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Endocrinology, doi:10.1210/en.2005-1651
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Endocrinology Vol. 147, No. 7 3249-3259
Copyright © 2006 by The Endocrine Society

DAX-1 Expression Is Regulated during Mammary Epithelial Cell Differentiation

Luisa A. Helguero, Malin Hedengran Faulds, Carola Förster, Jan-Åke Gustafsson and Lars-Arne Haldosén

Department of Biosciences and Nutrition, Karolinska Institutet NOVUM, SE-141 86 Huddinge, Sweden

Address all correspondence and requests for reprints to: Dr. Lars-Arne Haldosén, Department of Biosciences and Nutrition, Karolinska Institutet NOVUM, SE-141 86 Huddinge, Sweden. E-mail: lars-arne. haldosen{at}biosci.ki.se.

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} (ER{alpha}) and ß (ERß) 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 17ß-estradiol, the ER{alpha}-selective agonist 4,4',4'-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol, or the ERß-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.




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