From the Laboratories of Reproductive and
Developmental Toxicology and ¶ Structural Biology, NIEHS,
National Institutes of Health, Research Triangle Park, North Carolina
27709
Received for publication, April 14, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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The two known estrogen receptors,
ER The estrogenic effects of a variety of structurally diverse
endogenous and xenobiotic compounds are mediated through the estrogen receptors (ER Diethylstilbestrol (DES) is a known carcinogen, which is oxidatively
metabolized to a variety of metabolites with varying degrees of
hormonal activity (34). Indenestrol A (IA) is a metabolite derived from
DES that has high binding affinity for ER The discovery of ER To expand our knowledge about the molecular determinants of the
stereoselectivity of ER Materials and Biochemicals--
Media, serum, supplements,
enzymes, and chemicals were purchased from Sigma.
Cell Culture--
Ishikawa and MDA-MB 231 (MDA) were cultured in
Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10 and 5% fetal bovine serum (FBS), respectively, and
penicillin/streptomycin. For MDA, 5 µg/ml insulin was added to the
culture medium. Human hepatoma HepG2 were maintained in minimum
essential medium (MEM) supplemented with 10% FBS, 1 mM
sodium pyruvate, 2 mM glutamine, and 0.1 mM
non-essential amino acids in flasks coated with 0.1% gelatin. Cells
were cultured at 37 °C in a 5% CO2-humidified atmosphere.
Expression Vectors and Reporter Constructs--
For the
generation of cell lines stably expressing mouse (m) or human (h) ER Stable Transfection of Human Cells with the cDNAs of
h/mER RNase Protection Assay (RPA)--
For the generation of
riboprobe templates for hER Analysis of ER Protein Expression Using Western
Blotting--
Nuclear extracts were prepared as described previously
with modifications (9, 10). Aliquots containing 100 µg of protein were analyzed on 10% Tris-glycine polyacrylamide gels (NOVEX, San
Diego, CA) and electroblotted onto nitrocellulose membranes (Hybond-ECL, Amersham Biosciences) as described previously (8). The
membrane was probed with either 1 µg/ml H222 ER Transient Transfection and Transactivation Assay--
Cells were
seeded on 24-well plates 15 h prior to transfection in phenol
red-free DMEM/F12 supplemented with 5% dextran-coated charcoal
stripped FBS (DCC/FBS). The reporter plasmids were transfected in
DMEM/F12 supplemented with 1% DCC/FBS using FuGENE 6 according to the
manufacturer's protocol. Each well received 0.5 µg of reporter plasmid and 0.01 µg pRL-CMV (Renilla luciferase for
normalization; Promega, Madison, WI). A firefly luciferase reporter
driven by three copies of the vitellogenin estrogen response element
(3×ERE-Luc) and a reporter containing the human complement 3 gene (C3)
promoter (C3-Luc; kindly provided by D. McDonnell, Duke University,
Durham, NC) were used to measure ER transcriptional activity (11). For cotransfection of wild-type or mutated ER with the luciferase reporter
vectors, 0.09 µg of receptor plasmid, 0.4 µg of reporter, and 0.01 µg of Renilla luciferase normalization vector were used per well. After transfection, cells were incubated in medium
supplemented with 5% DCC/FBS as described above with DES,
17 Mammalian Two-hybrid Assays--
For mammalian two-hybrid
assays, HepG2 (human hepatoma) cells were plated in 24-well plates
(coated with 0.1% gelatin) 24 h prior to transfection. DNA was
introduced into cells using FuGENE 6. In standard transfections, 0.5 µg of reporter 5×-Gal4-TATA-Luc, containing 5 binding sites for the
yeast Gal4 transcription factor, 0.09 µg of receptor (either
pVP16-hER Ligand Binding Studies--
Ligand binding was analyzed by
competition of the test compound against 1 nM
17 Site-directed Mutagenesis--
The exchange of one amino acid in
the LBD of hER Statistical Analysis--
Statistical analyses were performed
using Student's t test (unpaired, one-tailed). Statistical
significance is indicated by asterisk (p Generation of Human Cell Lines with Stable Expression of ER IA-R and IA-S Show Distinct Abilities to Activate ER IA-R Has a Low Binding Affinity to ER Mutagenesis Studies Reveal That One Residue in the LBD Is Critical
for the Stereoselective Ligand Activation of ER
To determine whether this one residue might be responsible for
differences in the IA-R activation profile of ER
We then tested the ability of DES, E2, IA-S, and
IA-R to activate wild-type and mutated ER Mutagenesis of the Mismatched Residue in the hER Previously, our laboratory and others (5, 22-25) determined that
ER We showed in an earlier report that IA-S is a strong ER However, although both IA enantiomers function as agonists of both ER
subtypes, each ligand differed markedly with regard to its potency as
an activator of ER The fact that IA-R is a weak agonist for ER Recent studies analyzed the conformational changes that occur in ER We performed studies to test this hypothesis through experiments
pinpointing the molecular determinants responsible for the differences
in transactivation between ER Several studies have analyzed the molecular determinants of
ligand-dependent activation of ER In conclusion, this series of studies has revealed that IA-R
is a potency-selective agonist for ER and ER
, are hormone-inducible transcription factors that have
distinct roles in regulating cell proliferation and differentiation.
Previously, our laboratory demonstrated that ER
exhibits
stereoselective ligand binding and transactivation for several
structural derivatives and metabolites of the synthetic estrogen
diethylstilbestrol. We have previously described the properties of
indenestrol A (IA) enantiomers on ER
. In the study presented here,
the estrogenic properties of the S and R
enantiomers of IA, IA-S and IA-R,
respectively, were expanded to examine the activity in different cell
and promoter contexts using ER
and ER
. Using human cell lines
stably expressing either ER
or ER
, we found that IA-S
was a more potent activator of transcription than IA-R
through ER
in human endometrial Ishikawa and breast MDA-MB 231 (MDA)
cells. Interestingly, IA-R was more potent on ER
when
compared with ER
in MDA, but not in Ishikawa cells, and
IA-R exhibited equally low binding affinities to ER
and
ER
in vitro in contrast to its cell
line-dependent preferential activation of ER
. Alignment
of the protein structures of the ligand-binding domains of ER
and
ER
revealed one mismatched residue, Leu-384 in ER
and Met-283 in
ER
, which may be responsible for making contact with the methyl
substituent at the chiral carbon of IA-S and
IA-R. Mutagenesis and exchange of this one residue showed
that the binding of IA-R and IA-S was not
affected by this mutation in ER
and ER
. However, in
transactivation studies, IA-R showed higher potency in
activating L384M-mutated ER
and wild-type ER
compared with
wild-type ER
and M283L-mutated ER
in all cell and promoter
contexts examined. Furthermore, IA-R-bound ER
L384M and wild-type ER
displayed enhanced interactions with the
nuclear receptor interaction domains of the coactivators SRC-1 and
GRIP1. These data demonstrate that a single residue in the ligand-binding domain determines the stereoselectivity of ER
and
ER
for indenestrol ligands and that IA-R shows cell type selectivity through ER
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ER
),1
which function as ligand-inducible transcription factors for genes
involved in cell growth, proliferation, and differentiation (1).
Studies using knock-out mice for the two ER subtypes (
ERKO and
ERKO, respectively) have revealed that each receptor plays a unique
role in estrogen biology in a wide variety of target tissues (2).
Furthermore, in vitro studies indicated that ER
and ER
display marked differences in binding affinity and activation by
natural and synthetic ER ligands (3, 4). Interestingly, although ER
shows lower binding affinity for and activation by endogenous
estrogens, several xenoestrogens preferentially bind and activate ER
(3). These observations have prompted further studies aimed at
elucidating the molecular mechanisms of action of ER
and ER
, and
the search for ER subtype-selective agonists that could be used to
evaluate the physiological roles of each receptor.
but weak biological
activity (35). IA exists as a racemic mixture of enantiomers,
IA-S and IA-R (36), which have a methyl
substitution on the chiral carbon (Fig. 1). Previously, our laboratory
demonstrated that IA-R displays a lower binding affinity and
transactivation potency on ER
than IA-S (5), which may
help explain the differential biological activity and allow the study
of the stereoselectivity of ligand binding and transcriptional
responses of ERs.
prompted us to investigate whether both ER
subtypes would exhibit the same stereoselectivity for the IA
enantiomers. Human ER
and ER
share high sequence homology (97%)
in the central DNA-binding domain, but only moderate conservation (60%) in the C-terminal ligand-binding domain (LBD) and activation function-2 (AF-2). Since AF-2 mediates the ligand-dependent
transcriptional activities of both ER subtypes (1), it is possible that
structural differences within this region could account for differences
in the specificities of ER
and ER
for several estrogenic
chemicals (4, 6).
and ER
, we have characterized the ability
of IA-S and IA-R to function as
agonists/antagonists of the two ER subtypes. For the study of ER
subtype-specific activation, stable cell lines were established by
transfecting MDA-MB 231 (human breast adenocarcinoma; MDA) and Ishikawa
(human uterine adenocarcinoma) cells with human or mouse ER
or
ER
. Using these cell models and in vitro binding assays,
the ability of the IA enantiomers to bind ER
and ER
, activate
ERE-mediated transcription, and regulate coactivator recruitment was
studied and compared between the two ER subtypes. Alignment of the LBDs
of ER
and ER
revealed one mismatched amino acid (Leu-384 and
Met-283, respectively) that may be responsible for making contact with
the methyl substituent at the chiral carbon of IA-S and
IA-R. This observation suggested that this residue could
account for the functional differences observed between the two ER
subtypes, a hypothesis that was further investigated in this study.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or ER
, the cDNAs of mER
(GenBankTM accession
M38651), mER
(U81451, Ref. 7), hER
(M12674, cDNA kindly
provided by D. McDonnell, Duke University, Durham, NC) or hER
(AF051427, cDNA kindly provided by D. McDonnell, Duke University,
Durham, NC) were subcloned into the mammalian expression plasmid
pcDNA3 (Invitrogen, San Diego, CA) using standard cloning
procedures. The pcDNA3 vector carries the neomycin resistance gene
that allows selection of transfected cells with geneticin (G418).
/
--
MDA and Ishikawa cells were
seeded in 6-well plates for stable transfection. Transfection of cells
was performed with the FuGENE 6 reagent according to the
manufacturer's protocol with 0.5 µg of DNA and 1 µg of FuGENE 6 (Roche Diagnostics Corp.) per well in DMEM/F12 supplemented with 1%
FBS. Three days after transfection with the pcDNA3 vector
containing either the mER
, mER
, hER
, or hER
cDNA, cells
were trypsinized and seeded in 60-mm tissue culture plates. Stable
clones were selected by adding 200-400 µg/ml G418 to the culture
medium. After 4 weeks of selection, 10-20 clones each for mER
,
mER
, hER
, or hER
in MDA and Ishikawa were isolated and
cultured separately in medium with 100 µg/ml G418. Control cell lines
were established by transfecting parental MDA and Ishikawa lines with
the empty pcDNA3 vector.
, a fragment corresponding to base pairs
(bp) 1785-2092 of the cDNA and for hER
, a fragment
corresponding to bp 1820-2011 of the cDNA were subcloned into
pDP18 (Ambion, Austin, TX), respectively. A human cyclophilin probe
(Ambion) was used as an internal control. Antisense riboprobes were
generated from the linearized templates using the Maxiscript kit
(Ambion) and [32P]CTP (Amersham Biosciences). RPAs were
performed using the HybSpeed kit from Ambion according to the
manufacturer's instructions (Ambion). 10 µg of total RNA of each
sample, isolated with the Trizol reagent according to the
manufacturer's protocol (Invitrogen), was analyzed. 5 µg of RNA
isolated from untreated MCF-7 (human breast carcinoma) cells and BG-1
(ovarian carcinoma) cells were used as positive controls for hER
and
hER
, respectively. RPAs were performed exactly as described
previously (8).
antibody (kindly
provided by G. Greene, University of Chicago, Chicago, IL) or 2 µg/ml
anti-ER
antibody (06-629, Upstate Biotechnology, Lake Placid, NY).
Immunocomplexes were detected with a horseradish peroxidase-conjugated
anti-rat (ER
) or anti-rabbit (ER
) IgG secondary antibody at 1:300
dilution (Oncogene, Cambridge, MA) and an ECL chemiluminescence kit
(Amersham Biosciences).
-estradiol, IA-S, or IA-R (final
concentration of vehicle ethanol 1%, v/v) for 20 h. Luciferase
assays were performed using the Dual-Luciferase reporter assay system
according to the manufacturer's protocols (Promega). Each value was
normalized to the Renilla luciferase control, and each data
point generated is the average of duplicate determinations. All
experiments were repeated at least three times with consistent results.
, pVP16-hER
L384M, pVP16-hER
, or pVP16-hER
M283L),
0.5 µg of Gal4DBD-coactivator fusion (12, 13) (pM-SRC-1 (NR-box) or
pM-GRIP1 (NR-box); plasmids kindly provided by D. McDonnell, Duke
University, Durham, NC), and 0.01 µg of the pRL-CMV
Renilla luciferase normalization vector were used for each
well. All transfections were performed in triplicate. Prior to
transfection, cells were washed with phosphate-buffered saline, and 200 µl of phenol red-free MEM containing 5% DCC/FBS was added to each
well. Cells were incubated with the DNA/FuGENE 6 mix for 6 h;
receptor ligands in 200 µl of phenol red-free MEM were then added to
the cells and incubated for 20 h. Luciferase assays were performed
using the Dual-Luciferase reporter assay system according to the
manufacturer's protocols. Each value was normalized to the
Renilla luciferase control, and each data point generated is
the average of triplicate determinations. All experiments were repeated
three times.
-[3H]estradiol (71 Ci/mmol, PerkinElmer Life
Sciences). Reactions were performed with 1 µl of in vitro
translated wild-type or mutated ER
and ER
, respectively, in 100 µl of TEGM buffer (10 mM TRIS, 1.5 mM EDTA, 3 mM EGTA, 3 mM MgCl2, 10%
glycerol). ERs were in vitro translated with the
TNT kit (Promega) using the pcDNA3 expression plasmids
according to the manufacturer's protocol. Samples were incubated for
15 h at 4 °C. Unbound 17
-[3H]estradiol was
removed by adding DCC (0.25% charcoal, 0.025% dextran). Samples were
spun at 3000 × g, and remaining radioactivity contained in the supernatant was measured in a scintillation counter. Nonspecific binding was measured in the presence of 500× excess of
unlabeled estradiol. Scatchard analyses were performed with 0.1-2.5
nM 17
-[3H]estradiol.
and hER
was accomplished using the QuikChange
site-directed mutagenesis kit according to the manufacturer's protocol
(Stratagene). The expression plasmids pcDNA3-hER
and
pcDNA3- hER
were used to generate point mutations in the LBD.
ER
L384 M was generated by exchanging the codon for leucine at
position 384 for a methionine. The primers used for mutagenesis were:
forward primer, 5'-CTAGAATGTGCCTGGATGGAGATCCTGATG (mutated
codon in bold) and reverse primer,
5'-CATCAGGATCTCCATCCAGGCACATTCTAG-3' (mutated codon in
bold). ER
M283L was generated by exchanging the codon for methionine
at position 283 for a leucine. The primers used for mutagenesis were:
forward primer, 5'-GGAGAGCTGTTGGCTAGAGGTGTTAATGATG-3' (mutated codon in bold) and reverse primer,
5'-CATCATTAACACCTCTAGCCAACAGCTCTCC-3' (mutated codon in
bold). The mutations in the plasmids used in the mammalian two-hybrid
assay, pVP16-hER
L384M and pVP16-hER
M283L were created exactly
as described above using the pVP16-hER
and pVP16-hER
plasmids
(kindly provided by D. McDonnell, Duke University, Durham, NC) as
templates for mutagenesis.
0.05).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
ER
--
ER
and ER
were introduced into the ER-negative human
mammary MDA and endometrial Ishikawa cell lines (14-16) to obtain
in vitro models to study the ligand-mediated transcriptional
responses of ER
and ER
. For the generation of ER subtype and
cell-specific models, each cell line was transfected with expression
plasmids containing either the murine (m) or human (h) ER
or ER
cDNAs. Clonal cell lines with stable and comparable expression of
the respective ER were established. The expression of ER
or ER
mRNA and protein in each cell line was confirmed by RNase
protection assay (RPA) and Western blotting. The mRNA expression
levels were between 1 and 4% of the expression of the housekeeping
gene cyclophilin for mouse and human ER
or ER
expressed in MDA
and Ishikawa cells (data not shown). The parental cell lines, as well
as control cell lines stably transfected with the empty pcDNA3
vector, lacked endogenous ER
and ER
mRNA expression (data not
shown). One cell clone for each cell line expressing functional mouse
or human ER
or ER
was selected by assessing its estrogen
responsiveness in transactivation assays using a reporter construct
that contained three copies of the vitellogenin consensus estrogen
response element (ERE, 3×ERE-Luc) as reporter (data not shown).
However, we were not able to establish a cell clone of the MDA line
with expression of functional hER
.
and ER
in Human Breast and Endometrial Cell Lines--
We first examined the
ability of DES, E2, and the IA enantiomers IA-R
and IA-S (Fig. 1) to activate
transcription through ER
and ER
in dose response experiments.
Transactivation studies were performed in human MDA breast and Ishikawa
endometrial cell lines using the 3×ERE-Luc (containing 3 copies of the
vitellogenin A2 ERE) and C3-Luc (containing the natural complement 3 (C3)) reporter constructs (11, 17). The parental MDA and Ishikawa cell
lines, as well as cells transfected with empty pcDNA3 vector, lacked any ligand-inducible transactivation of the 3×ERE or C3 reporters, confirming the absence of endogenous functional ER
and
ER
in these cell lines (data not shown). IA-S and
IA-R, like E2 and DES, were agonists of ER
and ER
in all cell and promoter contexts examined. In contrast to
the weak agonism of IA-R on ER
, this compound showed a
high potency to activate both murine and hER
in MDA cells (Table
I). In Ishikawa cells, IA-R was less
potent than IA-S in transactivation of ER
(EC50: 4.0-8.0 nM for IA-R versus
0.2-0.5 nM for IA-S; see Table
II), but showed a slight, statistically
significant preference for hER
over hER
on the 3×ERE reporter
(EC50: 4.0 ± 3.6 nM for ER
versus 9.3 ± 1.1 nM for ER
; see Table
II). Interestingly, IA-R showed no marked difference in
activation of ER
versus ER
in Ishikawa cells on the C3
promoter, whereas IA-R was a significantly more potent activator of
transcription through ER
compared with ER
on both the 3×ERE and
C3 reporters in MDA cells, indicating a degree of cell type specificity
influencing gene expression by the different ER subtypes (see Tables I
and II).
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Fig. 1.
Chemical structures of diethylstilbestrol
(DES), 17 -estradiol
(E2), and the indenestrol A (IA) enantiomers
IA-S and IA-R.
Potency of ER ligands in MDA-MB 213 (MDA) cells stably expressing
mER, mER
, or hER
Potency of ER ligands in Ishikawa cells stably expressing mER,
mER
, hER
, or hER
Although It Is a Potent
ER
Agonist--
The finding that IA-R was a less potent activator
of ER
-mediated transcription compared with IA-S in MDA
and Ishikawa cells (Tables I and II) confirmed data previously obtained
in a yeast-based ER system (5). In addition, IA-R was a more
potent activator of ER
in MDA and partly in Ishikawa cells compared
with ER
, which suggests that IA-R might also display a
higher binding affinity for ER
as compared with ER
. Competition
binding studies with in vitro transcribed and translated
hER
or hER
and 3H-labeled E2 confirmed
our previous report (5) that IA-R has a low binding affinity
for ER
(Table III). Inconsistent with
the transactivation data, however, IA-R also showed low
binding affinity for ER
, although both IA-S and
IA-R exhibited slightly higher binding affinities for ER
than for ER
(Table III).
Relative binding affinities (RBA) of ER ligands to ER and ER
and ER
--
The
differences in IA-R transactivation and binding of ER
as
compared with ER
prompted us to examine differences in the LBD of
the two ER subtypes that could contribute to the distinct pharmacology
of IA-R on ER
and ER
. The structures predicted by
x-ray crystallographic studies of the LBDs of ER
and ER
provide a
potential answer to this question (18, 19). A structural model of the
superimposition of the IA isomers with DES and genistein bound to the
LBD of ER
and ER
, respectively, predicts that several residues
within the LBD of ER
and ER
could make contact with the methyl
substituent at the chiral carbon of IA (Fig.
2) (18, 19). From these selected
residues, only leucine 384 (Leu-384) in ER
and the analogous
methionine 336 (Met-336) in ER
are mismatched based on the alignment
of the reported protein sequences of rodent and human ER
and ER
(18) (Fig. 2). The ER
cDNA used in this study represents the
short form of human ER
(GenBankTM accession no.
AF051427) and position 283 is identical to position 336 of the
full-length hER
cDNA used for the alignment in Fig. 2.
Furthermore, the complete sequence of the ligand-binding domain of the
"short" hER
is identical to the full-length hER
cDNA (20,
21). Notably, the residues Leu-384 and Met-283 are among those that may
be involved in contact with the IA ligand when bound to the LBD.
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Fig. 2.
Superimposition of IA-S
(navy) and IA-R (green) molecules
with genistein (red) bound to the LBD of
hER . Structural model of a part of the
aligned LBD of hER
(blue) and hER
(red) drawn according to data presented in Refs. 18 and 19
using Molscript with the coordinates from the Brookhaven protein data
bank (accession 2ERD and 2ERT). Superimposition of the ligands and
numbering of residues was done based on the crystal structural data of
genistein bound to the LBD of hER
(18).
compared with ER
, the mismatched residues in ER
and ER
were exchanged, i.e. Leu-384 in hER
was mutated to a methionine (ER
L384M) and Met-283 in hER
to a leucine (ER
M283L). We first
determined whether the resulting ER mutants bound the IA enantiomers
with different affinities than the wild-type ERs. Scatchard analysis of
competition binding studies with in vitro transcribed and
translated receptors revealed that wild-type and mutated ERs have
comparable binding constants (Table IV).
Using competition binding studies we then determined the affinities of
DES, E2, IA-S, and IA-R for wild-type
and mutated ER
and ER
(Table V).
Relative binding affinities (RBA) compared with DES (set to 100)
revealed no significant differences between the mutated and the
wild-type ERs (Table V, ER
versus ER
L384M and ER
versus ER
M283L) for any ligand tested, indicating
that the receptor binding affinity of the IA enantiomers was
not affected by these mutations.
Binding affinities of radioactively labeled 17-estradiol to
wild-type and mutated ER
and ER
RBA of investigated compounds to wild-type and mutated ER and ER
and ER
. MDA
and Ishikawa cells were transiently transfected with the cDNAs of
wild-type or mutated hER
or hER
. Transactivation assays were used
as described above to measure receptor
activity on the 3×ERE and the C3 promoters (Tables VI and
VII, Fig. 3). The cell
line-dependent activation profile of IA-R
observed in the stably ER expressing
cell lines (Tables I and II) was
confirmed by results obtained in cells transiently expressing ER
(Tables VI and VII). Fig. 3 shows the dose-response curves obtained on
the C3 promoter in MDA cells. A compilation of all data obtained from
transactivation assays performed in MDA and Ishikawa cells is given in
Tables VI and VII, respectively. Specifically, these data confirmed
that IA-R was a more potent activator of ER
than of ER
in MDA cells on both promoters examined (Table VI). However, in
Ishikawa cells IA-R was only slightly more potent in
activating ER
compared with ER
on the 3×ERE, but displayed no
difference in potency between ER
and ER
on the C3 promoter (Table
VII). Consistent with their binding affinities, E2, DES,
and IA-S showed no significant differences in their abilities to
activate wild-type or mutated ER
and ER
in MDA and Ishikawa cells
(Fig. 3 and Tables VI and VII). In contrast, the L384M mutation of
ER
rendered IA-R more active compared with wild-type
ER
(note the shift in the IA-R induced transcriptional
potency of wild-type ER
compared with mutated ER
in Fig.
3B). Consistent with this observation, the ER
mutant
M283L exhibited lower potency than wild-type ER
when treated with
IA-R (note the shift in the IA-R induced transcriptional activity of wild-type ER
compared with mutated ER
in Fig.
3D). Furthermore, these effects were statistically
significant in MDA cells on both promoters examined and in Ishikawa
cells on the 3×ERE reporter (Tables VI and VII). Also, IA-R
was a significantly less potent activator of the ER
mutant M283L
than wild-type ER
on the C3 promoter in Ishikawa cells. However,
although the L384M mutation of ER
rendered IA-R consistently more
potent compared with wild-type ER
in Ishikawa cells in all performed
experiments, this effect failed to reach statistical significance
(EC50: 1.7 ± 1.2 nM for ER
L384M
versus 5.0 ± 4.4 nM for ER
,
p = 0.1; see Table VII). The nature of these results
suggests that these alterations were not apparently dependent on the
gene regulatory sequence nor the cell type, but most likely a result of
an intramolecular effect on the ER protein.
Potency of ER ligands in MDA cells transiently transfected with
wild-type and mutated ER and ER
Potency of ER ligands in Ishikawa cells transiently transfected with
wild-type and mutated ER and ER
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Fig. 3.
Comparison of transactivation of wild-type
versus mutated ER in MDA cells. Cells were
transiently transfected with the cDNA of wild-type hER
(solid diamonds and squares), wild-type hER
(solid diamonds and squares), hER
L384M
mutants (open diamonds and squares), or hER
M283L mutants (open diamonds and squares)
together with the C3-Luc reporter and pRL-CMV Renilla
luciferase normalization vector. Transcriptional activity of wild-type
versus mutant ER
(A and B) or
wild-type versus mutant ER
(C and
D) was compared. Following transfection, cells were treated
with E2 or DES (A and C) or with the
IA enantiomers IA-S and IA-R (B and
D) at the indicated concentrations for 20 h. After
20 h, cells were harvested and dual luciferase assays were
performed. Each value was normalized to the internal luciferase
control. Note that IA-R (B and D) is a
more potent activator of mutated ER
and wild-type ER
compared
with wild-type ER
and mutated ER
. Shown is a representative
experiment, with each data point being the average of duplicate
determinations. Experiments were repeated at least three times with
consistent results.
and hER
LBD
Influences the Interaction of the IA-R-bound Receptors with
Coactivators--
The observation that methionine 283 in hER
enhanced activation by IA-R suggested that this residue
could be important in coactivator recruitment by the
IA-R-liganded receptor. To test this hypothesis, mammalian
two-hybrid assays were performed to demonstrate the interaction of
hER
, hER
L384M, hER
, and hER
M283L with the nuclear
receptor-interacting regions (NR-boxes) of the coactivators SRC-1 and
GRIP1. Previously, GAL4-DBD fusions of the SRC-1 and GRIP1 NR-boxes
were shown to interact with ER
and ER
in an
agonist-dependent manner, consistent with known receptor-coactivator interactions (25). In the current study, as
expected, the interactions between the Gal4DBD-SRC-1 and Gal4DBD-GRIP-1 NR-box fusions, and each receptor were enhanced by the addition of the
agonists DES, E2, IA-S, and IA-R
(Fig. 4, A-D). The
interaction of SRC-1 with wild-type and mutant hER
or wild-type and
mutant hER
was similar for the DES, E2, and
IA-S bound receptors (Fig. 4, A and
B), in correlation with the transactivation data (Tables VI
and VII, Fig. 3). Notably, the interaction of SRC-1 in the presence of
IA-R, which displayed a higher potency on hER
L384M and
hER
compared with hER
and hER
M283L in transactivation studies
(Tables VI and VII, Fig. 3), was enhanced when the methionine residue
was present in hER
(hER
L384M) and in hER
(wild-type). GRIP1
binding to the IA-R-bound hER
L384M and hER
receptors
was also clearly increased (Fig. 4, C and D).
None of the receptor or coactivator fusion proteins were capable of
activating the 5×-Gal4-TATA-Luc reporter vector when tested together
with the empty Gal4-DBD or pVP16 vectors (data not shown). Taken
together, the results from these studies indicate that methionine 283 in hER
plays an important role in IA-R activation through
facilitating the recruitment of coactivators to the ligand-bound
receptor, as illustrated for these types of ligand agonists.
View larger version (37K):
[in a new window]
Fig. 4.
Mutagenesis of the one mismatched residue in
the hER and hER
LBD
influences the interaction of the IA-R-bound receptors
with coactivators. Mammalian two-hybrid assays were used to
quantitate the interaction of hER
, hER
L384M, hER
, and hER
M283L with the coactivators SRC-1 and GRIP1. For these experiments,
constructs containing the SRC-1 and GRIP receptor interaction domains
fused to the Gal4 transcription factor DNA-binding domain (pM-SRC-1
(NR) and pM-GRIP1 (NR)) were used together with constructs containing
either the hER
, hER
L384M, hER
, or hER
M283L cDNA fused
in-frame to the VP16 activation domain. HepG2 cells were transiently
transfected with the 5×-Gal4-TATA-Luc reporter, the pRL-CMV
normalization plasmid and either pM-SRC-1 (A and
B) or pM-GRIP1 (C and D), together
with pVP16-hER
, pVP16-hER
L384M, pVP16-hER
, or
pVP16-hER
M283L. Following transfection, cells were treated with
vehicle (veh) or 100 nM of E2, DES,
or the indenestrol enantiomers IA-S or IA-R.
After 20 h, cells were harvested and dual luciferase assays were
performed. Each value was normalized to the internal luciferase
control. Shown is a representative experiment with each data point
being the average of triplicate determinations. Experiments were
repeated three times with consistent results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
exhibits stereoselective ligand activation and identified potential molecular determinants for ER
ligand-dependent
activation and stereoselectivity. With the discovery of ER
(3, 4) the question emerged as to whether ER
also exhibits stereoselective ligand activation; this may explain the activity of the DES-type compounds as noted in the present studies and may elucidate the functional properties of ER
. Complementary to this mechanistic question is the search for ER subtype-specific agonists or antagonists that would permit the identification of distinct activities and roles
for ER
and ER
in estrogen target tissues. Katzenellenbogen and
co-workers (26) were the first to determine the stereoselectivity of
ER
using a chiral tetrahydrocrysene (THC). They found that the
enantiomer R,R-THC activates ER
but is an
antagonist on ER
, whereas S,S-THC is an
agonist on both ER
and ER
(26).
agonist, whereas IA-R displayed only weak agonistic activity
in a reconstituted ER transcription system in yeast (8). In the study
described here, we further explored the activity profile of these DES
derivatives by determining the potency of the enantiomers of IA to
activate ER
and ER
in different cell and promoter contexts. For
analysis of cell type specificity, we generated human mammary and
endometrial cell lines that stably express mouse or human ER
or
ER
. In transactivation studies using the cell lines generated, IA-S and IA-R exhibited agonistic and partial
agonistic properties, respectively, through ER
and ER
for all
cell and promoter contexts examined. This demonstrated that both IA
enantiomers have agonistic properties similar to E2 and
DES, but with differing degrees.
or ER
. The IA enantiomer IA-R was a
more potent activator of ER
compared with ER
in human mammary MDA
cells. In endometrial Ishikawa cells, IA-R showed higher
potency of activation on mER
than mER
. However, both hER
and
hER
showed a similar ability to activate transcription from the C3
reporter in the presence of IA-R, whereas IA-R
was a more potent activator of hER
than hER
on the more sensitive 3×ERE reporter. Taken together, these results indicated that
IA-R could function as a potency and cell type-selective
ER
agonist in MDA cells when compared with the endometrial Ishikawa
cell line. Data reported here also indicated that ER
bound to
IA-S is transcriptionally active to a similar degree in both
mammary and endometrial cells. These findings support the notion that the activity of the ER can be dependent on the cellular context, as
highlighted by the cell type-specific mixed agonist/antagonist activity
of the selective ER modulator tamoxifen through ER
(27).
in all cell
and promoter contexts examined suggests that either IA-R has
a low binding affinity for ER
and/or the conformation of
IA-R-liganded ER
does not favor activation.
IA-R exhibited comparably low binding affinities for ER
and ER
, which might be responsible for the weak activation of ER
by IA-R. However, the low binding affinity of
IA-R for ER
is not predictive of its higher potency to
activate ER
in MDA cells as compared with ER
. Paige et
al. (28) used affinity-selected peptides to show that ER
and
ER
assume ligand-specific conformations, which are likely to
modulate the activity of the liganded receptors. It is therefore
reasonable to assume that a conformational change could cause the
differential potencies of the activation of ER
versus
ER
by IA-R, i.e. IA-R induces a
conformational change permissive of ligand-dependent
activation principally of ER
, but only minimally of ER
.
and ER
upon binding of the ER subtype-specific THC ligands (29, 30).
Kraichely et al. (29) showed that the THCs induced a
distinct conformation of the receptors concomitant with quantitative differences in coactivator recruitment. They concluded that the ligand-dependent conformational change favors association
of the receptor with coactivators and through that interaction causes transactivation by the ER. This mode of action is mechanistically described by the tripartite model of steroid hormone receptor action
(31). This model takes into account the ligand binding affinity, the
conformation of liganded receptor, and the association of the receptor
with coregulator molecules, i.e. coactivators. In line with
this model, we hypothesized that IA-R permits
transcriptional activation of ER
but not of ER
, due to induction
of a conformation resulting in altered coactivator recruitment.
and ER
when bound by
IA-R. Comparison of the amino acid sequence of the LBD of
ER
and ER
revealed one mismatched residue within the LBD, Leu-384 in ER
and Met-283 in ER
that is likely to be proximal to the methyl substituent of the chiral carbon in IA based on the crystal structure data of liganded ER
and ER
(18, 19). Given this information, we speculated that this residue could interfere with the
bound IA, inducing a distinct conformational change either facilitating
or impeding activation by ER. Indeed, when this residue was exchanged
in ER
and ER
, the potency of activation of mutated ER
L384M
and ER
M283L by IA-R changed accordingly: IA-R
was a more potent activator of transcription of ER
L384M compared with wild-type ER
and also a less potent activator of mutated ER
M283L compared with wild-type ER
. Importantly, this shift in
activation of IA-R was independent of the cellular and
promoter context tested and, furthermore, the activities of
IA-S, as well as of DES and E2 were not markedly
affected by the Leu/Met mutational exchange. This suggests that
IA-R has a unique ligand structure that is sensitive to this
residue in the binding pocket and subsequently affects receptor
conformation. As expected by the low binding affinity of
IA-R observed for ER
and ER
, this mutation did not affect the binding affinities of IA-R or IA-S to
ER
and ER
, supporting the concept of dissociation of ligand
binding affinity from biological transcriptional activity. The residues
Leu-384 in ER
and Met-283 in ER
are therefore likely to be
important molecular determinants for the ER subtype-specific activity
of IA-R.
(5, 22-25, 32, 33).
In most of these reports, mutations of residues in helices 11 and 12 of
the AF-2 domain were described, and results indicated reduced DNA
and/or ligand binding accompanied by weaker transactivation. Studies on
the enantiomers of indenestrol showed that none of the ER
mutants
analyzed reversed the stereoselectivity of ER
(5, 22). In the latter
study and in results presented here, the higher potency of
IA-S to induce ER
activity was accompanied by a higher
binding affinity of IA-S to ER
as compared with
IA-R (36). Here, we showed that Leu-384 in helix 6 of hER
impedes the activation mediated by IA-R, independent of its
binding affinity. Feng et al. (33) mutated the residues of
lysine 362 in helix 3, valine 376 in helix 5, or glutamic acid 542 in
helix 12 of the hER
LBD. All three mutations independently resulted
in diminished transcriptional activity and coactivator binding (33).
Similar results were reported for mER
; Mak et al. (25)
mutated residues in the LBD and showed that the hydrophobic surface in
the LBD, specifically Leu-376 in mER
, which corresponds to leucine
379 in hER
, is required for binding of the coactivator SRC-1 and subsequent transactivation. In this report, we examined and compared the putative molecular determinants of the ligand specificity of both
ER
and ER
. The leucine 384 residue of ER
impeded
transactivation by IA-R, but not by other ligands, and
therefore might also impede recruitment of coactivators only when
IA-R is bound. In contrast, methionine 283 in the LBD of
ER
enhanced transactivation induced by IA-R, which might
be due to a conformational change leading to increased interactions
with coactivators. Indeed, the interaction of SRC-1 and GRIP1 in the
presence of IA-R, which was a more potent activator of
hER
L384M and hER
compared with hER
and hER
M283L, was
enhanced when the methionine residue was present in hER
(hER
L384M) and in wild-type hER
. Taken together, this agrees with our
suggested mechanism of IA-R activation: that
IA-R, albeit with low binding affinity, induces a
conformational change in ER
that facilitates
ligand-dependent activation, whereas IA-R bound
to ER
is unable to induce a favorable conformation and impedes activation.
in a cell type-specific manner, and a single residue in the LBD of ER
and ER
modulates their transcriptional activity in a cell type-independent fashion. Analysis of conformational changes and crystallographic and structural analysis of wild-type and mutated ER
and ER
bound to subtype specific agonists would further extend our understanding of the molecular mechanisms and structural determinants of ligand-specific transcriptional activity of ER
and ER
.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank members of the Receptor Biology Section for critical discussion and editing of the manuscript. We gratefully acknowledge Dr. D. P. McDonnell (Duke University, Durham, NC) for kindly providing several plasmids used in these studies.
![]() |
FOOTNOTES |
---|
* This work was supported by a Grant from the Deutsche Forschungsgemeinschaft (Mu 1490/1) (to S. O. M.).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 may be addressed: Merck KGaA, Institute of Toxicology, P.O. Box 64271, Darmstadt, Germany. Fax: 49-6151-72-91-8517; E-mail: stefan.o.mueller@merck.de.
To whom correspondence may be addressed: NIEHS, MD B3-02, 111 TW Alexander Dr., P.O. Box 12233, Research Triangle Park, NC 27709. Fax: 919-541-0696; E-mail: korach@niehs.nih.gov.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M203578200
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ABBREVIATIONS |
---|
The abbreviations used are:
ER, estrogen
receptor;
FBS, fetal bovine serum;
ERE, estrogen response element;
DES, diethylstilbestrol;
DCC, dextran-coated charcoal;
IA, indenestrol A;
THC, tetrahydrocrysene;
E2, 17-estradiol;
m, murine;
h, human;
MEM, minimal essential medium;
NR, nuclear receptor-interacting
regions;
RPA, RNase protection assay;
LBD, ligand-binding domain;
DBD, DNA-binding domain.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Hall, J. M.,
Couse, J. F.,
and Korach, K. S.
(2001)
J. Biol. Chem.
276,
36869-36872 |
2. |
Couse, J. F.,
and Korach, K. S.
(1999)
Endocrinol. Rev.
20,
358-417 |
3. |
Kuiper, G. G.,
Lemmen, J. G.,
Carlsson, B.,
Corton, J. C.,
Safe, S. H.,
van der Saag, P. T.,
van der Burg, B.,
and Gustafsson, J. A.
(1998)
Endocrinology
139,
4252-4263 |
4. |
Kuiper, G. G. J. M.,
Carlsson, B.,
Grandien, K.,
Enmark, E.,
Haggblad, J.,
Nilsson, S.,
and Gustafsson, J.
(1997)
Endocrinology
138,
863-870 |
5. | Kohno, H., Bocchinfuso, W. P., Gandini, O., Curtis, S. W., and Korach, K. S. (1996) J. Mol. Endocrinol. 16, 277-285[Abstract] |
6. |
Barkhem, T.,
Carlsson, B.,
Nilsson, Y.,
Enmark, E.,
Gustafsson, J.,
and Nilsson, S.
(1998)
Mol. Pharmacol.
54,
105-112 |
7. |
Couse, J. F.,
Lindzey, J. K.,
Grandien, K.,
Gustafsson, J.-A.,
and Korach, K. S.
(1997)
Endocrinology
138,
4613-4621 |
8. |
Mueller, S. O.,
and Korach, K. S.
(2001)
J. Androl.
22,
652-664 |
9. |
Chaidarun, S. S.,
and Alexander, J. M.
(1998)
Mol. Endocrinol.
12,
1355-1366 |
10. | Schreiber, E., Matthias, P., Mueller, M. H., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Medline] [Order article via Infotrieve] |
11. |
Norris, J. D.,
Fan, D.,
Kerner, S. A.,
and McDonnell, D. P.
(1997)
Mol. Endocrinol.
11,
747-754 |
12. |
Hall, J. M.,
and McDonnell, D. P.
(1999)
Endocrinology
140,
5566-5578 |
13. | Chang, W. Y., and Prins, G. S. (1999) Prostate 40, 115-124[CrossRef][Medline] [Order article via Infotrieve] |
14. | Ignar-Trowbridge, D. M., Teng, C. T., Ross, K. A., Parker, M. G., Korach, K. S., and McLachlan, J. A. (1993) Mol. Endocrinol. 7, 992-998[Abstract] |
15. | Soule, H. D., Maloney, T. M., Wolman, S. R., Peterson Jr, W. D., Brenz, R., McGrath, C. M., Russo, J., Pauley, R. J., Jones, R. F., and Brooks, S. C. (1990) Cancer Res. 50, 6075-6086[Abstract] |
16. | Cailleau, R., Young, R., Olive, M., and Reeves, W. J., Jr. (1974) J. Natl. Cancer Inst. 53, 661-674[Medline] [Order article via Infotrieve] |
17. | Norris, J. D., Fan, D. J., Wagner, B. L., and McDonnell, D. P. (1996) Mol. Endocrinol. 10, 1605-1616[Abstract] |
18. |
Pike, A. C. W.,
Brzozowski, A. M.,
Hubbard, R. E.,
Bonn, T.,
Thorsell, A. G.,
Engstrom, O.,
Ljunggren, J.,
Gustafsson, J. K.,
and Carlquist, M.
(1999)
EMBO J.
18,
4608-4618 |
19. | 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] |
20. | Moore, J. T., McKee, D. D., Slentz Kesler, K., Moore, L. B., Jones, S. A., Horne, E. L., Su, J. L., Kliewer, S. A., Lehmann, J. M., and Willson, T. M. (1998) Biochem. Biophys. Res. Commun. 247, 75-78[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Enmark, E.,
PeltoHuikko, M.,
Grandien, K.,
Lagercrantz, S.,
Lagercrantz, J.,
Fried, G.,
Nordenskjold, M.,
and Gustafsson, J. A.
(1997)
J. Clin. Endocrinol. Metab.
82,
4258-4265 |
22. |
Bocchinfuso, W. P.,
and Korach, K. S.
(1997)
Mol. Endocrinol.
11,
587-594 |
23. |
Valentine, J. E.,
Kalkhoven, E.,
White, R.,
Hoare, S.,
and Parker, M. G.
(2000)
J. Biol. Chem.
275,
25322-25329 |
24. | Lonard, D. M., Nawaz, Z., Smith, C. L., and O'Malley, B. W. (2000) Mol. Cell 5, 939-948[Medline] [Order article via Infotrieve] |
25. |
Mak, H. Y.,
Hoare, S.,
Henttu, P. M.,
and Parker, M. G.
(1999)
Mol. Cell. Biol.
19,
3895-3903 |
26. |
Sun, J.,
Meyers, M. J.,
Fink, B. E.,
Rajendran, R.,
Katzenellenbogen, J. A.,
and Katzenellenbogen, B. S.
(1999)
Endocrinology
140,
800-804 |
27. | McDonnell, D. P. (1999) Trends Endocrinol. Metab. 10, 301-311[CrossRef][Medline] [Order article via Infotrieve] |
28. |
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 |
29. |
Kraichely, D. M.,
Sun, J.,
Katzenellenbogen, J. A.,
and Katzenellenbogen, B. S.
(2000)
Endocrinology
141,
3534-3545 |
30. | Tyulmenkov, V. V., and Klinge, C. M. (2000) Arch. Biochem. Biophys. 381, 135-142[CrossRef][Medline] [Order article via Infotrieve] |
31. | Katzenellenbogen, J. A., O'Malley, B. W., and Katzenellenbogen, B. S. (1996) Mol. Endocrinol. 10, 119-131[Medline] [Order article via Infotrieve] |
32. | Mahfoudi, A., Roulet, E., Dauvois, S., Parker, M., and Wahli, W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4206-4210[Abstract] |
33. |
Feng, W.,
Ribeiro, R. C. n. J.,
Wagner, R. L.,
Nguyen, H.,
Apriletti, J. W.,
Fletterick, R. J.,
Baxter, J. D.,
Kushner, P. J.,
and West, B. L.
(1998)
Science
280,
1747-1749 |
34. | Korach, K. S., Metzler, M., and McLachlan, J. A. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 468-471[Abstract] |
35. | Korach, K. S., Metzler, M., and McLachlan, J. A. (1979) J. Biol. Chem. 254, 8963-8968[Medline] [Order article via Infotrieve] |
36. |
Korach, K. S.,
Chae, K.,
Levy, L. A.,
Duax, W. L.,
and Sarver, P. J.
(1989)
J. Biol. Chem.
264,
5642-5647 |