Proteasome-Mediated Proteolysis of Estrogen Receptor: A Novel Component in Autologous Down-Regulation
Elaine T. Alarid,
Natalie Bakopoulos and
Natalia Solodin
The Department of Physiology University of
Wisconsin-Madison Madison, Wisconsin 53706
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ABSTRACT
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Regulation of estrogen receptor (ER)
concentration is a key component in limiting estrogen responsiveness in
target cells. Yet the mechanisms governing ER concentration in the
lactotrope cells of the anterior pituitary, a major site of estrogen
action, are undetermined. In this study, we used a lactotrope cell
line, PR1, to explore regulation of ER protein by estrogen. Estrogen
treatment resulted in an approximate 60% decrease in ER steady state
protein levels. Suprisingly, the decline in ER protein was apparent
within 1 h of estrogen treatment and occurred in the absence of
protein synthesis and transcription. Direct regulation of ER protein
was further confirmed by pulse chase analysis, which showed that ER
protein half-life was shortened from greater than 3 h to 1 h
in the presence of estrogen. The estrogen-induced degradation of ER
protein could be prevented by pretreatment with peptide aldehyde
inhibitors of pro-teasome protease whereas inhibitors of calpain
and lysosomal proteases were ineffective. Inhibition of proteasome
activity maintained ER protein at a level equivalent to control cells
not stimulated with estrogen but increased estrogen-binding activity by
1.75-fold. Proteolytic regulation of ER by the proteasome is not
limited to pituitary lactotrope cells but is also operational in MCF-7
breast cancer cells, suggesting that this may be a common regulatory
pathway used by estrogen. These studies describe a nongenomic action of
estrogen that involves nuclear ER: rapid proteolysis of ER protein via
a proteasome-mediated pathway.
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INTRODUCTION
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Steroid hormone action is, in large part, controlled by cellular
receptor concentration. Unlike peptide hormone receptors, which are
single components of complex signal transduction cascades, steroid
receptors transmit signal directly to DNA. As ligand-activated
transcription factors, they bind regulatory elements in responsive
genes and, along with coactivators or corepressors, control
transcription. Although other proteins are involved, the receptor is
the limiting factor that dictates the magnitude of the steroid
response. In cell lines engineered to overexpress glucocorticoid
receptor (GR), there is a linear relationship between the amount of GR
and transcriptional activation of target genes (1). Similar studies of
estrogen receptor (ER) indicate that physiological levels of ER limit
estrogen transcriptional activity well below the cellular capacity to
respond to estrogen (2). This pivotal role of steroid receptors makes
them a focal point in regulating steroid hormone function.
The level of steroid receptors in cells changes with varying
physiological states. In most cases, the primary endocrine regulator is
the ligand itself. In an autoregulatory feedback loop, estrogen induces
a decline in both ER protein and mRNA (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Several mechanisms have
been proposed to explain how estrogen controls ER levels, most of which
focus on regulation at the level of RNA. Studies using Rat-1
fibroblasts stably transformed with ER (Rat 1+ER) (7) and MCF-7 breast
cancer cells (11, 12) support both transcriptional and
posttranscriptional mechanisms. The focus on transcriptional mechanisms
is based on the assumption that decreased protein concentration is
largely a consequence of decreased steady state levels of mRNA.
ER regulation by estrogen has been documented in a number of systems,
yet little is known about mechanisms governing ER regulation in
estrogen target tissues outside of breast cancer cell lines and uterus.
To examine ER regulation in the pituitary, we took advantage of the
recent derivation of a lactotrope cell line named PR1. The PR1 cell
line was derived from an estrogen-induced lactotrope hyperplasia in
F344 rats (14). It exhibits unique sensitivity to estrogen with a high
affinity for estradiol [dissociation constant (Kd) =
10-11 M (15)]. Like MCF-7 cells and other
model systems, we observed that estrogen induces a decrease in ER
protein levels in PR1 cells. However, within the first 12 h, ER
protein levels decrease without a concomitant decline in mRNA levels.
The rapid loss of ER protein in the absence of changes in ER mRNA
suggested that ER protein may be regulated independently of
transcription. Utilizing a short time frame of estrogen exposure, we
are able to isolate changes in ER protein levels away from changes in
RNA. This permits the exploration of regulatory mechanisms directly
controlling ER protein. Here we report that estrogen stimulates
degradation of ER protein via a proteasome-mediated proteolytic
mechanism.
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RESULTS
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Estrogen Action on ER in Pituitary Lactotrope Cells
Study of the regulation of ER in the pituitary has been hampered
by the lack of a model system that possesses endogenous ER and that
exhibits robust responses to estrogen. PR1 cells, like the hyperplasia
from which they were derived, are extremely sensitive to estrogen and
are stimulated to proliferate at doses of estradiol as low as
10-14 M (15). We capitalized on the
hypersensitivity of these cells to examine the mechanism(s) governing
estrogen regulation of ER in lactotrope cells. Total ER protein levels
were monitored by Western blot analysis of whole-cell extracts. Time
course (Fig. 1
, A and B) and dose
response (Fig. 1
, C and D) experiments indicate that 10-10
M 17ß-estradiol (E2) is sufficient to elicit
an approximate 50% decrease in ER protein levels at 1 and 2 h.
These results were confirmed using primary anti-ER antibodies directed
against the hinge and C-terminal regions to demonstrate that the loss
of signal was not due to epitope masking (data not shown). Moreover,
the identical results with different antibodies suggest that this
decline is representative of total ER protein and not an
epitope-specific pool. To further verify that the decrease in protein
was specific to ER, blots were reprobed with antibody directed against
the ubiquitous protein, I
B
, which is not regulated by estrogen.
I
B
protein levels were unchanged in the presence and absence of
E2 and serve as a loading control for total protein content
(Fig. 1A
, lower panel).

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Figure 1. Effect of Estrogen on Total ER Protein Levels in
PR1 Lactotrope Cells
A, Time course of ER response to estrogen. Cells were treated for
the indicated length of time with 10 nM 17ß-estradiol
(E2). After treatment cells were lysed directly in SDS
sample buffer, and proteins were separated by gel electrophoresis in a
7.5% acrylamide gel. Protein was transferred to nylon membrane, and
Western analysis was performed with antibody to rat ER. A
representative Western blot analysis of ER protein in total cell
extract is shown in the upper panel. The lower
panel shows the same Western reprobed with anti-I B
antibody as a loading control. B, Quantification of time course
analysis of ER response was performed by laser densitometry. Cumulative
data from four independent experiments are shown. Relative ER levels
are represented by the mean ± SEM relative to
EtOH-treated controls. Statistical analysis by ANOVA followed by
Students paired t test indicate that E2
treatment results in a significant decrease in ER levels,
P < 0.01. C, Dose response of estrogen on ER
protein levels. Equivalent numbers of PR1 cells were treated for 2
h with varying doses of E2 as indicated. A representative
Western analysis of total ER protein in whole-cell extract is shown. D,
Quantification of dose-response analysis was performed by laser
densitometry. Data represent the mean ± SEM for three
independent experiments relative to untreated controls.
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Analysis of autologous down-regulation of ER protein in MCF-7 and Rat
1+ER cells suggested that estrogen reduces ER primarily by decreasing
steady state ER mRNA levels (3, 7, 8, 9, 10, 11, 12, 13). In contrast, Northern blot
analysis of ER mRNA in PR1 cells shows that mRNA levels do not change
within 2 h of E2 treatment (see below, Fig. 5
, lanes 1
and 2). These results suggested that estrogen may induce a rapid
decrease in ER protein that is independent of ER synthesis. To test
this possibility, ER protein levels were examined in the presence of
inhibitors of protein synthesis and transcription. Figure 2A
shows that halting de novo
protein synthesis with a series of inhibitors can decrease ER protein
levels relative to untreated controls. However, in no case was the
decline in ER levels equivalent to that induced by estrogen.
Furthermore, the addition of inhibitors of either protein synthesis
(Fig. 2A
) or transcription (Fig. 2B
) failed to prevent estrogen-induced
down-regulation. In Fig. 2B
(left panel) and in subsequent
figures, ER appears as a doublet. The appearance of the doublet is
spurious from gel to gel but is only present in estrogen-treated
groups. The higher molecular weight form may, therefore, represent an
estrogen-dependent posttranslational modification such as
phosphorylation. Neither form of ER is preferentially lost in response
to estrogen. These data indicate that the mechanism employed to elicit
a rapid loss of receptor protein does not require de novo
protein synthesis or transcription.

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Figure 5. ER mRNA Levels Do Not Change in Response to
E2 or ALLnL
PR1 cells were pretreated with DMSO or ALLnL (100 µM) for
30 min followed by treatment with either EtOH or E2 (10
nM). After treatment, 20 µg of total RNA were isolated
and separated by electrophoresis in a 1% formaldehyde gel. A,
Representative Northern analysis of 20 µg of total RNA hybridized
with radiolabeled probe for human ER (upper panel) or
mouse GAPDH (lower panel). B, ER mRNA levels were
measured by PhosphoImager analysis of three independent experiments. ER
mRNA levels were normalized to GAPDH mRNA levels in the same lane to
correct for loading differences. Data are presented relative to the
untreated DMSO/EtOH (DMSO) control, which is arbitrarily set at 1.
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Figure 2. Inhibition of Protein Synthesis and Transcription
Do Not Effect Rapid Turnover of ER Protein
A, PR1 cells (1 x 106) were aliquoted into
Optimem media after 3 days in estrogen-free conditions. Protein
synthesis was inhibited by a 30-min preincubation with 10 µg/ml of
each of the designated inhibitor: cyclohexamide (C), puromycin (P),
anisomycin (A), and emetine (E). After preincubation, cells were
treated with either ethanol (EtOH) or 10 nM E2
for 2 h. Total cell extract was obtained by lysing cells directly
in sample buffer. The entire sample was analyzed by SDS-PAGE followed
by Western analysis with antibody directed against rat ER. Shown is a
representative Western blot (left), and the
quantification of three independent experiments by laser densitometry
(right) The data represent the results of three
independent experiments. B, Cells (1 x 106) were
preincubated with 100 µM of transcriptional inhibitor,
5,6-DRB, or DMSO for 30 min before treatment with 10 nM
E2 for 1 and 2 h. ER protein was analyzed as described
in panel A. Laser densitometric measurement of total ER levels is shown
to the right and represents the mean ±
SEM relative to controls for three independent experiments.
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ER Protein Is Degraded in Response to Estrogen
The half-life of ER protein is estimated at approximately 35 h
(16, 17, 18). Pulse chase analysis of metabolically labeled ER in
whole-cell extracts (Fig. 3
) indicates
that ER half-life in PR1 cells is greater than 3 h but is within a
similar time range as the half-life reported in breast and uterus. This
result supports the notion that decreased ER synthesis cannot account
for the rapid decline in ER protein steady state levels since
presynthesized ER levels do not change significantly within 2 h.
In addition, the data imply that estrogen may induce degradation of ER
protein. Figure 3
illustrates that the
half-life of liganded ER is approximately 1 h. Nonspecific bands
that coimmunoprecipitate with ER are not regulated by estrogen and
serve as an internal control. This 3-fold change in ER turnover rate is
greater than that reported in MCF-7 cells measured by density shift
technique and binding of [3H]tamoxifen aziridine
(16, 18) and contrasts with previous studies in uterine cells (17, 19).
These findings show that estrogen can directly control ER protein by
inducing proteolysis and further highlight the importance of receptor
protein regulation in lactotrope cells.

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Figure 3. Estrogen Induces Degradation of ER Protein
Pulse chase analysis was performed as described in Materials and
Methods. Cells were labeled with
35S-methionine for 2 h and were subsequently chased
for the indicated length of time in complete phenol red-free media with
10% stripped serum in the absence (-) or presence (+) of 10
nM E2. ER was isolated by immunoprecipitation
and analyzed by SDS-PAGE. B, ER level experiments were quantified with
a PhosphoImager and Imagequant software. Relative ER levels were
determined as a percentage of the EtOH-treated group before chase (time
0). Data represent the mean relative ER values of three independent
experiments.
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Inhibitors of Proteasome-Mediated Proteolysis Prevent ER
Turnover
A number of previous studies have addressed potential proteases
that may degrade ER (20, 21, 22, 23), but no one protease has emerged as a
definitive candidate for mediating estrogen-induced down-regulation.
The role of three major intracellular proteases (calpain, proteasome,
and lysosomal enzymes) was examined by blocking protease activity with
a series of inhibitors (Fig. 4A
). High
concentrations of each inhibitor were used intentionally to ensure that
any potential effects might be noticed. The multicatalytic proteasome
is responsible for degradation of the majority of cellular proteins.
Rock et al. (24) demonstrated that proteasome peptidase
activity could be blocked by exposure to peptide aldehyde inhibitors
including MG115 and N-acetyleucylleucylnorleucinal (ALLnL).
Pretreatment of PR1 cells with MG132, a more potent derivative of
MG115, and ALLnL prevented estrogen-induced loss of ER. In contrast,
calpeptin and the peptide inhibitor
N-acetylleucylleycylmethional (ALLM), which preferentially
block calpain activity, failed to prevent ER degradation. E64D and
NH4CL, which inhibit cysteine proteases and lysosomal
function, respectively, were also without effect. These results suggest
that proteasome activity may regulate ER response to estrogen. However,
protease inhibitors do not exhibit strict specificity. In the case of
the proteasome, the rank order of potency of peptide aldehyde
inhibitors directly reflects their specific activity against proteasome
function (24). Thus, the relative effectiveness of these inhibitors can
be used as a measure of specificity for the proteasome. Dose response
experiments were performed with MG132 and ALLnL to determine their
relative potencies (Fig. 4B
). As a control, similar experiments were
conducted with the calpain inhibitor, calpeptin. Examination of data in
Table 1
indicates that MG132 was the most
effective, preventing ER degradation to a dose of 0.03
µM. ALLnL was less effective but blocked ER degradation
between 100 uM and 12.5 µM.
L-1-Tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a
serine protease inhibitor that weakly inhibits proteasome activity,
partially prevented ER proteolysis at a high dose of 12.5 µg/ml (Fig. 4A
). Calpeptin was ineffective at all doses tested. The order of
potency exhibited by these inhibitors in our studies (MG132 >
ALLnL) is identical to that previously reported by Rock et
al. (24). In addition, the weak activity of TPCK against
proteasome function is reflected in its partial antagonist action at
high doses. These data support the conclusion that estrogen can
regulate ER protein directly through a proteasome-mediated pathway. In
addition, the ability of the MG132 and ALLnL to increase ER levels
relative to controls suggests that the proteasome may also be involved
in basal turnover of receptor.

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Figure 4. Proteasome Inhibitors Prevent ER Degradation
A, Equivalent number of PR1 cells ((1 x 106/ml) were
pretreated with DMSO, MG132 (50 µM), ALLnL (100
µM), ALLM (100 µM), E64D (50 µg/ml), TPCK
(12.5 µg/ml), or NH4Cl (50 mM) for 30 min.
Samples were then treated for an additional 2 h with EtOH (lane 1)
or 10 nM E2. After treatment, cells were lysed
in SDS sample buffer, and the entire sample was subjected to SDS-PAGE
and Western analysis for ER. B, PR1 cells were treated as in panel A
with the designated concentrations of MG132 (upper
panel), Calpeptin (upper panel), and ALLnL
(lower panel). ER levels were visualized after Western
analysis of whole-cell lysates.
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To ensure that proteasome inhibitors were acting directly on ER
protein, steady state levels of ER mRNA were examined in the presence
and absence of ALLnL and E2. Figure 5A
shows a representative autoradiogram
of a Northern blot hybridized with a radiolabeled probe of human ER
cDNA. To control for equivalent loading, blots were rehybridized with
probe for glyceraldehyde phosphate dehydrogenase (GAPDH). The
results of multiple Northern analyses are presented
quantitatively in Fig. 5B
. Our data show that neither pretreatment with
proteasome inhibitor nor treatment with E2 alters steady
state levels of ER mRNA within the first 2 h of treatment. To
further verify that the action of the inhibitor was limited to ER
protein, pulse chase experiments were performed (Fig. 6
). As shown in the more detailed time
course of ER half-life (Fig. 3
), estrogen induces greater than 50%
loss of ER within 2 h. We, therefore, chose the 2-h time point to
examine the effect of preventing proteasome function on
estrogen-induced shortening of ER half-life. In confirmation of our
previous results, estrogen treatment causes a dramatic decrease in ER
half-life. Addition of proteasome inhibitor maintained receptor levels
comparable to those of controls in both the presence and absence of
estrogen. Collectively, these results show that inhibition of
proteasome function prevents estrogen-induced proteolysis of ER
protein.

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Figure 6. Blocking Proteasome Function Prevents ER Turnover
PR1 cells were labeled with [35S]methionine for
2 h. After labeling, cells were washed in complete phenol red-free
RPMI media with 10% stripped serum and pretreated with DMSO or ALLnL
(100 µM) for 30 min (time 0). Cells were subsequently
chased for 2 h in complete medium with 10% stripped serum in the
absence (-) or presence (+) of 10 nM E2.
Samples were controlled for EtOH content. ER was isolated by
immunoprecipitation and analyzed by SDS-PAGE. B, ER levels from three
separate pulse chase experiments were quantified by PhosphoImager
analysis. Data are presented as a percentage of the DMSO-treated group
before the chase (t = 0).
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Preventing ER Down-Regulation Increases Estrogen-Binding
Activity
Examination of our Western and pulse chase analysis suggests that
prevention of ER down-regulation with proteasome inhibitors maintains
receptor content at a level equivalent to that found in the cells not
exposed to estrogen (Figs. 3
, 4
, and 6
). Whole-cell estrogen uptake
assays were performed to determine what effect this may have on
estrogen-binding activity. For comparison, parallel samples were
evaluated by both binding assay and Western analysis. Since binding
data requires incubation with [3H]estradiol, there can be
no measure of ER in the absence of estrogen. Therefore, ER in
unstimulated cells is represented by Western only. In accordance with
our previous data, Fig. 7A
illustrates
that total ER protein content in cells pretreated with proteasome
inhibitor is qualitatively similar to those in nontreated cells. This
level is higher than those pretreated with solvent since
[3H]estradiol induces degradation of receptor protein.
Surprisingly, pretreatment with proteasome inhibitor increased specific
binding by 1.75-fold relative to those pretreated with
dimethylsulfoxide (DMSO) (Fig. 7B
). In light of the Western analysis,
the binding activity in cells pretreated with ALLnL more closely
reflects the receptor content in unstimulated cells. This implies that
inclusion of a proteasome inhibitor increases estrogen-binding activity
not by increasing receptor number but by maintaining it at the level of
nonstimulated cells. These findings suggests that since ER protein is
down-regulated during the procedure time required to measure specific
binding, the resultant measurement may only account for receptor that
remains after rapid proteolysis has taken place. They further suggest
that binding measurements in the absence of proteasome inhibitors may
underestimate cellular content of ER.

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Figure 7. ALLnL Increases ER-Binding Activity
Whole-cell estrogen uptake assays were performed as described in
Materials and Methods. PR1 cells (1 x
106/ml) were preincubated with DMSO or ALLnL (100
µM) for 30 min. Cells were then incubated with
[3H]estradiol (2 nM) alone or with 200-fold
excess of unlabeled DES (0.4 µM) for 2 h. A,
Parallel sets of samples were lysed in SDS sample buffer and subjected
to Western blot analysis for ER levels. Lane 1 shows control sample
that is preincubated with DMSO and treated for 2 h with EtOH. B,
After incubation with radiolabeled ligand, cells were washed
extensively with PBS/1% BSA and lysed with EtOH. ER-binding activity
was determined by scintillation counting of the EtOH lysate. Specific
binding was determined by the subtraction of nonspecific (+DES) from
total (+E2 alone). Specific binding data are presented for
three independent experiments consisting of duplicate samples for each
treatment group. Relative binding activity is shown in
parentheses.
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Proteasome-Mediated Proteolysis of ER Is not Unique to Lactotrope
Cells
Autologous down-regulation of ER has been examined in a
number of systems including uterine cells (17, 19), MCF-7 breast cancer
cells (3, 4) (9, 10, 11, 12), and cells stably transformed with expression
plasmids of mouse (13) and human ER (7). The majority of these studies
focus on estrogens action after several hours, sometimes days, of
stimulation. The rapid response to estrogen exhibited by the PR1 cells
revealed the role of proteasome-mediated proteolysis in the regulation
of ER protein. To determine whether proteolytic regulation of ER is
unique to lactotropes, we examined whether ALLnL affected estrogen
regulation of ER protein in MCF-7 breast cancer cells, a model system
in which down-regulation has been studied extensively. Using an
identical treatment regimen, as described for the PR1 lactotropes, the
effects of blocking proteolysis on ER protein levels in MCF-7 cells was
examined. Western analysis shown in Fig. 8
illustrates that pretreatment with
ALLnL prevented down-regulation of ER protein in response to estrogen
exposure. The demonstration that this mechanism functions in both PR1
cells and MCF-7 cells shows that proteolytic regulation of ER extends
beyond rat lactotrope cells to human breast cancer cells and suggests
that it may be a common regulatory mechanism used by estrogen to
control ER protein.

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Figure 8. Proteolytic Regulation of ER in MCF-7 Cells
MCF7 cells (1 x 106/ml) were treated for the
indicated length of time with (+E2) or without (-E2) 10
nM E2 after 30 min preincubation with DMSO or
ALLnL (100 µM). Cells were lysed in SDS sample buffer,
and the total sample was subjected to SDS PAGE. Western analysis was
performed using antibody (SR1000) directed against an epitope in the
hinge region of human ER. A, Representative Western analysis; B,
quantification of three independent experiments. The lower molecular
weight nonspecific band seen in panel A serves as an internal loading
control.
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DISCUSSION
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Estrogen induces down-regulation of its receptor as part of a
classical endocrine feedback loop. The mechanism of this regulation has
been studied previously in a number of model systems. Work conducted in
estrogen target cells, such as breast and uterus, and in cells
expressing exogenous ER implicates both direct transcriptional
repression and posttranscriptional mechanisms (7, 9, 11, 12, 13). In line
with the transcriptional regulatory role of estrogen, earlier reports
focus on changes in ER mRNA levels after several hours and days of
stimulation. To our surprise, down-regulation of ER protein in PR1
lactotrope cells occurred rapidly in 1 h. The rate of ER protein
loss observed in PR1 cells is faster than previously reported changes
in ER mRNA levels in other model systems. Indeed, our results indicate
that estrogen induces the loss of ER protein independent of protein
synthesis and transcription. This observation prompted us to reexamine
the regulatory mechanisms governing autologous down-regulation of ER,
not from the perspective of estrogens action on transcription, but
from the perspective of potential actions of estrogen directly on ER
protein.
Western analysis of steady state levels showed a rapid loss of total ER
protein content in response to estrogen (Fig. 1
). We used pulse chase
analysis to directly demonstrate that estrogen induces degradation of
ER protein and shortens its half-life from greater than 3 h to
1 h. Previous studies that examine the effect of estrogen on ER
half-life report conflicting results. For example, while estrogen
shortens ER half-life in MCF-7 cells (4, 16), it has no effect in
primary uterine cells (17, 19) and lengthens ER half-life in COS cells
transfected with mouse ER (25). This variation may simply reflect
differences in model systems. It may also, however, reflect differences
in methodology. ER levels are commonly determined based on specific
estrogen binding. With the exception of studies by Dauvois et
al. (25), all reported measurements of ER half-life have been
based on binding assays. Comparison of estrogen-binding activity and
total ER levels detectable by Western blot (Fig. 7
) shows a discrepancy
between the total number of ERs present in the cell and the number
determined by binding assay. Our studies indicate that within the
length of time necessary to measure specific estrogen binding (16), a
portion of ER protein is degraded. This is supported by early studies
of Horwitz and McGuire (6) who demonstrated that the number of
ligand-bound sites extracted from the nucleus drops rapidly after 30
min. Consequently, the ER level estimated by binding assays may not
take into account ER that is lost during incubation with radiolabeled
ligand. The underestimation of ER in the absence of estrogen could
account for the lack of measurable difference in ER half-life in the
presence of estrogen. The advantage of pulse chase is that
[35S]methionine labeling permits the direct examination
of a presynthesized pool of ER protein before and after treatment with
estrogen, and it does not require binding studies to assess receptor
levels.
Throughout these studies, we focused on total ER content by analyzing
ER in whole-cell extracts. Although ER is a nuclear protein, when
cellular fractions are prepared to distinguish cytoplasmic and nuclear
components, ER can be artifactually extracted with cytoplasmic
proteins. Estrogen induces undefined changes in the biochemical
properties of ER that prevent extraction of ER with hypotonic buffers.
This process, referred to as "transformation," can be characterized
by an increase in ER in the nuclear fraction upon stimulation with
estrogen. It is possible that changes in ER solubility may account for
the increased half-life of liganded ER reported for mouse ER (25) since
the analysis was performed on lysate extracted with high salt. Earlier
studies of ER regulation consider "cytoplasmic" and nuclear ER
separately. This confounds interpretation of the results in light of
the knowledge that ER is exclusively nuclear (26). To simplify our
interpretation, we chose to examine whole-cell lysate, which eliminates
any changes in ER levels associated with transformation or the
extraction process.
We provide evidence that estrogen induces proteolysis of ER via a
proteasome-mediated pathway in both the pituitary and MCF-7 breast
cancer cell lines. Proteolysis is an important regulatory strategy
governing a number of processes, including cell cycle regulation,
signal transduction, antigen presentation, and protein quality control.
In particular, the proteasome pathway has been implicated as a major
protease responsible for the turnover of most proteins in the cell
(24). Among those proteins are transcriptional regulators, including
p53 (27), MyoD (28, 29), cJun (30), yeast MAT
2 (31), and the steroid
family corepressor N-CoR (32). In most cases, substrate recognition by
the proteasome requires the attachment of multiple ubiquitin moieties
to proteins targeted for degradation. Nirmala and Thampan (33)
demonstrated that ER is ubiquitinated in an estrogen-dependent manner
in normal goat uterus (33). Further, the ubiquitin-activating enzyme,
UBA, and ubiquitin-conjugating enzymes, UBCs, can promote in
vitro degradation of ER protein (34). Consistent with
ubiquitin-dependent proteasome function, ubiquitination would serve as
a targeting signal to direct ER to the proteasome. Once ER is within
the multicatalytic enzyme, it would then be degraded into small
peptides, which may account for the lack of observable protein
intermediates associated with ER processing (16, 17). This proteolytic
mechanism can account for the rapid loss of ER protein that precedes
changes in ER RNA.
The threshold nature of this response is illustrated by both dose
response and binding studies. Examination of the Western blots shown in
Fig. 7A
shows that the addition of 200-fold excess of
diethylstilbestrol (DES) had no greater effect on ER levels beyond
E2 alone. Dose analysis indicates that significant
down-regulation of ER protein does not occur at doses lower than
10-10 M E2. This is a saturable
dose of E2 and is sufficient to occupy 100% of ER in the
PR-1 cells (15). Although it remains possible that the detection method
is not sensitive enough to measure loss of a small amount of receptor,
doses of E2 sufficient to occupy 50% of the receptors,
i.e. 10-11 M E2, failed
to evoke a corresponding decrease in ER protein. Furthermore, levels of
estrogen sufficient to occupy 100% of receptors do not result in the
total loss of ER protein. The lack of correlation between receptor
occupancy and amount of ER degraded suggests that ligand binding itself
does not target ER for degradation. Since proteasomes are estimated to
comprise 1% of the total soluble cellular protein, it is unlikely that
they are a limiting component (24). It is possible that downstream
events may be required to manifest this response.
Estrogen treatment cannot completely deplete cells of ER protein.
Approximately 40% of ER protein remains despite exposure to large
doses of estradiol. It is interesting to speculate that certain ER
molecules may be resistant to degradation. It is unlikely that the
remaining ER represents receptor in a subpopulation of cells since the
PR-1 cells do not proliferate significantly (15) and are most likely
synchronized when maintained in an estrogen-free environment for 3
days. Moreover, based on molecular weight and epitope recognition, the
remaining ER is not ERß (35) or truncated estrogen receptor products
(TERPs) (36), additional ER species reported to be present in
pituitary cells. The question remains what distinquishes ER that is
destined for degradation from ER that is not. In several cases of
proteasome-regulated proteolysis, alterations in the biochemical and
physical properties of proteins serve as signals to induce
ubiquitination and degradation. The antiestrogen ICI induces rapid
degradation of ER (25, 37). In PR1 cells, this appears to operate
through a proteasome-mediated mechanism similar to E2
(E. T. Alarid, unpublished observation). Dauvois et al.
(20) suggest that ICI disrupts nuclear-cytoplasmic shuttling and
promotes cytoplasmic accumulation in certain cells. Interestingly,
cytoplasmic ER did not appear to degrade, suggesting that nuclear
localization may be a requirement for the degradation process.
Regulation of MyoD protein, a skeletal muscle transcription factor, is
controlled in part by ubiquitin-proteasome-mediated degradation (28, 29). Abu Hatoum et al. (28) recently demonstrated that
binding of MyoD to its cognate DNA response element stabilizes MyoD
protein and generates a complex that is resistant to proteasome
degradation. DNA binding may likewise confer resistance to ER protein.
Degradation of many proteins is additionally regulated by
phosphorylation. Activation of the NF-
B signaling cascade by several
extracellular stimuli requires phosphorylation-dependent degradation of
I
B
by the ubiqutin proteasome pathway (38). The functional role
of phosphorylation of ER is not as yet clearly defined. Specific
protein-protein interactions also influence susceptibility to
proteasome-mediated degradation. Work by Whitesell and Cook (39)
demonstrates that changes in composition of the protein complex
associated with GR in the cytoplasm can result in rapid turnover of the
GR by the proteasome. Involvement of the coding sequence in
down-regulation of ER has been demonstrated previously (7, 13). Further
evaluation of the sequence requirement for proteasome-mediated
degradation of ER may predicate posttranslational modifications, such
as cellular compart-mentalization, DNA binding, phosphorylation, or
specific protein interactions, that may contribute to ER fate.
We demonstrate that estrogen can regulate ER protein in the absence of
transcription and protein synthesis in the pituitary. ER protein
regulation can, therefore, be added to the growing number of estrogen
actions that do not involve ER-mediated transcription. Predominant
among those activities is the activation of signal transduction
cascades. Estrogen has been shown to activate MAPK (40) and ERK
activity (41) and to lead to the accumulation of second messenger
molecules including cAMP (42), inositol phosphate (41, 43, 44), and
calcium (45). It has been hypothesized that these nonclassical
mechanisms of estrogen action may be mediated through putative
membrane-bound receptors that are derived from the same coding
transcript (41) and are recognized by the same antibodies as the
nuclear ER (41, 46). In the case of ER protein regulation, estrogens
action involves nuclear, not membrane, ERs. Membrane-bound ERs make up
less than 3% of the protein product from nuclear ER transcript (41).
Since a significant proportion of ER is down-regulated in response to
estrogen, it is most likely that nuclear ER is responsible for
mediating estrogens action. To our knowledge, proteasome-mediated
proteolysis of ER is the first identification of a nongenomic mechanism
of estrogen action that involves nuclear ERs. Identification of
this novel mechanism of estrogen action introduces the possibility
for further exploration of nuclear signaling events induced by estrogen
that do not involve transcriptional activation.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
PR1 and MCF-7 cells were grown in high glucose DMEM (Mediatech,
Herndon, VA) supplemented with 10% FBS (HyClone Laboratories, Inc., Logan UT), 1 mM sodium pyruvate,
1000 U/ml penicillin, and 1000 mg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD). Culture conditions were
maintained at 10% CO2 and 37 C in a water-jacketed
incubator (Forma Scientific, Inc., Marietta, OH). Cells were passaged
using either trypsinization or physical dislodgment with media.
Inhibitor and Estrogen Stimulation
Before treatment, cells were washed with PBS and cultured at 5%
CO2 in phenol red-free and estrogen-free Optimem media
(Life Technologies, Inc.) for a minimum of 3 days. While
identical results are observed in cells maintained in media with
charcoal-stripped serum, we choose to utilize this defined medium to
minimize variation that may be associated with the stripping protocol.
On the day of treatment, cells were washed with PBS and collected by
dispersion with PBS followed by centrifugation. Cell pellets were then
resuspended in Optimem that was preequilibrated at 37 C at 5%
CO2 and distributed into 1 ml aliquots of 106
cells per tube. In experiments utilizing inhibitors, samples were
pretreated with the designated inhibitors for 30 min at 37 C while
gently rotating. After pretreatment, cells were exposed to
17ß-estradiol (E2; Sigma Chemical Co.,
St. Louis, MO) at various doses and for varying lengths of time as
indicated in the figure legends. During treatment, the cells were kept
at 37 C and rotated continuously. Protease inhibitors tested included
ALLnL, ALLM, Calpeptin, TPCK, ethyl(+)-(2S,
3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)
butylcarbamoyl]-2-oxiranecarboxylate (E64D), MG132, and
NH4Cl. Controls consisted of pretreatment with DMSO
(Sigma Chemical Co.) and treatment with ethanol (EtOH),
the solvents for the inhibitors and estradiol, respectively. For
practical purposes, ALLnL was used as the preferential proteasome
inhibitor. In experiments using protein synthesis inhibitors
(cyclohexamide, puromycin, anisomycin, emetine) and transcription
inhibitor [5,6-dichloro-1-b-ribofuranosyl benzimidazole (DRB)], cells
were pretreated for 30 min as described above before a 2-h treatment
with 10 nM E2. All inhibitors were purchased
from Sigma Chemical Co. except MG132 and TPCK, which were
gifts from Dr. Shigeki Miyamoto.
Western Blot Analysis
Upon termination of experiments, the cells were pelleted by
centrifugation, washed with PBS, and lysed immediately in 2x SDS
sample buffer (120 mM Tris-base, 20% glycerol, 2% SDS,
2% ß-mercaptoethanol, bromophenol blue, pH 6.8) to yield whole-cell
extracts. Whole-cell extracts were boiled and electrophoresed in a
7.5% or 10% SDS-PAGE gel. Proteins were electrophoretically
transferred using a Trans-blot Cell (Bio-Rad Laboratories, Inc., Richmond, CA) to nylon membrane (Immobilon-P,
Millipore Corp., Bedford, MA) in a Tris-glycine transfer
buffer with 20% methanol. The membranes were preblocked in a solution
of 5% milk, 0.02% sodium azide, 0.2% Tween 20 in PBS. Membranes were
then incubated overnight in the same solution containing primary
antibody. The primary antibodies used to detect ER were an anti-ER
antibody no. 715 (47) directed against a peptide within the hinge
region (amino acids 270284) of the rat ER and anti-ER antibodies
directed against the hinge (amino acids 287300; SR1000), and
C-terminal (amino acids 582595; SR1010) regions of the human ER
(Stressgen, Vancouver, British Columbia, Canada). Antibody to
I
B
(C21-Santa Cruz Biotechnology, Inc., Santa Cruz,
CA) was used to visualize protein not regulated by estrogen and as a
loading control. Antibody dilution curves were performed with all
primary antibodies to ensure that saturating concentrations were used
in the first probe reaction. Blots were washed in PBS containing 0.2%
Tween (PBST) before incubation with secondary antibodies conjugated to
horseradish peroxidase (HRP) diluted in the identical solution without
sodium azide. HRP-conjugated secondary antibodies used were directed
against rabbit or mouse IgG (Amersham Pharmacia Biotech,
Arlington Heights, IL) as appropriate. After washing in PBST,
the signal was visualized using the enhanced chemiluminescence (ECL)
detection method (Amersham Pharmacia Biotech) and exposed
to x-ray film.
Northern Blot Analysis
Total RNA was isolated from cells using phenol-chloroform
extraction as described previously (48). Twenty micrograms of total RNA
were electrophoresed in a 1% agarose gel containing formaldehyde and
transferred to nylon membrane (Genescreen; NEN Life Science Products, Boston, MA) (49). The RNA was immobilized to
the membrane by UV cross-linking (Bio-Rad Laboratories, Inc.). Prehybridization and hybridization of the membranes were
performed in a hybridization oven (Robbins Scientific Corp., Sunnyvale,
CA) at 55 C in a 25% formamide solution. The blots were probed
with 32P-radiolabeled cDNA fragments of the human ER, and
mouse GAPDH. Blots were stripped between hybridizations in a boiling
solution containing 1% glycerol, 2 mM EDTA, and 0.5% SDS
for a minimum of 10 min. Signal was quantified with a PhosphoImager
using Imagequant software (Molecular Dynamics, Inc.,
Sunnyvale, CA). Expression level of ER mRNA was determined by
normalizing values to those of the loading control, GAPDH. Values
obtained for the untreated DMSO control were set at 1. The data are
presented as the mean ± SD of the ER mRNA level
relative to the DMSO control for three independent experiments.
Whole-Cell Estrogen Uptake Assay
PR-1 cells that were maintained in Optimem for a minimum of 3
days were aliquoted into microcentrifuge tubes at a concentration of
2 x 106/ml in fresh medium that was preequilibrated
to 37 C at 5% CO2. The cells were pretreated for 30 min
with either DMSO or ALLnL at 37 C while rotating. After pretreatment, 2
nM [3H]estradiol (New England Nuclear/Dupont,
Boston, MA)was added to all samples. To account for nonspecific
binding, 0.4 µM DES (Sigma Chemical Co.) was
added in addition to 2 nM [3H]estradiol in a
parallel set of samples. During the 2-h treatment period, cells were
kept at 37 C and were rotated continuously. All samples were controlled
for equivalent amounts of ethanol. Cells were harvested by
centrifugation and were washed two times with 1% BSA in PBS at 4 C.
The final pellet was resuspended in ethanol and counted in a
scintillation counter. Specific binding was calculated by the
subtraction of nonspecific from total binding. Samples within
individual experiments were performed in duplicate. The data are
presented as the mean + SE of three independent
experiments. To compare total ER protein content to ER binding,
whole-cell extract from a parallel set of samples was examined by
Western blot analysis as described above.
Pulse Chase
Estrogen-deprived PR1 cells were rinsed twice in RPMI media
lacking phenol red, methionine, and cysteine (RPMI-,
Life Technologies, Inc.). Cells were incubated for 45 min
in RPMI- supplemented with L-glutamine, sodium
pyruvate, nonessential amino acids, and 5% stripped serum (50) that
had been dialyzed overnight against 0.9% NaCl. Metabolic labeling with
[35S]methionine was conducted for 2 h at a
concentration of 1 mCi/107 cells. After labeling, cells
were washed with complete phenol red-free RPMI media containing 10%
stripped serum. ALLnL and E2 treatment was performed as
described above for 1, 2, or 3 h in complete phenol red-free RPMI
medium containing 10% stripped serum. Treated cells were lysed in a
solution consisting of 10 mM Tris, pH 7.5, 150
mM NaCl, 1 mM EDTA, and 0.4% NP40, and ER was
immunoprecipitated using antirat ER antibody and protein A sepharose
(Pharmacia Biotech, Piscataway, NJ).
Immunoprecipitate was analyzed by SDS-PAGE. 35S-labeled ER
was visualized by autoradiography, and relative values of ER protein
were determined with a PhosphoImager using Imagequant software
(Molecular Dynamics, Inc.). Data are presented as a
percentage of the DMSO control group before exposure to E2.
Values represent three independent experiments.
 |
ACKNOWLEDGMENTS
|
---|
We are indebted to Dr. Dipak Sarkar for the gift of PR1 cells
and to Dr. Jack Gorski for antibodies against rat ER and for thoughtful
discussions throughout the course of these investigations. For critical
reading of this manuscript, we thank Drs. Pamela Mellon and Shigeki
Miyamoto.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Elaine T. Alarid, Ph.D., Department of Physiology, 120 Service Memorial Institute, 1300 University Avenue, Madison, Wisconsin 53706. e-mail: alarid@physiology.wisc.edu.
This work was supported by NIH Grant K01 CA-79090 to E.T.A.
Received for publication March 4, 1999.
Revision received May 10, 1999.
Accepted for publication May 25, 1999.
 |
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