Helguero Luisa A, Hedengran Faulds Malin, Förster Carola, Gustafsson Jan-Ake, Haldosén Lars-Arne
Department of Biosciences and Nutrition, Karolinska Institutet NOVUM, SE-141 86 Huddinge, Sweden.
Endocrinology. 2006 Jul;147(7):3249-59. doi: 10.1210/en.2005-1651. Epub 2006 Apr 20.
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.
在最近的研究中，我们发现DAX-1（剂量敏感性性别反转/先天性肾上腺发育不全关键区域，位于X染色体上）在小鼠乳腺上皮细胞系HC11中表达。在本研究中，我们聚焦于整个小鼠乳腺上皮细胞分化过程中DAX-1表达及亚细胞定位的调控，以及其在小鼠乳腺中的激素调节。在含表皮生长因子（EGF）的培养基中生长的增殖性HC11细胞，经蛋白质免疫印迹法和定量实时PCR检测，表达极低水平的DAX-1，而在撤除EGF并诱导分化后，DAX-1表达增加。用PD 098059抑制MAPK通路，即使在有EGF存在的情况下，也会导致DAX-1水平升高。利用共聚焦显微镜，我们发现随着细胞分化，DAX-1的细胞质水平增加。在3月龄处女小鼠的乳腺腔细胞中，DAX-1染色呈核型。在假孕小鼠中观察到核质模式，在产后6天的泌乳小鼠乳腺中发现细胞质模式。用雌激素反应元件-荧光素酶报告基因检测法以及对雌激素受体调控基因受体相互作用蛋白140 kDa进行定量实时PCR，评估了DAX-1对HC11乳腺上皮细胞中内源性表达的雌激素受体α（ERα）和β（ERβ）转录活性的影响。将人DAX-1与HC11细胞共转染，可抑制由17β-雌二醇、ERα选择性激动剂4,4',4'-(4-丙基-(1H)-吡唑-1,3,5-三基)三苯酚或ERβ选择性激动剂2,3-双(4-羟基苯基)-丙腈诱导的雌激素反应元件-报告基因及受体相互作用蛋白140 kDa的表达。总之，撤除EGF诱导分化后DAX-1表达增加，且DAX-1降低了HC11细胞对雌激素的反应。需要进一步研究以确定DAX-1在HC11细胞分化调控中是否也很重要。
