Human prolactin gene promoter regulation by estrogen: convergence with tumor necrosis factor-alpha signaling

Estrogens have been implicated in the regulation of prolactin gene expression in man, although previous studies have not defined the molecular mechanism whereby estradiol activates the human prolactin gene promoter (hPrl). We found that estradiol induced a reproducible 1.8-fold activation of the hPrl gene promoter, using pituitary GH3 cells stably transfected with a 5000-bp hPrl promoter fragment linked to luciferase reporter gene. This activation was blocked by treatment with estrogen receptor (ER) antagonists 4-hydroxytamoxifen and ICI-182,780. Promoter deletion and mutagenesis experiments identified a functional estrogen response element (ERE) sequence 1189 bp upstream of the transcription start site that was responsible for estrogen-mediated promoter activation. This site differed from the consensus ERE sequence by two base pairs, one in each half-site. This ERE was identified to be functional through binding ERalpha in EMSAs. Chromatin immunoprecipitation assays confirmed ERalpha binding to this sequence in vivo in the absence of ligand, with increased recruitment when cells were cultured in the presence of estradiol. When cells were treated with both estradiol and TNFalpha, we observed synergistic activation of the hPrl promoter, which was mediated by the -1189-bp ERE. Mutagenesis of this ERE abolished the promoter-activating effect not only of estradiol but also of TNFalpha. These data suggest a novel, promoter-specific signaling interaction between estrogen and TNFalpha signaling, which is likely to be important for prolactin regulation in vivo.