Repression of cAMP-Induced Expression of The Mouse P450 17
-Hydroxylase/C17-20 Lyase Gene (Cyp17) by Androgens
María Burgos-Trinidad1,
Geri L. Youngblood2,
Medardo R. Maroto,
Arno Scheller,
Diane M. Robins and
Anita H. Payne
Department of Obstetrics and Gynecology (M.B.-T., G.L.Y.,
M.R.M., A.H.P.) and The Reproductive Sciences Program (M.B.-T.,
D.M.R., A.H.P.) Department of Human Genetics (A.S., D.M.R.)
Department of Biological Chemistry (G.L.Y., A.H.P.) The University
of Michigan Ann Arbor, Michigan 48109
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ABSTRACT
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In primary cultures of mouse Leydig cells,
testosterone represses the cAMP-induced de novo synthesis
of P450 17
-hydroxylase/C17-20 lyase (P450c17) protein and the
accumulation of P450c17 mRNA, via an androgen receptor (AR)-mediated
mechanism. To examine the mechanism by which androgens repress the
cAMP-induced expression of the mouse Cyp17 gene, constructs
containing 5'-flanking sequences of the mouse Cyp17 linked
to the chloramphenicol acetyltransferase (CAT) reporter gene were
cotransfected into MA-10 tumor Leydig cells with a mouse AR expression
plasmid. In the presence of dihydrotestosterone, the cAMP-induced
expression of a reporter construct containing -1021 bp of
Cyp17 promoter sequences was repressed. In contrast, no
repression by dihydrotestosterone was observed when the -1021 bp
Cyp17-CAT construct was cotransfected with a human AR
expression plasmid missing the second zinc finger of the DNA-binding
domain, indicating that DNA binding is involved in AR-mediated
repression of Cyp17 expression. Analysis of deletions of
the -1021 bp fragment demonstrated that -346 bp of 5'-flanking region
of the mouse Cyp17 promoter are sufficient to confer
androgen repression of the cAMP-induced expression of
Cyp17. Deoxyribonuclease I footprinting analysis indicated
that the AR interacts with sequences between -330 and -278 bp of the
Cyp17 promoter. This region overlaps with the previously
identified cAMP-responsive region located between -346 and -245 bp of
the Cyp17 promoter. These results suggest that AR-mediated
repression involves binding of the AR to sequences in the
cAMP-responsive region of the Cyp17 promoter, possibly
interfering with the binding of the protein(s) that mediate cAMP
induction of Cyp17.
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INTRODUCTION
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The biosynthesis of testosterone in Leydig cells requires
the activities of the cytochrome P450 enzyme, 17
-hydroxylase/C17-20
lyase (P450c17). This enzyme is a single polypeptide catalyzing two
sequential reactions, the hydroxylation of the C21 steroid progesterone
at carbon 17, followed by the cleavage of the C17-20 bond to produce
androstenedione, the immediate precursor of testosterone (1). Studies
from this laboratory, using primary cultures of mouse Leydig cells,
demonstrated that cAMP is essential for the induction of P450c17 enzyme
activity (2), de novo synthesis of P450c17 protein (3, 4),
and the expression of P450c17 mRNA (5). The synthesis of P450c17 ceases
in the absence of cAMP (3). The cAMP-mediated induction of P450c17 mRNA
requires newly synthesized proteins (5). Furthermore, testosterone,
which is produced by the Leydig cells upon stimulation of the cells
with cAMP, negatively regulates cAMP induction of P450c17 activity (6),
de novo synthesis (4), and levels of mRNA (5). When
testosterone production is inhibited by the addition of
aminoglutethimide (AG), an inhibitor of cholesterol metabolism, the
cAMP-induced increase in P450c17 enzyme activity, de novo
synthesis (4), and the expression of P450c17 mRNA are enhanced (5). The
repression caused by testosterone can be mimicked by the addition of
the androgen agonist, mibolerone, and prevented by the addition of the
androgen antagonist, hydroxyflutamide (4). The synthetic
glucocorticoid, dexamethasone, and estradiol have no effect on cAMP
induction of P450c17 mRNA levels (5). These results indicate that
testosterone represses cAMP-induced synthesis and mRNA accumulation of
mouse P450c17 via an androgen receptor (AR)-mediated mechanism.
To investigate the molecular mechanisms involved in the regulation of
mouse P450c17 by cAMP and steroid hormones in Leydig cells, the
structural gene encoding P450c17 was previously isolated and
characterized (7). In transient transfections of MA-10 tumor Leydig
cells with Cyp17 promoter-driven reporter constructs, a
region necessary for cAMP induction of the gene was localized to
between -346 and -245 bp relative to the transcription initiation
site and is referred to as the cAMP-responsive region or CRR (8). No
functional cAMP response element was identified in this fragment,
indicating that cAMP-induction of Cyp17 is not mediated by
the cAMP-response element-binding protein (8). Gel mobility shift
assays using nuclear extracts from cAMP-treated MA-10 cells
demonstrated that cAMP induced a protein or proteins that binds
specifically to the CRR of Cyp17 (9). The present study was
designed to examine the mechanism(s) by which androgens, via the AR,
negatively regulate the cAMP-induced expression of the Cyp17
gene.
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RESULTS
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To examine whether mouse MA-10 tumor Leydig cells contain
endogenous AR, cells were transiently transfected with the
2X(HRE)TATACAT reporter plasmid (see Materials and Methods)
and treated for 12 h with the androgen, dihydrotestosterone (DHT).
Treatment of the transfected cells with DHT did not increase
chloramphenicol acetyltransferase (CAT) activity relative to control,
indicating that MA-10 cells do not contain endogenous AR (Fig. 1
). In contrast, cotransfection of 2X(HRE)TATACAT with
the mouse AR (mAR) expression vector resulted in a minor increase in
the basal expression of 2X(HRE) TATACAT, which was markedly increased
by treatment with DHT. Similar results were obtained using a different
androgen-responsive CAT reporter construct, the 2X(HRE)tkCAT,
containing two copies of a hormone response element (HRE) from the
sex-limited protein (Slp) gene, in front of the thymidine kinase (tk)
promoter. In addition, a hormone-binding assay was used to examine for
the presence of endogenous AR in MA-10 cells, by measuring specific
binding of the androgen agonist [3H]R1881 (Table 1
). MA-10 cells exhibited specific binding of
[3H]R1881 in the absence of transfected AR expression
plasmid, indicating that MA-10 cells do contain endogenous AR. However,
from the functional data presented above (Fig. 1
), endogenous AR
appears nonfunctional or alternatively, the concentration is too low to
mediate induction of androgen-responsive reporter constructs, or
androgen repression of the cAMP-induced expression of the
Cyp17-CAT constructs.

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Figure 1. MA-10 Tumor Leydig Cells Do Not Contain Functional
ARs
MA-10 cells were transfected with 5 µg of the 3X(HRE)tkCAT or 5 µg
of the 2X(HRE)TATACAT reporter plasmids in the presence or absence of
the mAR expression plasmid (0.5 µg). All plates received the same
total amount of DNA by adding appropriate amounts of the parent vector
pCMV5, lacking the mAR sequences. After 24 h of incubation, cells
were untreated or treated with 0.1 µM DHT for 12 h
as indicated, and the relative amount of CAT activity was determined as
described in Materials and Methods.
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Repression of cAMP Induction of Cyp17 Gene Expression
by Androgens
To examine the effect of androgens on the cAMP-induced
expression of Cyp17, a construct containing -1021 bp of
5'-flanking sequences of the mouse Cyp17 promoter in front
of the CAT reporter gene (-1021 bp Cyp17-CAT) was
cotransfected into MA-10 cells with increasing amounts of mAR
expression vector, and cells were treated with cAMP or cAMP plus DHT.
In the absence of mAR, DHT had no significant effect on the
cAMP-induced CAT expression. Increasing amounts of the mAR caused a
dose-dependent repression of the cAMP-induced expression of the -1021
bp Cyp17-CAT construct in the absence of DHT (Fig. 2
). Addition of DHT to cAMP-treated cells resulted in a
further decrease in CAT expression. Similar results were obtained when
the -1021 bp Cyp17-CAT construct was cotransfected with
increasing amounts of a rat androgen receptor (rAR) expression vector
(10). These results demonstrate that repression of cAMP-induced
expression of P450c17 mRNA and protein synthesis reflects repression at
the level of transcription and is mediated by the androgen receptor and
that -1021 bp of 5'-flanking sequences are sufficient to mediate
repression of Cyp17-CAT expression by the mAR.

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Figure 2. Effect of mAR on cAMP-Induced Expression of the
-1021-bp Cyp17-CAT Construct
MA-10 cells were transiently transfected with the -1021-bp
Cyp17-CAT construct (5 µg) and increasing amounts of a
mAR expression plasmid, as indicated. All plates received the same
total amount of DNA as described in Fig. 1 . After 24 h, all the
cells were treated with 100 µM aminoglutethimide (AG) in
the absence or presence of 500 µM cAMP or cAMP plus 0.1
µM DHT. The relative CAT activity of -1021 bp
Cyp17-CAT in the presence of cAMP and in the absence of
mAR was arbitrarily set at 100. Values represent the averages of
duplicate plates from a representative experiment.
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A Functional DNA-Binding Domain (DBD) of the AR Is Required to
Mediate Repression
To investigate whether the DBD of the AR is necessary for
androgen-mediated repression of cAMP-induced expression of
Cyp17, the effect of a human AR (hAR)-DBD mutant expression
plasmid on the cAMP-induced expression of Cyp17 was
examined. The hAR-DBD mutant (DBDm) is missing the second zinc finger
of the DBD (11). This mutant has normal androgen-binding affinity and
localizes in the nucleus but has reduced DNA binding and failed to
activate transcription of an androgen-responsive reporter gene (11).
MA-10 cells were cotransfected with the -1021 bp Cyp17-CAT
construct and increasing amounts of the DBDm expression plasmid (Fig. 3
). The DBDm did not repress the cAMP-induced expression
of -1021 bp Cyp17-CAT in the absence or presence of DHT. In
contrast, the wild type hAR repressed the cAMP-induced expression of
-1021 bp Cyp17-CAT to basal levels of expression when cells
were treated with DHT (Fig. 3
). The DBDm was also tested in
cotransfections with the 2X(HRE)TATACAT construct, and no increase in
CAT expression of this construct was observed when cells were treated
with DHT (data not shown), thus confirming that the DBDm cannot mediate
induction of a hormone-responsive reporter gene. Interestingly, in
contrast to the mAR or the rAR (10), the hAR did not exhibit nearly the
same extent of androgen-independent repression of Cyp17-CAT
expression even at 2 µg of hAR expression vector. However, the extent
of ligand-independent repression at a given concentration of mAR was
found to be variable (compare Fig. 2
and Fig. 4
).

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Figure 3. DNA Binding Is Involved in AR-Mediated Repression
of Cyp17-CAT Expression
MA-10 cells were transiently transfected with the -1021-bp
Cyp17-CAT construct (5 µg) and increasing amounts of
the hAR expression plasmid or the hAR-DBDm expression plasmid, as
indicated. All plates were corrected to the same total amount of DNA
with the parent vector pCMV5. After 24 h, cells were treated with
100 µM AG in the absence or presence of 500
µM cAMP or cAMP plus DHT. The relative CAT activity of
the -1021-bp Cyp17-CAT in the presence of cAMP and in
the absence of receptor expression plasmid was arbitrarily set at 100.
Values represent the mean ± SD from duplicate plates
in three separate experiments.
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Figure 4. Localization of Cyp17-Regulatory
Regions Mediating Androgen Repression
MA-10 cells were transiently transfected with the indicated
Cyp17-CAT constructs (5 µg), in the absence (-) or
presence (+) of 0.5 µg of the mAR expression plasmid. All cultures
were corrected for the same amount of DNA as described in Fig. 1 . All
cultures were treated for 12 h with 100 µM AG in the
absence or presence of 500 µM cAMP or cAMP plus DHT. The
relative CAT activity of -1021 bp Cyp17-CAT in the
presence of cAMP and in the absence of receptor expression plasmid was
arbitrarily set at 100. Values represent the average ± the range
of duplicate plates in two separate experiments.
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To demonstrate that the DBDm was expressed to a similar extent as wild
type hAR in transfected cells, the amount of each receptor expressed in
MA-10 cells was determined by measuring the specific binding of the
androgen agonist [3H]R1881 (Table 1
). The transiently
transfected DBDm was expressed in MA-10 cells, although the level of
[3H]R1881 bound is about 50% of that observed with the
wild type hAR. It is unlikely that this difference in expression could
account for the complete loss of repression since even up to 2 µg
DBDm expression plasmid failed to show any effect, compared with 0.25
µg hAR. The hAR has the highest affinity for R1881. Dexamethasone
failed to inhibit binding of [3H]R1881. Estradiol
competes for [3H]R1881 binding with 50% inhibition at
100-fold molar excess concentration, in agreement with previous reports
(12). Taken together, these results indicate that a functional DBD of
the AR is required to observe repression and suggest the involvement of
DNA binding in AR-mediated repression of Cyp17-CAT
expression.
Localization of the DNA Sequences in the Cyp17 Promoter
Required for Androgen Repression
To identify sequences in the Cyp17 promoter that
mediate androgen repression of cAMP induction of Cyp17, CAT
reporter constructs containing different lengths of 5'-flanking
sequences of the Cyp17 gene were transiently transfected
into MA-10 cells in the absence or presence of a mAR expression
plasmid. Cells were treated for 12 h with cAMP or cAMP plus DHT.
The cAMP-induced expression of each of the constructs was inhibited by
about 3040% in the presence of mAR (Fig. 4
).
Treatment with DHT resulted in a further decrease in CAT expression of
each of the constructs, to near basal levels of expression. These
results indicate that the first -346 bp of Cyp17 are
sufficient to bring about ligand-dependent repression by the mAR of
cAMP-induced Cyp17-CAT expression.
Localization of AR-Binding Sites in the Cyp17 Gene
To determine whether AR can interact directly with sequences
within the first -346 bp of the Cyp17 promoter and to
identify the specific sequences involved in mediating repression,
deoxyribonuclease I (DNase I) footprinting experiments were performed
using baculovirus extract containing AR. The results shown in Fig. 5A
indicate that AR interacts with sequences within the
Cyp17 promoter. Two footprints were observed when the
Cyp17 promoter fragment (-346 to -72) was incubated with
AR (Fig. 5A
). The first region protected, androgen response element 1
(ARE-1), is found between -330 and -313 and comprises the sequence
5'-AATTATTAACTGTGCAGC-3', and the second androgen-response element
(ARE-2) lies between -306 and -278 and has the sequence
5'-GACATTACAGCACGCACTCTGAAACCTTG-3'. A more pronounced protection
of these two regions was observed with higher amounts of AR (Fig. 5A
).
Both sequences share little homology with the consensus sequences for
typical ARE or negative glucocorticoid response element-like sequences
(13). Under identical experimental conditions AR protected a region in
the Slp-enhancer fragment (Fig. 5B
) encompassing tandem HRE-like
sequences (14, 15). The protected fragments in both the
Cyp17 promoter and the Slp-enhancer fragment were
specifically competed by a double-stranded oligonucleotide containing a
single copy of HRE-3 from the Slp gene (16). These results indicate
that AR interacts with sequences within the -346 and -245-bp CRR
region of the Cyp17 promoter (8). Binding of AR to this
region may overlap or interfere with the binding of transcription
factor(s) essential for cAMP induction of Cyp17 gene
expression.

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Figure 5. Localization of Potential AR-Binding Sites in the
Cyp17 Promoter
A, A 275-bp fragment of the Cyp17 promoter (-346 to
-72 bp) was end-labeled with 32P and used to analyze AR
binding in DNase I protection assays as described in Materials
and Methods. Radiolabeled DNA was incubated in the absence
(lane 1) or in the presence of 25 (lane 2), 50 (lanes 3 and 6), 75
(lane 4), or 100 (lane 5) µg of baculovirus extracts containing AR or
100 µg (lane 7) of extract from noninfected Sf9 cells. Lane 6
contains HRE competitor oligonucleotide at 500-fold over the
concentration of probe. B, A 270-bp fragment containing 160 bp from the
androgen-responsive enhancer of the mouse Slp gene and including
several sequences similar to HREs, was end-labeled and incubated in the
absence (lane 1) or in the presence of 40 (lane 2) or 50 (lanes 3 and
4) µg of baculovirus extracts containing AR or 50 µg Sf9 cell
extract (lane 5). Lane 4 contains HRE competitor at 500-fold over the
concentration of probe. Lane M is the A+G sequencing reaction of the
radiolabeled DNA fragments. The numbers on the left side
of the figures correspond to nucleotide sequence in the
Cyp17 gene (A) or the Slp gene (B). Protected regions
are indicated by lines on the right side.
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DISCUSSION
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The expression of P450c17 is essential for testosterone
biosynthesis in Leydig cells. Previous studies have shown that
expression of P450c17 is absolutely dependent on cAMP (3), and
androgens negatively regulate the cAMP-induced expression of P450c17
via an AR-mediated mechanism (4). In the present study we sought to
investigate the molecular mechanism(s) by which androgens repress the
cAMP-induced expression of the mouse Cyp17 gene. The
steroid-binding data presented in these studies indicates that MA-10
cells contain endogenous AR, consistent with previous results
demonstrating the presence of AR in purified rat Leydig cells (17).
However, androgens failed to induce CAT activity from
androgen-responsive reporter constructs, indicating that the amount of
endogenous AR present in MA-10 cells is not sufficient to mediate
induction of androgen-reponsive genes or that the receptor is not
functional. Therefore, to examine whether AR was involved in mediating
androgen repression, MA-10 cells were cotransfected with AR expression
vectors and Cyp17 promoter constructs. Herein, we report
that in cotransfection studies androgens repress the cAMP-induced
expression of Cyp17-CAT reporter constructs. We have shown
that -346 bp of the Cyp17 promoter are sufficient to confer
androgen repression of cAMP-induced Cyp17 expression and
that a functional DBD of the AR is required to observe repression.
Evidence is presented indicating that the AR interacts directly with
sequences within the region between -346 and -245 bp upstream of the
start site of transcription, which has been demonstrated previously to
be required for cAMP induction of the Cyp17 gene (8).
Interestingly, androgen-independent repression of cAMP-induced
expression of Cyp17 was observed in MA-10 cells transfected
with the mAR or the rAR (10), but not with the hAR even at 2 µg hAR
expression vector. Ligand-independent repression by the mAR (18) and
the hAR (19) has been reported previously and attributed to elevated
levels of receptor interacting with DNA in the absence of ligand,
combined with competition for basal transcription factors by the
cytomegalovirus (CMV) promoter (19). Since the hAR, mAR, and rAR were
expressed via the same CMV promoter, competition for transcription
factors by the CMV promoter is unlikely to account for any species
difference. However, the extent of ligand-independent repression
observed may reflect differences in the levels of expression of the
different AR plasmids transfected into MA-10 cells, rather than an
intrinsic species-type difference between the receptors.
The mechanism(s) by which steroid hormones repress gene transcription
appears to be different from the mechanisms involved in activation (13, 20, 21). Several mechanisms have been proposed to explain
transcriptional repression based on investigations involving the
glucocorticoid receptor (GR), which has been shown to repress
expression of a variety of genes. These mechanisms include interactions
with negative glucocorticoid response elements (nGRE); these sequences
differ from positive GRE sequences, which mediate enhancement of
transcription (22, 23, 24), interaction of the GR with promoter elements
preventing or interfering with the binding of positive transcription
factors (25, 26), and protein-protein interactions between GR and other
factors required for transcriptional activation (27, 28, 29, 30). nGREs differ
from the positive GREs not only in sequence but between each other, so
that no clear consensus exists (13). In the first mechanism, the
receptor is thought to bind nGRE with a lower affinity than the
positive GREs or in a different conformation, resulting in decreased
transcriptional activity (22). In addition, a nGRE may be negative in
some tissues and act as a positive response element in others as is the
case of the proliferin gene where positive or negative regulation is
determined by the relative amounts of c-jun and
c-fos (AP-1), which differ in different cell types (31). The
second mechanism was first proposed for the human glycoprotein hormone
-subunit gene (25). The interaction of the GR with sequences
adjacent to the cAMP-response element (CRE) blocked the binding of the
transcription factor, the cAMP-response element-binding protein, thus
preventing transcriptional activation. Further studies demonstrated
that this repression requires the DBD of GR (26). However, subsequent
studies have shown that DNA binding of GR to the glycoprotein hormone
-subunit gene may not be necessary for repression (28), even though
purified GR interacts with the promoter in vitro (25). As
examples of the third mechanism, inhibition of the collagenase I
induction by glucocorticoids involves repression of AP-1 activity by GR
involving protein-protein interactions between the DBD of GR and the
DNA-binding region of the jun monomer (30). In contrast, inhibition of
rat PRL by glucocorticoids does not appear to require the DBD of GR but
involves protein-protein interactions between GR and other
transcription factors resulting in repression (27). Any of these
mechanisms could contribute or account for the negative regulation of
Cyp17 expression by androgens.
Androgens have been documented to repress mRNA levels of several genes,
including the testosterone-repressed prostate message TRPM-2 (32, 33)
and transforming growth factor ß (34). In the human prostate tumor
cell line LNCaP, AR mRNA expression is down-regulated by androgen
treatment (35). However, it is not known whether this repression is
mediated by a direct action of the activated AR on the transcription of
these genes. With regard to negative gene regulation meditated by AR,
there are only three examples in which transfection and DNA-binding
data have been presented. The expression of the mAR gene is induced by
cAMP and repressed by androgens (18). Clay et al. (19) have
shown that repression of the glycoprotein hormone
-subunit gene by
androgen may involve direct binding of AR to the proximal promoter
(19). They proposed that binding of AR may block the binding of a
transcription factor. In contrast, androgen repression of the
low-affinity neurotrophin receptor (p75) does not require a direct
binding of the AR with specific DNA elements (36), even though an
intact DBD of the receptor was required to observe repression.
Our data demonstrate that binding of the AR to the Cyp17
promoter is essential for mediating androgen repression of
cAMP-induction of Cyp17 transcription. Deletion of the
second zinc finger of the DBD of the hAR abolished the ability of AR to
repress the cAMP-induced Cyp17-CAT expression. However, the
possibility that deletion of the second zinc finger of the AR-DBD
disrupts some unknown protein-protein interactions that accounts for
the loss of repression, rather than disruption of AR-DNA binding,
cannot be excluded at this time. To provide additional evidence for the
role of DNA binding, we examined whether AR can interact directly with
sequences within this region by DNase I footprinting experiments. AR
binds in vitro to two distinct DNA sites in the
Cyp17 promoter. The interaction of AR with Cyp17
DNA sequences was weak, relative to the high-affinity interaction of AR
with the consensus HREs from the Slp gene (14, 15). However,
interaction of AR with negative AREs (nAREs) may be weaker than with
positive AREs (22). In addition, computer analysis of the first -346
bp of Cyp17 did not identify any putative ARE (37) or
nGRE-like motifs in this region (13), and thus the sequences protected
by AR share no sequence homology with reported consensus HREs.
Together, these results suggest a role of DNA binding in the androgen
repression of Cyp17 expression. However, mutagenesis studies
are necessary to demonstrate that DNA binding of receptor demonstrated
in vitro, is also required in vivo.
The molecular mechanism(s) by which the AR represses the cAMP-induced
expression of Cyp17 is not yet clear. cAMP is absolutely
essential for Cyp17 expression (38), and previous studies
have demonstrated that the CRR of the Cyp17 promoter is
located between -346 and -245 bp (8). This 101-bp fragment overlaps
with the DNA sequences recognized by AR in DNase I footprinting. The
protein(s) required for Cyp17 expression in Leydig cells, as
well as the specific sequences within the CRR that mediate the
cAMP-induced expression of Cyp17, remain to be identified.
Taken together, our results suggest that androgens can inhibit the
expression of Cyp17 by a mechanism involving the binding of
AR to regions in the Cyp17 promoter in a manner that
interferes or prevents the binding of factors required for cAMP
activation of Cyp17 gene expression.
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MATERIALS AND METHODS
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Materials
DL-Aminoglutethimide was obtained from Aldrich
(Milwaukee, WI). Waymouths MB752/1 medium and horse serum were
obtained from GIBCO-BRL Life Technology (Gaithersburg, MD).
8-Bromo-cAMP, HEPES, testosterone, estradiol, and dexamethasone were
obtained from Sigma (St. Louis, MO). 5
-DHT was obtained from Sigma.
Unlabeled R1881 and [3H]methyltrienolone
[17
-methyl-3H]R1881, 80 Ci/mmol) were obtained from
DuPont-New England Nuclear (Boston, MA).
Plasmids
CAT reporter plasmids containing different length fragments of
the mouse Cyp17 promoter have been described previously (8).
The pGRE2CAT reporter plasmid [henceforth referred to as
2X(HRE)TATACAT], which contains two copies of a GRE consensus sequence
derived from the aminotransferase gene in front of the adenovirus E1b
TATA sequence, was a gift from Dr. John A. Cidlowski (39). The
3X(HRE)tkCAT reporter plasmid contains a trimer of HRE-3 of the mouse
Slp gene fused to the herpes simplex virus thymidine kinase (tk)
promoter (14). The mAR expression plasmid in the pcDNAI/neo expression
vector was obtained from Dr. Don J. Tindall (40) and subcloned into the
pCMV5 expression vector. The hAR expression vector containing the
full-length AR cDNA in the pCMV5 expression plasmid (41) and the
hAR-DBDm missing the second zinc finger of the receptor (11) were a
gift from Dr. Frank S. French and Dr. Elizabeth M. Wilson. The rAR
recombinant baculovirus was generously provided by Dr. Olli A.
Jänne (42). Spodoptera frugiperda (Sf9) insect cells
were infected with the recombinant baculovirus, and extracts were
prepared as previously described (43).
Cell Culture and Transfections
MA-10 mouse tumor Leydig cells (a generous gift from Dr. Mario
Ascoli) were grown in Waymouths MB 752/1 medium containing 15% horse
serum, 20 mM HEPES, and gentamicin. For transfections,
cells were plated in 60- mm dishes at approximately 1 x
106 cells per dish 4048 h before transfection. Four hours
before transfection, cells were fed fresh media and transfected in
duplicate with the indicated amounts of DNA plasmids by the calcium
phosphate precipitation method (44). Calcium phosphate DNA precipitates
to be used for several plates were prepared in one solution and equally
distributed over all plates (11 µg DNA/plate). Empty vector was added
to maintain the same total amount of pCMV5 expression plasmid per
transfection. Four hours after addition of the DNA precipitate, cells
were treated with 15% glycerol (23 min), the plates were washed with
PBS to remove the glycerol, and fresh medium containing 15%
charcoal-stripped serum was added to all plates. After 24 h of
incubation, cells were treated with fresh media containing
charcoal-stripped serum and 100 µM aminoglutethimide (AG)
plus or minus the indicated factors for 12 h. AG and steroids were
added from methanolic stock solutions, and the final concentration of
methanol was 0.4% in all treatment media. AG was added to block the
production of progesterone, which is produced by the MA-10 cells upon
treatment of cells with cAMP. Progesterone can bind to the AR although
with lower affinity than the androgen, DHT. MA-10 cells do not express
the Cyp17 gene even after treatment of the cells with cAMP
(38) and therefore do not produce testosterone. Cells were treated with
500 µM cAMP, a concentration that results in maximal
induction of expression of Cyp17-CAT constructs in MA-10
cells (8). Cell extracts were assayed for CAT activity by measuring the
amount of [3H]acetylated chloramphenicol produced during
a 2-h incubation as previously described (8). All cultures were
cotransfected with 4 µg SV2-ß-gal to correct for transfection
efficiency. Results are expressed relative to ß-galactosidase
activity.
Androgen-specific binding of hAR and hAR-DBDm was determined in MA-10
cells transiently expressing the recombinant AR using a whole
cell-binding assay previously described, with minor modifications (45).
Cells were transfected with 5 µg of the indicated AR expression
plasmid (hAR or DBDm), or with the parent vector lacking the AR
sequences (pCMV5), as previously described. Control plates received no
DNA. Four hours after addition of the DNA precipitate, cells were
treated with 15% glycerol (23 min), the plates were washed with PBS
to remove the glycerol, and fresh medium containing 15%
charcoal-stripped serum was added to all plates. Forty hours after
transfection, the medium was removed and the cells were washed twice
with warm PBS and once with serum-free Waymouths MB 752/1 media.
Cells were incubated for 2 h with 5 nM
[3H]methyltrienolone (R1881) in duplicate dishes in the
presence or absence of 100-fold molar excess unlabeled steroids.
Labeling medium was removed, and the cells were washed three times with
cold PBS containing 0.1% BSA. The cells were harvested in 2% SDS,
10% glycerol, and 10 mM Tris, pH 6.8, and radioactivity
was determined by scintillation counting. Binding is expressed as a
percentage of [3H]R1881 bound in the absence of unlabeled
steroid. Values represent the average of duplicate plates.
DNase I Footprinting Assay
A 275-bp fragment of the Cyp17 promoter corresponding
to bases -346 to +72 bp was labeled at the 5'-end with T4
polynucleotide kinase and [
-32P]ATP (7000 Ci/mmol, ICN
Pharmaceuticals, Costa Mesa, CA) and purified on a 5% polyacrylamide
gel. A 270-bp fragment containing 160 bp from the androgen-responsive
enhancer of the mouse Slp gene, and which includes several sequences
similar to HREs (14, 15, 16), was labeled as above and used as a positive
control. DNAse I footprinting assays were performed as described (15)
with the following modifications. Extracts from noninfected SF9 cells
or baculovirus extracts containing AR (43) were preincubated with 1
µg poly(deoxyinosinic-deoxycytidylic acid) for 20 min on ice in
footprinting buffer containing 20 mM HEPES (pH 7.9), 20%
glycerol, 100 mM KCl, 2 mM dithiothreitol, 0.2
mM EDTA, 0.05% Nonidet P-40, 0.5 mM
phenylmethylsulfonyl fluoride, 50 nM DHT, 5
µM leupeptin, 5 µM pepstatin, and 0.3
µM aprotinin. Where indicated, preincubations contained a
specific 30-bp competitor oligonucleotide corresponding to one copy of
HRE-3 from the Slp enhancer (16) at 500-fold molar excess over the
concentration of probe. Radiolabeled DNA (30,000 cpm,
0.5-2 ng) was
added (50 µl final reaction volume) followed by a 1-h incubation on
ice. MgCl2 and CaCl2 were added to a final
concentration of 2 mM, before DNase I digestion with 188 ng
DNase I (Worthington Biochemicals, Freehold, NJ) for 5 min on ice.
Naked DNA was cleaved with 54 ng DNase I. Reactions were terminated by
addition of 85 µl DNase I stop solution [100 mM Tris (pH
8.0), 100 mM NaCl, 1% sodium lauroyl sarcosine, 10
mM EDTA, 100 µg/ml proteinase K, and 25 µg/ml sheared
salmon sperm DNA] and incubation at 37 C for 30 min and then 80 C for
2 min followed by phenol/chloroform extraction and ethanol
precipitation. The digestion products were resolved on 8%
polyacrylamide gel containing 7 M urea. The Maxam-Gilbert
A+G sequencing reaction of the radiolabeled fragments run in an
adjacent lane was used to locate the footprinted regions (46).
 |
ACKNOWLEDGMENTS
|
---|
The authors are grateful to Dr. E. M. Wilson and Dr. F. S.
French for generously providing the hAR, rAR, and the hAR-DBDm
expression vectors; to Dr. D. J. Tindall for the mAR expression vector;
to Dr. O. A. Jänne for the recombinant baculovirus rAR; to Dr. J.
A. Cidlowski for the pGRE2CAT plasmid; and to Dr. Mario Ascoli for
providing the MA-10 cells. We thank Shelly F. Bender and Christa B.
Williams for technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Anita H. Payne, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, 300 Pasteur Drive, Stanford, California 94305-5317.
This work was supported by NIH Grants HD-08358, HD-17916 (to A.H.P.),
and GM-31546 (to D.M.R.) and by the National Research Service Award
HD-07672 (to M.B.-T.) and P30-HD-18258 (to the Molecular Biology Core
of the Reproductive Sciences Program).
1 Current address: University of Michigan Medical Center, 5562 MSRBII,
1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0678. 
2 Current address: Lineberger Cancer Center, CB 7295, University of
North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599. 
Received for publication June 25, 1996.
Revision received September 12, 1996.
Accepted for publication October 7, 1996.
 |
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