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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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DAX-1 (DOSAGE-SENSITIVE SEX reversal/adrenal hypoplasia congenita critical region on the X chromosome; NROB1) is an atypical member of the nuclear receptor family. It shares structural homologies with the classical nuclear receptors, such as a putative ligand binding domain localized in the C-terminal region, but it lacks the zinc-finger type DNA binding domain and cannot directly bind to promoter response elements of target genes. Throughout DAX-1 protein structure, three LXXLL motifs (NR boxes) can be identified, which mediate direct protein-protein interactions with the C-terminal ligand binding domain/AF-2 domain of nuclear receptors (1), such as estrogen receptors (ERs) (2).

Since 1994, when DAX-1 was first described and mapped to the chromosome region Xp21 (3), research on DAX-1 has focused on its role as an anti-steroidogenic factor whose physiological functions were established as a transcriptional corepressor of steroidogenic factor 1 (SF-1) in the induction of steroidogenic genes, such as cytochrome P450 steroid hydroxylases, steroidogenic acute regulatory protein, and aromatase (Cyp19) (4, 5, 6). DAX-1 is expressed in tissues directly involved in steroid hormone production and reproductive function, e.g. adrenal cortex, Leydig, and Sertoli cells in the testis and theca, and granulosa cells in the ovary (7). However, DAX-1 functions extend beyond regulation of SF-1-dependent genes as it also inhibits ligand-dependent transactivation by agonist-bound nuclear receptors like ER{alpha} and ERß (2), androgen receptor (AR) (8, 9), and progesterone receptor (PR) (9).

Evidence supports the role of DAX-1 as a differentiating factor. During development of the human adrenal gland, DAX-1 represses steroidogenic gene expression of definitive zone cells, allowing their proliferation and differentiation into glomerulosa, fasciculate, and reticularis (7). DAX-1 is also an important factor in sex determination, shifting the balance to the default female differentiation program (7). Recently the Wnt-4/ß-catenin pathway was found to positively regulate DAX-1 expression in the female developing gonad (10) and in testicular Sertoli and Leydig cells (11). Wnt-4 also plays an essential role in mouse mammary gland side-branching through early pregnancy, mediating progesterone effects during morphogenesis that determine cell fate and promote cell survival and proliferation at several stages during mammary gland development (12).

HC11 mouse mammary epithelial cell line is a well-established model to study cell differentiation. It was cloned from COMMA-1D cell line that was obtained from mammary glands of BALB/c mice in mid-pregnancy (13). HC11 cells can be differentiated in vitro by manipulating the growth factor and hormone conditions. In this model, epidermal growth factor (EGF) affects laminin organization and assembly, which is necessary for lactogenic hormone responsiveness (14). When HC11 cells are grown in medium with high serum concentrations and EGF, laminin is detected in intracellular granules. If confluent cells are exposed to low serum medium in the absence of EGF, laminin is deposited in fibrils in the extracellular space. Grown under these conditions, the cells become competent to respond to the lactogenic hormones dexamethasone, insulin, and prolactin (DIP-treatment), which in turn induce the expression of ß-casein.

In a recent study, we found that when HC11 cells enter the differentiation program, i.e. in the absence of EGF, 17ß-estradiol (E2)-induced transcription of an estrogen response element (ERE)-reporter gene was repressed. We also found that, in these cells, loss of ERE-reporter activity correlated with the up-regulation of DAX-1 nuclear levels (15). To our knowledge, this was the first report on DAX-1 expression in mammary epithelial cells. In the present study, we have further investigated DAX-1 regulation by EGF and also analyzed its subcellular localization as HC11 cells progressed from a proliferating to differentiated state. Furthermore, we have also studied DAX-1 expression in mouse mammary glands from different differentiation states and found it highly expressed in pseudopregnant mammary glands.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents, hormones, and antibodies
RPMI 1640 medium without phenol-red, L- glutamine, trypsin-EDTA, and gentamycin were purchased from Gibco, Invitrogen Corp. (Paisley, Scotland, UK). EGF, insulin, ovine prolactin, dexamethasone, E2, fetuin, PD098059, DAPI, and 3,3'-diaminobenzidine tetrahydrochloride were obtained from Sigma (St. Louis, MO). Human transferrin was from Roche Diagnostics (Mannheim, Germany). Fetal bovine serum (FBS) was from Integro (Dieren, Holland). 4,4',4'-(4-Propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT) was kindly provided by KaroBio (Huddinge, Sweden) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) was purchased from Tocris (Bristol, UK). The hormones were diluted in absolute ethanol to a stock concentration of 10 µM. The primary antibodies used to detect DAX-1 are described in Table 1Go and were: rabbit polyclonal antihuman DAX-1 (K17; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antihuman DAX-1 (H-300; Santa Cruz Biotechnology), mouse monoclonal antihuman DAX-1 (2ZH7431H; R&D Systems, Tokyo, Japan), mouse polyclonal antimouse DAX-1 (A0179; produced upon demand by The University of Texas, Southwestern Medical Center, Dallas, TX) and mouse monoclonal antihuman DAX-1 (2F4; a kind gift from Dr. E. Lalli). Total and activated MAPK/ERK were detected with anti-p44/p42 MAPK and anti-phospho-p44/p42 MAPK (Thr202/Tyr204) antibodies, respectively (9102 and 9101S; Cell Signaling Technology, Beverly, MA). ER{alpha} was detected with a rabbit polyclonal antimouse ER{alpha} antibody (MC-20; Santa Cruz Biotechnology), the specificity of which has been described earlier (16).


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  		TABLE 1. Antibodies tested

 
Cell culture
HC11 mammary epithelial cells were routinely grown in complete medium (RPMI 1640, 10% FBS, L- glutamine, 5 µg/ml insulin, 10 ng/ml EGF, and 50 µg/ml gentamicin), and proliferating and confluent cells were obtained under these growth conditions. When cells reached confluence, the medium was changed to –EGF medium (RPMI 1640, 2% FBS, 5 µg/ml insulin, and 50 µg/ml gentamicin) and competent cells were obtained after 48 h of growth in this medium. To induce differentiation of competent cells, they were treated for 72 h with –EGF medium containing 100 nM dexamethasone and 1 µg/ml ovine prolactin (DIP-treatment).

Nuclear and cytosolic extracts
Cells were grown in 10-cm plates and treated as described in Cell culture, washed with cold PBS, scraped into 1.5 ml tubes, and spun down. Pellets were dissolved in lysis buffer [10 mM Tris (pH 7.4), 10 mM NaCl, and 6 mM MgCl2] and kept on ice for 5 min. After a second centrifugation, pellets were resuspended in buffer lysis buffer containing 1 mM DTT, 0.4 mM PMSF, and 1 mM Na3VO4 and homogenized using a glass homogenizer. After centrifugation, supernatants were collected and later concentrated in Microcon centrifuge columns (Amicon, Bedford, MA). This concentrate was used as cytosolic fraction. The nuclear pellets were resuspended in three volumes of buffer C [20% glycerol, 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.4 mM PMSF, and 1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 0.2 U/ml aprotinin]. After 20 min on ice, the samples were centrifuged at 14,000 rpm for 10 min at 4 C, and the supernatants obtained were used as nuclear extracts. Protein concentrations were determined with Bradford reagent (Bio-Rad, Hercules, CA).

Whole-cell extracts
HC11 cells were grown in 10-cm plates and treated as described in Cell culture. Cells were washed with cold PBS, collected in a 1.5-ml tube, and pelleted by centrifugation at 4 C for 2 min. Supernatants were discarded and pellets were frozen in liquid nitrogen. Pellets were thawed and resuspended in lysis buffer [400 mM NaCl, 10 mM HEPES (pH 7.4), 1.5 mM MgCl2, 0.1 mM EGTA, 5% (vol/vol) glycerol, 1.5 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 1 mM sodium molybdate, and 1 mM sodium orthovanadate]. The lysate was kept on ice for 20 min and centrifuged at 14,000 rpm for 10 min at 4 C. Protein concentration was determined with Bradford reagent (Bio-Rad).

Western blotting
SDS-solubilizing buffer was added to 50 µg protein of nuclear or 75 µg of cytosolic protein of whole-cell extracts, and the samples were boiled for 3 min. Proteins were separated on a 10% SDS-polyacrylamide gel and transferred onto a PVDF membrane by semidry blotting. The membranes were blocked for 1 h in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl (TBS), containing 5% (wt/vol) milk protein. After washing twice with TBS for 5 min, the membranes were incubated overnight with the primary antibodies in TBS plus 0.5% Tween 20 (TTBS) [2ZH7431H or A0179 antibodies, 1/500 (vol/vol)] or in TTBS-2.5% (wt/vol) milk protein [K-17 antibody, 1/1000 (vol/vol)]. Anti-total or phospho p44/p42 MAPK antibody was used diluted 1/500 (vol/vol) in TTBS-2.5% (wt/vol) milk protein. Membranes were then washed twice with TTBS for 10 min, after which the corresponding secondary antibodies coupled to horseradish peroxidase (Sigma, diluted 1:5000 in TTBS) were added. Immunoreactivity was detected with the enhanced chemiluminescence kit (ECL plus; Amersham Pharmacia Biotech, Buckinghamshire, UK). As control for equal protein loading and equal gel to membrane transfer, the membranes were stained with Ponceau S (Sigma) and blots were stained with antimouse ß-actin antibody (GTX 28227; GeneTex, San Antonio, TX). The experiments were repeated at least four times. The densitometry of the bands was done using Quantity one software (Bio-Rad). Each sample was normalized to ß-actin content and, thereafter, values were related to those obtained for proliferating cells, which were arbitrarily set to one. Differences between stages of differentiation and proliferating cells were calculated with one-way ANOVA and Dunnet’s post test.

RNA extraction and quantitative real-time PCR (qPCR)
Total RNA was extracted and purified using RNAeasy kit (Qiagen, Hilden, Germany). Briefly, 1 x 106 cells were disrupted using guanidine isothiocyanate and homogenized with a syringe and needle. RNA was purified using a silica-gel membrane included in the kit. Isolation and amplification of DAX-1 and receptor-interacting protein 140 kDa (RIP-140) cDNA was carried out in two steps. First, cDNA was synthesized from 1 µg total RNA using Superscript II reverse transcriptase and random primers (Invitrogen Life Technologies). Later, 11 ng of total cDNA was amplified in an ABI 7500 thermocycler (Applied Biosystems, Foster City, CA) using TaqMan Universal Mastermix following the manufacturer’s protocol (Applied Biosystems). The primer pairs and probe for mouse DAX-1 were designed with Primer Express software (Applied Biosystems) taking base pairs –25 to +240 of the mouse DAX-1 gene as the template. The probe was designed using 6-carboxyfluorescein and TaqMan MGB probe as a quencher. Primers were: sense 5'-CTACTGATGAGCGCGAAGCA-3', and antisense, 5'-AAGCGCACCTCTCGCTCTT-3'. The sequence of the probe was 5'-AGCACGCGTCTCAG-3'. Human DAX-1 (hDAX-1) and mouse RIP-140 cDNAs were amplified using TaqMan gene expression assays (Applied Biosystems, Warrington, UK). The variation in amplification efficiency was assessed using GAPDH gene as an internal control (TaqMan gene expression assays; Applied Biosystems). The quantification method used was from a standard curve. In the case of mouse DAX-1, three independent experiments were carried out in duplicate and after normalizing to the internal control, the values from proliferating stage were used as the calibrator. Differences between different stages of differentiation and proliferating cells were calculated with one-way ANOVA and Dunnet’s posttest. In the case of RIP-140 two independent experiments were carried out in triplicates.

Laser confocal fluorescence microscopy
HC11 cells were cultured on sterile 18- x 18-mm glass coverslides. DAX-1 subcellular localization was evaluated throughout differentiation of HC11 cells and at different time points after EGF withdrawal. Each cellular stage (proliferating, confluent, competent, or differentiated) was obtained by manipulating the growth conditions as described in Cell culture. To study effects after EGF withdrawal, cells were grown in complete medium until confluent and then exposed to –EGF medium for 2, 8, 18, 24, or 48 h. After rinsing twice in PBS, cells were fixed in 10% buffered formalin and washed again with PBS. Cells were permeabilized in 0.5% Triton-PBS for 30 min, unspecific binding was blocked by incubation in blocking solution for 1 h (10% FBS in 0.1% Tween-PBS). Incubation with the primary antibodies proceeded overnight at room temperature. Antibodies were diluted 1/200 in 0.1% Tween-PBS. After three washes with PBS, the corresponding secondary antibodies coupled to Cy3, tetramethylrhodamine isothiocyanate, or fluorescein isothiocyanate (Sigma) were added (1/250 in blocking solution) for 1 h. Cells then were washed four times with PBS and nuclei were stained with DAPI (Sigma; 1/10,000 vol/vol).

Immunohistochemistry
Mammary glands from 3-month-old virgin, pseudo-pregnant and 6-d lactation mice were fixed in 4% paraformaldehyde/PBS overnight and paraffin embedded. Paraffin sections (4 µm) were dewaxed in xylene and rehydrated through decreasing ethanol to water. Endogenous peroxidase was blocked by incubation for 30 min with a solution of 1% hydrogen peroxide, and antigen retrieval was performed by microwaving the sections in 10 mM citrate buffer (pH 6.0) for 20 min at 800 W. Tissue sections were incubated for 1 h at 4 C with 10% goat serum diluted in PBS. DAX-1 antibodies were diluted 1/100 in PBS containing 3% BSA. Sections were incubated with antibodies overnight at 4 C. The primary antibody was omitted as a negative control. Sections were rinsed in PBS before addition of the secondary antibody, and the avidin-biotin complex method was used to visualize the signal (Vector Labs, Burlingame, CA). Briefly, sections were incubated with the corresponding biotinylated secondary antibody (1/200 in PBS) for 2 h at room temperature, rinsed in PBS, and incubated with avidin-horseradish peroxidase for 1 h. After thorough washing in PBS, sections were developed with 3,3'-diaminobenzidine tetrahydrochloride (DakoCytomation, Glostrup, Denmark) and slightly counterstained with methyl green (Sigma). Sections were dehydrated through an ethanol series, followed by xylene and mounted in Pertex (Histolab, Gothenburg, Sweden).

Transient transfections
HC11 cells were seeded at a density of 5 x 105 cells/ml and grown in complete medium. At 60% confluence, transfections were carried out using FuGene reagent (2 µl/1 µg Fugene/pSG5-DAX-1 ratio; Roche Diagnostics) following the manufacturer’s suggestions.

Luciferase gene reporter assays
Cells were seeded in 24-well plates 24 h before transfection. One hundred nanograms of reporter plasmid 3xERE-TATA-Luc together with 20 ng human-pSG5-DAX-1 were used. Ten nanograms of plasmid expressing placenta alkaline phosphatase was included as a control for transfection efficiency. The transfection medium was changed after 24 h to complete medium with or without 1 or 10 nM E2, the ER{alpha}-selective agonist PPT, or the ERß-selective agonist DPN. After 24 h, cells were lysed in buffer containing 25 mM Tris-HCl, pH 7.8, 1 mM EDTA, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, and 2 mM dithiothreitol. Luciferase activity was measured with the LucScreen system (Tropix; PerkinElmer, Wellesley, MA) using a ß-max apparatus (Wallac, Gaithersburg, MD). The results are presented as mean of fold induction ± SD of three experiments performed in triplicate.

Cell counting
HC11 cells were seeded in 24-well plates, and 24 h later, 900 ng of pSG5-hDAX-1 were transfected. After 24 h, cells were rinsed in PBS, then serum-free medium (SFM: RPMI 1640, L-glutamine, 5 µg/ml insulin, 2 mg/ml fetuin, 10 µg/ml transferrin, and 50 µg/ml gentamicin) with or without 10 nM PPT or DPN was added. Forty-eight hours later, cells were detached with trypsin-EDTA and counted in a Bürker chamber. Cell number index was calculated compared to the untreated control arbitrarily set to 1. The results are presented as mean ± SE of three independent experiments carried out in quadruplicate. Statistical significance between transfected and untransfected groups was evaluated with Student’s t test.

Animals
Female C57BL/6 female mice were fed ad libitum and kept under a 12-h light, 12-h dark cycle. Ovaries and mammary glands from 3-month-old virgin, pseudo-pregnant, and 6-d lactation mice were used. Pseudo-pregnancy was induced by treatment of the animals with one ip injection of 10 U pregnant mare serum gonadotropin (PMSG; Sigma) followed by one dose of human chorionic gonadotropin (hCG) in saline, 3 d before excision of the glands. All animal experimentation was conducted in accordance with accepted standards of humane animal care as outlined by The Stockholm South Ethical Committee of the Swedish National Board for Laboratory Animals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DAX-1 is regulated through mammary epithelial cell differentiation
The specificity of four commercial anti-DAX-1 antibodies (Table 1Go) was tested by Western blotting and immunocytochemistry. Mouse ovary was used as positive control while T47-D (human breast cancer) and HeLa (human cervix adenocarcinoma) cells, which express no or very low levels of DAX-1 mRNA (Dr. E. Treuter, personal communication) were used as negative controls. As shown in Fig. 1AGo, K-17, 2ZH7431H (from now on referred to as 2ZH7), and A0179 antibodies detected a 50-kDa band corresponding to the predicted DAX-1 molecular mass in the positive ovary control. This band was not observed in HeLa or T47-D cells. H-300 antibody detected several bands but none was 50 kDa, thus, this antibody was not used in our experiments (data not shown). A band of a slightly higher molecular mass than 50 kDa in ovarian extracts was also observed. Besides DAX-1, K-17 antibody detected, with high affinity, a band approximating 65 kDa that seems to be unspecific.


Figure 1
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  		FIG. 1. Specificity of DAX-1 antibodies. A, Seventy-five micrograms of protein from whole-cell extracts were analyzed by Western blotting using K-17, 2ZH7, and A0179 antibodies. Mouse ovary was used as positive control. HeLa and T47-D cell lines were used as negative controls. B, DAX-1 expression throughout differentiation of HC11 mouse mammary epithelial cells. Seventy-five micrograms of whole-cell extracts (left panel) or 50 µg of nuclear extracts (right panel) were analyzed by Western blot using the indicated antibodies. ß-Actin was used as control for loading efficiency. The figures are representative of at least four independent experiments. C, Densitometric analysis of results obtained with 2ZH7 antibody. Filled columns show results obtained with whole-cell extracts and empty bars show results from nuclear extracts. After normalization of band intensity to ß-actin content, relative intensity units were calculated setting the values of proliferating stage to 1. Mean ± SD is shown. *, P < 0.05; **, P > 0.01. D, qPCR analysis of DAX-1 RNA. The bar graph shows pooled results from four independent experiments carried out in duplicates. After normalization with GAPDH gene, relative units were calculated, arbitrarily setting proliferation values to 1.

 
DAX-1 expression was studied in HC11 cells throughout differentiation. Whole-cell extracts were evaluated using K-17 and 2ZH7 antibodies. DAX-1 increased as cells entered the differentiation program, that is in competent (–EGF) and differentiated (–EGF, DIP-treated) cells compared with proliferating and confluent cells (+EGF) (Fig. 1BGo, left panel). As shown previously (15), the same regulation was observed in nuclear extracts (right panel). A quantitative analysis of DAX-1 intensity is shown in Fig. 1CGo where it is evident that when cells acquired the capacity to proceed through differentiation, DAX-1 levels increased (P < 0.01). To further study whether the higher levels of nuclear and total DAX-1 were due to a regulation of the protein stability or to increased mRNA levels, qPCR was carried out. The results indicate that DAX-1 mRNA levels increased about 4-fold as cells became competent, and although partially decreased, remained higher in fully differentiated cells compared with those grown in EGF-containing medium (Fig. 1DGo). This implies that, throughout HC11 cell differentiation, total cellular content of DAX-1 protein increased due to higher levels of mRNA, and that DAX-1 was recruited to the nucleus.

Next, DAX-1 subcellular localization was studied using laser confocal fluorescence microscopy. The specificity of the antibodies was controlled using HeLa and T47-D cell lines as negative controls. Figure 2AGo shows merged images where nuclei were stained with DAPI (blue), and the antibody signal is shown in red (K-17) or green (2ZH7, A0179). In both cell lines stained with K-17, nuclear (magenta) and cytosolic signal (only in HeLa cells, red) were observed. In contrast, 2ZH7 did not stain HeLa cells and showed very low reactivity localized in the nucleus of few T47-D cells (light blue). With A0179 antibody, both cell lines showed weak staining in the cytoplasm. Another antibody, 2F4, was a kind gift from Dr. E. Lalli and has been used in immunohistochemistry, immunofluorescence, and Western blotting studies (Table 1Go) (17, 18). It was reported to give low cytosolic background staining (18). In accordance with this, we observed a similar staining pattern as that shown with A0179 (data not shown). Given the fact that, by Western blotting, DAX-1 was not detected by any of the antibodies in HeLa or T47-D cells, we conclude that, in immunofluorescence studies, K-17 antibody might be cross-reacting with other nuclear epitopes and, therefore, we excluded it from our study.


Figure 2
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  		FIG. 2. A, DAX-1 cellular localization throughout HC11 mammary epithelial cell differentiation. DAX-1 detected by immunofluorescence in HeLa and T47-D cells. The antibodies used were rabbit polyclonal K-17 and antirabbit IgG coupled to tetramethylrhodamine isothiocyanate (red); mouse monoclonal 2ZH7 or mouse polyclonal A0179 and antimouse IgG coupled to fluorescein isothiocyanate. Nuclei were stained with DAPI (blue). Merged images are shown. Magnification is x40. The figure is representative of two independent experiments. B, HC11 cells were seeded on glass microscopy slides and grown in medium + EGF (proliferating), EGF (competent), or –EGF + DIP (differentiated). After fixation, immunocytochemistry was carried out using 2ZH7 or 2F4 antibodies (green). Arrowheads indicate nuclear staining; the arrows indicate cytoplasmic staining on the sides where cells are in close contact with each other. Nuclei were stained with DAPI (shown to the right of each panel in gray). Magnification is x63.

 
DAX-1 subcellular localization was studied in HC11 cells entering the differentiation program using 2ZH7 and 2F4 antibodies (Fig. 2BGo). The results obtained with A0179 were similar to those obtained with 2F4 antibody (data not shown). In proliferating and confluent cells (+EGF, data not shown), cells were stained with low intensity. In this case, the signal was located in the nucleus (arrowhead). In competent cells (–EGF), the overall staining was higher and the fluorescent signal was variable between the cells. In the case of 2F4 antibody, the staining pattern was mainly nuclear with some signal in the cytosol (arrow). The staining pattern observed with 2ZH7 antibody in competent cells was nuclear. In differentiated cells (–EGF, +DIP) the signal in the cytoplasm increased. However, the picture observed with 2ZH7 compared with 2F4 was somewhat different because, with 2ZH7, the signal was redistributed throughout the cell, whereas with 2F4, it was mainly detected in the cytoplasm in areas of cell-cell interaction (white arrow). Because DAX-1 is known to shuttle from nucleus to cytoplasm and 2ZH7 antibody detects an epitope in the N-terminal region while both 2F4 and A0179 detect an epitope in the middle of the primary structure of the protein, we speculate that when HC11 cells are treated with the lactogenic stimuli (DIP) and become differentiated, DAX-1 may interact with other nuclear protein/proteins, which mask the epitope detected by 2F4 and A0179 in the nucleus. As shown by Western blot, the increase in cytoplasmic intensity of DAX-1 is also due to an increase in the content of DAX-1 protein levels in this fraction (Fig. 3Go).


Figure 3
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  		FIG. 3. DAX-1 cytosolic levels throughout differentiation of HC11 mouse mammary epithelial cells. Fifty micrograms of cytosolic extracts were analyzed by Western blot using 2ZH7 antibody. ß-Actin was used as control for loading efficiency. The figure is representative of two experiments.

 
Negative regulation of DAX-1 expression: nuclear levels of DAX-1 are suppressed by inhibition of p44/p42 MAPK activation
DAX-1 expression and nuclear localization after EGF withdrawal were studied in more detail throughout a period from 0.5–48 h after EGF withdrawal. RNA was extracted from cells grown with or without EGF and DAX-1 mRNA was analyzed by qPCR. After 0.5 h, DAX-1 RNA increased and remained high until 48 h (Fig. 4AGo). Nuclear DAX-1 levels were analyzed by Western blotting (Fig. 4BGo). DAX-1 increased from 2 h onward. Under these conditions, total p44/p42 MAPK levels remained constant while activated p44/p42 MAPK decreased. The possible role of MAPK pathway in mediating the negative effect of EGF on DAX-1 nuclear levels was studied using the specific MEK inhibitor PD 098059 (19). HC11 cells grown with EGF (+EGF) were treated with 1 or 10 µM PD 098059 for 24 h or with 50 µM PD 098059 for 30 min or 2, 8, and 24 h. Nuclear DAX-1 increased in a dose- and time-dependent manner (Fig. 4Go, left and right panels, respectively). Under these conditions, total p44/p42 MAPK levels remained constant while activated p44/p42 MAPK decreased. These results indicate that if activation of p44/p42 MAPK is inhibited, DAX-1 nuclear levels increase. At late time points, p44/p42 MAPK levels returned to initial values. We speculate that this effect could be due to decreased levels of MAPK phosphatases influenced by serum factors.


Figure 4
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  		FIG. 4. DAX-1 levels are negatively regulated by EGF. A, qPCR of DAX-1 RNA. Cells were grown in the presence of EGF (0 h), then the medium was changed to medium without the growth factor, and cells were incubated for the indicated time points until 48 h when they are competent. The bar graph shows pooled results from three independent experiments carried out in duplicates. After normalization with GAPDH gene, relative units were calculated, arbitrarily setting proliferation values to 1. Differences between the groups and proliferating cells were calculated using one-way ANOVA and Dunnet’s post test. **, P < 0.05. B, Fifty micrograms of nuclear extracts were analyzed by Western blotting using 2ZH7 antibody. Cells were grown in medium containing EGF (0 h), then the medium was changed to medium without the growth factor and cells were incubated for the indicated time points until 48 h when they are competent. The same extracts were blotted with anti-phospho p44/p42 MAPK and total p44/p42 MAPK. C, Cells grown in EGF-containing medium (+EGF) were treated with 1 or 10 µM of the MEK inhibitor PD 098059 for 24 h, or with 50 µM of PD 098059 for the indicated time points. Competent and differentiated cells were used as positive controls. DAX-1 was detected with K-17 antibody. Note the unspecific band approximating 60 kDa showing that lanes were loaded equally. To control that MEK was efficiently blocked; extracts were blotted with anti-phospho p44/p42 MAPK and total p44/p42 MAPK. The figures are representative of two independent experiments.

 
Subcellular localization of DAX-1 after EGF withdrawal for the same time was analyzed by confocal microscopy of cells stained with 2F4 antibody. Most of the cells were stained in the nuclei, although fluorescence was also detected in the cytoplasm after 8 h of EGF depletion (data not shown).

DAX-1 is differentially expressed through differentiation of mouse mammary gland
Ovary sections were used as positive control for 2ZH7, A0179, K17, and 2F4 antibodies. With 2ZH7, A0179, and 2F4, few granulosa cells and all stromal cells were stained in the cytoplasm. On the other hand, K-17 antibody stained both the nucleus of granulosa cells (arrow) and cytoplasm of stromal cells (arrowhead; supplemental Fig. 1A). Given the fact that K-17 antibody detects a nuclear epitope in DAX-1-negative T47-D and HeLa cells, we have excluded it from our analysis. However, because it is the only commercial antibody used in immunohistochemistry, our results with K-17 in the mouse mammary gland are included as a supplemental figure (supplemental Fig. 1B).

Regulation of DAX-1 expression in 3-month-old virgin, PMSG/hCG-treated (pseudopregnant) and 6-d lactating mammary glands was evaluated by immunohistochemistry (Fig. 5Go). A qualitative analysis shows an increase of DAX-1-positive cells in PMSG/hCG-treated compared with virgin tissues stained with 2ZH7, A0179, and 2F4 antibodies (the last one not shown). In mammary glands from virgin mice, the signal was localized to the nucleus of some luminal cells, whereas, in PMSG/hCG-treated glands, the signal was stronger and detected in the nucleus and cytoplasm of luminal cells comprising almost the entire duct. In mammary glands from 6-d lactating mice, DAX-1 was detected only in the cytoplasm of both luminal and lobular epithelium (marked D and AL, respectively). These results indicate that DAX-1 localization is under the influence of the hormonal regulation that occurs during pregnancy and lactation.


Figure 5
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  		FIG. 5. DAX-1 expression is regulated in mouse mammary gland. Paraffin-embedded tissues were processed for immunohistochemistry as described in Materials and Methods. Three-month-old virgin, PMSG/hCG-treated (pseudopregnant) and 6-d lactating mammary glands were stained with 2ZH7 and A0179. Background staining due to unspecificity of the secondary antibodies is shown in F (antimouse IgG) and C (antirabbit IgG). In lactating mammary glands, D indicates the position of ducts and AL indicates the localization of alveoli. Nuclei were counterstained with methyl green. The bar indicates 10 µm, magnification is x40.

 
Given the fact that, in lactating mammary gland, DAX-1 was found in the cytoplasm and, taking into consideration the repressor role of DAX-1 previously reported with respect to ERs (2), AR (8), and PR (9), we hypothesized a repressor role for DAX-1 on the activity of ERs, which could explain the estrogen insensitivity of the mammary gland during late pregnancy and lactation (20).

DAX-1 inhibits ER{alpha} and ERß activation of ERE promoter
We have shown earlier, in HC11 mammary epithelial cells, which endogenously express ER{alpha} and ERß (15, 16), that EGF is necessary for E2-stimulated ERE-promoter activity (15). Because DAX-1 levels increased upon EGF withdrawal, we hypothesized that this orphan receptor could have an inhibitory role on the transcriptional activity of ER. Thus, HC11 cells grown in the presence of EGF were transiently cotransfected with hDAX-1 and 3xERE-TATA-Luc reporter gene. Luciferase activity was measured 24 h later. Overexpression of hDAX-1 abrogated the transcriptional activity induced by E2 on endogenously expressed ERs (Fig. 6AGo, compare columns 2 and 3 vs. 4 and 5). Also, hDAX-1 inhibited reporter activity induced by the ER{alpha}-selective ligand PPT (columns 6 and 7 vs. 8 and 9) and the ERß-selective ligand DPN (columns 10 and 11 vs. 12 and 13) to the same extent as for E2, indicating that this negative regulation is exerted on both endogenously expressed ERs.


Figure 6
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  		FIG. 6. Repression of endogenous ER transcriptional activity at an ERE promoter by hDAX-1. A, HC11 cells growing in EGF-containing medium were transiently cotransfected with pSG5-hDAX1 and 3xERE-TAT-Luc reporter gene; 24 h later, cells were treated with 1 or 10 nM E2, PPT, or DPN in medium plus EGF. After 24 h, cells were lysed and luciferase activity was measured. Mean ± SD of three independent experiments carried out in triplicates is shown. B, qPCR of the ERE-regulated endogenous gene RIP-140. HC11 cells were transiently transfected with pSG5-hDAX-1 as described in Materials and Methods. Twenty-four hours after transfection, the medium was changed to SFM with or without 10 nM PPT or DPN and incubation proceeded for another 24 h. The experiment is representative of two, each carried out in triplicates. Values were normalized to those of GAPDH; the bar graph shows the mean ± SE. Differences between transfected and untransfected samples in each group were calculated with one-tailed Student’s t test. *, P < 0.05; ***, P < 0.001.

 
To study further whether inhibition of the 3xERE-regulated reporter gene by overexpression of hDAX-1 also occurs on endogenous promoters, gene expression of an E2-regulated gene was measured by qPCR. RIP-140 is a transcriptional corepressor that harbors a perfect ERE consensus site in its promoter (21) and is up-regulated by E2, PPT, and DPN treatment of MCF-7 breast cancer cells (21, 22). After transient transfection of HC11 cells with pSG5-hDAX-1, hDAX-1 mRNA levels were measured by qPCR and found to be increased up to 4000-fold (data not shown). RIP-140 gene expression was induced by 30 and 12% by PPT and DPN, respectively (Fig. 6BGo). On the other hand, when hDAX-1 was overexpressed, RIP-140 gene transcription was significantly reduced by 34% in PPT or DPN-treated cells (P < 0.05). These results indicate that DAX-1 functions as a corepressor of ERs on endogenous ERE-regulated promoters. It is worth mentioning that, in cells grown in the absence of any ligand (SFM), hDAX-1 had a positive effect on RIP-140 expression. Whether this effect was due to a competition by DAX-1, of inhibitory cofactors sitting on RIP-140 promoter under basal conditions, remains to be studied.

Effects of ER{alpha}- and ERß-selective ligands on HC11 cell number are reversed by DAX-1 overexpression
Recently, we reported that ER{alpha} and ERß have opposing activities on HC11 cell growth (16). Upon PPT stimulation, HC11 cells proliferate while DPN inhibits cell proliferation and stimulates apoptosis. This results in either increased or decreased cell number, respectively. To study how overexpression of DAX-1 would affect cell number, we used a similar experimental setting as shown in Fig. 6Go in which we transiently transfected with pSG5-hDAX-1 and treated the cells with PPT or DPN for 48 h. In this case, DAX-1 overexpression reversed the growth stimulatory effect of PPT and the growth inhibitory effect of DPN (Fig. 7Go). Similar results were observed in the breast cancer cell line T47-D where DAX-1 expression had no significant effect in untreated cells but inhibited PPT stimulation by 37% (data not shown). Taken together, these results indicate that DAX-1 represses ER{alpha} and ERß activity, thus influencing ER signaling on cell growth. In the absence of ligand (SFM), a slight inhibitory effect was observed in DAX-1-transfected cells. Because under these conditions, we also observed an effect on the RIP-140 promoter we speculate that this biological response could be the result of competition of other cofactors by DAX-1, which would lead to changes in gene transcription patterns, thus influencing proliferation or apoptosis.


Figure 7
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  		FIG. 7. DAX-1 over-expression modulates the effect of PPT and DPN on HC11 cell number. HC11 cells were transiently transfected with pSG5-hDAX-1 and grown in SFM alone or with 10 nM PPT or DPN. After 48 h incubation, cells were counted in a Bürker Chamber. Mean ± SE from three independent experiments carried out in quadruplicates is shown. Results were analyzed with one-tailed Student’s t test. a, P < 0.001; b, P < 0.05 vs. untreated SFM. *, P = 0.051; **, P < 0.05 vs. not transfected DPN or PPT-treated cells, respectively.

 
Endogenously expressed DAX-1 and ER{alpha} colocalize in competent cells
Confocal laser scanning microscopy was used to investigate whether endogenous DAX-1 and ER{alpha} colocalize in HC11 cells (Fig. 8Go). We used the antibody 2ZH7 to detect DAX-1 and MC-20 to detect ER{alpha}. In competent cells (–EGF), DAX-1 (green) was predominantly detected in the nucleus, while, as reported earlier (16), ER{alpha} (red) was also detected in the cytoplasm (arrow). Both proteins colocalized in the cytoplasm (yellow). After 2 h of treatment with 10 nM E2, an increase in nuclear staining of both DAX-1 and ER{alpha} was observed at sites of colocalization in some cells (yellow, arrowhead). The ER{alpha}-selective ligand PPT had a more pronounced effect inducing even more nuclear colocalization. These results are in agreement with those of Holter et al. (8), who showed that in Cos-7 cells transiently transfected with hDAX-1 and AR, both proteins colocalized mainly in the cytoplasm, but they shuttled from cytoplasm to nucleus upon addition of 10 nM dihydrotestosterone.


Figure 8
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  		FIG. 8. DAX-1 and ER{alpha} colocalize in competent HC11 cells. HC11 cells were seeded on glass microscopy slides and cultured as described in Materials and Methods to achieve the competent stage. They were treated with 10 nM E2 or PPT in SFM for 2 h. After fixation, cells were stained with mouse monoclonal anti-DAX-1 antibody (2ZH7, green) and rabbit polyclonal anti-ER{alpha} antibody (MC-20, red). Magnification, x63. This experiment was repeated twice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Orphan nuclear receptor DAX-1 plays key roles in mammalian sex development, reproduction, and steroidogenesis (7). Although studies have focused on its functional activity as repressor of transcription factors such as SF-1, ER, AR, and PR, there are few reports on how DAX-1 expression is regulated. Yu et al. (23) studied the promoter of the murine DAX-1 gene and found that it is positively regulated by SF-1 and inhibited by chicken ovalbumin upstream promoter-transcription factor. A search with MatInspector software on the DAX-1 promoter sequence provided by the authors revealed potential specificity protein 1, activator protein 1, nuclear factor {kappa}B, signal transducer and activator of transcription, and c-Jun binding sites, thus, suggesting that several signaling pathways may regulate DAX-1 expression. In a previous report using mammary epithelial HC11 cells, we found that EGF regulates DAX-1 nuclear protein levels in a negative manner (15). Therefore, the aim of this study was to further investigate and characterize DAX-1 expression throughout differentiation of mouse mammary epithelial HC11 cells and normal mammary gland.

Low DAX-1 levels were observed in HC11 cells proliferating in medium containing EGF. Both Western blotting and qPCR analysis showed that when cells were induced to differentiate in –EGF medium, total DAX-1 levels increased. Furthermore, we showed that EGF, probably through activation of p42/p44 MAPK, lowers the level of DAX-1 mRNA and decreases the levels of DAX-1 protein in the nucleus. Opposite to our findings, are those of Amsterdam and colleagues (24, 25), who described a positive effect of MAPK pathway on DAX-1 as well as SF-1 expression, describing a mechanism that may account in part for the desensitization to gonadotropin action observed in granulosa cells from human and rat primary cultures.

As shown by immunofluorescence, DAX-1 signal increased in the cytoplasm of competent and differentiated cells. The finding that, from 8 h onward of EGF withdrawal, DAX-1 is also found in the cytoplasm might indicate that this protein is regulated at some stage of the differentiation program of HC11 cells. The cytoplasmic pattern we observed in competent and differentiated cells stained with 2F4 antibody is similar to that described by Battista et al. using the same antibody, where in fetal adrenal cells DAX-1 nucleocytoplasmic localization was modulated by the substrate on which these cells were cultured (18). As mentioned in the introductory section, HC11 cells secrete their own extracellular matrix, and the extracellular deposition of laminin induced by EGF withdrawal was shown to be one key factor for the differentiation process of these cells (14). Further studies are needed to investigate whether DAX-1 subcellular localization is modulated by the microenvironment.

DAX-1 is predominantly expressed in adrenal gland, ovary, testes, hypothalamus, and pituitary (7). To our knowledge, there are no reports on DAX-1 expression in normal mammary gland. In this study, we found that DAX-1 staining increased in nucleus and cytoplasm of PMSG/hCG-treated compared with 3-month-old virgin mammary glands. We also observed a different pattern in 6-d lactating glands where DAX-1 was found mostly in the cytoplasm, suggesting that the protein shuttles as cells differentiate (as shown also in HC11 cells). These results could point out DAX-1 as an important factor in mammary gland development and lactation.

Only one report using K-17 antibody shows expression of DAX-1 in human breast tumors (26). The results presented in that study showed a positive correlation between DAX-1 expression, AR and ERs. The authors also found an increase in DAX-1 levels in invasive carcinoma compared with benign breast lesions and carcinoma in situ. Similar observations were reported using the same antibody in human common ovarian carcinoma where a positive correlation was found between DAX-1 immunoreactivity and tumor grade and shortened survival (27). On the other hand, in benign prostatic hyperplasia, DAX-1 expression is strongly reduced compared with normal human prostate suggesting a limiting role of DAX-1 in modulating AR activity in this tissue (9).

In a recent study in HC11 cells, we found that up-regulation of DAX-1 correlated with the loss of activity of ERE-reporter gene (15). In this work, we were able to demonstrate that overexpression of hDAX-1 in HC11 cells was sufficient to block the transcriptional activity of endogenous ERs at an ERE promoter. Furthermore, we were able to demonstrate that upon E2 stimulation both ER{alpha} and DAX-1 colocalize in the nucleus of competent cells. Future studies will provide information on the nature of this inhibition. That is, whether DAX-1 inhibition of ER{alpha} transcriptional activity is mediated by a direct mechanism through protein-protein interaction or indirectly by recruiting other corepressor proteins such as NCoR (28) or RIP-140 (29) and competing for binding with coactivators (30). Both mechanisms have been described in transfected systems.

It has been known for years that, during lactation, the mammary gland is nonresponsive to estrogens, as judged from E2-induced cell proliferation and PR expression (20). The results presented in this study suggest that DAX-1 inhibits ligand-bound ER activity at ERE promoters by probably sequestering ER to specific regions and/or by favoring ER interaction with other transcriptional corepressors.

Interestingly, DAX-1 inhibits ER-induced RIP-140 gene transcription. From transfection experiments, it has been suggested that RIP-140 binds to both SF-1 and DAX-1 and modulates their activities on the steroidogenic acute regulatory promoter (29, 31). If this holds true at physiological levels, increase in one corepressor (DAX-1) could have an effect on ER signaling by influencing expression of a second cofactor (RIP-140, known to be a corepressor of ER{alpha} and ERß activity). In proliferating HC11 cells overexpressing DAX-1, the final outcome was a reversion of PPT and DPN effect on ER{alpha} and ERß modulation of cell number. The results presented in this study indicate that expression of DAX-1 is likely to influence proliferation by reversing ER effect.

In summary, in this report, we describe for the first time, DAX-1 expression in the mouse mammary gland. We found that, in mammary epithelial cells, EGF, through activation of p44/p42 MAPK, regulates DAX-1 in a negative manner. In addition, DAX-1 overexpression inhibited ER{alpha} and ERß activities on ERE-regulated reporter and an endogenous gene. These results correlated with a DAX-1 repressive effect on PPT and DPN in cell growth. We show that, under agonist stimulation, endogenous DAX-1 and ER{alpha} colocalize in the nucleus of competent cells. Further studies are needed to determine whether DAX-1 modulation of ER signaling could participate in the regulation of mammary gland differentiation during pregnancy and lactation. One interesting possibility that deserves further study is whether EGF could play a dual role in maintaining mammary epithelial cells in a proliferative state by activating ER{alpha} in a ligand-independent manner through the AF-1 domain, and by simultaneously inhibiting DAX-1 expression.


   Acknowledgments
 
We are grateful to Dr. E. Treuter for pSG5-DAX-1 plasmid and for sharing his knowledge and antibodies with us. We thank Dr. E. Lalli for 2F4 antibody and Drs. M. Warner and G. Cheng for providing the mouse mammary gland and ovary slides.


   Footnotes
 
This work was supported by the Swedish Cancer Fund, Magnus Bergvalls Foundation, KaroBio AB, and Karolinska Institutet.

Present address for C.F.: Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstrasse 6, D-97070 Würzburg, Germany.

Disclosure: L.A.H., M.H.F., C.F., L.-A.H., and J.-Å.G. have nothing to declare.

First Published Online April 20, 2006

Abbreviations: AR, Androgen receptor; DAX-1, dosage-sensitive sex reversal/adrenal hypoplasia congenita critical region on the X chromosome; DIP, dexamethasone, insulin, and prolactin; DPN, 2,3-bis(4-hydroxyphenyl)-propionitrile; E2, 17ß-estradiol; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; FBS, Fetal bovine serum; hCG, human chorionic gonadotropin; hDAX-1, human DAX-1; PMSG, pregnant mare serum gonadotropin; PPT, 4,4',4'-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol; PR, progesterone receptor; qPCR, quantitative real-time PCR; RIP-140, receptor-interacting protein 140 kDa; SF-1, steroidogenic factor 1; SFM, serum-free medium.

Received December 23, 2005.

Accepted for publication April 10, 2006.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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