title: DAX-1 expression is regulated during mammary epithelial cell differentiation.
abstract: In recent studies, we have found that DAX-1 (dosage-sensitive sex reversal/adrenal hypoplasia congenita critical region on the X chromosome) is expressed in the mouse mammary epithelial cell line HC11. In this study, we focused on the regulation of DAX-1 expression and subcellular localization throughout mouse mammary epithelial cell differentiation and its hormonal regulation in the mouse mammary gland. Proliferating HC11 cells grown in epidermal growth factor (EGF)-containing medium, expressed very low levels of DAX-1 as detected by Western blotting and quantitative real-time PCR, whereas, upon EGF withdrawal and induction of differentiation, DAX-1 expression increased. Inhibition of MAPK pathway with PD 098059 resulted in increased DAX-1 levels even in the presence of EGF. Using confocal microscopy, we showed that DAX-1 cytoplasmic levels increased as cells differentiated. DAX-1 staining was nuclear in luminal cells of mouse mammary glands from 3-month-old virgin mice. A nucleo-cytoplasmic pattern was observed in pseudopregnant mice and a cytoplasmic pattern was found in mammary glands from 6-d lactating mice. The influence of DAX-1 on transcriptional activity of endogenously expressed estrogen receptors alpha (ERalpha) and beta (ERbeta) in HC11 mammary epithelial cells was evaluated with an estrogen response element-luciferase reporter assay and by quantitative real-time PCR of the ER-regulated gene receptor-interacting protein 140 kDa. Cotransfection of HC11 cells with human DAX-1 inhibited estrogen response element-reporter and receptor-interacting protein 140 kDa expression induced by 17beta-estradiol, the ERalpha-selective agonist 4,4',4'-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol, or the ERbeta-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile. In summary, DAX-1 expression increased upon differentiation induced by EGF withdrawal, and DAX-1 decreased response to estrogens in HC11 cells. Further studies are needed to determine whether DAX-1 is also important in regulation of differentiation of HC11 cells.
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet NOVUM, SE-141 86 Huddinge, Sweden.
authors.forename: Luisa A
authors.initials: LA
authors.lastname: Helguero
authors.forename: Malin
authors.initials: M
authors.lastname: Hedengran Faulds
authors.forename: Carola
authors.initials: C
authors.lastname: Förster
authors.forename: Jan-Ake
authors.initials: JA
authors.lastname: Gustafsson
authors.forename: Lars-Arne
authors.initials: LA
authors.lastname: Haldosén
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D008321:Mammary Glands, Animal / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis* / Q000502:physiology*; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000502:physiology*
chemical_list: D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C539965:NR0B1 protein, human; C539966:Nr0b1 protein, mouse; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins; D004815:Epidermal Growth Factor