title: Absence of ligand-independent transcriptional activation of the estrogen receptor via the estrogen response element in pituitary lactotrophs in primary culture.
abstract: The estrogen receptor (ER) is a ligand-activated transcription factor that enhances gene expression by binding to specific regulatory DNA sequences called estrogen response elements (EREs). In some cell lines, the ER is also activated in a ligand-independent manner by multiple signaling pathways. In this study, we developed a novel adenovirus-mediated assay for promoter activation, termed LASETA, which we then used to examine whether ligand-independent activation of the ER occurred in normal pituitary lactotrophs in primary culture. In the LASETA adenovirus vector, the loxP-flanked stop sequence was deleted by prolactin (PRL) promoter-regulated expression of Cre recombinase. This led to lactotroph-specific expression of a reporter gene driven by an ERE-containing promoter. Estrogen-induced expression of the reporter protein luciferase in LASETA was specific for lactotrophs and was ER-dependent. LASETA was shown to be reliable even with varying Cre recombinase expression levels, which were caused by changes in PRL promoter activity. Using LASETA, we observed no change in ERE-mediated ER activity in the absence of estrogen after treatment of normal lactotrophs with agents such as insulin-like growth factor-1, epidermal growth factor, the adenylate cyclase activator forskolin, the extracellular signal-regulated kinase kinase inhibitor U0126, and the protein kinase A inhibitor H89. The ERE-mediated ligand-independent ER activity was induced by the growth factors and forskolin in the somatolactotroph tumor cell line GH4C1 cells. These results suggest that ERE-mediated ligand-independent activation of ER does not occur in normal lactotrophs in primary culture, and is a phenomenon likely restricted to transformed cells.
authors.affiliation: Department of Physiology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan.
authors.forename: Maho
authors.initials: M
authors.lastname: Ishida
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Mitsui
authors.forename: Michi
authors.initials: M
authors.lastname: Izawa
authors.forename: Jun
authors.initials: J
authors.lastname: Arita
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001971:Bromocriptine / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D005260:Female; D000077267:Fulvestrant; D017930:Genes, Reporter / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D052682:Lactotrophs / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D008024:Ligands; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology; D011388:Prolactin / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D011960:Receptors, Estrogen / Q000502:physiology*; D020218:Response Elements / Q000235:genetics*; D013449:Sulfonamides / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology*; D014161:Transduction, Genetic
chemical_list: D002070:Butadienes; D004965:Estrogen Antagonists; D007546:Isoquinolines; D008024:Ligands; D009570:Nitriles; D047428:Protein Kinase Inhibitors; D011960:Receptors, Estrogen; D013449:Sulfonamides; C113580:U 0126; D005576:Colforsin; D000077267:Fulvestrant; D001971:Bromocriptine; D004958:Estradiol; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D011388:Prolactin; D008156:Luciferases; D013937:Thymidine Kinase; C045073:Cre recombinase; D019426:Integrases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide