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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS and DISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß and STAT5. ER 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 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 |
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TOPABSTRACTINTRODUCTIONRESULTS and DISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 
-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 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 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
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 and ERß and members of the non-related
STAT family of transcription factors.
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RESULTS and DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS and DISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß Ligand Dependently Decrease STAT5-Mediated
Transcriptional Activity
, 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 and/or ERß could influence the
transcriptional activity of oPRL-activated STAT5. 293 cells were
transiently transfected with expression plasmids for PRLR, STAT5A,
ER, 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. 1B). However, when
increasing amounts of ER were cotransfected with STAT5A, an ER
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, suggesting that the repressive mechanism
of ER is E2 dependent (Fig. 1B). ER 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. 1B). 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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or ERß together with the ß-casein reporter construct (Fig. 1C
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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 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 or ERß. In conclusion, these experiments show that
ligand-activated ER 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 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, or
ERß plasmids. The reporter activity was, as expected, induced in the
presence of STAT5A or STAT5B in response to oPRL (Fig. 2). When the ER 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
and ERß specifically influence STAT5 transcriptional activity.
Furthermore, this shows that the results obtained in our previous
experiments, described in Fig. 1, 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 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 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, and ERß. The results
depicted in Fig. 3 show that the protein
expression levels of STAT5A, ER, or ERß remained stable under the
different hormonal treatments (Fig. 3, A, B, and C, respectively). A
Western blot with nuclear extracts of 293 cells transfected with PRLR,
STAT5A, ER, 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 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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STAT5A Interacts Directly with ER 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 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 and ERß were subsequently used together with in
vitro translated GST-STAT5A in coimmunoprecipitation assays with
an antibody directed against GST. ER and ERß were successfully
coprecipitated with GST-STAT5A as shown in Fig. 4, lanes 1 and 2. Neither ER nor ERß
was precipitated by the GST antibody in the absence of GST-STAT5A (Fig. 4, lanes 5 and 6). Lanes 3 and 4 show 20% of the volume of in
vitro translated ER and ERß, respectively, used in the
coprecipitation experiments. These results demonstrate a direct
physical interaction occurring between STAT5A and ER and ERß
in vitro, which may be part of the negative effect of the
ERs on STAT5 transcriptional activity.
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or ERß
N-Terminal Domains and ERß contain a ligand-independent AF-1 in the N
terminus (A/B domain). For ER, 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 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 and ERß (outlined in Fig. 5A) 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 (
182) or ß
( 93) (Fig. 5B). The cells were then treated with E2 or oPRL alone
or in combination. Increasing amounts of both ER 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 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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and ERß
are dispensable for the cross-talk with STAT5A, we tested other
deletion mutants of ER and ERß in cotransfection experiments to
delineate which functional domains of the ERs participate in the
repression of STAT5A activity. ERA/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. 6A). In 293 cells, cotransfection of
ERA/BC or ERßA/BC together with STAT5A, had no effect on the
STAT5A-mediated induction of reporter activity (Fig. 6B). These results
indicate that the A/BC domains of ER 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 and ERß were fused to the DBD of the yeast factor
Gal4 (added for protein stability; constructs outlined in Fig. 6C).
Surprisingly, the Gal4-DEF and Gal4-ß DEF chimeras, when
cotransfected with STAT5A, could not repress ß-casein reporter
activity (Fig. 6D). 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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and ERß Mediates the Physical Interaction with
STAT5A and ERß depicted in
Fig. 6, A and C. In vitro translated and radiolabeled
ERA/BC and ERßA/BC were successfully coprecipitated with the
antibody directed against GST, only in the presence of GST-STAT5A (Fig. 7A, lanes 1 and 3). In contrast, no
interaction between STAT5A and ERDEF or ERßDEF was detected (Fig. 7B). Since we have demonstrated that the ER 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 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. 1 and 5. Furthermore, mere physical interaction
between the ER 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. 6B. Thus, we
hypothesize that the DBD of ER 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 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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or ERß DBD Interaction Is Specific were fused
to the DEF domains of ER-related receptor 2 (ERR2) (/2DEF) and the
A/BC domains of ERR2 were fused to the DEF domains of ER (2/DEF)
(constructs schematically presented in Fig. 8A). 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/ERR2 chimeras were cotransfected together with STAT5A, PRLR, and
the ß-casein reporter in 293 cells. The results depicted in Fig. 8B
show that the 2/DEF chimera had no notable repressive effect on
STAT5A activity indicating specificity in ER/STAT5 contacts. Neither of
the reciprocal /2DEF chimera showed any significant repression of
STAT5A activity compared with wild-type ER (Fig. 8C).
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/ERR2 chimeras were performed. The constructs
containing the A/BC domains of ER and the LBD of ERR2 (/2DEF)
were able to interact with STAT5A, whereas the 2/DEF could not (Fig. 8D). Apparently the /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 LBD.
The results from the transient transfection experiment (Fig. 8B)
indicate that a high concentration of the 2/DEF chimera showed a
slight repressive effect on STAT5-induced activity; however, compared
with the effect of a lesser concentration of wild-type ER, 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 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 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS and DISCUSSIONMATERIALS AND METHODSREFERENCES |
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(mER), used for transfections of mammalian
cells, was made through digestion of pSP72-mER (39)
with restriction enzymes BamHI and EcoRI and
insertion of the obtained fragment, containing the full coding sequence
of mER, into BamHI/EcoRI-digested pSG5. To
generate the mER/mERR2 swap, a StuI/EcoRI
fragment of pSP72-mouse ERR2 (mERR2) (25) was replaced
with the StuI/EcoRI fragment from pSP72-mER,
exchanging nt 470-1302 of mERR2 with nt 728–1,800 of mER. The
resulting sequence encodes the A/BC domain of mERR2 linked to the DEF
domain of mER. The pSP72-mER/mERR2DEF swap was made through
exchange of the StuI/EcoRI fragment of
pSP72-mER with the StuI/EcoRI fragment from
pSP72-mERR2, substituting nt 728–1,800 of mER with nt 470–1,302 of
mERR2. The resulting sequence encodes the A/BC domain of mER linked
to the DEF domain of mERR2. For transfections in mammalian cells the
pSP72-/2DEF and the pSP72–2/DEF were digested with
HindIII and EcoRI, and the fragments were
inserted into HindIII/EcoRI-digested pSG5-mERß
(39), thus generating pSG5-/2DEF and pSG5–2/DEF.
These constructs were characterized by Western blotting with antibodies
directed against the N terminus and C terminus of ER, 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), ERA/BC, ERßA/BC,
ER182, ERß93 (41), GAL4-/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 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 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 (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 (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, 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AF-2, Activation function-2; DBD, DNA-binding
domain; ERR2, ER-related receptor 2; GAS, interferon -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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TOPABSTRACTINTRODUCTIONRESULTS and DISCUSSIONMATERIALS AND METHODSREFERENCES |
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activation site-related element. Mol Endocrinol 8:1528–1536
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