Activation of Estrogen Receptor-
by the Heavy Metal Cadmium
Adriana Stoica,
Benita S. Katzenellenbogen and
Mary Beth Martin
Department of Biochemistry and Molecular Biology (A.S., M.B.M.)
Vincent T. Lombardi Cancer Center Georgetown University
Washington, DC, 20007
Department of Molecular and Integrative
Physiology (B.S.K.) University of Illinois Urbana, Illinois
61801
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ABSTRACT
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Previous studies from this laboratory have shown
that the heavy metal cadmium (Cd) mimics the effects of estradiol in
estrogen-responsive breast cancer cell lines. To understand the
mechanism by which cadmium activates estrogen receptor-
(ER-
),
the ability of cadmium to bind to and activate wild-type and various
mutants of ER-
was examined. When tested in transient cotransfection
assays in COS-1 cells, cadmium concentrations as low as
10-11 M activated
ER-
. Scatchard analysis employing either purified human recombinant
ER-
or extracts from ER-containing MCF-7 cells demonstrated that
109Cd binds to the ER with an equilibrium
dissociation constant of approximately 4 to 5 x
10-10 M. Cadmium also
blocks the binding of estradiol to ER-
in a noncompetitive manner
(Ki = 2.96 x
10-10 M), suggesting
that the heavy metal interacts with the hormone-binding domain of the
receptor. To study the role of the hormone-binding domain in cadmium
activation, COS-1 cells were transiently cotransfected with GAL-ER, a
chimeric receptor containing the DNA-binding domain of the
transcription factor GAL4 and the hormone-binding domain of ER-
, and
a GAL4-responsive reporter gene. Treatment of the transfected cells
with either 10-6 M
cadmium or 10-9 M
estradiol resulted in a 4-fold increase in reporter gene activity. The
effect of cadmium on the chimeric receptor was blocked by the
antiestrogen, ICI-164,384, suggesting that cadmium activates ER-
through an interaction with the hormone-binding domain of the receptor.
Transfection and binding assays with ER-
mutants identified C381,
C447, E523, H524, and D538 as possible interaction sites of cadmium
with the hormone-binding domain of ER-
.
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INTRODUCTION
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Breast cancer is the most common malignancy affecting women and is
the leading cause of death in women between the ages of 35 and 45 (1).
The estimated lifetime breast cancer risk for women in the United
States is 1 in 9 (1). Epidemiological studies suggest that endocrine
factors play an important underlying role in the etiology of breast
cancer. Prominent common risk factors include age, age at menarche and
menopause, age at first full-term pregnancy, and family history of
breast cancer (2). Depending on the age at diagnosis, menarche at age
11 or earlier compared with age 13 or greater confers up to a doubling
of breast cancer risk (3, 4). This increased risk is due to the larger
lifetime number of normal menstrual cycles (5). In contrast, risk
decreases with earlier age at menopause, for both natural and
surgically induced menopause, reflecting the postmenopausal decrease in
levels of estrogen and progesterone (6, 7). The primacy of hormonal
factors in the epidemiology of breast cancer reflects the control of
proliferation by estrogens (8, 9).
Because the estrogen receptor (ER) is a critical mediator of growth,
molecules that can bind to and activate the ER can potentially increase
the risk of breast cancer. A number of natural and man-made chemicals
in the environment possess estrogenic activity and, therefore, may pose
a health risk. Data presented in this paper and in a previously
published study from this laboratory (10) suggest that the heavy metal,
cadmium, is a new environmental estrogen. In that study, cadmium was
shown to mimic the effects of estradiol in the estrogen-responsive
breast cancer cell line, MCF-7. Treatment with cadmium resulted in an
increase in cell growth, an increase in the steady state levels of
progesterone receptor, pS2, and cathepsin D, and a decrease in the
steady state level of estrogen receptor-
(ER-
). The changes in
steady state levels of protein and mRNA of these genes were due to
changes in transcription that were blocked by the antiestrogen,
ICI-164,384. Transfection assays also demonstrated that the effects of
cadmium were mediated by ER-
.
The goal of the present study was to gain insight into the mechanism by
which cadmium activated ER-
. To achieve this goal, the ability of
cadmium to bind to and activate wild-type ER and mutant forms of the
receptor was investigated. The results presented herein demonstrate
that low concentrations of cadmium activate ER-
through an
interaction with the hormone-binding domain of the receptor. The metal
binds with high affinity and blocks estradiol binding to the receptor.
The interaction of cadmium with the receptor appears to involve several
amino acids in the hormone-binding pocket of the receptor, suggesting
that the metal may form a coordination complex with the hormone-binding
domain and thereby activate the receptor.
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RESULTS
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Interaction of Cadmium with the Hormone-Binding Domain of the
ER
To determine the dose-response effectiveness of cadmium in
stimulating ER activity, the human ER-
gene and an
estrogen-responsive chloramphenicol acetyltransferase (CAT) reporter
construct under the control of the mouse mammary tumor virus (MMTV)
promoter, in which the glucocorticoid response element was replaced
with two estrogen response elements (11), were transiently
cotransfected into COS-1 cells. After transfection, the cells were
treated for 24 h with either CdCl2
(10-12 to 10-6
M) or estradiol (10-9 and
10-8 M). The amount of
chloramphenicol acetyltransferase (CAT) activity was then measured,
expressed as percent conversion, and normalized to the amount of
ß-galactosidase activity (Fig. 1
). As
expected, treatment with estradiol resulted in an approximately 4-fold
increase in CAT activity. Treatment with 10-12
M to 10-6 M cadmium also
stimulated expression of CAT by approximately 2- to 6-fold, suggesting
that low doses of cadmium activated ER-
.

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Figure 1. The Ability of Cadmium to Activate ER-
Wild-type ER- was transiently cotransfected into COS-1 cells.
The transfected cells were treated with either estradiol
(10-9 or 10-8
M) or CdCl2
(10-10 to 10-6
M) for 24 h. CAT activity was measured as described in
Materials and Methods. The results were
normalized to B-galactosidase activity and expressed as percent of CAT
activity in untreated cells. E2, Estradiol; Cd,
cadmium chloride. The values are the mean of five experiments
(±SD).
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To identify the region of ER-
involved in activation by cadmium,
chimeric receptors containing the hormone-binding domain of either
ER-
or the GR were employed (12). These chimeric receptors consist
of the DNA-binding domain of the yeast transcription factor GAL-4 and
the hormone-binding domain of either ER-
(GAL-ER) or the GR
(GAL-GR). Stimulation of transcription by GAL-ER or GAL-GR from a
GAL-4-responsive reporter gene requires either estradiol or
dexamethasone, respectively. When the chimeric receptor GAL-ER and the
GAL-4-CAT reporter construct were transiently cotransfected into COS-1
cells and the cells were treated with 10-6
M cadmium or 10-9 M
estradiol there was an approximately 4-fold increase in CAT activity
(Fig. 2
). Addition of the estrogen antagonist ICI 164,384 (5 x
10-7 M) blocked both the cadmium and
the estradiol-induced increase in transcription by 75%
(P = 0.0075) and 91%, respectively. Cadmium has been
shown to block the binding of dexamethasone to the GR (13). Since
cadmium interacts with the hormone-binding domain of the GR, GAL-GR was
employed as a control; the results are compared in Fig. 2
. Cadmium and estradiol had no effect in
cells transfected with GAL-GR, while 10-9
M dexamethasone induced an approximately 4-fold
increase in CAT activity. The dexamethasone-induced increase was
inhibited by cadmium, demonstrating an antagonistic effect of the metal
on the GR. Taken together, these results suggest that cadmium interacts
with the hormone-binding domain of ER-
to activate the receptor.
Although cadmium interacts with the hormone-binding domain of the GR,
it does not activate the GR.

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Figure 2. The Ability of Cadmium to Activate GAL-ER
GAL-ER and GAL-GR chimeric genes and a GAL4-CAT reporter construct were
transiently cotransfected into COS-1 cells. The transfected cells were
treated with 10-9 M estradiol,
10-9 M dexamethasone, or 10-6
M cadmium in the presence and absence of 5 x
10-7 M ICI-164,384. CAT activity was measured
as described in Materials and Methods. The results were
normalized to the B-galactosidase activity and expressed as percent of
CAT activity in untreated cells. The results represent the mean of four
experiments (±SD). E2, Estradiol; Cd, cadmium
chloride; Dex, dexamethasone; ICI, ICI-164,384.
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Binding of Cadmium to the ER
To determine whether cadmium blocked estradiol binding to the
ER-
, the effects of the heavy metal on hormone binding were measured
using a single-dose ligand- binding assay. Extracts from MCF-7 cells
were treated with various concentrations of cadmium
(10-12 to 10-5
M) for 1 h. The ability of the ER to bind hormone was
then assayed by incubating the extract with 10-8
M [3H]estradiol in the presence or
absence of a 200-fold molar excess of diethylstilbestrol for 18 h
at 4 C. As shown in Fig. 3a
, cadmium
blocked the binding of estradiol to the receptor. Hormone binding
decreased with increasing cadmium concentration. Hormone binding also
decreased with increasing preincubation time with the heavy metal (Fig. 3b
, P = 0.0193). Maximal inhibition was seen after a
30-min preincubation with 10-6
M cadmium. In contrast to cadmium, preincubation
with high concentrations of zinc (10-6 to
10-4 M
ZnSO4) for 1 h did not influence hormone
binding to ER-
(Fig. 3b
and data not shown). These results
demonstrate a degree of specificity for the action of cadmium in
blocking the binding of estradiol to ER-
.

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Figure 3. Effects of Cadmium on Estradiol Binding to ER-
A, Effect of cadmium concentration on estradiol binding. Cytosolic
extracts from MCF-7 cells were treated with various concentrations of
cadmium chloride (10-12 to 10-5
M) for 1 h. The ability of the ER to bind hormone was
assayed with 10- 8 M
[3H]estradiol in the presence or absence of a 200-fold
molar excess of DES for 18 h at 4 C. The amount of specific
binding of [3H]estradiol was determined as described in
Materials and Methods and expressed as percent of
untreated cells. Results represent the mean value of six
experiments ± SD. B, Effect of cadmium preincubation
time on estradiol binding. Cytosolic extracts were treated either with
10-6 M cadmium chloride for 0, 15, 30, or 60
min or with 10-5 M zinc chloride for 0 or 60
min before the determination of specific estradiol (10- 8
M) binding as described above. Results represent the mean
value of five experiments ± SD. E2,
Estradiol; Cd, cadmium; Zn, zinc.
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To determine whether cadmium altered the binding affinity of
estradiol to the receptor, a multiple-dose ligand-binding assay was
performed. Recombinant human ER-
was incubated with
10-10, 10-9, or
10-8 M cadmium chloride and various
concentrations of [3H] estradiol
(10-12 to 10-7
M) in the presence or absence of a 200-fold molar excess of
diethylstilbestrol (DES). The affinity and binding capacity of the
receptor were determined according to the method of Scatchard (19)
(Fig. 4
). In the absence of cadmium, estradiol bound to
the receptor with an equilibrium dissociation constant
(Kd) of 2.92 x 10-10
M (±0.34, n = 3). In the presence of cadmium
chloride, the dissociation constant of estradiol was unchanged, but the
number of binding sites decreased (Table 1
). These data demonstrate that cadmium
competes with estradiol for binding to ER-
in a noncompetitive
manner since it does not alter the binding affinity of hormone for the
receptor.

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Figure 4. Effects of Cadmium on the Binding of Estradiol to
Recombinant Human ER-
Recombinant ER- (4 x 10-9 M) was
incubated with vehicle or 10-10, 10-9, or
10-8 M cadmium for 1 h at 4 C.
[3H]Estradiol (10-12 to 10-7
M) in the presence or absence of a 200-fold molar excess of
DES was added for 18 h at 4 C. The binding affinity and binding
capacity of ER- were determined according to the method of Scatchard
as described in Materials and Methods. A, Scatchard plot
of a representative binding assay. , Control; ,
10-10 M cadmium; , 10-9
M cadmium; , 10-8 M cadmium. B,
Saturation curves of the [3H]estradiol binding to ER- .
BT, Total binding; BN, nonspecific binding; BS, specific binding. The
experiment was repeated three times.
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To characterize the binding of cadmium to the ER, Scatchard analysis
was performed using radioactive cadmium, 109Cd,
and recombinant ER-
. Recombinant human ER-
was incubated with
various concentrations of 109Cd
(10-12 to 10-7
M) in the presence and absence of a 200-fold molar excess
of CdCl2; the results are shown in Fig. 5
. Interestingly, cadmium bound to ER-
with an
affinity similar to that of estradiol for the receptor. The
dissociation constant for cadmium binding is approximately 5 x
10-10 M (±1.5, n =
3). The binding of cadmium to the ER-
in MCF-7 cells was also
measured and compared with the binding to recombinant receptor. A
similar dissociation constant was obtained (Kd =
4.5 x 10-10 M (±
2.1, n = 3, data not shown) when the binding of cadmium to ER-
was measured in whole-cell extracts. These results demonstrated that
there was no significant difference in the affinity of binding of
either cadmium or estradiol to the ER. Scatchard analysis also showed
that ER-
contained approximately 1.1 cadmium-binding site.
Cadmium Binding to and Activation of ER Mutants
Cadmium is a transition metal that is capable of forming a
coordination complex either directly or indirectly with many different
amino acids including cysteines. The hormone-binding domain of the ER
contains four cysteines at positions C381, C417, C447, and C530. To
test the role of these cysteines in the interaction with cadmium, each
cysteine was mutated to alanine (14). The cysteine mutants (C381A,
C417A, C447A, and C530A), as well as the quadruple mutant
(C381A-C417A-C447A-C530A), were then transiently cotransfected with an
estrogen-responsive CAT construct into COS-1 cells, and the cells were
treated with either 10-6 M cadmium
chloride or 10-9 M estradiol. The
amount of CAT activity was measured, expressed as percent conversion,
and normalized to the amount of ß-galactosidase activity (Fig. 6a
). After treatment of the cysteine
mutants with cadmium, there was an approximately 4-fold increase in CAT
activity of C417A and an approximately 8-fold increase with C530A. In
contrast to the effects observed with mutants C417A and C530A, cadmium
failed to activate the mutants C381A and C447A and the quadruple mutant
C381A-C417A-C447A-C530A, suggesting that cysteines C381 and
C447 are involved in cadmium activation of the ER. To demonstrate that
the mutation of cysteine to alanine did not interfere with the activity
of the receptor, the transiently transfected cells were treated with
estradiol. After hormone treatment, there was an approximately 4- to
5-fold increase in CAT activity with all of the mutants. These results
corroborate previous studies employing these mutants, which demonstrate
that mutation of cysteines to alanine in the hormone-binding domain
does not alter the ability of estradiol to transactivate the receptor.
To determine whether the cysteine mutants were capable of binding
cadmium, the mutants were expressed in COS-1 cells, and specific
binding of 109Cd was determined using a
whole-cell binding assay (Table 2
). The
dissociation constants for binding of cadmium to the cysteine
mutants C381A and C447A were 5 x 10- 9
M and 3 x10-9 M,
respectively.

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Figure 6. The Ability of Cadmium to Activate Wild-Type and
Mutants of ER-
A, Wild-type ER- , the cysteine mutants C381A, C417A, C447A, C530A,
and the quadruple mutant C381A C417A C447A C530A were transiently
cotransfected with an estrogen-responsive CAT construct into COS-1
cells. The cells were treated with either 10-9
M estradiol or 10-6 M cadmium
chloride. CAT activity was measured as described in Materials
and Methods. The results were normalized to B-galactosidase
activity and expressed as percent control of wild-type ER- . Data
represent the mean value of three experiments ± SD.
B, Wild-type ER- and ER- mutants E390Q, E523Q, E523A, H524A, and
D538N were transiently cotransfected with an estrogen-responsive CAT
construct into COS-1 cells, and the cells were treated as described
above. Results represent the mean value of three experiments ±
SD. , Control;
, estradiol; ,
cadmium.
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In addition to cysteines, cadmium interacts with other amino acids
including histidine, glutamic acid, and aspartic acid. To identify
other possible metal interaction sites in the hormone-binding domain,
glutamic acids E380 and E523, histidine H524, and aspartic acid D538
were mutated (15, 16, 17). The ability of cadmium and estradiol to activate
these mutants was also tested in transiently transfected COS-1 cells
(Fig. 6b
). Cadmium did not activate mutants E523Q, E523A, H524A, or
D538N but activated E380Q, resulting in a 4.5-fold increase in CAT
activity; this suggests that glutamic acid E523, aspartic acid D538,
and histidine H524 may also play a role in the interaction of cadmium
with the ER. As expected, estradiol treatment of all four mutants
resulted in an approximately 4- to 5-fold increase in CAT activity,
suggesting that mutation of these amino acids did not interfere with
the activity of the ER. The ability of cadmium and estradiol to bind to
these mutants was also determined using a whole-cell binding assay
(Table 2
). In contrast to the cysteine mutants, there was no detectable
binding of cadmium to mutants E523Q, H524A, and D538N. However, the
latter three mutants were capable of binding estradiol with high
affinity but at a lower affinity than the wild-type receptor.
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DISCUSSION
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A previously published study from this laboratory showed that the
heavy metal cadmium mimicked many of the biological effects of
estradiol in breast cancer cells (10). In this study, we provide
evidence that activation of the ER occurs as a result of a
high-affinity interaction of the metal with the hormone-binding domain
of the receptor. The results of the present study demonstrate that
cadmium activates a chimeric receptor containing the DNA-binding domain
of the yeast transcription factor GAL4 and the hormone-binding domain
of ER-
. The ability of cadmium to activate the chimeric receptor is
blocked by an antiestrogen. Interaction of the metal with the receptor
also inhibits the binding of estradiol to the receptor. Mutational
analysis identified several amino acids as potential metal interaction
sites, suggesting that cadmium activates ER-
through the formation
of a coordination complex within the hormone-binding domain of the
receptor.
The ER is a member of a superfamily of transcription-regulating
proteins that bind zinc. The binding of zinc to cysteines in the
DNA-binding domain of ER-
results in the formation of a protein
motif referred to as a zinc finger (18). Other metals have been shown
to substitute for zinc in the zinc finger of ER-
and to influence
the binding of the DNA-binding domain to an estrogen response element
(19). The replacement of zinc with either nickel or copper inhibits the
binding of the DNA-binding domain to an estrogen response element,
whereas replacement of zinc with either cadmium or cobalt has no effect
on specific DNA binding. In addition to the DNA-binding domain, metals
have been shown to bind to the hormone-binding domain of receptors and
to block binding of the ligand. In the case of ER-
, calcium has been
shown to reversibly block the binding of estradiol (20). Interaction of
arsenite, cadmium, and selenite with cysteines in the hormone-binding
domain of the GR has also been shown to inhibit the binding of
dexamethasone to the receptor (13). Results presented in this study
suggest that the interaction of cadmium with ER-
is similar to the
interaction of the metal with the GR, i.e. cadmium binds
with high affinity to the hormone-binding domain of ER-
and blocks
the binding of estradiol. Interaction of cadmium with the ER also
appears to involve cysteines, specifically cysteines 381 and 447. In
addition to cysteines, the metal appears to interact with glutamic acid
523, histidine 524, and aspartic acid 538. Although the interaction of
cadmium with the ER results in activation of the receptor, this does
not appear to be the case for the GR.
Metals serve several different functions in proteins including
participation in catalytic reactions and stabilization of protein
structure. Through interactions with different amino acids, metals may
promote local folding, as in the case of the zinc finger, or assembly
of different regions of the protein into one domain (21). Cadmium is a
heavy metal that is capable of forming a coordination complex with a
number of different amino acids (reviewed in Ref. 22). It is found in
both tetrahedral and octahedral arrangements with the side chains of
cysteine, aspartic acid, histidine, and glutamic acid, and, to a lesser
extent, with serine. In addition to amino acid side chains, cadmium
interacts with the terminal nitrogen of serine, the peptide oxygen of
phenylalanine, lysine, arginine, asparagine, histidine, glutamine, and
water. Mutational analysis of ER-
revealed several amino acids in
the hormone-binding domain (C381, C447, E523, H524, and D538) as
potential sites of interaction with the metal. The exact role of these
amino acids in interaction and activation of the ER by cadmium remains
unknown. It is possible that these amino acids participate directly in
the formation of the metal-binding site or indirectly in the
recruitment of cadmium to the binding site. However, the latter appears
unlikely. If the function of these amino acids is to recruit the metal
to its binding site, then mutation of any one of these amino acids
would not alter cadmium binding and activation of the receptor.
Mutation of either E523, H524, or D538 resulted in the complete loss of
binding and activation, suggesting that these amino acids play a more
integral role in the interaction and activation of the receptor than
recruitment. When the cysteines were mutated to alanines, the ability
of cadmium to bind to the receptor was not altered, but the ability of
cadmium to transactivate the receptor was lost, suggesting that the
interaction of the metal with these cysteines influences the formation
of an active conformation of the receptor.
Similar to other receptors (23, 24, 25, 26, 27, 28, 29), the hormone-binding domain of the
ER contains 12
-helices (H1H12) folded into a three-layered
antiparallel
-helical sandwich. The central core layer contains
three
- helices (H5/6, H9, and H10) sandwiched between two
additional layers of helices composed of H14, H7, H8, and H11. The
central core of the hormone-binding domain is flanked by helix H12. It
has been proposed that the hormone-binding domain functions in a manner
similar to a mouse trap (23). Upon binding, the ligand induces a
conformational change resulting in the formation of a salt bridge
between H4 and H12, repositioning helix H12 over the central core and
consequently entrapping the hormone. Ultimately, the repositioning of
helix H12 results in the formation of a transcriptionally active
receptor. The amino acids identified as playing a role in the
interaction of cadmium with ER-
are located on helices H4, H8, and
H11 and at the interface of the loop and H12. Cysteines 381 and 447 are
located on helices H4 and H8, respectively. Glutamic acid 523 and
histidine 524 are located on helix H11 and are in close proximity to
the ligand. Aspartic acid 538 is located at the loop-H12 interface and
is also reasonably close to the ligand. It is possible that interaction
of cadmium with these amino acids brings different helices of the
hormone- binding domain into close proximity. The interaction of
cadmium with these amino acids may mimic the effects of estradiol by
repositioning H12 similar to the repositioning observed upon hormone
binding. This model remains to be tested. It also remains to be
determined whether cadmium interacts directly or indirectly with these
amino acids through water molecules.
Cadmium is a heavy metal with no known physiological function. Human
exposure to the metal occurs primarily through dietary sources,
cigarette smoking, and, to a lesser degree, drinking water (30, 31). In
newborns, the amount of cadmium found in the body is negligible, but by
age 30, the body burden may reach 30 mg. Cadmium has a biological
half-life estimated to range from 10 to 30 yr (32), which may account
for the significant accumulation of the metal in the body. In
nonsmokers, the kidney concentration of cadmium is approximately 1520
µg/g wet weight, while in smokers, the concentration doubles to
3040 µg/g wet weight. Interestingly, the human mammary gland, an
estrogen target organ, also contains high concentrations of cadmium (31
µg/g) (33) suggesting that the metal may be a potential risk factor
for breast cancer.
In this study, we provide evidence that the heavy metal cadmium is an
environmental estrogen. At low concentrations, the metal mimics the
effects of estradiol in transient transfection assays. It binds with
high affinity to the hormone-binding domain of ER-
and blocks the
binding of estradiol to the receptor. Binding to the ER appears to
involve several amino acids, suggesting that cadmium activates the
receptor through the formation of a coordination complex with specific
residues in the hormone-binding domain.
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MATERIALS AND METHODS
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Transient Transfection Assays
A low-temperature and low-pH calcium phosphate method was
employed to transfect COS-1 cells (34). COS-1 cells were plated at a
density of 3 x 106 cells per 150-mm dish in
phenol red-free improved MEM (IMEM) containing 10%
charcoal-treated calf serum (CCS) for 24 h. The cells were
transfected with 120 µg of DNA containing 15 µg of an ER-
expression vector (wild type or mutant, as described below), 75 µg of
a reporter construct, 6 µg of ß-galactosidase, and salmon sperm
carrier DNA. Sixteen to 18 h after transfection, the precipitate
was washed off, and the cells were replenished with phenol red-free
IMEM containing 10% CCS in the presence or absence of
10-9 M estradiol or
10-6 M cadmium chloride. The cells
were harvested 24 h later and CAT activity was measured as
described previously (10). CAT activity was expressed as the percent
conversion of chloramphenicol to its acetylated forms and was
normalized to the activity of ß-galactosidase. The increase in CAT
activity in response to treatment is expressed relative to untreated
controls. Statistical significance was determined by Students
t test.
Expression vectors for the wild-type ER-
(pRER-wt) and the amino
acid mutants (C381A, C417A, C447A, C530A, E523A, D538N, H524A) are
described elsewhere (14, 15, 16, 17). For these transient transfection assays,
the estrogen-responsive reporter construct, pbCAT-(S)MERE (11), was
obtained from Dr. D. El Ashry (Lombardi Cancer Center, Georgetown
University, Washington, DC). The chimeric receptors, GAL-ER and GAL-GR,
and the reporter plasmid 17M2GCAT are also described elsewhere
(12).
ER Binding Assays
The ability of cadmium to block estradiol binding to the ER-
was determined in cell extracts from MCF-7 cells that were maintained
in phenol red-free IMEM containing 5% CCS for 2 days. After 2 days in
estrogen-depleted medium, the cells were lysed by sonication in a
high-salt buffer containing 10 mM Tris, pH 7.5, 1.5
mM EDTA, 5 mM sodium molybdate, 0.4
M KCl, 1 mM monothioglycerol, 2 mM
leupeptin. The homogenate was incubated on ice for 30 min and
centrifuged at 100,000 x g for 1 h at 4 C (35).
The protein concentration of the cell extract was determined by the
Bradford method. Unless indicated otherwise, cell extracts were
preincubated on ice for 60 min with various concentrations of
CdCl2 (10-12 to
10-5 M).
[3H]Estradiol, 10-8
M, was then added in the presence and absence of
a 200-fold molar excess of diethylstilbestrol (DES) and incubated at 4
C for 1618 h. Free steroid was removed by the addition of 5%
dextran-coated charcoal. The amount of radioactivity was measured by
scintillation counting. Specifically bound complexes were calculated by
subtracting nonspecific binding from total binding.
The ability of cadmium to bind to the ER was determined using
recombinant human ER-
(PanVera Corp., Madison, WI) and MCF-7 cell
extracts. Recombinant ER-
(4 x 10- 9
M) was incubated with various concentrations of
109Cd (10-12 to
10-7 M, specific activity 90
µCi/µg cadmium chloride, Amersham Pharmacia Biotech,
Piscataway, NJ) in the presence and absence of a 200-fold molar excess
of CdCl2 for 1618 h at 4 C. Free radioactivity
was removed by adding 5% dextran-coated charcoal. In the whole-cell
binding assay, 5 x 105 MCF-7 cells were
plated into six-well dishes in IMEM containing 5% CCS. The medium was
subsequently replaced with phenol red-free IMEM containing 5% CCS.
After 2 days in estrogen-depleted medium, the cells were incubated for
1618 h at 4 C with various concentrations of
109Cd (10-12 to
10-7 M) in the presence and absence
of a 200-fold molar excess of CdCl2. Free
radioactivity was removed by washing. The cells were disrupted by four
freeze-thaw cycles, and the amount of 109Cd bound
to the ER was measured by scintillation counting. The data were
analyzed by the method of Scatchard (36). To determine whether cadmium
alters the binding affinity of estradiol for the ER, recombinant human
ER-
was incubated with 10-10,
10-9, or 10-8
M cadmium and various concentrations of
[3H] estradiol (10-12 to
10-7 M) in the presence or absence
of a 200-fold molar excess of DES. The assay conditions and data
analysis are described above.
To measure the binding of cadmium and estradiol to ER mutants, COS-1
cells were plated into six-well dishes and transiently transfected with
either wild-type or mutant ER-
. The transfected cells were incubated
at 4 C for 16 h with various concentrations of either
109Cd (10-12 to
10-7 M) or
[3H]estradiol (10-12 to
10-7 M) in the presence or absence
of a 200-fold molar excess of CdCl2 or DES,
respectively. Free radioactivity was removed by aspiration, and the
cells were disrupted by four freeze-thaw cycles. The amount of bound
radioactivity was quantitated by scintillation spectrophotometry, and
the data were analyzed as described above.
 |
ACKNOWLEDGMENTS
|
---|
We thank Prof. P. Chambon for providing estrogen receptor
mutants, Dr. M. E. Lippman for helpful discussions, and Drs. E.
Davidson, J. Katzenellenbogen, M. Danielsen, and B. Wolfe for critical
reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Mary Beth Martin, Lombardi Cancer Center, E411 Research Building, 3970 Reservoir Road, NW, Washington, DC 20007.
This work was supported by NIH Grant CA-70708 (to M.B.M.), Cancer
Research Foundation of America (to A.S.), The Susan G. Komen Foundation
(to A.S.), and NIH Grant CA-18119 (to B.S.K.).
Received for publication July 13, 1999.
Revision received December 28, 1999.
Accepted for publication January 5, 2000.
 |
REFERENCES
|
---|
-
American Cancer Society 1993 Cancer facts and figures
1993. American Cancer Society, Atlanta, GA
-
Lippman ME 1985 Endocrine responsive cancers of man. In:
Williams RH (ed) Textbook of Endocrinology. WB Saunders, Philadelphia,
pp 13091326
-
Kampert JB, Whittemore AS, Paffenbarger Jr RS 1988 Combined
effect of child-bearing, menstrual events, and body size on
age-specific breast cancer risk. Am J Epidemiol 128:962979[Abstract]
-
Pike MC, Henderson BE, Casagrande JT, Rosario I, Gray GE 1981 Oral contraceptive use and early abortion as risk factors for breast
cancer in young women. Br J Cancer 43:72[Medline]
-
Henderson BE, Ross RK, Judd HL, Krailo MD, Pike MC 1985 Do
regular ovulatory cycles increase breast cancer risk? Cancer 56:1206[Medline]
-
Trichopoulos D, MacMahon B, Cole P 1972 Menopause and breast
cancer risk. J Natl Cancer Inst 48:605613[Medline]
-
Pike MC, Spicer DV, Dahmoush L, Press MF 1993 Estrogens,
progestogens, normal breast cell proliferation, and breast cancer risk.
Epidemiol Rev 15:1735[Medline]
-
King RJB 1993 Estrogen and progestin effects in human breast
carcinogenesis. Breast Cancer Res Treat 27:315[Medline]
-
Freiss G, Prebois C, Vignon F 1993 Control of breast cancer
cell growth by steroids and growth factors: interactions and
mechanisms. Breast Cancer Res Treat 27:5768[Medline]
-
Garcia-Morales P, Saceda M, Kenney N, Kim N, Salomon DS,
Gottardis MM, Solomon HB, Sholler PF, Jordan VC, Martin MB 1994 Effect
of cadmium on estrogen receptor levels and estrogen-induced responses
in human breast cancer cells. J Biol Chem 269:1689616901[Abstract/Free Full Text]
-
El-Ashry D, Chrysogelos SA, Lippman ME, Kern FG 1996 Estrogen
induction of TGF-
is mediated by an estrogen response element
composed of two imperfect palindromes. J Steroid Biochem Mol Biol 59:261269[CrossRef][Medline]
-
Webster NJG, Green S, Jin JR, Chambon P 1988 The
hormone-binding domains of the estrogen and glucocorticoid receptors
contain an inducible transcriptional activation function. Cell 54:199207[Medline]
-
Simons Jr SS, Chakraborti PK, Cavanaugh AH 1990 Arsenite and
cadmium(II) as probes of glucocorticoid receptor structure and
function. J Biol Chem 265:19381945[Abstract/Free Full Text]
-
Reese JC, Katzenellenbogen BS 1991 Mutagenesis of cysteines in
the hormone binding domain of the human estrogen receptor. J Biol
Chem 266:1088010887[Abstract/Free Full Text]
-
Wrenn CK, Katzenellenbogen BS 1993 Structure-function analysis
of the hormone binding domain of the human estrogen receptor by
region-specific mutagenesis and phenotypic screening in yeast. J
Biol Chem 268:2408924098[Abstract/Free Full Text]
-
Pakdel F, Katzenellenbogen BS 1992 Human estrogen receptor
mutants with altered estrogen and antiestrogen ligand discrimination.
J Biol Chem 267:34293437[Abstract/Free Full Text]
-
Pakdel F, Reese JC, Katzenellenbogen BS 1993 Identification of
charged residues in an N-terminal portion of the hormone-binding domain
of the human estrogen receptor important in transcriptional activity of
the receptor. Mol Endocrinol 7:14081417[Abstract]
-
Danielsen M 1991 Structure and Function of the Glucocorticoid
Receptor. Academic Press Ltd, London, pp 3978
-
Predki PF, Sarkar B 1992 Effect of replacement of "zinc
fingers" zinc on estrogen receptor DNA interactions. J Biol Chem 267:58425846[Abstract/Free Full Text]
-
Maaroufi Y, Hardouze AB, LeClercq G 1997 Decrease of hormone
binding capacity of estrogen receptor by calcium. J Recept Signal
Transduction Res 17:833853[Medline]
-
Hughes MN 1981 The Inorganic Chemistry Of Biological
Processes, ed 2. Wiley, Chichester, UK
-
Rulisek L, Vondrasek J 1998 Coordination geometries of
selected transition metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+)
in metalloproteins. J Inorg Biochem 71:115127[CrossRef][Medline]
-
Wurta JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D,
Gronemeyer H 1996 A canonical structure for the ligand-binding domain
of nuclear receptors. Nat Struct Biol 3:8794[Medline]
-
Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H,
Moras D 1996 Crystal structure of the RAR-
ligand-binding domain
bound to all-trans retinoic acid. Nature 378:681689[CrossRef]
-
Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear
receptor RXR-
. Nature 375:377382[CrossRef][Medline]
-
Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T,
Engstrom O, Ohman L, Greene GL, Gustafsson J, Carlquist M 1997 Molecular basis of agonism and antagonism in the estrogen receptor.
Nature 389:753758[CrossRef][Medline]
-
Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD,
Fletterick RJ 1995 A structural role for hormone in the thyroid hormone
receptor. Nature 378:690697[CrossRef][Medline]
-
Tanenbaum DM, Wang Y, Williams SP, Sigler PB 1998 Crystallographic comparison of the estrogen and progesterone
receptors ligand binding domain. Proc Natl Acad Sci USA 95:59986003[Abstract/Free Full Text]
-
Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA,
Greene GL 1998 The structural basis of estrogen receptor/coactivator
recognition and the antagonism of this interaction by tamoxifen. Cell 95:927937[Medline]
-
Gartell MJ, Craun JC, Podrebarae DS, Gunderson ER 1986 Pesticides, selected elements and other chemicals in infant and toddler
total diet samples. October 1980March 1982. J Assoc Off Anal Chem 69:123145[Medline]
-
Gartell MJ, Craun JC, Podrebarae DS, Gunderson ER 1986 Pesticides, selected elements and other chemicals in adult total diet
samples. October 1980March 1982. J Assoc Off Anal Chem 69:146161[Medline]
-
International Agency for Research on Cancer 1976 IARC
Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man
(Cadmium, Nickel, Some Expoxides, Miscellaneous Industrial Chemicals,
General Considerations on volatile Anaesthetics), ed 11. World Health
Organization pp 4864
-
Antila E, Mussalo-Rauhamaa H, Kantola M, Atroshi F,
Westermarck T 1996 Association of cadmium with human breast cancer. Sci
Total Environ 186:251256[CrossRef][Medline]
-
Chen C, Okayama H 1987 High-efficiency transformation of
mammalian cells by plasmid DNA. Mol Cell Biol 7:27452752[Medline]
-
Stoica A, Saceda M, Fakhro A, Solomon HB, Fenster BD, Martin
MB 1997 The role of transforming growth factor-B in regulation of
estrogen receptor expression in the MCF-7 breast cancer cell line.
Endocrinology 138:14981505[Abstract/Free Full Text]
-
Scatchard G 1949 The attractions of protein for small
molecules and ions. Ann NY Acad Sci 51:660672