Coactivator and Corepressor Regulation of the Agonist/Antagonist Activity of the Mixed Antiestrogen, 4-Hydroxytamoxifen
Carolyn L. Smith,
Zafar Nawaz and
Bert W. OMalley
Department of Cell Biology, Baylor College of Medicine,
Houston, Texas 77030-3498
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ABSTRACT
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Mixed antiestrogens, such as
4-hydroxytamoxifen (4HT), act as either partial agonists or antagonists
of estrogen receptor (ER) function in a tissue-, cell-, and
promoter-specific manner, suggesting that intracellular factors
modulate their ability to regulate transcription. To determine whether
coactivators and corepressors have the capacity to modulate the
relative agonist/antagonist activity of 4HT, ER-dependent gene
expression was measured in the absence or presence of expression
vectors for SRC-1 (steroid receptor coactivator-1) or SMRT (silencing
mediator of retinoic acid and thyroid hormone receptors). In Hep G2
cells in which 4HT is an agonist, exogenous SRC-1 enhanced estradiol
(E2)- and 4HT-stimulated
transcription in a dose-dependent manner, while SMRT overexpression
strongly reduced basal and 4HT-stimulated gene expression with no
effect on E2 activity. These observations were
not cell- or promoter-specific inasmuch as similar results were
obtained in HeLa cells under conditions in which 4HT is an antagonist.
A protein-protein interaction assay indicated that the full-length ER
binds to SMRT in vitro. To assess whether relative
coactivator and corepressor expression within a given cell could
modulate the balance of 4HT agonist/antagonist activity, SRC-1 and SMRT
were coexpressed. SMRT overexpression blocked SRC-1 coactivation of
4HT-stimulated gene expression and preferentially inhibited 4HT agonist
activity whether or not exogenous SRC-1 was present. The cumulative
data in this model system indicate that the relative expression of
coactivators and corepressors can modulate 4HT regulation of ER
transcriptional activity and suggest they could contribute to the
tissue-specific ability of mixed antiestrogens to activate or inhibit
ER-mediated gene expression.
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INTRODUCTION
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The estrogen receptor (ER) is a member of a superfamily of
transcription factors that serve as nuclear receptors for small
hydrophobic ligands (1, 2). Estrogen binding to its receptor induces
the ligand-binding domain to undergo a characteristic conformational
change, whereupon receptor dimerizes, binds to DNA, and subsequently
stimulates gene expression (3, 4, 5, 6). Two distinct activation functions
(AFs) contribute to the ERs transcriptional activity. The first, AF1,
is located within the amino-terminal portion of the receptor whereas
the second, hormone-dependent AF2 is located in the latter half of the
molecule overlapping the ligand-binding domain (6, 7). The ability of
these regions to contribute to ER transcriptional activity varies with
the cell and promoter examined. In some contexts, individual activation
domains are the major determinants, but in most cases AF1 and AF2
synergize with one another to stimulate ER trans-activation
of gene expression (6, 8, 9).
In addition to hormone, the ER binds ligands that serve as
antiestrogens. The pure (type II) antiestrogens, exemplified by ICI
164,384 and ICI 182,780, are unable to activate the ER in nearly all
instances and efficiently antagonize ER function (10, 11). In contrast,
mixed antiestrogens, such as trans-4-hydroxytamoxifen (4HT),
inhibit ER activity in a selective manner and may even activate
transcription under certain conditions (12). The latter group of
antihormones engender a conformational change in the ligand-binding
domain distinct from that induced by estradiol (E2) and are
thought to inhibit the activity of the hormone-dependent AF2 but not
AF1 (3, 13, 14). The biocharacter (agonist versus antagonist
activity) of mixed antiestrogens varies among different tissues, cells
and promoters, but it may also deviate within a given biological
context. For example, tamoxifen, the metabolic precursor of 4HT, is
initially an antagonist of MCF-7 breast cancer cells grown in nude mice
but eventually stimulates tumor growth via an ER-agonistic action that
can be blocked by the pure antiestrogen, ICI 164,384, and is not
dependent upon the host animal (15, 16, 17).
When steroid receptors are occupied with agonists, the AF2 domain is
thought to serve as a focal point for interaction with coactivator
proteins, which are thought to act as adapters to the general
transcriptional machinery and modulators of chromatin structure
(18, 19, 20). In addition, it is hypothesized that the distinct,
antiestrogen-induced conformation of the ligand-binding domain does not
promote AF2-coactivator interactions and thereby impedes the ability of
the DNA-bound receptor to activate transcription (21). However, the ER
can stimulate transcription via its AF1 domain, and in some contexts
4HT is a relatively good ER agonist (8), suggesting that a mechanism(s)
must exist to enable the ER and the general transcription machinery to
productively associate under these conditions. In addition, the
acquisition of agonist activity by 4HT in cells in which it formerly
was an antagonist suggests that the differential expression of
cell-specific factors may facilitate a shift in 4HT biocharacter.
Therefore, to test whether increased expression of a positively acting
factor could enhance the agonist activity of a mixed antiestrogen, we
examined the ability of a coactivator for the steroid receptor
superfamily, steroid receptor coactivator-1 (SRC-1; 22 to
stimulate ER transcriptional activity in cell/promoter contexts where
4HT is either an agonist or antagonist of ER function. We found that
SRC-1 overexpression stimulated the agonist activity of 4HT, but was
unable to reverse the antagonist action of this antiestrogen. We also
have demonstrated that the silencing mediator of retinoic acid and
thyroid hormone receptors (SMRT) protein (23), identified as a
corepressor of several members of the nuclear receptor superfamily
including the thyroid hormone receptor (TR) and retinoic acid receptor
(RAR), could negatively influence the ability of a mixed antiestrogen
to activate ER-dependent gene expression.
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RESULTS
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SRC-1 Increases ER Transcriptional Activity Stimulated by 4HT
In Hep G2 (human hepatocellular carcinoma) cells, the mixed
antiestrogen 4HT acts as a relatively good ER agonist (8). To determine
whether coactivators could contribute to this transcriptional activity,
the ability of SRC-1 to enhance the ER-dependent transcription of a
synthetic target gene was examined. A human ER (hER) expression vector,
pSVMT-wER, and a pC3-Luc synthetic target gene, which consists of a
portion (-1807 to +58) of the promoter for the third component of
human complement (C3) fused to the firefly luciferase (Luc) gene, were
transiently transfected into Hep G2 cells with or without an expression
vector for SRC-1. As expected, 4HT was a relatively good ER agonist in
the absence of exogenous coactivator (8, 24) and, under our assay
conditions, 100 nM 4HT stimulated ER transcriptional
activity to an extent similar to that achieved by 10 nM
E2. Although 4HT can stimulate transcription via the AP-1
transcription factor (25), estrogen-dependent expression of the
pC3-Luc target gene is mediated strictly via the ER and three imperfect
estrogen response elements (EREs) located within the C3 promoter (24).
When increasing amounts (0
1000 ng) of an expression vector for human
SRC-1 were cotransfected into these cells, basal, estrogen- and
4HT-stimulated gene expression was increased in a dose-dependent manner
(Fig. 1A
). In contrast, the type II antiestrogen ICI 164,384
did not activate transcription of the C3 target gene in these cells,
and exogenous SRC-1 expression had little or no effect on ER
transcriptional activity in the presence of this pure antagonist.

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Figure 1. SRC-1 Increases ER Transcriptional Activity
Stimulated by 4HT and E2 in Hep G2 Cells
A, Hep G2 cells were transfected with 1 µg pSVMT-wER and 2.5 µg
pC3-Luc in the presence of increasing concentrations of (0, 250, 500,
750, or 1000 ng) of an expression vector (pBK-SRC-1) for SRC. The total
amount of DNA transfected into each well was adjusted to 4.5 µg with
pBK-CMV. B, Hep G2 cells were transfected with 1 µg pSVMT-wER and 2.5
µg TK-C3ER1&2-Luc reporter gene with 1 µg pBK-CMV (solid
bars) or 1 µg pBK-SRC-1 (open bars). In both
panels, estrogen-stimulated activity measured in the absence of
exogenous SRC-1 was defined as 100. Data are presented as the
average ± SEM of three experiments. Cells were
treated with either vehicle (basal), 10 nM E2,
100 nM 4HT, or 100 nM ICI 164,384.
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The 5'-flanking region of the C3 gene contains binding sites for a
number of transcription factors, including the CCAAT/enhancer binding
protein (26, 27), and to determine whether SRC-1 enhanced transcription
via the imperfect EREs located within this promoter (24) and not other
transcription factor-binding sites, the expression of a modified target
gene, TK-C3ER1&2-Luc, consisting of the three imperfect EREs of the C3
gene linked to the heterologous thymidine kinase promoter, was
examined. Both E2 and 4HT increased transcription of
TK-C3ER1&2-Luc, and exogenous SRC-1 further enhanced gene expression
3-fold (Fig. 1B
). In parallel experiments, cotransfection of an
identical amount of SRC-1 expression vector increased E2-
and 4HT-stimulated expression of the intact C3 promoter (pC3-Luc) by
5-fold (see Fig. 1A
), indicating that the ability of SRC-1 to
coactivate ER-dependent transcription may be influenced by promoter
context.
Previous studies have demonstrated that SRC-1 (also known as
p160) binds to the carboxy-terminal portion of the hER (amino acids
282595), but not to a shorter ER mutant (
534) lacking the last 61
amino acids (21). This deleted region encompasses sequences required
for AF2 function (28), and it has been suggested that SRC-1 may be a
mediator of this estrogen-dependent activation domain (21).
Introduction of three amino acid substitutions (D538A/E542A/D545A) to
the ERs ligand-binding domain disrupts AF2 activity but not receptor
dimerization or hormone-binding affinity (28). To determine whether an
intact AF2 domain is required for SRC-1 coactivation of ER-dependent
transcription, HepG2 cells were transfected with expression vectors for
wild type (pRST7-hER) or AF2 mutant
(pRST7-hER-3x) ER and the pC3-Luc target gene, and
transcriptional activity was assessed in the absence and presence of
exogenous SRC-1. In accordance with a previous report (8), these
three-point mutations significantly decreased 4HT agonist activity but
had little effect on E2-stimulated transcription of pC3-Luc
in HepG2 cells. When SRC-1 expression levels were increased,
transcription of the target gene by estrogen-activated wild type or AF2
(D538A/E542A/D545A) mutant ER was enhanced to a similar extent (Fig. 2
and data not shown) and indicates that an intact AF2
domain is not required for SRC-1 activity in this cell and promoter
context.

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Figure 2. SRC-1 Enhances the Transcriptional Activity of an
AF2 Mutant ER
Hep G2 cells were transfected with 1 µg pRST7-hER-3x and
2.5 µg pC3-Luc in the presence of 1 µg pBK-CMV (solid
bars) or 1 µg pBK-SRC-1 (open bars).
Estrogen-stimulated luciferase activity measured in the absence of
exogenous SRC-1 was defined as 100. Data are presented as the
average ± SEM of three experiments. Cells were
treated with either vehicle (basal), 10 nM E2,
or 100 nM 4HT.
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In HeLa (human cervical carcinoma) cells, 4HT exhibits little agonist
activity and effectively inhibits E2-stimulated gene
expression (29). To assess the ability of SRC-1 to contribute to ER
transcriptional activity under conditions where 4HT is an antagonist,
HeLa cells were transfected with a simple synthetic target gene,
ERE-E1b-CAT, consisting of an ERE upstream of the E1b TATA box and the
chloramphenicol acetyltransferase (CAT) gene, and a wild type hER
expression vector (pSVMT-wER) with or without 1 µg of an expression
vector for SRC-1. Under these assay conditions, estrogen increased CAT
activity by
6-fold, and ectopic SRC-1 expression further stimulated
gene expression by 13-fold (Fig. 3
). In contrast to the data
obtained from Hep G2 cells, 4HT was a poor agonist in HeLa cells, and
exogenous SRC-1 stimulated reporter gene expression by only
4-fold
in cells treated with vehicle or 4HT. As expected, ER transcriptional
activity was very low in the presence of the pure antiestrogen ICI
164,384, whether or not exogenous SRC-1 was present.

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Figure 3. SRC-1 Preferentially Increases Estrogen-Stimulated
ER Transcriptional Activity in HeLa Cells
HeLa cells were transfected with 1 µg pSVMT-wER and 2.5 µg
ERE-E1b-CAT in the presence of 1 µg pBK-CMV (solid
bars) or 1 µg pBK-SRC-1 (open bars). CAT
activity measured in cells treated with E2 in the absence
of exogenous SRC-1 was defined as 100. Data represent the average
± SEM of five experiments. Cells were treated with either
vehicle (basal), 1 nM E2, 100 nM
4HT, or 100 nM ICI 164,384 (ICI).
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SRC-1 Enhances Ligand-Independent Activation of ER
In the absence of exogenous ligand, the hER can be activated by
dopamine receptor agonists of the D1 subtype (29, 30) and
cholera toxin/3-isobutyl-1-methylxanthine (IBMX) [agents that increase
intracellular cAMP concentrations (31)]. To determine whether SRC-1
has the potential to contribute to ER transcriptional activity
initiated by ligand-independent signaling pathways, HeLa cells were
transfected with an ER expression vector and the ERE-E1b-CAT target
gene and stimulated with forskolin (an activator of adenylate cyclase)
and IBMX (a phosphodiesterase inhibitor). Elevation of intracellular
cAMP levels by this treatment stimulated ER transcriptional activity by
5-fold, and ectopic SRC-1 expression further increased gene expression
an additional 6-fold (Fig. 4
) indicating that SRC-1 can
significantly increase ER transcriptional activity in the absence of
ligand when cells are appropriately stimulated. In parallel
experiments, SRC-1 increased estrogen-stimulated activity by
4-fold
(Fig. 4
). To ensure that forskolin/IBMX-induced transcription was
ER-dependent, CAT gene expression was assessed in the presence of the
pure antiestrogen ICI 164,384 with or without exogenous SRC-1, and no
significant activity was observed.

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Figure 4. SRC-1 Stimulates the Ligand-Independent Activation
of ER Transcriptional Activity
HeLa cells were transfected with 1 µg pSVMT-wER and 2.5 µg
ERE-E1b-CAT in the presence of 0.5 µg pBK-CMV (solid
bars) or 0.5 µg pBK-SRC-1 (open bars). ER
transcriptional activity determined in the presence of E2
and absence of exogenous SRC-1 was defined as 100. Data are presented
as the average ± SEM of three experiments. Cells were
treated with vehicle (basal), 1 nM E2, 5
µM forskolin and 50 µM IBMX (F/I), 100
nM ICI 164,384 (ICI), 100 nM 4HT, or
combinations thereof.
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As previously demonstrated (29, 32), 4HT does not antagonize ER
transcriptional activity stimulated by dopamine or cAMP-signaling
pathways, and the resulting gene expression is greater than that
achieved by 4HT and either agent alone (Fig. 4
). Since 4HT is normally
an antagonist in these cells (29), these ligand-independent activation
pathways also encourage a shift in the balance of this mixed
antiestrogens activity from antagonist to agonist. The ability of
exogenous SRC-1 to further enhance forskolin/IBMX- stimulated ER
activity in the presence of 4HT (
4-fold) suggests that this
coactivator also contributes to this mechanism of gene expression.
The Corepressor, SMRT, Inhibits 4HT Agonist Activity
It is clear that in comparison to estrogens, the relative ability
of 4HT to activate ER-dependent transcription varies between cell and
tissue types (14, 24, 33), and it has been postulated that cellular
coregulatory proteins contribute to the differential biocharacter of
4HT (12). Although SRC-1 stimulated 4HT agonist activity in Hep G2
cells, it was unable to convert 4HT from an antagonist to agonist in
HeLa cells. Therefore, the possibility that a corepressor protein may
help to define the agonist/antagonist balance of 4HT activity was
examined. In HepG2 cells, where 4HT is a relatively good ER agonist,
exogenous SMRT did not decrease E2-dependent transcription
(Fig. 5A
). However, SMRT attenuated 4HTs agonist activity
(>70%) with the result that the ability of 4HT to activate
transcription was significantly attenuated in comparison to estrogen.
SMRT also decreased basal ER activity by 60%. In contrast, SMRT had
little effect on the already low activity of ER in the presence of the
pure antiestrogen ICI 164,384. Overall changes in gene expression are
unlikely to account for SMRT inhibition of 4HT-stimulated ER activity
because exogenous SMRT did not influence ß-galactosidase activity
expressed from a SV40- or cytomegalovirus (CMV)-regulated constitutive
expression vector (data not shown). When SMRT was expressed ectopically
in HeLa cells, it did not inhibit estrogen-stimulated expression of the
ERE-E1b-CAT reporter gene (Fig. 5B
). However, it further decreased the
low 4HT agonist activity observed in these cells. Taken together, these
data indicate that ectopic expression of this corepressor decreases 4HT
agonist activity whether it is weak (HeLa cells) or relatively strong
(Hep G2 cells).

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Figure 5. SMRT Preferentially Inhibits 4HT-Stimulated ER
Transcriptional Activity
A, Hep G2 cells were transfected with 1 µg pSVMT-wER and 2.5 µg
pC3-Luc in the presence of increasing concentrations (0, 250, 500, 750,
or 1000 ng) of an expression vector (pAB gal-SMRT) for SMRT. Total
DNA transfected into each well was adjusted to 4.5 µg with pAB gal.
Cells were treated with vehicle (basal), 10 nM
E2, 100 nM 4HT, or 100 nM ICI
164,384. B, HeLa cells were transfected with 1 µg pSVMT-wER and 2.5
µg ERE-E1b-CAT reporter gene with increasing concentrations of
pAB gal-SMRT (0, 500, 1000, or 2500 ng). The total DNA transfected
into each well was adjusted to 6 µg with pAB gal. Cells were
treated with vehicle (basal), 1 nM E2, or 100
nM 4HT. In both panels, ER transcriptional activity in the
presence of E2 and absence of exogenous SMRT were defined
as 100. Data represent the average ± SEM of at least
three experiments.
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Interaction of ER and SMRT
Since SMRT altered 4HT-stimulated ER activity in transient
transfection assays, the ability of ER and SMRT to physically interact
was assessed in vitro by
glutathione-S-transferase (GST) pull-down assay. A fusion
protein consisting of GST fused to the amino terminus of full-length
hER was incubated with 35S-labeled, in vitro
translated SMRT protein (amino acids 291495) in the absence or
presence of E2 and tamoxifen. Virtually no SMRT protein was
retained by GST alone (Fig. 6
). In comparison to the input
lane, significant levels of SMRT were retained by GST-ER regardless of
the presence of estrogen or antiestrogen. Thus, SMRT and ER interact
in vitro, but in a hormone-independent manner.

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Figure 6. In Vitro Interaction of GST-ER with SMRT
35S-labeled SMRT (amino acids 291495) was tested for
interaction with GST alone (lane 2) or GST-ER in the presence of
vehicle (lane 3), 1 µM E2 (lane 4), or 1
µM tamoxifen (lane 5). The arrow
(right) indicates the position of SMRT. Lane 1 shows
25% of the input [35S]SMRT used in these assays.
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SRC-1 Overexpression Does Not Reverse SMRT Inhibition of 4HT
Agonist Activity
The above studies indicated that both SRC-1 and SMRT have the
potential to modulate ER-dependent gene expression stimulated by 4HT.
To determine whether perturbation of coactivator and corepressor levels
within a given cell could alter 4HT-induced ER transcriptional
activity, various combinations of expression vectors for SRC-1 and SMRT
were introduced into HepG2 cells and target gene expression was
assessed. Ectopic expression of SMRT substantially decreased
4HT-induced transcription of the pC3-Luc reporter gene in comparison to
E2 (Fig. 7
; compare lanes 5 and 6 to 2 and 3).
Increasing SRC-1 expression in these cells did not restore 4HT agonist
activity relative to E2, even when 50% less SMRT
expression vector was cotransfected (compare lanes 11 and 12 to 5 and
6). As anticipated, in the absence of exogenous SMRT, ectopic SRC-1
increased the ER transcriptional activity induced by E2 and
4HT equally well. Thus, within a given cell, elevated expression of
SMRT shifts the balance of 4HT activity from an agonist to antagonist,
and overexpression of SRC-1 was unable to reverse this process.
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DISCUSSION
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SRC-1 Stimulates 4HT Agonist Activity
Mixed antiestrogens, such as 4HT, regulate ER transcriptional
activity in a tissue-, cell-, and promoter-dependent manner.
Accumulating evidence suggests that the differential ability of partial
antagonists to modify gene expression cannot be accounted for by
alterations in the ligand-receptor complex alone, but also must take
into consideration coregulator (coactivator and corepressor) proteins
that regulate ER interactions with the general transcriptional
machinery and chromatin (12). Therefore, a putative coactivator and
corepressor of the steroid receptor superfamily were tested to
determine whether these coregulators have the potential to modify the
biocharacter of a mixed antiestrogen in cell-based, ER-dependent
transcription assays. Ectopic SRC-1 expression strongly enhanced
estrogen-stimulated expression of two different target genes
(ERE-E1b-CAT and pC3-Luc, respectively) in HeLa and Hep G2 cells and
indicates that the ability of SRC-1 to enhance ER transcriptional
activity is not cell or promoter restricted. In contrast, SRC-1 only
weakly augmented 4HTs low agonist activity in HeLa cells, but
enhanced 4HT-stimulated transcriptional activity to an extent
equivalent to that of estrogen in HepG2 cells, indicating that ER
liganded with either a full or partial agonist is an equivalent
substrate for SRC-1 coactivation in the latter promoter and cellular
context. Therefore, SRC-1 has the potential to contribute to ER
transcriptional activity stimulated by estrogen or 4HT, but this
coactivator is not a dominant determinant of the agonist
versus antagonist activity of this antihormone.
AF2 Is Not Required for SRC-1 Coactivation
Steroid receptor coactivator-1 (p160) was postulated to act as a
coactivator/mediator for the AF2 domain of steroid receptor superfamily
members (22), and it was predicted that SRC-1 would not interact
functionally with the ER when liganded with antiestrogens (21).
However, the demonstration that SRC-1 enhanced 4HT-stimulated,
AF1-dependent ER transcriptional activity in Hep G2 cells and
efficiently coactivates the E2-stimulated transcriptional
activity of an AF2-defective ER mutant (D538A/E542A/D545A) suggests
that this coactivator may act through regions in addition to AF2. In
support of this, a recently published study utilizing a modified
mammalian two-hybrid approach in Chinese hamster ovary cells indicated
that SRC-1 facilitated the physical interaction between the
amino-terminal (containing AF1) and carboxy-terminal (containing AF2)
regions of ER (34). Furthermore, SRC-1 binds to an ER
534 mutant when
examined in the context of the full-length receptor (35) instead of the
ligand-binding domain alone (21). The remaining studies reporting a
lack of 4HT-dependent interaction between SRC-1 (p160) and ER were
conducted with the ligand-binding domain alone or in MCF-7 cells in
which 4HT is typically a weak agonist or antagonist (21) and AF1
contributions to SRC-1 interaction(s) with the ER were not likely to
have been assessed.
SRC-1 and Ligand-Independent Activation of the ER
The coactivation of forskolin/IBMX-stimulated ER transcriptional
activity by SRC-1 in the absence of exogenous ligand suggests that a
hormone-independent mechanism exists to facilitate a functional
interaction of ER and this coactivator. Furthermore, the ability of
ligand-independent signaling pathways to shift 4HT activity from
antagonist to agonist and thereby enable SRC-1 to strongly enhance 4HT
activity in a cell/promoter context where this antiestrogen is
typically a relatively poor agonist, supports this hypothesis. Because
all agents (i.e. dopamine, cAMP, growth factors) capable of
ligand-independently activating the ER initiate or alter the activity
of an intracellular signal transduction cascade and presumably kinases
and/or phosphatases (reviewed in 36 , it is possible that
phosphorylation of the ER and/or SRC-1 may contribute to their ability
to functionally interact with one another or with other accessory
transcription factors (e.g. CREB binding protein; CBP)
necessary for steroid receptor-dependent transcription (37).
Interestingly, CBP serves as a coactivator for numerous, diverse
transcription factors (e.g. CREB, Elk-1, c-Jun, c-Myb,
c-Fos), and at least some of these interactions are dependent and/or
enhanced by the phosphorylation of the site-specific activator
(38, 39, 40, 41, 42).
SMRT Regulation of 4HT Biocharacter
Since SRC-1 was unable to alter the agonist/antagonist balance of
4HT activity, SMRT was tested for its ability to alter 4HTs
agonist/antagonist activity to determine whether a corepressor has the
potential to regulate mixed antiestrogen activity. In both
cell/promoter contexts tested, ectopic expression of SMRT decreased the
ability of 4HT to activate transcription while having little or no
effect on estrogen-stimulated gene expression; this suggests that full
agonists enable the ER to overcome corepressor function, while partial
agonist/antagonists do not. Although the experiments presented in this
paper used a SMRT expression vector lacking the coding region for the
first 28 amino acids, they have been repeated with another expression
vector (pCMX-SMRT) that directs the expression of a SMRT isoform
containing the authentic amino terminus with essentially identical
results (data not shown). This indicates that the first 28 amino acids
of this corepressor are not required to modulate 4HTs ability to
activate ER-dependent gene expression.
The corepressor SMRT was reported to bind to TR and RAR in the absence,
but not the presence, of their cognate ligands via a portion of the
hinge region referred to as the CoR box (23, 43, 44). Although there is
no homology between the CoR box and any region of the ER, three other
structural motifs have been implicated in the association between
corepressors and two orphans of the steroid receptor superfamily,
Rev-ErbA and COUP-TF1; none displays significant homology to the CoR
box or the ER (45, 46, 47). Thus, dissimilar receptor sequences facilitate
interaction between different nuclear receptor superfamily members and
SMRT. Although SMRT preferentially bound to a GST-ER fusion protein in
comparison to GST alone, no hormone-dependent interaction was observed,
and this could reflect differences between in vitro and
in vivo binding conditions. For example, if
hormone-dependent, ER-coactivator interactions contribute to the
displacement/inactivation of corepressor in vivo,
differences in ER-SMRT in vitro interactions are not likely
to be observed. Alternatively, SMRT may interact with the
hormone-independent AF1 domain of ER. Importantly, our data should not
be used to implicate SMRT itself as the authentic, native corepressor
for the ER. It is equally likely that another unidentified protein(s)
regulates ER transcriptional activity with improved specificity and/or
affinity.
Roles of Potential ER Corepressors
Although heat shock protein interactions contribute to maintaining
the ER in a transcriptionally inactive state within the cell (48),
under certain conditions ER is present within the nucleus and able to
bind DNA in the absence of ligand (49), suggesting that another
mechanism may inhibit this receptors basal transcriptional activity.
For instance, corepressors could recruit histone deacetylase activity
to a target gene promoter and thereby maintain chromatin in an inactive
state, directly interact with and inhibit the general transcription
complex formed at the promoter (TATA), inhibit functional
ER-coactivator interactions, or block the transcriptional synergism
between AF1 and AF2 required for full ER activity in most cell/promoter
contexts (6, 8, 9). Thus, it is postulated that the unliganded ER is
bound to a corepressor(s) that either dissociates or is inactivated
upon estrogen binding; the resulting conformational change would enable
the ER to preferentially associate with coactivator(s) and directly
and/or indirectly encourage the general transcriptional machinery to
activate target gene expression. In contexts where they act as
antagonists, mixed antiestrogens may induce a ligand-binding domain
conformation that enables the receptor to retain its ability to
interact with corepressor(s) and/or decreases its affinity for SRC-1
such that corepressors are not efficiently inactivated. Alternatively,
if the mixed antiestrogen-occupied ER simultaneously binds coactivators
and corepressors under these conditions, the repressor domain(s) of
corepressors may inhibit ER transcriptional activity by blocking the
activation function of coactivators.
The agonist activity of 4HT may manifest itself in tissues and cells
where corepressor expression is low, ER transcriptional activity is
stimulated by corepressor-insensitive coactivators and/or cell-specific
corepressors are unable to bind to ER liganded with mixed
antiestrogens. In support of the concept that an authentic ER
corepressor may exist is the demonstration of a repressor domain within
the ERs ligand-binding domain (amino acids 370470) that functions
independently of heat shock protein interaction (50). Furthermore,
there is a precedent for the existence of a corepressor(s) for
"steroid" as opposed to a orphan (Rev-ErbA) or type II (TR and RAR)
receptor. A repressor domain has been mapped within the extreme carboxy
terminus of the progesterone receptor, and it appears to require an
additional cellular factor(s) to inhibit transcription (51).
Implications of Coregulators for 4HT Biocharacter
Collectively, these data accumulated in model transfection assays
indicate that cellular coactivators and corepressors have the potential
to contribute to the overall ability of a mixed antiestrogen to
regulate ER target gene expression. At the tissue/organ mRNA level, the
coactivators and corepressors examined to date appear to be
ubiquitously expressed (22, 52, 53, 54, 55, 56), and presumably most, if not all,
cells contain both forms of coregulators. However, the relative
expression levels of known coactivators and corepressors have not been
assessed, and it is likely that novel coregulators of the nuclear
receptor superfamily remain to be identified. It is possible that
authentic ER corepressor proteins may be expressed at relatively high
levels in cells where 4HT is an antagonist and/or coactivators are
present in comparatively large amounts in contexts where 4HT is an
agonist. It is also likely that promoters contribute to the relative
balance of 4HT agonist/antagonist activity by imposing spatial
constraints on the ability of ER and coactivators/corepressors to make
protein-protein interactions.
Tamoxifen, in its role as an antiestrogen, is an important endocrine
therapy for the treatment of breast cancer and more recently is being
tested as a chemopreventative agent in women with a high risk of
developing this disease (57). However, it also exerts estrogen-like
effects in bone, liver and urogenital tissues; of which some
(e.g. an increased occurrence rate of endometrial cancer)
are clearly not beneficial (58). Our data highlights the necessity to
understand the role that native intracellular factors play in the
interpretation of tamoxifen/4HT biocharacter in cells/tissues that
endogenously express ER and provides a theoretical rationale for the
contribution of coregulatory proteins to the tissue-specific and
gene-specific activity of ER agonists and antagonists.
 |
MATERIALS AND METHODS
|
---|
Chemicals
17ß-Estradiol (E2) and tamoxifen were obtained
from Sigma Chemical Company (St. Louis, MO). The antiestrogens, ICI
164,384 and ICI 182,780, and 4HT were gifts from Alan Wakeling (Zeneca
Pharmaceuticals, Macclesfield, UK) and D. Salin-Drouin (Laboratoires
Besins Iscovesco, Paris, France), respectively. Forskolin and IBMX were
purchased from Research Biochemicals International (Natick, MA).
Plasmid DNAs
The mammalian expression vectors for full-length hER (pSVMT-wER)
and the AF2 (D538A/E542A/D545A) mutant ER (pRST7-hER-3x)
and its corresponding wild type ER counterpart (pRST7-hER)
have been described previously (8, 29). The synthetic target genes,
ERE-E1b-CAT (29), pC3-Luc (8), and TK-C3ER1&2-Luc (24) have been used
in previous studies. Mammalian expression vectors for SMRT
(pAB
galSMRT or pCMX-SMRT) and SRC-1 (pBK-SRC-1) and the
corresponding parent vectors (pAB
gal and pBK-CMV) were described
previously (22, 23, 47), as was the baculoviral expression vector,
pGST-hER (3).
The in vitro transcription vector for SMRT,
pT7-SMRT(291495), was constructed as follows. First, human skeletal
muscle poly(A)+ RNA (Clontech, Palo Alto, CA) was reverse
transcribed using the primer, 5'-GCTGGCATGTTCCTGCACCG-3', and this
material was used as a template cDNA for PCR amplification using the
primers, 5'-AGCTGACGTCGACGCCTCGTG-3' and 5'-CTGCACCGCCTGGCTTCTAT-3'.
The resulting product was cloned into the TA cloning vector, pCR3
(Invitrogen, Carlsbad, CA) to produce pCR3-SMRT(5651289).
Subsequently, this vector was digested with EcoRV and
BglII and ligated with the EcoRI (filled) to
BglII fragment of pGAD10-SMRT(11921495) to yield the
pCR3-SMRT(5651495) vector. The cDNA insert of pGAD10-SMRT(11921495)
was isolated from a brain cDNA library by yeast two-hybrid assay using
the ligand-binding domain of the human TR as bait. The
SalI-XhoI fragment of pCR3-SMRT(5651495) was
isolated, repaired with Klenow DNA polymerase, and cloned into the
NcoI-EcoRI (filled) site of the pT7ßSal vector
(59) to produce pT7-SMRT(5651495). Next, human skeletal muscle was
reverse transcribed using the primer, 5'-GTGCGGGACTTGGCGATCT-3', and
the resulting cDNA was amplified by PCR with the primers,
5'-AAGATTCCGAGCTCTGCTAC-3' and 5'-CACGAGGCGTCGACGTCAGC-3'. This PCR
product was TA Cloned (Invitrogen) to create pCR3-SMRT(29564).
Finally, to construct pT7-SMRT(291495), the SalI fragment
of pCR3-SMRT(29564) was inserted into the SalI site of
pT7-SMRT(5651495).
Cell Culture and Transfections
HeLa and HepG2 cells were routinely maintained in DMEM
supplemented with 10% FBS. Twenty-four hours before transfection,
3 x 105 HeLa or 8 x 105 HepG2 cells
were seeded per well of a six-well multiwell dish in phenol red-free
DMEM containing 5% dextran-coated charcoal-stripped serum. Cells were
transfected with the indicated DNAs using Lipofectin (Life
Technologies, Grand Island, NY) according to the manufacturers
guidelines. Six hours later, the DNA/Lipofectin mixture was removed and
cells were fed with phenol red-free media containing 5% stripped serum
and the hormone treatments indicated in the figure legends. Twenty-four
hours thereafter, cells were harvested and extracts were assayed for
CAT (29) or luciferase activity using the Luciferase Assay System
(Promega, Madison, WI). Duplicate samples were measured in each
experiment, and data are presented as the average ±
SEM of at least three experiments.
Protein-Protein Interaction by GST Pull-down Assay
The full-length hER was expressed as a GST fusion protein in a
baculovirus expression system in the presence of ethanol (vehicle), 1
µM estradiol, or 1 µM tamoxifen and
purified using glutathione-Sepharose affinity chromatography as
described previously (60). Radiolabeled SMRT (amino acids 291495) was
produced from the pT7-SMRT(291495) vector with the TNT-Coupled
Reticulocyte Lysate System for in vitro transcription and
translation as recommended by the manufacturer (Promega).
To assess protein-protein interactions, equivalent levels of GST alone
or GST-hER (as assessed by Coomassie staining) were incubated with
glutathione-Sepharose 4B beads (Pharmacia, Piscataway, NJ) in NENT
buffer (20 mM Tris, pH 8.0 containing 100 mM
NaCl, 1 mM EDTA, and 0.5% NP-40) containing either
ethanol, 1 µM E2, or 1 µM
tamoxifen for 60 min at room temperature. Subsequently, the supernatant
was removed and the beads were washed twice with NENT buffer. Eight
microliters of 35S-labeled SMRT were incubated with the
beads in NENT buffer containing vehicle, estrogen, or tamoxifen for
2 h at room temperature. Beads were washed five times with NENT
buffer, dried, resuspended in 50 µl SDS-PAGE loading buffer, resolved
by 7.5% SDS-PAGE, and visualized by fluorography.
 |
ACKNOWLEDGMENTS
|
---|
The authors acknowledge Donald McDonnell for providing the
pC3-Luc, pTK-C3ER1+2-Luc, pRST7-hER, and pRST7-hER-3x plasmids and Ron
Evans for furnishing the pCMX-SMRT expression vector. We also thank
Yayun Zheng and members of the Cell and Organ Culture Core for
technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Carolyn L. Smith, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.
A portion of this work was presented at the 10th International Congress
of Endocrinology, San Francisco, CA, June 1215, 1996.
This work was supported by NIH Grant DK-48896 (to B.W.O.).
Received for publication February 14, 1997.
Accepted for publication March 18, 1997.
 |
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