Androgen Suppression of GnRH-Stimulated Rat LHß Gene Transcription Occurs Through Sp1 Sites in the Distal GnRH-Responsive Promoter Region
Denis Curtin,
Shannon Jenkins,
Nicole Farmer,
Alice C. Anderson,
Daniel J. Haisenleder,
Emilie Rissman,
Elizabeth M. Wilson and
Margaret A. Shupnik
Departments of Pharmacology (D.C.), Internal Medicine (S.J., N.F.,
A.C.A., D.J.H., M.A.S.), and Biology (E.R.), University of Virginia,
Charlottesville, Virginia 22908; and Department of Pediatrics and
Department of Biochemistry and Biophysics (E.M.W.), University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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ABSTRACT
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Steroids may regulate LH subunit gene transcription by modulating
hypothalamic GnRH pulse patterns or by acting at the pituitary
gonadotrope to alter promoter activity. We tested direct pituitary
effects of the androgen dihydrotestosterone (DHT) to modulate the rat
LHß promoter in transfected LßT2 clonal gonadotrope cells and in
pituitaries of transgenic mice expressing LHß-luciferase. The LHß
promoter (-617 to +44 bp)-luciferase construct was stimulated in
LßT2 cells 7- to 10-fold by GnRH. Androgen treatment had little
effect on basal promoter activity but suppressed GnRH stimulation by
approximately 75%. GnRH stimulation of LHß was also suppressed by
DHT in isolated pituitary cells from male or female mice with
functional nuclear ARs, but not in male littermates with mutant AR.
GnRH stimulation of the LHß promoter requires interactions between a
complex distal response element containing two specificity protein-1
(Sp1) binding sites and a CArG box, and a proximal element with
two bipartite binding sites for steroidogenic factor-1 and early
growth response protein-1 (Egr-1). DHT effectively suppressed promoter
constructs with an intact distal response element. The distal response
element does not bind AR, but AR reduces Sp1 binding to this region.
Glutathione-S-transferase pull-down studies demonstrated
direct interactions of AR with Sp1, which requires the DNA-binding
domain of AR, and weaker interactions with Egr-1. We conclude that
androgen suppression of the rat LHß promoter occurs primarily through
direct interaction of AR with Sp1, with some possible role through
binding to Egr-1. These interactions result in interference with
GnRH-stimulated gene transcription by reducing cooperation between the
distal and proximal GnRH response elements.
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INTRODUCTION
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EXPRESSION OF THE pituitary gonadotropin
subunit genes are regulated by several physiological signals. These
include pulsatile release of GnRH from the hypothalamus, which
regulates the three gonadotropin genes (
, LHß, and FSHß) in a
subunit-specific, frequency-dependent manner, as well as gonadal
steroids and peptide hormones (1, 2, 3, 4, 5, 6). The sex steroids,
including E, progesterone, and T, stimulate or inhibit gonadotropin
gene transcription by acting on the hypothalamus or the pituitary
(5, 6, 7, 8). At the hypothalamic level, steroids may alter GnRH
pulse patterns and thus indirectly regulate gonadotropin gene
transcription (9, 10). Alternatively, steroids may act
directly on the pituitary gonadotrope to modulate either basal or
GnRH-stimulated gene transcription rates (8, 11).
In rats, both E and T suppress the castration-induced rise in
gonadotropin gene transcription (7, 12), although the
mechanisms for these steroid effects have not been completely defined.
Data from transgenic mice bearing promoter-reporter transgenes for the
human glycoprotein
-subunit, rat LHß subunit, and bovine LHß
subunit promoters suggest that E suppresses gene activity of these
constructs primarily by feedback at the hypothalamus to alter GnRH
pulse patterns, rather than acting directly at the gonadotrope
(13, 14, 15). In contrast, androgens appear to act at least
partially at the level of the gonadotrope to regulate both basal gene
transcription and the responses to GnRH. Human
-subunit gene
promoter activity was directly suppressed by androgens in transient
transfection studies (16). Although the human
-subunit promoter can bind AR directly, androgen suppression is
mediated through gene elements distinct from the receptor binding site
(17). Transcription suppression is proposed to occur
through protein-protein interactions between transcription factors
binding to the promoter in these regions, and the DNA binding and
ligand-binding domains of the AR (17).
The rat LHß promoter is stimulated by GnRH through complex distal and
proximal response elements that interact functionally for full
responsiveness (18, 19, 20, 21, 22, 23). In vivo, the interplay
between androgens and GnRH stimulation is complex. Low physiological
(pM) levels of androgens are required for GnRH
stimulation of rat LHß mRNA levels in female rats treated with
phenoxybenzamine to clamp endogenous GnRH pulses, whereas higher
(nM) androgen levels invariably suppress
stimulation by exogenous GnRH (24). In these
studies, we examined the direct effects of the
nonaromatizable androgen, dihydrotestosterone (DHT), to modulate the
basal or GnRH-stimulated transcription of the rat LHß promoter in
cultured pituitary cells from transgenic animals and clonal gonadotrope
cells. Transient transfection studies were performed in LßT2 cells, a
clonal gonadotrope line that expresses the endogenous LHß and
-subunit genes and the GnRH receptor (25). Androgen
treatment directly suppressed the response of the LHß promoter to
GnRH in pituitary cells, and this effect required both the AR and
upstream GnRH-responsive regions in the LHß gene.
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RESULTS
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Direct Suppression of the Transfected LHß Promoter by DHT
The effects of steroid hormones on the activation of the LHß
promoter were investigated using a luciferase reporter construct
containing the GnRH-responsive LHß promoter region between -617 and
+44 bp. This construct contains both the proximal and distal
GnRH-responsive elements described by several investigators
(18, 19, 20, 21, 22, 23). Transfected cells were treated for 24 h
with 1 nM concentrations of steroid followed by 6 h of
treatment with 10 nM GnRH. GnRH alone stimulated the LHß
promoter approximately 10-fold over control cells. E treatment
increased the GnRH response somewhat to 14-fold, while DHT decreased
the GnRH stimulation to approximately 3-fold (Fig. 1A
). Suppression of the GnRH response was
specific for androgens as it was observed only with DHT, or with T in
separate experiments (not shown), but not with thyroid hormone, E, or
progesterone. Little effect on basal promoter activity was seen with
any hormone. Suppression of the GnRH effect was observed with DHT
concentrations of 10 pM and higher, and was maximally
effective at 1 nM, in reasonable agreement with the
affinity of the nuclear androgen receptor for DHT (Fig. 1B
).

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Figure 1. Suppression of LHß Promoter Activity by DHT
A, LßT2 cells were transfected with 3 µg of -617 LHß-luciferase
reporter construct and treated with 1 nM E (E2), DHT,
T3, or progesterone (P) for 24 h. Some wells in each
steroid treatment group were then treated with 10 nM GnRH
for 6 h. Luciferase activity was measured in cell extracts, and
normalized activity (ALU) is represented as the mean ±
SEM for three experiments each with three wells per group.
*, P < 0.05; **, P < 0.01
GnRH vs. untreated or steroid controls. B, LßT2 cells
were treated with 10 nM GnRH (horizontal
arrow) and varying concentrations of DHT (1 pM
to100 nM) for 24 h. Data are expressed as in panel A
for three experiments with three wells per group in each experiment. *,
P < 0.05; **, P < 0.01
vs. GnRH alone.
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GnRH Receptor mRNA Levels
One potential reason for androgen suppression of GnRH effects
could be a reduction in GnRH receptors. Treatment of LßT2 cells with
either DHT, E, or GnRH under the same conditions used for transfection
assays was followed by measurement of GnRH receptor (GnRH-R)
mRNA (Fig. 2
). None of the steroid
treatments resulted in a decrease in GnRH-R mRNA levels when compared
with control. The level of GnRH-R mRNA was not suppressed but was
slightly greater with DHT treatment when compared with control
(Fig. 2
).

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Figure 2. GnRH-R mRNA Levels in LßT2 Cells After Steroid
Treatment
LßT2 cells were treated with 1 nM DHT or E (E2) for
24 h or 10 nM GnRH for 6 h. Total RNA was
isolated and GnRH-R mRNA was quantified by dot blot analysis and by
calculations from an RNA standard curve spotted on the same filter.
Results are presented as the mean ± SEM for three
experiments, with six to seven samples per group. *,
P < 0.05 vs. untreated controls.
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Requirement for AR
We next tested whether androgen suppression of GnRH-stimulated
LHß promoter activity could be observed in normal gonadotropes, and
whether the androgen receptor was required for the suppression of the
GnRH stimulation response by DHT. To perform these studies, we used
previously characterized transgenic animals that express the
LHß-luciferase reporter transgene in their pituitaries
(14). The LHß promoter driving luciferase activity
contains both distal and proximal GnRH responsive regions. Pituitary
luciferase activity, driven by the LHß promoter, was stimulated when
the animals were castrated and was suppressed when animals were treated
in vivo with steroids or a GnRH antagonist
(14). Thus, the transgene responded identically to the
endogenous LHß gene in rat pituitaries as measured by transcription
run-off assays.
Male mice bearing the LHß-luciferase transgene were bred to
heterozygous testicular feminization (Tfm) female mice, which have a
frameshift mutation in the AR gene on one of their X chromosomes
(38). This breeding resulted in male progeny which all
expressed LHß-luciferase in their pituitaries, but which had either a
functional wild-type or mutant AR. Female littermates also expressed
LHß-luciferase in their pituitaries, but were either homozygous for
wild-type AR or heterozygous for the AR mutation. Pituitary cells from
both groups of male mice and from female mice homozygous for wild-type
AR were cultured and then treated in vitro with GnRH in the
absence and presence of DHT.
As shown in Fig. 3
, GnRH stimulated LHß
promoter activity in cultured pituitary cells from male mice with
wild-type AR (WT-M) or mutant AR (Tfm-M), or in pituitary cells from
female mice with wild-type AR (WT-F). DHT treatment suppressed the
GnRH-stimulatory response in pituitary cells containing wild-type AR
from males or females. In contrast, DHT treatment had no effect on the
GnRH response in pituitary cell cultures from Tfm males (Fig. 3
, middle panel). Thus, DHT can exert a suppressive effect on
the GnRH response in normal gonadotropes, and this effect requires
wild-type AR.

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Figure 3. DHT Suppressive Effects in LHß-Luciferase
Transgene Activity in Transgenic Mouse Pituitary Cells
Male transgenic mice bearing the LHß-luciferase transgene were
crossed with heterozygous female Tfm mice bearing one
mutant AR gene. Male and female offspring of this cross were genotyped
for the presence of wild-type (WT) or mutant (Tfm) AR. Pituitary cells
isolated from both groups of males (WT-M and Tfm-M) and from females
with only wild-type AR (WT-F) were treated in vitro with
media alone (Con), 1 nM DHT, 10 nM GnRH, or
both. Normalized luciferase activity is expressed as the mean ±
SEM for 5 experiments, with 914 determinations per group.
**, P < 0.01 vs. untreated
controls.
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The Upstream GnRH-Responsive Element Is Required for Complete
Androgen Suppression
We next investigated which part of the LHß promoter was
necessary for the DHT suppression of GnRH-stimulated promoter activity.
The rat LHß promoter contains two composite GnRH-responsive regions
that cooperate to confer GnRH stimulation (18, 19, 20, 21, 22, 23). The
distal region (-456 to -350 bp) contains two specificity protein-1
(Sp1) sites, including an overlapping Sp1/CArG box site at the 5'-end,
while the proximal region (-112 to -50 bp) contains two bipartite
binding sites for SF1 and Egr-1, separated by a Ptx-1 binding site
(Fig. 4
). Mutation of individual elements
(5'Sp1, CArG box, and 3'Sp1) in the distal GnRH-responsive region
interfere severely with GnRH stimulation, but multiple mutations or
deletion of this region permits the proximal sites to function
effectively (18, 19). The ability of DHT to suppress the
GnRH stimulation of three constructs, including the entire -617 bp
promoter (-617), the same promoter with mutated CArG and 3'Sp1 sites
(CG/Spmut), and a deletion construct in which all distal sites
(-245) were removed was tested.

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Figure 4. Response of Transfected LHß-Luciferase Constructs
to GnRH and DHT
The LHß promoter contains two GnRH responsive elements including a
distal region (-456 to -350 bp) comprised of two Sp1 binding sites
and an overlapping Sp1/CArG box and a proximal region (-112 to -50
bp) including one Ptx-1, two SF1, and two Egr-1 binding sites.
Transfected constructs include the full-length (-617 to + 44 bp)
LHß-luciferase promoter, the -617 promoter construct with mutations
in Sp1 and CArG as shown (CG/Spmut), and a construct (-245) with the
upstream region deleted. LßT2 cells were transfected and treated with
1 nM DHT for 24 h alone or in combination with 10
nM GnRH for 6 h. Normalized luciferase activity is
expressed as the mean of four experiments with three wells/group in
each experiment. *, P < 0.05; **,
P < 0.01 vs. untreated controls. a,
**, P < 0.01 for GnRH vs. GnRH plus
DHT.
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The intact -617 to +44-bp promoter construct was stimulated 8-fold by
GnRH, and this stimulation was severely diminished by DHT (Fig. 4
). In
contrast, the CArG/Sp1 mutant and the -245-bp promoter were stimulated
by GnRH approximately 4.4- and 3.5-fold, respectively, and this
response was unaffected or only slightly suppressed by DHT. This
indicates that the distal response region plays a critical role in
mediating DHT suppression of the GnRH response.
AR Does Not Bind Directly to the LHß Promoter
The sequence of the distal regulatory region of LHß does not
contain any consensus steroid receptor binding sites or any obvious
androgen response elements. We investigated whether AR could bind
directly to this region using EMSAs. Two oligonucleotides representing
the 5'Sp1/CArG region or the 3'Sp1 region of the distal GnRH-responsive
region were used as probes with nuclear proteins from LßT2 cells
treated with DHT (Fig. 5
). Both gene
regions bind several proteins. The most slowly migrating complex (at
arrow) for each oligonucleotide probe contains Sp1, as
previously demonstrated by a change in the mobility of these complexes
by the addition of Sp1 antibody, and by the formation of identical
complexes with recombinant Sp1 (18). AR protein does not
directly bind to these gene regions, as the addition of anti-AR
antibody did not supershift or eliminate any of the observed
DNA-protein complexes from LßT2 cells. Similarly, recombinant AR did
not bind to the LHß gene regions (as in Fig. 8
), but did bind to an
androgen response element (ARE) (not shown).

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Figure 5. AR Does Not Bind to the Upstream Response Region in
the LHß Promoter
Oligonucleotides representing the upstream GnRH response element
containing the overlapping Sp1/CArG binding sites (5'Sp1CArG) or the
second Sp1 element (3'Sp1) were end-labeled with
[ -32P]ATP. Probes were incubated with nuclear proteins
isolated from LßT2 cells treated with 1 nM DHT (LßT2 in
figure) and with antibody to the N terminus of the AR (AR-Ab) in some
reactions, and then separated on an acrylamide gel. Lanes with no
protein added are labeled minus (-). The position of complexes
previously demonstrated to contain Sp1 are also indicated. A
representative gel is shown from one of seven separate experiments.
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Figure 8. AR Reduces Sp1 Binding to the Distal
GnRH-Responsive Region
The oligonucleotide representing the 3'Sp1 region of the LHß promoter
was end-labeled with [ -32P]ATP and incubated with
recombinant AR protein (lanes 24), DHT-treated LßT2 nuclear protein
(lane 5), or AR in increasing concentrations with DHT-treated LßT2
proteins (lanes 68). The samples were electrophoresed for 4 h
and subjected to autoradiography. AR concentrations used were as
follows: lanes 24, 0.607 µg/µl; lane 6, 0.103 µg/µl; lane 7,
0.207 µg/µl; and lane 8, 1.035 µg/µl. Lane 1 contains no
protein and is labeled minus (-), and lanes 3 and 4 contain anti-AR
antibody and anti-Sp1 antibody, respectively. A representative gel is
shown from one of five independent experiments.
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AR Binds to Sp1
Because functional studies indicated that DHT effects were
mediated via the distal GnRH response region, and AR did not bind
directly to this DNA, we investigated the potential for AR interactions
with transcription factors binding to the GnRH-responsive LHß
promoter regions. Given that there are two Sp1 binding sites in the
distal GnRH-responsive region of the LHß promoter required for full
GnRH activation, we first examined the potential role of Sp1 in DHT
suppression of GnRH stimulation by performing
glutathione-S-transferase (GST) pull-down experiments with a
GST-Sp1 fusion protein. Full-length AR, steroidogenic factor 1 (SF-1),
and Pit-1 protein were in vitro translated with
[35S]-labeled methionine and used in pull-down
experiments with GST-Sp1. Labeled AR specifically bound to GST-Sp1
(Fig. 6A
), on average 20- to 30-fold
greater that to GST alone in eight separate experiments. In contrast,
neither SF-1 nor Pit-1 bound significantly to GST-Sp1 (Fig. 6B
).

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Figure 6. AR Specifically Interacts with Sp1 in GST Pull-Down
Experiments
A, In vitro translated
[35S]methionine-labeled AR was incubated with recombinant
proteins (1 µg) of GST alone (GST), GST-Sp1 (Sp1), or GST-Egr-1
(Egr-1). Bound proteins are shown on the autoradiogram. The migration
of labeled AR alone (0.5 µl of lysate) and AR binding in the absence
(-) or presence (+) of 1 nM DHT to recombinant proteins (2
µl lysate) was detected by autoradiography. The arrow
indicates migration of AR. B and C, Pull-down results using GST alone
and GST-Sp1 (B) or GST-Egr-1 (C) constructs incubated with in
vitro translated [35S]methionine-labeled SF-1 and
Pit-1. In vitro translated proteins and GST were in
identical quantities as in panel A, and the migration position of each
translated protein is indicated by an arrow.
Representative gels are shown from one of eight independent
experiments.
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Because there was a trend for DHT suppression of the GnRH response in
transfection studies with the -245 LHß-luciferase construct
containing only the proximal GnRH-responsive element (Fig. 4
) and
because Egr-1 is clearly critical in basal and GnRH-stimulated promoter
activity (19, 20, 21, 22, 23), we tested the potential for AR to
interact with Egr-1. Labeled AR consistently bound to GST-Egr-1 less
strongly than to GST-Sp1 (Fig. 6A
) in eight separate experiments, with
average binding to GST-Egr-1 approximately 25% (ranging from 12 to
39%) that of binding to GST-Sp1 under the same film exposure
conditions. This correlated with the milder suppression of the
LHß-luciferase construct containing Egr-1, but not Sp1, sites (Fig. 4
). As with GST-Sp1, neither SF1 nor Pit-1 bound specifically to
GST-Egr-1 (Fig. 6C
).
The AR DBD Is Required for Interactions with Sp1 and Egr-1
To determine which region of the AR was required for interactions
with Sp1 and Egr-1, several different AR constructs were tested in
pull-down experiments with GST-Sp1 and GST-Egr-1 (Fig. 7
). Labeled constructs containing the
entire AR (AR), the N-terminal region and the DNA binding domain or DBD
(AR-N), or the DBD and C-terminal region of AR (AR-C), all bound to
both GST-Sp1 and GST-Egr-1. In contrast, an AR protein containing the
C-terminal and N-terminal regions, but no DBD (AR
DBD), failed to
bind to GST-Sp1 or GST-Egr-1. Thus, the DBD region of the receptor is
critical for binding of the AR protein to these transcription factors,
and this was observed in four independent
experiments.

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Figure 7. The DBD of AR Is Required for Interactions with Sp1
and Egr-1
Top panel, Schematic representation of the four AR
proteins that were in vitro translated and labeled with
[35S]methionine for use in GST pull-down experiments. AR
is the full-length receptor, AR-N includes aa 1660 of the amino
terminus, AR-C includes aa 507919 of the carboxyl terminus, and
AR DBD is the full-length receptor with aa 538614 deleted.
Middle and bottom panels, Autoradiograms of proteins
bound in GST pull-down experiments with GST-Sp1 and GST-Egr-1,
respectively. The arrows designate the migration
position of each translated AR protein in the input lanes.
Representative gels are shown from one of four separate experiments.
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AR Reduces Sp1 Binding to the LHß Distal GnRH-Responsive
Region
To determine whether interactions between AR and transcription
factors binding to the LHß promoter could have functional
consequences in the context of the promoter, we performed gel shift
studies with LßT2 nuclear proteins and recombinant AR. We
concentrated on the 3'Sp1 binding region of the LHß distal
GnRH-responsive region, because the mutation of this Sp1 site largely
eliminates the androgen suppression of the -617 LHß-luciferase
construct (Fig. 4
). As shown, recombinant AR did not bind to this DNA
(Fig. 8
, lanes 24). Nuclear proteins
from LßT2 cells bound to this DNA with characteristic Sp1-containing
bands (lane 5). Addition of recombinant AR at increasing concentrations
reduced Sp1 binding to probe in a concentration-dependent manner (lanes
68).
Sp1 Cotransfection Abrogates DHT Suppression of GnRH
Stimulation
Cotransfection studies were performed to investigate the effects
of additional Sp1 on DHT suppression of the GnRH-stimulated 617 bp
LHß-luciferase promoter in LßT2 cells. Cotransfection of Sp1
overcame the suppressive effects of DHT in the presence of GnRH, and
promoter activity was restored to levels approaching that of GnRH alone
(Fig. 9
).

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Figure 9. Sp1 Cotransfection Restores GnRH Stimulation of
LHß-Luciferase Promoter
LßT2 cells were transfected with 3 µg of the -617 LHß-luciferase
promoter construct and with 3 µg CMV-Sp1 as indicated and treated
with 1 nM DHT for 24 h alone or in combination with 10
nM GnRH for 6 h. Normalized luciferase activity from
cell extracts was plotted as a function of percentage of control for
three separate experiments, with three wells per group in each
experiment. **, P < 0.01 vs.
control.
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DISCUSSION
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We have found that androgens specifically suppress GnRH
stimulation of the rat LHß gene promoter. This response does not
occur through a consensus binding site for AR in the promoter but does
require AR in normal gonadotropes. Complete suppression requires
an intact distal GnRH response region, containing the Sp1 and
CArG binding sites. A promoter construct in which the distal response
region is mutated is not suppressed by DHT, while a deletion construct
containing only the proximal GnRH response region with binding sites
for Egr-1 and SF-1 is only partially suppressed. We postulate that the
androgen response occurs primarily via protein-protein interactions
between the AR and proteins binding directly to the distal response
region, or proteins that otherwise interact with this site. Our studies
suggest that Sp1 plays a primary role in this regard. This does not
preclude some physiological role for Egr-1 in this response, and AR
interactions with both proteins may be required for complete
suppression in vivo.
Both androgens and estrogens have been shown to modulate the response
of the gonadotrope to GnRH; however, both the direction and the
mechanism of these steroid responses appear to be different. For
example, in vivo E can act on the hypothalamus to alter GnRH
pulses (9), but also enhances pituitary responses to GnRH
(26, 27, 28). This may be at least partially due to increases
in GnRH receptor mRNA and protein noted after chronic E treatment
in vivo, a stimulation that is amplified by the stimulatory
feedback of GnRH on its own receptor levels (26, 27).
However, other investigators have reported enhanced GnRH
transcriptional responses and lower basal transcription for the human
-subunit promoter in transfection studies with pituitary cells from
female rats treated with E (11). This enhancement resulted
from decreased phosphorylation of CREB in E-treated rats, presumably
via altered GnRH pulse patterns, and subsequent restoration of
phosphorylation and enhanced transcriptional responses when GnRH was
restored (28). Thus, the majority of E-mediated responses
on the gonadotrope genes appear to result from effects mediated through
the hypothalamic effects on GnRH. We did not observe significant
effects of E on either GnRH-R mRNA, possibly due to our fairly short
(24 h) treatment period, or on the LHß promoter stimulation to an
acute GnRH challenge.
In contrast, androgens have a direct effect on gonadotrope gene
transcription at the pituitary level. We have shown that androgen
suppression of LHß occurs in both clonal cell lines and normal
pituitary gonadotropes and requires an intact AR. There is little
effect of androgen treatment on GnRH-R mRNA, either in our studies or
in vivo (26). However, suppressive effects of
androgens on human
-subunit, as well as the rat and bovine LHß,
promoter activity have been observed in transient transfection studies
(16, 17, 29). All of these genes appear to be suppressed
by interactions of the AR with other transcription factors, rather than
by direct binding to DNA. The human
-subunit promoter does contain
an ARE, and binding of the AR to the DNA has been demonstrated
(16). However, transcriptional suppression by androgens
does not occur through this DNA region, but rather via the
basal
element and tandem cAMP response element sites contained elsewhere in
the human
-subunit promoter (17). The suppression
requires the DNA and ligand binding regions of the AR, although the DBD
alone was sufficient to suppress transcription in transfection studies.
Suppression of the
-subunit gene is proposed to occur by AR
interactions with proteins binding to the cAMP response element
and
basal element sites. The specific proteins involved on
the
-subunit gene have yet to be defined, but are unlikely to be Sp1
and Egr-1, and androgen suppression of the two LH subunit genes thus
occurs through divergent gene elements. In at least one other gene,
GnRH itself, androgen appears to suppress gene transcription not by AR
binding to DNA but via protein-protein interactions through as many as
three promoter sites that bind to potential neuronal-specific
transcription factors (29).
Promoter deletion/mutation studies from our laboratory
(18) and that of Kaiser et al.
(19) demonstrated that full GnRH stimulation of the rat
LHß gene promoter requires functional cooperation between the distal
Sp1/CArG region (-456 to -350 bp) and the proximal tripartite
enhancer region (-112 to -50 bp) containing binding sites for Egr-1,
SF-1, and Ptx-1. The mechanism for this cooperation is at present
unknown but may include direct physical communication between the
distal and proximal response regions, possibly through a looping
mechanism bringing the two response regions into proximity
(19). Physical contact could then occur either directly
between transcription factors or via a cofactor that binds both
regions. In either scenario, binding of the AR to Sp1 or to Sp1 and
Egr-1, could interfere with functional cooperation between the regions,
either by preventing binding of the proposed cofactor or by disrupting
direct interactions between transcription factors.
Stimulation of the bovine LHß gene by GnRH occurs primarily via a
proximal promoter element containing SF-1 and Egr-1 sites, rather than
by cooperation between multiple response elements (30).
Androgen suppression requires the Egr-1 and SF-1 binding region of the
promoter, suggesting that AR binding to proteins such as Egr-1 or SF-1
may play a critical role in this context. In contrast, androgen
suppression through a similar region in the rat LHß gene is not
predominant, although a partial decrease of transcription occurs with a
short construct (-245 bp) containing only these sites. These
functional data correlate with the weaker although significant
association of Egr-1 and AR in our GST pull-down assays. We have found
that the greatest androgen repression of the rat LHß gene occurs via
the distal GnRH-responsive region of the gene containing the Sp1 sites,
and that cotransfection of Sp1 can abrogate the suppressive response.
These data correlate with our biochemical findings that Sp1 and AR
directly interact in vitro, and that this interaction
reduces Sp1 binding to the distal GnRH-responsive region of the
promoter. AR reduction of Sp1 binding to DNA, in addition to AR binding
to Egr-1, would then cooperate to reduce or abolish the functional
cooperativity of the two GnRH-responsive regions in the rat LHß
promoter.
Functional interaction between AR and Sp1 has also been documented in
the cyclin-dependent kinase inhibitor p21 gene (31), which
is stimulated rather than suppressed by androgen treatment. The p21
promoter contains an ARE and six Sp1 sites. Deletion of the ARE does
not eliminate androgen stimulation, which only occurs upon mutation of
specific Sp1 sites. Direct AR-Sp1 interactions were demonstrated by
mammalian one-hybrid analysis and by coimmunoprecipitation
(31). Direct interactions of AR and Sp1 on this gene
promoter were not demonstrated, but it is unlikely that AR would reduce
Sp1 binding in this context. Thus, AR may modulate Sp1-mediated
transcriptional activation by several mechanisms, functionally
requiring direct binding of Sp1, but not AR, to DNA. Interestingly, E
stimulation of several genes is conferred by Sp1 sites and occurs by
binding of ER to Sp1 rather than to DNA. In those studies, EMSAs do not
detect a unique ER
-Sp1 complex, and antibodies to ER
do not
eliminate or supershift DNA-protein complexes; rather, Sp1 binding
intensity to DNA is enhanced (32, 33).
Overall, these studies demonstrate a direct effect of DHT on the
pituitary gonadotrope to suppress LHß promoter stimulation by GnRH.
This mechanism might play an important role in modulating gonadotropin
gene responses to hypothalamic stimulation when androgen levels are
high and could be an important aspect of steroid feedback on this axis.
In light of recent studies demonstrating similar actions of androgens
on the human
-subunit, bovine LHß, and GnRH genes (16, 17, 29, 30), this mechanism appears to be a common and significant
means of regulation of hypothalamic-pituitary function.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Transfection Studies
The clonal gonadotrope cell line, LßT2 cells
(24), which express GnRH, ERs, ARs, and both LH subunits
and secrete LH, were originally obtained from Dr. Pamela Mellon
(University of California San Diego). Immunoblots performed with mouse
and rat pituitary and LßT2 cell extracts suggest that AR levels are
similar. Cells were grown in phenol red-free DMEM with resin-stripped
10% FBS and antibiotics and were plated in 35-mm wells at a density of
11.5 x 106 cells per well 1620 h before
CaPO4 transfection. Each well was transfected
with 3 µg of reporter vector as indicated for 16 h, washed, and
treated with 10 nM GnRH for 6 h before collection and
measurement of luciferase activity. Steroids as indicated were included
during the transfection period and the GnRH treatment period, for a
total treatment of 24 h. In several experiments, a cytomegalovirus
(CMV)-ß-galactosidase vector (0.3 µg/well) was included to
normalize for luciferase activity, and in all cases luciferase activity
was normalized for protein. After transfection, cells were washed with
PBS and lysates were collected in 250 µl lysis buffer (Promega Corp., Madison, WI), vortexed, centrifuged for 1 min, and
assayed in a Turner 20e luminometer (Turner Designs, Mountain View,
CA). Protein concentrations were determined by the colorimetric assay
from Bio-Rad Laboratories, Inc. (Hercules, CA). Hormones
were obtained from Sigma (St. Louis, MO). Data for
normalized luciferase activity are presented as the mean ±
SEM for six wells per group, compared with untreated
controls, and each experiment was performed between 3 and 6 times with
equivalent results.
LHß-luciferase reporter vectors have been described elsewhere
(18). For most studies, the construct containing the
promoter region from -617 to +44 bp relative to the transcriptional
start site, and both GnRH-responsive elements, was used. A related
mutant construct (CGm3'Sp1 m), in which point mutations in the CArG box
and 3'Sp1 site within the distal GnRH response element (-456 to -350
bp) were introduced into the -617 to +44 promoter region, was used to
determine the effects of androgen on the distal element in the
context of the entire LHß promoter. In some experiments the
deletion construct from -245 to +44 bp, containing only the proximal
GnRH response element, was also tested. In additional experiments, the
-617 to +44 LHß-luciferase promoter construct was cotransfected with
a CMV-Sp1 expression construct (a generous gift of Dr. Randall Urban,
University of Texas Medical Branch, Galveston, TX), as indicated, to
determine whether additional Sp1 could rescue the suppressive effect of
androgens on the promoter. Total DNA was normalized with vector
alone.
GnRH-R mRNA Measurements
LßT2 cells were grown in T75 flasks treated for 24
h with DHT or E (both at 1 nM concentrations) in the
presence or absence of 10 nM GnRH for 6 h, as for
transfection studies. After treatment, cells were washed with PBS and
collected in guanidinium isothiocyanate, and total RNA was isolated as
previously described (35). GnRH-R mRNA was measured by a
quantitative dot blot procedure previously described (27).
GnRH-R sense strand RNA was synthesized by in vitro
transcription with Riboprobe Gemini kit (Promega Corp.). A
standard curve of sense RNA (501000 pg/dot) was spotted on each
nitrocellulose filter along with cellular RNA samples (10 µg/sample),
and a sample of pooled rat pituitary RNA was included as a
positive control and as a measure of filter-filter variability.
Hybridization was performed with a saturating amount (1 ng cDNA/µg
RNA) of labeled cDNA. GnRH-R mRNA levels were calculated by linear
regression using the sense RNA standard curve. Results were expressed
as picograms of GnRH-R mRNA per 100 µg cellular DNA.
Transgenic Animal Studies
Transgenic mice bearing the LHß-luciferase transgene have been
described previously, and the isolated pituitary cells from these
animals have been demonstrated to respond to GnRH in culture
(36). LHß-luciferase mice were bred in the C57/B6J
background, the same background as the Tfm carrier females
heterozygous for the AR mutation on the X chromosome (37, 38). The female Tfm heterozygotes were purchased from
The Jackson Laboratory (Bar Harbor, ME), and bred to male
LHß-luciferase males. Tail DNA of these progeny were tested by PCR
analysis for AR receptor status, in addition to phenotypic gonadal
laparotomy. Two groups of male siblings, all expressing
LHß-luciferase, and expressing either wild-type or mutant AR, were
selected for further study. In addition, female mice homozygous for
wild-type AR were also selected for comparison with the males.
Pituitaries from mice were collected after 8 wk of age, and the
isolated cells were cultured and treated with 1
nM DHT and/or 10 nM GnRH,
as for transfected cells. Cells representing one pituitary equivalent
were incubated in one 35-mm well. After treatment, luciferase activity
was measured as for transfected cells, and results are expressed as
luciferase activity/mg protein. Data are plotted as the mean ±
SEM for 5 experiments, and each group contains
914 individual wells.
Nuclear Proteins and EMSAs
Nuclear proteins were isolated from LßT2 cell nuclei by the
method of Dignam et al. (39). The extraction
buffer [20 mM HEPES, pH 7.3, 0.6
M KCl, 20 µg/ml ZnCl2,
0.2 mM EGTA, 0.5 mM
dithiothreitol (DTT)] contained protease inhibitors (10 µg/ml each
aprotinin, antipain, chymostatin, leupeptin, and 1 µg/ml pepstatin).
After ultracentrifugation at 100,000 x g, supernatant
proteins were subjected to chromatography on a Sephadex G column with
buffer (20 mM HEPES, pH 7.3, 1.5
mM MgCl2, 0.15
M KCl, 0.5 mM DTT, and
protease inhibitors). For EMSAs, nuclear protein (46 µg) was
incubated with labeled DNA (50,000100,000 cpm) and buffer
containing final concentrations of 10 mM
Tris-HCl, pH 7.5, 4% glycerol, 1 mM
MgCl2, 0.5 mM EDTA, 0.5
mM DTT, to mM NaCl, and 1 µg poly(dI-dC). Final
volumes were 1520 µl, and final salt concentrations were adjusted
to 100125 mM KCl. Samples were incubated on ice
for 45 min and subjected to electrophoresis on a 5% acrylamide 1x
Tris-borate EDTA gel for 1.5 h (18).
Recombinant AR used in EMSA studies was isolated from Sf9 cells
(40). A sample of Sf9 cells was mixed in lysis buffer (20
mM Tris, pH 8, 350 nM NaCl, 10% glycerol, 10
mM imidazole, 10 µg/ml leupeptin, 10 µg/ml pepstatin,
10 µg/µl aprotinin, 1 mM phenylmethylsulfonylfluoride,
1 µM DHT, and 1 mM DTT), freeze-thawed three
times, and spun at 100,000 x g for 30 min and
supernatant was collected. Total protein was quantified using BCA
protein assay (Pierce, Rockford, IL), and AR
content was compared against AR that had been column purified using a
metal affinity resin (Talon from CLONTECH Laboratories, Inc., Palo Alto, CA). AR content was assessed by immunoblot
analysis using AR (N-20) antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Complimentary oligonucleotides representing rat LHß gene sequences
for EMSA included wild-type sequences representing the 5'Sp1/CArG
box region and 3'Sp1 site. The wild-type sense strand
sequence for 5'Sp1/CArG was
(5'-GCTAAACCACACCCATTTTTGGACCCAATCCAGGCATCC-3').
The oligonucleotide representing the wild-type 3'Sp1 site was
(5'-GCTGGGCGAGGGGCGGCGCCCACCTC-3'). Double-stranded DNA containing one
copy of the LHß promoter was end-labeled with
[
-32P]ATP and purified from a 6% acrylamide
gel. The wild-type sense strand sequence that was used as a
representative androgen response element was ARE
(5'-GAAGTCTGGTACAGGGTGTTCTTTTTG-3'). This oligonucleotide was labeled
by the same method as above. EMSA experiments using the LHß promoter
oligonucleotides and AR were run on a 5% acrylamide 1x Tris-borate
EDTA gel for 3 h. Antibodies used were AR (N-20) and Sp1 from
Santa Cruz Biotechnology, Inc. Recombinant AR was
incubated with DNA under the same conditions as for nuclear proteins
and was added simultaneously with nuclear proteins in some
experiments.
GST Pull-Down Experiments
BL21 bacterial cells were transformed with constructs expressing
GST, GST-Sp1, or GST-Egr-1. Luria Broth (100 ml) containing 50 µg/ml
ampicillin was inoculated with 1 ml of bacteria and incubated in an
orbital shaker at 37 C. Bacteria were grown to
A600 = 0.5, induced with 0.1 mM
isopropyl ß-thiogalactopyranoside, and shaken overnight at room
temperature. The bacterial pellet was resuspended in 5 ml of buffer
containing 50 mM Tris, pH 7.5, 0.5 mM EDTA, 300
mM NaCl, 10 mg/ml lysozyme, and 1 mM DTT. One
hundred microliters of 10% Nonidet P-40 were added, and after 10 min,
the lysate was frozen at -70 C in an ethanol bath. Lysate was thawed
at room temperature and then incubated for 1 h in 5 ml of buffer
containing 1.5 M NaCl, 12 mM
MgCl2, 5 µg deoxyribonuclease I, 10 µg/ml
leupeptin, 1 µg/ml pepstatin A, and 0.1 mM
phenylmethylsulfonylfluoride. Lysates were passed through a 20-gauge
needle and centrifuged for 30 min at 7,500 x g.
Soluble lysate was conjugated with glutathione beads
(Sigma) overnight at 4 C. Beads were washed with PBS, and
protein concentrations were assessed after electrophoresis on 12%
polyacrylamide denaturing gels by Coomassie Blue stain and
immunoblotting (GST-Sp1 and GST-Egr-1) with Sp1 and Egr-1 antibody from
Santa Cruz Biotechnology, Inc. For pull-down experiments,
approximately 1 µg of GST fusion protein was used in each sample
incubation. In steroid treatment groups, 1 nM DHT
was added and equivalent volumes of ethanol were added to untreated
samples. BSA (20 µg/ml) was added to each incubation containing
[35S]methionine-labeled (0.04 mCi/50-µl
reaction) in vitro translated proteins
(TNT Rabbit Reticulocyte
Transcription/Translation Kit; Promega Corp.). Labeled
proteins included full-length AR (amino acids 1919), amino-terminal
AR (AR-N, aa 1660), carboxy-terminal AR (AR-C, aa 507919), and the
DBD-deleted AR (AR
DBD, aa 1919,
aa 538614). Total volume was
adjusted to 150 µl with GST wash buffer (10 mM
MgCl2, 150 mM KCl, 20
mM HEPES, 10% glycerol, and 0.12% Nonidet
P-40). Beads and proteins were incubated for 1.5 h at 4 C and then
centrifuged and washed four times in GST wash buffer. Beads were
resuspended in 10 µl of SDS loading buffer and boiled for 5 min.
Proteins were electrophoresed on SDS containing 10% acrylamide gels at
150 V, along with standard molecular weight markers (Benchmark,
Life Technologies, Inc., Gaithersburg, MD). Gels
containing [35S]methionine-labeled proteins
were dried and exposed to film for 2472 h at -70 C.
 |
ACKNOWLEDGMENTS
|
---|
We thank Ms. Savera Shetty for technical assistance with the
transgenic mice.
 |
FOOTNOTES
|
---|
Address all correspondence and requests for reprints to: Margaret
A. Shupnik, Ph.D., Box 8800578, 7141 Multistory Building OMS,
Department of Internal Medicine/Endocrinology, University of Virginia
Medical Center, Charlottesville, Virginia 22908.
This work was supported by National Institute of Child Health and Human
Development/National Institutes of Health through cooperative agreement
(U54-HD-28934) as part of the Specialized Cooperative Centers Program
in Reproduction Research though both an individual research project
(M.A.S. and D.J.H.) and the Molecular Core at the University of
Virginia, and at the University of North Carolina at Chapel Hill
(U54-HD-35041). We also acknowledge additional support from the
National Institutes of Health (R01 MH01349).
Abbreviations: ARE, Androgen response element; CMV,
cytomegalovirus; DBD, DNA-binding domain; DHT, dihydrotestosterone;
DTT, dithiothreitol; Egr-1, early growth response protein-1;
GnRH-R, GnRH receptor; GST,
glutathione-S-transferase; SF-1, steroidogenic
factor 1; Sp1, specificity protein-1.
Received for publication December 14, 2000.
Accepted for publication July 20, 2001.
 |
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