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Molecular Endocrinology 18 (2): 412-421
Copyright © 2004 by The Endocrine Society

Estrogen Receptor Functional Activity Changes during Differentiation of Mammary Epithelial Cells

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

Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden

Address all correspondence and requests for reprints to: Lars-Arne Haldosén, Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden. E-mail: Lars-Arne.Haldosen{at}mednut.ki.se.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mammary gland development involves complex cycles of proliferation, differentiation, and morphogenesis, regulated by hormones including estrogens, prolactin (PRL), and epidermal growth factor (EGF). The mouse mammary epithelial cell line HC11 has been shown to be valuable for investigations of differentiation of mammary gland. In this study, we show that HC11 cells express estrogen receptor (ER){alpha} and ERß proteins at all developmental stages. We have established two different stable HC11 cell lines; H-estrogen response element (ERE) containing an ERE-reporter and H-Bc containing a ß-casein reporter. Transcription of the ERE-reporter was activated only in proliferating cells in the presence of EGF. When the cells entered the differentiation program, in the absence of EGF, estradiol-induced transcription of the ERE reporter was repressed, and similar results were obtained when MAPK signaling was inhibited in proliferating cells. We propose that these findings are related to changes in ER corepressor levels, regulated by EGF. We also report that the ß-casein reporter was activated in terminally differentiated cells and that this induction was effectively repressed by estradiol treatment. Finally, we show a physical interaction between endogenous ER{alpha} and signal transducer and activator of transcription 5 in differentiated HC11 cells. In summary, our results show that ER functional activity changes during differentiation of HC11 cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MAMMARY GLAND DEVELOPMENT represents a complex program of cell proliferation, differentiation, and morphogenesis, regulated by various hormones and growth factors including estrogens, prolactin (PRL), and epidermal growth factor (EGF). With the onset of puberty, estrogens initiate the maturation of the mammary gland, and, together with progesterone, the development of ductal epithelia. Estrogens mediate their action through the estrogen receptors {alpha} and ß (ER{alpha} and ERß), belonging to the nuclear receptor superfamily of transcription factors, which regulate transcription in association with coregulators via binding to DNA enhancer elements located in the promoter regions of target genes. Mice devoid of ER{alpha} proteins show a rudimentary structure of the mammary gland whereas the ERß-/- mice show no difference in morphology compared with the mammary glands of wild-type littermates (1). However, ultrastructural analysis of the lactating gland of ERß -/- mice showed larger alveoli, reduced amount of secretory epithelium, increased interepithelial distance, and reduced extracellular matrix and lamina basalis (2).

PRL is a pituitary peptide hormone of significant importance in the mammary gland where it regulates growth and differentiation of epithelial cells and upholds milk production. Upon PRL-binding, PRL receptor dimerizes, and a receptor-associated tyrosine kinase, Janus activated kinase 2 (JAK2), is activated by transphosphorylation (3, 4). JAK2, in turn, phosphorylates different intracellular signal mediators, among them two members of the transcription factor family of signal transducers and activators of transcription (STAT), STAT5A and STAT5B (5). When activated, via cytokines such as interferons or peptide hormones (PRL and GH), STATs translocate to the nucleus and promote expression of target genes by direct binding to DNA via {gamma}-interferon activated sequences. Two {gamma}-interferon-activated sequences are located in the promoter of the milk protein-encoding ß-casein gene, regulated by STAT5 (6, 7). Mice with targeted disruption of STAT5A or STAT5B show distinctive phenotypes: STAT5A knock-out females cannot lactate due to incomplete terminal differentiation of the secretory epithelial cells in the mammary gland (8), whereas mice lacking STAT5B are capable of lactating, but at an insufficient level to sustain pups (9).

The EGF family of ligands are expressed throughout mammary development, promoting proliferation and/or differentiation (10, 11). These hormones act through binding to transmembrane tyrosine kinase receptors, the ErbB family of receptors, which consists of four members. Ligand binding stimulates receptor dimerization, activation of the receptor tyrosine kinase domain, and tyrosine phosphorylation of the receptor leading to recruitment of Src homology 2 (SH2)-domain-containing proteins to the receptor and their activation by phosphorylation. These events result in activation of several intracellular signaling pathways, among them the MAPK pathway (12). EGF has also been shown to stimulate phosphorylation of ER, resulting in ligand-independent activation (13, 14, 15), and to mediate estrogen induction of end bud formation (16). Furthermore, EGF and estrogen have been shown to synergize in stimulating cell proliferation by up-regulation of EGF receptor (17).

We have previously demonstrated, by using a transient transfection model, a negative influence of ligand-activated ER{alpha} and ERß on STAT5A and STAT5B transcriptional activity as a result of direct protein-protein interaction (18). We wanted to further study the ER/STAT5 interaction in a cell culture model system in which the two forms of ER and STAT5A and B are expressed endogenously. For this purpose we have used the mouse mammary epithelial cell line HC11, which has retained some in vivo-like characteristics and, as we have shown earlier, expresses STAT5A and B (19). HC11 cells are grown to a confluent state in the presence of EGF. After removal of EGF for 48 h and a reduction of serum levels, the cells are considered competent for stimulation with the lactogenic hormones, glucocorticoid, insulin, and PRL (DIP), resulting in milk protein synthesis (20). In the present study, we show that HC11 cells express ER{alpha} and ERß proteins at all the different cellular stages of development, i.e. proliferation, confluence, and terminal differentiation. This protein expression was slightly up-regulated by stimulation with E2 throughout the whole differentiation process. We have further established two different stable HC11 cell lines; H-ERE containing an ERE-reporter and H-Bc containing a ß-casein reporter. The results from these cells revealed that the ERs were able to activate transcription of the ERE-reporter only when the cells were exposed to EGF. In terminally differentiated cells, when EGF was removed from the medium, the ERs were unable to induce the ERE-reporter upon E2 treatment. Blocking EGF-induced MAPK signaling with an inhibitor resulted in a dose-dependent down-regulation of E2-induced ERE-reporter activity. We further propose that this repression of ER transcriptional activity in differentiating HC11 cells is related to increased expression levels of the corepressors dosage-sensitive sex reversal adrenal hypoplasia congenita, critical region on the X chromosome gene (DAX-1) and short heterodimer partner (SHP). Also, inhibition of MAPK signaling in proliferating cells increased the amount of DAX-1 and SHP expressed in the nucleus. Interestingly, in terminally differentiated HC11 cells, i.e. cells treated with lactogenic hormones, transcription of the ß-casein promoter was potently down-regulated by E2, and this down-regulation was inhibited by ER antagonists. Furthermore, coprecipitation assays showed that ER and STAT5 physically interact in terminally differentiated cells.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Expression Levels of ER{alpha} and ERß during Different Developmental Stages in HC11 Cells
We wanted to follow up our findings on ER{alpha} and ERß down-regulation of STAT5-induced transcription using transient transfections in human embryonic kidney 293 cells (18). The mouse mammary epithelial cell line HC11 has been shown to be a valuable tool for studies of different steps in the differentiation process. We have earlier shown endogenous expression of STAT5A and B in this cell line (19). We started by examining the expression levels of ER{alpha} and ERß proteins at the different stages of HC11 cellular development by Western blot analysis of nuclear extracts. Samples were taken at proliferative, confluent, competent, and terminally differentiated stages. The expression levels of both ER{alpha} and ERß remained largely constant during the different cellular stages (Fig. 1Go, A and B). This observation is in keeping with that of Schams et al. (21) showing by Western blot that ER proteins are expressed at all stages of mammary gland development. Interestingly, both of the ER proteins were slightly up-regulated when cells were treated with 10 nM E2, 24 h before harvesting, through the whole differentiation program. ERß expression in terminally differentiated cells, on the other hand, was slightly down-regulated by E2 treatment.



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  		Fig. 1. ER{alpha} and ERß Expression Levels in HC11 Cells during Different Developmental Stages

HC11 cells were cultured, with or without E2-treatment, as described in Materials and Methods, and nuclear extracts were made at proliferative, confluent, competent, and terminally differentiated stages. Proteins were separated by SDS-PAGE, and Western blot analyses were performed with m{alpha}ER{alpha}-(panel A) and B, m{alpha}ERß-(panel B) specific antibodies.

 
ER Transcriptional Activity during Proliferation and Differentiation of HC11 Cells
We wanted to analyze ER transcriptional activity during the different developmental stages of HC11 cells. For this reason, a stable cell line of HC11 with a 3xestrogen response element (ERE)-TATA-Luc reporter (H-ERE) was established. The cells were grown in complete, phenol-red free medium in the presence of EGF. The cells were then treated with increasing concentrations of E2 (0.1, 1, and 10 nM) or 10 nM E2 together with 10 nM ICI 164,384, 24 h before the cells were harvested and luciferase activity was determined. As shown in Fig. 2Go, a dose-dependent increase of the ERE-reporter activity could be seen upon E2 treatment, and this activity was repressed by addition of ICI 164,384. These results show that ERs are transcriptionally active in the proliferative phase of HC11 cells.



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  		Fig. 2. E2 Activates an ERE-Reporter in Proliferating HC11 Cells

H-ERE stable cells were grown in phenol red-free medium in the presence of EGF. The transcriptional responses to increasing concentrations of E2 (0.1, 1, and 10 nM) or 10 nM E2 in combination with 10 nM ICI 164,384 were determined. All experiments were made in triplicate at least three times, and data are presented as mean of fold induction ± SD. Activity of the reporter without E2 treatment was arbitrarily set to 1.

 
To continue the investigations on ER transcriptional activation, H-ERE cells at the predifferentiated stage were analyzed. The cells were grown to confluence as described above, after which EGF was removed from the medium and the serum level was reduced. After 48 h, the cells were treated with 10 nM E2 for an additional 24 h before the cells were harvested. As a control, cells were treated for 24 h with 10 nM E2 in the presence of EGF. When EGF was removed from the medium and the cells were in a predifferentiated state, a significantly reduced E2-stimulated ERE reporter activity was detected (Fig. 3AGo). EGF is known to induce transcriptional activity of ER{alpha} in a ligand-independent way through serine phosphorylation of ER activation function 1 (13, 14). This does not seem to be the case in HC11 cells as ERE-reporter activity was not significantly different in cells exposed or not exposed to EGF (Fig. 3AGo, first and third bars).



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  		Fig. 3. E2-Induced ERE-Reporter Activity Is Significantly Reduced in Predifferentiated Stage and Completely Abolished in Terminally Differentiated Cells

H-ERE stable cells were grown in phenol-red free medium in the presence of EGF. When the cells reached confluent state, EGF was removed from the medium. A, After 48 h incubation, the cells were treated with 10 nM E2 for 24 h and assayed for ERE-reporter activity. B, After a 48-h period, the cells were either treated with 10 nM E2 for 24 h and assayed for reporter activity or treated with 10 ng/ml EGF for 48 h followed by 10 nM E2 treatment for 24 h and then assayed for reporter activity. C, After a 48-h period, the cells were treated for 72 h with 10-7 M dexamethasone and 1 µg/ml ovine PRL, insulin was already present in medium (DIP). Cells were then treated with 0.1, 1, or 10 nM E2 for 24 h and assayed for reporter activity. Cells treated with 10 nM E2 for 24 h in the presence of EGF were included as positive control. All the experiments were made in triplicate at least three times, and data are presented as mean of fold induction ± SD. Activity without E2 treatment was arbitrarily set to 1.

 
We then wanted to investigate whether it was possible for predifferentiated H-ERE cells to regain E2-stimulated reporter activity as seen in proliferating cells. First, EGF was withdrawn from confluent cells for 48 h after which EGF was reintroduced to the medium for additional 48 h. Cells were then treated with E2 for 24 h, or left untreated, and reporter activity was measured. H-ERE cells at proliferating and predifferentiated stages were used as controls. As shown in Fig. 3BGo, addition of EGF for 48 h to predifferentiated cells only slightly increased E2-stimulated reporter activity (compare bars 3 and 4 with bars 5 and 6). Thus, at least with respect to ER transcriptional activity, full reversal of the differentiation process in HC11 cells initiated by removal of EGF is not possible.

Because removal of EGF caused a potent decrease in ligand-activated ERE-reporter activity, we determined whether rendering the cells terminally differentiated would affect ER activity. Confluent H-ERE cells were grown in the absence of EGF for 48 h and then treated with 10-7 M dexamethasone, 1 µg/ml ovine PRL (insulin was already present in the medium) with or without increasing concentrations of E2 (0.1, 1, and 10 nM). After 72 h, the cells were harvested and luciferase activity was measured. Proliferating E2-treated cells in the presence of EGF were included in the experiment as a positive control. In terminally differentiated cells, the ERE-reporter was not responsive to E2 treatment (Fig. 3CGo). EGF is known to be a potent mitogen for mammary epithelial cells (10, 11). If EGF activity is inhibited, E2-induced expression of progesterone receptors and proper development of terminal end buds are blocked (16). In conclusion, our results indicate that when mammary epithelial cells start the differentiation process, in our model when EGF is removed, ERs are transcriptionally inactive and are no longer able to induce ligand-dependent transcription from an ERE-reporter.

Influence of MAPK Pathway on E2-Induced ERE-Reporter Activity
EGF signaling in the cell is mediated through the EGF receptor. After ligand binding, the receptor dimerizes and tyrosine phosphorylation occurs due to intrinsic kinase activity of the receptor. These phosphorylated residues represent docking sites for adapter proteins, such as Shc and Grb. These, in turn, activate different downstream pathways, among others the MAPK pathway (12). In an earlier report we have demonstrated that withdrawal of EGF from HC11 cells increased expression of STAT5, i.e. cells became predifferentiated and competent to respond to lactogenic hormone treatment (19). We have also shown that inhibition of MAPK signaling in EGF-exposed cells increased STAT5 expression. Our conclusion was that EGF-activated MAPK signaling inhibits predifferentiation and represses STAT5 expression. We then found it interesting to investigate whether inhibition of MAPK signaling in EGF-exposed H-ERE cells could decrease E2-stimulated ERE-reporter activity.

H-ERE cells, at proliferating stage, were pretreated with increasing concentrations of the MAPK kinase (MEK)-inhibitor PD98059 (0.1, 1, and 10 µM) 30 min before E2 stimulation. The results show a dose-dependent down-regulation of the E2-induced ERE-reporter activity in PD98059 pretreated cells compared with cells with only E2 treatment (Fig. 4Go, compare bars 3, 5, and 7 with bar 8). When pretreated with the MAPK inhibitor (10 µM), the E2-induced reporter activity is completely abolished. Interestingly, in cells pretreated with only PD98059, an increase in basal activity of the ERE-promoter could be observed when low concentrations (0.1 and 1 µM) of the inhibitor were used (Fig. 4Go, bars 2 and 4). The reason for this is at present unclear. PD98059 has been stated to be a specific MEK inhibitor with an in vitro IC50 value for MEK1 and MEK2 of 2–7 µM and 50 µM, respectively (22). One explanation could be that, at low concentrations, PD98059 inhibits an unknown serine/threonine kinase that represses ER transcriptional activity. Further studies are needed to resolve this issue.



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  		Fig. 4. Inhibition of MAPK Pathway Decreases E2-Induced Transcription from an ERE-Reporter in HC11 Cells in the Presence of EGF

H-ERE stable cells were grown in phenol red-free medium in the presence of EGF. Thirty minutes before 10 nM E2 stimulation, the cells were pretreated with increasing concentrations of PD98059 (0.1, 1, and 10 µM). Proliferating E2-stimulated cells without pretreatment were added as positive control. All experiments were made in triplicate at least three times, and data are presented as mean of fold induction ± SD. Activity of the reporter without E2 treatment was arbitrarily set to 1.

 
Coregulator Expression during Differentiation of HC11 Cells
ER regulation of transcription occurs in concert with coregulators. These are cellular factors recruited to complement nuclear receptor (NR) function upon endocrine signals. They can be divided into two subclasses: coactivators, mediating NR stimulation of transcriptional activity, and corepressors, having the opposite function (23). We found it of interest to investigate whether the decrease in ER functional activity during differentiation of HC11 cells might be related to changes in NR cofactor status. To investigate this, we analyzed expression of two known ER corepressors, SHP (25) and DAX-1 (24) as well as a known ER coactivator, steroid receptor coactivator-1 (SRC-1) (26). Western blot analysis of nuclear extracts from proliferating, confluent, competent, and terminally differentiated HC11 cells was performed. An increase in expression levels of the two corepressors DAX-1 and SHP was seen upon withdrawal of EGF, i.e. when the cells start to differentiate (Fig. 5Go, A and B; compare lanes 1 and 2 with 3 and 4). The expression level of the coactivator SRC-1 was almost identical in proliferating, confluent, and competent HC11 cells, although an increase in SRC-1 expression level was seen in terminally differentiated cells (Fig. 5CGo). Thus, a possible explanation for the decreased ER transcriptional activity seen in differentiated HC11 cells could be increased expression of ER corepressors rather than decreased expression of the coactivator SRC-1.



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  		Fig. 5. Expression Levels of Corepressors SHP and DAX-1 Are Up-Regulated in Differentiating Cells

Nuclear extracts of HC11 cells in proliferating, confluent, competent, and terminally differentiated stages were prepared and separated by SDS-PAGE. Western blot analyses were performed using anti-SHP (panel A), anti-DAX-1 (panel B), and anti-SRC-1 (panel C) antibodies.

 
MAPK pathway seems to be an important regulator for ER signaling in HC11 cells. To investigate whether this pathway influences expression of corepressors SHP and DAX-1, increasing concentrations of MEK inhibitor PD98059 (1 and 10 µM) were added to proliferating, i.e. EGF-exposed, HC11 cells for 24 h. Western blot analyses were performed on nuclear extracts. As a control, levels of SHP and DAX-1 in nuclear extracts from competent and terminally differentiated HC11 cells were analyzed. The results, depicted in Fig. 6AGo (SHP) and B (DAX-1), show dose-dependent up-regulation of corepressor levels in proliferating cells treated with PD98059 (Fig. 6Go, A and B; compare lanes 2 and 3 with lane 1). Also, by inhibiting MAPK signaling in proliferating cells, the amount of corepressors present in the nucleus increased to levels similar to those in differentiated cells. Thus, in HC11 cells, corepressor levels seem to be dependent on MAPK signaling. Although increased expression of SHP and DAX-1 could explain decreased ER transcriptional activity in differentiated HC11 cells, further studies are needed to show that SHP and DAX-1 actually interact with ER in these cells.



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  		Fig. 6. Inhibition of MAPK Signaling in Proliferating HC11 Cells Increases the Amount of SHP and DAX-1 in the Nucleus

Nuclear extracts from proliferating HC11 cells, treated with 1 or 10 µM PD98059 or left untreated, and from competent and terminally differentiated HC11 cells were analyzed by Western blot using anti-SHP (panel A) and anti-DAX-1 (panel B) antibodies.

 
Influence of E2 on ß-Casein Reporter Activity in Terminally Differentiated HC11 Cells
STAT signaling and milk protein expression have been extensively studied in HC11 cells. To further study the impact of estrogen on STAT signaling and terminal differentiation in the mammary gland, a stable HC11 cell line containing a ß-casein reporter (H-Bc) was established. First, the cells were seeded and grown to a confluent state in the presence of EGF. After removal of EGF and a reduction in serum levels, the cells were treated with 10-7 M dexamethasone and 1 µg/ml ovine PRL (insulin was already present in the medium). The cells were then harvested after 24, 48, and 72 h and assayed for ß-casein reporter activity. A time-dependent increase in ß-casein reporter activity could be seen (Fig. 7AGo). As described above, we have earlier shown, by using a transient transfection model, that ER{alpha} and ERß repress STAT5 transcriptional activity on a ß-casein reporter in a ligand-dependent manner (18). Now we wanted to examine whether this is also the case for the HC11 model system. H-Bc cells were grown and treated as described above. At competent stage, cells were treated with dexamethasone and ovine PRL, with or without addition of an increasing concentrations of E2 (0.1, 1, and 10 nM) and 10 nM E2 in combination with ICI 164,384 (1 and 10 nM) or tamoxifen (1 and 10 nM) for a 72-h period. The results, depicted in Fig. 7BGo, show a dose-dependent decrease in ß-casein reporter activity when the cells were cotreated with E2 (Fig. 7BGo, compare bars 3, 4, and 5 to bar 2). Increasing concentrations of ER antagonists ICI 164,384 and tamoxifen inhibited the E2-induced repression of the STAT5-induced transcriptional activity (Fig. 7BGo, bars 6 and 7 and 8 and 9, respectively). This suggests that the negative influence of estrogen on PRL signaling is receptor mediated and also occurs in mammary epithelial cells with endogenous expression of ER and STAT5.



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  		Fig. 7. E2 Inhibits the Transcriptional Activity of the ß-Casein Reporter Induced by Lactogenic Hormones in Terminally Differentiated Cells

A, H-Bc stable cells were grown to a confluent state in the presence of EGF. Then EGF was removed from the medium for 48 h, and the cells were treated with 10-7 M dexamethasone and 1 µg/ml ovine PRL for 24, 48, and 72 h, after which ß-casein reporter gene activity was measured. B, H-Bc stable cells were terminally differentiated as described above. The cells were then treated with increasing concentrations of E2 (0.1, 1, and 10 nM) or 10 nM E2 and increasing concentrations of ICI 164,384 and tamoxifen (1 and 10 nM). Reporter gene activity was then measured as described in Materials and Methods. All experiments were performed in triplicate at least three times, and data are presented as mean of fold induction ± SD. Activity of the reporter without hormonal treatment was arbitrarily set to 1.

 
Two earlier reports have shown that E2 can decrease PRL- and GH-induced STAT5 tyrosine phosporylation (27, 28). In one of these papers, E2 increased expression of suppressor of cytokine signaling 2 (27), explaining the reduced GH-induced JAK2 and finally STAT5 phosphorylation. To investigate whether reduced ß-casein reporter activity in E2-treated H-ERE cells could be explained by E2 reduction of STAT5 phosphorylation, HC11 cells were terminally differentiated and treated with E2 for 24 h or left untreated. Nuclear extracts were then analyzed by Western blotting with an antibody against tyrosine-phosphorylated STAT5. As can be seen in Fig. 8Go, E2 treatment of terminally differentiated HC11 cells did not decrease the level of tyrosine-phosphorylated STAT5. The discrepancy between our results and the earlier reports are at present unknown but could be due to differences in the experimental models (e.g. cell lines used).



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  		Fig. 8. E2 Treatment Does Not Decrease PRL-Induced Tyrosine Phosphorylation of STAT5 in HC11 Cells

HC11 cells were terminally differentiated as described in Materials and Methods. The cells were then treated with 10 nM E2 for 24 h or left untreated, after which nuclear extracts were prepared. Proteins were separated by SDS-PAGE, and Western blot analysis was performed using an antiphospho-STAT5 antibody.

 
ER{alpha} and STAT5 Protein-Protein Interaction in Terminally Differentiated HC11 Cells
In our recent study we found ER{alpha} and ERß to coprecipitate with STAT5A in pull-down experiments in vitro (18). To investigate whether endogenously expressed ERs and STAT5 in HC11 cells form a complex, immunoprecipitation assays were performed. HC11 cells were grown to a confluent state in the presence of EGF. Then EGF was removed from the medium for 48 h and the serum level was lowered, after which the cells were stimulated with 10-7 M dexamethasone and 1 µg/ml ovine PRL. After 72 h, the cells were treated with 10 nM E2 for an additional 24 h, or left untreated, after which whole-cell extracts were made and immunoprecipitated with an antibody against STAT5. A Western blot assay was performed using an anti-ER{alpha} antibody. Interestingly, the protein-protein interaction between ER{alpha} and STAT5 was not ligand dependent, but E2 treatment enhanced the interaction (Fig. 9Go, lanes 1 and 3). Lanes 2 and 4 show immunoprecipitation in the absence of STAT5 antibody. This result demonstrates that a direct physical contact between ER{alpha} and STAT5 proteins occurs in terminally differentiated cells.



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  		Fig. 9. Endogenously Expressed ER{alpha} and STAT5 Proteins Interact in Terminally Differentiated HC11 Cells

Whole-cell extract from terminally differentiated HC11 cells, either treated for 24 h with 10 nM E2 or left untreated, was incubated for 20 min on ice with an antibody directed against STAT5. Protein A Sepharose, diluted 1:1 in PBS, was then added to the mix and incubated at room temperature. Complexes were washed with PBS-Tween, eluted with SDS buffer, and separated on a 7.5% polyacrylamide gel. Western blot experiment was performed using an ER{alpha} antibody. Lanes 2 and 4 show immunoprecipitation in the absence of STAT5 antibody.

 
In conclusion, in HC11 cells, ER functional activity changes during differentiation of mammary epithelial cells. We further propose that the mechanism behind the down-regulation of ER transcriptional activity in differentiating HC11 cells could be due to an up-regulation of corepressors. In this setting, EGF and MAPK pathways are critical regulators of ER functional activity. Our results do not support a direct effect of EGF/MAPK on ER, i.e. ligand-independent activation through serine phosphorylation. Regulation of ER functional activity is not only of interest for understanding estrogen signaling in normal mammary epithelial cells. In a high proportion of breast tumors, members of the ErbB family of receptors are overexpressed, resulting in constitutive signaling (29). Thus, an understanding of the effects of EGF signaling pathway may also help to understand mechanism(s) regulating estrogen responsiveness of breast tumors.

During lactation, the mammary gland is nonresponsive to estrogens, as judged from estrogen-induced cell proliferation and progesterone receptor levels (30). Hormonal contraceptives given to breast-feeding women should preferably contain only progestins and not estrogen, as the latter may decrease milk volume (31). Furthermore, E2 inhibits PRL-induced milk protein production in vitro (32). STAT5A has been shown to be important for mammary epithelial cells to resist regression and involution-mediated apoptosis (33). Upon withdrawal of lactogenic hormones, including PRL, epithelial cell death and tissue restructuring occurs in mammary gland (34). This involution process has been shown to be accelerated by E2 (35, 36). Results from our earlier study (18) and our present study suggest that the effects of estrogen described above could be explained by ER repression of STAT5 functional activity. We also show that E2 can have dual roles during differentiation of HC11 cells: 1) by directly activating transcription from an ERE or 2) indirectly, by repressing ß-casein promoter activity, depending on the cellular state of the cells. In summary, this study shows that the HC11 cell line constitutes an interesting model for studies on regulation of estrogen signaling in mammary epithelial cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culturing and Stable Transfections
HC11 mouse mammary epithelial cells were seeded and grown in phenol red-free RPMI 1640 medium (Invitrogen, San Diego, CA) with 10% (vol/vol) fetal calf serum (FCS), L-glutamine, 50 µg/ml gentamycin, 10 ng/ml EGF (Sigma Chemical Co., St. Louis, MO) and 5 µg/ml insulin (Sigma). Confluent cells were cultured in phenol red-free RPMI 1640, L-glutamine, 50 µg/ml gentamycin, 2% calf serum, and 5 µg /ml insulin for 48 h, resulting in cells competent to respond to lactogenic hormones. Cells were then treated for 72 h with the medium above containing 10-7 M dexamethasone (Sigma) and 1 µg/ml ovine PRL (Sigma) resulting in terminally differentiated cells. For stable transfections, HC11 cells were grown in 10-cm plates and transfected with 10 µg of either reporter plasmid ß-casein-Luc or 3xERE-TATA-Luc (18) using the calcium phosphate precipitation technique, previously described by Petersen and Haldosén (19). Stable clones were selected in 240 µg/ml geneticin. The stable cell line containing the ß-casein reporter was named H-Bc, and the cell line with the 3xERE reporter was named H-ERE.

Nuclear Extracts
Cells were grown in 10-cm plates, washed with cold PBS, scraped into Eppendorf tubes, and spun down. Pellets were dissolved in RSB (10 mM Tris, pH 7.4, 10 mM NaCl, and 6 mM MgCl2) and left on ice for 5 min. After a second centrifugation, pellets were resuspended in RSB buffer containing 1 mM dithiothreitol (DTT), 0.4 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM Na3VO4 and homogenized using a glass homogenizer. After centrifugation, pellets were resuspended in 3 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). After 20 min on ice, the samples were spun and the supernatants obtained were used as nuclear extracts. Protein concentrations were determined with Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, CA).

Whole-Cell Extracts
HC11 cells were grown in 10-cm plates and treated as described above. Cells were washed with cold PBS, collected in an Eppendorf tube, and pelleted by centrifugation at 4 C for 2 min. Supernatants were discarded and pellets were frozen in liquid nitrogen. After thawing, pellets were resuspended in buffer containing 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 and left on ice for 20 min. Supernatants obtained after 10 min centrifugation were used as whole-cell extract. Protein concentrations were determined with Bradford reagent (Bio-Rad Laboratories, Inc.).

Western Blotting
Sodium dodecyl sulfate (SDS)-solubilizing buffer was added to 25 µg protein of nuclear extracts, and the samples were boiled for 5 min. Proteins were separated on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane by semidry blotting. The membrane was blocked for 1 h in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl [Tris-buffered saline (TBS)], containing 5% (wt/vol) milk protein. After washing twice with TBS for 5 min, the membranes were incubated overnight with mouse anti-ER{alpha} [DAKO Corp., Carpinteria, CA; diluted 1:1000 in TBS containing 0.05% (vol/vol) Tween 20 (TTBS)], mouse anti-ERß (Upstate Biotechnology, Inc., Lake Placid, NY; diluted 1:200 in TTBS), rabbit anti-SHP serum [previously described by Johansson et al. (25); diluted 1:5000 in TTBS], rabbit anti-DAX-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; diluted 1:1000 in TTBS), rabbit anti-SRC-1 (Santa Cruz Biotechnology, Inc.; diluted 1:1000 in TTBS), or rabbit anti-phospho-STAT5 (New England Biolabs, Inc, Beverly, MA; diluted 1:1000 in TTBS). Membranes were then washed twice with TTBS for 5 min, after which secondary antibodies, goat antimouse IgG or goat antirabbit IgG, coupled to horseradish peroxidase (diluted 1:5000 in TTBS) were added. Immunoreactive bands were detected with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL). As control for equal protein loading and equal gel to membrane transfer, all membranes were stained with Ponceau red (Sigma Chemical Co.). All experiments were repeated at least three times.

Immunoprecipitation
Fifty micrograms of whole-cell extract from terminally differentiated HC11 cells, treated with or without E2, were incubated for 20 min on ice with an antibody directed against chicken anti-STAT5A (37). Thirty microliters of Protein A Sepharose, diluted 1:1 in PBS, were then added to the samples and incubated for an additional 30 min at room temperature. The beads were pelleted and washed three times with PBS/0.025% (vol/vol) Tween, after which bound proteins were eluted by incubation in 5x SDS-solubilizing buffer for 5 min at 100 C. Eluted proteins were separated on a 7.5% SDS-polyacrylamide gel. Western blot was performed using an mouse anti-ER{alpha} antibody (DAKO Corp., Carpinteria, CA) as described above.

Reporter Assays
Stably transfected cells were grown in 24-well plates and treated as indicated in figure legends. The cells were then lysed in 25 mM Tris-EDTA buffer, pH 7.8, 1 mM EDTA, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, and 2 mM DTT. Luciferase activity was measured with the LucScreen system (Tropix, Norwalk, CT) using a ß-max apparatus (Wallac). The results are presented as mean of fold induction ± SD of at least three experiments performed in triplicate.


   ACKNOWLEDGMENTS
 
We thank Dr. Bernd Groner for providing the ß-casein-Luc reporter and Dr. Eckhardt Treuter for the rabbit anti-SHP serum.


   FOOTNOTES
 
This work was supported by the Swedish Cancer Society, Karolinska Institutet, Magn. Bergvalls stiftelse, and the Novo Nordisk Foundation.

Abbreviations: DAX-1, Dosage-sensitive sex reversal adrenal hyperplasia congenita, critical region on the X chromosome gene-1; DTT, dithiothreitol; E2, estradiol; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; JAK2, Janus activated kinase 2; MEK, MAPK kinase; NR, nuclear receptor; PMSF, phenylmethylsulfonyl fluoride; PRL, prolactin; SDS, sodium dodecyl sulfate; SHP, short heterodimer partner; SRC, steroid receptor coactivator; STAT, signal transducer and activator of transcription; TBS, Tris-buffered saline; TTBS, TBS containing Tween 20.

Received for publication July 23, 2003. Accepted for publication October 29, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Bocchinfuso WP, Korach KS 1997 Mammary gland development and tumorigenesis in estrogen receptor knockout mice. J Mammary Gland Biol Neoplasia 2:323–34[CrossRef][Medline]
  2. Weihua Z, Andersson S, Cheng G, Simpson ER, Warner M, Gustafsson J-Å 2003 Update on estrogen signaling. FEBS Lett 546:17–24[CrossRef][Medline]
  3. Darnell JE, Jr, Kerr IM, Stark GR 1994 Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415–1421[Abstract/Free Full Text]
  4. Schindler C, Darnell Jr JE 1995 Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem 64:621–651[Medline]
  5. Ihle JN 1996 STATs: signal transducers and activators of transcription. Cell 84:331–334[CrossRef][Medline]
  6. Burdon TG, Maitland KA, Clark AJ, Wallace R, Watson CJ 1994 Regulation of the sheep ß-lactoglobulin gene by lactogenic hormones is mediated by a transcription factor that binds an interferon-{gamma} activation site-related element. Mol Endocrinol 8:1528–1536[Abstract]
  7. Schmitt-Ney M, Doppler W, Ball RK, Groner B 1991 ß-Casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor. Mol Cell Biol 11:3745–3755[Abstract/Free Full Text]
  8. Liu X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A, Hennighausen L 1997 Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev 11:179–186[Abstract/Free Full Text]
  9. Udy GB, Towers RP, Snell RG, Wilkins RJ, Park SH, Ram PA, Waxman DJ, Davey HW 1997 Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc Natl Acad Sci USA 94:7239–7244[Abstract/Free Full Text]
  10. Schroeder JA, Lee DC 1998 Dynamic expression and activation of ERBB receptors in the developing mouse mammary gland. Cell Growth Differ 9:451–464[Abstract]
  11. Sebastian J, Richards RG, Walker MP, Wiesen JF, Werb Z, Derynck R, Hom YK, Cunha GR, DiAugustine RP 1998 Activation and function of the epidermal growth factor receptor and erbB-2 during mammary gland morphogenesis. Cell Growth Differ 9:777–785[Abstract]
  12. Boonstra J, Rijken P, Humbel B, Cremers F, Verkleij A, van Bergen en Henegouwen P 1995 The epidermal growth factor. Cell Biol Int 19:413–430[CrossRef][Medline]
  13. Bunone G, Briand PA, Miksicek RJ, Picard D 1996 Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 15:2174–2183[Medline]
  14. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494[Abstract/Free Full Text]
  15. Tremblay A, Tremblay GB, Labrie F, Giguere V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß through phosphorylation of activation function AF-1. Mol Cell 3:513–519[CrossRef][Medline]
  16. Ankrapp DP, Bennett JM, Haslam SZ 1998 Role of epidermal growth factor in the acquisition of ovarian steroid hormone responsiveness in the normal mouse mammary gland. J Cell Physiol 174:251–260[CrossRef][Medline]
  17. Vanderboom RJ, Sheffield LG 1993 Estrogen enhances epidermal growth factor-induced DNA synthesis in mammary epithelial cells. J Cell Physiol 156:367–372[CrossRef][Medline]
  18. Faulds MH, Pettersson K, Gustafsson J-Å, Haldosen LA 2001 Cross-talk between ERs and signal transducer and activator of transcription 5 is E2 dependent and involves two functionally separate mechanisms. Mol Endocrinol 15:1929–1940[Abstract/Free Full Text]
  19. Petersen H, Haldosen LA 1998 EGF modulates expression of STAT5 in mammary epithelial cells. Exp Cell Res 243:347–358[CrossRef][Medline]
  20. Ball RK, Friis RR, Schoenenberger CA, Doppler W, Groner B 1988 Prolactin regulation of ß-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. EMBO J 7:2089–2095[Medline]
  21. Schams D, Kohlenberg S, Amselgruber W, Berisha B, Pfaffl MW, Sinowatz F 2003 Expression and localisation of oestrogen and progesterone receptors in the bovine mammary gland during development, function and involution. J Endocrinol 177:305–317[Abstract]
  22. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR 1995 PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270:27489–27494[Abstract/Free Full Text]
  23. McKenna NJ, O’Malley BW 2002 Minireview: nuclear receptor coactivators—an update. Endocrinology 143:2461–2465[Abstract/Free Full Text]
  24. Zhang H, Thomsen JS, Johansson L, Gustafsson J-Å, Treuter E 2000 DAX-1 functions as an LXXLL-containing corepressor for activated estrogen receptors. J Biol Chem 275:39855–39859[Abstract/Free Full Text]
  25. Johansson L, Bavner A, Thomsen JS, Farnegardh M, Gustafsson J-Å, Treuter E 2000 The orphan nuclear receptor SHP utilizes conserved LXXLL-related motifs for interactions with ligand-activated estrogen receptors. Mol Cell Biol 20:1124–1133[Abstract/Free Full Text]
  26. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract/Free Full Text]
  27. Leung KC, Doyle N, Ballesteros M, Sjogren K, Watts CK, Low TH, Leong GM, Ross RJ, Ho KK 2003 Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2. Proc Natl Acad Sci USA 100:1016–1021[Abstract/Free Full Text]
  28. Wyszomierski SL, Yeh J, Rosen JM 1999 Glucocorticoid receptor/signal transducer and activator of transcription 5 (STAT5) interactions enhance STAT5 activation by prolonging STAT5 DNA binding and tyrosine phosphorylation. Mol Endocrinol 13:330–343[Abstract/Free Full Text]
  29. Stern DF 2000 Tyrosine kinase signalling in breast cancer: ErbB family receptor tyrosine kinases. Breast Cancer Res 2:176–183[CrossRef][Medline]
  30. Shyamala G, Ferenczy A 1982 The nonresponsiveness of lactating mammary gland to estradiol. Endocrinology 110:1249–1256[Abstract]
  31. Kelsey JJ 1996 Hormonal contraception and lactation. J Hum Lact 12:315–318[Free Full Text]
  32. Kleinberg DL, Todd J, Babitsky G, Greising J 1982 Estradiol inhibits prolactin induced {alpha}-lactalbumin production in normal primate mammary tissue in vitro. Endocrinology 110:279–281[Abstract]
  33. Humphreys RC, Hennighausen L 1999 Signal transducer and activator of transcription 5a influences mammary epithelial cell survival and tumorigenesis. Cell Growth Differ 10:685–694[Abstract/Free Full Text]
  34. Atwood CS, Ikeda M, Vonderhaar BK 1995 Involution of mouse mammary glands in whole organ culture: a model for studying programmed cell death. Biochem Biophys Res Commun 207:860–867[CrossRef][Medline]
  35. Ambili M, Jayasree K, Sudhakaran PR 1998 60K gelatinase involved in mammary gland involution is regulated by ß-oestradiol. Biochim Biophys Acta 1403:219–231[Medline]
  36. Athie F, Bachman KC, Head HH, Hayen MJ, Wilcox CJ 1996 Estrogen administered at final milk removal accelerates involution of bovine mammary gland. J Dairy Sci 79:220–226[Abstract]
  37. Wood TJ, Sliva D, Lobie PE, Pircher TJ, Gouilleux F, Wakao H, Gustafsson J-Å, Groner B, Norstedt G, Haldosen LA 1995 Mediation of growth hormone-dependent transcriptional activation by mammary gland factor/Stat 5. J Biol Chem 270:9448–9453[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Nuclear Receptors:   DAX1  |  SHP  |  ERα  |  ERβ
Coregulators:   SRC-1
Ligands:   17β-Estradiol



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