Received for publication, December 4, 2002, and in revised form, February 6, 2003
Antiestrogen compounds exhibit a variety of
different effects in different tissues and are widely used for the
treatment of osteoporosis, breast cancer, and other diseases. Upon
examining the molecular mechanisms, we found that Smad4, a common
signal transducer in the bone morphogenetic protein
(BMP)/transforming growth factor-
(TGF-
) signaling pathway,
functions as a transcription corepressor for human estrogen receptor
(ER
). Endogenous ER
was co-immunoprecipitated with
Smad4, and the interaction was induced by antiestrogen ligands such as
tamoxifen, raloxifene, and droloxifen, which was confirmed in chromatin
immunoprecipitation assays. Smad4 and ER
form a complex when ER
binds to the estrogen-responsive element within the estrogen target
gene promoter. Importantly, the expression of Smad4 inhibits both
antiestrogen-induced luciferase activity and estrogen downstream target
gene transcription in breast cancer cells. Mapping of the interaction
domains indicates that the activation function 1 (AF1) domain of ER
is essential for its interaction with Smad4, while the MH1 domain and
linker region of Smad4 are essential for the interaction. Our findings represent a novel mechanism that TGF-
may regulate cell fate through
Smad4-mediated cross-talk with estrogen.
 |
INTRODUCTION |
Estrogen regulates cell proliferation, differentiation, motility,
and apoptosis in a variety of cell types (1, 2). The effects of
estrogen, including breast cancer, are mediated through its binding to
estrogen receptors (3). The estrogen receptor (ER),1 of which two isoforms
(
and
) have been identified, are members of the steroid/thyroid
hormone superfamily of nuclear receptors (4-8). ER
and ER
display distinct expression patterns and biological functions in
different tissues (9). Both of the two receptors are
ligand-dependent transcriptional activators and regulate
gene transcription either by binding directly to the
estrogen-responsive element with a consensus sequence of
5'-GGTCAnnnTGACC-3' (10) or interacting with other transcription
factors such as Sp1 and AP1 (10). Estrogen receptors recruit
coactivators and corepressors in the regulation of gene transcription.
Numerous coactivators have been shown to be associated with activated
ER, such as CBP/p300 (11-13), SRC-1 (14, 15), TIF2 (16), GRIP1 (17,
18), TIF1 (19, 20), and RIP140 (21, 22). Fewer corepressors have been
reported to date, among which SMRT (23, 24) and N-CoR (25, 26) are the
important ones. These cofactors may function as molecular gates to
enable integration of diverse signal transduction pathways at nuclear
receptor-regulated promoters (27).
A relatively new class of synthetic drugs known as selective estrogen
receptor modulators or SERMs are currently in use for treatment of
osteoporosis, breast cancer, and other hormone-dependent medical disorders (3). SERMs exhibit a wide range of estrogen-like and
antiestrogen actions based on the target tissue being studied (28). For
example, tamoxifen is an ER antagonist in breast tissue (29) but an ER
agonist in bone (30) and uterine (31) tissue. Raloxifene, a compound
related to tamoxifen, is also an ER antagonist in breast tissue;
however, it exerts agonist activity in bone but not uterine tissue
(28). It has been believed that novel and as yet unidentified cofactors
bind to the antiestrogen-modulated ER and are responsible for the
observed tissue-specific activity (10).
In contrast to estrogen promotion of the development of breast cancer,
transforming growth factor
(TGF-
) is known to inhibit the growth
of mammary epithelial cells and may play a protective role in mammary
carcinogenesis (32). TGF-
is the prototypic autocrine or paracrine
inhibitor of cell cycle progression in epithelial cells and appears to
directly antagonize the effects of many different mitogenic growth
factors (33, 34). Loss of responsiveness to TGF-
is believed to be a
major factor in tumor formation (35-43). Specific defects in TGF-
receptors (e.g. TGF-
-related signal transduction/gene
activation) and TGF-
-regulated cell cycle proteins have been
implicated in oncogenesis (35-43). Alterations of TGF-
signal
components have occurred in some breast cancer cell lines, and these
may contribute to tumor formation and proliferation (44-47).
Additionally, inhibition of breast cancer cell growth by tamoxifen is
mediated by TGF-
(44-48). TGF-
1 inhibition of ER
transcription expression occurs within 3 h in MCF-7 breast cancer
cells (49). These data suggest that cross-talk between estrogen
receptors and TGF-
signaling is critical in breast cancer development.
The signaling responses to TGF-
s are mediated by their intracellular
substrates, the Smad proteins (34, 50). Upon ligand binding, the
activated receptors directly phosphorylate regulatory Smads (R-Smads).
Phosphorylated R-Smads interact with Smad4, and the complex
translocates into the nucleus to regulate gene transcription. Smad4 is
the common signaling molecule shared in the TGF-
superfamily. Smad4/DPC4 is also known as a tumor suppressor (37),
mutations of which are frequently found in human cancers. Smad4
mutations account for half of pancreas cancers (37, 38) and more than 30% of invasive metastatic colorectal cancers (39) and other tumors.
Oncogene Jab1 mediates the degradation of Smad4 (51). It has been shown
that Smad1, Smad2, Smad3, and Smad4 all have physical interactions with
ER
and might be important for TGF-
regulation of ER
signaling
(52, 53). Among these Smads, Smad3 and Smad4 have also been studied in
another steroid receptor, the androgen receptor, and androgen
receptor-mediated signaling and important functions have been suggested
(54). However, the possible significance of Smad4 in estrogen
signaling has not been well assessed, and the detailed mechanism of the
cross-talk is not clear.
In this study, we examined the potential role of Smad4 in estrogen
signaling. We demonstrate that Smad4 interacts with ER
in
vivo and represses estrogen-induced transcriptional activity mediated through the ERE. Smad4 is incorporated in the ER
-DNA complex and might recruit other corepressors to the complex. Finally, we show that Smad4 strongly inhibits the transcription of various downstream genes of estrogen signaling. These findings suggest that
TGF-
may regulate cell fate through Smad4-mediated cross-talk with
estrogen and may provide a molecular basis to explain the wide range of
SERM-mediated, tissue-selective effects in breast cancer patients.
 |
EXPERIMENTAL PROCEDURES |
Constructs and Cell Culture--
In the yeast two-hybrid assay,
we cloned the full-length Smad4 coding sequence into pGBT9
(Clontech) to generate the pGBT9-Smad4 bait plasmid
in which Smad4 is fused with the Gal4 DNA binding domain. Likewise, the
human ER
was cloned into pACT2 (Clontech) and
fused with the VP16 activation domain (pACT2-hER
). The HA-tagged human ER
expression plasmid was cloned into a pCDNA3 vector
linked with HA at the amino terminus by PCR using pRST7-ER
(a gift
from the National Institutes of Health) as the template. The AF1 (E2), AF1 + DNA binding domain (DBD) (E1), DBD + AF2 (E3), and AF2 (E4) domains of ER
were cloned into the same pCDNA3 vector by PCR and
restriction enzyme digestion in BamHI and XhoI
sites. An estrogen-response element-containing reporter (3× ERE-TATA)
was a gift from Dr. Valerie Clack (Duke University Medical Center,
Durham, NC). The FLAG-tagged Smad4 expression plasmid was a gift from
Dr. Rik Derynk (University of California, San Francisco, CA). We cloned
the MH1, MH1 + linker, linker, MH2 + linker, and MH2 domains of Smad4
into a pCMV5 vector linked with FLAG at the carboxyl terminus by PCR. COS-1 cells were incubated in Dulbecco's modified Eagle's medium supplemented with antibiotics and 10% fetal bovine serum at 37 °C
in 5% CO2. MCF-7 and T47D cells were bought from the
American Type Culture Collection and maintained according to the
manufacturer's instructions.
Yeast Two-hybrid--
According to the manufacturer's
instruction (Clontech) and previous work (51), the
interaction was quantified by a liquid
-galactosidase assay when
ER
and Smad4 were coexpressed. pACT2 and pGBT9 vectors were also
examined as negative controls. The stimulation of
-galactosidase
activity indicates interaction between these two overexpressed proteins.
Coimmunoprecipitation Assay--
COS-1 cells were split and
plated at 1 × 106 cells per 100-mm dish and starved
with phenol red-free Dulbecco's modified Eagle's medium supplemented
with 10% charcoal-stripped fetal bovine serum (Cellgro) for 24 h.
HA-tagged hER
was cotransfected with FLAG-tagged Smad4 into COS-1
cells using Tfx-50 according to the manufacturer's instructions
(Promega). The cells were treated with different ligands as follows: 1 nM
-estradiol (E2); 100 nM
tamoxifen (Tam); 10 nM raloxifene (Ral); 10 nM
droloxifen (Dro); 2 ng/µl TGF-
1; or an ethanol (EtOH) vehicle
(control) for 24 h. Forty-eight hours after transfection, the
cells were lysed with 0.6 ml radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium
deoxycholate, 0.1% SDS, 2 mM EDTA, and 150 mM
NaCl) with freshly added inhibitors (0.01 mg/ml aprotinin, 0.01 mg/ml
leupeptin, 0.1 mM Na3VO4, and 0.1 mM phenylmethylsulfonyl fluoride). Anti-FLAG monoclonal
antibody (Sigma) was added into the 0.2-ml cell lysate of the protein
samples and rotated at 4 °C for 3 h. The protein complexes were
immunoprecipitated by protein G-Sepharose beads (Amersham Biosciences),
and the samples were loaded and run in a 7% SDS-PAGE gel. The
precipitates were transferred to a nitrocellulose membrane and
immunoblotted by anti-HA polyclonal antibody (Berkeley Antibody).
Likewise, the immunoprecipitation was also done with anti-HA polyclonal
antibody and immunoblotted with an anti-FLAG monoclonal antibody. The
truncated HA-hER
or FLAG-Smad4 expression plasmids were also
transfected into COS-1 cells, and co-immunoprecipitation was done with
the same method. MCF-7 or T47D cells were split and plated at 80%
confluence per 100-mm dish and starved with RPMI 1640 phenol red-free
medium supplemented with 10% charcoal-stripped fetal bovine serum
(Cellgro) for 24 h. The aliquots of cells were treated with
ligands and lysed in 0.6 ml of RIPA buffer (with freshly added
inhibitors) 24 h after treatment. Anti-Smad4 monoclonal antibody
(Santa Cruz Biotechnology) was added to 0.5-ml cell lysates for the
immunoprecipitation, and the immunoblotting was done with anti-hER
polyclonal antibody (Santa Cruz Biotechnology).
Transfection and Luciferase Assay--
To identify the function
of Smad4 on estrogen response element, 2× ERE-TATA was used as the
reporter plasmid. MCF-7 cells were split and plated at 5 × 104 cells per 24-well plate and starved with RPMI 1640 phenol red-free medium supplemented with 10% charcoal-stripped fetal
bovine serum (Cellgro) for 24 h. The cells were transfected using
Tfx-50 with 0.2 µg of luciferase reporter and 20 ng of the hER
expression plasmid. 50 ng of Smad4 expression plasmid or empty vector
was also cotransfected. Twenty-four hours after transfection, the aliquots of cells were treated with one of the ligands, i.e.
E2, Tam, Ral, Dro, and TGF-
1 or vehicle. Luciferase
activities were assayed 48 h after transfection using the
Dual-LuciferaseTM assay kit (Promega) according to the
manufacturer's instructions. Firefly luciferase activity
was assayed and normalized against Renilla luciferase
activity. Luciferase values shown in the figures are representative of
transfection experiments performed in triplicate from at least three
independent experiments. The standard deviations are shown in vertical lines.
Gel-shift Assay--
The expression plasmids of human ER
and
Smad4 were cotransfected into COS-1 cells. Forty-eight hours after the
transfection, the transfected cell lysate and an aliquot of
non-transfected cell lysate were collected and incubated with
radiolabeled ERE, which is the probe in gel-shift assays. The ERE DNA
oligomers were radiolabeled by a kinase reaction with a DNA polymerase
I large (Klenow) fragment and [
-32P]dCTP. Binding
reactions were preincubated for 20 min at 22 °C with indicated
lysates in 75 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10 mM Tris-HCl, pH 7.5, 6%
of bovine serum albumin, and 25 ng of poly(dIdC) in a volume of
19 µl. One microliter of DNA probe (0.5 ng, 50,000-100,000 cpm) was
added to each of the reactions. The reactions were subjected to
nondenaturing electrophoresis on a 4% polyacrylamide gel.
Chromatin Immunoprecipitation (ChIP) Assay--
The MCF-7 cells
were grown to 95% confluences in phenol red-free Dulbecco's modified
Eagle medium supplemented with 10% charcoal-dextran-stripped fetal
bovine serum for at least 3 days. Following the addition of ligands for
3 h, cells were washed and lysed according to the protocol from
Dr. Myles Brown (Harvard Medical School, Boston, MA).
Immunoprecipitation was performed overnight at 4 °C with specific
antibodies against ER
or Smad4. The precipitates were extracted and
heated as described (55). For PCR, (the sequences of specific primers
were given as a gift from Dr. Myles Brown) 1 µl from a 50-µl DNA
extraction and 21-25 cycles of amplification were used.
Principle of Real Time PCR--
A fluorescence signal from each
real time quantitative PCR reaction (Applied Biosystems, Foster City,
CA) is collected as normalized values plotted versus the
cycle number. Reactions are characterized by comparing threshold cycle
(C(t)) values. The C(t) is a value defined by the fractional cycle
number at which the sample fluorescence signal passes a fixed threshold
above base. Quantitative values are obtained from the C(t) numbers at which the increase in signal associated with an exponential growth of
PCR product starts to be detected (Applied Biosystems) according to the
manufacturer's manual. The
-actin gene is used as a control for the
endogenous RNA transcripts, and each sample was normalized by its
-actin content. The final result is expressed as relative fold by
comparing the amount of RNA of target gene to the
-actin gene, which
is determined by the equation 2
(C(t)target
C(t)
-actin).
Oligonucleotide Primers Design--
The target cDNA sequence
was evaluated using Primer3
(www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The sequences of primers are listed as follows:
-actin upper primer, 5'-AGA CTT
CGA GCA GGA GAT GG-3';
-actin lower primer, 5'-CGG ATG TCA ACG TCA
CAC TT-3'; CATD upper primer, 5'-TCT GTG GAG GAC CTG ATT GC-3'; CATD
lower primer, 5'-AGG TTG GAG GAG CCC GTG T-3'; EBAG9 upper primer,
5'-CGT CGG CTT GTA TAA CCT-3'; and EBAG9 lower primer, 5'-TTA ATT TCC
GTC CTC TGC-3'. The forward and reverse primers were designed to flank
at least one intron to prevent amplification of genomic DNA that
may be contained in samples.
RNA Extraction and PCR Amplification--
Total RNA was
extracted from T47D cells by using STAT-60 (TEL-TEST Inc.). The quality
of the RNA samples was determined by electrophoresis through agarose
gels and staining with ethidium bromide. The 18 and 28 S RNA bands were
visualized under UV light. Equal amounts of total RNA were
reverse-transcribed to cDNA by TaqMan® Reverse Transcription
Reagents (Applied Biosystems) according to manufacturer's protocol.
PCR was performed using the SYBR® Green PCR Master Mix (Applied
Biosystems). The amplification reactions were performed in 25 µl of
final volume containing 1× SYBR buffer, 0.4 µM primer
mixture, and 3 µl cDNA. To reduce variability between replicates,
PCR premixes, which contain all reagents except for cDNA, were
prepared and aliquoted into 0.2-ml thin-wall strip tubes (MJ Research,
Cambridge, MA). The thermal cycling conditions comprised an initial
denaturation step at 95 °C for 10 min and 40 cycles at 95 °C for
30s and 60 °C for 1min. For each set of primers, PCR thermal cycle
conditions have been optimized to achieve single band PCR product in
1% agarose gel with ethidium bromide staining. Experiments were
performed with duplicates for each data point. All of the PCR reactions
were performed using OPTICONTM DNA Engine (MJ
Research).
 |
RESULTS |
Smad4 Interacts with ER
in Vivo--
To study the potential
cross-talk between estrogen receptor and Smads, we first examined the
interaction between Smad4 and ER
in a yeast two-hybrid system
because Smad4/DPC4 has been identified as a candidate tumor
suppressor (37). Human ER
was cloned into the pACT2 plasmid, and
human Smad4 was cloned into the pGBT9 plasmid.
-galactosidase
activity was stimulated when both ER
and Smad4 were expressed (Fig.
1A), indicating Smad4
interaction with ER
in yeast. To examine the physical interaction in
mammalian cells, we performed a coimmunoprecipitation experiment in
COS-1 cells. HA-tagged ER
and FLAG-tagged Smad4 were cotransfected
into COS-1 cells in the presence of different ligands. The interaction
was detected by immunoprecipitation with anti-FLAG monoclonal antibody followed by immunoblotting with an anti-HA polyclonal antibody (Fig.
1B). We confirmed the result by immunoprecipitation with an
anti-HA antibody followed by immunoblotting with an anti-FLAG antibody
(Fig. 1C). The interaction was independent of
-estradiol E2 and antiestrogen ligands in both assays.

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Fig. 1.
Interaction between Smad4 and
ER . A, -galactosidase
activity was stimulated when both ER and Smad4 were expresseds,
indicating Smad4 interaction with ER . PACT2 and pGBT9 vectors were
also examined as negative controls. B, FLAG-tagged Smad4
(lanes 1 and 3-7) and HA-tagged hER
(lanes 2-7) or empty pCDNA3 vector (lanes 1 and 2) were cotransfected into COS-1 cells treated with
E2 (lane 4), Tam (lane 5), Ral
(lane 6), Dro (lane 7) or ethanol (EtOH) vehicle
(control) (lanes 1-3). Cell lysates were immunoprecipitated
by anti-FLAG M2 monoclonal antibody 48 h after transfection. The
precipitates were then immunoblotted with anti-HA polyclonal antibody.
The result was confirmed in panel C by immunoprecipitation
with anti-HA polyclonal antibody and immunoblotting with anti-FLAG
monoclonal antibody.
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To further confirm the endogenous protein interaction between Smad4 and
ER
in breast cancer cells, co-immunoprecipitation assays were
performed with MCF-7 cells and T47D cells. The aliquot of cells was
treated with one of the ligands as indicated in Fig. 2. The whole cell lysates were
immunoprecipitated with anti-Smad4 monoclonal antibody, and the
precipitates were immunoblotted with an anti-ER
polyclonal antibody.
The endogenous ER
and Smad4 were coimmuno- precipitated in MCF-7
cells as expected (Fig. 2A, lane 2), indicating
the interaction between these two endogenous proteins. Moreover, the
interaction was enhanced to different levels with the treatment of
different antiestrogen ligands (Fig. 2A, lanes
4-6). Unlike the case in MCF-7 cells, however, the endogenous interaction was not detectible in T47D cells even with E2
treatment (Fig. 2B, lanes 3-4), which can be due
to the lower expression level of endogenous Smad4 proteins in T47D
cells. Interestingly, only antiestrogen ligands induced the interaction
(Fig. 2B, lanes 5-7). Taken together, these
results suggest that the interaction between endogenous Smad4 and ER
proteins are differentially regulated by SERMs.

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Fig. 2.
Smad4 interacts with ER
in breast cancer cells. T47D cells (A), or MCF-7
cells (B), were treated with E2 (lane
4), Tam (lane 5), Ral (lane 6), Dro
(lane 7) or vehicle (lanes 1-3). Cell
lysates were immunoprecipitated by an anti-Smad4 monoclonal antibody
(Smad4) (lanes 1 and 3-7) or pre-immunized
antibody (Pre) (lane 2) (control) 48 h after
transfection. The precipitates were then immunoblotted with an
anti-hER polyclonal antibody.
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Smad4 Represses Transactivation by Estrogen--
To examine the
function of the interaction between Smad4 and ER
, a luciferase
reporter plasmid bearing two repeats of ERE (2× ERE-TATA) was
cotransfected into MCF-7 cells with ER
. The aliquots of cells were
treated with 17
-estradiol (E2), TGF-
1, or both. Fig.
3A shows that TGF-
1
inhibited E2-induced transcription activity as expected.
The ectopic expression of Smad4 exhibited a stronger inhibition of
E2-induced luciferase activity in comparison with TGF-
1.
The inhibition was further demonstrated in a dose-dependent manner (Fig. 3B). Because different SERMs exhibit
tissue-specific transcription activity, the antiestrogen compounds
tamoxifen, raloxifene, and droloxifen were also examined. As expected,
binding of these ligands to ER
did not result in luciferase
activation. In the presence of Smad4, however, the transcription levels
were suppressed by these antiestrogen ligands (Fig. 3C).
Raloxifene appears to be the most potent inhibitor of luciferase
activity, exhibiting ~3-fold higher levels of inhibition than the
control vehicle. Consistent with previous data that SERMs can affect
the interaction between Smad4 and ER
, these results indicate that Smad4 acts as an ER
transcription corepressor, and its repression activity is differentially regulated by different SERMs. Similar results were obtained in T47D cells (data not shown).

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Fig. 3.
Effect of Smad4 on
ER -mediated transcriptional activity. A
luciferase reporter plasmid containing estrogen response element (2×
ERE-TATA) was cotransfected with human ER into the MCF-7 cells.
A, the effect of Smad4 on ER transactivation by estrogen
was examined. The aliquot of cells was treated with E2,
TGF- 1, E2, and TGF- 1 or ethanol vehicle (control).
The values represent artificial units of luciferase activity after
normalization. B, increased amounts of Smad4 were
cotransfected, and luciferase activity was assayed. C, the
effect of Smad4 on ligand-mediated ER transactivation was examined.
The MCF-7 cells were cotransfected with 2× ERE-TATA reporter, ER
(open bar), ER and Smad4 (closed
bar). The aliquot of cells was treated with one of the four
ligands, i.e. E2, Tam, Ral, Dro or vehicle.
Luciferase activity was assayed and compared. D, the effect
of Smad4 on Smad3-enhanced ER transactivation was examined.
Increased amounts of Smad4 were cotransfected with fixed amount of
Smad3. The aliquot of cells was treated with E2 and
TGF- 1. Luciferase activity was assayed and analyzed as
described.
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Previous reports showed that ER-mediated transcriptional activation can
be enhanced by Smad3 (52, 53) by physical interaction between Smad3 and
ER
. Because TGF-
induces the formation of a Smad3 and Smad4
complex (58), we examined the effect of Smad4 on Smad3-enhanced ER
transactivation in MCF-7 cells. As shown in Fig. 3D, Smad3
increased E2 activity on ER
transcription in the
presence of TGF-
1. The addition of Smad4, however, can repress the
Smad3-enhanced ER
transactivation in a dose-dependent
manner. It is also noteworthy that the amounts of Smad4 used in this
assay are significantly lower than that of Smad3. Together, the results from luciferase assays indicate that Smad4 is a corepressor of ER
and suppresses Smad3-mediated ER
transactivation.
Smad4 Forms a Complex with ER
on DNA Element--
Because ER
can regulate gene transcription via direct binding to the ERE on the
target gene promoter, we tested whether Smad4 and ER
form a complex
on ERE using gel-shift assays. Smad4 and ER
expression plasmids were
cotransfected into COS-1 cells, and the complex of Smad4-ER
formed a
shift band (Fig. 4A,
lane 3) when compared with non-transfected COS-1 cell lysate
(Fig. 4A, lane 2). The addition of antibodies
against ER
or Smad4 supershifted the complex (Fig. 4A,
lanes 4 and 5), confirming that the complex contains Smad4 and ER
. This result argues against the idea that Smad4 interferes with the DNA binding of ER
. Genes regulated through direct ER binding include cathepsin D
(CATD) (59) and ER binding fragment-associated
antigen 9 (EBAG9) (50, 60). To confirm Smad4-ER
-DNA
complex formation in vivo, we applied a chromatin
immunoprecipitation assay (55) with the promoter region of
CATD containing ERE (55). MCF-7 cells were grown in the
absence of estrogen for at least 3 days followed by treatment with
vehicle or different ligands. The endogenous transcription complex
present on the ERE region of the promoter was determined by
immunoprecipitation using antibodies against Smad4 or ER
, followed
by semi-quantitative reverse transcriptions PCR with specific pairs of
primers spanning the ERE region. As shown in Fig. 4B, the
occupancy by both ER
and Smad4 indicates the formation of the
Smad4-ER
complex on the CATD gene promoter. Treatment with antiestrogen ligands increased the occupancy, which is consistent with our previous finding that SERMs regulate Smad4 interaction with
ER
and the repression activity of Smad4 on estrogen signaling.

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Fig. 4.
Smad4 forms a complex with
ER on DNA. A, Smad4 and ER
were cotransfected into COS-1 cells, and the cell lysates were
collected and incubated with radiolabeled ERE as a probe in the
presence or absence of anti-hER (lane 4) and/or
anti-Smad4 (lane 5) antibodies as indicated. The
Smad4/hER complexes are indicated in lanes 3-5.
B, MCF-7 cells were starved for 3 days and treated with
different ligands (E2, Tam, and Ral) for 3 h. The cell
lysates were collected and immunoprecipitated with anti-ER and
anti-Smad4 monoclonal antibodies, respectively. Semi-quantitative
reverse transcription PCR was performed with the precipitates and
specific primers for the CATD promoter region as indicated.
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Interaction Domains of Smad4 and ER
--
We mapped the
interaction domain(s) of both Smad4 and ER
. ER
has A to F domains
from the NH2 terminus to COOH terminus containing two
important activation function (AF) domains, AF1 and AF2 (61-63). A
series of truncated ER
constructs in HA-tagged pCDNA3 vector have been cloned, with deletion of either the AF1 or the AF2 domain as
shown in Fig. 5A. HA-tagged
ER
(E) or one of its four deletion mutants was cotransfected into
COS-1 cells with FLAG-tagged Smad4. The immunoprecipitation was
performed with either anti-FLAG antibody or anti-HA antibody, followed
by immunoblotting with anti-HA or anti-FLAG antibody, respectively. It
has been shown in Fig. 5B that the mutants E3 and E4 in
which AF1 domain is deleted are unable to bind Smad4, whereas E1 and E2
lacking either AF2 or AF2 + DBD retained the interaction. These results
indicate that AF1 domain is essential for the interaction of ER
with
Smad4, whereas the AF2 domain or the DBD is not required.

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Fig. 5.
Mapping the interaction domains of ER .
A, the protein structure of ER is shown. The
numbers indicate the locations of the amino acids at the boundaries of
each domain. Four deletion mutants used in the following experiments
are aligned. E, E1, E2, E3,
and E4 denote full-length ER , the AF1 domain + DBD, the
AF1 domain, DBD + the AF2 domain, and the AF2 domain, respectively.
B, the interaction of FLAG-Smad4 with various ER deletion
mutants was examined by immunoprecipitation with anti-FLAG or anti-HA
antibody followed by immunoblotting with anti-HA or anti-FLAG antibody
in COS-1 cells. The top two panels show the interaction, and
the bottom two panels show the expression of each protein as
indicated.
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We next determined the domain(s) of Smad4 which mediate(s) the
interaction with ER
, using MH1, linker (L), MH1 + linker (m1), MH2 + linker (m2), or MH2 (m3) truncated fragments of Smad4 (Fig. 6A). FLAG-tagged Smad4 (S4) or
one of its five fragments was cotransfected into COS-1 cells with
ER
. Cells were lysed and subjected to immunoprecipitation with
anti-FLAG antibody, followed by immunoblotting with anti-HA antibody.
As indicated in Fig. 6B, the MH1 domain and linker region were both sufficient to interact with ER
, whereas the MH2 domain was
unable to bind ER
. As expected, the MH2 domain could not inhibit
E2-induced transactivity whereas the MH1 domain and linker region retained the ability of inhibition (Fig. 6C).

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Fig. 6.
Mapping the interaction domains of
Smad4. A, the protein structure of Smad4 is shown. The
five deletion mutants used are aligned. S4, MH1,
m1, m2, m3, and L denote
full-length Smad4, the MH1 domain, the MH1 domain + linker, the MH2
domain + linker, and the MH2 domain and linker, respectively.
B, the interaction of ER with various Smad4 deletion
mutants was examined by immunoprecipitation using anti-FLAG antibody
followed by immunoblotting with anti-HA antibody. The top panel
shows the interaction, and the bottom two panels show
the expression of each protein as indicated. C, the Smad4
deletion mutants were cotransfected into MCF-7 cells with 2× ERE-TATA
luciferase reporter, and luciferase activity was assayed.
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Smad4 Inhibits Downstream Gene Expression of Estrogen
Signaling--
To further confirm the repression effects of Smad4 on
ER transactivation, we applied real time PCR to examine the mRNA
expression level of the ER target gene CATD and EBAG9. T47D cells were
transfected with Smad4 expression plasmids or empty vector as a
control. The aliquot of cells was treated with 100 nM
E2 for 24 h before RNA extraction and PCR assays. As
Fig. 7 shows, E2 can induce
the mRNA expression of both CATD (Fig. 7A) and EBAG9
(Fig. 7B). Ectopic Smad4 decreases the E2
induction of both genes without changing their basal expression levels,
confirming that Smad4 can suppress estrogen signaling in breast cancer
cells.

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|
Fig. 7.
Smad4 inhibits E2-induced gene
activation. T47D cells were plated in 6-well dishes and
transfected with Smad4 expression plasmids or empty pCDNA3 vector
(control plasmid). The aliquot of cells were treated with
E2 or ethanol vehicle ( ) as indicated. CATD
(A) and EBAG9 (B) expression was monitored by
reverse transcription and real time PCR. The expression of -actin
was also examined as control. The relative amounts of mRNA of CATD
and EBAG9 to -actin were calculated using the equation 2 (C
(t) target C (t) -actin). Experiments were performed in
triplicate for each data point, and standard deviations are shown in
vertical lines.
|
|
 |
DISCUSSION |
Estrogen regulates cell proliferation, differentiation, motility,
and apoptosis in a variety of different cell types. It plays important
roles not only in normal mammary gland cells but also in breast cancer
cells. Recent studies indicate that various ligands, including SERMs,
induce distinct conformational changes in the ER (64, 65). These
changes in ER may, in turn, alter the interactions of the receptor with
cell- and tissue-specific coactivating or corepressing transcription
factors (4, 10). Here we have identified Smad4 as an ER
corepressor
and have found that antiestrogens enhance the endogenous interactions
between Smad4 and ER
. This finding provides further evidence
to support the belief that ER conformational changes regulate the
interaction of ER with its cofactors and may also help to explain the
diversity of estrogen biological activity.
Smad4 is a common partner for the signaling molecules in the TGF-
pathway. Identification of Smad4 as an ER
corepressor provides a
mechanism of cross-talk between TGF-
and estrogen. Consistent with
the previous observation (52), our data also demonstrated that Smad3
enhanced ER-mediated transactivation. Interestingly, Smad4 can reverse
Smad3-enhanced gene transcription. Both Smad3 and Smad4 can interact
with ER
independently. It appears that Smad3 alone activates
ER-mediated gene transcription, whereas TGF-
or the Smad3 and Smad4
complex inhibit ER-mediated transcription. As we know, TGF-
induces
Smad3 phosphorylation, which forms a complex with Smad4 and
translocates into the nucleus in regulation of gene transcription. So
our results indicate that, in the presence of Smad4, TGF-
inhibits
ER-induced transactivation. When Smad4 is mutated or deleted in cancer
cells, TGF-
is no longer able to inhibit ER-induced gene
transcription and likely activates gene transcription instead. If the
findings are confirmed to be true in clinical samples, Smad4 may be
used for a therapeutic purpose.
As our data have shown, antiestrogens can induce the interaction
between endogenous Smad4 and ER
in T47D cells, which have a low
abundance of Smad3 mRNA (45), suggesting that Smad4 and ER
interaction is not likely to be dependent on Smad3. The MH2 domain of
Smad proteins are known for their interactions with other proteins
(67-69). However, our mapping data show that the Smad4 MH2 domain is
not involved in binding with ER
. Because the MH2 domain of Smad3 is
required for efficient physical and functional interaction with ER
(52,53), and the MH2 domains mediate homomeric and heteromeric complex
formation (67-69), it is possible that the interaction of Smad3 with
ER
is mediated through Smad4. Furthermore, Smad4 could also disrupt
the interaction between Smad3 and ER
by competing with the Smad3 MH2
domain binding site of ER
. In their recent work, Kang
et al. (54) have also demonstrated that Smad4 can decrease
the AR-Smad3 interaction and repress the Smad3-enhanced AR
transactivation. Nevertheless, there are many important questions that
remain to be addressed relevant to other R-Smads such as Smad2 and
Smad1 in bone morphogenetic protein (BMP) signaling.
The AF1 and AF2 domains are two important activation functional domains
of ER. AF1, which is localized in the NH2-terminal A/B
region, is believed to be constitutive in a cell- and promoter-specific manner (72), whereas AF2 resides in the COOH-terminal ligand-binding domain (LBD) (region E) and exerts estrogen-dependent
transcriptional activity by recruiting coactivators. These two domains
have been tested frequently in studying the regulation of ER function.
In mapping the ER
interaction domains with Smad4, our data
demonstrate that ER
N-terminal AF1 is sufficient to mediate ER
interaction with Smad4. Several lines of evidence indicate that ER
AF1 domain is under negative regulation. Corepressors such as N-CoR
bind to the tamoxifen-liganded ER
and inhibit AF1 activity in which the mechanism remains unknown (25, 26). It is also known that SERM
agonist effects stem from ER
AF1, which directly contacts coactivators. Interestingly, tamoxifen-liganded ER
enhances the activity of the progesterone receptor AF1, presumably by sequestration of active repressor complex, and overexpression of N-CoR reverses this
effect. It is clear that SERMs inhibit ER
action both by blocking
coactivator recruitment and promoting corepressor recruitment. Interaction of Smad4 with the ER
AF1 domain provides evidence to
explain SERM-liganded ER
transcription activity. The SERMs do not
behave the same in the presence of ER
(9), however. ER
also
inhibits ER
-dependent estrogen and tamoxifen responses in heterodimers, and it is believed that ER
homodimers respond to
SERMs differently.
We have examined potential interaction between ER
and Smad4 in
similar co-immunoprecipitation and gel shift assays. In comparison to
ER
, the interaction was not detectable, and Smad4 could not form a
complex with ER
on DNA (data not shown). ER
appears unresponsive to Smad4-mediated transcription repression induced by tamoxifen (data
not shown). Tamoxifen produces objective tumor shrinkage in advanced
breast cancer and reduces the risk of relapse in women treated for
invasive breast cancer (71). Clinically, breast cancer patients with
ER
tumors will initially benefit from tamoxifen treatment. However,
most of the patients eventually develop tamoxifen resistance, the
mechanism of which is not well understood, although several possible
mechanisms have been proposed (66, 70). It has been shown that ER
mRNA expression in primary breast tumors is up-regulated in
tamoxifen-resistant breast cancer patients (57). Clinical studies show
that the beneficial response of breast cancer to tamoxifen is related
to ER
, and ER
-positive cells are associated to elevated levels of
the proliferation markers Ki67 and cyclin A (56). Because the
resistance of breast cancer to tamoxifen therapy is correlated to the
presence of ER
(56, 57), it is likely that tamoxifen specifically
induces the interaction of ER
, instead of ER
, with some tumor
suppressors such as Smad4. Smad4 is able to regulate ER
-mediated
transcription activity by interacting with the other partner
(i.e. ER
) of the heterodimer. However, with the increased
ratio of expression level of ER
/ER
, the effect of Smad4 on ER
could be decreased; thus, the tamoxifen effect on transcription
repression would also be abolished. Likewise, the effects of Smad4 on
antiestrogen-mediated transcription can also be variable in cells with
a different ratio of ER
/ER
.
Taken together, we demonstrated an in vivo interaction
between Smad4 and ER
and identified the function of Smad4 as a
transcriptional corepressor for ER
. It would be a future focus to
investigate how the R-Smads and Smad4 are assembled in the
physiological context with mutually exclusive function of coactivation
and corepression. More detailed understanding of the regulation by
different SERMs may also be helpful in the design of novel selective
estrogen receptor modulators for the treatment of
hormone-dependent medical disorders such as breast cancer,
osteoporosis, and other pathological conditions.
We thank Dr. David Ke for the antiestrogen
compounds and Janice Walker for proofreading of the manuscript.
Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M212332200
The abbreviations used are:
ER, estrogen
receptor;
EBAG, ER binding fragment-associated antigen 9;
TGF-
, transforming growth factor-
;
SERM, selective estrogen receptor
modulator;
ERE, estrogen-response element;
Tam, tamoxifen;
Ral, raloxifene;
Dro, droloxifen;
E2,
-estradiol;
AF1, activation function 1;
DBD, DNA-binding domain;
MH1, Mad homology 1;
R-Smad, regulatory Smad;
HA, hemagglutinin;
CATD, cathepsin D.
1.
|
Mangelsdorf, D. J.,
Thummel, C.,
Beato, M.,
Herrlich, P.,
Schutz, G.,
Umesono, K.,
Blumberg, B.,
Kastner, P.,
Mark, M.,
and Chambon, P.
(1995)
Cell
83,
835-839[Medline]
[Order article via Infotrieve]
|
2.
|
Couse, J. F.,
and Korach, K. S.
(1999)
Endocr. Rev.
20,
358-417[Abstract/Free Full Text]
|
3.
|
Osborne, C. K.,
Zhao, H.,
and Fuqua, S. A.
(2000)
J. Clin. Oncol.
18,
3172-3186[Abstract/Free Full Text]
|
4.
|
Chen, H.,
Lin, R. J.,
Xie, W.,
Wilpitz, D.,
and Evans, R. M.
(1999)
Cell
98,
675-686[Medline]
[Order article via Infotrieve]
|
5.
|
Evans, R. M.
(1988)
Science
240,
889-895[Medline]
[Order article via Infotrieve]
|
6.
|
Beato, M.,
Herrlich, P.,
and Schutz, G.
(1995)
Cell
83,
851-857[Medline]
[Order article via Infotrieve]
|
7.
|
McKenna, N. J.,
Xu, J.,
Nawaz, Z.,
Tsai, S. Y.,
Tsai, M. J.,
and O'Malley, B. W.
(1999)
J. Steroid Biochem. Mol. Biol.
69,
3-12[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Klinge, C. M.
(2000)
Steroids
65,
227-251[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Hall, J. M.,
Couse, J. F.,
and Korach, K. S.
(2001)
J. Biol. Chem.
276,
36869-36872[Free Full Text]
|
10.
|
Shang, Y.,
and Brown, M.
(2002)
Science
295,
2465-2468[Abstract/Free Full Text]
|
11.
|
Chrivia, J. C.,
Kwok, R. P.,
Lamb, N.,
Hagiwara, M.,
Montminy, M. R.,
and Goodman, R. H.
(1993)
Nature
365,
855-859[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Kamei, Y.,
Xu, L.,
Heinzel, T.,
Torchia, J.,
Kurokawa, R.,
Gloss, B.,
Lin, S. C.,
Heyman, R. A.,
Rose, D. W.,
Glass, C. K.,
and Rosenfeld, M. G.
(1996)
Cell
85,
403-414[Medline]
[Order article via Infotrieve]
|
13.
|
Smith, C. L.,
Onate, S. A.,
Tsai, M. J.,
and O'Malley, B. W.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8884-8888[Abstract/Free Full Text]
|
14.
|
Onate, S. A.,
Tsai, S. Y.,
Tsai, M. J.,
and O'Malley, B. W.
(1995)
Science
270,
1354-1357[Abstract]
|
15.
|
Takeshita, A.,
Yen, P. M.,
Misiti, S.,
Cardona, G. R.,
Liu, Y.,
and Chin, W. W.
(1996)
Endocrinology
137,
3594-3597[Abstract]
|
16.
|
Voegel, J. J.,
Heine, M. J.,
Zechel, C.,
Chambon, P.,
and Gronemeyer, H.
(1996)
EMBO J.
15,
3667-3675[Abstract]
|
17.
|
Hong, H.,
Kohli, K.,
Garabedian, M. J.,
and Stallcup, M. R.
(1997)
Mol. Cell. Biol.
17,
2735-2744[Abstract]
|
18.
|
Hong, H.,
Kohli, K.,
Trivedi, A.,
Johnson, D. L.,
and Stallcup, M. R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4948-4952[Abstract/Free Full Text]
|
19.
|
Thenot, S.,
Henriquet, C.,
Rochefort, H.,
and Cavailles, V.
(1997)
J. Biol. Chem.
272,
12062-12068[Abstract/Free Full Text]
|
20.
|
Cavailles, V.,
Dauvois, S.,
L'Horset, F.,
Lopez, G.,
Hoare, S.,
Kushner, P. J.,
and Parker, M. G.
(1995)
EMBO J.
14,
3741-3751[Abstract]
|
21.
|
Le Douarin, B.,
Zechel, C.,
Garnier, J. M.,
Lutz, Y.,
Tora, L.,
Pierrat, P.,
Heery, D.,
Gronemeyer, H.,
Chambon, P.,
and Losson, R.
(1995)
EMBO J.
14,
2020-2033[Abstract]
|
22.
|
L'Horset, F.,
Dauvois, S.,
Heery, D. M.,
Cavailles, V.,
and Parker, M. G.
(1996)
Mol. Cell. Biol.
16,
6029-6036[Abstract]
|
23.
|
Horlein, A. J.,
Naar, A. M.,
Heinzel, T.,
Torchia, J.,
Gloss, B.,
Kurokawa, R.,
Ryan, A.,
Kamei, Y.,
Soderstrom, M.,
Glass, C. K.,
and Rosenfeld, M. G.
(1995)
Nature
377,
397-404[CrossRef][Medline]
[Order article via Infotrieve]
|
24.
|
Kurokawa, R.,
Soderstrom, M.,
Horlein, A.,
Halachmi, S.,
Brown, M.,
Rosenfeld, M. G.,
and Glass, C. K.
(1995)
Nature
377,
451-454[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Jackson, T. A.,
Richer, J. K.,
Bain, D. L.,
Takimoto, G. S.,
Tung, L.,
and Horwitz, K. B.
(1997)
Mol. Endocrinol.
11,
693-705[Abstract/Free Full Text]
|
26.
|
Chen, J. D.,
and Evans, R. M.
(1995)
Nature
377,
454-457[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
McKenna, N. J.,
Lanz, R. B.,
and O'Malley, B. W.
(1999)
Endocr. Rev.
20,
321-344[Abstract/Free Full Text]
|
28.
|
Black, L. J.,
Sato, M.,
Rowley, E. R.,
Magee, D. E.,
Bekele, A.,
Williams, D. C.,
Cullinan, G. J.,
Bendele, R.,
Kauffman, R. F.,
Bensch, W. R.,
Frolik, C. A.,
Termine, J. D.,
and Bryant, H. U.
(1994)
J. Clin. Invest.
93,
63-69[Medline]
[Order article via Infotrieve]
|
29.
|
Jordan, V. C.
(1992)
Cancer
70,
977-982[Medline]
[Order article via Infotrieve]
|
30.
|
Love, R. R.,
Mazess, R. B.,
Barden, H. S.,
Epstein, S.,
Newcomb, P. A.,
Jordan, V. C.,
Carbone, P. P.,
and DeMets, D. L.
(1992)
N. Engl. J. Med.
326,
852-856[Abstract]
|
31.
|
Kedar, R. P.,
Bourne, T. H.,
Powles, T. J.,
Collins, W. P.,
Ashley, S. E.,
Cosgrove, D. O.,
and Campbell, S.
(1994)
Lancet
343,
1318-1321[Medline]
[Order article via Infotrieve]
|
32.
|
Akhurst, R. J.,
and Derynck, R.
(2001)
Trends Cell Biol.
11,
S44-S51[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
Massague, J.,
and Chen, Y. G.
(2000)
Genes Dev.
14,
627-644[Free Full Text]
|
34.
|
Massague, J.
(1998)
Annu. Rev. Biochem.
67,
753-791[CrossRef][Medline]
[Order article via Infotrieve]
|
35.
|
Markowitz, S.,
Wang, J.,
Myeroff, L.,
Parsons, R.,
Sun, L.,
Lutterbaugh, J.,
Fan, R. S.,
Zborowska, E.,
Kinzler, K. W.,
Vogelstein, B.,
Brattain, M.,
and Wilson, J. K. V.
(1995)
Science
268,
1336-1338[Medline]
[Order article via Infotrieve]
|
36.
|
Grady, W. M.,
Rajput, A.,
Myeroff, L.,
Liu, D. F.,
Kwon, K.,
Willis, J.,
and Markowitz, S.
(1998)
Cancer Res.
58,
3101-3104[Abstract]
|
37.
|
Hahn, S. A.,
Schutte, M.,
Hoque, A. T.,
Moskaluk, C. A.,
da Costa, L. T.,
Rozenblum, E.,
Weinstein, C. L.,
Fischer, A.,
Yeo, C. J.,
Hruban, R. H.,
and Kern, S. E.
(1996)
Science
271,
350-353[Abstract]
|
38.
|
Goggins, M.,
Shekher, M.,
Turnacioglu, K.,
Yeo, C. J.,
Hruban, R. H.,
and Kern, S. E.
(1998)
Cancer Res.
58,
5329-5332[Abstract]
|
39.
|
Miyaki, M.,
Iijima, T.,
Konishi, M.,
Sakai, K.,
Ishii, A.,
Yasuno, M.,
Hishima, T.,
Koike, M.,
Shitara, N.,
Iwama, T.,
Utsunomiya, J.,
Kuroki, T.,
and Mori, T.
(1999)
Oncogene
18,
3098-3103[CrossRef][Medline]
[Order article via Infotrieve]
|
40.
|
Cui, W.,
Fowlis, D. J.,
Bryson, S.,
Duffie, E.,
Ireland, H.,
Balmain, A.,
and Akhurst, R. J.
(1996)
Cell
86,
531-542[Medline]
[Order article via Infotrieve]
|
41.
|
Hata, A.,
Shi, Y.,
and Massague, J.
(1998)
Mol. Med. Today
4,
257-262[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
He, W.,
Cao, T.,
Smith, D. A.,
Myers, T. E.,
and Wang, X. J.
(2001)
Oncogene
20,
471-483[CrossRef][Medline]
[Order article via Infotrieve]
|
43.
|
Zhu, Y.,
Richardson, J. A.,
Parada, L. F.,
and Graff, J. M.
(1998)
Cell
94,
703-714[Medline]
[Order article via Infotrieve]
|
44.
|
Brattain, M. G.,
Ko, Y.,
Banerji, S. S.,
Wu, G.,
and Willson, J. K.
(1996)
J. Mammary Gland Biol. Neoplasia
1,
365-372[Medline]
[Order article via Infotrieve]
|
45.
|
Pouliot, F.,
and Labrie, C.
(1999)
Int. J. Cancer
81,
98-103[CrossRef][Medline]
[Order article via Infotrieve]
|
46.
|
Kalkhoven, E.,
Roelen, B. A.,
de Winter, J. P.,
Mummery, C. L.,
van den Eijnden-van Raaij, AJ,
van der Saag, P. T.,
and van der, B. B.
(1995)
Cell Growth & Differ.
6,
1151-1161[Abstract]
|
47.
|
Sun, L.,
Wu, G.,
Willson, J. K.,
Zborowska, E.,
Yang, J.,
Rajkarunanayake, I.,
Wang, J.,
Gentry, L. E.,
Wang, X. F.,
and Brattain, M. G.
(1994)
J. Biol. Chem.
269,
26449-26455[Abstract/Free Full Text]
|
48.
|
Perry, R. R.,
Kang, Y.,
and Greaves, B. R.
(1995)
Br. J. Cancer
72,
1441-1446[Medline]
[Order article via Infotrieve]
|
49.
|
Stoica, A.,
Saceda, M.,
Fakhro, A.,
Solomon, H. B.,
Fenster, B. D.,
and Martin, M. B.
(1997)
Endocrinology
138,
1498-1505[Abstract/Free Full Text]
|
50.
|
Watanabe, T.,
Inoue, S.,
Hiroi, H.,
Orimo, A.,
Kawashima, H.,
and Muramatsu, M.
(1998)
Mol. Cell. Biol.
18,
442-449[Abstract/Free Full Text]
|
51.
|
Wan, M.,
Cao, X.,
Wu, Y.,
Bai, S.,
Wu, L.,
Shi, X.,
Wang, N.,
and Cao, X.
(2002)
EMBO Rep.
3,
171-176[Abstract/Free Full Text]
|
52.
|
Matsuda, T.,
Yamamoto, T.,
Muraguchi, A.,
and Saatcioglu, F.
(2001)
J. Biol. Chem.
276,
42908-42914[Abstract/Free Full Text]
|
53.
|
Yamamoto, T.,
Saatcioglu, F.,
and Matsuda, T.
(2002)
Endocrinology
143,
2635-2642[Abstract/Free Full Text]
|
54.
|
Kang, H. Y.,
Huang, K. E.,
Chang, S. Y.,
Ma, W. L.,
Lin, W. J.,
and Chang, C.
(2002)
J. Biol. Chem.
277,
43749-43756[Abstract/Free Full Text]
|
55.
|
Shang, Y.,
Hu, X.,
DiRenzo, J.,
Lazar, M. A.,
and Brown, M.
(2000)
Cell
103,
843-852[Medline]
[Order article via Infotrieve]
|
56.
|
Jensen, E. V.,
Cheng, G.,
Palmieri, C.,
Saji, S.,
Makela, S.,
Van Noorden, S.,
Wahlstrom, T.,
Warner, M.,
Coombes, R. C.,
and Gustafsson, J. A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
15197-15202[Abstract/Free Full Text]
|
57.
|
Speirs, V.,
Malone, C.,
Walton, D. S.,
Kerin, M. J.,
and Atkin, S. L.
(1999)
Cancer Res.
59,
5421-5424[Abstract/Free Full Text]
|
58.
|
Derynck, R.,
Zhang, Y.,
and Feng, X. H.
(1998)
Cell
95,
737-740[Medline]
[Order article via Infotrieve]
|
59.
|
Augereau, P.,
Miralles, F.,
Cavailles, V.,
Gaudelet, C.,
Parker, M.,
and Rochefort, H.
(1994)
Mol. Endocrinol.
8,
693-703[Abstract]
|
60.
|
Tsuchiya, F.,
Ikeda, K.,
Tsutsumi, O.,
Hiroi, H.,
Momoeda, M.,
Taketani, Y.,
Muramatsu, M.,
and Inoue, S.
(2001)
Biochem. Biophys. Res. Commun.
284,
2-10[CrossRef][Medline]
[Order article via Infotrieve]
|
61.
|
Kumar, V.,
Green, S.,
Staub, A.,
and Chambon, P.
(1986)
EMBO J.
5,
2231-2236[Abstract]
|
62.
|
Kumar, V.,
Green, S.,
Stack, G.,
Berry, M.,
Jin, J. R.,
and Chambon, P.
(1987)
Cell
51,
941-951[Medline]
[Order article via Infotrieve]
|
63.
|
Tora, L.,
White, J.,
Brou, C.,
Tasset, D.,
Webster, N.,
Scheer, E.,
and Chambon, P.
(1989)
Cell
59,
477-487[Medline]
[Order article via Infotrieve]
|
64.
|
Norris, J. D.,
Paige, L. A.,
Christensen, D. J.,
Chang, C. Y.,
Huacani, M. R.,
Fan, D.,
Hamilton, P. T.,
Fowlkes, D. M.,
and McDonnell, D. P.
(1999)
Science
285,
744-746[Abstract/Free Full Text]
|
65.
|
Shiau, A. K.,
Barstad, D.,
Loria, P. M.,
Cheng, L.,
Kushner, P. J.,
Agard, D. A.,
and Greene, G. L.
(1998)
Cell
95,
927-937[Medline]
[Order article via Infotrieve]
|
66.
|
Fuqua, S. A.,
Schiff, R.,
Parra, I.,
Friedrichs, W. E.,
Su, J. L.,
McKee, D. D.,
Slentz-Kesler, K.,
Moore, L. B.,
Willson, T. M.,
and Moore, J. T.
(1999)
Cancer Res.
59,
5425-5428[Abstract/Free Full Text]
|
67.
|
Itoh, S.,
Itoh, F.,
Goumans, M. J.,
and Ten Dijke, P.
(2000)
Eur. J. Biochem.
267,
6954-6967[Abstract/Free Full Text]
|
68.
|
Lagna, G.,
Hata, A.,
Hemmati-Brivanlou, A.,
and Massague, J.
(1996)
Nature
383,
832-836[CrossRef][Medline]
[Order article via Infotrieve]
|
69.
|
Wu, R. Y.,
Zhang, Y.,
Feng, X. H.,
and Derynck, R.
(1997)
Mol. Cell. Biol.
17,
2521-2528[Abstract]
|
70.
|
Speirs, V.,
and Kerin, M. J.
(2000)
Br. J. Surg.
87,
405-409[CrossRef][Medline]
[Order article via Infotrieve]
|
71.
|
Muss, H. B.
(1992)
Breast Cancer Res. Treat.
21,
15-26[Medline]
[Order article via Infotrieve]
|
72.
|
Berry, M.,
Metzger, D.,
and Chambon, P.
(1990)
EMBO J.
9,
2811-2818[Abstract]
|