Cross-Talk Between ERs and Signal Transducer and Activator of Transcription 5 Is E2 Dependent and Involves Two Functionally Separate Mechanisms

Malin Hedengran Faulds, Katarina Pettersson, Jan-Åke Gustafsson and Lars-Arne Haldosén

Department of Medical Nutrition, Karolinska Institute, NOVUM, S-14186 Huddinge, Sweden

Address all correspondence and requests for reprints to: Dr. Lars-Arne Haldosen, Department of Medical Nutrition, Karolinska Institute, 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
 
Steroid hormone receptors and signal transducers and activators of transcription (STAT) factors constitute two distinct families of transcription factors activated by different signaling pathways. In previous reports, cross-talk between STAT5 and several steroid receptors has been demonstrated. We investigated putative cross-talk between ER{alpha} and ERß and STAT5. ER{alpha} and ERß were found to potently repress PRL-induced STAT5 transcriptional activity on a ß-casein promoter construct in a ligand-dependent manner. This down-regulation was found to rely on direct physical interaction between the ERs and STAT5, mediated via the ER DNA-binding domain (DBD). The contact between the ER DBD and STAT5 is highly specific; the interaction is abolished if the ER{alpha} DBD is replaced with the DBD of a closely related steroid receptor. The physical interaction, however, is insufficient to confer the repression of STAT5 activity, which in addition requires the ligand-activated C-terminal part of the ERs, although these domains are not in direct contact with STAT5. Negative cross-talk between ERs and STAT5 is thus mediated via several functionally separated domains of the ERs. Our findings may enhance the understanding of mechanisms of regulation of the different hormonal signaling pathways occurring during different functional events in tissues coexpressing ERs and STAT5.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS and DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGENS AND PRL are hormones important for proper function of the mammary gland at various developmental stages. Estrogens are required for the maturation of the mammary gland that takes place during puberty, and, together with progesterone, for proliferation of the mammary ductal epithelia early in pregnancy. In addition, estrogens influence cellular proliferation and differentiation in a number of other tissues, including uterus, ovary, testis, and prostate (1). The physiological action of estrogens are mediated by the ER{alpha} and ERß (2, 3) belonging to the nuclear receptor superfamily of transcription factors, which regulate transcription via direct binding to DNA enhancer elements located in the promoter regions of target genes. The ERs and other members of the nuclear receptor family have a functionally and evolutionary conserved structure composed of a central DNA binding domain (DBD) which utilizes two Zn-finger motifs in recognition and binding of specific DNA sequences, referred to as estrogen response elements. The DBD shows the highest degree of similarity between different members of the nuclear receptor family. The ligand binding domain (LBD) is located C-terminally of the DBD and contains a ligand-dependent transactivation function [activation function-2 (AF-2)]. This multifunctional region is also involved in receptor dimerization, cofactor interaction, and interaction with the heat shock protein-chaperone complex. The N-terminal A/B-domain of the ERs, which is the most variable region between different nuclear receptors, harbors a transactivation function (AF-1) that can act autonomously in the absence of ligand, but can also synergize with the ligand-activated AF-2. Binding of E2 or related compounds to the ERs results in a conformational change allowing the receptor to interact with coactivators and the general transcription machinery, thus influencing the transcription rate of target genes.

PRL is a peptide hormone, important especially in the mammary gland where it regulates growth and differentiation of epithelial cells and upholds milk production. When PRL binds to the PRL receptor (PRLR), the receptor dimerizes and a receptorassociated tyrosine kinase, Janus activated kinase 2, is activated by transphosphorylation (4, 5). Janus activated kinase 2, in turn, phosphorylates different intracellular signal mediators, among them two members of the transcription factor family of STAT (signal transducers and activators of transcription), STAT5A and STAT5B (6). The STAT factors constitute a family of seven different proteins, STAT1–4, -5A, -5B, and -6 (7), which in their latent state reside in the cytoplasm. Upon activation, via cytokines such as interferons or peptide hormones (PRL, GH), the STATs translocate to the nucleus and promote expression of target genes by direct binding to DNA via interferon {gamma}-like sequence (GAS) elements. Two GAS elements are located in the promoter of the milk protein-encoding ß-casein gene, which is regulated by STAT5A and STAT5B (8, 9). The two isoforms STAT5A and STAT5B are encoded by separate genes but show a sequence similarity of >90% and have been demonstrated to display similar function in terms of gene regulation (10, 11). Mice with targeted disruption of STAT5A or STAT5B manifest distinctive phenotypes; STAT5A knock-out females are unable to lactate due to incomplete terminal differentiation of the secretory epithelial cells in the mammary gland (12), while mice lacking STAT5B are capable of lactating, but at an insufficient level to sustain pups (13). These differences in function of STAT5A and STAT5B are probably due to tissue- and time-dependent differential expression of STAT5A and 5B at different stages of mammary gland development during pregnancy and lactation.

Previous reports have demonstrated cross-talk occurring between STATs and several nuclear receptors, the interaction between STAT5A and the GR being the most extensively studied (14, 15). GR has been shown to synergistically enhance STAT5A-induced transcription of ß-casein gene promoter constructs in transient transfection experiments of mammalian cells. STAT5A apparently recruits GR to the promoter, thereby directing the strong transactivation function located in the N terminus of GR, to supplement the weaker one of STAT5A (16, 17). Synergistic activity between STAT5 and the PRs and MRs has also been reported on the ß-casein promoter, although to a lesser extent than in the case of GR, whereas the AR appears to have no effect on STAT5A transcriptional activity. Previous reports have, in contrast, shown a negative influence of ER{alpha} on STAT5A (15).

We have examined putative ER/STAT5 cross-talk in detail, and in the present study we demonstrate a negative influence of both ER{alpha} and ERß on STAT5A and STAT5B transcriptional activity. The repression of STAT5 activity by the ERs is ligand-dependent and appears to involve two separate mechanisms. Using different deletion mutants of the ER{alpha} and ERß, we have delineated the functional domains within the ERs required for efficient down-regulation of STAT5 transcriptional activity. We present evidence for a direct physical contact between the ERs and STAT5, which appears to be a prerequisite for repression to take place. Furthermore, we show that the physical interaction is mediated via the ER DBD but the ligand-activated LBD of the ERs is essential for efficient down-regulation of STAT5 activity. Taken together, our results demonstrate a potent negative cross-talk between the nuclear receptors ER{alpha} and ERß and members of the non-related STAT family of transcription factors.


    RESULTS and DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS and DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ER{alpha} and ERß Ligand Dependently Decrease STAT5-Mediated Transcriptional Activity
We wanted to examine cross-talk between the ERs and the STAT5 factors, and decided to use a mammalian cell-based transient cotransfection assay. The human embryonic kidney (HEK) 293 cell line, which lacks endogenous expression of ERs, has been shown to sustain functional activity of transfected ER and we, in turn, evaluated this cell line as a model system for STAT5 transcriptional activity. 293 cells were transiently transfected with a luciferase reporter construct containing 300 nucleotides (nts) from the ß-casein promoter which present two GAS elements, together with expression plasmids encoding the PRL receptor (PRLR) and STAT5A or STAT5B (PRLR was included to transduce the PRL signal from the cell surface). The cells were treated with 1 µg/ml of ovine PRL (oPRL) or vehicle for 24 h and harvested, and luciferase activity was determined. As shown in Fig. 1AGo, the activity of the ß-casein reporter was increased in the presence of oPRL when cells were cotransfected with either STAT5A or STAT5B, demonstrating full functional activity of the STAT5 factors in this cell system. We then turned to investigate whether ER{alpha} and/or ERß could influence the transcriptional activity of oPRL-activated STAT5. 293 cells were transiently transfected with expression plasmids for PRLR, STAT5A, ER{alpha}, or ERß together with the ß-casein luciferase reporter construct. STAT5A was as previously able to activate the ß-casein reporter construct in response to oPRL, and the addition of E2 had no effect on the reporter gene expression (Fig. 1BGo). However, when increasing amounts of ER{alpha} were cotransfected with STAT5A, an ER{alpha} dose-dependent decrease in transcriptional activity was observed in the presence of oPRL and E2 in combination. PRL alone did not induce the inhibitory effects of ER{alpha}, suggesting that the repressive mechanism of ER{alpha} is E2 dependent (Fig. 1BGo). ER{alpha} repressed STAT5A activity down to merely 15% of what was obtained with STAT5A alone. Similar results were obtained when increasing amounts of ERß were cotransfected with STAT5A, and the cells were treated with oPRL and E2 in combination (Fig. 1BGo). Again a requirement for E2 was evident for repression by ERß of STAT5A to occur, as treatment of the cells with oPRL alone did not have any effect on STAT5A activity. These results also demonstrate that ER/STAT5A cross-talk is not ER subtype specific.



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Figure 1. E2-Activated ER{alpha} and ERß Down-Regulate STAT5 Induced Transcriptional Activity

A, 293 HEK cells were transfected with 100 ng ß-casein luciferase reporter together with plasmids encoding PRLR, STAT5A, or STAT5B, each 20 ng. The transcriptional responses to 1 µg/ml of oPRL or vehicle were determined. All experiments were done in triplicate at least three times, and data are presented as mean of fold induction ± SD. Activity of reporter plasmid alone without hormone treatment was arbitrarily set to 1. B, 293 HEK cells were transfected with 100 ng ß-casein reporter, 20 ng PRLR, 20 ng STAT5A, and increasing amounts of ER{alpha} or ERß (10, 50, and 100 ng, respectively). The transcriptional responses to 10 nM E2, 1 µg/ml of oPRL, or the two in combination were determined. Further analysis as described in panel A. C, 293 HEK cells were transfected with 100 ng ß-casein luciferase reporter together with plasmids encoding PRLR, STAT5B, and ER{alpha} or ERß expression plasmids, each 20 ng. Hormonal treatments and analysis were as described in panel B.

 
To investigate whether cross-talk between PRL and E2 signaling pathways was restricted to the 5A subtype of the STAT factors, we transiently transfected 293 cells with expression plasmids for PRLR, STAT5B, ER{alpha}, or ERß together with the ß-casein reporter construct (Fig. 1CGo). STAT5B potently activated the ßcasein reporter in response to oPRL, and consistently, addition of E2 did not have any effect, similar to the observations with STAT5A. When ER{alpha} or ERß was cotransfected with STAT5B, the transcriptional activity was reduced in the presence of oPRL and E2. PRL alone was again unable to induce the inhibitory effects of ER{alpha} or ERß. In conclusion, these experiments show that ligand-activated ER{alpha} and ERß can repress the functional activity of both isoforms of STAT5.

The ß-casein promoter used in the luciferase reporter construct is known to be targeted by other transcription factors than STAT5, e.g., Yin Yang 1, C/EBP, and GR (18, 19, 20). To determine whether the E2-induced repressive effect of ER{alpha} and ERß on the ß-casein reporter in the experiments described above was specifically targeting STAT5 activity, we tested a luciferase reporter construct containing three repeats of a 9-bp element related to the GAS family of STAT response elements exclusively bound by STAT5, pSPI-GLE-Luc (21). 293 cells were transiently transfected with the pSPI-GLE-Luc reporter, PRLR, STAT5A, STAT5B, and ER{alpha}, or ERß plasmids. The reporter activity was, as expected, induced in the presence of STAT5A or STAT5B in response to oPRL (Fig. 2Go). When the ER{alpha} and ß constructs were cotransfected together with STAT5 and cells were treated with E2 and oPRL in combination, a substantial decrease in STAT5A or STAT5B-induced reporter activity was observed, demonstrating that ER{alpha} and ERß specifically influence STAT5 transcriptional activity. Furthermore, this shows that the results obtained in our previous experiments, described in Fig. 1Go, B and C, were not due to repressive action of ER on other transcription factors targeting the ß-casein promoter. Since both STAT5A and STAT5B were similarly influenced by ER{alpha} and ERß, we restricted subsequent experiments to STAT5A. To ensure that repression of STAT5A transcriptional activity was not due to altered levels of expressed STAT5A by the coexpression of ER{alpha} and ERß and/or different hormonal treatments, we performed Western blot analysis of whole cell extracts from 293 cells cotransfected with expression plasmids for PRLR, STAT5A, ER{alpha}, and ERß. The results depicted in Fig. 3Go show that the protein expression levels of STAT5A, ER{alpha}, or ERß remained stable under the different hormonal treatments (Fig. 3Go, A, B, and C, respectively). A Western blot with nuclear extracts of 293 cells transfected with PRLR, STAT5A, ER{alpha}, and ERß, with an antibody against phosphorylated STAT5, was also performed to ensure that nuclear translocation and phosphorylation levels of STAT5 were not altered in the presence of ER{alpha} or ERß or treatment with E2. The results show no significant decrease of the amount of STAT5A present in the nucleus when treated with the combination of E2 and oPRL compared with oPRL alone (data not shown).



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Figure 2. E2-Activated ER{alpha} and ERß Exclusively Down-Regulate STAT5-Induced Transcriptional Activity

293 HEK cells were transfected with 100 ng pSPI-GLE-Luc reporter and PRLR, STAT5A, and ER{alpha} or ERß, each 20 ng. The transcriptional responses to 10 nM E2, 1 µg/ml of oPRL, or the two in combination were determined. All experiments were performed in triplicate at least three times, and data are presented as mean of fold induction ± SD.

 


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Figure 3. Repression of STAT5A Transcriptional Activity Is Not a Consequence of Reduced STAT5A Expression Levels

Protein levels of STAT5A and ER{alpha} and ß were examined after transfection of 293 HEK cells and treatment with E2, oPRL, or a combination of both. Western blotting was performed with a monoclonal antibody against ER{alpha} and polyclonal antibodies against STAT5A and ERß as indicated.

 
To rule out that ER-mediated repression of STAT5A activity was not a nonspecific feature of any steroid receptor, we transiently transfected 293 cells with the ß-casein reporter and expression plasmids for STAT5A and PRLR together with GR. The results obtained show that 293 cells were able to support the enhanced effect of GR on STAT5A transcriptional activity described in COS 7 cells (data not shown).

STAT5A Interacts Directly with ER{alpha} and ERß in Vitro
Considering that cross-talk between STAT5A and other members of the nuclear receptor family appears to be mediated via a direct interaction, we wanted to investigate whether the inhibitory effect of ER on STAT5 functional activity occurred through direct protein-protein contact. We therefore performed immunoprecipitation experiments with STAT5A and ER{alpha} or ERß. For this, we generated a plasmid fusion construct of STAT5A coupled to glutathione S-transferase (GST), which could be in vitro translated in rabbit reticulocyte lysate. In vitro translated and radiolabeled ER{alpha} and ERß were subsequently used together with in vitro translated GST-STAT5A in coimmunoprecipitation assays with an antibody directed against GST. ER{alpha} and ERß were successfully coprecipitated with GST-STAT5A as shown in Fig. 4Go, lanes 1 and 2. Neither ER{alpha} nor ERß was precipitated by the GST antibody in the absence of GST-STAT5A (Fig. 4Go, lanes 5 and 6). Lanes 3 and 4 show 20% of the volume of in vitro translated ER{alpha} and ERß, respectively, used in the coprecipitation experiments. These results demonstrate a direct physical interaction occurring between STAT5A and ER{alpha} and ERß in vitro, which may be part of the negative effect of the ERs on STAT5 transcriptional activity.



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Figure 4. ER{alpha} and ERß Directly Interact with STAT5A in Vitro

Lysate containing GST-STAT5A, in vitro translated in the presence of nonradioactive methionine, was incubated for 20 min with lysate containing ER{alpha} or ß, in vitro translated in the presence of radioactive methionine (lanes 1 and 2). After addition of an antibody directed against GST, protein A Sepharose (1:1 slurry with PBS) was added for 20 min. Formed complexes were washed with PBS/Tween, eluted with SDS buffer at 100 C, and separated on a 15% polyacrylamide gel. Dried gel was analyzed with autoradiography. Lanes 3 and 4 show 20% of lysate input (ip) of ER{alpha} or ß, respectively. Lanes 5 and 6 show as negative controls, i.e., precipitation in the absence of GST-STAT5A.

 
ER Repression of STAT5 Activity Is Independent of ER{alpha} or ERß N-Terminal Domains
ER{alpha} and ERß contain a ligand-independent AF-1 in the N terminus (A/B domain). For ER{alpha}, this domain has been demonstrated both to be able to function autonomously and to synergize with the ligand-dependent AF-2 located in the LBD (22). Separate regions of the ER{alpha} A/B domain have been shown to be involved in E2-dependent activity and agonistic response to tamoxifen (23). To investigate whether the ER AF-1 was involved in the repressive effect on STAT5 transcriptional activity, N-terminal deletion mutants of ER{alpha} and ERß (outlined in Fig. 5AGo) were used together with STAT5A in transfection experiments in 293 cells. The cells were cotransfected with the ß-casein reporter construct, PRLR, STAT5A, and increasing amounts of the N-terminal deletion mutants of ER{alpha} ({Delta} 182) or ß ({Delta} 93) (Fig. 5BGo). The cells were then treated with E2 or oPRL alone or in combination. Increasing amounts of both ER{alpha} and ß A/B deletion mutants down-regulated STAT5A transcriptional activity in an E2-dependent manner, similar to what was observed with the full-length ERs. This experiment shows that the repressive effect of ER{alpha} and ERß on STAT5A activity occurs independently of their respective N-terminal domains. These results demonstrate, furthermore, that ERmediated repression of STAT5A functional activity constitutes a distinct mechanism from the synergistic effect of GR on STAT5A transcriptional activity, where N terminus was shown to be indispensable (16).



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Figure 5. The N Termini of ER{alpha} and ERß Are Not Required for Down-Regulation of STAT5A Transcriptional Activity

A, Schematic picture of ER{alpha} and ß N-terminal deletion mutants. B, 293 HEK cells were transfected with 100 ng ß-casein luciferase reporter construct, 20 ng of each PRLR, STAT5A, and increasing amounts (10, 50, and 100 ng, respectively) of plasmid vectors expressing the N-terminal deletion mutants ER{alpha}{Delta}182 or ERß{Delta}93. The transcriptional responses to 10 nM E2, 1 µg/ml oPRL, or the two in combination were determined. All experiments were done in triplicate at least three times, and data are presented as mean of fold induction ± SD.

 
Two Separate Mechanisms Are Required for Efficient ER-Mediated Down-Regulation of STAT5 Transcriptional Activity
Based on our finding that the N-termini of ER{alpha} and ERß are dispensable for the cross-talk with STAT5A, we tested other deletion mutants of ER{alpha} and ERß in cotransfection experiments to delineate which functional domains of the ERs participate in the repression of STAT5A activity. ER{alpha}A/BC and ERßA/BC constructs contain the AF-1 and the DBD but lack the DEF domain containing the LBD and the AF-2 (schematically presented in Fig. 6AGo). In 293 cells, cotransfection of ER{alpha}A/BC or ERßA/BC together with STAT5A, had no effect on the STAT5A-mediated induction of reporter activity (Fig. 6BGo). These results indicate that the A/BC domains of ER{alpha} and ß are unable to impose a down-regulation of STAT5A transcriptional activity. We accordingly speculated that repression of STAT5A function required the ER DEF domains. To investigate this, we tested deletion mutants where the DEF domains of ER{alpha} and ERß were fused to the DBD of the yeast factor Gal4 (added for protein stability; constructs outlined in Fig. 6CGo). Surprisingly, the Gal4-{alpha}DEF and Gal4-ß DEF chimeras, when cotransfected with STAT5A, could not repress ß-casein reporter activity (Fig. 6DGo). Taken together, these results suggest that efficient down-regulation of STAT5A activity by the ERs probably necessitates participation of more than one domain of ER.



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Figure 6. The Combination of DBD and LBD of ERs Is Required in Down-Regulation of STAT5A Transcriptional Activity

A, Schematic picture of ER{alpha} and ß A/BC constructs. B, 293 HEK cells were transfected with 100 ng ß-casein luciferase reporter construct, 20 ng of both PRLR- and STAT5A-expressing plasmids and increasing amounts (10, 50, and 100 ng, respectively) of the {alpha}/ß A/BC constructs. The transcriptional responses to 10 nM E2, 1 µg/ml oPRL, or the two in combination were determined. All experiments were performed in triplicate at least three times, and data are presented as mean of fold induction ± SD. C, Schematic picture of ER{alpha} and -ß DEF constructs. D, 293 HEK cells were transfected with 100 ng ß-casein luciferase reporter construct, 20 ng of both PRLR and STAT5A, and increasing amounts (10, 50, and 100 ng, respectively) of DEF constructs of ER{alpha} or -ß as described in Materials and Methods. Hormonal treatments and analysis are as described in panel B.

 
The DBD of ER{alpha} and ERß Mediates the Physical Interaction with STAT5A
To map the domain(s) of the ERs involved in the physical interaction with STAT5A, we performed coprecipitation experiments with the different ER deletion constructs of ER{alpha} and ERß depicted in Fig. 6Go, A and C. In vitro translated and radiolabeled ER{alpha}A/BC and ERßA/BC were successfully coprecipitated with the antibody directed against GST, only in the presence of GST-STAT5A (Fig. 7AGo, lanes 1 and 3). In contrast, no interaction between STAT5A and ER{alpha}DEF or ERßDEF was detected (Fig. 7BGo). Since we have demonstrated that the ER{alpha} and ERß A/B domains are not involved in the repressive action on STAT5A functional activity, these results suggest that the physical interaction between STAT5A and the ERs is mediated via the DBD (or C-domain) of ER{alpha} and ERß, whereas no direct physical contact occurs between STAT5A and the respective ER DEF domains. However, the ER DEF domains must participate in the repression of STAT5A activity, considering the results from the transfection assays with the full-length or A/B domain-deleted ERs described in Figs. 1Go and 5Go. Furthermore, mere physical interaction between the ER{alpha} or ERß DBD and STAT5A is clearly not sufficient to repress STAT5A transcriptional activity, as evident from the results with the ER A/BC deletion mutants presented in Fig. 6BGo. Thus, we hypothesize that the DBD of ER{alpha} or ERß appears to confer physical interaction with STAT5A which allows, by proximity, an E2-dependent trans-repressive function located in the ER DEF domain to down-regulate STAT5A transcriptional activity. In a study by Stoecklin et al. (16) it was shown that a GR mutant with the DBD exchanged for the ER{alpha} DBD was able to cooperate with STAT5A in transcriptional induction, further supporting our conclusions on the physical interaction between ER DBD and STAT5A.



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Figure 7. The Physical Interaction Between STAT5A and ERs Is Mediated via the DBD

A, Twenty-five microliters of lysate containing GST-STAT5A, in vitro translated in the presence of nonradioactive methionine, were incubated with 25 µl {alpha}- or ßA/BC, in vitro translated, and radiolabeled (lanes 1 and 3). After addition of an antibody directed against GST, protein A-Sepharose was added. Formed complexes were washed with PBS/Tween, eluted with SDS buffer at 100 C, and separated on a 15% polyacrylamide gel. Dried gel was analyzed with autoradiography. Lanes 2 and 4 show 20% of lysate input of {alpha}A/BC or ßA/BC, respectively. Lane 5 shows negative control, i.e., precipitation in the absence of GST-STAT5A. B, Twenty-five microliters of lysate containing GST-STAT5A were incubated with 25 µl of lysate containing {alpha}DEF or ßDEF, respectively (lanes 1 and 3). Reactions were carried out as described in panel A. Lanes 2 and 4 show 20% of lysate input of {alpha}DEF or ßDEF, respectively. Lanes 5 and 6 are included as negative controls, i.e., precipitation in the absence of GST-STAT5A.

 
STAT5A and ER{alpha} or ERß DBD Interaction Is Specific
Our results from the transient transfection experiments with the Gal4 ER-DEF chimeras described above demonstrated that the contact between the ER DBDs and STAT5A was, to some extent, specific, since the Zn-finger containing Gal4 DBD was unable to substitute for the ER DBD in mediating a physical interaction. To further investigate the specificity of the interaction between the ERs and STAT5A, a set of chimeric receptors was generated; the A/BC domains of ER{alpha} were fused to the DEF domains of ER-related receptor 2 (ERR2) ({alpha}/2DEF) and the A/BC domains of ERR2 were fused to the DEF domains of ER{alpha} (2/{alpha}DEF) (constructs schematically presented in Fig. 8AGo). The ERRs are the closest relatives to the ERs in the nuclear receptor superfamily, and the amino acid identity of ERR2 and the ERs is approximately 65% in the DBD and 35% in the LBD (24). ERR2 has been shown to bind to estrogen response element sequences but is not activated by estrogens, and no ligand has currently been identified (25, 26). The ER{alpha}/ERR2 chimeras were cotransfected together with STAT5A, PRLR, and the ß-casein reporter in 293 cells. The results depicted in Fig. 8BGo show that the 2/{alpha}DEF chimera had no notable repressive effect on STAT5A activity indicating specificity in ER/STAT5 contacts. Neither of the reciprocal {alpha}/2DEF chimera showed any significant repression of STAT5A activity compared with wild-type ER{alpha} (Fig. 8CGo).



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Figure 8. The Contact Between STAT5A and the DBD of the ERs Is Specific

A, Schematic picture of ER{alpha}/ERR2 chimeras. B, 293 HEK cells were transfected with 100 ng ß-casein luciferase reporter construct, 20 ng of both PRLR and STAT5A, and increasing amounts (10, 50 and 100 ng, respectively) of the ERR2/{alpha}DEF chimera. The transcriptional responses to 10 nM E2, 1 µg/ml oPRL, or the two in combination were determined. All experiments were performed in triplicate at least three times and data are presented as mean of fold induction ± SD. C, 293 HEK cells were transfected with 100 ng ß-casein luciferase reporter construct, 20 ng of both PRLR and STAT5A, and increasing amounts (10, 50, and 100 ng, respectively) of the ER{alpha}/2DEF chimera. Hormonal treatments and analysis were as described in panel B. D, Twenty-five microliters of lysate containing in vitro translated GST-STAT5A were incubated with 25 µl of lysate containing ER{alpha} or ER{alpha}/ERR2 constructs, in vitro translated in the presence of radioactive methionine (lane 1, 2, and 3, respectively). Reactions were carried out as described in Fig. 7AGo. Lanes 4, 5, and 6 show 20% of lysate input (ip) (ER{alpha}, 2/{alpha}DEF or {alpha}/2DEF, respectively). ER{alpha} precipitated in the absence of GST-STAT5A is included as a negative control (lane 7).

 
To further test the hypothesis that the DBD of the ERs mediates the direct contact with STAT5, coprecipitation experiments with GST-STAT5A together with the ER{alpha}/ERR2 chimeras were performed. The constructs containing the A/BC domains of ER{alpha} and the LBD of ERR2 ({alpha}/2DEF) were able to interact with STAT5A, whereas the 2/{alpha}DEF could not (Fig. 8DGo). Apparently the {alpha}/2DEF chimera can physically interact with STAT5A but the ERR2 DEF is unable to repress STAT5A activity (which could be argued to be due to the lack of ligand). On the other hand, the DBD of the ERR2 is unable to mediate a strong physical interaction with STAT5A, thus preventing efficient repression from the ER{alpha} LBD. The results from the transient transfection experiment (Fig. 8BGo) indicate that a high concentration of the 2/{alpha}DEF chimera showed a slight repressive effect on STAT5-induced activity; however, compared with the effect of a lesser concentration of wild-type ER{alpha}, this effect is minor. Perhaps, ERR2 A/BC might interact weakly with STAT5A but with an affinity too low to detect in our coprecipitation system. These results further confirm that to exert an efficient repressive effect on STAT5 transcriptional activity, 1) several domains of ERs are needed and 2) the ERs need to be ligand activated. These results also demonstrate a high degree of specificity in the contact between STAT5A and the DBD of the ERs.

By cotransfection experiments in 293 HEK cells and in vitro precipitation experiments we have shown that ER{alpha} and ERß repress oPRL-induced STAT5 transcriptional activity in a ligand-dependent manner. Our data indicate that this repression occurs as a consequence of a direct physical contact between ER{alpha} or ERß and STAT5. Furthermore, our data suggest that the physical interaction does not by itself confer down-regulation of STAT5 activity, but that in addition the ligand-activated C-terminal part of the ERs is required. Negative cross-talk between the ERs and the STAT5 factors thus involves two separate mechanisms mediated by different domains of the ERs.

At present, the physiological significance of negative cross-talk between the ERs and STAT5 is not understood but could be of importance in tissues where PRL and estrogens both exert important functions, such as mammary gland, ovary, testis, and prostate. The mammary gland has been shown to express both ER subtypes as well as STAT5A and STAT5B (10, 27, 28).

In mammary gland, STAT5A is important for terminal differentiation, i.e., during late pregnancy and lactation (12). During lactation the mammary gland is nonresponsive to estrogens, as measured as estrogeninduced cell proliferation and PR levels (29). However, hormonal contraceptives given to breast-feeding women should preferably contain only progestin and not E, as the use of the latter may decrease milk volume (30). Thus, it is possible that some specific PRL-regulated mammary gland processes during lactation are negatively influenced by estrogens. Furthermore, E2 has been shown to inhibit PRL-induced milk protein production in vitro (31). Also, mammary epithelial cells in nonpregnant animals express milk proteins during estrus but not in diestrus and proestrus (32), periods with low, increasing, and high E levels, respectively (33). During estrus, higher levels of activated STAT5 are detected as compared with diestrus (34). Whether the examples above are an outcome of ER-repressive action on STAT5-regulated milk protein expression remains to be studied. STAT5A has also been shown to be important for mammary epithelial cells to resist regression and involution-mediated apoptosis (35). Upon withdrawal of lactogenic hormones, including PRL, decreased milk synthesis, epithelial cell death, and tissue restructuring occurs in mammary gland (36). This involution process has been shown to be accelerated by E administration (37, 38). At present it can only be speculated whether this estrogenic effect is through ER-repressive action on STAT5 functional activity.

Not much is known about how STAT factors communicate with other transcription factors and the transcriptional machinery. Thus, at present, it can only be speculated which molecular mechanisms are involved in ER-mediated repression of STAT5 activity. One possibility could be that ER binds to STAT5A and inhibits necessary contact between STAT5 and other transcription factors. Further studies are needed to resolve this issue.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS and DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs
pSG5-mouse ER{alpha} (mER{alpha}), used for transfections of mammalian cells, was made through digestion of pSP72-mER{alpha} (39) with restriction enzymes BamHI and EcoRI and insertion of the obtained fragment, containing the full coding sequence of mER{alpha}, into BamHI/EcoRI-digested pSG5. To generate the mER{alpha}/mERR2 swap, a StuI/EcoRI fragment of pSP72-mouse ERR2 (mERR2) (25) was replaced with the StuI/EcoRI fragment from pSP72-mER{alpha}, exchanging nt 470-1302 of mERR2 with nt 728–1,800 of mER{alpha}. The resulting sequence encodes the A/BC domain of mERR2 linked to the DEF domain of mER{alpha}. The pSP72-mER{alpha}/mERR2DEF swap was made through exchange of the StuI/EcoRI fragment of pSP72-mER{alpha} with the StuI/EcoRI fragment from pSP72-mERR2, substituting nt 728–1,800 of mER{alpha} with nt 470–1,302 of mERR2. The resulting sequence encodes the A/BC domain of mER{alpha} linked to the DEF domain of mERR2. For transfections in mammalian cells the pSP72-{alpha}/2DEF and the pSP72–2/{alpha}DEF were digested with HindIII and EcoRI, and the fragments were inserted into HindIII/EcoRI-digested pSG5-mERß (39), thus generating pSG5-{alpha}/2DEF and pSG5–2/{alpha}DEF. These constructs were characterized by Western blotting with antibodies directed against the N terminus and C terminus of ER{alpha}, respectively. For immunoprecipitation studies, the EcoRI/HindIII fragment from pFASTBac-HTP-STAT5A (Petersen, H., unpublished) was inserted into the pSG5-GST-mERß expression vector (39) digested with BamHI and HindIII (excising the mERß coding sequence); the EcoRI of the mSTAT5A site and BamHI of the vector site were filled in with Klenow fragment to allow blunt-ended ligation. pSPI-GLE-Luc, containing three tandem GAS sequences (21), ß-casein-Luc (40), PCMV-PRLR, pME18S-STAT5A, and STAT5B used for transfection studies have been described previously. The various ER constructs have also been described elsewhere: pSG5/pSP72-mERß wild type (39), ER{alpha}A/BC, ERßA/BC, ER{alpha}{Delta}182, ERß{Delta}93 (41), GAL4-{alpha}/ß-DEF (42), and pSP72-mERR2 (39).

Cell Culture and Transient Transfections
Cells from the HEK cell line 293 were cultured in a 1:1 mix of Ham’s Nutrient mixture F12 (F12, Life Technologies, Inc., Gaithersburg, MD) and DMEM (Life Technologies, Inc.) with 7.5% (vol/vol) FCS, 0.5% (vol/vol) nonessential amino acids (Life Technologies, Inc.) and 100 U penicillin/ml and 100 µg streptomycin/ml. Cells were seeded in 24-well plates for reporter assays and 10-cm plates for whole-cell extracts, 24 h before transfection. Transfections were carried out using Lipofectin reagent (Life Technologies, Inc.) and performed as suggested by the manufacturer in a serum- and antibiotic-free mix of 1:1 F12 and phenol-red free DMEM. One hundred nanograms of reporter plasmid ß-casein-Luc or pSPI-GLE-Luc together with 20 ng pCMV-PRLR and 20 ng pME18S-STAT5A or B together with 20 ng of each of the plasmids containing ER{alpha} or ERß constructs were used as indicated in figure legends. For titration experiments, 100 ng of reporter were transfected together with 20 ng of PRLR, 20 ng of STAT5, and ER constructs in 10, 50, and 100 ng amounts. Ten nanograms of plasmid expressing placental alkaline phosphatase were included as a control for transfection efficiency. The transfection medium was changed after 24 h to a phenol-red free 1:1 mix of F12 and DMEM containing 7.5% (vol/vol) dextran-coated charcoal-treated FCS, 0.5% (vol/vol) nonessential amino acids, and 100 U penicillin/ml and 100 µg streptomycin/ml. The hormones E2 (10 nM), oPRL (1 µg/ml), a combination of both, or vehicle (0.1% ethanol) were added as indicated in the figures. After 24 h, cells were 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 dithiothreitol. Luciferase activity was measured with the LucScreen system (Tropix, Perkin-Elmer Corp., Norwalk, CT) using a ß-max apparatus (Wallac, Inc., Gaithersburg, MD). The results are presented as mean of fold induction ± SD of at least three experiments performed in triplicate.

Whole-Cell Extracts
293 cells were transfected, as described above, with 100 ng pSG5-mER{alpha} or ß, together with pCMV-PRLR and pME18S-STAT5A. After 24 h, cells were stimulated for 20 min with 10 nM E2 or 1 µg/ml oPRL or a combination of both. Cells were washed with cold PBS, collected in an Eppendorf tube, and pelleted by centrifugation at 4 C for 10 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 phenylmethylsulfonyl fluoride, 1 mM sodium molybdate, and 1 mM sodium orthovanadate and left on ice for 20 min. Supernatants obtained after an additional 10 min centrifugation were collected as whole-cell extract. Protein concentrations were determined with Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, CA).

Western Blotting
SDS-solubilizing buffer was added to 25 µg protein of whole-cell 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 one of the following antibodies: rabbit anti-STAT5A [Santa Cruz Biotechnology, Inc., Santa Cruz, CA; diluted 1:2,000 in TBS containing 0.05% (vol/vol) Tween 20 (TTBS)], mouse anti-ER{alpha} (DAKO Corp., Carpinteria, CA; diluted 1:1,000 in TTBS), rabbit anti-ERß (Upstate Biotechnology, Inc., Lake Placid, NY; diluted 1:200 in TTBS), rabbit anti-phospho-STAT5 (Cell Signaling Technology, Beverly, MA; diluted 1:1,000 in TTBS) or rabbit anti-ER{alpha} (Novocastra, Newcastle upon Tyne, UK; diluted 1:1,000 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 1:5,000 in TTBS), were added. Immunoreactive bands were detected with an enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech, Arlington Heights, IL).

In Vitro Translation and Immunoprecipitation
Plasmids (1 µg) were in vitro transcribed/translated in the TNT-coupled RRL system (Promega Corp.) with either Sp6 or T7 polymerase, according to the manufacturer‘s instructions. Twenty-five microliters of lysate, from each reaction described above, were used for immunoprecipitation experiments. Twenty-five microliters of lysate containing GST-STAT5A, translated in the presence of nonradioactive methionine, were mixed with 25 µl of lysate containing wild-type ER{alpha}, ERß, or different mutant ERs, as indicated in the figure legends. All ERs were translated in the presence of [35S]-methionine. Samples were incubated for 20 min on ice, after which an antibody directed against GST was added. The samples were incubated for an additional 20 min. Sixty microliters of protein A-Sepharose, diluted 1:1 in PBS, were then added to the mix and incubated for an additional 30 min at room temperature. The beads were pelleted and washed three times with PBS/0.025% 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 15% SDS-polyacrylamide gel. Dried gels were analyzed by autoradiography.


    ACKNOWLEDGMENTS
 
We thank Drs. Roland Ball, Alice L.-F. Mui, and Bernd Groner for providing the constructs containing PRLR, STAT5A, STAT5B, and ß-casein-Luc reporter.


    FOOTNOTES
 
This work was supported by grants from the Novo Nordisk Foundation, the Swedish Cancer Society, and Karolinska Institutet.

Abbreviations: AF-2, Activation function-2; DBD, DNA-binding domain; ERR2, ER-related receptor 2; GAS, interferon {gamma}-like sequence; GST, glutathione-S-transferase; HEK, human embryonic kidney; LBD, ligand-binding domain; nt, nucleotide; oPRL, ovine PRL; PRLR, PRL receptor; STAT, signal transducer and activator of transcription; TBS, Tris-buffered saline; TTBS, Tween 20-TBS.

Received for publication May 9, 2001. Accepted for publication July 23, 2001.


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