Mutant and Wild-Type Androgen Receptors Exhibit Cross-Talk on Androgen-, Glucocorticoid-, and Progesterone-Mediated Transcription
Paul M. Yen,
Ying Liu,
Jorma J. Palvimo,
Mark Trifiro,
Jeannie Whang,
Leonard Pinsky,
Olli A. Jänne and
William W. Chin
Division of Genetics (P.M.Y., Y.L., J.W., W.W.C.) Department of
Medicine Brigham and Womens Hospital and Harvard Medical
School Boston, Massachusetts 02115
Divisions of Genetics
and Endocrinology (M.T., L.P.) Department of Medicine Sir
Mortimer B. Davis-Jewish General Hospital Lady Davis Research
Institute for Medical Research McGill University, Montreal,
Quebec H3T 12E2 Canada
Institute of Biomedicine (J.J.P,
O.A.J.) Department of Physiology University of Helsinki
FIN-00014, Helsinki, Finland
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ABSTRACT
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Androgen, glucocorticoid, and progesterone
receptors (ARs, GRs, and PRs) often can regulate transcription via
composite hormone response elements in target genes. We have used
artificial and natural mutant ARs from patients with androgen
resistance to study their effects on dominant negative activity on wild
type AR, GR, and PR function on mouse mammary tumor virus (MMTV) and
tyrosine aminotransferase (TAT) promoters. Artificial ARs that
contained internal deletions within the amino-terminal region had
minimal transcriptional activity but blocked ligand-mediated
transcription by wild type AR. Mutants containing deletions of the
DNA-binding and ligand-binding domains had minimal or weak dominant
negative activity. We then tested the ability of wild type and mutant
ARs to modulate GR- and PR-mediated transcriptional activity. The
amino-terminal deletion mutants exerted dominant negative effects on
GR- and PR-mediated activity, both in the absence and presence of
testosterone. Surprisingly, wild type AR, which had approximately 20%
of the maximal transcriptional activity of GR on the MMTV promoter,
also had dominant negative activity on dexamethasone-regulated
transcription mediated by GR. This dominant negative activity likely
involves DNA binding because a point mutation in the DNA-binding domain
abrogated such activity of an amino-terminal deletion mutant.
Additionally, natural human AR mutants from patients with androgen
resistance, which do not bind either DNA or ligand, did not block
dexamethasone-mediated transcription. In summary, these studies suggest
that mutant and wild type ARs can display dominant negative activity on
other steroid hormone receptors that bind to a composite hormone
response element. This cross-regulation may be important in regulating
maximal transcriptional activity in tissues where these receptors are
coexpressed and may contribute to the phenotype of patients with
steroid hormone resistance.
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INTRODUCTION
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Steroid hormone receptors can recognize common hormone response
elements (HREs). In particular, androgen, glucocorticoid, and
progesterone receptors (ARs, GRs, and PRs) can regulate transcription
via the mouse mammary tumor virus promoter (MMTV) (1, 2, 3, 4). This promoter
region between -187 to -69 contains two palindromic HREs separated by
40 bp and two single downstream half-sites arranged as direct repeats.
Functional studies have shown the upstream palindrome is sufficient for
transcriptional activation by GR, and the proximal palindromic element
and two half-sites also can mediate transcriptional activation by GR
(4, 5). Deletion analysis has shown that dexamethasone, progesterone,
and testosterone can regulate transcription via nucleotide sequences
between -201 and -69 of the promoter (4, 5). DNAase I footprinting
analyses of the promoter region showed nearly identical patterns for GR
and PR, suggesting that each of the receptors binds to common sites
coinciding with the palindrome and half-site sequences (6). Methylation
interference studies also demonstrated that GR and PR make similar
contacts with nucleotide sequences within these composite HREs (4, 7).
Similar observations were found comparing AR, GR, and PR binding to the
HRE sequence of the tyrosine aminotransferase (TAT) gene (8, 9).
Although there have been studies examining the transcriptional
activity of AR, GR, and PR on the MMTV promoter, there have been
limited studies examining cross-talk and dominant negative activity by
steroid hormone receptors on this or other composite response elements.
Recently, mutant ARs have been shown to exert dominant negative
activity on wild type AR-mediated transcriptional activity using a
reporter containing two consensus androgen response elements (AREs)
(10). In particular, deletion of a subregion within the the
amino-terminal domain abrogated ligand-mediated transcriptional
activity by AR, suggesting that there is an activation factor (AF)
domain within this region similar to the AF-1 region of PR and estrogen
receptor, and the
1 region of GR (3, 10, 11). Additionally, these
deletion mutants displayed dominant negative activity on wild type AR
function. In contrast, deletions of the DNA-binding and ligand-binding
domains had little effect on dominant negative activity.
In this study, we investigated a battery of artificial truncation
and deletion mutants as well as natural AR mutants for their dominant
negative activity on wild type AR, GR, and PR transcription on the MMTV
promoter. Our studies demonstrate that the AF-1 deletion mutant of AR
had virtually no transcriptional activity but exhibited strong dominant
negative activity on AR, GR, and PR-1 (longer PR isoform, also known as
PRB) activity. Surprisingly, wild type AR also had dominant negative
activity on GR and PR-1 transcription. Studies with natural and
artificial AR mutants strongly suggest that DNA binding by wild type
and mutant AR is important in mediating the dominant negative activity.
These results also suggest that there is cross-talk among steroid
hormone receptors that may be important for target gene regulation in
tissues where these receptors are coexpressed. Additionally, they
indicate that receptor mutations in patients with androgen resistance
may affect the functions of other receptors (e.g. GR) and
thereby contribute to the patient phenotype in tissues where AR and GR
are expressed.
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RESULTS
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We first examined a battery of AR deletion mutants (Fig. 1A
) for their effects on the transcriptional activity of
the MMTV promoter in CV-1 cells. Wild type AR (WT AR) mediated
approximately a 125- to 175-fold increase in transcriptional activity
over vector alone in the presence of testosterone (Figs. 1B
and 2
).
This increase appeared to be maximal as transfection of 3-fold more AR
expression plasmid gave a similar fold-induction (data not shown).
Deletion of amino acids 38296 (
38-296) and 46408 (
46-408) in
the amino-terminal region almost completely abrogated ligand-dependent
activation, whereas deletion of amino acids 40147 (
40-147) had
minimal effect. Of note, these mutants have similar ligand- and
DNA-binding affinities as WT AR (9, 10). Thus, similar to previous
findings with a consensus ARE (10), these data suggest that there is a
potent AF region between amino acids 147 and 296. Deletion of the
DNA-binding domain (
557-610) and the most carboxy-terminal 115 amino
acids (
788-902) also completely abrogated transactivation. As a
control for appropriate intracellular localization, cells transfected
with mutant receptors were immunostained with anti-AR antibody. Such
studies showed that these receptors are localized mainly in the nucleus
(U. Karvonen, P. J. Kallio, O. A. Jänne, and J. J. Palvimo,
manuscript submitted). Jenster et al. (12) also previously
showed nuclear localization of comparable human AR mutants.

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Figure 1. AR Mutants and Their Transcriptional Activities
A, Rat AR mutants used in these studies. B, Transcriptional activity
via the MMTV promoter by wild type AR and AR deletion mutants. CV-1
cells were transfected with 0.1 µg expression vector encoding WT AR
or AR deletion mutants, 1.7 µg MMTV-luciferase reporter, and 1.0 µg
RSV-ß galactosidase control vector. Cells were treated with
10-6 M testosterone for 48 h and
harvested, and cell lysates were prepared and analyzed for luciferase
activity as described in Materials and Methods.
Luciferase activity was normalized to fold basal luciferase activity
with 1-fold basal activity defined as reporter activity with empty
expression vector in the absence of hormone. Data are expressed as
means ± SD (n = 4). *, point mutant; AD,
amino-terminal domain deletions; DBD, DNA-binding domain deletion; and
LBD, ligand-binding domain (carboxy terminus) deletion.
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Figure 2. Dominant Negative Activity by AR Mutants on Wild
Type AR-Mediated Transcription
CV-1 cells were transfected with 0.1 µg expression vector encoding WT
AR and 0.3 µg AR deletion mutants, 1.7 µg MMTV-luciferase reporter,
and 1.0 µg RSV-ß galactosidase control vector. In some samples, pSG
vector was added so that each sample contained the same amount of DNA.
Cells were treated with 10-6 M testosterone
for 48 h and harvested, and cell lysates were prepared and
analyzed for luciferase activity as in Fig. 1 . Data are expressed as
means ± SD (n = 4).
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We next examined the dominant negative activity on WT AR function by
the transcriptionally inactive AR deletion mutants. Deletion mutants
46-408 and
38-296 completely blocked WT AR transctivation at 1:1
and 3:1 ratios of cotransfected mutant AR/WT AR expression plasmids
(Fig. 2
, and data not shown). The DNA-binding domain
deletion mutant had no effect, and the carboxy-terminal deletion mutant
had a weak effect on WT AR function at a 3:1 expression plasmid ratio.
Taken together, these data are in agreement with those on a consensus
ARE (10) and suggest that although amino-terminal deletion mutants of
AR lose their ability to transactivate in the presence of testosterone,
they exhibit potent dominant negative activity on WT AR function.
Since GR also is a potent transcriptional activator via the MMTV
promoter, we investigated the dominant negative activity of these
mutants on GR-mediated transactivation in the absence or presence of
testosterone. GR had a maximal transcriptional activity of
approximately 1000-fold in the presence of dexamethasone (Fig. 3
). AR deletion mutants
46-408 and
38-296 had
potent dominant negative activity on GR-mediated transcriptional
activation, both in the presence or absence of testosterone.
Surprisingly, wild type AR and
40-147 also blocked GR-mediated
transcriptional activation, almost to the level of AR alone with
testosterone. For WT AR,
46-408, and
40-147, addition of
testosterone further augmented dominant negative activity. In contrast
to the other AR mutants and WT AR,
557-610 and
788-902 had weaker
dominant negative activity on GR-mediated transactivation. The weak
dominant negative activity by
557-610 may be due to squelching of
common cofactors important for GR-mediated transcription. We also
examined the effects of these mutants on the transcriptional activity
of the more potent PR isoform, PR-1, and observed similar results with
wild type AR and the AR mutants (data not shown).

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Figure 3. Dominant Negative Activity by WT AR and AR Mutants
on GR-Mediated Transcription
CV-1 cells were transfected with 0.1 µg expression vector encoding GR
and 0.3 µg WT AR and AR deletion mutants, 1.7 µg MMTV-luciferase
reporter, and 1.0 µg RSV-ß galactosidase control vector. In some
samples, pSG vector was added so that each sample contained the same
amount of expression vector. Cells were treated with 10-6
M testosterone and dexamethasone as indicated for 48 h
and harvested, and cell lysates were prepared and analyzed for
luciferase activity as in Fig. 1 . Data are expressed as means ±
SD (n = 4). dex, Dexamethasone.
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To compare further the dominant negative activity of WT AR with
46-408, we performed dose-response studies in which the amount of GR
expression plasmid was constant, and increasing amounts of AR
expression plasmid were cotransfected (Figs. 4
). At a
3:1 ratio of AR:GR expression plasmid, AR blocked GR-mediated
transcription to about 20% maximal transcription whereas a 1:1 ratio
of
46-408:GR expression plasmid blocked transcription to about the
same level. Increasing amounts of
46-408 almost completely blocked
GR-mediated transcription. Interestingly, when low amounts of AR and
46-408 expression plasmids were cotransfected (1:1), there was a
greater testosterone-dependent effect on dominant negative
activity.
To examine whether the dominant negative activity of
46-408 required
DNA binding, we examined whether a double AR mutant containing the same
internal deletion and a mutation of the fourth coordinating cysteine of
the first zinc finger (
46-408 C562G) was able to mediate this
effect. The latter mutation has been shown to abrogate DNA binding of
several nuclear hormone receptors, including AR (Refs. 1315 and J. J.
Palvimo and O. A. Janne, unpublished results). In contrast to
46-408, this mutation was unable to block GR-mediated transcription,
suggesting that DNA binding is important in mediating this effect (Fig. 5A
). We also examined AR/GR cross-talk by examining this
effect using a reporter containing the TAT promoter and observed
similar effects with
46-408 and
46-408 C562G (Fig. 5B
).
To examine further the mechanism for dominant negative activity
by AR, we used two natural mutant human ARs from patients with androgen
resistance to study their effects on AR- and GR-mediated transcription
(Fig. 6
). One of these mutants (AR DBDmut) contains a
point mutation in the second zinc finger of the DNA-binding domain and
has lost the ability to bind DNA, and the other mutant (AR LBDmut) has
a point mutation in the ligand-binding domain and binds testosterone
poorly (16, 17). AR DBDmut was unable to mediate ligand-dependent
transcriptional activity; however, when cotransfected with wild type
human AR (WT hAR), it surprisingly enhanced ligand-mediated
transcriptional activity to a greater extent than that of WT AR (Fig. 6A
). These findings suggest that the AR DBDmut either titrated out a
repressor of AR function or, more likely, WT hAR and DBDmut may form
heterodimers that are potent transcriptional activators. Studies using
two other DNA-binding natural human AR mutants (R614H and
614) also
showed similar enhancement of WT AR transcriptional activity (Ref. 18
and L. Pinsky, unpublished results). The AR LBDmut had no
transcriptional activity or dominant negative activity.

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Figure 6. Transcriptional Activity and Dominant Negative
Activity of Natural AR Mutants
A, Transcriptional activity of AR mutants. CV-1 cells were transfected
with indicated amounts of hAR, DBDmut, and LBDmut in pRSV, with 1.7
µg MMTV-luciferase reporter, and 1.0 µg RSV-ß galactosidase
control vector. Cells were treated -/+ 10-6 M
testosterone for 48 h, harvested, and analyzed for luciferase
activity as in Fig. 4 . B, Dominant negative activity by AR mutants on
GR-mediated transcriptional activity. CV-1 cells were transfected with
0.1 µg hGR and 0.5 µg hAR, DBDmut, and LBDmut in pRSV, with 1.7
µg MMTV-luciferase reporter, and 1.0 µg RSV-ß galactosidase
control vector and treated with 10-6 M
dexamethasone and/or testosterone. pRSV vector was added to some
samples so that each sample contained the same amount of DNA. Samples
were harvested, assayed, and analyzed as in Fig. 4 . For AR, 100%
maximal activity = transcriptional activity with 0.1 µg hAR
expression vector in the presence of 10-6 M
testosterone. For GR, 100% maximal activity is defined the same as in
Fig. 4 . Data are expressed as means ± SD (n =
3).
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We also tested the dominant negative activity of these mutants on
GR-mediated transactivation (Fig. 6B
). We observed that WT AR
maintained good dominant negative activity, whereas AR DBDmut and
LBDmut did not. These findings further support the role of DNA
binding in mediating dominant negative activity. In combination with
the earlier findings (Figs. 3
and 4
), they also suggest that AR may
modulate GR-mediated transactivation in tissues where both receptors
are coexpressed. In support of this possibility, we also examined the
expression of rat AR and human GR in CV-1 cells and showed that they
are similarly expressed in CV-1 cells using a whole cell ligand-binding
assay (Table 1
). Moreover, they suggest that natural
mutant ARs may have impaired dominant negative activity on GR-mediated
transactivation (Fig. 6B
).
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Table 1. Whole Cell Ligand-Binding Studies of CV-1
Cells Cotransfected with AR, GR, and AR + GR Expression
Plasmids
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DISCUSSION
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These studies describe several novel features of AR function.
First, we have demonstrated the critical role of an amino-terminal
subregion for ligand-dependent transactivation (amino acids 147296).
In contrast to other steroid hormone receptors (3, 4), this AF-1 domain
appears to be a major contributor to ligand-mediated transcription.
Second, we have generated potent dominant negative inhibitors of AR,
GR, and PR function when this subregion is deleted. Moreover, WT AR had
dominant negative activity on GR function. Given the observation that
the PR isoform, PR-2, has potent dominant negative activity on GR, AR,
and PR-1 activity (Refs. 1921 and P. M. Yen, unpublished results),
these findings suggest that coexpression of these steroid hormone
receptors not only may allow multiple ligands to regulate target gene
expression but also may enable mutual modulation of target gene
transcription by these receptors. In general, when two steroid hormone
receptors are coexpressed in saturating amounts and both ligands are
present, the resultant transcriptional activity will approach the
transcriptional activity of the less potent receptor. Recently,
putative coactivators for nuclear hormone receptors called SRC-1 and
TIF2 have been identified (22, 23). Interestingly, SRC-1 augments
transcriptional activation by PR-1, but had minimal effects on GR,
suggesting that some coactivators for steroid hormone receptors may be
receptor-specific. Differential expression of receptor-specific
coactivators in tissues and cells could contribute to differences in
transcriptional activation observed among different steroid hormone
receptors in certain target genes. Our observation of different levels
of maximal transcriptional activation by steroid hormone receptors on
the MMTV promoter in CV-1 cells (GR>PR-1>AR>PR-2) would be
consistent with this model.
Several lines of evidence suggest that the mechanism of dominant
negative activity by AR and AR mutants likely involves competitive DNA
binding to the composite response element. First, our studies with AR
truncation and deletion mutants show that deletion of the DNA-binding
domain greatly weakens dominant negative activity on AR- and PR-
mediated transactivation and weakens dominant negative activity on
GR-mediated transactivation. Second, a point mutation of the DBD of the
potent dominant negative inhibitor,
46-408, abrogates DNA binding
and its effect on GR-mediated transcription. Third, our results using a
natural AR mutation, which only contains a single amino acid deletion
in the DNA-binding domain, further demonstrates the importance of the
integrity of the DNA-binding domain on mediating dominant negative
activity of GR-mediated transactivation. Fourth, we have observed only
weak dominant negative activity by AR or AR
46-408 on thyroid
hormone receptor- or estrogen receptor-mediated transactivation (P. M.
Yen and Y. Liu, unpublished results), suggesting that squelching of a
common coactivator such as SRC-1 is unlikely. Last, WT AR, AR
46-408, and GR can bind to the TAT HRE with similar affinities (9, 24, 25, 26), suggesting that they may have similar DNA-binding properties
for the MMTV and TAT HREs, and thus could serve as competitors for DNA
binding. Taken together, the foregoing data favor a model in which
transcriptionally less active AR or inactive mutant ARs compete with GR
and PR-1 for binding to HREs. Titration of a common cofactor that
interacts with the DNA-binding domains of AR and GR is less likely
based on these experiments but cannot be excluded. Additionally, it is
possible that AR and AR mutants may form heterodimers with GR or PR-1
similar to AR/
46-408 dimers, mineralocorticoid receptor/GR dimers,
and PR DBD/GR DBD dimers reported previously (9, 10, 27, 28).
Interestingly, Bamberger et al. (29) recently showed that an
alternative splice variant of GR, GRß, which does not bind
dexamethasone and has dominant negative activity on wild type GR
transcriptional activity, may utilize a similar mechanism by competing
with wild type GR for binding to the HRE. However, it should be noted
that we have not observed AR/PR-1 heterodimer formation in solution or
on DNA in coimmunoprecipitation experiments (J. Whang and P. M. Yen,
unpublished results).
It also is interesting that AR and AR mutants had dominant negative
activity on GR- and PR-mediated transcription in the absence or
presence of testosterone. Control experiments showed that
10-6 M dexamethasone and progesterone did not
stimulate transcriptional activity by AR (Y. Liu and P. M. Yen,
unpublished results), suggesting that these hormones did not account
for testosterone-independent dominant negative activity by AR.
Immunostaining experiments have shown that unliganded rat AR and human
AR are localized in the nuclei of transfected cells (Ref. 12 and U.
Karvonen, P. J. Kallio, O. A. Jänne, and J. J. Palvimo,
manuscript submitted). Additonally, it has been shown that unliganded
AR can bind to AREs in vitro with similar kinetics and
affinity as liganded AR (9, 10). Ikonen et al. (30) also
showed that unliganded AR expressed in COS cells also could bind to an
ARE in vitro. Given these findings, it is possible that both
unliganded and liganded AR compete for DNA binding to HREs in these
cotransfection studies. Of note, we observed testosterone-dependent
effects on AR and
46-408 dominant negative activity when lower
amounts of expression plasmid were used (Fig. 4
), suggesting that at
lower receptor concentrations, unliganded AR may be complexed with
endogenous heat shock proteins and unable to bind DNA.
We also examined AR- and GR-mediated transcription using a reporter
plasmid containing the TAT promoter and upstream sequences. These
studies showed that AR,
46-408, and
46-408 C562G had similar
effects on GR-mediated transcription as observed with the
MMTV-luciferase reporter. These findings suggest that cross-talk
between AR and GR may occur on different target genes that utilize
different promoters. Additionally, whole cell ligand-binding studies
showed that AR and GR are similarly expressed in cotransfection studies
of CV-1 cells. These findings then would suggest that AR modulation of
GR activity can occur at relatively low amounts of receptors per cell
and does not require excessive amounts of AR expression. The AR and GR
expression level is similar to endogenous receptor levels in cells and
tissues in which both receptors are coexpressed (31, 32, 33, 34, 35).
Another potential consequence for this cross-talk between AR and GR is
that patients with androgen resistance may be more sensitive to
glucocorticoids in certain tissues than normal patients due to lack of
dominant negative activity by mutant ARs. Studies of urinary free
cortisol collections or graded dexamethasone suppression tests, in
patients with androgen resistance, or target gene responsiveness to
dexamethasone in Tfm mice may shed light on the physiological
significance of this cross-talk (36). Our functional studies thus raise
the possibility that mutations in one receptor may modulate the
function(s) of another receptor when there is cross-talk between the
receptors, particularly in tissues in which AR and GR are coexpressed
(e.g. skin, bone, and prostate).
The foregoing studies, as well as the development of estrogen receptor
mutations that have dominant negative activity (37), raise the
possibility of using mutant steroid hormone receptors in transgenic
animals to study the effects of steroid hormone receptors in growth,
development, metabolism, and oncogenesis. These receptors could even be
targeted to specific tissues, as has been recently shown with dominant
negative retinoic acid receptors (38, 39), and provide an alternative
to knockout mice for studying loss of steroid hormone receptor
function. However, careful studies of potential cross-talk among
steroid hormone receptors need to be performed before such models can
be used. Finally, cross-talk among related receptors in regulating
target gene expression via composite HREs may be yet another mechanism
for fine control of gene expression as shown here as well as by that
recently demonstrated in the regulation of the proliferin gene by GR
and mineralocorticoid receptor (40). This cross-talk may modulate
ligand-dependent transcription in tissues where AR, GR, and PR-1 are
coexpressed. Understanding the interplay among these different
receptors should prove to be an exciting and interesting area for
future studies.
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MATERIALS AND METHODS
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Preparation of Vectors
Previously constructed pSG expression vectors encoding rat AR
and various deletions mutants (
40-147,
38-296,
46-408,
557-610,
788-902, and
46-408 C562G) (10, 41) and pSV40
expression vectors encoding human AR and natural AR mutants (DBDmut Phe
at amino acid position 582 was deleted, and LBDmut containing an amino
acid substitution of Arg to Cys at amino acid position 773) (16, 17)
and human GR, PR-1, and PR-2 in pSG (Dr. P. Chambon, INSERM,
Strasbourg, France) were used (42). The pRSV-GR and MMTV-luciferase
reporter plasmid containing the MMTV promoter region and luciferase
cDNA (pMTV-luc) (Dr. R. Evans, Salk Institute, La Jolla, CA) (26) and
the TAT-chloramphenicol acetyltransferase (CAT) reporter plasmid
containing the minimal TAT promoter and 3.0 kb upstream sequences also
were used in some experiments (Dr. S. Stoney Simons, Jr., NIH,
Bethesda, MD) (43). Clones were isolated, sequenced, prepared, and
purified by affinity chromatography (QIAGEN, Chatsworth, CA) before
being used in transfections.
Cotransfection Studies
CV-1 cells were grown in DMEM/10% FCS. The serum was stripped
of steroid hormones by incubating with charcoal for 12 h at 4 C
and constant mixing with 5% (wt/vol) AG1-X8 resin (Bio-Rad, Richmond,
CA) twice for 12 h at 4 C before ultrafiltration. The cells were
transfected with expression (0.1 µg) and reporter (1.7 µg) plasmids
as well as a RSV-ß-galactosidase control plasmid (1 µg) (44) in
3.5-cm plates using the calcium-phosphate precipitation method (45).
Cells were grown for 48 h in the absence or presence of
10-6 M dexamethasone, progesterone, or
testosterone (Sigma), and harvested. Cell extracts were analyzed for
both luciferase (46) and ß-galactosidase (44) activities to correct
for transfection efficiency. Except where indicated, the corrected
luciferase activities of untreated samples were normalized to the
luciferase activities of samples containing vector alone in the absence
of ligand (1-fold basal). In experiments studying TAT-CAT reporter
activity, chloramphenicol acetyltransferase enzyme was measured using a
CAT ELISA kit (Boehringer-Mannheim, Mannheim, Germany).
For the whole cell ligand-binding studies, 7.5 µg expression plasmid
(pSG-hGR, pSG-rAR) were cotransfected into confluent 10-cm plates
containing CV-1 cells. [3H]Dexa-methasone and
[3H]mibolerone binding in whole cells was prepared and
measured as described previously (10).
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ACKNOWLEDGMENTS
|
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The authors would like to thank Dr. Pierre Chambon (INSERM,
Strasbourg, France) for the GR, PR-1, and PR-2 expression plasmids, Dr.
Ronald Evans (Salk Institute, La Jolla, CA) for the pMTV-luc reporter
plasmid, and Dr. S. Stoney Simons, Jr. (NIH, Bethesda, MD) for the
TAT-CAT reporter plasmid, and Drs. Remco Spanjaard and Anath
Shalev (Harvard Medical School, Boston, MA) for helpful
discussions.
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FOOTNOTES
|
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Address requests for reprints to: Paul M. Yen, G.W. Thorn Research Building, Room 907, Brigham and Womens Hospital, 20 Shattuck Street, Boston, Massachusetts 02115.
This work was supported by NIH Grant K080K02186 and a Clinical
Research Grant from The March of Dimes Foundation (P.M.Y.) and The
Medical Research Council of The Academy of Finland, The Finnish
Foundation for Cancer, and The University of Helsinki (J.J.P. and
O.A.J.), and grants from Medical Research Council, Canada, and FRSQ,
Québec (M.I.T. and L.P.).
Received for publication June 25, 1996.
Revision received October 31, 1996.
Accepted for publication November 11, 1996.
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REFERENCES
|
---|
-
Majors J, Varmus HE 1983 A small region of mouse mammary
tumor virus long terminal repeat confers glucocorticoid hormone
regulation on a linked heterologous gene. Proc Natl Acad Sci USA 80:58665870[Abstract]
-
Chandler VL, Maler BA, Yamamoto KR 1983 DNA sequences bound
specifically glucocorticoid receptor in vitro render a
heterologous promoter hormone responsive in vivo. Cell 33:489499[Medline]
-
Gronemeyer H 1991 Transcription activation by estrogen and
progesterone receptors. Annu Rev Genet 25:89123[CrossRef][Medline]
-
Truss M, Beato M 1993 Steroid hormone receptors: interaction
with deoxyribonucleic acid and transcription factors. Endocr Rev 14:459479[Abstract]
-
Ham J, Thomson A, Needham M, Webb P, Parker M 1988 Characterization of response elements for androgens, glucocorticoids,
and progestins in mouse mammary tumour virus. Nucleic Acids Res 16:52635277[Abstract]
-
Ahe D, Janich S, Scheidereit C, Renkawitz R, Schutz G, Beato
M 1985 Glucocorticoid and progesterone receptors bind to the same sites
in two hormonally regulated promoters. Nature 313:706709[Medline]
-
Cairns C, Gustafsson J-A, Carlstedt-Duke J 1991 Identification of protein contact sites within the
glucocorticoid/progestin response element. Mol Endocrinol 5:598604[Abstract]
-
Truss M, Chalepakis G, Beato M 1990 Contacts between
steroid hormone receptors and thymines in DNA: an interference method.
Proc Natl Acad Sci USA 87:71807184[Abstract]
-
Kallio PJ, Palvimo JJ, Mehto M, Jänne OA 1994 Analysis
of androgen receptor-DNA interactions with receptor proteins produced
in insect cells. J Biol Chem 269:1151411522[Abstract/Free Full Text]
-
Palvimo JJ, Kallio PJ, Ikonen T, Mehto M, Jänne O 1993 Dominant negative regulation of trans-activation by the rat androgen
receptor: roles of the N-terminal domain and heterodimer formation. Mol
Endocrinol 7:13991407[Abstract]
-
Jenster G, van der Korput HA, Trapman J, Brinkman AO 1995 Identification of two transcription activation units in the N-terminal
domain of the human androgen receptor. J Biol Chem 270:73417346[Abstract/Free Full Text]
-
Jenster G, van der Korput HA, van Vroonhoven C, van der Kwast
TH, Trapman J, Brinkman AO 1991 Domains of the human androgen receptor
involved in steroid binding, transcriptional activation, and
subcellular localization. Mol Endocrinol 5:13961404[Abstract]
-
Nagaya T, Madison LD, Jameson JL 1992 Thyroid hormone receptor
mutants that cause resistance to thyroid hormone: evidence for receptor
competition for DNA sequences in target genes. J Biol Chem 27:1301413019
-
Yen PM, Wilcox EC, Hayashi Y, Refetoff S, Chin WW 1995 Studies
on the repression of basal transcription (silencing) by artificial and
natural human TRß mutants. Endocrinology 136:28452851[Abstract]
-
Yen PM, Liu Y, Sugawara A, Chin WW 1996 Vitamin D receptors
repress basal transcription and exert dominant negative activity on
triiodothyronine-mediated transcriptional activity. J Biol Chem 271:1091010916[Abstract/Free Full Text]
-
Beitel LK, Prior L, Vasilou DM, Gottlieb B, Kaufman M,
Lambroso R, Alvarado C, McGillivray B, Trifiro MA, Pinsky L 1994 Complete androgen insensitivity due to mutations in the probable
alpha-helical segments of the DNA-binding domain in the human androgen
receptors. Hum Mol Genet 3:2127[Abstract]
-
Prior L, Bordet S, Trifiro MA, Mhatre A, Kaufman M, Pinsky L,
Wrogeman K, Belsham DD, Pereira F, Greenberg C 1992 Replacement of
arginine 773 by cysteine or histidine in the human androgen receptor
causes complete androgen insensitivity with different receptor
phenotypes. Am J Hum Genet 51:14355[Medline]
-
Kazemi-Esfariani P, Beitel LK, Kaufman M, Gottlieb B, Alvarado
C, Trifiro M, Pinsky L, Evidence for transcriptional superactivity of
human steroid receptors by interaction with DNA-binding-deficient
androgen receptors. Program of the 76th Annual Meeting of The Endocrine
Society, Anaheim, CA, 1994 (Abstract 738)
-
Vegeto E, Shahbaz MM, Wen DX, Goldman ME, OMalley BW,
McDonnell DP 1993 Human progesterone receptor A form is a cell- and
promoter-specific repressor of human progesterone receptor B function.
Mol Endocrinol 7:12441255[Abstract]
-
Tung L, Mohamed MK, Hoeffler JP, Takamoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone B-receptors activation
transcription without binding to progesterone response elements and are
dominantly inhibited by A-receptors. Mol Endocrinol 7:12561259[Abstract]
-
McDonnell DP, Goldman ME 1994 RU486 exerts
antiestrogenic activities through a novel progesterone receptor A
form-mediated mechanism. J Biol Chem 269:1194511949[Abstract/Free Full Text]
-
Onate SA, Tsai SY, Tsai M-J, OMalley BW 1995 Sequence
and characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Voegel JJ, Heine MJS, Zechel C, Chambon P, Grone-meyer H 1996 TIF2, a 160 kD transcriptional mediator for the ligand-dependent
actiation AF-2 of nuclear receptors. EMBO J 15:36673675[Abstract]
-
Rodriguez R, Carson MA, Weigel NL, OMalley BW, Schrader WT 1989 Hormone-induced changes in the in vitro DNA-binding
activity of the chicken progesterone receptor. Mol Endocrinol 3:356362[Abstract]
-
Cairns C, Gustafssson J-A, Carlstedt-Duke J 1991 Identification of protein contact sites within the
glucocorticoid/progestin response element. Mol Endocrinol 5:598604[Abstract]
-
Dahlman-Wright K, Wright APH, Gustafsson J-A 1992 Determinants of high-affinity DNA binding by the glucocorticoid
receptor: evaluation of receptor domains outside the DNA-binding
domain. Biochemistry 31:90409044[Medline]
-
Liu W, Wang J, Sauter NK, Pearce D Steroid receptor
heterodimerization demonstrated in vitro and in
vivo. Proc Natl Acad Sci USA 92:1248012484
-
Trapp T, Rupprecht R, Castren M, Reul JMHM, Holsboer F 1994 Heterodimerization between mineralocorticoid and glucocoricoid
receptor: a new principle of glucocorticoid acton in the CNS. Neuron 13:14571462[Medline]
-
Bamberger CM, Bamberger AM, deCastro M, Chrousos GP 1995 Glucocorticoid receptor beta, a potential endogenous inhibitor of
glucocorticoid action in humans. J Clin Invest 95:24352541[Medline]
-
Ikonen T, Palvimo JJ, Kallio PJ, Renkainen P, Janne OA 1994 Stimulation of androgen-regulated transactivation by modulators of
protein phosphorylation. Endocrinology 135:13591366[Abstract]
-
Horwitz KB, Costlow ME, McGuire WL 1975 MCF-7: a human breast
cancer cell line with estrogen, androgen, progesterone, and
glucocorticoid receptors. Steroids 26:786795
-
Norris JS, Kohler PO 1977 The coexistence of androgen and
glucocorticoid receptors in the DDT1 cloned cell line. Endocrinology 100:613618[Abstract]
-
Snochowski M, Dahlberg E, Gustafsson JA 1980 Characterization
and quantification of the androgen and glucocorticoid receptors in
cytosol from rat skeletal muscle. Eur J Biochem 111:603616[Abstract]
-
Davies P, Rushmere NK 1990 Association of glucocorticoid
receptors with prostate nuclear sites for androgen receptors and with
androgen response elements. J Mol Endocrinol 5:117127[Abstract]
-
Masuyama A, Ouchi Y, Sato F, Hosoi T, Nakamura T, Orimo H 1992 Characteristics of steroid hormone receptors in cultured MC3T3-El
osteoblastic cells and effect of steroid hormones on cell
proliferation. Calcif Tissue Int 51:376381[Medline]
-
Yarbrough WG, Quarmby VE, Simental JA, Joseph DR, Sar M,
Lubahn DB, Olsen KL, French FS, Wilson EM 1990 A single base mutation
in the androgen receptor gene cause androgen insensitivity in the
testicular feminized rat. J Biol Chem 265:88938900[Abstract/Free Full Text]
-
Ince BA, Schodin DJ, Shapiro DJ, Katzenellenbogen BS 1995 Repression of endogenous estrogen receptor activity in MCF-7
human breast cancer cells by dominant negative estrogen receptors.
Endocrinology 136:31943199[Abstract]
-
Saitou M, Sugal S, Tanaka T, Shimouchi K, Fuchs E, Narumiya S,
Kakizuka A 1995 Inhibition of skin development by targeted expression
of a dominant-negative retinoic acid receptor. Nature 374:159162[CrossRef][Medline]
-
Imakado S, Bickenbach JR, Bundman DS, Rothnagel JA, Attar PS,
Wang X-J, Walczak VR, Wisniewski S, Pote J, Gordon JS, Heyman RA, Evans
RM, Roop DS 1995 Targeting expression of a dominant-negative retinoic
acid receptor mutant in the epidermis of transgenic mice results in
loss of barrier function. Genes Dev 9:317329[Abstract]
-
Pearce D, Yamamoto KR 1993 Mineralocorticoid and
glucocorticoid receptor activities distinguished by non-receptor
factors at a composite response element. Science 259:11611165[Medline]
-
Kallio PJ, Poukka H, Moilanen A, Janne OA, Palvimo JJ 1995 Androgen receptor-mediated transcriptional regulation in the absence of
direct interaction with a specific DNA element. Mol Endocrinol 9:10171028[Abstract]
-
Kastner P, Bocquel M-T, Turcotte B, Garnier J-M, Horwitz
KB, Chambon P, Gronemeyer H 1990 Transient expression of human and
chicken progesterone receptors does not support alternative
translational initiation from a single mRNA as the mechanism generating
two receptor isoforms. J Biol Chem 265:1216312167[Abstract/Free Full Text]
-
Oshima H, Simons Jr SS 1992 Modulation of transcription factor
activity by a distant steroid modulatory element. Mol Endocrinol 6:416428[Abstract]
-
Edlund T, Walker MD, Barr PJ, Rutter WJ 1985 Cell specific
expression of the rat insulin genes: evidence for role of two distinct
5' flanking sequences. Science 230:912916[Medline]
-
Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: A
Laboratory Manual, ed. 2. Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY
-
DeWet JR, Wood KV, DeLuca M, Helsinki DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells.
Mol Cell Biol 7:725737[Medline]