Distinguishing Androgen Receptor Agonists and Antagonists: Distinct Mechanisms of Activation by Medroxyprogesterone Acetate and Dihydrotestosterone
Jon A. Kemppainen,
Elizabeth Langley1,
Choi-iok Wong,
Kathy Bobseine,
William R. Kelce2 and
Elizabeth M. Wilson
Laboratories for Reproductive Biology and the Departments of
Pediatrics (J.A.K., E.L., C-i.W., E.M.W.), and Biochemistry and
Biophysics (E.L., E.M.W.) University of North Carolina Chapel
Hill North Carolina 27599
Endocrinology Branch (K.B.,
W.R.K.) Reproductive Toxicology Division National Health and
Environmental Effects Research Laboratory United States
Environmental Protection Agency Research Triangle Park, North
Carolina 27711
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ABSTRACT
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Natural and pharmacological androgen receptor (AR)
ligands were tested for their ability to induce the AR
NH2-terminal and carboxyl-terminal (N/C)
interaction in a two-hybrid protein assay to determine whether N/C
complex formation distinguishes in vivo AR agonists from
antagonists. High-affinity agonists such as dihydrotestosterone,
mibolerone, testosterone, and methyltrienolone at concentrations
between 0.1 and 1 nM induce the N/C interaction
more than 40-fold. The lower affinity anabolic steroids, oxandrolone
and fluoxymesterone, require concentrations of 10100
nM for up to 23-fold induction of the N/C
interaction. However no N/C interaction was detected in the presence of
the antagonists, hydroxyflutamide, cyproterone acetate, or RU56187, at
concentrations up to 1 µM, or with 1
µM estradiol, progesterone, or
medroxyprogesterone acetate; each of these steroids at 1500
nM inhibited the dihydrotestosterone-induced
N/C interaction, with medroxyprogesterone acetate being the most
effective. In transient and stable cotransfection assays using the
mouse mammary tumor virus reporter vector, all ligands displayed
concentration-dependent AR agonist activity that paralleled induction
of the N/C interaction, with antagonists and weaker agonists failing to
induce the N/C interaction. AR dimerization and DNA binding in mobility
shift assays and AR stabilization reflected, but were not dependent on,
the N/C interaction. The results indicate that the N/C interaction
facilitates agonist potency at low physiological ligand concentrations
as detected in transcription, dimerization/DNA binding, and
stabilization assays. However the N/C interaction is not required for
agonist activity at sufficiently high ligand concentrations, nor does
its inhibition imply antagonist activity.
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INTRODUCTION
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Androgen receptor (AR) function is required for male sex
development in the fetus, virilization at puberty, and maintenance of
reproductive function in the adult. Interruption of these processes by
pharmacological androgen antagonists or environmental endocrine
disruptors can cause incomplete masculinization of the fetus or
possibly reduced male fertility later in life (1, 2). Overstimulation
of the prostate by androgen agonists may promote prostate cancer (3).
Experimental approaches to identify and distinguish AR agonists from
antagonists would aid in the classification of environmental and
pharmaceutical chemicals since ligand-binding affinity alone does not
necessarily reflect biological potency, and transient transcriptional
assays can be hampered by the complexity of the systems.
Previous studies from this laboratory identified an AR
NH2-terminal and carboxyl-terminal (N/C) interaction that
requires high-affinity androgen binding (4). The androgen-induced N/C
interaction is inhibited by the androgen antagonist hydroxyflutamide.
These results raised the possibility that an N/C interaction is
required for AR agonist activity and that its interruption is a
prerequisite for antagonist activity. Similar studies with the estrogen
receptor revealed a ligand-dependent N/C interaction that predicted
parallel dimerization (5). Recent studies on AR suggest that its N/C
interaction is intermolecular and results in the formation of an
antiparallel homodimer (6). A feature common to both models is the
requirement for high-affinity agonist binding to promote the N/C
interaction. In the present report we tested the requirement for the AR
N/C interaction in relation to AR dimerization, DNA binding, and
transcriptional activity in transient and stable cotransfection assays
to distinguish the activities of several natural and pharmaceutical
agonists and antagonists. The results suggest that at higher
concentrations, certain weak AR agonists such as medroxyprogesterone
acetate (MPA) activate AR through a mechanism that does not involve the
N/C interaction, although potent agonists capable of AR activation at
low ligand concentrations induce the N/C interaction.
Furthermore, inhibition of the N/C interaction does not necessarily
reflect the activity of an antagonist.
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RESULTS
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Ligand Binding Affinities
Binding affinities of ligands listed in Table 1
are indicated as apparent
equilibrium binding constant (Kd) determined by Scatchard
analysis as previously reported, inhibition constant Ki of
endogenous AR in rat prostate extracts, or the concentration
required for 50% inhibition of [3H]methyltrienolone
(R1881) binding to recombinant AR in transfected COS cells (Table 1
).
Relative competitive binding affinities for [3H]R1881 in
transfected COS cells were mibolerone
R1881 >
RU56187 > dihydrotestosterone (DHT)
MPA
progesterone > estradiol (E2) > cyproterone
acetate > testosterone
oxandrolone
fluoxymesterone > hydroxyflutamide. Chemical structures of
several of the ligands are shown in Fig. 1
. Binding of DHT and testosterone was
weaker in the COS cell assay when compared with apparent equilibrium
binding affinities in tissue cytosols (Table 1
) likely resulting from
partial metabolism during the 2-h 37 C incubation. Analysis of COS
cells transfected with the parent plasmid lacking the AR sequence
showed binding only of [3H]progesterone. Lack of binding
of [3H]R1881 and [3H]mibolerone in the
absence of AR expression suggested that this endogenous binding
activity was not due to the progesterone receptor (7, 8, 9, 10). Inhibition
constants for the anabolic steroids oxandrolone (Ki 62
nM) and fluoxymesterone (Ki 44 nM)
were less than that of hydroxyflutamide (Ki 175
nM), but about 100 times greater than the equilibrium
binding constant (Kd) for high affinity agonists such as
DHT. Competitive binding by MPA and RU56187 were similar to DHT,
although the reported Kd values for MPA were slightly
greater than that for DHT (Table 1
).
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Table 1. Summary of Human AR Ligand Binding Affinities,
Ligand-Induced N/C Interaction, and Transcriptional Activation and
Inhibition
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N/C Interaction
Induction of AR N/C complex formation was determined in a
two-hybrid protein assay using Chinese hamster ovary (CHO) cells as
previously described (4, 6). Relative ligand potency between 0.1
nM and 1 µM was DHT
mibolerone
> testosterone
R1881 > oxandrolone >
fluoxymesterone (Fig. 2
). No N/C
interaction was detected with MPA, RU56187, E2,
progesterone, hydroxyflutamide, or cyproterone acetate up to
concentrations of 1 µM. Induction of the N/C interaction
did not correlate with relative binding affinities (Fig. 2
and Table 1
). The relatively high-affinity ligands, MPA and RU56187, failed to
promote the N/C interaction, whereas the lower affinity anabolic
steroids, oxandrolone and fluoxymesterone, induced the N/C interaction.
The inability of MPA to induce the N/C interaction was not limited to
CHO cells, as it was also ineffective in monkey kidney CV1 or COS cells
where 5-fold induction of the N/C interaction was observed with 1
nM DHT (data not shown).

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Figure 2. Ligand Dependence of the AR N/C Interaction
Determined in a Two-Hybrid Protein Assay in CHO Cells
CHO cells were transfected with GALD-H and VPAR1660 human AR fusion
protein expression vectors and the G5E1b-luciferase reporter vector as
described in Materials and Methods. Cells were incubated
with increasing concentrations of DHT, T (0.1100 nM),
methyltrienolone (R1881), mibolerone (MIB) (0.110 nM),
oxandrolone (OXAND), fluoxymesterone (FLUOXY) (0.1100
nM), MPA, RU56187, E2, progesterone (PROG),
hydroxyflutamide (OH-FL), and cyproterone acetate (CA) (0.11000
nM) as indicated. Shown are the optical luciferase units,
and above the bars, the fold induction relative to the
activity determined in the absence of ligand. The data are
representative of at least three independent experiments.
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Inhibition of the DHT-induced N/C interaction by hydroxyflutamide
reported previously (4) raised the possibility that this inhibition may
be necessary for and indicative of androgen antagonist activity.
Because MPA is a weak AR agonist in vivo (11, 12, 13), it was
surprising that MPA at concentrations as low as 10 nM
blocked the DHT-induced N/C interaction and was about 50 times more
potent than hydroxyflutamide as an inhibitor (Fig. 3
and Table 1
). RU56187 was a slightly
less potent inhibitor of the N/C interaction than MPA, exhibited a high
AR equilibrium binding affinity, and is reported to have antagonist
activity in vivo (14, 15). Ligands with less inhibitory
activity than MPA or RU56187 at concentrations between 50 and 500
nM were hydroxyflutamide, cyproterone acetate,
E2, and progesterone (Fig. 3
and Table 1
). The anabolic
steroids, oxandrolone and fluoxymesterone, and the potent androgen
agonists, DHT, mibolerone, testosterone, and R1881, showed little or no
inhibition of the DHT-induced N/C interaction.

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Figure 3. Inhibition of the DHT-Induced AR N/C Interaction
Determined in the Two-Hybrid Protein Interaction Assay in CHO Cells
CHO cells were transfected with the AR fusion expression vectors and
the G5E1b-luciferase reporter vector as described in Materials
and Methods and incubated with and without 1 nM DHT
or in the presence of 1 nM DHT with increasing
concentrations of the indicated ligands (abbreviations as in Fig. 2
legend). Optical luciferase units are shown with the fold induction
relative to the activity determined in the absence of ligand indicated
above the bars. The data are representative of at least
three independent experiments.
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To investigate the possibility that MPA induces an AR
carboxyl-terminal/carboxyl-terminal (C/C) interaction, we tested GALD-H
with VPD-H in the two-hybrid assay. VPD-H contains the VP16
transactivation domain linked as a fusion protein to the AR hinge and
steroid-binding domain amino acid residues 624919 and was used
previously to demonstrate lack of a C/C interaction induced by DHT (4).
Neither MPA nor DHT induced a C/C interaction in this assay more than
2-fold (results not shown).
Transcriptional Activation
Agonist and antagonist activities were determined in CV1
cells transiently transfected with a mouse mammary tumor virus
(MMTV)-luciferase reporter and full-length human AR expression vectors.
Ligands with more than 10-fold agonist activity at 0.001 nM
were DHT, mibolerone, and R1881 (Fig. 4
and Table 1
). Similar induction was achieved by 0.01 nM
testosterone, 0.1 nM MPA, and 1 nM oxandrolone
or fluoxymesterone. Cyproterone acetate, progesterone, E2,
and RU56187 induced luciferase activity at concentrations between 10
and 100 nM, but transcriptional activity remained low at
100 nM hydroxyflutamide, the latter requiring
concentrations of 110 µM for agonist activity in this
assay (16). Agonist potency, therefore, tended to parallel the
ligand-induced N/C interaction. Lack of an N/C interaction induced by
MPA is associated with 100-fold higher MPA concentrations necessary for
transcriptional activity compared with DHT.

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Figure 4. Ligand-Dependent AR-Mediated Transcriptional
Activation of the MMTV-Luciferase Reporter Vector in CV1 Cells
CV1 cells were transfected with 0.1 µg pCMVhAR full-length human AR
expression vector and 5 µg MMTV-luciferase reporter vector using
calcium phosphate as described in Materials and Methods.
Transfected cells were incubated with increasing concentrations of the
indicated ligands (abbreviations as in Fig. 2 legend). Shown is the
fold induction of luciferase activity relative to the activity
determined in the absence of ligand. The data are representative of at
least three independent experiments.
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When transcriptional activity was tested in the same cells (CHO cells)
used for the N/C two-hybrid assay but using the MMTV-luciferase
reporter, 10- to 100-fold higher MPA concentrations were also required
relative to DHT. In CV1 cells transfected with the MMTV-luciferase
reporter and the pCMV5 parent plasmid lacking AR sequence, there was no
induction of luciferase activity by MPA or any other ligand tested,
ruling out the possibility that MPA activity was mediated through an
endogenous receptor or altered luciferase expression by a nonreceptor
mechanism. A luciferase reporter vector with two copies of the MMTV
glucocorticoid response element separated by a 29-bp linker derived
from pMTV29VTM (17) and cloned into pT81Luc (18) also had greater
agonist activity with DHT relative to MPA (data not shown).
Antagonist activity of the ligands was tested in CV1 cells by
coincubation with 0.1 nM DHT. Hydroxyflutamide was the most
effective antagonist with about 50% inhibition at 100 nM
(Fig. 5
). Cyproterone acetate was
slightly less effective, and RU56187 had some inhibitory activity but
decreased in effectiveness at higher concentrations. Antagonist
activity was also observed with increasing concentrations of
progesterone and E2, but none was observed with MPA (Fig. 5
) or with the high-affinity agonists or the anabolic steroids (results
not shown). Thus, except for MPA, at least partial inhibition of
DHT-induced transcriptional activity correlated with inhibition of the
DHT-induced N/C interaction.

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Figure 5. AR Antagonist Activity in CV1 Cells
CV1 cells were transfected with 0.1 µg pCMVhAR and 5 µg
MMTV-luciferase reporter vector as described in Materials and
Methods and incubated in the presence and absence of 0.1
nM DHT or in the presence of 0.1 nM DHT with
increasing concentrations of the indicated ligands (abbreviations as in
Fig. 2 legend). Shown is the fold induction relative to activity
determined in the absence of DHT. The data are representative of at
least three experiments.
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The inability of MPA to induce the N/C interaction but have agonist
rather than antagonist activity in transient transcription assays and
in vivo (11, 12, 13, 19) prompted us to investigate whether two
AR mutants that cause severe androgen insensitivity might be
selectively activated by MPA in transient cotransfection assays. The
mutants, V889M and R752Q, each retain high-affinity equilibrium binding
of [3H]R1881 (20, 21, 22) but are defective in the N/C
interaction (6). V889M had a similar blunted response to both DHT and
MPA in the MMTV-luciferase reporter assay in CV1 cells, whereas R752Q
required about 10-fold higher concentrations of MPA (10 nM)
relative to DHT to induce transcription (data not shown). Thus, neither
mutant defective in the N/C interaction was efficiently activated
by MPA or DHT, suggesting that these regions of the ligand-binding
domain are important in AR activation by both steroids.
The weak in vivo AR agonist activity reported for MPA
(11, 12, 13, 19) is reflected in MMTV-luciferase assays by the requirement
for higher MPA concentrations relative to DHT for reporter gene
activation. Nevertheless, the agonist activity of MPA was surprising
considering that MPA inhibits the N/C interaction better than most
antagonists. We therefore tested a CHO cell line in which the
MMTV-luciferase reporter and human AR expression vectors were stably
integrated in the genome using pcDNA3.1/Zeo vector with the zeocin gene
and the human AR-coding sequence (K. Bobseine and W. R. Kelce,
unpublished data). Cell lines such as this were used previously to
distinguish agonist activities not detected by transient transfection
(24). DHT at 0.1 nM stimulated luciferase activity 2-fold
while MPA required a 10-fold higher concentration for similar induction
(data not shown). A greater overall response to MPA (10-fold)
compared with DHT (6-fold) likely resulted from MPA activation of
endogenous glucocorticoid receptor since coincubation with 500
nM hydroxyflutamide inhibited MPA-activated gene
transcription about 50% and DHT activity by 95%, but had no effect on
induction by dexamethasone (data not shown). The AR-mediated MPA
response was therefore similar to that of DHT, but required
higher steroid concentrations as observed in the transient assays. The
chromatin arrangement of the reporter gene seemed to have little effect
on the relative concentration-dependent gene activation by DHT and
MPA.
Dimerization and DNA Binding
Because binding of baculovirus expressed full-length AR to
androgen response element DNA requires exposure of Sf9 cells to
androgen and is inhibited by coincubation with the antagonist
hydroxyflutamide (25), we tested the activity of these ligands to
promote AR DNA binding in vitro. DNA binding of full-length
AR was observed with 50 nM DHT, mibolerone, MPA,
oxandrolone, fluoxymesterone (Fig. 6A
),
or 50 nM R1881 or testosterone (Fig. 6B
). Concentrations of
DHT or R1881 less than 50 nM reduced AR DNA binding
probably due to insufficient saturation of baculovirus-expressed AR
(data not shown). AR DNA binding was observed at 1 µM
RU56187 or hydroxyflutamide but was barely detectable with 1
µM progesterone, E2, or cyproterone acetate
(Fig. 6
).

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Figure 6. Ligand-Dependent DNA Binding of Full-Length Human
AR Expressed in Baculovirus
Sf9 cells expressing human AR were incubated with increasing
concentrations of ligands as described in Materials and
Methods. Cells were extracted in high-salt buffer, dialyzed to
reduce the salt content, and incubated with 32P-labeled
androgen response element DNA as described in Materials and
Methods. Shown is a reproduction of films in which the free
32P-labeled oligo bands were removed from the bottom. In
panels A and B, 50, 250, and 1000 nM of the indicated
ligands were added in lanes 118 (abbreviations as in Fig. 2 legend).
Lane 19 contains extracts from cells left untreated with recombinant
virus or ligand. The AR-32P-oligo complex typically
migrates as a double band (indicated with arrows) with
the upper more slowly migrating band being predominant. In the absence
of ligand the AR-32P-oligo complex is not detected (25 ).
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Dimerization and DNA binding of baculovirus-expressed AR
NH2- and carboxyl-terminal fragments that contain the
DNA-binding domain were also shown previously to distinguish androgen
agonists and antagonists (25). While the NH2-terminal and
DNA-binding domain fragment AR1660 (not shown) and the DNA- binding
and carboxyl-terminal fragment AR507919 (Fig. 7
, lanes C) each homodimerize and bind
DNA independently of hormone, agonists are required for dimerization
and DNA binding of the N/C complex and an antagonist such as
hydroxyflutamide inhibits this DHT-induced DNA binding (25, 26). Since
both fragments contain the DNA-binding domain, dimerization could be
mediated by the DNA-binding domain and/or by the N/C interaction.
Results of this assay (25) and others (22) nevertheless predicted the
AR N/C interaction, which was later confirmed in the two-hybrid
interaction assay (4).

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Figure 7. Ligand Dependence of the AR
NH2-Terminal and Carboxyl-Terminal Fragment Dimerization
and DNA Binding in DNA Mobility Shift Assays
Human AR NH2-terminal DNA-binding domain fragment AR1660
(N) and DNA ligand-binding domain-carboxyl terminal fragment AR507919
(C) were expressed separately in Sf9 cells. Cells expressing C were
incubated at the indicated ligand concentrations. After high-salt
extraction and dialysis to lower the salt concentration, 20 µg total
protein of N or C or 10 µg each of the N- and C-terminal fragments
were combined and analyzed in DNA mobility shift assays as described in
Materials and Methods. Cells expressing C were incubated
with 50 nM DHT, mibolerone (MIB), testosterone (T),
and R1881 (shown in panel A) along with 50, 500, and 1000
nM oxandrolone (OXAND) and fluoxymesterone (FLUOXY). R1881
(50 nM) is repeated in panels B and C as a control along
with 50, 500, and 1000 nM MPA, RU56187, and cyproterone
acetate (CA) (panel B), and the same concentrations of E2,
progesterone (Prog), and hydroxyflutamide (OH-FL) (panel C). Shown are
reproductions of films where the free 32P-oligo band was
cut from the bottom. Migration of the N + C and C complexes is
indicated by arrows.
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Dimerization and DNA binding of the AR fragments were observed at 50
nM DHT, mibolerone, testosterone, and R1881 (Fig. 7A
, C+N)
but required 0.51 µM oxandrolone or fluoxymesterone for
similar activity. MPA-induced dimerization and DNA binding of the two
AR fragments were somewhat less efficient than DHT but similar to the
anabolic steroids (Fig. 7
, A and B). Only slightly weaker DNA binding
of the N/C hybrids was detected using cyproterone acetate and
E2, whereas 1 µM RU56187 or 1
µM progesterone was required for DNA binding, and
essentially no DNA binding was detected with 1 µM
hydroxyflutamide (Fig. 7
and Table 2
).
Binding of the homodimer C fragment alone was most effective with
mibolerone, testosterone, and R1881; it was not detected with 50
nM DHT, required 500 nM MPA, and was weak to
undetectable with the anabolic steroids and other ligands (Fig. 7
, lanes C). Similar high-level AR expression was observed by immunoblot
analysis of full-length AR or the AR507919 fragment after the
different hormone treatments and expression levels were independent of
the extent of AR dimerization and DNA binding (data not shown).
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Table 2. Summary of AR Stabilization and Half-Times of
[3H]Ligand Dissociation, AR DNA Binding, and
in Vivo Activity of ligands
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Thus, MPA, cyproterone acetate, E2, and progesterone at
concentrations between 50 nM and 1 µM induce
dimerization and DNA binding of the NH2- and
carboxyl-terminal AR fragments, where both fragments
contain the AR DNA-binding domain (Fig. 7B
). Yet none of these ligands,
except for MPA, promote DNA binding of full-length AR and none induce
the N/C two-hybrid interaction (Fig. 7A
). The effectiveness of these
ligands to induce dimerization and DNA binding of the AR
NH2- and carboxyl-terminal fragments in mobility shift
assays is confounded by the presence of the dimerization region in the
DNA-binding domain. We therefore attempted to address the contribution
of the AR DNA-binding domain in the two-hybrid assay by testing the
interaction of a fusion protein comprised of the GAL4 DNA-binding
domain and AR DNA and ligand-binding domains (GAL-AR507919) with
VPAR1660 or VPAR. However, lack of an interaction in this and
additional experiments using GAL-AR (GAL4 DNA-binding
domain-full-length AR fusion protein) suggests that the presence of two
DNA-binding domains in a fusion protein (i.e. from GAL4 and
AR) interferes with hybrid formation.
AR Stabilization
One property that has distinguished androgen agonists from
antagonists is their ability to stabilize AR against degradation (27).
It was therefore of interest to determine the concentration dependence
of AR stabilization by MPA, RU56187, and the anabolic steroids.
Transfected COS cells were incubated with
[35S]methionine/cysteine and increasing
concentrations of ligands, followed by chase periods with unlabeled
methionine for 27 h as previously described (27). The results
shown for several ligands in Fig. 8
and
summarized in Table 2
indicate that more than 100 nM MPA
was required to increase the AR degradation half-time to almost 5
h at 37 C (Fig. 8C
). A similar degree of AR stabilization was achieved
by 1 nM DHT, mibolerone, or R1881, 5 nM
testosterone (Table 2
), or 10 nM fluoxymesterone or
oxandrolone (Fig. 8
, B and D). Cyproterone acetate and progesterone
stabilized AR only at 1 µM (Table 2
), and almost no AR
stabilization was observed with 1 µM RU56187 (Fig. 8A
),
hydroxyflutamide, or E2 (Table 2
). The results tend to
parallel the MMTV-luciferase and DNA-binding activities in that ligands
that efficiently stabilize AR are more effective agonists. It is
noteworthy that the lower affinity anabolic steroids, oxandrolone and
fluoxymesterone, promote the N/C interaction and stabilize AR at
concentrations of 510 nM, concentrations only slightly
higher than those required for the high-affinity agonists, DHT,
mibolerone, R1881, and testosterone. However, higher concentrations of
the anabolic steroids were required for DNA binding of the AR
fragments, perhaps reflecting the lower AR binding affinity for these
ligands.

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Figure 8. Ligand-Dependent AR Stabilization
Full-length AR was expressed in COS cells from pCMVhAR as described in
Materials and Methods and incubated in the presence of
[35S]methionine/cysteine for 30 min at 37 C followed by
incubation with unlabeled methionine medium for increasing times in the
presence of the indicated concentrations of ligand. Samples were
extracted in RIPA buffer and AR was immunoprecipitated and analyzed on
SDS polyacrylamide gels as previously described (22 ). Scanning the
exposed films resulted in optical density readings of the AR bands,
which migrated at approximately 114 kDa. Shown are the optical density
measurements on a semilog scale. Approximate half-times of AR
degradation at 37 C at the indicated ligand concentrations are shown on
the figures for RU56187 (panel A), fluoxymesterone (panel B), MPA
(panel C), and oxandrolone (panel D).
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Ligand Dissociation Rates
Because only few ligands could be obtained in
3H-labeled form, dissociation rates could not be determined
for MPA, oxandrolone, fluoxymesterone, cyproterone acetate, or
hydroxyflutamide. Nevertheless, we determined that
[3H]RU56187 dissociates rapidly from AR with a
t1/2 of 5 min at 37 C (Fig. 9
) compared with t1/2 of
2.53.5 h for [3H]R1881 (Fig. 9
), [3H]DHT,
and [3H]mibolerone (Table 2
). A rapid dissociation rate
for [3H]estradiol (t1/2 0.67 h) (Table 2
) was also observed. The results raise the possibility that rapid
ligand dissociation is associated with the lack of an N/C interaction
and with in vivo antagonist activity.

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Figure 9. Dissociation Rates of [3H]R1881 and
[3H]RU56187 from Human AR
COS cells were transfected with pCMVhAR and incubated with 5
nM [3H]R1881 or 5 nM
[3H]RU56187 for 2 h at 37 C followed by the addition
of a 10,000-fold molar excess of unlabeled ligands as described in
Materials and Methods. Cells were harvested at
increasing time intervals, and radioactivity was determined. The data
are shown on a semilog plot, and the half-time of dissociation is
indicated: t1/2 2.5 ± 0.5 h for
[3H]R1881 and t1/2 5 ± 1 min for
[3H]RU56187.
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DISCUSSION
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Failure of MPA to induce the N/C interaction suggests that during
AR dimerization, DNA binding, and gene activation, MPA activates AR by
a mechanism different from other agonists. With the other ligands
tested, agonist activity correlated with induction of the N/C
interaction and antagonist activity with its inhibition. MPA is a weak
androgen in vivo (see below) which perhaps relates to its
inability to induce the N/C interaction. Induction of the N/C
interaction by high-affinity AR agonists appears to contribute to their
biological potency at low physiological concentrations. Lack of
induction of the N/C interaction may account for the high MPA
concentrations required to stabilize AR, which would contribute to a
reduced biological potency as an androgen agonist.
The apparent discrepancy in ligand potency between maximal induction in
the N/C assay using the GAL4-AR carboxyl-terminal and the VP16-AR
NH2-fragment fusion proteins (0.11 nM)
vs. agonist potency of full-length AR in the luciferase
assay (0.0010.01 nM) probably reflects deletion of the
NH2-terminal domain. We demonstrated previously that
although the AR ligand-binding domain retains high-affinity binding
after deletion of the NH2-terminal region, the ligand
dissociation rate increases 5- to 7-fold, and androgen no longer
stabilizes this truncated receptor as it does full-length AR (22).
Thus, higher ligand concentrations are likely required for the N/C
interaction between AR fragments than between monomers of full-length
AR.
Equilibrium dissociation constants (MPA, 1.72.9 nM; DHT,
0.92.6 nM) and saturation binding capacities (MPA,
107249 fmol/mg protein; DHT, 42257 fmol/mg protein) for MPA and DHT
binding to AR were similar when measured in rat pituitary and
hypothalamic extracts (28). However, direct measurement of in
vivo bioactivity classifies MPA as a weak androgen. MPA increases
the synthesis of ß-glucuronidase in mouse kidney but only at 100-fold
higher doses relative to testosterone (11, 12). In the androgen-
insensitive Tfm mouse, ß-glucuronidase activity did not increase,
indicating that gene activation in response to MPA is AR mediated (12).
MPA doses up to 1000 times higher than testosterone were required to
increase ventral prostate weight in castrated rats (13). High-dose (0.9
mg/day) MPA was less effective than low-dose DHT (0.2 mg/day) in
stimulating the synthesis of rat prostatic binding protein mRNA, and
the effects of MPA were inhibited by flutamide (19), again indicating
that its in vivo activity is AR mediated. MPA, rather than a
metabolite, was shown to bind AR, and its low in vivo
androgenic activity correlated with reduced nuclear uptake (29, 30).
MPA was reported to dissociate rapidly from AR (13, 31), although these
studies did not account for possible degradation of the MPA-AR complex.
X-ray crystal analysis indicates that MPA has an inverted 1ß,2
half-chair conformation of the A-ring resulting from steric strain by
the 6
-methyl group that restricts side chain flexibility (32). This
predicted rigid structure of MPA is in contrast to the flat, flexible
structure of methyltrienolone (R1881), which can undergo large shape
changes (33). It is conceivable that ligand flexibility facilitates the
conformational changes required for the AR N/C interaction.
Acetate derivatives of steroids often have slower metabolic breakdown
rates, making them candidates for use in hormone therapy (34). MPA,
available for clinical use as Provera or Depo-Provera, has been used in
the treatment of sexual precocity; its progestin and weak androgen
effects inhibit pituitary gonadotropin secretion and lower
gonadal steroid production (35). Stimulation of the growth of pubic
hair in female patients without a significant slowdown in skeletal
maturation (35) suggested weak androgenic activity of large doses of
MPA (200300 mg every 710 days). 21-Hydroxylated metabolites of MPA
bind the glucocorticoid receptor and suppress pituitary secretion of
ACTH. Because of its progestational effect, MPA was formerly
used to prevent spontaneous abortion. However, prenatal exposure to MPA
was reported to cause mild clitoral hypertrophy and posterior labial
fusion in the female and hypospadias in the male (36, 37, 38, 39),
contraindicating its use during pregnancy. The virilizing effect of MPA
in the female fetus can be explained by its androgenic activity.
Antiandrogen effects of MPA in the male fetus could result from
competition for DHT binding to AR and subsequent insufficient agonist
activity. Anogenital distance, a measure of antiandrogen activity in
rodents (1), was lengthened in females and shortened in males exposed
to MPA during fetal development. More recently, MPA was approved for
use in the United States as an injectable contraceptive based on its
effectiveness in suppressing gonadotropin secretion, inhibiting
follicular maturation and preventing ovulation. Doses of 150 mg im
every 6 weeks to 3 months lack androgen effects in the adult
female.
MPA has also been used in breast cancer therapy (40, 41). In an MFM-223
mammary cancer cell line that has high levels of AR, but low levels of
estrogen, progesterone, and glucocorticoid receptors, cell
proliferation was inhibited by 1 nM DHT or 10
nM MPA (42), indicating the AR agonist effect of MPA
inhibits breast cancer cell growth. Response rates of breast cancer
patients to MPA therapy correlated with higher AR levels (43). The
antiproliferative activity of DHT and MPA on breast cancer cells was
attributed to increased 17ß-hydroxysteroid dehydrogenase activity,
which promotes increased oxidation of estradiol to the weak estrogen,
estrone (44).
Agonists vs. antagonist activity is influenced by
metabolism, binding affinity, association and dissociation rates, and
ligand-induced receptor conformation, stabilization, dimerization, DNA
binding, and interactions with associating proteins. Clearly,
equilibrium binding affinity is of limited usefulness in predicting
in vivo bioactivity unless combined with measurements of
ligand dissociation rates. AR binding affinity of RU56187 is similar to
that for DHT, yet RU56187 is an antagonist in vivo (14, 15).
The anabolic steroids, oxandrolone and fluoxymesterone, have high
inhibition constants for binding, yet induce the N/C interaction and
stabilize AR at relatively low ligand concentrations and are AR
agonists in vivo. Oxandrolone induces male- specific liver
P450 enzymes (45). Oxandrolone and fluoxymesterone are structurally
related 17
-alkylated synthetic anabolic steroids used clinically to
promote weight gain, stimulate growth of the bone matrix, and improve
libido and sexual performance (46). In low doses oxandrolone (47, 48)
or fluoxymesterone (49) accelerate linear growth in children with
constitutional growth delay and Turners syndrome (50, 51).
Evidence from crystal structure analysis of the retinoic acid
receptor-
(52), thyroid hormone receptor (53), and estrogen receptor
(54) indicates that hormone binding causes helix 12 [helix 11 in
retinoid X receptor-
(55)] at the carboxyl terminus to undergo a
conformational change closing down over the ligand-binding pocket. For
the estrogen receptor, binding of the antagonist raloxifene prevents
alignment of helix 12 over the binding pocket (54). MPA binding to AR
may distort the position of helix 12 causing an increased rate of
ligand dissociation and interference with the N/C interaction. Proper
closure of helix 12 might be expected to slow ligand dissociation from
the pocket and form a new interface for the N/C interaction. Alignment
of helix 12 by MPA binding may differ from that induced by potent
agonists or antagonists, a distortion that may account for the high MPA
concentrations required to stabilize AR.
Part of the discrepancy between ligand binding affinity and agonist and
antagonist activities relates to differences in ligand binding
kinetics. Association and dissociation rate kinetics can be fast or
slow for high-affinity ligands. Slow dissociation of the most potent AR
agonists, DHT, mibolerone, and R1881, is associated with AR
stabilization at low ligand concentration. Fast dissociating ligands
such as RU56187 fail to stabilize AR and have agonist activity in
transcriptional activation assays but are antagonists in
vivo. Mutations in the AR hormone-binding domain at valine 889 and
arginine 752 cause severe androgen insensitivity, increase the rate of
dissociation of bound androgen without altering high-affinity
equilibrium binding (22), disrupt the N/C interaction (6), and cause
loss of AR stabilization by low ligand concentrations (22). These
mutations likely increase the rate of ligand dissociation and AR
degradation by preventing helix 12 from closing the binding pocket and
interfering with the N/C interaction. Rapid ligand dissociation could
reduce in vivo agonist activity and enhance dose-dependent
antagonist activity as suggested previously for some antiestrogens and
antiandrogens (13).
The most reliable in vitro indicators of in vivo
AR antagonist activity therefore appear to be failure of a ligand to
stabilize AR against degradation at steroid concentrations of 500
nM or more and an inability to induce AR DNA binding. DNA
binding itself, however, appears to be a poor indicator of agonist
potency. In vivo agonist activity is best reflected by a
slow dissociation rate of bound ligand, AR stabilization at low ligand
concentrations (
10 nM), and induction of the N/C
interaction. Concentrations at which a ligand activates AR in
MMTV-luciferase assays can indicate agonist potency. MPA is an agonist
in transient transcription assays but requires 100-fold higher
concentrations than DHT. A similar shift in in vitro
sensitivity to DHT results from certain AR missense mutations that
cause partial or complete androgen insensitivity (56), indicating the
critical importance of AR activation by low ligand concentrations.
However, even though acetate derivatives of steroids have increased
metabolic half-lives, it cannot be ruled out that the in
vivo pharmacology of MPA limits its bioavailability to the AR.
A model for androgen-induced AR dimerization suggests an antiparallel
orientation of monomers interacting through the DNA-binding domain and
a ligand-dependent N/C interaction (4). Similar studies with the
estrogen receptor (5) and AR fragments expressed in yeast (57) predict
a parallel interaction model, and studies on solution dimerization of
the human progesterone receptor favor a parallel model (58). More
recent studies on AR made use of androgen insensitivity mutations in
the steroid-binding domain that do not interfere with high-affinity
equilibrium binding of androgen but increased the dissociation rate of
bound androgen and disrupted the N/C interaction. Placement of the
mutations in different AR fragments allowed assessment of directional
dimerization in association with AR transcriptional activation, and the
results were consistent with an antiparallel activated AR dimer model
(6). Lack of an interaction between the ligand-binding domains bound to
MPA or DHT argue against a parallel dimer model for AR with either
ligand. Taken together the results suggest that the N/C interaction is
required for potent in vivo agonists to be effective at low
concentrations, but is not required for AR DNA binding in
vitro or weak in vivo agonist activity at higher ligand
concentrations. Formation of the N/C interaction likely contributes to
in vivo potency by stabilizing AR at low ligand
concentrations.
 |
MATERIALS AND METHODS
|
---|
Ligand Binding and Dissociation
Reagents were obtained as previously reported (22) with MPA and
other steroids from Sigma Chemical Co. (St. Louis, MO) and RU56187 from
Roussel Uclaf. Relative equilibrium binding was determined in COS cell
competitive binding assays using [3H]R1881. Monkey kidney
COS cells (3.5 x 105 cells per well of six-well
plate) were transiently transfected using diethylaminoethyl
(DEAE)-dextran and 1 µg pCMVhAR full-length AR expression vector per
well. Cells were maintained in 10% calf serum and DMEM for 36 h
and labeled for 2 h at 37 C with 5 nM
[3H]R1881 in the presence and absence of increasing
concentrations of unlabeled ligands. Cells were washed with PBS and
harvested in 2% SDS, 10% glycerol, and 10 mM Tris, pH
6.8, and radioactivity was determined by scintillation counting.
Dissociation rate kinetics were determined in COS cells transfected as
described above using 3 µg pCMVhAR/well. Transfected cells were
incubated with 5 nM [3H] ligand for 2 h
followed by the addition of a 10,000-fold molar excess of unlabeled
ligand. After increasing times, cells were washed with PBS and
harvested in 0.5 ml of the SDS buffer above. Radioactivity was
determined by scintillation counting. Apparent inhibition constants
(Ki) for hydroxyflutamide, oxandrolone, and fluoxymesterone
were determined using rat prostate cytosols prepared from tissue
obtained 24 h after castration, extracted in binding buffer as
previously described (1, 59), and incubated for 20 h at 4 C with
0.520 nM [3H]R1881 with or without
increasing concentrations of unlabeled ligands between 0.1 and 1
µM. Apparent Ki values were determined using
double reciprocal plots and slope-replot analysis.
N/C Luciferase Assay
Recombinant fusion proteins included GALD-H which contained the
Saccharomyces cerevisiae GAL4 DNA-binding domain amino acid
residues 1147 linked in frame with human AR steroid-binding domain
amino acid residues 624919. VPAR1660 contained the herpes simplex
virus VP16 transactivation domain amino acid residues 411456 linked
in frame with AR NH2-terminal and DNA-binding domain amino
acid residues 1660 (4). CHO cells (0.4 x 106 cells
per 6-cm dish) were transfected using DEAE-dextran and 1 µg GALD-H, 1
µg VPAR1660, and 5 µg G5E1b-luciferase per plate, the latter
containing five GAL-4 DNA-binding sites (60). DNA was added to 0.42 ml
H20 plus 0.5 ml 2xTBS (0.14 M NaCl, 3
mM KCl, 1 mM CaCl2, 0.5
mM MgCl2, 0.9 mM
NaH2PO4, and 25 mM Tris-HCl, pH
7.4), and then 0.11 ml DEAE-dextran (0.5%) was added, after which the
mixture was added to the aspirated plates and incubated for 1 h at
37 C. Plates were aspirated and 4 ml
-MEM containing 10% calf
serum, penicillin/streptomycin, and 20 mM HEPES, pH 7.2,
were added and incubated at 37 C for 3 h followed by a 4-min 15%
glycerol shock in
-MEM. Cells were washed twice with 4 ml TBS, and 4
ml 0.2% calf serum-
- MEM media were added. The medium was changed
24 and 48 h later to serum-free medium and ligands were added.
Cells were washed 4 h after the last addition with 4 ml PBS and
harvested in 0.5 ml lysis buffer (Ligand Pharmaceuticals Inc.,
San Diego, CA). Luciferase light units were measured on a Monolight
2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA).
To test for inhibitory activity, cells were incubated with 1
nM DHT in the presence of increasing concentrations of
ligands. Decreases due to toxicity of up to 1 µM ligand
were monitored using an AR fusion plasmid GAL-A1 (4) coding for a
constitutively active fusion protein containing AR
NH2-terminal residues 1503 linked to the GAL-4
DNA-binding domain. Decreases in constitutive transcriptional activity
after exposure were minimal.
MMTV-Luciferase Assay
Monkey kidney CV1 cells (0.4 x 106 cells per
6-cm dish) were transfected 24 h after plating using calcium
phosphate with 100 ng human AR expression vector pCMVhAR and 5 µg
MMTV-luciferase reporter vector per plate. DNA is added to 0.28
M NaCl, 1.5 mM Na2HPO4,
0.05 M HEPES, pH 7.11 (0.14 ml/plate; 0.28
M NaCl, 1.5 mM Na2HPO4,
50 mM HEPES, pH 7.12); 0.25 M CaCl2
(0.14 ml/plate) was added dropwise with vortexing and incubated 5 min,
followed by the addition of DMEM-H media containing 10% calf serum,
penicillin, streptomycin, and 20 mM HEPES, pH 7.2 (0.8
ml/plate) to minimize particle size, and then incubated for 15 min. The
mixture was added to the aspirated plates followed by the addition of 3
ml DMEM-H containing 10% calf serum and incubated for 4 h at 37
C. Cells are washed twice with TBS, and 4 ml phenol red-free medium
containing 0.2% calf serum was added. Ligands were added and cells
were harvested and assayed as described above for the N/C luciferase
assay.
DNA Mobility Shift Assay
Spodoptera frugiperda (Sf9) cells plated at 3.5
x 106 cells per 6-cm dish or 1 x 107
cells per 10-cm dish were infected for 45 h at 27 C at
multiplicity of infection of 15 with AR recombinant
baculovirus in Autographa californica nuclear polyhedrosis
virus (AcMNPV) coding for full-length human AR, AR1660
coding for the NH2-terminal, DNA-binding, and hinge regions
(amino acid residues 1660), and AR507919 coding for the DNA- and
steroid-binding domains (amino acid residues 507919) (25). The
indicated concentrations of ligands were added 24 h and again
4 h before cell harvest. Cells were washed once in PBS at 4 C,
pelleted, and resuspended in 0.15 ml/6-cm dish or 0.4 ml/10-cm dish in
high-salt extraction buffer containing 0.5 M NaCl, 1
mM EDTA, 1 mM dithiothreitol, 10% glycerol, 10
mM Tris, pH 7.4 with protease inhibitors, 40
µM leupeptin, 5 µM aprotinin, 10
µM pepstatin A, 2 mM Pefabloc, 5
mM benzamidine, and 10 mM
-amino-n-caproic acid. Cells were frozen and thawed three
times, incubated on ice for 40 min, and microfuged for 15 min.
Supernatants were dialyzed against the above buffer except containing
25 mM KCl and 0.5 mM phenylmethylsulfonyl
fluoride as the only protease inhibitor. The reaction mixture contained
approximately 20 µg total cell protein of either full-length AR,
AR1660 (N), or AR507919 (C), or when combined, 10 µg total
protein each for extracts of N and C. The reaction mix also contained 4
µg of poly dI-dC, 80 µg BSA, and DNA-binding buffer (25
mM KCl, 10% glycerol, 0.2 mM EDTA, 1
mM dithiothreitol, 10 mM Tris-HCl, pH 7.5) to a
final volume of 20 µl. 32P-labeled oligonucleotides
(0.20.3 ng, 20,00025,000 cpm) were added and incubated for 1 h
on ice. Annealing oligos
5'-CGACCAGAGTACGTGATGTTCTCAGG-3' with
AccI-5' compatible end and
5'-GATCCCTGAGAACATCACGTACTCTGGT-3' with 3'
BamHI compatible end were 32P-labeled using the
Klenow fragment of DNA polymerase. The androgen response element
(underlined) derives from the 0.5-kb first intron fragment
of the rat C3 prostatein gene (61). Before electrophoresis, 2 µl
0.2% bromophenol blue were added, and the 5% nondenaturing acrylamide
gel was preelectrophoresed at 100 V for 30 min at 4 C. Samples are
electrophoresed at 150 V for 4 h at 4 C. Gels are dried under
vacuum at 80 C for 1 h and exposed to Biomax MR x-ray film
(Eastman Kodak, Rochester, NY) at -80 C.
AR Stabilization
Full-length AR was expressed from pCMVhAR (8 µg) in COS cells
(1.2 x 106 cells/10-cm dish) transfected using
DEAE-dextran. After 48 h, cells were incubated in methionine-free
medium for 20 min followed by the addition of methionine-free medium
containing 100 µCi [35S]L-methionine/cysteine (PRO-MIX,
Amersham, >1000 Ci/mmol) in vitro labeling mix. Cells were
incubated for increasing times in the presence of the indicated
concentrations of ligands, washed twice with PBS, and harvested in RIPA
buffer and immunoprecipitated using AR52 antipeptide AR antibody as
previously described (22).
 |
ACKNOWLEDGMENTS
|
---|
We are grateful for the technical assistance of K. Michelle
Cobb, Christy Lambright, and De-Ying Zang; to Frank S. French for
helpful discussions and reading the manuscript; and D. Gallet and D.
Martini at Hoechst Marion Roussel (Roussel Uclaf) for labeled and
unlabeled RU56187.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. E. M. Wilson, Laboratories for Reproductive Biology, CB 7500, Room 374, Medical Sciences Research Building, University of North Carolina, Chapel Hill North Carolina 27599. emw{at}med.unc.edu
This work was supported by Grants HD-16910 and IU54-HD-35041 from the
National Institute of Child Health and Human Development Center for
Population Research, and by ES-08265 from the National Institute of
Environmental Health Sciences.
1 Present address: Departamento de Biotecnología, Instituto de
Investigaciones Biomédicas, Universidad Nacional Autónoma
de México, México, D. F. México. 
2 Present address: Monsanto Company, 645 South Newstead Avenue, St.
Louis, Missouri 63110. 
Received for publication May 20, 1998.
Revision received November 4, 1998.
Accepted for publication December 1, 1998.
 |
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