptional mechanism that mediates E2-induced hPRLR expression. E2 stimulated transcriptional activity in MCF7A2 cells transfected with PIII lacking an estrogen responsive element, and increased hPRLR mRNA and protein. The abolition of the E 2 effect by mutation of Sp1 or C/EBP elements that bind Sp1/Sp3 and C/EBPβ within PIII indicated the cooperation of these transfactors in E2-induced transcription of the hPRLR. DNA affinity protein assay showed that E2 induced estrogen receptor α (ERα) binding to Sp1/Sp3 and C/EBPβ DNA-protein complexes. The ligand-binding domain of ERα was essential for its physical interaction with C/EBPβ, and E2 promoted this association, and its DNA binding domain was required for transactivation of PIII. Co-immunoprecipitation studies revealed tethering of C/EBPβ to Sp1 by E2-activated ERα. Chromatin immunoprecipitation analysis showed that E2 induced recruitment of C/EBPβ, ERα, SRC1, p300, pCAF, TFIIB, and Pol II, with no change in Sp1/Sp3. E2 also induced promoter-associated acetylation of H3 and H4. These findings demonstrate that an E2/ERα, Sp1, and C/EBPβ complex with recruitment of coactivators and TFIIB and Pol II are required for E2-activated transcriptional expression of the hPRLR through PIII. Estradiol produced in breast stroma and adipose tissue, which are major sources of estrogen in post-menopausal women, could up-regulate hPRLR gene expression and stimulate breast tumor growth.