From the Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
Received for publication, November 9, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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The estrogen receptor mediates breast cell
proliferation and is the principal target for chemotherapy of breast
carcinoma. Previous studies have demonstrated that the estrogen
receptor binds to calmodulin-Sepharose in vitro. However,
the association of endogenous calmodulin with endogenous estrogen
receptors in intact cells has not been reported, and the function of
the interaction is obscure. Here we demonstrate by
co-immunoprecipitation from MCF-7 human breast epithelial cells that
endogenous estrogen receptors bind to endogenous calmodulin. Estradiol
treatment of the cells had no significant effect on the interaction.
However, incubation of the cells with tamoxifen enhanced by 5-10-fold
the association of calmodulin with the estrogen receptor and increased
the total cellular content of estrogen receptors by 1.5-2-fold. In
contrast, the structurally distinct calmodulin antagonists
trifluoperazine and CGS9343B attenuated the interaction between
calmodulin and the estrogen receptor and dramatically reduced the
number of estrogen receptors in the cell. Neither of these agents
altered the amount of estrogen receptor mRNA, suggesting that
calmodulin stabilizes the protein. This hypothesis is supported by the
observation that, in the presence of Ca2+, calmodulin
protected estrogen receptors from in vitro proteolysis by
trypsin. Furthermore, overexpression of wild type calmodulin, but not a
mutant calmodulin incapable of binding Ca2+, increased the
concentration of estrogen receptors in MCF-7 cells, whereas transient
expression of a calmodulin inhibitor peptide reduced the estrogen
receptor concentration. These data demonstrate that calmodulin binds to
the estrogen receptor in intact cells in a
Ca2+-dependent, but estradiol-independent,
manner, thereby modulating the stability and the steady state level of
estrogen receptors.
Alterations in intracellular free Ca2+ concentrations
are frequently translated into cellular events via the Ca2+
sensor protein, calmodulin. Calmodulin, an acidic effector protein that
regulates multiple intracellular processes (1), has an essential role
in cell proliferation and cell cycle progression (2-4). Inactivation
of calmodulin blocks the cell cycle (4, 5), whereas overexpression of
calmodulin in a mouse mammary cell line increases the rate of cell
division (4). In addition, calmodulin is directly involved in DNA
synthesis (2). Interestingly, calmodulin concentrations are increased
in several malignancies, including those of the liver (6), lung (7),
keratinocytes (8), and breast (9-11). Whether the increased calmodulin
contributes to neoplastic transformation or is a consequence of the
altered cellular homeostasis that occurs during malignancy has not been ascertained.
It has been reported that calmodulin associates in vitro in
a Ca2+-dependent manner with the estrogen
receptor isolated from rat uterus cytosol (12, 13), and that this
interaction stimulates binding of the estrogen receptor to the estrogen
response element (13). The estrogen receptor, which regulates the
expression of specific genes and participates in breast cell
proliferation (14), is the principal target for chemotherapy of breast
carcinoma (15). Several lines of evidence implicate a role for
Ca2+ and calmodulin in mediating the actions of estrogen as
follows. (i) Estrogen induces Ca2+ fluxes in breast
carcinoma cells (16); (ii) Ca2+ channel antagonists inhibit
the proliferation of breast carcinoma cells (17); (iii) tamoxifen, the
primary chemotherapeutic agent used to treat breast cancer, deregulates
intracellular Ca2+ homeostasis (18); (iv) sustained
increased intranuclear Ca2+ concentrations induce DNA
fragmentation and apoptosis of breast cancer cells in a
calmodulin-dependent manner (19); (v) calmodulin stimulates
tyrosine phosphorylation and activation of the estrogen receptor (20);
and (vi) calmodulin is required for the formation of the estrogen
receptor-estrogen response element complex and for activation of an
estrogen responsive promotor (21). Together these findings suggest that
Ca2+/calmodulin could be a participant in the mitogenic
effects of estrogen. Therefore, we set out to examine the physiological
significance of the interaction of calmodulin with the estrogen receptor.
Materials--
The antibody to calmodulin was described
previously (22). The anti-estrogen receptor antibody was obtained from
Santa Cruz. Calmodulin-Sepharose was purchased from Amersham Pharmacia
Biotech. Affi-Gel was from Bio-Rad. Tissue culture reagents were
obtained from Life Technologies, Inc. Fetal bovine serum was from
BioWhittaker. Restriction enzymes were purchased from New England
Biolabs. Purified human estrogen receptor was from PanVera,
Ca2+-free pig brain calmodulin was obtained from Ocean
Biologics, and sequencing grade
L-1-tosylamide-2-phenylethyl chloromethyl ketone -treated
trypsin was obtained from Promega. All other reagents were of standard
analytical grade.
Cell Culture and Lysis--
MCF-7 human breast epithelial cells
were grown to 80% confluence in Dulbecco's modified Eagle's medium
with 10% fetal bovine serum. To examine the effect of estradiol, the
medium was replaced with phenol red-free Dulbecco's modified Eagle's
medium, and the fetal bovine serum was treated with dextran-coated
charcoal (23). Where indicated, cells were treated with estradiol,
tamoxifen, or the calmodulin antagonists trifluoperazine
(TFP)1 or CGS9343B. The
concentrations and incubation times are indicated in the figure
legends. To lyse the cells, the medium was removed, cells were washed
three times with phosphate-buffered saline (145 mM NaC1, 12 mM Na2HPO4, and 4 mM
NaH2PO4, pH 7.2) and 1 ml of lysis buffer (50 mM Tris-base, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 0.1 mg/ml leupeptin, aprotonin, and
pepstatin containing 1 mM CaCl2 or 1 mM EGTA) was added. The lysates were collected and quick-frozen in methanol/solid CO2.
Construction of Plasmids and Transfection--
The mammalian
expression vector, POPI3, was purchased from Stratagene. An
oligonucleotide linker flanked by NotI and
AflII-containing XbaI, SmaI, and
BlpI cleavage sites was fashioned and inserted into the
NotI and AflII sites of POPI3. This introduced
XbaI, SmaI and BlpI sites in the
polylinker region to form POPI3-Xb. A puromycin resistance gene was
removed from pBabe-puro (24) with BamHI and ClaI.
The fragment was treated with S1 nuclease to form blunt ends. POPI3 was
digested with AatII and treated with calf intestinal
phosphatase to prevent self ligation. The puromycin gene was ligated
into the AatII site to form POPI3-Xb-puro.
The vector containing the cDNA for mammalian calmodulin was
obtained from Dr. A. Persechini (University of Rochester, Rochester, NY) and was designated pMZ-xCaM1 (25). To excise the calmodulin cDNA for insertion into POPI3-Xb-puro, the PstI site of
pMZ-xCaM1 was changed to an AflII site by insertion of an
oligonucleotide linker containing AflII and SalI
sites flanked by PstI. The vector was digested with
NcoI, then treated with Klenow enzyme and deoxynucleotides to form a blunt end, and finally, digested with AflII. To
accommodate the fragment, POPI3-Xb-puro was digested with
AflII and XbaI. The fragment was ligated with the
vector to form POPI3-Xb-puro-mCaM.
The vector containing the cDNA for mutant calmodulin that does not
bind Ca2+ (CaM
A synthetic gene encoding the calmodulin binding sequence of myosin
light chain kinase inserted in the eukaryotic expression vector pSVL
(27) was kindly provided by Dr. John Dedman (University of Cincinnati,
Cincinnati, OH). Transient transfection of this construct into
mammalian cells neutralizes calmodulin function (27).
Vectors were transiently transfected into MCF-7 cells by the calcium
phosphate precipitation method. After 6 h, the medium was replaced
with 10% glycerol in phosphate-buffered saline for 3 min. Cells were
washed three times with phosphate-buffered saline and returned to
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Unless indicated otherwise, cells were lysed 48 h
post-transfection.
Calmodulin-Sepharose Chromatography--
Cell lysates,
normalized for total protein, were incubated for 2 h at 4 °C
with 2 ml of calmodulin-Sepharose in the presence of 1 mM
CaCl2 or 1 mM EGTA. Samples were washed five
times in lysis buffer containing 1 mM CaCl2 or
1 mM EGTA, resolved by SDS-PAGE, and immunoblotting was
performed as described below.
Immunoprecipitation and Immunoblotting--
Equal amounts of
protein lysate were immunoprecipitated with anti-calmodulin monoclonal
antibody linked to Affi-Gel as described previously (28). Samples were
washed five times in lysis buffer, resolved by SDS-PAGE, and
transferred to polyvinylidene difluoride (PVDF) membrane. Immunoblots
were probed with anti-estrogen receptor or anti-calmodulin antibody.
Complexes were visualized with a horseradish peroxidase-conjugated
secondary antibody and developed by enhanced chemiluminescence.
For immunoprecipitation of pure proteins, 1 µg of estrogen receptor
was incubated with 1 µM tamoxifen or an equal volume of vehicle at 4 °C. After 30 min, 5 µg of calmodulin was added in lysis buffer (final volume of 300 µl) containing 1 mM
CaC12 or 1 mM EGTA, and incubation was
continued for 30 min. Samples were immunoprecipitated with
anti-calmodulin antibody and processed by Western blotting as described above.
In Vitro Proteolysis of the Estrogen Receptor--
Pure estrogen
receptor (8 µg) was incubated at 4 °C with 10.5 µg calmodulin
(5-fold molar excess) or 10.5 µg of myoglobin in 125 µl of 50 mM Tris, pH 7.6, containing 1 mM
Ca2+ or 1 mM EGTA. After 20 min,
L-1-tosylamide-2-phenylethyl chloromethyl ketone-treated
trypsin (1:100 trypsin:estrogen receptor, w/w) was added, and samples
were incubated at 25 °C. At the time points indicated in the figure
legend, 30-µl aliquots were removed from the reaction mixture and
immediately added to boiling SDS. Samples were resolved by 12%
SDS-PAGE, and proteins were detected with Coomassie Blue R-250. The
extent of digestion, expressed relative to undigested protein, was
quantified by laser-scanning densitometry from Coomassie Blue-stained gels.
Isolation of RNA and Northern Blot Analysis--
Total RNA was
isolated from MCF-7 cells with RNAzol B (Teltest, Inc.). Two micrograms
of RNA was mixed with denaturing buffer (60% deionized formamide, 7%
formaldehyde, 1× borate buffer, 6% glycerol, 0.02% bromphenol blue,
and 0.02% xylene cyanol), resolved on a 1% agarose, 3% formaldehyde
gel in borate buffer (5 mM boric acid, 5 mM
sodium borate, 10 mM sodium sulfate, and 1 mM
EDTA, pH 8.0), transferred onto a nitrocellulose membrane, and
cross-linked with UV light. The estrogen receptor cDNA (kindly
provided by Dr. P. Yen, National Institutes of Health) was removed from
pcDNA3 with EcoRI and purified by low melt agarose.
Approximately 20 ng of estrogen receptor cDNA was labeled with
[32P]dCTP using a random primer labeling kit obtained
from Life Technologies, Inc. Northern blots were probed with
radiolabeled cDNA, and estrogen receptor mRNA was identified by autoradiography.
Miscellaneous Methods--
Protein determinations were performed
using the DC protein assay from Bio-Rad. RNA was quantified by
spectrophotometry. Densitometry of the bands on the gels, the
autoradiographs, and the enhanced chemiluminescence signals was
performed with NIH-Image. Statistical significance was evaluated by
Student's t test using InStat software (GraphPad Software,
Inc.).
Binding of Estrogen Receptors to Calmodulin--
The interaction
of calmodulin with the estrogen receptor was initially characterized in
MCF-7 breast epithelial cells by calmodulin-Sepharose chromatography.
This approach demonstrated that calmodulin binds to the estrogen
receptor (Fig. 1B),
corroborating previous observations with cytosol from rat uterus (12,
13). Binding of the estrogen receptor to calmodulin-Sepharose was
abrogated when Ca2+ was chelated with EGTA (data not
shown). To evaluate the effect of estradiol and tamoxifen on the
association of calmodulin with the estrogen receptor, we first examined
the level of estrogen receptors in cells incubated with each agent
(Fig. 1A). Estradiol alone decreased the concentration of
estrogen receptors by 50%, whereas tamoxifen increased the amount of
estrogen receptor in the cell lysate by 1.5-2-fold; inclusion of
estradiol during cell culture prevented the augmentation by tamoxifen
(Fig. 1A). Analysis by calmodulin-Sepharose chromatography
revealed that estradiol treatment did not substantially alter the
binding of estrogen receptors to calmodulin (Fig. 1B). In
contrast, 1 µM tamoxifen enhanced binding of the estrogen
receptor to calmodulin by 5-10-fold (Fig. 1B). The enhanced
binding induced by tamoxifen is substantially greater than the
1.5-2-fold increase in estrogen receptor content produced by
tamoxifen. When cells were treated with both tamoxifen and estradiol,
the increased binding of estrogen receptors to calmodulin produced by
tamoxifen was attenuated (Fig. 1B). Under the conditions
tested, neither estradiol (data not shown) nor tamoxifen (Fig.
2) influenced the amount of calmodulin in
the cells nor the amount of calmodulin immunoprecipitated.
Co-immunoprecipitation of Estrogen Receptors with Calmodulin and
the Effect of Tamoxifen on the Interaction--
Although the in
vitro interaction of estrogen receptors with calmodulin-Sepharose
has been described (12, 13), the binding of estrogen receptors to
endogenous calmodulin in intact cells has not been previously reported.
Binding of endogenous calmodulin to the estrogen receptor was revealed
by co-immunoprecipitation using anti-calmodulin monoclonal antibody
(Fig. 1C). Analogous to the data obtained with
calmodulin-Sepharose, co-immunoprecipitation demonstrated that
estradiol did not significantly alter the interaction between the
estrogen receptor and calmodulin. Although fewer estrogen receptors
co-immunoprecipitated with calmodulin in cells stimulated with
estradiol than in control cells (Fig. 1C, compare
lanes 1 and 2), the magnitude of the decrease was
similar to that produced by estradiol in cell lysates (Fig.
1A). These data indicate that estradiol does not
significantly modulate the binding of the estrogen receptor to
calmodulin. By contrast, tamoxifen substantially enhanced in a
dose-dependent manner the interaction between calmodulin and the estrogen receptor (Fig. 2A). The maximum increase
was observed at 0.25-0.5 µM tamoxifen. Although
tamoxifen also increased the total amount of estrogen receptor in the
cells, the magnitude of the increase was substantially less than the
magnitude of the increase in the amount of estrogen receptor that
co-immunoprecipitated with calmodulin (compare Figs. 2, A
and B). Moreover, the maximum increase in total estrogen
receptors was detected at 0.5-1 µM tamoxifen, a
concentration slightly higher than that at which maximum binding was
observed. Neither the total amount of calmodulin in the cell nor the
amount of calmodulin immunoprecipitated was significantly altered by
treatment (Fig. 2).
To verify that the tamoxifen-induced augmentation of the interaction
between calmodulin and the estrogen receptor is direct, in
vitro analysis with pure proteins was performed. Pure estrogen receptors were preincubated with tamoxifen or vehicle, followed by
incubation with calmodulin. The amount of estrogen receptor that
co-immunoprecipitated with calmodulin was increased approximately 2-fold by tamoxifen (Fig. 1D). Chelation of Ca2+
with EGTA revealed that the binding of estrogen receptors to calmodulin
is Ca2+-dependent (Fig. 1D).
Effect of Calmodulin Antagonists on the Interaction between
Estrogen Receptors and Calmodulin--
To examine the function of the
interaction of calmodulin with the estrogen receptor, cells were
incubated with two structurally distinct, cell-permeable calmodulin
antagonists, TFP (29) or CGS9343B (30). Exposure to 10 µM
TFP or 40 µM CGS9343B for 16 h reduced the total
number of estrogen receptors in the cells to 50% of control and
virtually undetectable levels, respectively (Fig.
3A). These concentrations of
the antagonists had no effect on cell viability (data not shown).
CGS9343B induced a dose-dependent decrease in the number of
estrogen receptors; this reduction was not modulated by estradiol (Fig.
3B). The effect of the calmodulin antagonists was specific
for estrogen receptors as they did not alter the amount of cellular
IQGAP1 (data not shown), a major calmodulin-binding protein in breast
epithelial cells (28).
To examine the effects of the antagonists on the interaction between
calmodulin and the estrogen receptor, cells were incubated with each
calmodulin antagonist for a short time period. Treatment of MCF-7 cells
with TFP or CGS9343B for 30 min altered neither the total amount of
estrogen receptor nor of calmodulin in the cell lysate (Fig.
4B). By contrast, the amount
of estrogen receptor bound to calmodulin was decreased by both TFP and
CGS9343B (Fig. 4A). Compared with vehicle, the relative
amount of estrogen receptor that co-immunoprecipitated with calmodulin
was 0.42 ± 0.04 and 0.38 ± 0.04 (mean ± S.E.,
n = 3; p < 0.001) for TFP and
CGS9343B, respectively (Fig. 4C).
Effect of Calmodulin Antagonists on Estrogen Receptor Gene
Expression--
Taken together, our data demonstrate that stimulation
and inhibition of the interaction of calmodulin with the estrogen
receptor increases and reduces the total cellular content of estrogen
receptors, respectively. These results indicate that calmodulin
influences either the expression or stability of the estrogen receptor.
To discriminate between these two possibilities, Northern blot analysis was compared among MCF-7 cells treated for 16 h with vehicle, tamoxifen, or CGS9343B (Fig. 5). Neither
compound significantly altered the amount of estrogen receptor
mRNA, indicating that calmodulin does not modulate estrogen
receptor gene expression. Thus, calmodulin binding most likely
stabilizes the estrogen receptor against proteolysis. This hypothesis
was evaluated both in vitro with purified proteins and
in intact cells.
Effect of Calmodulin on Proteolysis of the Estrogen Receptor in
Vitro--
In vitro proteolysis revealed that the estrogen
receptor was very sensitive to digestion by
L-1-tosylamide-2-phenylethyl chloromethyl ketone-treated
trypsin. Incubation at 37 °C (1:7 trypsin:estrogen receptor, w/w)
resulted in complete disappearance of the full-length receptor within 1 min (data not shown). Analysis at 25 °C with a very low ratio of
trypsin to estrogen receptor (1:100, w/w) was required to detect
limited proteolysis (Fig. 6). Because
Ca2+ regulates the binding of calmodulin to the estrogen
receptor (see Fig. 1D), the effect of calmodulin on
proteolysis of the estrogen receptor was performed both in the presence
and absence of Ca2+. In the presence of Ca2+,
calmodulin reduced the susceptibility of the estrogen receptor to
proteolysis by trypsin. The rates of disappearance of intact estrogen
receptor and the appearance of degradation products were substantially
reduced by Ca2+/calmodulin (Fig. 6A). Myoglobin,
which has a molecular mass close to that of calmodulin, was used as a
control protein. Chelation of Ca2+, which substantially
decreases the activity of trypsin (31), markedly slowed the rate of
digestion of the estrogen receptor (compare the samples with myoglobin
in Figs. 6, A and B). In the absence of
Ca2+, the protective effect of calmodulin on proteolysis of
the estrogen receptor by trypsin was substantially attenuated (Fig.
6B).
Overexpression of Calmodulin or Calmodulin-binding Peptide--
To
further evaluate the ability of calmodulin to protect the estrogen
receptor in intact cells, MCF-7 cells were transiently transfected with
a mammalian expression vector directing the expression of wild type
calmodulin or a mutant calmodulin that is incapable of binding
Ca2+, CaM
Targeted expression of an inhibitor peptide derived from myosin light
chain kinase can neutralize the function of calmodulin in the nucleus
of transiently transfected mammalian cells (27). Transient transfection
of this peptide into MCF-7 cells decreased by ~50% the total amount
of estrogen receptor in the cells after 48 h (Fig. 7B).
Thus, selectively increasing or decreasing the relative amount of
functional calmodulin resulted in a concomitant increase and decrease,
respectively, of estrogen receptor levels in the cells.
Our data clearly document an interaction between endogenous
calmodulin and endogenous estrogen receptors in human breast epithelial cells. The binding is regulated by Ca2+, which is required
for the interaction. Incubation of MCF-7 cells for 16 h with
estradiol or the antiestrogen tamoxifen decreased and increased,
respectively, the total amount of estrogen receptor in the cells (see
Figs. 1 and 2 and Refs. 32 and 33). Moreover, concentrations of
tamoxifen as low as 0.25 µM substantially increased the
binding of calmodulin to the estrogen receptor. Tamoxifen acts as a
calmodulin antagonist in vitro (34-36) and has been
reported to inhibit the binding of the estrogen receptor to calmodulin (12). Our data differ from the last report. Several factors may account
for the discrepant results. The studies of Bouhoute and Leclercq (12)
were carried out on rat uterine cytosol by adding tamoxifen after cell
lysis, at the time of mixing with calmodulin-Sepharose. In our
analyses, intact MCF-7 human breast epithelial cells were incubated
with tamoxifen, the tamoxifen was removed before the cells were lysed,
and calmodulin was immunoprecipitated. This strategy revealed that
pretreatment of MCF-7 cells with therapeutic tamoxifen concentrations
increased binding of estrogen receptors to calmodulin. Our experimental
technique represents the physiological conditions associated with the
use of tamoxifen as a chemotherapeutic agent in patients. Moreover, our
approach examined interactions between physiologically relevant
concentrations of calmodulin and estrogen receptors. Tissue differences
are also likely to be relevant. Tamoxifen is an estrogen receptor
antagonist in breast, whereas in the uterus it is an estrogen receptor
agonist (37). Thus, it is possible that cofactors for the estrogen
receptor in the different cell types may alter the interaction of
tamoxifen-bound estrogen receptors with calmodulin.
Interestingly, as was observed with purified proteins (21), estradiol
did not alter the binding of estrogen receptors to calmodulin. Thus,
estradiol and tamoxifen, both of which bind to the estrogen receptor,
produce different effects on the interaction between calmodulin and the
estrogen receptor. Although the calmodulin binding domain of the
estrogen receptor has not been identified, these apparently discrepant
data can be interpreted in the context of recently solved crystal
structures (38). The conformation of the human estrogen receptor ligand
binding domain bound to tamoxifen is distinct from that bound to
estradiol (38). Similar findings were obtained using peptides, leading
to the suggestion that different estrogen receptor-ligand complexes
could contact different proteins within the cell (39). It seems
reasonable to infer from our data that the conformation of
tamoxifen-bound estrogen receptor has increased affinity for
calmodulin, whereas estrogen does not change the conformation of the
calmodulin binding domain of the receptor. Another possible, but less
likely, explanation is that tamoxifen alters the conformation of
calmodulin, thereby increasing binding to the estrogen receptor.
We also documented that the binding of calmodulin directly effects the
stability and therefore the steady state level of estrogen receptors.
This finding was demonstrated both in vitro with purified estrogen receptors and in intact cells. Two complementary strategies were adopted to establish this finding in intact cells. In the first,
we observed that two cell-permeable calmodulin antagonists, namely
CGS9343B and TFP, attenuated calmodulin binding to the estrogen
receptor and dramatically reduced the cellular content of estrogen
receptors. In particular, CGS9343B lowered in a
dose-dependent manner the amount of estrogen receptors in
MCF-7 cells to virtually undetectable levels at 40 µM
CGS9343B. Calmodulin antagonists are frequently used to evaluate the
role of calmodulin in cellular functions (40, 41), but at high
concentrations have been reported to alter the functions of other
proteins. To minimize nonspecific interactions, we used relatively low
concentrations of the antagonists, namely 10 µM TFP and
40 µM CGS9343B. Analysis performed with purified proteins
in vitro revealed that TFP had an IC50 of 12.5 µM for inhibition of calmodulin-stimulated cAMP
phosphodiesterase, whereas CGS9343B was specific for calmodulin at 1000 µM (30), a concentration 25-fold higher than the highest
concentration used in this study. Moreover, the two structurally
distinct compounds produced similar effects on estrogen receptor
concentrations, further decreasing the likelihood of a nonspecific effect.
To validate the results obtained with the calmodulin antagonists, we
employed a second approach. The amount of available intracellular calmodulin was increased and decreased by transient expression of the
cDNA for calmodulin or the cDNA of a calmodulin-binding peptide, respectively. Increasing intracellular calmodulin
concentrations led to estrogen receptor accumulation in MCF-7 cells,
whereas the inhibitor peptide mimicked the effect of the calmodulin
antagonists and decreased estrogen receptor content. Consistent with
the binding and in vitro proteolysis data, analysis with
CaM The mechanism by which calmodulin protects the estrogen receptor from
proteolysis is not known. The interaction of calmodulin with targets
frequently results in a conformational change in the target protein
(1). Thus, the direct binding to calmodulin probably induces a tertiary
conformation of the estrogen receptor that restricts access to its
normal proteolytic cleavage sites. This model is supported by the
in vitro trypsin digestion data presented in Fig. 6.
Additional mechanisms may be operative in intact cells. For example,
calmodulin could modulate the interaction between the estrogen receptor
and other proteins. In addition to binding DNA hormone response
elements, estrogen receptors bind to adaptor proteins that modulate
their function (43, 44). These include the steroid receptor coactivator
(SRC) family, receptor interacting proteins (RIP), and members of the
heat shock protein (hsp) family, including hsp90 (43, 45). Although
previously unclear, the role of hsp90 in the regulation of the estrogen
receptor appears to be a molecular chaperone effect that may be
important for the correct folding of the receptor (46). Interestingly, hsp90 also binds calmodulin (47, 48), and this interaction prevents
hsp90 from binding to F-actin (47). Moreover, recent evidence documents
that calmodulin facilitates the effects of hsp90 on the dissociation of
endothelial nitric-oxide synthase from caveolin-1 (49). It is therefore
possible that synergistic interactions between calmodulin and hsp90
could stabilize the estrogen receptor.
The estrogen receptor is involved in breast cell proliferation, and
exogenous estrogens have been implicated in the development of breast
carcinoma (50). Importantly, the estrogen receptor is the primary
target for chemotherapy of breast tumors (15). Therefore, a clear
understanding of the function and the regulation of the estrogen
receptor is a prerequisite for optimal pharmacotherapy of breast
carcinoma. Reports from several groups link calmodulin and the estrogen
receptor. For example, binding to calmodulin enhances the affinity of
the estrogen receptor for the estrogen response element (13), and
calmodulin has a fundamental role in estradiol-regulated gene
expression in breast carcinoma cells (21). The concentration of
calmodulin in cells is tightly regulated. Overexpression of calmodulin
cDNA increases calmodulin mRNA 20-50-fold, whereas calmodulin
protein concentrations are only 2-4-fold higher (4, 51). Nevertheless,
higher concentrations of calmodulin are present in rapidly growing
cells, and increased intracellular concentration of calmodulin have
been reported in several malignancies (6-10), including breast
carcinoma (9-11). These data, coupled with the findings presented here
that calmodulin enhances the stability of the estrogen receptor,
suggest that calmodulin may be a component of estrogen-induced cell
proliferation. Calmodulin antagonists, which inhibit growth of human
breast carcinoma cell lines (52) and augment antiestrogen therapy (53,
54), could be potentially useful in the treatment of breast carcinoma
in patients.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ca) was a gift from Dr. K. Beckingham
(Rice University, Houston, TX) and was designated pRK5 (26). To excise
the calmodulin cDNA for insertion into POPI3-Xb-puro, pRK7 was
first digested with BlpI, then treated with S1 nuclease to
form a blunt end, and finally, digested with XbaI. To
accommodate the fragment, POPI3-Xb-puro was digested with
BlpI, treated with S1 nuclease to form a blunt end, and then
digested with XbaI. The fragment was ligated with the vector
to form POPI3-Xb-puro-CaM
Ca.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Binding of calmodulin to the estrogen
receptor. MCF-7 cells, treated for 16 h with either vehicle,
10 nM estradiol (E2), 1 µM
tamoxifen (Tam), or both agents, were washed three times as
described under "Experimental Procedures." A, cells were
lysed, and equal amounts of protein were resolved by SDS-PAGE,
transferred to PVDF membranes, and probed with anti-estrogen receptor
antibody. Complexes were visualized by enhanced chemiluminescence.
B, equal amounts of protein from MCF-7 cell lysates were
incubated with calmodulin (CaM)-Sepharose in the presence of
1 mM CaCl2. After washing the beads, proteins
were resolved by SDS-PAGE and processed as described above.
C, equal amounts of protein were immunoprecipitated
(IP) with anti-calmodulin antibody ( CaM),
described under "Experimental Procedures." Samples were processed
as described under B above. The position of migration of the
estrogen receptor (ER) is indicated. Data are representative
of at least two independent experimental determinations. D,
pure estrogen receptor was incubated with 1 µM tamoxifen
or vehicle. After 30 min, 5 µg of calmodulin was added in the
presence of 1 mM Ca2+ or 1 mM EGTA.
Samples were immunoprecipitated with anti-calmodulin antibody and
processed as described under B above.
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Fig. 2.
Immunoprecipitation of the estrogen receptor
with calmodulin. MCF-7 cells, treated for 16 h with either
vehicle or the indicated concentrations of tamoxifen (Tam),
were lysed, and equal amounts of protein were immunoprecipitated
(IP) with anti-calmodulin monoclonal antibody
(panel A) or applied directly to SDS gels (panel
B) as described under "Experimental Procedures." Proteins were
resolved by SDS-PAGE, transferred to PVDF membranes, and probed
with anti-estrogen receptor or anti-calmodulin antibody. The
positions of migration of the estrogen receptor (ER) and
calmodulin (CaM) are indicated. Data are representative of
two independent experimental determinations.
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Fig. 3.
Effect of calmodulin antagonists on estrogen
receptor content. A, MCF-7 cells, treated for 16 h
with vehicle, 10 µM TFP, or 40 µM CGS9343B,
were lysed, and equal amounts of protein were applied directly to the
gel. Proteins were resolved by SDS-PAGE, transferred to PVDF membranes,
and probed with anti-estrogen receptor antibody. The position of
migration of the estrogen receptor (ER) is indicated.
B, MCF-7 cells were incubated with the indicated
concentrations of CGS9343B in the absence (Control) or
presence of estradiol (E2) for 16 h. Equal
amounts of protein lysate were resolved by SDS-PAGE, transferred to
PVDF membranes, and probed with anti-estrogen receptor antibody. The
relative amount of estrogen receptor was quantified. The results,
presented in the bottom panel, are expressed as percentages
of the value of the untreated cells (no CGS9343B). Note that, compared
with control cells, the amount of estrogen receptor in
estradiol-treated cells is reduced. Data are representative of at least
two independent experimental determinations.
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Fig. 4.
Effect of calmodulin antagonists on the
interaction between calmodulin and the estrogen receptor. MCF-7
cells were treated for 30 min with vehicle, 25 µM TFP or
40 µM CGS9343B. Cells were lysed, and equal amounts of
protein were immunoprecipitated (IP) with anti-calmodulin
monoclonal antibody ( CaM) (panel A) or applied
directly to SDS gels (panel B) as described under
"Experimental Procedures." Proteins were resolved by SDS-PAGE,
transferred to PVDF membranes, and probed with anti-estrogen receptor
or anti-calmodulin antibody. The positions of migration of the estrogen
receptor (ER) and calmodulin (CaM) are indicated.
Data are representative of three independent experimental
determinations. C, the relative amount of estrogen receptor
that co-immunoprecipitated with calmodulin in A was
quantified. The results represent means ± S.E., relative to
control (vehicle), of three independent experimental determinations. *,
significantly different from no treatment (p < 0.001).
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Fig. 5.
Estrogen receptor mRNA levels in MCF-7
cells. MCF-7 cells were treated for 16 h with vehicle, 1 µM tamoxifen (Tam), or 40 µM
CGS9343B. Total RNA was isolated and quantified by spectrophotometry,
and equal amounts were resolved on agarose/formaldehyde gels. Northern
blot analysis was performed using radiolabeled estrogen receptor
cDNA as a probe. A representative autoradiograph is shown with the
position of migration of the estrogen receptor mRNA (ER
mRNA) indicated.
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Fig. 6.
Limited proteolysis of estrogen
receptors. Pure estrogen receptor (8 µg) was incubated with 10.5 µg of calmodulin (CaM, ) or 10.5 µg of myoglobin
(Mb,
) in the presence of 1 mM
Ca2+ (panel A) or 1 mM EGTA
(panel B). Proteins were incubated with
L-1-tosylamide-2-phenylethyl chloromethyl ketone-treated
trypsin at 25 °C for 0, 1, 2, or 5 min. Samples were resolved by
12% SDS-PAGE and stained with Coomassie Blue. The positions of
migration of molecular mass standards are indicated on the left,
whereas those of intact estrogen receptor (ER), degradation
products, calmodulin (CaM), and myoglobin (Mb)
are indicated on the right. Lower panels depict optical density scans
of full length estrogen receptors. The optical density at time 0 was
set at 1. Representative experiments of at least two independent
determinations are shown.
Ca (26). Immunoblotting with anti-calmodulin
antibodies revealed that the concentration of calmodulin (wild type and
mutant) in transfected cells was 1.5-2-fold higher than that in cells transfected with vector alone (Fig.
7A, lower panel).
As noted earlier, the binding of calmodulin to the estrogen receptor is dependent upon Ca2+; CaM
Ca should therefore not alter
the stability of the estrogen receptor. Probing immunoblots of cell
lysates with anti-estrogen receptor antibody demonstrated a 1.69 ± 0.27-fold (mean ± S.E., n = 3) increase in the
amount of estrogen receptor in cells overexpressing wild type
calmodulin (Fig. 7A). By contrast, overexpression of CaM
Ca had no effect on the amount of estrogen receptor (Fig. 7A).
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Fig. 7.
Effect of overexpression of calmodulin or
calmodulin-binding peptide in MCF-7 cells on levels of the estrogen
receptor. A, MCF-7 cells were transiently transfected
with a mammalian expression vector, POPI3, containing nothing
(V), wild type calmodulin (CaM), or a mutant
calmodulin incapable of binding
Ca2+(CaM Ca). An equal aliquot of
each lysate was resolved by SDS-PAGE, transferred to PVDF membranes,
and probed with anti-estrogen receptor or anti-calmodulin antibody. The
positions of migration of the estrogen receptor (ER) and
calmodulin (CaM) are indicated. Data are representative of
two independent experimental determinations. B, MCF-7 cells
were transiently transfected with pSVL vector alone (V) or
pSVL containing a calmodulin-binding peptide (CaMBP). Equal
amounts of protein lysate were resolved by SDS-PAGE and transferred to
PVDF membranes, and blots were probed with anti-estrogen receptor
antibody. Two independent experiments are depicted.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ca revealed that Ca2+ binding to calmodulin was
required for the protective effect on the estrogen receptor.
Interestingly, a direct positive correlation has been reported between
the concentrations of calmodulin and estrogen receptors in human breast
tumors (42).
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ACKNOWLEDGEMENTS |
---|
We thank Drs. E. Moret and B. Schmid (Novartis, Switzerland) for the gift of CGS9343B, A. Persechini (University of Rochester) and K. Beckingham (Rice University) for the calmodulin cDNA constructs, J. Dedman (University of Cincinnati) for the calmodulin-binding peptide construct, and P. Yen (National Institutes of Health) for estrogen receptor cDNA. We also thank Alvaro E. Rojas and Ann Marie Bynoe for the preparation of the manuscript and Dr. Michael Briggs for help with the figures.
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FOOTNOTES |
---|
* This work was supported in part by a grant from the National Institutes of Health (to D. B. S.) and a Breast Cancer Research Grant from the Massachusetts Department of Public Health (to J. L. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Praecis Pharmaceuticals, Inc., One Hampshire St.,
Cambridge, MA 02139.
§ To whom correspondence should be addressed: Brigham and Women's Hospital, Thorn 530, 75 Francis St., Boston, MA 02115. Tel.: 617-732-6627; Fax: 617-278-6921; E-mail: dsacks@rics.bwh.harvard.edu.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M010238200
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ABBREVIATIONS |
---|
The abbreviations used are:
TFP, trifluoperazine;
PVDF, polyvinylidene difluoride;
PAGE, polyacrylamide
gel electrophoresis;
CaMCa, mutant calmodulin incapable of binding
Ca2+;
hsp, heat shock protein.
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REFERENCES |
---|
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---|
1. | Cohen, P., and Klee, C. (1988) Calmodulin , Elsevier Science Publishing Co., Inc., New York |
2. | Reddy, G. P. V., Reed, W. C., Sheehan, E. L., and Sacks, D. B. (1992) Biochemistry 31, 10426-10430[Medline] [Order article via Infotrieve] |
3. | Gratzer, W. B., and Baines, A. J. (1988) in Calmodulin (Cohen, P. , and Klee, C. B., eds) , pp. 329-340, Elsevier Science Publishing Co., Inc., New York |
4. | Rasmussen, C. D., and Means, A. R. (1987) EMBO J. 6, 3961-3968[Abstract] |
5. | Natsukari, N., Zhang, S. P., Nichols, R. A., and Weiss, B. (1995) Neurochem. Int. 26, 465-476[CrossRef][Medline] [Order article via Infotrieve] |
6. | Wei, J.-W., Morris, H. P., and Hickie, R. A. (1982) Cancer Res. 42, 2571-2574[Abstract] |
7. | Liu, G. X., Sheng, H. F., and Wu, S. (1996) Br. J. Cancer 73, 899-901[Medline] [Order article via Infotrieve] |
8. | Grief, F., Soroff, H. S., Albers, K. M., and Taichman, L. B. (1989) Eur. J. Cancer Clin. Oncol. 25, 19-26[Medline] [Order article via Infotrieve] |
9. | Singer, A. L., Sherwin, R. P., Dunn, A. S., and Appleman, M. M. (1976) Cancer Res. 36, 60-66[Abstract] |
10. | Tuccari, G., Rizzo, A., and Barresi, G. (1993) Eur. J. Histochem. 37, 57-64 |
11. | Chun, K. Y., and Sacks, D. B. (2000) in Calcium: The Molecular Basis of Calcium Action in Biology and Medicine (Pochet, R. , Donato, R. , Haiech, J. , Heizmann, C. , and Gerke, V., eds) , pp. 541-563, Kluwer Academic Publishers, Dordrecht, The Netherlands |
12. | Bouhoute, A., and Leclercq, G. (1994) Biochem. Pharmacol. 47, 748-751[Medline] [Order article via Infotrieve] |
13. | Bouhoute, A., and Leclercq, G. (1995) Biochem. Biophys. Res. Commun. 208, 748-755[CrossRef][Medline] [Order article via Infotrieve] |
14. | Walker, R. A., and Varley, J. M. (1993) Cancer Surveys 16, 31-57[Medline] [Order article via Infotrieve] |
15. | Lykkesfeldt, A. E. (1996) Acta Oncol. (Stockh.) 35 Suppl. 5, 9-14 |
16. | Dickson, R. B., and Lippman, M. E. (1987) Endocrine Rev. 8, 29-43[Medline] [Order article via Infotrieve] |
17. | Taylor, J. M., and Simpson, R. U. (1992) Cancer Res. 52, 2413-2418[Abstract] |
18. | Jain, P. T., and Trump, B. F. (1997) Anticancer Res. 17, 1167-1174[Medline] [Order article via Infotrieve] |
19. | Bellomo, G., Perotti, M., Taddei, F., Mirabelli, F., Finardi, G., Nicotera, P., and Orrenius, S. (1992) Cancer Res. 52, 1342-1346[Abstract] |
20. | Migliaccio, A., Rotondi, A., and Auricchio, F. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 5921-5925[Abstract] |
21. |
Biswas, D. K.,
Reddy, P. V.,
Pickard, M.,
Makkad, B.,
Pettit, N.,
and Pardee, A. B.
(1998)
J. Biol. Chem.
273,
33817-33824 |
22. | Sacks, D. B., Porter, S. E., Ladenson, J. H., and McDonald, J. M. (1991) Anal. Biochem. 194, 369-377[Medline] [Order article via Infotrieve] |
23. |
Tcholakian, R. K.,
Jones, G. M.,
Sanborn, B. M.,
Rodriguez-Rigau, L. J.,
and Reimondo, G. G.
(1980)
Clin. Chem.
26,
101-106 |
24. |
Pear, W. S.,
Nolan, G. P.,
Scott, M. L.,
and Baltimore, D.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8392-8396 |
25. |
Persechini, A.,
Blumenthal, D. K.,
Jarrett, H. W.,
Klee, C. B.,
Hardy, D. O.,
and Kretsinger, R. H.
(1989)
J. Biol. Chem.
264,
8052-8058 |
26. |
Maune, J. F.,
Klee, C. B.,
and Beckingham, K.
(1992)
J. Biol. Chem.
267,
5286-5295 |
27. |
Wang, J.,
Campos, B.,
Jamieson, G. A.,
Kaetzel, M. A.,
and Dedman, J. R.
(1995)
J. Biol. Chem.
270,
30245-30248 |
28. |
Joyal, J. L.,
Annan, R. S.,
Ho, Y. D.,
Huddleston, M. E.,
Carr, S. A.,
Hart, M. J.,
and Sacks, D. B.
(1997)
J. Biol. Chem.
272,
15419-15425 |
29. | Motohashi, N. (1991) Anticancer Res. 11, 1125-1164[Medline] [Order article via Infotrieve] |
30. | Norman, J. A., Ansell, J., Stone, G. A., Wennogle, L. P., and Wasley, J. W. F. (1987) Mol. Pharmacol. 31, 535-540[Abstract] |
31. | Burrell, M. M. (1993) Enzymes of Molecular Biology , Humana Press Inc., Totowa, NJ |
32. | Kiang, D. T., Kollander, R. E., Thomas, T., and Kennedy, B. J. (1989) Cancer Res. 49, 5312-5316[Abstract] |
33. | Pink, J. J., and Jordan, V. C. (1996) Cancer Res. 56, 2321-2330[Abstract] |
34. | Lam, H.-Y. P. (1984) Biochem. Biophys. Res. Commun. 118, 27-32[Medline] [Order article via Infotrieve] |
35. | Gulino, A., Barrera, G., Vacca, A., Farina, A., Ferretti, C., Screpanti, I., Dianzani, M. U., and Frati, L. (1986) Cancer Res. 46, 6274-6278[Abstract] |
36. | Hardcastle, I. R., Rowlands, M. G., Houghton, J., Parr, I. B., Potter, G. A., and Jarman, M. (1995) J. Med. Chem. 38, 241-248[Medline] [Order article via Infotrieve] |
37. | McDonnell, D. P. (2000) J. Soc. Gynecol. Invest. 7, S10-S15[CrossRef][Medline] [Order article via Infotrieve] |
38. | Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927-937[Medline] [Order article via Infotrieve] |
39. |
Paige, L. A.,
Christensen, D. J.,
Gron, H.,
Norris, J. D.,
Gottlin, E. B.,
Padilla, K. M.,
Chang, C.-Y.,
Ballas, L. M.,
Hamilton, P. T.,
McDonnell, D. P.,
and Fowlkes, D. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3999-4004 |
40. |
Apodaca, G.,
Enrich, C.,
and Mostov, K. E.
(1994)
J. Biol. Chem.
269,
19005-19013 |
41. |
Taules, M.,
Rodriguez-Vilarrupla, A.,
Rius, E.,
Estanyol, J. M.,
Casanovas, O.,
Sacks, D. B.,
Perez-Paya, E.,
Bachs, O.,
and Agell, N.
(1999)
J. Biol. Chem.
274,
24445-24448 |
42. | Krishnaraju, K., Murugesan, K., Vij, U., Kapur, B. M. L., and Farooq, A. (1990) Br. J. Cancer 63, 346-347 |
43. | Katzenellenbogen, J. A., O'Malley, B. W., and Katzenellenbogen, B. S. (1996) Mol. Endocrinol. 10, 119-131[Medline] [Order article via Infotrieve] |
44. | Tsai, M.-J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Renoir, J. M.,
Radanyi, C.,
Faber, L.,
and Baulieu, E. E.
(1990)
J. Biol. Chem.
265,
10740-10745 |
46. |
Aumais, J. P.,
Lee, H. S.,
Lin, R.,
and White, J. H.
(1997)
J. Biol. Chem.
272,
12229-12235 |
47. |
Nishida, E.,
Koyasu, S.,
Sakai, H.,
and Yahara, I.
(1986)
J. Biol. Chem.
261,
16033-16036 |
48. |
Minami, Y.,
Kawasaki, H.,
Suzuki, K.,
and Yahara, I.
(1993)
J. Biol. Chem.
268,
9604-9610 |
49. |
Gratton, J.-P.,
Fontana, J.,
O'Connor, D. S.,
Garcia-Cardenas, G.,
McCabe, T. J.,
and Sessa, W. C.
(2000)
J. Biol. Chem.
275,
22268-22272 |
50. | Hulka, B. S., and Stark, A. T. (1995) Lancet 346, 883-887[Medline] [Order article via Infotrieve] |
51. | Li, Z., Joyal, J. L., and Sacks, D. B. (2000) Biochemistry 39, 5089-5096[CrossRef][Medline] [Order article via Infotrieve] |
52. | Wei, J.-W., Hickie, A., and Klaassen, D. J. (1983) Cancer Chemother. Pharmacol. 11, 86-90[Medline] [Order article via Infotrieve] |
53. | Frankfurt, O. S., Sugarbaker, E. V., Robb, J. A., and Villa, L. (1995) Cancer Lett. 97, 149-154[CrossRef][Medline] [Order article via Infotrieve] |
54. | Newton, C. J., Eycott, K., Green, V., and Atkin, S. L. (2000) J. Steroid Biochem. Mol. Biol. 73, 29-38[CrossRef][Medline] [Order article via Infotrieve] |