From the Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 18, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The steroid hormone estrogen elicits biological
effects in cells by binding to and activating the estrogen receptor
(ER). Estrogen binding induces a conformational change in the receptor, inducing nuclear translocation and transcriptional activation of ER.
The ubiquitous Ca2+-binding protein calmodulin has
been shown to interact directly with ER and enhance its stability. To
further elucidate the functional sequelae of the association between
calmodulin and ER, we examined the effect on ER transcriptional
activation of specifically inhibiting calmodulin. The cell-permeable
calmodulin antagonist CGS9343B prevented estrogen-induced
transcriptional activation by ER, without altering basal transcription.
The inhibition was dose-dependent and independent of the
time of estrogen stimulation. To validate these findings, calmodulin
function was also neutralized by targeted expression of a specific
inhibitor peptide. By inserting localization signals, the inhibitor
peptide was selectively targeted to different subcellular domains.
Inactivation of calmodulin function in the nucleus virtually eliminated
estrogen-stimulated ER transcriptional activation. By contrast, when
membrane calmodulin was specifically neutralized,
estrogen-stimulated transcriptional activation by ER was only slightly
attenuated. Importantly, the inhibitor peptides did not significantly
reduce the amount of ER in the cells. Together, these data demonstrate
that calmodulin is a fundamental component of ER transcriptional activation.
The classic steroid hormone estrogen promotes the proliferation of
both normal and malignant breast epithelial cells and shortens the cell
cycle. Estrogen mediates its biological effects in cells through the
estrogen receptor (ER),1 a
member of the nuclear receptor family of ligand-dependent
transcription factors (reviewed in Refs. 1 and 2). Analogous to other steroid hormone receptors, ER is an intracellular transcription factor
composed of six domains. Estrogen binding to the C-terminal hormone-binding domain induces conformational changes in ER, thereby promoting its dimerization and nuclear localization. The DNA-binding domain of the activated ER binds to DNA sequences, termed estrogen response elements, found in the regulatory regions of target
genes. Several factors, including coactivators, corepressors, and
integrator proteins, are important in ER-mediated transcription
(reviewed in Refs. 3 and 4). It is becoming apparent that
transcriptional regulation requires the recruitment by ER of multiple,
distinct proteins that cooperate to achieve the required response (3). These factors can alter the magnitude of cellular responses to estrogen. There are yet additional factors that modulate ER function. For example, ER interacts with members of the heat-shock protein family
(1), and dissociation of heat-shock protein seems to be necessary for
ER to activate transcription. One of the major roles of ligand binding
is to change the nature of protein-protein interactions between steroid
receptors and other proteins (2). Conversely, other proteins can alter
the state of ER independent of ligand binding. For example,
phosphorylation of ER by several protein kinases, including a
calmodulin-stimulated kinase, modulates ER transcriptional activation
(5).
Calmodulin, a ubiquitous modulator of Ca2+ signaling (6),
regulates the function of multiple, diverse proteins (7, 8). A
substantial body of evidence supports a role for Ca2+ and
calmodulin in estrogen action (Ref. 9, and references therein). For
example, calmodulin binds to ER in a
Ca2+-dependent manner (9, 10) and is required
for formation of the ER-estrogen response element complex (11). In
addition, calmodulin stimulates 17- Materials--
Tissue culture reagents were purchased from
Invitrogen and fetal bovine serum (FBS) was obtained from Biowhittaker.
Charcoal-treated FBS was from Cocalico Biologicals, Inc. MCF-7 and T47D
breast epithelial cells as well as COS-7 green monkey kidney cells
were obtained from the American Type Culture Collection.
pcDNA3-CaMBP4-Flag (calmodulin-binding peptide with
C-terminal-tagged Flag) was kindly provided by Drs. Marcia Kaetzel,
Thomas Freeman, and John Dedman (University of Cincinnati). ERE3-TK-Luc
reporter was a generous gift from Dr. Myles Brown (Dana-Farber Cancer
Institute). CGS9343B was generously donated by Drs. E. Moret and B. Schmid (Novartis, Basel, Switzerland). Permanox plastic eight-well
chamber culture slides were from Nalge Nunc International. FuGENE 6 was
purchased from Roche Molecular Biochemicals. Polyvinylidene difluoride
(PVDF) membrane was purchased from Millipore Corporation. pEYFP-Mem
vector was purchased from Clontech. pRL-TK plasmid
was from Promega.
Antibodies--
Anti-ER Plasmid Construction--
A synthetic gene that encodes the
myosin light chain kinase calmodulin-binding sequence was used (15).
The Flag-tagged construct, which comprises four tandem
calmodulin-binding peptide (CaMBP) repeats, is termed CaMBP4-Flag. The
pcDNA3.1-CaMBP4-Flag was used as template. A 356-bp fragment that
contains the four CaMBP repeats and a C-terminal-tagged Flag fusion
protein was amplified by PCR and BsrG1 sites were designed
at both ends for insertion into pEYFP-Mem
(Clontech) to produce a membrane-targeted
construct. The oligonucleotides used in PCR were
5'-CGCTGTACATCGAGTCTAGCGCCACCATG-3' and
3'-CGCTGTACAGGATCCTTATCACTTGTCATC-5'. pEYFP-Mem encodes a fusion
protein that consists of the N-terminal 20 amino acids of neuromodulin
and a yellow-green fluorescent variant of the enhanced green
fluorescent protein (EYFP). The neuromodulin fragment contains a signal
for post-translational palmitoylation that targets EYFP to membranes.
To label CaMBP4-Flag with EYFP, the pEYFP-Mem plasmid was cut with
BsrG1, and the CaMBP4-Flag was inserted and ligated with T4
DNA ligase. The construct was named CaMBP/m. Because the CaMBP
localizes in the nucleus (15), CaMBP4-Flag was inserted into pEYFP
lacking any localization sequences to develop the nuclear targeted
construct, termed CaMBP/n. The sequence of all constructs was confirmed
by restriction mapping and DNA sequencing. All plasmids were purified
using the Qiagen DNA Purification Kit (Qiagen) following the
instructions provided by the manufacturer.
Cell Culture and Transfection--
MCF-7 and COS-7 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% (v/v) FBS. T47D cells were grown in RPMI 1640 medium supplemented
with 10% (v/v) FBS. MCF-7 and T47D cells were plated in 100-mm dishes
(for Western blots) or 96-well plates (for measurement of
transcription); COS-7 cells were plated in 12-well dishes. DNA was
transiently introduced into cells 24 h after plating using FuGENE
6 according to the manufacturer's instructions. When transfecting
cells in 100-mm dishes, 4 µg of CaMBP/m, CaMBP/n, or EYFP-Mem vector
was used. When measuring transcriptional activity in MCF-7 and T47D
cells, transfections were performed in triplicate with 200 ng of total DNA per well, comprising 10 ng of pRL-TK (which encodes Renilla reniformis luciferase, used as an internal control for
transfection efficiency), 40 ng of ERE3-TK-Luc reporter, and 150 ng of
CaMBP/m, CaMBP/n, or EYFP-Mem vector. For COS-7 cells, 0.65 µg of
total DNA, containing 0.1 µg of pcDNA3-ER Luciferase Reporter Assay--
Equal numbers of cells were lysed
in 50 µl (for 96-well plates) or 200 µl (for 12-well plates)
Passive Lysis Buffer (Promega), and luciferase activity was measured
using the dual luciferase reporter assay (Promega), essentially as
described previously (16). Briefly, light emission from firefly
luciferase activity was measured using a 300-650-nm photomultiplier
tube in a Turner Design 20/20 DLReady luminometer for 12 s. Stop & Glo reagent was added to quench the firefly luciferase, and
Renilla (control) luciferase activity in the same sample
tube was then measured for an additional 12 s. Firefly luciferase
activities were normalized for transfection efficiency to the
Renilla luciferase internal control. Where indicated, cells
were incubated with CGS9343B or an equal volume of ethanol (vehicle).
The concentrations and incubation times are indicated in the figure
legends. The concentrations of CGS9343B used did not reduce the
viability of the cell lines used in this study (data not shown). In
addition, E2 did not enhance the signal in cells
transfected with the TK-Luc plasmid lacking estrogen response element
(data not shown).
Immunoprecipitation and Immunoblotting--
Cells were lysed in
buffer A (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1 mM CaCl2, 0.1% Triton X-100, 0.1% protease
inhibitor mixture (Sigma), and 1 mM phenylmethylsulfonyl
fluoride (Sigma)) and equal amounts of protein lysate were resolved
directly by SDS-PAGE or immunoprecipitated with anti-Flag M2 agarose
affinity beads. Samples were washed five times in buffer A, resolved by SDS-PAGE, and transferred to PVDF membrane. Immunoblots were probed with anti-ER Immunofluorescence Staining--
MCF-7 cells, grown on Permanox
plastic slides, were transiently transfected with 0.8 µg CaMBP/m,
CaMBP/n or EYFP-Mem empty vector using FuGENE 6. After 24 h,
slides were processed for immunocytochemisty essentially as described
previously (17). Slides were incubated for 1 h with mouse
anti-Flag or anti-calmodulin monoclonal antibody, washed four times
with phosphate buffered saline (145 mM NaCl, 12 mM Na2HPO4, 4 mM
NaH2PO4, pH 7.2), and then incubated with tetramethyl rhodamine isothiocyanate-labeled goat anti-mouse IgG for
1 h and mounted with Aqua Polymount (Polysciences, Inc.).
Digital micrographs were acquired using a Zeiss Axiovert S100
microscope with the MRC-1024 Confocal Imaging System (Bio-Rad), and
were imported into a Dell Power Edge 2200 computer for processing using
the Lasersharp 3.0 program (BioRad). Confocal data were converted to
TIFF files. Data were obtained from multiple cells in at least three
different fields from multiple wells, each from at least two
independent experimental determinations.
Miscellaneous--
Densitometry of enhanced chemiluminescence
signals was performed using the Scion Image software for PC
(Scion Corporation). Protein concentrations were determined with the
detergent-compatible protein assay (Bio-Rad). Statistical
significance was assessed by Student's t test using InStat
software (GraphPad Software, Inc.). Cell viability was assessed by
monitoring exclusion of trypan blue.
Calmodulin Antagonist Reduces ER The Calmodulin Antagonist CGS9343B Inhibits the Transcriptional
Activity of ER--
In addition to its stabilizing effect on ER (9),
calmodulin is required for formation of the ER-ERE complex (11). The latter data suggest that calmodulin may modulate transcriptional activation by ER. To examine this hypothesis, MCF-7 cells were transiently transfected with an ER-responsive reporter plasmid and
incubated with or without CGS9343B. E2-stimulated ER
transcription in MCF-7 cells by 4-5-fold (Fig.
2). Exposure of cells to 40 µM CGS9343B for 16 h completely eliminated
E2-induced transcription, without altering basal
transcription (Fig. 2A). More detailed analysis revealed
that the inhibition produced by CGS9343B was dose-dependent, with E2 stimulation essentially
abolished at 40 µM CGS9343B (Fig. 2B). The
abrogation of E2-stimulated transcription by CGS9343B could
be caused by reduction in ER, disruption of the association of
calmodulin with ER (9), or another mechanism. To evaluate the first
possibility, E2-stimulated transcription was examined at
different time intervals. E2-enhanced transcriptional activity in a time-dependent manner (Fig. 2C).
Neither E2 nor CGS9343B significantly altered transcription
at 0 h. As seen with 16 h of incubation, CGS9343B completely
prevented enhancement of transcription by E2 at all time
points (Fig. 2C). Note that incubation with CGS9343B for
8 h reduced ER
To confirm the biological relevance of our observations,
analysis was performed in T47D cells, another ER
To attempt to eliminate the possibility that the inhibition of
transcriptional activity of ER by CGS9343B may have been
caused by a decrease in receptor abundance, transfected ER Development of CaMBPs to Specifically Inhibit Calmodulin in
Selected Subcellular Domains--
CGS9343B is reported to be a
specific antagonist for calmodulin at concentrations up to 1 mM (18), a concentration 25-fold higher than the highest
concentration used in this work. Nevertheless, caution should always be
exercised in interpreting results obtained with antagonists. Therefore,
we adopted a complementary strategy to inhibit calmodulin. Transient
transfection of an inhibitor peptide derived from muscle myosin
light-chain kinase into mammalian cells blocks calmodulin function
(15). The CaMBP was tagged with Flag and EYFP. To discriminate between
the interaction of calmodulin and ER in the nucleus with the
interaction in the plasma membrane, the EYFP-CaMBP-Flag construct was
selectively targeted to subcellular regions. The constructs are termed
CaMBP/m and CaMBP/n for membrane- and nuclear-targeted versions,
respectively. The peptides were characterized before evaluation in ER
transcription assays. To verify calmodulin binding, Flag-tagged CaMBP/m
and CaMBP/n were transfected into cells and lysates were
immunoprecipitated with anti-Flag affinity gel. Probing the resultant
Western blots for calmodulin demonstrated that both CaMBP/n and CaMBP/m
specifically bind endogenous calmodulin, with essentially the same
affinity (Fig. 5A). Probing
the immunoprecipitates for yellow fluorescent protein revealed that
equal amounts of CaMBP are present (Fig. 5A). The EYFP-Mem
vector is not seen on the blot (Fig. 5A, top) because it lacks Flag, but it was present in the lysates (data not
shown).
The subcellular localization of CaMBP/m and CaMBP/n in
MCF-7 cells was assessed by immunocytochemistry. EYFP-Mem vector
(containing yellow fluorescent protein and the membrane-targeting
sequence) was expressed at the plasma and intracellular membranes (Fig. 5B, left). CaMBP/m had a distribution virtually
identical to that of the vector alone; it was expressed both at the
plasma membrane and in the cytoplasm (Fig. 5B,
center). By contrast, CaMBP/n was expressed almost
exclusively in the nucleus (Fig. 5B, right). The
merged images verify that the EYFP plasmids express the Flag-tagged peptides.
CaMBPs Attenuate ER Transcriptional Activation--
We next
examined the effect on ER transcription of neutralizing calmodulin
function in different subcellular domains. As shown in Fig.
6A, transient transfection
into MCF-7 cells of CaMBP/n (which neutralizes nuclear calmodulin)
eliminated E2-induced ER transcriptional activation.
Inhibiting calmodulin function in the extranuclear regions of the cell
with CaMBP/m had a much less dramatic effect. When membrane calmodulin
function was neutralized, E2 readily increased ER
transcriptional activation, reaching a level only 24% below that
attained in vector-transfected cells (Fig. 6A). Neither
CaMBP/m nor CaMBP/n significantly altered basal ER transcriptional
activity (data not shown). Importantly, in contrast to the reduction in
ER
Our results suggest that by blocking nuclear calmodulin function,
CaMBP/n reduces transcriptional activation by ER. The attenuation of ER
transcription by inhibiting the association of calmodulin with ER at
the membrane was less anticipated. The mechanism is unknown. A membrane
ER has been demonstrated, but this receptor is not believed to induce
transcription (22), making it unlikely that this could account for the
effect of CaMBP/m. Although CaMBP/m did not reduce total ER, the amount
of ER in the nucleus could be lower. Alternatively, CaMBP/m could alter
the cellular distribution of calmodulin, reducing the amount of nuclear
calmodulin; this could decrease ER transcriptional activation. Studies
are underway to identify the mechanism.
During the preparation of this manuscript, Pedrero et al.
(21) showed that the calmodulin antagonist W7 reduced by 74%
E2-stimulated ER transcription in breast epithelial cells.
However, no evidence was presented in that study that the inhibition of
transcription was independent of the reduction in ER protein produced
by calmodulin antagonists. Moreover, W7 lacks specificity and inhibits
calmodulin-independent enzymes, such as protein kinase A and protein
kinase C (23). Our study is not subject to these caveats. We inhibited
calmodulin function by two independent strategies, namely
with CGS9343B, believed to be a specific calmodulin antagonist (18),
and a specific calmodulin target peptide. Importantly, we examined
transcription under conditions in which the amount of ER was not
significantly reduced. Together, our data document that disruption of
the interaction between calmodulin and ER prevented the latter from
activating transcription in response to E2.
First reported almost 20 years ago (24), the participation of
calmodulin in estrogen function has become the focus of renewed interest (Ref. 9, and references therein). Calmodulin binds to ER in
intact cells independently of E2, thereby modulating ER
stability and steady state levels (9). Moreover, calmodulin is an
integral component of the ER-estrogen response element complex (11,
25). The data presented here demonstrate that an interaction between
calmodulin and ER in the nucleus is required for
E2-stimulated ER transcriptional activation. The molecular
mechanism by which calmodulin facilitates ER transcription is unknown.
Calmodulin has been shown to modulate the activity of a number of
nuclear proteins, several of which are involved in transcription. For example, Ca2+/calmodulin-dependent kinases regulate
gene transcription by altering coactivator function (26). Furthermore,
calmodulin binds to members of the basic helix-loop-helix transcription
factors, modifying their DNA binding (27). Recently, a family of
calmodulin-binding transcription activators was identified (28). It is
not known whether calmodulin directly binds a transcription
activator or has another role in ER transcription. Our previous results
imply that calmodulin alters the tertiary conformation of ER (9). One
could envisage that this would alter the ability of ER to interact with
coactivators and/or corepressors, altering transcription. Regardless of
the mechanism, our data contribute to deciphering the intricate
meshwork of ER signaling pathways. In addition, they further explain
the prior observations that calmodulin antagonists inhibit the growth
of breast cell lines (29) and synergistically amplify antiestrogen
therapy (30).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-estradiol (E2)
binding to ER, inducing tyrosine phosphorylation and activation of the
ER (12). Recent evidence from our laboratory indicates that endogenous
ER binds to endogenous calmodulin, thereby stabilizing ER (9). Together with the report that calmodulin antagonists inhibit the growth of human
breast carcinoma cell lines (13), these finding suggest that
Ca2+/calmodulin may participate in ER signaling pathways.
Therefore, we set out to examine whether calmodulin modulates the
transcriptional activation of ER.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(Ab-15) antibodies were manufactured
by Neomarkers. Anti-ER
and anti-Flag antibodies were from Upstate
Biotechnology. Anti-calmodulin monoclonal antibodies have been
characterized previously (14). Anti-green fluorescent protein
antibodies and anti-Flag M2 agarose affinity beads were purchased from
Clontech and Sigma, respectively. Tetramethyl
rhodamine isothiocyanate-labeled goat anti-mouse IgG was purchased from
Jackson ImmunoResearch Laboratories, Inc. Anti-mouse Ig, horseradish
peroxidase-conjugated secondary antibodies were from Amersham Biosciences.
, 0.5 µg of
ERE3-TK-Luc reporter, and 50 ng of pRL-TK, was used. Six hours after
transfection, the medium was replaced with phenol red-free culture
medium containing 10% charcoal-treated FBS. Twenty-four hours later,
E2 or an equal amount of vehicle was added to the cells in
the absence or presence of CGS9343B. Cells were incubated for the times
indicated in the figure legends, lysed, and processed as described below.
, anti-ER
, anti-calmodulin (14), or anti-green fluorescent protein antibodies. Complexes were visualized with the
appropriate horseradish peroxidase-conjugated secondary antibody and
developed by enhanced chemiluminescence.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Protein Level in MCF-7
Cells--
We demonstrated previously that incubation of MCF-7 cells
with calmodulin antagonists for 16 h reduced the amount of ER
(9). ER
was not examined. Therefore, we incubated MCF-7 cells with the cell-permeable calmodulin antagonist CGS9343B (18-20) for
different time intervals. Examination of equal amounts of protein
lysate by Western blotting revealed that ER
was decreased in a
time-dependent manner (Fig.
1A). Our prior analysis showed
that the reduction in ER
seemed to be caused primarily by calmodulin
stabilization of the ER
protein (9). This finding is supported by
reverse transcription-PCR, which demonstrated that transcription of the ER
gene is not reduced by CGS9343B (data not shown). In contrast to
the reduction in ER
protein levels, CGS9343B had no effect on the
amount of ER
in the cells (Fig. 1B). These data are
consistent with the recent observation that ER
does not bind to
calmodulin (21). Therefore, all further analyses were restricted to
ER
. Note that CGS9343B did not reduce the cell viability in any cell lines examined in this study at the concentrations used in this work
(data not shown).
View larger version (20K):
[in a new window]
Fig. 1.
Effect of the calmodulin antagonist CGS9343B
on ER content. MCF-7 cells were incubated with 40 µM
CGS9343B for the indicated time periods. After lysis, equal amounts of
protein were resolved by SDS-PAGE, transferred to PVDF, and membranes
were probed for ER (A) or ER
(B). The
relative amounts of ER
and ER
were quantified by densitometry.
The results, presented in the graphs, are expressed relative
to 0 h. A representative experiment is shown.
by only 19% (Fig. 1), far less than its effect
on transcription. Together, these results suggest that the absence of
E2-stimulated ER transcription is not caused merely by a
reduction in ER; an interaction of calmodulin with ER seems necessary
for transcriptional activation.
View larger version (18K):
[in a new window]
Fig. 2.
E2-induced
transcriptional activity in MCF-7 cells was inhibited by the
calmodulin antagonist CGS9343B. MCF-7 cells were transiently
co-transfected with ERE3-TK-Luc and pRL-TK as described under
"Experimental Procedures." pRL-TK was used to normalize for
transfection efficiency. After lysis, luciferase activity was
determined by luminometry. In all cases, lysates were prepared from
equivalent numbers of cells. A, cells were treated with
vehicle (EtOH) or 10 nM E2 for 16 h in the
absence or presence of 40 µM CGS9343B. Results are
expressed relative to cells treated with vehicle alone, which was set
as 1. *, significantly different from E2-stimulated ER
transcription (p < 0.05). B, cells were
treated as described in A, except that the concentration of
CGS9343B was varied. C, cells were treated with vehicle
(EtOH) (clear bars) or 10 nM E2 in
the absence (gray bars) or presence (black bars)
of 40 µM CGS9343B for the indicated times. Results are
expressed relative to cells treated with vehicle alone, which was set
as 1. Significantly different from vehicle: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Significantly different from E2-stimulated ER
transcription: , p < 0.05;
, p < 0.01;
, p < 0.001. For all, data are the
means of at least three separate experiments, each performed in
triplicate. Means ± S.E. are shown.
-positive cell line. Analogous to the observations in MCF-7 cells, incubation of T47D cells
with 40 µM CGS9343B completely prevented enhancement of transcription by E2 (Fig.
3A). Incubation with CGS9343B
for both 8 and 16 h produced essentially identical results. The
magnitude of the inhibition of transcription produced by CGS9343B was
substantially greater than the extent of the reduction of ER
protein
in T47D cells, which was 44-48% (Fig. 3B). Note that
although E2 reduced ER
protein, the magnitude of the
reduction produced by CGS9343B was independent of E2. These
findings mimic our prior observations in MCF-7 cells (9).
View larger version (10K):
[in a new window]
Fig. 3.
CGS9343B inhibited
E2-induced transcriptional activity in T47D
cells. A, T47D cells were transiently cotransfected with
ERE3-TK-Luc and pRL-TK and lysates were prepared as described in the
legend to Fig. 2. Cells were treated with vehicle or 10 nM
E2 for 8 h (white bars) or 16 h
(black bars) in the absence or presence of 40 µM CGS9343B. Results are expressed relative to cells
treated with vehicle alone, which was set as 1. *, significantly
different from E2-stimulated ER transcription
(p < 0.001). Data are from four separate experiments,
each performed in triplicate. Means ± S.E. are shown.
B, T47D cells were treated with 40 µM CGS9343B
in the absence or presence of 10 nM E2 for
16 h. Equal amounts of lysate were resolved by SDS-PAGE,
transferred to PVDF, and the blot was probed for ER . The relative
amounts of ER
were quantified by densitometry. The results,
presented in the graphs, are expressed relative to vehicle alone. A
representative experiment of three separate determinations is
shown.
was also examined. ER
was cotransfected into COS-7 cells with the luciferase reporter gene. Consistent with its effects on endogenous ER, CGS9343B completely inhibited E2-stimulated transcription of
transfected ER (Fig. 4A).
Although COS-7 cells do not have endogenous ER
or ER
and might
not contain all the components necessary for ER degradation, CGS9343B
reduced transfected ER
in COS-7 cells by approximately the same
extent as the reduction observed with endogenous ER (Fig.
4B).
View larger version (10K):
[in a new window]
Fig. 4.
CGS9343B inhibited
E2-induced transcriptional activity of
transfected ER . A, COS-7 cells were
transiently cotransfected with pcDNA3-ER
, ERE3-TK-Luc, and
pRL-TK as described under "Experimental Procedures." 24 h
later, cells were treated with vehicle or 10 nM
E2 for 16 h in the absence or presence of 40 µM CGS9343B and subsequently assayed for luciferase
activity. Results are expressed relative to cells treated with vehicle
alone, which was set as 1. *, significantly different from
E2-stimulated ER transcription (p < 0.05).
Data are the means of three separate experiments, each performed in
duplicate. Means ± S.E. are shown. B, aliquots of the
lysates used in the transcription assay were cleared by centrifugation
and equal amounts of protein were analyzed by Western blotting. The
relative amounts of ER
were quantified by densitometry. The results,
presented in the graphs, are expressed relative to vehicle alone. A
representative experiment of three separate determinations is
shown.
View larger version (22K):
[in a new window]
Fig. 5.
Characterization of specific CaMBPs.
A, MCF-7 cells were transiently transfected with EYFP-Mem
vector alone (v), CaMBP/n (n), or CaMBP/m
(m). Mock transfected cell lysate was used as control
(lys). 48 h after transfection, cells were lysed in
buffer containing 1 mM CaCl2 and equal amounts
of lysate were immunoprecipitated (IP) with anti-Flag
affinity gel. Eluted proteins were resolved by SDS-PAGE and transferred
to PVDF. The blot was probed for yellow fluorescent protein to identify
the CaMBPs (top) and calmodulin (CaM)
(bottom). B, MCF-7 cells, transfected as
described in A above, were processed for immunocytochemistry
as detailed under "Experimental Procedures." Cells were probed with
anti-Flag antibody (Flag) and visualized with tetramethyl
rhodamine isothiocyanate-labeled secondary antibody, which fluoresces
red (top). Yellow fluorescent protein
(YFP) is shown in the center (green).
Merged images are presented in the bottom. Yellow indicates
colocalization. Data are representative of at least three experimental
determinations.
produced by CGS9343B, neither CaMBP/n nor CaMBP/m significantly
changed the amount of ER
in MCF-7 cells (Fig. 6B).
Similarly, the CaMBPs had no effect on the amount of calmodulin.
Therefore, these data indicate that the effect of calmodulin in ER
transcriptional activation is independent of its effect on ER
stability.
View larger version (13K):
[in a new window]
Fig. 6.
CaMBP reduced
E2-induced transcriptional activity without
altering the amount of ER or calmodulin. A, MCF-7 cells
were transiently transfected with EYFP-Mem vector alone ( ), CaMBP/n
(n), or CaMBP/m (m), and cotransfected with
ERE3-TK-Luc and pRL-TK. Cells were treated with vehicle or 10 nM E2 for 16 h and assayed for luciferase
activity as described under "Experimental Procedures." Results are
expressed as fold stimulation produced by E2 relative to
vehicle control. Data represent the means ± S.E. of at least six
separate experiments, each performed in triplicate. *, significantly
different from vector (p < 0.05);
, significantly
different from CaMBP/n (p < 0.05);
, significantly
different from vehicle (p < 0.05). B, MCF-7
cells were transiently transfected with EYFP-Mem vector alone
(v), CaMBP/n (n), or CaMBP/m (m) and
lysed 48 h later. Equal amounts of protein were resolved by
SDS-PAGE, transferred to PVDF, and probed for ER
and calmodulin. The
positions of migration of ER
and calmodulin (CaM) are
indicated. The relative amounts of ER and calmodulin were quantified by
densitometry. The results, presented in the graphs, are
expressed relative to vector and represent the mean and range of two
independent experimental determinations. The error in the last
calmodulin bar is too small to be visible.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to the following for generously donating reagents: Dr. Myles Brown (Dana Farber Cancer Institute) for ERE3-luciferase reporter plasmids; Drs. Marcia Kaetzel, Thomas Freeman, and John Dedman (University of Cincinnati) for the pcDNA3.1-CaMBP-Flag plasmid; and Drs. E. Moret and B. Schmid (Novartis, Basel, Switzerland) for CGS9343B. We thank Michelle Lowe at the Brigham and Women's Confocal Core Facility for expert assistance with confocal microscopy.
![]() |
FOOTNOTES |
---|
* This work was supported in part by United States Army Grant DAMD 17-02-1-0305 and National Institutes of Health Grant CA93645 (to D. B. S.).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.
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, November 4, 2002, DOI 10.1074/jbc.M210708200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ER, estrogen
receptor;
E2, 17--estradiol;
FBS, fetal bovine serum;
Luc, luciferase;
PVDF, polyvinylidene difluoride;
CaMBP, calmodulin-binding peptide;
CaMBP/m, EYFP-CaMBP-Flag membrane
expression construct;
CaMBP/n, EYFP-CaMBP-Flag nuclear expression
construct;
EYFP, yellow-green fluorescent variant of the enhanced green
fluorescent protein;
TK, thymidine kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Tsai, M. J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486[CrossRef][Medline] [Order article via Infotrieve] |
2. | Dickson, R. B., and Stancel, G. M. (2000) J. Natl. Cancer. Inst. Monogr. 135-145 |
3. |
McKenna, N. J.,
Lanz, R. B.,
and O'Malley, B. W.
(1999)
Endocr. Rev.
20,
321-344 |
4. | Xu, L., Glass, C. K., and Rosenfeld, M. G. (1999) Curr. Opin. Genet. Dev. 9, 140-147[CrossRef][Medline] [Order article via Infotrieve] |
5. |
MacGregor, J. I.,
and Jordan, V. C.
(1998)
Pharmacol. Rev.
50,
151-196 |
6. | Cohen, P., and Klee, C. (1988) Calmodulin , Elsevier, New York |
7. | 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 |
8. | Chin, D., and Means, A. R. (2000) Trends Cell Biol. 10, 322-328[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Li, Z.,
Joyal, J. L.,
and Sacks, D. B.
(2001)
J. Biol. Chem.
276,
17354-17360 |
10. | Bouhoute, A., and Leclercq, G. (1992) Biochem. Biophys. Res. Commun. 184, 1432-1440[Medline] [Order article via Infotrieve] |
11. |
Biswas, D. K.,
Reddy, P. V.,
Pickard, M.,
Makkad, B.,
Pettit, N.,
and Pardee, A. B.
(1998)
J. Biol. Chem.
273,
33817-33824 |
12. | Migliaccio, A., Rotondi, A., and Auricchio, F. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 5921-5925[Abstract] |
13. | Strobl, J. S., and Peterson, V. A. (1992) J. Pharmacol. Exp. Ther. 263, 186-193[Abstract] |
14. | Sacks, D. B., Porter, S. E., Ladenson, J. H., and McDonald, J. M. (1991) Anal. Biochem. 194, 369-377[Medline] [Order article via Infotrieve] |
15. |
Wang, J.,
Campos, B.,
Jamieson, G. A., Jr.,
Kaetzel, M. A.,
and Dedman, J. R.
(1995)
J. Biol. Chem.
270,
30245-30248 |
16. |
Briggs, M. W., Li, Z.,
and Sacks, D. B.
(2002)
J. Biol. Chem.
277,
7453-7465 |
17. |
Kim, S. H., Li, Z.,
and Sacks, D. B.
(2000)
J. Biol. Chem.
275,
36999-37005 |
18. | Norman, J. A., Ansell, J., Stone, G. A., Wennogle, L. P., and Wasley, J. W. (1987) Mol. Pharmacol. 31, 535-540[Abstract] |
19. |
Joyal, J. L.,
Burks, D. J.,
Pons, S.,
Matter, W. F.,
Vlahos, C. J.,
White, M. F.,
and Sacks, D. B.
(1997)
J. Biol. Chem.
272,
28183-28186 |
20. |
Li, Z.,
Kim, S. H.,
Higgins, J. M.,
Brenner, M. B.,
and Sacks, D. B.
(1999)
J. Biol. Chem.
274,
37885-37892 |
21. |
Garcia Pedrero, J. M.,
Del Rio, B.,
Martinez-Campa, C.,
Muramatsu, M.,
Lazo, P. S.,
and Ramos, S.
(2002)
Mol. Endocrinol.
16,
947-960 |
22. | Levin, E. R. (1999) Trends Endocrinol. Metab. 10, 374-377[CrossRef][Medline] [Order article via Infotrieve] |
23. | Sugimura, M., Sato, T., Nakayama, W., Morishima, Y., Fukunaga, K., Omitsu, M., Miyamoto, E., and Shirasaki, Y. (1997) Eur. J. Pharmacol. 336, 99-106[CrossRef][Medline] [Order article via Infotrieve] |
24. | Flandroy, L., Cheung, W. Y., and Steiner, A. L. (1983) Cell Tissue Res. 233, 639-646[Medline] [Order article via Infotrieve] |
25. | Bouhoute, A., and Leclercq, G. (1995) Biochem. Biophys. Res. Commun. 208, 748-755[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Corcoran, E. E.,
and Means, A. R.
(2001)
J. Biol. Chem.
276,
2975-2978 |
27. |
Onions, J.,
Hermann, S.,
and Grundstrom, T.
(1997)
J. Biol. Chem.
272,
23930-23937 |
28. |
Bouche, N.,
Scharlat, A.,
Snedden, W.,
Bouchez, D.,
and Fromm, H.
(2002)
J. Biol. Chem.
277,
21851-21861 |
29. | Wei, J. W., Hickie, R. A., and Klaassen, D. J. (1983) Cancer Chemother. Pharmacol. 11, 86-90[Medline] [Order article via Infotrieve] |
30. | 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] |