Differential Roles for Signal Transducers and Activators of Transcription 5a and 5b in PRL Stimulation of ER
and ERß Transcription
Jonna Frasor,
KyungSoo Park,
Michael Byers,
Carlos Telleria,
Toshio Kitamura,
Li-yuan Yu-Lee,
Jean Djiane,
Ok-Kyong Park-Sarge and
Geula Gibori
Department of Physiology and Biophysics (J.F., C.T., G.G.),
University of Illinois at Chicago, Chicago, Illinois 60612; Department
of Physiology (K.P., M.B., O.-K.P.-S.), University of Kentucky,
Lexington, Kentucky 40536; Department of Hemopoietic Factors (T.K.),
University of Tokyo, Tokyo 108-8639, Japan; Departments of Medicine,
Molecular and Cell Biology, and Immunology (L.-Y.Y.-L.), Baylor College
of Medicine, Houston, Texas 77030-3411; and Department of Biologie
Cellulaire (J.D.), Institut National de Recherche Agronomique,
Jouy-en-Josas, F-78350 France
Address all correspondence and requests for reprints to: Geula Gibori, 835 South Wolcott, M/C 901, Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612. E-mail:
ggibori{at}uic.edu
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ABSTRACT
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PRL has been shown to stimulate mRNA expression of both ER
and
ERß in the rat corpus luteum and decidua of pregnancy. To investigate
whether PRL may stimulate ER expression at the level of transcription
and which transcription factors may mediate this stimulation, we have
cloned the 5'-flanking regions of both rat ER genes. A constitutively
active PRL receptor (PRL-RCA) stimulated both ER
and
ERß promoter activity, indicating that PRL is acting to stimulate ER
transcription. Putative signal transducer and activator of
transcription (Stat)5 response elements were identified at -189 in the
ER
promoter and at -330 in the ERß promoter. Mutation of these
response elements or overexpression of dominant negative Stat5
prevented stimulation of ER
and ERß promoter activity, indicating
that PRL regulation of ER expression requires both intact Stat5 binding
sites as well as functional Stat5. Interestingly, either Stat5a or
Stat5b could stimulate ER
transcription while stimulation of ERß
occurred only in the presence of Stat5b. Through mutational analysis, a
single nucleotide difference between the ER
and ERß Stat5 response
elements was shown to be responsible for the lack of Stat5a-mediated
stimulation of ERß. These findings indicate that PRL stimulation of
ER expression occurs at the level of transcription and that PRL
regulation of ER
can be mediated by either Stat5a or Stat5b, while
regulation of ERß appears to be mediated only by Stat5b.
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INTRODUCTION
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IN THE PREGNANT rat, E2 is a potent tropic
hormone, which stimulates both progesterone biosynthesis and luteal
cell hypertrophy (1). However, the stimulatory effect of
E2 depends upon previous exposure of the corpus luteum to PRL or
PRL-related hormones from placental origin (2). This
prerequisite was shown to be due to PRL stimulation of E2 binding
activity and mRNA levels for both ER
and ERß (2, 3).
In addition to the corpus luteum, PRL has been shown to stimulate E2
binding activity or mRNA levels in the rat decidua (4),
mammary gland (5), and liver (6). The
mechanism of PRL action on ER expression, however, is not known.
Although PRL has been shown to activate multiple signaling pathways,
including MAPK (7, 8, 9, 10, 11), PKC
(12), c-src
(13, 14, 15, 16), and PI3K (17, 18, 19, 20), the major and
most comprehensively studied pathway activated by PRL is the janus
kinase 2/signal transducer and activator of transcription 5
(Jak2/Stat5) pathway. PKC
may be involved in PRL regulation of
relaxin expression in the rat corpus luteum (12) whereas
PI3K and/or MAPK may regulate PIM-1 expression in Nb2 cells
(11). However, the mechanisms of by which PRL regulates
gene expression through these pathways are not fully understood. In
contrast, PRL has clearly been shown to regulate gene transcription
through the Jak2/Stat5 pathway. This pathway has been implicated in the
regulation of numerous genes by PRL, including milk proteins in the
mammary gland (21),
2-macroglobulin in the corpus
luteum (22), sodium-dependent bile acid cotransporter in
rat liver (23), the CIS gene promoter in COS cells
(24), the 3ß-hydroxysteroid dehydrogenase
gene promoter in HeLa cells (25), the PRL receptor
(PRL-R) gene in insulin-producing INS-1 cells (26),
and the aP2 promoter in NIH-3T3 cells (27).
In the general Jak/Stat signaling paradigm, the PRL-R dimerizes upon
ligand binding (28). This causes activation of the
tyrosine kinase, Jak2, which undergoes autophosphorylation and
subsequently phosphorylates the receptor on tyrosine residues
(28, 29). The phosphorylated tyrosines on the receptors
and Jak2 become docking sites for the SH2 domains of Stat proteins
(30, 31). Jak2 can thus phosphorylate and activate the
recruited Stat proteins. The phosphotyrosine residues on the Stat
proteins can serve as docking sites for the SH2 domain of another Stat
protein so that Stats can either homo- or heterodimerize and
translocate to the nucleus (32). By binding to cognate
response elements located upstream of their responsive genes, Stat
proteins can interact with basal transcriptional machinery and thereby
regulate transcription (33).
Two forms of Stat5, Stat5a and Stat5b, were shown to transduce PRL
signaling (33, 34). Although encoded by different genes,
they are approximately 95% homologous at the protein level. These
proteins contain a single conserved tyrosine residue in the C terminus
(Y694 in Stat5a and Y699 in Stat5b), which becomes phosphorylated by
Jak2 in response to PRL and is necessary for regulation of gene
transcription (33, 34). Both Stat5a and Stat5b recognize
the same DNA binding site, or GAS site (
-interferon-activating
sequence; TTCNNNGAA), and can mediate PRL-induced transcription
(34). Stat5a and Stat5b have been knocked out, either
independently or together, and several key differences between these
two transcription factors were observed (35, 36). Without
Stat5a, mammary gland maturation and function is impaired, while male
patterns of liver function appear to be disrupted when Stat5b is absent
(35, 36, 37). The reproductive phenotype in these mice is not
clear. In the single Stat5a or Stat5b knockouts, no reproductive
defects were observed, while the double knockout was infertile
(36). However, in another Stat5b knockout, the ability to
maintain pregnancy was reduced (35).
One of the major functions of PRL in luteal function is to stimulate ER
expression and thereby maintain luteal responsiveness to E2
(2). To examine whether this stimulation occurs at the
level of transcription, a 2-kb genomic fragment of the rat ERß
promoter region was isolated and sequenced. The rat ER
promoter was
also cloned. Sequence analysis has revealed that both contain putative
Stat5 response elements. Both promoters were found to be stimulated by
PRL and to require intact Stat5 binding sites and functional Stat5.
However, PRL stimulation of ERß could be mediated by Stat5b only. The
lack of ERß responsiveness to Stat5a was found to be due to a single
nucleotide difference in the ERß Stat5 response element.
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RESULTS
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Our laboratory has previously shown, using semiquantitative
RT-PCR, that PRL stimulates both ER
and ERß mRNA levels in corpora
lutea of pregnant rats and in primary cultures of luteinized granulosa
cells (3). Since this method does not provide information
as to the differential levels of expression between two genes, we have
used quantitative real-time RT-PCR. Known amounts of rat ER
and
ERß cDNA were used to generate standard curves for analysis of mRNA
levels in experimental samples amplified in parallel reactions (Fig. 1A
). In corpora lutea on d 7 of
pregnancy, approximately 45,000 copies of ER
and 550 copies of ERß
were detected in samples corresponding to 1 ng of RNA (Fig. 1B
). In
rats, hypophysectomized on d 3 of pregnancy, ER
and ERß levels
were reduced to approximately 30% of the control levels. Sustained
treatment with PRL induced a 3.5-fold induction of ER
expression and
a 2-fold induction of ERß expression. In contrast to corpora lutea,
in which there was approximately 70 times more ER
than ERß,
luteinized granulosa cells cultured for 72 h expressed only 8
times more ER
than ERß, with approximately 850 copies of ER
and
120 copies of ERß per ng of starting RNA (Fig. 1C
). After a 12-h
treatment with PRL, a 2.4-fold increase in ER
and a 1.8-fold
increase in ERß expression was observed. These findings confirm that
PRL can stimulate both ER
and ERß mRNA expression and further
demonstrate a much higher level of ER
than ERß in corpora lutea of
pregnancy and in primary luteinized granulosa cells.

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Figure 1. Standard Curves for Real-Time Quantitative PCR
A, Known amounts of ER and ERß cDNA, ranging from 103
to 107 copies/µl and 103 to 106
copies/µl, respectively, were amplified as described in
Materials and Methods. The crossing point represents the
number of cycles required to reach a chosen level of fluorescence, at
which all standards and samples were in the linear range of
amplification. The crossing point was then plotted against the number
of copies of cDNA/µl, and the linear regression equation through the
data points was used to determine the number of copies of ER or
ERß in reverse transcribed RNA samples. B, Real-time quantitative PCR
was carried out for ER and ERß using mRNA from corpora lutea of
pregnant rats, hypophysectomized rats, and hypophysectomized rats
treated with PRL. C, Real-time quantitative PCR was carried out for
ER and ERß using mRNA from primary luteinized granulosa cells that
had been treated with 1 µg/ml PRL for 12 h.
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To investigate whether PRL can regulate ER expression at the level of
transcription, the ER
and ERß 5'-flanking regions were cloned, and
promoter- reporter constructs were prepared as described in
Materials and Methods. A putative Stat5 response element
(5'-TTCTAGGAA-3'), which represents a perfect consensus Stat5 binding
site, was located at -180 bp in the ER
promoter region. In
addition, a putative Stat5 response element
(3'-TTCTGGTAA-5') with one nucleotide difference
(underlined) from the consensus sequence was located at
-330 bp in the ERß promoter region (Fig. 2
). CHO cells were transfected with
either the ER
or ERß promoter-luciferase reporter constructs
(ER
-luc, ERß-luc) together with an expression vector for a
constitutively active PRL-R (PRL-RCA)
(38). Control cells were transfected with an expression
vector for PRL-RL, which, in the absence of any
exogenous PRL treatment, served as a control. The active PRL-R has
previously been shown to signal to the ß-casein promoter in much the
same way as PRL acting through the long form of its receptor but in the
absence of exogenous PRL treatment (38). The presence of
PRL-RCA caused a marked phosphorylation of both
Stat5a and Stat5b (Fig. 3A
) and induced a
10-fold stimulation of ER
-luc activity (Fig. 3B
, left)
and a 5-fold stimulation of ERß-luc activity (Fig. 3B
, right), indicating that PRL can induce phosphorylation of
Stat5a and Stat5b as well as regulate both ER
and ERß expression
at the level of transcription.

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Figure 2. Rat ERß Gene Promoter Region
The rat ERß 5'-flanking region was cloned as described in
Materials and Methods. This regulatory region of the
ERß gene spans from -2,023 bp to +46 bp and contains a putative
Stat5 response element at -330 bp (underlined and
labeled). The second gene-specific primer used for cloning
the ERß promoter is underlined.
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Figure 3. Effect of PRL-RCA on Stat5
Phosphorylation and ER and ERß Promoter Activity in CHO Cells
A, CHO cells were cultured in six-well plates and transfected with 2
µg/well expression vectors for PRL-RCA or
PRL-RL (control). Forty-eight hours after the start of
transfection, WCE were prepared and Stat5a and Stat5b were
immunoprecipitated using specific antibodies to each form of Stat5.
Western blotting was performed first using an antibody, which
recognizes the tyrosine-phosphorylated form of both Stat5a and Stat5b
(Y694 and Y699, respectively). The blots were stripped and reprobed
using the same antibodies for immunoprecipitation. B, Cells were
transfected as described in panel A with the addition of 0.5 µg/well
ER -luc or ERß-luc. Luciferase activity was measured in each well
and normalized to the total protein level within that well. The
experiment was repeated three times with triplicate wells for each
group. The data represent the combined mean ± SEM for
all three experiments.
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To examine whether PRL-RCA activation of Stat5
was required for its stimulatory effect on ER
and ERß promoter
activity, CHO cells were transfected with expression vectors for a
dominant negative Stat5a (DN-5a) or a mutant Stat5b (Mut-5b). The DN-5a
contains a C-terminal deletion in the transactivation domain and is
transcriptionally inactive (39). The Mut-5b contains a
four-amino acid substitution in the DNA binding domain, which prevents
it from entering the nucleus, binding DNA, and therefore regulating
transcription (40, 41). Both of these Stat5 expression
vectors prevented PRL-RCA stimulation of ER
and ERß promoter-driven luciferase activity (Fig. 4
), indicating that
PRL-RCA regulation of both ER
and ERß
transcription requires a functional Stat5.

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Figure 4. Effect of Dominant Negative Stat5 on
PRL-RCA Stimulation of ER and ERß Promoter Activity in
CHO Cells
CHO cells were cultured and transfected as described for Fig. 3 . In
addition, each well was transfected with 1 µg/well DN-Stat5a or
Mut-Stat5b. Luciferase activity was measured in each well and
normalized to the total protein level within that well. The experiment
was repeated three times with triplicate wells for each group. The data
represent the combined mean ± SEM for all three
experiments.
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The ability of PRL-RCA to regulate ER
-luc and
ERß-luc was also studied in COS cells. In contrast to CHO cells, COS
cells express very low levels of endogenous Stat5a and Stat5b, which
could not be detected by Western analysis (Fig. 5A
, lanes 1 and 2). When COS cells were
transfected with expression vectors for either Stat5a or Stat5b, high
levels of Stat5 expression were detected and both became
phosphorylated in response to PRL-RCA (Fig. 5A
, lanes 3 and 4). We next examined the capacity of each Stat5 to
transactivate the ER
and ERß promoters. As shown in Fig. 5B
, ER
promoter activity was stimulated 2-fold by
PRL-RCA in the presence of either Stat5a or
Stat5b. In contrast, ERß promoter activity was stimulated by
PRL-RCA only in the presence of Stat5b (Fig. 5B
).
In the presence of Stat5a, PRL-RCA had little or
no effect on ERß-driven reporter activity. These findings provide
additional evidence that PRL can regulate ER expression at the level of
transcription. In addition, it appears that ER
is responsive to
either Stat5a or Stat5b while ERß may be responsive only to
Stat5b.
Because these studies in both CHO and COS cells consist of a
reconstructed PRL signaling pathway, we questioned whether PRL utilizes
the same pathway to regulate expression of the endogenous ER
and
ERß genes. To address this question, primary luteinized granulosa
cells were used. PRL is known to induce phosphorylation of Stat5 in
these cells; however, it is not known whether this is due to Stat5a or
Stat5b activation (22, 42). Primary cells were cultured
for 72 h and then treated with PRL for 5 min. After
immunoprecipitation and Western blotting, it was found that primary
luteinized granulosa cells express both Stat5a and Stat5b and both
become highly phosphorylated in response to PRL (Fig. 6A
). To examine the possibility that
Stat5a and Stat5b can differentially affect stimulation of endogenous
ER
and ERß mRNA, primary luteinized granulosa cells were
transfected with expression vectors for constitutively active Stat5a or
Stat5b (CA-5a or CA-5b). These constitutively active Stat5s were
generated by random mutagenesis and found to contain two mutations, one
in the DNA binding domain (H299R) and one in the transactivation domain
(S711F). They have been shown to be constitutively phosphorylated,
located in the nucleus, and capable of binding DNA and regulating gene
transcription in the absence of any cytokine stimulation
(43). CA-5a induced a 4-fold stimulation of endogenous
ER
mRNA while CA-5b stimulated ER
mRNA expression 2-fold (Fig. 6B
). In contrast, ERß expression was stimulated 2-fold only in the
presence of CA-5b. These results confirm our findings that Stat5b, and
not Stat5a, can mediate regulation of ERß while stimulation of ER
can be mediated by either Stat5a or Stat5b.
Because both ER promoters contain putative Stat5 response elements, we
next examined whether these were essential for regulation by PRL.
Mutations were made to each promoter so that the Stat5 response
elements were no longer capable of binding Stat5. Stimulation of both
ER
and ERß promoter-driven luciferase activity by
PRL-RCA was completely prevented by mutation to
the Stat5 response elements (Fig. 7
),
indicating that PRL stimulation of ER expression requires intact Stat5
DNA binding sites. Because the ER
and ERß promoter Stat5 response
elements contain a 1-bp difference (GAA for ER
and TAA for ERß),
we next investigated whether this single nucleotide could explain the
differential responsiveness of ER
and ERß to Stat5a. A single
nucleotide mutation was made to the ER
promoter
(ER
-M1) so that it resembled the ERß
response element (GAA to TAA), and the corresponding mutation was made
to ERß (ERß-M1) so that it resembled the
ER
response element (TAA to GAA). The mutated ER
promoter
(ER
-M1) containing the ERß Stat5 response
element was still stimulated by PRL-RCA in the
presence of Stat5a or Stat5b, although the degree of stimulation was
markedly reduced (Fig. 8A
). Of great
interest was our finding that a single nucleotide mutation to the Stat5
response element of the ERß promoter (ERß-M1)
rendered ERß now highly responsive to Stat5a (Fig. 8B
). In addition,
stimulation of the mutated ERß promoter was significantly increased
in the presence of Stat5b as well (Fig. 8B
). Based on these results, it
appears that the nonconsensus Stat5 binding site in the ERß promoter
both prevents its responsiveness to Stat5a and limits its
responsiveness to Stat5b.

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Figure 7. Role of Putative Stat5 Response Elements in
PRL-RCA Stimulation of ER and ERß Promoter Activity
COS cells were transfected with 0.5 µg/well wild-type, mutated, or
promoter-less reporter constructs for ER -luc (A) or ERß-luc (B).
In addition, each well was transfected with 0.5 µg ß-gal, 1 µg
Stat5b, and 2 µg PRL-RCA or PRL-RL (control).
Forty-eight hours after the start of transfection, luciferase activity
was measured in each well and normalized ß-gal activity within that
well. The experiment was repeated four times with triplicate wells for
each group. The data represent the combined mean ±
SEM for all four experiments.
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DISCUSSION
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In the rat corpus luteum of pregnancy, PRL is known to stimulate
expression of both ER
and ERß mRNA, leading to a functionally
significant increase in the number of E2 binding sites (3, 44). Results from our current studies indicate that PRL causes
this increase in ER expression at the level of transcription, which is
mediated by the transcription factor Stat5. Using real-time
quantitative RT-PCR, we provide evidence that ER
mRNA expression is
far more abundant in both corpora lutea and luteinized granulosa cells
than is ERß. This is expected since previous work from Dr.
Park-Sarges group (45) has shown that the LH surge
specifically down-regulates ERß expression but has no effect on ER
levels. Also, Dr. Joanne Richards laboratory (46) found
low expression of ERß mRNA and no ERß DNA binding activity in
luteinized granulosa cells. In addition, although the ERß knockout
mouse is subfertile, it is capable of supporting a full-term pregnancy,
suggesting that luteal function is not compromised in the absence of
ERß and that ER
is sufficient to sustain pregnancy
(47). Nevertheless, PRL is capable of stimulating
expression of both ER
and ERß at the level of transcription.
Although ERß may not be crucial for luteal function, the ability of
PRL to stimulate ERß may be important in other PRL target tissues. In
addition, multiple hormones and cytokines are capable of activating
Stat5 and may therefore be capable of regulating ER expression through
a similar pathway.
Perhaps the most intriguing finding from these studies was that ER
and ERß appear to be regulated differently by Stat5a and Stat5b. In
COS and primary luteinized granulosa cells, the ER
promoter and
endogenous gene were responsive to both Stat5a and Stat5b. In contrast,
in each of the models used, the findings support the conclusion that
ERß is far more responsive to Stat5b than to Stat5a. In CHO cells,
dominant negative expression vectors for both Stat5a and Stat5b were
capable of preventing stimulation of both ER
and ERß. This finding
suggests then that the DN-5a may prevent stimulation of ERß by
forming heterodimers with endogenous Stat5b. Alternatively,
overexpression of these Stats may be capable of preventing the
endogenous Stat5 from being activated through competition for Jak2
substrate binding sites. This lack of stimulation by Stat5a on ERß
expression, however, may not be an issue in the corpus luteum since the
major Stat5 expressed is Stat5b (data not shown). Whether PRL can or
cannot regulate ERß transcription in other tissues, such as the
mammary gland, where Stat5a plays a major role in PRL signaling,
remains to be investigated. The differential regulation of ER
and
ERß by Stat5a and Stat5b may be one mechanism that contributes to the
tissue-specific pattern of ER expression.
The lack of ERß stimulation by Stat5a was attributed to a single
nucleotide in the Stat5 response element, which both prevented ERß
responsiveness to Stat5a and limited its responsiveness to Stat5b. When
this single nucleotide was introduced into the ER
response element,
responsiveness to both Stat5a and Stat5b was significantly reduced.
These results are somewhat expected since a nonconsensus binding site
should be less effective at driving gene expression than would be a
consensus site. Also, when this single nucleotide was mutated in ERß,
so that it resembled the consensus ER
Stat5 response element, ERß
transcription in the presence of either Stat5a and Stat5b was markedly
increased. These findings could explain, in part, why ERß expression
is so much lower in the corpus luteum than is ER
. However, these
results do not explain the complete lack of ERß responsiveness to
Stat5a. If this nucleotide were the sole explanation, then it would be
expected that ER
would be unresponsive to Stat5a once it too
had the same nucleotide as ERß. This suggests that within the
ER
promoter some additional regulatory region may be capable of
enhancing Stat5a action. It is possible that the differential action of
Stat5a and Stat5b on these two promoters may be explained by the
ability of Stat5 to form stable tetramers through protein-protein
interactions involving a tryptophan residue, which is conserved in all
Stats and a lysine residue in the Stat5 N-terminal domain
(48). Interestingly, only Stat5a tetramers and not Stat5b
tetramers were shown to bind the multiple GAS sites in the CIS gene
promoter (24). In this same promoter, Stat5b
preferentially bound as a dimer. The study of Stat5a dimer and tetramer
DNA binding sites revealed that Stat5a tetramers could bind to a wider
range of nonconsensus sites, which a Stat5a dimer could not bind. A
spacing of 6 bp between tandem GAS sites was the preferred distance for
Stat5a tetramer binding (49). Also, it appears that two
full GAS sites are not completely necessary for Stat5a tetramer binding
since one of the sites could be replaced with a GAS half-site
(24). Although we have not found additional GAS sites or
half-sites in either promoter, it is possible that some nonconsensus
site in the ER
promoter could be essential for its responsiveness to
Stat5a while the ERß promoter may not be capable of supporting Stat5a
tetramer binding.
In conclusion, it is clear that PRL regulation of ER expression is at
the level of transcription and that Stat5 mediates this regulation.
Furthermore, the Stat5 response elements within the ER
and ERß are
necessary for this stimulation. Our data also demonstrate that ER
and ERß are differentially responsive to Stat5a and Stat5b and that a
single nucleotide in the ERß promoter can explain its lack of
responsiveness to Stat5a. And finally, this increase in ER
transcription represents a functional stimulation of ER expression
since only a modest increase of E2 binding sites by PRL in the corpus
luteum is sufficient to render the corpus luteum responsive to E2.
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MATERIALS AND METHODS
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Materials
PMSG, human CG, DMEM/F12 (1:1), DMEM, horseradish peroxidase
conjugated secondary antibodies, and all other reagent grade chemicals
were obtained from Sigma (St. Louis, MO). OptiMem,
LipofectAMINE,
-MEM medium, and Trizol were purchased from
Life Technologies, Inc. (Gaithersburg, MD). The Advantage
RT-for-PCR kit and the chemiluminescence ß-gal substrate were from
CLONTECH Laboratories, Inc. (Palo Alto, CA). FBS was from
HyClone Laboratories, Inc. (Logan, UT). DNA Master SYBR
Green I was purchased from Roche Molecular Biochemicals
(Indianapolis, IN). Trypsin-EDTA, antibiotics, and antimycotics were
from Mediatech (Herndon, VA). Antibodies to Stat5a, Stat5b, and
phosphorylated Stat5a/5b were from Upstate Biotechnology, Inc. (Lake Placid, NY). Protein A/G agarose beads and the
enhanced chemiluminescence detection reagents were obtained from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The
luciferase assay substrate and reporter lysis buffer were
purchased from Promega Corp. (Madison, WI).
Animal Models
Pregnant and immature female Sprague Dawley rats were obtained
from Sasco Animal Labs (Madison, WI) and housed under controlled
conditions of light and temperature with free access to standard rat
chow and water. All experiments were conducted in accordance with the
principles and procedures of the NIH Guide for the Care and Use of
Laboratory Animals and were approved by the Institutional Animal Care
and Use Committee.
To determine the effect of PRL on ER expression, mRNA was
obtained from corpora lutea from pregnant rats that had either been
left intact, hypophysectomized, or hypophysectomized and treated with
PRL as previously published (3). To obtain primary
luteinized granulosa cells, follicular development was induced in
immature rats (2426 d of age) by injection of 15 IU PMSG ip. An
ovulatory dose of human CG (10 IU, ip) was given 48 h later.
Luteinized granulosa cells were harvested and cultured as previously
described (42). Transfection of primary cells was carried
out in OptiMem using LipofectAMINE according to the manufacturers
directions.
Real-Time, Quantitative RT-PCR
RNA from tissue and cell cultures was isolated using Trizol
according to the manufacturers instructions. Reverse transcription
was carried out using reagents from the Advantage RT-for-PCR kit
according to the manufacturers instructions. One microgram of total
RNA was used for the reverse transcription reaction, and the product
was diluted to a final volume of 100 µl by adding diethyl
pyrocarbonate-treated H2O. To generate
standard curves for quantitative PCR, rat ER
and ERß cDNA,
which was kindly provided by Dr. Maruyama (50), were
diluted to concentrations ranging from 103 to
107 copies/µl. Five-microliter aliquots of
standards or diluted reverse transcription products were combined with
2 µl 10x DNA Master SYBR Green I, 1.6 µl
MgCl2 (3 mM final concentration), and
specific primers for rat ER
or ERß (0.5 µM final
concentration). The primers used have been previously published
(3). Reactions were carried out in glass capillary tubes
in a total volume of 20 µl. The DNA Master SYBR Green I mix contains
Taq DNA polymerase, reaction buffer, deoxynucleotide
triphosphate, 10 mM
MgCl2, and SYBR Green I dye, which is a specific
fluorescence dye for double-stranded DNA. PCR reactions were performed
in the Roche Lightcycler instrument and the accompanying
software was used for data analysis (Roche Molecular Biochemicals, Mannheim, Germany). After a 2-min denaturation,
PCR cycles were carried out as follows: 0 sec at 95 C, 10 sec at the
annealing temperature, and 15 sec at 72 C. For ER
, 40 cycles at an
annealing temperature of 63 C were used; for ERß, 35 cycles at an
annealing temperature of 69 C were used. At the end of each cycle, the
amount of double-stranded DNA was monitored by measuring the level of
SYBR Green I fluorescence. After the completion of all cycles, a level
of fluorescence was selected at which all of the standards and samples
were within the linear range of amplification. The crossing point, or
the number of cycles necessary for each sample or standard to obtain
the selected level of fluorescence, was calculated using the
Roche Lightcycler software. Based on these crossing
points, a standard curve was generated, and the number of ER
or
ERß copies was calculated for each sample. The data presented
represent the number of copies of ER
or ERß in 1 ng of total
RNA.
Cloning of the ER
and ERß Promoters
The 5'-flanking region of the rat ER
gene was cloned using
the rat PromoterFinder DNA Walking kit (CLONTECH Laboratories, Inc.). Five different genomic libraries were generated by
digesting genomic DNA with 5 different restriction enzymes, namely
EcoRV, ScaI, DraI, PvuII,
and SspI followed by ligation with a specifically designed
PromoterFinder adapter. The ER
promoter region was amplified by
nested PCR using the five different genomic libraries as templates and
two sets of primers designed according to the published sequence of the
ER
promoter (51). The first PCR reaction was carried
out using the following primers: 5'-CCACTCATAAATCTCTT- GGTAACGGC-3'
and 5'-GAAGGAAGGAATGTGCTCGAATGATC-3'. A second PCR reaction was carried
out using product from the first reaction and the following primers:
5'-CTGGGGTTGCAATTAGTC-ATTTAGGC-3' and
5'-TCGCGAATTCGAGTGGCGCGGTGTGTGATCAAG-3'. The second primer also
included an attached EcoRI site for subsequent cloning. All
five sources of genomic DNA yielded an amplified product of the
expected size (880 bp). The PCR products were pooled, and the internal
KpnI site at -769 in the ER
promoter region and the
added EcoRI site were used to subclone the PCR product into
the pBluescript DNA vector (Stratagene, La Jolla, CA).
Subcloning of the ER
promoter region into the pGL3-basic luciferase
reporter vector (Promega Corp.) was carried out utilizing
the KpnI and BglII sites in the pGL3 vector and
the KpnI and BamHI sites in the pBluescript
vector. Both strands of the ER
promoter generated from different
colonies were sequenced. Sequence analysis revealed two differences
from the originally published sequence (G to C at -494, A to G at
-346) (51).
To isolate the regulatory region of the rat ERß gene, we used the
touchdown PCR amplification approach using the GenomeWalker kit
(CLONTECH Laboratories, Inc.) according to the
manufacturers procedure. Two gene-specific primers were designed
against the most 5'-end of sequences of the rat ERß mRNA
(gene-specific primer 1: 5'-AAGCTGCAAAGATTACCCACGACTA-3' and
gene-specific primer 2: 5'-GACTAACGGATGTTAGTGCGTCTT-3')
(52). Thus, the expected gene-regulatory DNA would contain
46 bp of the 5'-end of the ERß mRNA. The primary PCR amplification
was carried out using the combined GenomeWalker libraries (1 µl) and
the primer set of gene-specific primer 1 and adapter primer 1, under
PCR conditions of 72 C for 4 min (7 cycles) and 67 C for 4 min (33
cycles). The secondary PCR amplification was carried out using 1 µl
of the diluted primary PCR products (1:100) and the primer set of
gene-specific primer 2 and adapter primer 2, under PCR conditions of 72
C for 4 min (5 cycles) and 67 C (22 cycles). This procedure yielded two
prominent PCR fragments (
1 and
2 kb) that were subsequently
isolated, subcloned into PCR2.1 T/A overhang vector (CLONTECH Laboratories, Inc.), and sequenced using M13 forward and
backward primers. Both contained the adapter 2 sequences at their
5'-end and the gene-specific primer 2 at their 3'-end. The inserts of
these clones were isolated by restriction digestion using
EcoRV/SpeI and subsequent fill-in reactions with
Klenow, and inserted into the SmaI arms of the pUBT-luc
vector (53). For these studies, the
1
kb promoter region of the rat ERß gene was used.
Mutations to ER
and ERß Promoters
The first set of mutations made to the ER
and ERß promoters
consisted of six and five nucleotides, respectively, being changed to
abolish the Stat5 binding sites. Oligonucleotide primers for these
mutations were made as follows (mutated nucleotides
underlined): ER
5'-GCCAAGGGGGCTGGAGTTTCTTGATATCATGCTGA-TTCTAGTGGTGCTACTGCCG
-3' and ERß
5'-ATTACTGCTTATTTCGGTGCTATGA-TATCAACCCGGGGCCTGGCCCATGC-3'.
The second set of mutations made to the ER
and ERß promoters
consisted of a single nucleotide being changed. The consensus Stat5
site of the ER
promoter (TTCnnnGAA) was changed so that it resembled
the Stat5 response element of the ERß promoter (TTCnnnTAA). The
nonconsensus ERß Stat5 response element (TTCnnnTAA) was mutated so
that it would resemble the ER
Stat5 response element (TTCnnnGAA).
Oligonucleotide primers for these mutations were made as follows
(mutated nucleotides underlined): ER
5'-GGCTGGAGTTTCTTCTAGTAAT-GCTGATTCTAGTGG-3' and ERß
5'-CGGTGCTATTCCCAGAACCCGG-GGCCTGG-3'. All mutations were
made using the QuikChange Site-directed Mutagenesis kit according to
the manufacturers directions (Stratagene). The presence
of the correct mutations was confirmed by DNA sequencing.
Culture and Transfection of CHO and COS Cells
CHO and COS cells were routinely cultured in
-MEM and
DMEM/F12 (1:1), respectively. All media were supplemented with 10%
FBS, 100 IU/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml
Amphotericin. Cultures were carried out at 37 C in
a 5% CO2, humidified atmosphere. For transient
transfections, 100,000 cells were seeded per well in six-well plates
and cultured for 24 h. Both CHO and COS cells were transfected
using calcium phosphate DNA precipitation and were approximately 50%
confluent at the start of transfection (54). In general, a
total of 45 µg DNA were transfected per well, and the total amount
of DNA was equalized with empty vector when necessary. Twenty-four
hours after the start of transfection, media were changed to standard
culture media supplemented with 1% FBS, and cells were cultured for an
additional 24 h at 5% CO2. The entire
length of the experiments was standardized to 48 h from the start
of transfection.
Reporter Assays
Luciferase and ß-galactosidase (ß-gal) activities were
measured by first preparing cell lysates in 1x reporter lysis buffer.
Luciferase activity driven by the ER
or ERß promoter was measured
by combining lysate with Firefly luciferase assay substrate and
measuring luminescence for 10 sec on a Lumat LB 9507 Luminometer (EG&G
Berthold, Oak Ridge, TN). As a control, cells transfected with
the ER
or ERß promoter were cotransfected with an expression
vector for ß-gal. ß-gal Activity was measured in a separate aliquot
of lysate by incubating with a luminescent ß-gal substrate for 1
h at room temperature and then measuring luminescence for 5 sec. The
luciferase activity was normalized to ß-gal activity within the same
well. In experiments done in CHO cells, luciferase activity was
normalized to total protein levels in each well because of inconsistent
ß-gal expression.
Immunoprecipitation and Western Blotting
Whole cell extracts (WCE) from primary luteinized granulosa
cells and cell lines were prepared by lysing cells in RIPA buffer (1x
PBS, 1% Nonidet, 0.5% sodium deoxycholate, 0.1% SDS) containing 1
µM sodium orthovanadate, 10 µg/ml phenylmethylsulfonyl
fluoride, and 30 µl/ml aprotinin. For immunoprecipitation, 500 µg
of WCE were incubated with 4 µl anti-Stat5a or anti-Stat5b antibodies
for 1 h at 4 C. Protein A/G agarose beads were added, and the
mixture was incubated overnight at 4 C on a rocking platform. The beads
were washed four times in PBS, resuspended in 2x electrophoresis
buffer, and boiled for 5 min. For Western blots performed on WCE,
protein was diluted in an equal volume of 2x electrophoresis buffer
and boiled for 5 min. Twenty microliters of immunoprecipitated protein
or 20 µg of WCE were separated on a 10% SDS-PAGE gel and transferred
to a nitrocellulose membrane. Western blotting was performed using
protocols provided with the Stat5 antibodies.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Drs. George Kuiper and Jan-Ake Gustafsson for
sharing the information on the mouse promoter sequences before their
publication and Dr. Alice Mui for the dominant negative Stat5a.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grants HD-11119 (to G.G.), HD-12356 (to
G.G.), HD-36879 (to O.-K.P.-S.), and HD-01135 (to O.-K.P.-S.).
Abbreviations: CA-5a, CA-5b, Constituitively activated Stat5a
and Stat5b; DN-5a, dominant negative Stat5a; ß-gal,
ß-galactosidase; GAS,
-interferon-activating sequence; Jak2, janus
kinase 2; Mut-5b, mutant Stat5b; PRL-R, PRL receptor;
PRL-RCA , constitutively active PRL-R; Stat5, signal
transducer and activator of transcription 5; WCE, whole-cell
extracts.
Received for publication May 23, 2001.
Accepted for publication August 28, 2001.
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