Transcriptional Regulation by a Naturally Occurring Truncated Rat Estrogen Receptor (ER), Truncated ER Product-1 (TERP-1)
Derek A. Schreihofer,
Eileen M. Resnick,
Ann Y. Soh and
Margaret A. Shupnik
Department of Internal Medicine Division of Endocrinology and
Metabolism University of Virginia Health Sciences Center
Charlottesville, Virginia 22908
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ABSTRACT
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Truncated estrogen receptor product-1 (TERP-1) is
a naturally occurring rat estrogen receptor (ER) variant transcribed
from a unique start site and containing a unique 5'-untranslated region
fused to exons 58 of ER
. TERP-1 is detected only in the pituitary,
and TERP-1 mRNA levels are highly regulated during the estrous cycle,
exceeding those of the full-length ER
on proestrus. These data
suggest that TERP-1 may play a role in estrogen- regulated feedback in
the pituitary. We examined the ability of TERP-1 to modulate gene
transcription in transiently transfected ER-negative (Cos-1) and
ER-positive pituitary (
T3 and GH3) cell lines. In Cos-1 cells
transiently cotransfected with TERP-1 and either ER
or ERß, low
levels of TERP-1 (ratios of < 1:1 with ER) enhanced transcription
of model promoters containing estrogen response elements by an average
of 3- to 4-fold above that seen with ER alone. At higher concentrations
of TERP-1 (> 1:1 with ER) transcription was inhibited. TERP-1 also had
a biphasic action on transcription in the
T3 and GH3 pituitary cell
lines, although the stimulatory action was less pronounced. TERP-1
actions were dependent on ligand-activated ER as TERP-1 did not bind
estradiol in transfected Cos-1 cells or in vitro, and
estrogen antagonists prevented the stimulatory effects of TERP-1.
Coimmunoprecipitation studies suggest that TERP-1 does not bind with
high affinity to the full-length ER
. However, TERP-1 may compete
with ER for binding sites of receptor cofactors because steroid
receptor coactivator-1 (SRC-1) rescued the inhibitory actions of
TERP-1. The ability of TERP-1 to both enhance and inhibit ER-dependent
promoter activity suggests that TERP-1 may play a physiological role in
estrogen feedback in the rat pituitary.
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INTRODUCTION
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Estrogen acts in many tissues to alter gene expression through
specific nuclear receptors. These receptors bind estrogen, dimerize,
and modulate gene transcription through interactions with estrogen
response elements (EREs) in the promoter regions of estrogen-regulated
genes (1). As a member of the steroid/thyroid hormone superfamily of
nuclear transcription factors, the estrogen receptor (ER) is composed
of several distinct functional domains. These include a
ligand-independent transactivation function at the N terminus (AF-1), a
DNA-binding domain (DBD) that allows dimerization of ERs and binding to
EREs in estrogen-regulated genes, and a ligand-binding domain (LBD)
near the C terminus (1, 2, 3). The LBD is important for both
ligand-activated transactivation through the AF-2 region and
dimerization of ERs (1, 2). The DBD and LBD both play a role in nuclear
localization of ERs (1, 2).
In addition to the recently described ER isoform ERß (4), several
variants of the ER
have been identified in human breast cancer
(5, 6, 7) and tumor cell lines (7, 8). These variants have contained point
mutations (5, 9), exon deletions (7, 8, 10), and C terminus truncations
(6). Some of these variants can have transcriptional effects (9, 10).
However, except in rare cases these variant ERs represent only a small
proportion of ER present in cells (8). Further, there have been no
reports of specific modulation of the levels of these splice variants
or mutant receptors in normal tissue. In rats, a number of ER variants
have been identified in reproductive tissues (11, 12, 13). At least some of
these rat ERs are highly regulated throughout the estrous cycle and can
exceed the levels of full-length ER
under physiological conditions
(11, 12, 13).
We identified one such ER
variant mRNA and protein in female rat
pituitaries (11). This variant, truncated ER product-1 (TERP-1),
encodes a protein that is truncated at the N terminus. The transcribed
mRNA contains exons 58 of the full-length ER
and a 31-bp unique
sequence upstream of exon 5, indicating a unique transcriptional start
site and promoter. Two in-phase translational start codons are present
in exon 5, and immunoblot analysis confirms that TERP-1 protein is
translated in vivo in the pituitary (13), in transfected
cells, and in vitro. TERP-1 mRNA expression is also highly
regulated by estrogen levels in vivo (11). In male rats and
in ovariectomized female rats, pituitary TERP-1 mRNA levels are very
low to undetectable as determined by Northern analysis and ribonuclease
(RNase) protection, but treatment with estradiol for 13 days
increases TERP-1 mRNA up to 7- to 10-fold (11, 13, 14). A unique
property of TERP-1 is its regulation during the rat estrous cycle, when
it is stimulated at least 50-fold, which suggests possible important
regulatory actions for this ER receptor variant under physiological
conditions. Although TERP-1 is essentially undetectable during early
diestrus, mRNA levels exceed ER
on proestrus (13). This timing of
expression suggests that TERP-1 could modulate estrogen actions around
the proestrus gonadotropin/PRL surge. In the pituitary, ER
is
present primarily in lactotropes and gonadotropes (14, 15, 16). Recent
evidence that TERP-1 is present in ER-containing lactotropes (12, 17),
and possibly gonadotropes (18), further supports a role in estrous
cyclicity in the pituitary. This tissue-specific expression and
regulation of TERP-1 distinguishes it from other ER splice variants or
mutants.
Although the physiological role of TERP-1 is unknown at the
present time, its regulation by estrogen in the pituitary and timing of
expression during the estrous cycle suggests that it may play a role in
estrogen feedback in pituitary cells. One possible mechanism for such
feedback effects would be that TERP-1 could alter expression of
estrogen-regulated genes either alone or in concert with ER
or
ERß. Because TERP-1 lacks a DBD and a portion of the LBD (based on
the predicted translational start site and protein size), it was
unclear whether TERP-1 could affect the transcription of
estrogen-regulated genes. If TERP-1 is able to influence transcription
of estrogen-regulated genes through EREs, it is likely that full-length
ERs are a necessary component in TERP-1 actions. In the present study
we examined the ability of TERP-1 to alter the transcription of model
and natural estrogen-responsive promoters in cell culture and the
necessity of full-length ERs and estrogen for TERP-1s actions. We
found that TERP-1 could influence the transcription of
estrogen-regulated genes. Although TERP-1 alone had no effect on
transcription, it had a biphasic action on ERE-containing promoters in
the presence of full-length ERs. Low ratios of TERP-1 to ER (<1:1)
enhanced transcription, whereas higher ratios were inhibitory. The
stimulatory effects were both promoter and cell type dependent, whereas
the inhibitory actions of TERP-1 were observed in all cell types.
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RESULTS
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TERP-1 Protein Expression and Estrogen Binding
Protein levels of ER
and TERP-1 were assessed in calcium
phosphate-transfected Cos-1 cells by immunoblotting. For comparison,
in vitro translated ER
and TERP-1 are also shown (Fig. 1
). ER
was clearly detected as a 64-
to 66-kDa protein, and TERP-1 was detected as a pair of doublets at
2224 kDa, which probably represent translation from two methionines
(amino acids 393 and 408) in exon 5. In transfected Cos-1 cells, ER
and TERP-1 proteins of this size were also detected (Fig. 1
). The
larger protein, corresponding to the 5'-translational start site, is
preferred in both transfected cells and normal pituitary cells (13).
TERP-1 protein was clearly detectable in cell lysates when
concentrations of 500 ng or greater were transfected. The inability to
detect TERP-1 protein at lower transfection concentrations may be due
to high turnover of the protein or the sensitivity of our antibody at
low molar concentrations of TERP-1. However, several such experiments
consistently demonstrated that levels of full-length ER
(1 µg)
were relatively constant, even with increasing amounts of TERP-1
protein. Based on immunoblotting analysis, both full-length ER
and
transfected TERP-1 were at much lower levels in GH3 cells as compared
with Cos-1 cells, even with high transfection concentrations (data not
shown).

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Figure 1. ER Protein Expression in Transfected Cos-1 Cells
ER (1 µg) was transfected into Cos-1 cells in DMEM/10% NCS alone
or with 0.1, 1, or 4 µg of TERP-1 by calcium phosphate. Cell lysates
were collected 48 h later, and 100 µg of protein were subjected
to electrophoresis on a 12% SDS-containing polyacrylamide gel. Western
analysis was performed with enhanced chemiluminescence using the C1355
rat C terminus ER antibody. The first two lanes contain in
vitro translated (IVT) ER and TERP-1 for comparison. The
upper panel shows a 10-min exposure, and the
lower panel shows a 2-h exposure of TERP-1 from the same
blot. Migration positions of mol wt markers are shown on the
left.
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The predicted protein sequence of TERP-1 contains most of the ligand-
binding domain of ER
(predicted amino acids 393600 of rat ER
),
and thus TERP-1 might exert some transcriptional effects through
estrogen binding. We assessed estrogen binding in vitro and
in transfected Cos-1 cells using either 1 µg of ER
or 1 µg of
TERP-1. ER
and TERP-1 were in vitro translated using
coupled transcription-translation from rabbit reticulocyte lysate.
Lysates (10 µl) were incubated with 0.05 pmol
[3H]-estradiol (E2) and 5 pmol of unlabeled
diethylstilbestrol (DES), and binding was determined using the
hydroxylapatite method (19). E2 binding by ER
was
readily displaced by DES, whereas TERP-1 showed no specific binding
(Fig. 2A
). TERP-1 did not alter the
ability of ER
to bind E2 when they were coincubated with
[3H]E2 and DES. Binding was determined in
transfected Cos-1 cells with a competition assay using
[3H]E2 and varying amounts of unlabeled
E2. Parallel experiments with ER
and TERP-1 were
performed in duplicate, and analysis of binding curves generated in two
separate experiments showed that ER
-transfected cells specifically
bound [3H]E2 with an affinity of 0.18
nM (Fig. 2B
). In agreement with the in vitro
experiments, TERP-1 did not bind E2 (Fig. 2B
).
TERP-1 Effects on Transcription in Cell Lines
We first tested TERP-1 transcriptional effects in the ER-negative
Cos-1 cell line using model promoters containing two consensus
vitellogenin EREs upstream of either a TATA box (2EREpGL2) or the
thymidine kinase promoter (Vit6luc) fused to a luciferase reporter. As
shown in Fig. 3A
, cotransfection of 1
µg of an expression vector for full-length rat ER
and treatment of
transfected cells with 10 nM E2 stimulated
2EREpGL2 approximately 7-fold and Vit6luc approximately 2.5-fold.
Cotransfection of 1 µg ER
and 100 ng of TERP-1 increased
expression of 2EREpGL2 and Vit6luc above that seen with ER
alone.
The effect is specific to ER-mediated transcription, as TERP-1 did not
alter the expression of a promoter lacking an ERE, the rat glycoprotein
-subunit (Fig. 3A
). However, the response was influenced by the
promoter context, as TERP-1 enhancement of
E2/ER
-stimulated transcription was greater for the
Vit6luc reporter than the 2EREpGL2 (Fig. 3A
). TERP-1 also enhanced
ERß-stimulated transcription of Vit6luc in Cos-1 cells, but this
response was less than for ER
(Fig. 3A
). The effect of TERP-1 on
transcription of both model promoters was biphasic. Figure 3B
shows an
example of this response with the 2EREpGL2 promoter in Cos-1 cells.
Stimulatory responses with TERP-1 occurred at transfected TERP-1 to ER
ratios of <1:1. The amount of transfected TERP-1 needed for optimal
stimulation of ER-mediated transcription was not absolute, possibly due
to efficiency of transfection or resulting levels of TERP-1 protein
translated. However, the amount of TERP-1 that enhanced transcription
was directly related to the amount of ER
, increasing with higher
levels of ER
transfected (data not shown). TERP-1 to ER ratios of
1:1 had either no net effect on E2/ER-stimulated
transcription or were inhibitory (Fig. 3B
), and TERP-1 to ER ratios
>1:1 inhibited ER-induced transcription (Fig. 3B
).

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Figure 3. Effect of TERP-1 on Transcription in Transfected
Cells
A, Representative experiments showing the effect of 100 ng TERP-1 on
ER - and ERß-stimulated transcription of reporter genes in
transiently transfected Cos-1 cells. Open bars represent
transcriptional responses of ERE-containing reporters (1 µg of
2EREpGL2 or Vit6luc) and a reporter lacking an ERE ( 3luc) to 1 µg
transfected ER or ERß in the absence of E2.
Hatched bars represent the same conditions after 6
h of treatment with 10 nM E2. Solid
bars represent 6 h of E2 treatment in cells
cotransfected with reporter, ER, and 100 ng TERP-1. In each case,
E2 treatments were normalized to the untreated condition
and represent the mean ± SD of triplicate
measurements. B, TERP-1 dose response in transfected Cos-1 cells using
the 2EREpGL2 reporter (1 µg) cotransfected with 1 µg ER and
increasing concentrations of TERP-1 (1 ng to 10 µg). The open
bar represents transfection in the absence of E2,
and solid bars represent 6 h treatment with 10
nM E2 normalized to the untreated condition.
Each bar represents the mean ± SEM of
three independent experiments with individual experiments performed in
triplicate. C, Representative experiments showing the effect of 100 ng
or 1 µg TERP-1 on ERE-containing reporters (1 µg Vit6luc or PRLluc)
in T3 and GH3 pituitary cell lines. Bars are the same
as in panel A, with the addition of stippled bars to
represent the effect of 1 µg of TERP-1. T3 cells were treated with
or without 10 nM E2 for 6 h, GH3
cells for 24 h. In all cases, total transfected DNA was normalized
with the pcDNA empty vector. In this and subsequent figures rALU refers
to luciferase activity in arbitrary light units corrected for total
lysate protein and normalized to the untreated condition (nt).
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Physiologically, TERP-1 is highly expressed in the pituitary, and given
its induction by E2 we expect TERP-1 protein to be present
in lactotropes and gonadotropes. Recent experiments confirm that TERP-1
mRNA is present in lactotropes (12, 17). Therefore, we examined the
ability of TERP-1 to alter transcription in pituitary cell lines that
contain endogenous ER. In the presence of 10 nM
E2, the endogenous ER modestly stimulated transcription of
model promoters in the mouse
T3 gonadotrope cell line, and TERP-1
enhanced E2/ER-stimulated transcription of both Vit6luc and
2EREpGL2 above levels achieved with the endogenous ER alone (Fig. 3C
).
Transfection of 1 µg or more of TERP-1 was inhibitory (Fig. 3C
). In
the presence of 10 nM E2, the endogenous ER
stimulated transcription of the model promoters in the rat
somatolactotrope GH3 cell line less than 2-fold (Fig. 3C
). Transfection
of 100 ng of TERP-1 failed to enhance this effect (Fig. 3C
), although
higher concentrations of TERP-1 were inhibitory (Fig. 3C
). Therefore,
we also tested TERP-1 effects on a natural pituitary promoter in a
pituitary cell line, and we used a rat PRL reporter (PRLluc) for this
purpose. This PRL promoter construct contains 2.5 kb of the
physiological promoter with a composite ERE that requires the
transcription factor Pit-1 for optimal E2 induction (20).
The addition of 10 nM E2 increased expression
of PRLluc 5-fold (Fig. 3C
). As we observed in Cos-1 cells using model
promoters, TERP-1 enhanced this estrogen-induced effect at low
concentrations of transfection (Fig. 3C
). However, the stimulatory
effect of TERP-1 on PRLluc was much smaller, averaging 50% (Fig. 3C
).
Transfection of 1 µg of TERP-1 failed to enhance
E2/ER-stimulated transcription (Fig. 3C
), and higher doses
of TERP-1 were inhibitory (data not shown).
Role of Hormone and ER in the TERP-1 Transcriptional Response
To test the requirement for full-length ER
and hormone in
mediating the effects of TERP-1, stimulation of the Vit6luc promoter
was measured in Cos-1 cells. Transfection of TERP-1 at concentrations
up to 2 µg in the absence of ER
had no influence on promoter
activity, demonstrating a dependence on ER-induced transcription (Fig. 4
). TERP-1s stimulatory actions were
also dependent on estrogen. Both the selective ER antagonist
4-hydroxy-tamoxifen and the antiestrogen ICI 182,780 antagonized
TERP-1-enhanced E2-stimulated transcription in Cos-1 cells
(Fig. 5
). These data demonstrate a
dependence on ligand-activated ER for TERP-1s stimulatory
actions.

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Figure 5. Effect of Estrogen Antagonists on TERP-1-Induced
Transcription
Representative experiment showing the effect of antiestrogens on
TERP-1-induced enhancement of E2/ER-stimulated
transcription in transiently transfected Cos-1 cells. Cells were
transfected with 1 µg Vit6luc reporter and 1 µg ER with or
without 10 ng TERP-1. After 16 h, cells were washed and
placed in serumless DMEM. Cells were treated for 18 h with 1
nM E2 (solid bars),
E2 and 1 µM 4-hydroxytamoxifen
(hatched bar), or E2 and 1 µM
ICI 182,780 (stippled bar). Values represent means
± SD of triplicate measures normalized to the untreated
condition (open bar). In all cases total transfected DNA
was normalized with the pcDNA empty vector.
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Based on its predicted structure, TERP-1 contains one of two
heterodimerization regions of the full-length receptor and may interact
directly with full-length ER
. Coimmunoprecipitation studies of ER
and TERP-1 were performed with both in vitro translated
proteins and transfected Cos-1 cells with essentially the same results.
Because TERP-1 contains identical sequences to the C terminus of ER
,
the proteins were distinguished by using the hinge antibody ER715,
which recognized only the full-length ER
, and an epitope-tagged
construct of TERP-1 (TERP-FLAG). TERP-FLAG contains the eight-amino
acid FLAG epitope at the C terminus of TERP-1 and can modulate ER
transcriptional activity in Cos-1 cells (not shown). As shown in Fig. 6
, immunoprecipitation studies failed to
show a direct interaction between ER
and TERP-FLAG (upper
panel), although coprecipitation of ER
and ERß could be
demonstrated under these conditions (bottom panel). Thus,
under these stringent conditions, TERP-1 does not appear to interact
directly with ER
.
Interaction of TERP-1 with Steroid Receptor Coactivator-1
(SRC-1)
Although TERP-1 does not bind estrogen and does not appear to have
a stable direct interaction with the full-length ER
, it only has
transcriptional effects in the presence of a ligand-activated
full-length ER. Therefore, it probably acts through other
protein-protein interactions. One possible mechanism is titration of
inhibitory cofactors such as steroid corepressors or stimulatory
coactivators such as SRC-1 (21) receptor interacting protein 140
[RIP140 (22)]. Experiments with the corepressors N-COR (nuclear
receptor corepressor) and SMRT (silencing mediator of retinoic
acid and thyroid hormone receptor) suggest that they do not affect
estrogen-stimulated transcription by ER
(Ref. 23 and our unpublished
results), a requirement for TERP-1 actions. However, TERP-1 may
interact with a coactivator such as SRC-1, either directly or by
competing with SRC-1 for binding partners. We tested the ability of
SRC-1 to influence TERP-1 effects on ER
-mediated transcription.
SRC-1 enhanced E2/ER
-stimulated transcription of Vit6luc
in transfected Cos-1 cells in a dose-dependent manner (Fig. 7A
). Cotransfection of low levels of
TERP-1 (100 ng) did not further enhance SRC-1s effects on
E2/ER
-stimulated transcription (Fig. 7A
). However,
increasing levels of SRC-1 overcame the inhibitory action of higher
levels (25 µg) of cotransfected TERP-1 (Fig. 7B
). These data
suggest that TERP-1 may compete with ER
for binding sites on SRC-1
or similar cofactors, thus limiting the extent of ER
activation.

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Figure 7. Effect of SRC-1 on TERP-1-Induced Stimulation and
Inhibition of ER -Stimulated Transcription
A, The effect of 100 ng TERP-1 on SRC-1-enhanced
E2/ER -stimulated transcription of Vit6luc in transiently
transfected Cos-1 cells. All cells were transfected with 1 µg Vit6luc
and 1 µg ER . Open bars represent transcriptional
responses of Vit6luc in the absence of E2. Solid
bars represent the same conditions after 6 h of treatment
with 10 nM E2. Hatched bars
represent cells transfected with 1 µg Vit6luc, 1 µg ER , and
increasing concentrations of SRC-1 (0.1, 0.25, and 1 µg) treated for
6 h with 10 nM E2. Stippled
bars represent cells transfected with reporter, ER , SRC-1,
and 100 ng TERP-1. In each case, E2 treatments were
normalized to the untreated condition and represent the mean ±
SEM of four experiments performed in duplicate. B, The
effect of SRC-1 on TERP-1 inhibition of E2/ER -stimulated
transcription of Vit6luc in transiently transfected Cos-1 cells. All
cells were transfected with 1 µg Vit6luc and 1 µg ER .
Open bars represent transcriptional responses of Vit6luc
in the absence of E2. Solid bars represent
the same conditions after 6 h of treatment with 10 nM
E2. Hatched bars represent cells transfected
with 1 µg Vit6luc, 1 µg ER , 2 µg TERP-1, and increasing
concentrations of SRC-1 (0, 0.1, 0.25, and 1 µg) treated for 6 h
with 10 nM E2. In all cases the SRC-1 parental
pBKcmv empty vector was used to normalize for changes in SRC-1, and
pcDNA was used to normalize for total DNA transfected.
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DISCUSSION
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Previously, we identified a truncated ER mRNA and protein from
female rat pituitaries (11). This truncated ER product-1 (TERP-1) cDNA
encodes a unique 31-bp 5'-end and the intact exons 58 of the rat
ER
. Thus, TERP-1 contains most of domain E and all of domain F but
has no AF-1 region or DBD. Although two potential in-phase start codons
are present near the N terminus of exon 5 and result in two transcripts
in vitro, our studies in transfected cells suggest that the
first ATG (residue 393) is used preferentially in Cos-1 cells (Fig. 1
),
in agreement with our observations in normal pituitary cells (13).
TERP-1 expression in vivo is localized to the pituitary,
where it may play a role in estrogen-regulated gene expression (11).
High TERP-1 mRNA levels are induced by estrogen in castrate male and
female rats and during the estrous cycle under conditions where the
full-length ER
is modulated only slightly (13, 14). Recent reports
and our unpublished observations demonstrate that TERP-1 is localized
to pituitary cells that express ER
, namely lactotropes and
gonadotropes (12, 17, 18). This suggests that TERP-1 is dependent on
ligand-bound ER for expression as well as biological activity. Thus,
TERP-1 could provide a level of cellular feedback to estrogen-sensitive
genes in the pituitary. Our observation that TERP-1 modulated
transcription from both heterologous and physiological promoters in
pituitary cell lines supports this hypothesis.
Transcriptional activation by naturally occurring truncated or mutant
steroid receptors is not uncommon, and the interaction of receptor
isoforms may provide an important level of gene regulation.
Interestingly, many of these interactions do not require an intact DBD
or binding to a hormone response element. Recently a third PR
isoform, PR-C, has been identified (24). Like TERP-1, PR-C is amino
truncated and lacks the first zinc finger of the DBD. PR-C is also able
to enhance PR-A- and PR-B-induced transcription, although it has no
transactivating capacity alone. Other superfamily members also have
actions that do not require the complete receptor. For example, the LBD
of the 9-cis-retinoic acid receptor (RXR) alone can enhance
transcriptional activation by the orphan receptor Nurr1 (25).
Furthermore, although it has no DBD, the orphan receptor SHP has an LBD
and dimerizes with other superfamily members, including ER
and
ERß, to inhibit transcription (26, 27). Mutations of the first zinc
finger in the glucocorticoid receptor (GR) DBD inhibit DNA binding to
glucocorticoid response elements but allow ligand-dependent
activation of interleukin-6, a promoter the intact GR suppresses (28).
In addition, ER
can activate transcription from elements other than
EREs by interacting with other transcription factors, and variant ER
forms may play a role in this response. For example, tamoxifen-bound
ER
can interact with Jun/Jun or Fos/Jun dimers to enhance
transcription driven by AP1 (29), and this effect does require an
intact DBD. The estrogen antagonist raloxifene also stimulates
ERß-induced transcription through interactions with Fos and Jun at
AP1 sites (30). These mechanisms of transcriptional activation by
steroid receptors demonstrate that various unique promoter sequences
may be identified by the combination of receptor isoforms and ligands
present in the cell.
The mechanisms by which TERP-1 influences estrogen-regulated gene
transcription clearly require a ligand-activated ER. TERP-1 does not
bind estrogen itself, but modulates ER
or ERß stimulated
transcription in the presence of estrogen, while antiestrogens prevent
TERP-1 effects. TERP-1 effects are not a general transcriptional
phenomenon, as a promoter that does not contain an ERE was not affected
by the addition of TERP-1. TERP-1 effects on ER
and ERß might
occur through direct interactions with the activated full-length
receptors, or by interactions with coactivator or corepressor proteins
that themselves influence ER activity. The inhibition of
TERP-1-enhanced E2-stimulated gene activation by
antiestrogens is consistent with either or both possibilities.
Fawell et al. (31) localized the dimerization domain of the
mouse ER
in the C terminus. Specifically, amino acids 507518 are
necessary for ligand-induced dimerization, and the homologous region is
present in TERP-1. Heterodimerization between receptor isoforms has
been noted for several nuclear receptors. The resultant dimers can have
transcriptional properties in which the activity of one isoform
predominates or may result in modified or enhanced activities. For
example, the progesterone receptor isoforms PR-A and PR-B both
stimulate transcription from PREs, but have different
agonist/antagonist profiles (32). When PR-A/PR-B heterodimers form,
PR-A effects predominate on some promoters, but not others (32).
Similarly, ER
and ERß form heterodimers, and ER
activity can
predominate in a promoter- dependent manner (33, 34). A C-terminal
truncated form of ER
, resulting from an exon 5 deletion, was
recently reported to stimulate activated ER-dependent promoter activity
in osteosarcoma cells (35). This variant can bind DNA, and may form
heterodimers on DNA with full-length ER
, but cannot bind hormone
itself. Thus, the enhanced activity of the heterodimer in the
osteosarcoma context may occur through enhanced receptor interactions
with the transcriptional machinery or altered interactions with other
accessory proteins. Our immunoprecipitation studies do not demonstrate
direct interactions between TERP-1 and ER
, even though ER
and
ERß formed heterodimers under these conditions. This result suggests
two possibilities. TERP-1 may interact with ER
, but under less
stringent conditions than tested here, or only as part of a
multiprotein complex that is not preserved under immunoprecipitation
conditions. Alternatively, TERP-1 may act as an intracellular buffer by
binding to cofactors that interact with the C-terminal portion of ER,
thus altering the levels of proteins available to interact with
full-length ER.
Several steroid receptor cofactors, including those with positive
(coactivators) and inhibitory (corepressors) effects on transcription
have been identified (36). ER-stimulated transcription may be enhanced
by several coactivators such as steroid-receptor coactivator-1 [SRC-1,
(21)], the closely related ER-associated protein, ERAP160 (37, 38),
and receptor-interacting protein 140, RIP140 (39). Such proteins
interact with the ER LBD in a ligand-dependent manner, but do not
enhance transcription without ER. Our studies demonstrate that
transfected human SRC-1 can enhance rat ER
activity, as has been
noted for the human (40) and mouse (34) ER. Low levels of TERP-1 did
not increase ligand-activated ER
-stimulated transcription above
levels observed with SRC-1 alone. However, the enhancement of ER
activity by TERP-1 may occur by titration of corepressor proteins. Such
proteins have been identified for ER
(41), PR (41), and TR (42, 43).
A potential novel ER-specific repressor, which binds to the LBD of the
ER without the hinge region, has also recently been described from MCF7
breast cancer cells (44) and could represent one of many possible
cell-specific cofactors influencing ER action. The interaction of
TERP-1 with corepressor proteins is consistent with our results in that
binding of estrogen and SRC-1 alone may reduce the influence of
corepressors on ER-mediated transcription to the point that TERP-1s
effects are negligible.
Increasing concentrations of SRC-1 can overcome the inhibitory effect
of TERP-1. One potential mechanism is a direct interaction between
TERP-1 and SRC-1. Evidence for such an interaction comes primarily from
studies of TRß. Recent data from Feng et al. (45) shows
that mutations in amphipathic helices 3, 5, 6, and 12 of TRß diminish
the binding of glucocorticoid receptor-interacting protein 1 (GRIP1)
and SRC-1. The same mutations reduced transactivation by TRß (45).
Three similar mutations in helices 3, 5, and 12 of human ER
decreased GRIP1 binding and hormone-dependent transactivation (45). The
authors suggest that all three regions are needed for coactivator
binding and full receptor activity. TERP-1 contains only one of these
regions, helix 12. However, Jeyakumar et al. (46) recently
demonstrated that a 20-amino acid peptide (a.a. 437456) of TRß
reduced SRC-1 binding to TRß by 80%. Thus, TERP-1 might be able to
serve an analogous inhibitory role in certain cell contexts. Cell and
promoter-dependent responses we observed could result from the relative
contribution of such cofactors to ER-mediated transcription of
individual promoters and the relative concentration of specific
cofactors, ER and TERP-1, in different cell lines.
The expression and regulation of TERP-1 in vivo suggests
that it may play a role in estrogen feedback in the female rat
pituitary during the estrous cycle. The present study demonstrates that
low ratios of TERP-1:ER enhance E2-stimulated gene
expression in cell lines transiently transfected with ER
or ERß
and in cell lines expressing endogenous ER
. However, TERP-1 inhibits
E2-stimulated gene expression at higher ratios. TERP-1
appears to modulate transcription of model promoters and a
physiological promoter in an estrogen- and ER-dependent manner. The
ability of TERP-1 to both enhance and inhibit transcription allows for
the possibility that the dynamic regulation of TERP-1 during the
estrous cycle leads to both enhancement and inhibition of estrogen
actions in the pituitary. It is important to note that in
E2-treated rats TERP-1 mRNA levels are similar to those
observed during proestrus, and TERP-1 protein is readily detected in
the pituitaries of E2-treated rats at near- molar
equivalence with ER
(13). In the present study, TERP-1 protein
levels in transiently transfected Cos-1 cells were well below the
levels of ER
, even when greater amounts of TERP-1 plasmid were
transfected (Fig. 1
). These observations suggest that in
vivo TERP-1 may play a predominantly inhibitory role. The results
of the present study suggest that TERP-1 may act via protein-protein
interactions rather than direct high-affinity interactions with ER.
Thus, the truncated receptor could act as a buffer to modulate the
levels of coactivators and corepressors available for interaction with
the full-length ER, and thereby modify its transcriptional
activity.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Transfections
The monkey kidney Cos-1, ER-negative cell line was maintained in
DMEM supplemented with 10% newborn calf serum (NCS), 100 U/ml
penicillin, and 100 µg/ml streptomycin. Cells were plated in
phenol-red free DMEM/3% charcoal- stripped NCS in 30-mm wells and
grown to 6080% confluence before transfection by the calcium
phosphate method (47). Each well was transfected with a total of 5 µg
DNA for 1417 h, washed with PBS, and treated with hormone or drugs
for 68 h before being lysed and collected for assessment of
luciferase activity. The ER-containing GH3 rat somatolactotrope cell
line was maintained in DMEM/10% NCS and transfected by the
diethylaminoethyl (DEAE)-Dextran method (48). Cells were plated in
phenol-red free DMEM/10% stripped NCS in 60-mm wells and allowed to
grow to 4050% confluence before transfection. Media were removed,
and cells were washed with PBS before 2 h of treatment with DNA in
DEAE-dextran. After removal of DEAE-dextran, cells were treated for 2
min with 10% dimethylsulfoxide in HEPES-buffered saline, washed with
PBS, and treated with hormones or drugs in media for 24 h. AlphaT3
(
T3) cells (49), an ER-expressing gonadotropin cell line of murine
origin, were maintained in DMEM/10% FCS and plated and transfected
with calcium phosphate as described for Cos cells. Cell culture media
and sera were purchased from GIBCO-BRL (Gaithersburg, MD).
E2 and 4-hydroxytamoxifen were obtained from Sigma Chemical
Co. (St. Louis, MO). The antiestrogen ICI 182,780 was kindly provided
by Dr. Richard Santen (gift to Dr. Santen from Dr. Alan Wakeling,
Zeneca Pharmaceuticals, Macclesfield, England). Details of specific
experiments are provided in Results and in figure
legends.
Plasmids
TERP-1 and full-length rat ER
cDNAs were isolated from female
rat pituitaries by 5'-rapid amplification of cDNA ends (11) and
cloned into a pcDNA expression vector (Invitrogen Corp., San Diego, CA)
under the control of the cytomegalovirus (CMV) promoter. The
full-length rat ERß clone in the pCMV5 expression vector was obtained
from Dr. Deborah Lannigan. The SRC-1 expression vector was obtained as
a gift from Dr. Bert OMalley. All reporter plasmids contained the
luciferase reporter gene. Two simple reporter plasmids containing model
estrogen-responsive promoters were used in these studies.
(ERE)2-vit-TK-luc (Vit6luc) contained two vitellogenin consensus EREs
and the thymidine kinase promoter. 2EREpGL2 was kindly provided by Dr.
Deborah Lannigan and contained two consensus EREs upstream of a TATA
box minimal promoter. One physiological estrogen-responsive reporter
plasmid, PRL luc Link V2 (PRLluc, gift of Dr. Richard N. Day),
containing 2.5 kb of the rat PRL promoter was also used. A rat
glycoprotein
-subunit promoter construct (-411 to +77 bp) cloned in
our laboratory [
3-luc (50)] and lacking EREs was used as a control
for ERE dependence. To normalize the total plasmid DNA concentrations
used in transfections, the empty pcDNA vector was used. In experiments
with SRC-1, the pBKcmv empty vector was used to normalize total DNA
(21). Luciferase activity was assessed on a Turner-20E luminometer
using a Promega (Madison, WI) luciferase assay kit, and samples were
normalized by assessing lysate protein with Bio-Rad (Hercules, CA)
protein dye. Data are presented as relative arbitrary light units,
normalized to the estrogen-negative condition. Values represent the
mean ± SD of triplicate samples, unless otherwise
noted.
Estrogen Binding Assay
Estrogen binding of ER
and TERP-1 was assessed in
vitro and in transfected Cos-1 cells. ER
and TERP-1 were
in vitro translated using coupled transcription-translation
from rabbit reticulocyte lysate (TNT kit, Promega). Lysates (10 µl)
were incubated with 0.05 pmol [3H]E2 and 5
pmol of unlabeled DES in triplicate at 4 C for 4 h. Bound and free
E2 were separated using the hydroxylapatite method (19),
and 200-µl aliquots in 10 ml of ReadySafe scintillation fluid
(Beckman Instruments, Fullerton, CA) were counted in an LKB Rackbeta
scintillation counter (LKB Instruments, Rockville, MD). We assessed
estrogen binding in calcium phosphate-transfected Cos-1 cells using
either 1 µg of ER
or 1 µg of TERP-1. Cells were plated in
DMEM/3.3% charcoal-stripped NCS in 30-mm wells. Media were changed
daily for 3 days before transfection. Cells were transfected for
16 h. Media were removed, the cells washed with PBS, and wells
filled with serumless, phenol-red free DMEM. Cells were incubated for
an additional 24 h before estrogen binding was assessed.
Estrogen binding was determined with a competition assay. Cells were
treated with 1 nM [2,4,5,6-3H]E2
(New England Nuclear, specific activity = 114 Ci/mmol) and varying
amounts of unlabeled E2. Cells were incubated for 1 h
at 37 C, rinsed twice with PBS/0.1% BSA and twice with PBS, and
collected in 10:1 Tris-EDTA (pH 8). Cells were then sonicated for 20
sec with a Fisher sonic dismembrator, and 200 µl of each extract were
added to 10 ml of scintillation fluid (ReadySafe; Beckman) and counted
in an LKB Rackbeta scintillation counter. Specific binding (labeled
E2 - 1 µM unlabeled E2) was
calculated, corrected for total protein, and analyzed by Scatchard
plots.
Immunoblot Analysis
TERP-1 and ER
protein expression in transfected cells was
determined by immunoblot analysis using a rabbit polyclonal antibody
(C1355) generated against the final 14 C-terminal amino acids of the
rat ER
. The antibody and conditions for Western analysis were
determined using in vitro translated TERP-1 and ER
and
have been described previously (13). For analysis of cellular
expression, Cos-1 cells were transfected with 1 µg of ER
and
varying concentrations of TERP-1 by calcium phosphate for 4 h and
allowed to incubate 48 h in DMEM/10% NCS. In some experiments GH3
cells were also analyzed after transfection with TERP-1. Western
analysis was performed with enhanced chemiluminescence (Amersham,
Arlington heights, IL) using the C1355 antibody at 1:7500 and an
horseradish peroxidase-conjugated donkey antirabbit IgG secondary
antibody (1:500; Amersham).
Immunoprecipitations
Because TERP-1 contains the identical amino acid sequence as the
C terminus of ER
, it was necessary to differentiate TERP-1 from
ER
by epitope tagging. PCR was used to add the eight-amino acid FLAG
epitope (Eastman-Kodak, Rochester, NY) to the TERP-1 sequence. The
resulting PCR product was ligated into the PCR2.1 cloning vector
(Invitrogen), sequenced, and then subcloned into the pcDNA expression
vector. Proteins were synthesized from ER
, ERß, TERP-1, and
TERP-FLAG plasmids (1 µg each construct alone, or in combination) by
in vitro translation using coupled transcription-translation
from rabbit reticulocyte lysate (TNT kit, Promega). Each reaction also
contained 40 µCi [35S]methionine (New England Nuclear;
1175 Ci/mmol), and 10 nM E2. In some
experiments, different ER protein isoforms were cotranslated in the
same reactions. Results were similar or identical to results with
individually translated proteins. Each immunoprecipitation reaction
contained 6 µl of translated proteins, 1% BSA, and 50 µl of a 2x
slurry of either anti-FLAG M2 affinity gel (VWR), or protein A
Sepharose (Pharmacia, Piscataway, NJ) linked to either ER C1355
(C-terminal antibody) or ER715 (gift of Dr. Jack Gorski). ER715
recognizes the hinge region of ER
and will recognize only
full-length receptor. Beads and antibody were first brought to a 2x
slurry with 1.5% Nonidet-P40 (NP-40) in PBS, pH 7.6. The proteins,
beads, and antibody were incubated at 4 C for 1 h. After
incubation, beads were washed four times with 1.5% NP-40 in PBS and
resuspended in 25 µl of 1.5% NP-40 in PBS and 25 µl of loading
buffer (100 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 0.2%
bromphenol blue). Samples were boiled for 3 min and subjected to
electrophoresis on a 12% acrylamide gel for 4 h at 150 V. Gels
were dried and exposed to film overnight.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to acknowledge the excellent technical
assistance of Alice Anderson and Anthony Lim at the Core for Studies in
Molecular and Cellular Research at the University of
Virginia.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Margaret A. Shupnik, Department of Internal Medicine, Division of Endocrinology and Metabolism, Box 578, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: MAS3X{at}VIRGINIA.EDU
This work was supported by NIH Grants RO1 HD-25719 (M.A.S.), F32
HD-08219 (D.A.S.), a training grant for Diabetes and Hormone Action at
the University of Virginia (T32 DK-07320; D.A.S.), training grant T32
GM-07055 (E.M.R.) and the Center for Research in Reproduction
(U54-HD28934).
Received for publication October 27, 1997.
Revision received October 8, 1998.
Accepted for publication October 21, 1998.
 |
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