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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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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). 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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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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). 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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). 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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), 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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) 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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). As shown, recombinant AR did not bind to this DNA
(Fig. 8, lanes 2–4). 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
6–8).
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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 (50–1000 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
9–14 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 (4–6 µg) was
incubated with labeled DNA (50,000–100,000 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 15–20 µl, and final salt concentrations were adjusted
to 100–125 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 1–919), amino-terminal
AR (AR-N, aa 1–660), carboxy-terminal AR (AR-C, aa 507–919), and the
DBD-deleted AR (ARDBD, aa 1–919, aa 538–614). 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 24–72 h at -70 C.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit gene
despite the absence of a high affinity binding site for the estrogen
receptor. Mol Endocrinol 5:725–733-subunit gene by androgen may involve
direct binding of the androgen receptor to the proximal promoter.
J Biol Chem 268:13556–13564-subunit gene by androgen receptor occurs
independently of DNA binding but requires the DNA and ligand binding
domains of the receptor. Mol Endocrinol 11:1497–1506GSU and LHß genes through distinct
protein-protein interactions. Proc 82nd Annual
Meeting of The Endocrine Society, Toronto, Canada, 2000, Abstract 488
/ß)-dependent activation at GC-rich (Sp1) promoter
elements. J Biol Chem 278:5379–5387
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V. G. Thackray, S. M. McGillivray, and P. L. Mellon Androgens, Progestins, and Glucocorticoids Induce Follicle-Stimulating Hormone {beta}-Subunit Gene Expression at the Level of the Gonadotrope Mol. Endocrinol., September 1, 2006; 20(9): 2062 - 2079. [Abstract] [Full Text] [PDF]
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 X.-M. Ou, K. Chen, and J. C. Shih Glucocorticoid and Androgen Activation of Monoamine Oxidase A Is Regulated Differently by R1 and Sp1 J. Biol. Chem., July 28, 2006; 281(30): 21512 - 21525. [Abstract] [Full Text] [PDF]
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J. Pielecka and S. M. Moenter Effect of Steroid Milieu on Gonadotropin-Releasing Hormone-1 Neuron Firing Pattern and Luteinizing Hormone Levels in Male Mice Biol Reprod, May 1, 2006; 74(5): 931 - 937. [Abstract] [Full Text] [PDF]
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 J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, and C. Y. Bowers Somatotropic and Gonadotropic Axes Linkages in Infancy, Childhood, and the Puberty-Adult Transition Endocr. Rev., April 1, 2006; 27(2): 101 - 140. [Abstract] [Full Text] [PDF]
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W. M. Bryant, M. A. Gibson, and M. A. Shupnik Stimulation of the Novel Estrogen Receptor-{alpha} Intronic TERP-1 Promoter by Estrogens, Androgen, Pituitary Adenylate Cyclase-Activating Peptide, and Forskolin, and Autoregulation by TERP-1 Protein Endocrinology, January 1, 2006; 147(1): 543 - 551. [Abstract] [Full Text] [PDF]
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K. Kim, R. Barhoumi, R. Burghardt, and S. Safe Analysis of Estrogen Receptor {alpha}-Sp1 Interactions in Breast Cancer Cells by Fluorescence Resonance Energy Transfer Mol. Endocrinol., April 1, 2005; 19(4): 843 - 854. [Abstract] [Full Text] [PDF]
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 H. Yuan, A. Gong, and C. Y.F. Young Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells Carcinogenesis, April 1, 2005; 26(4): 793 - 801. [Abstract] [Full Text] [PDF]
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 J. D. Veldhuis, A. Bae, R. S. Swerdloff, A. Iranmanesh, and C. Wang Experimentally Induced Androgen Depletion Accentuates Ethnicity-Related Contrasts in Luteinizing Hormone Secretion in Asian and Caucasian Men J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1632 - 1638. [Abstract] [Full Text] [PDF]
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D.J. Haisenleder, L.L. Burger, K.W. Aylor, A.C. Dalkin, H.E. Walsh, M.A. Shupnik, and J.C. Marshall Testosterone Stimulates Follicle-Stimulating Hormone {beta} Transcription via Activation of Extracellular Signal-Regulated Kinase: Evidence in Rat Pituitary Cells Biol Reprod, March 1, 2005; 72(3): 523 - 529. [Abstract] [Full Text] [PDF]
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L L Burger, D J Haisenleder, A C Dalkin, and J C Marshall Regulation of gonadotropin subunit gene transcription J. Mol. Endocrinol., December 1, 2004; 33(3): 559 - 584. [Abstract] [Full Text] [PDF]
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J. Hong, I. Samudio, S. Liu, M. Abdelrahim, and S. Safe Peroxisome Proliferator-Activated Receptor {gamma}-Dependent Activation of p21 in Panc-28 Pancreatic Cancer Cells Involves Sp1 and Sp4 Proteins Endocrinology, December 1, 2004; 145(12): 5774 - 5785. [Abstract] [Full Text] [PDF]
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K. M. H. Doyle, D. L. Russell, V. Sriraman, and J. S. Richards Coordinate Transcription of the ADAMTS-1 Gene by Luteinizing Hormone and Progesterone Receptor Mol. Endocrinol., October 1, 2004; 18(10): 2463 - 2478. [Abstract] [Full Text] [PDF]
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J. S. Jorgensen, C. C. Quirk, and J. H. Nilson Multiple and Overlapping Combinatorial Codes Orchestrate Hormonal Responsiveness and Dictate Cell-Specific Expression of the Genes Encoding Luteinizing Hormone Endocr. Rev., August 1, 2004; 25(4): 521 - 542. [Abstract] [Full Text] [PDF]
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D. Curtin, H. A. Ferris, M. Hakli, M. Gibson, O. A. Janne, J. J. Palvimo, and M. A. Shupnik Small Nuclear RING Finger Protein Stimulates the Rat Luteinizing Hormone-{beta} Promoter by Interacting with Sp1 and Steroidogenic Factor-1 and Protects from Androgen Suppression Mol. Endocrinol., May 1, 2004; 18(5): 1263 - 1276. [Abstract] [Full Text] [PDF]
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T. J. Spady, R. Shayya, V. G. Thackray, L. Ehrensberger, J. S. Bailey, and P. L. Mellon Androgen Regulates Follicle-Stimulating Hormone {beta} Gene Expression in an Activin-Dependent Manner in Immortalized Gonadotropes Mol. Endocrinol., April 1, 2004; 18(4): 925 - 940. [Abstract] [Full Text] [PDF]
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D. J. Bernard Both SMAD2 and SMAD3 Mediate Activin-Stimulated Expression of the Follicle-Stimulating Hormone {beta} Subunit in Mouse Gonadotrope Cells Mol. Endocrinol., March 1, 2004; 18(3): 606 - 623. [Abstract] [Full Text] [PDF]
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L. L. Burger, D. J. Haisenleder, K. W. Aylor, A. C. Dalkin, K. A Prendergast, and J. C. Marshall Regulation of Luteinizing Hormone-{beta} and Follicle-Stimulating Hormone (FSH)-{beta} Gene Transcription by Androgens: Testosterone Directly Stimulates FSH-{beta} Transcription Independent from Its Role on Follistatin Gene Expression Endocrinology, January 1, 2004; 145(1): 71 - 78. [Abstract] [Full Text] [PDF]
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A. Belgorosky, C. Pepe, R. Marino, G. Guercio, N. Saraco, E. Vaiani, and M. A. Rivarola Hypothalamic-Pituitary-Ovarian Axis during Infancy, Early and Late Prepuberty in an Aromatase-Deficient Girl Who Is a Compound Heterocygote for Two New Point Mutations of the CYP19 Gene J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5127 - 5131. [Abstract] [Full Text] [PDF]
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S.-Z. Yang and S. A. Abdulkadir Early Growth Response Gene 1 Modulates Androgen Receptor Signaling in Prostate Carcinoma Cells J. Biol. Chem., October 10, 2003; 278(41): 39906 - 39911. [Abstract] [Full Text] [PDF]
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D. J. Haisenleder, H. A. Ferris, and M. A. Shupnik The Calcium Component of Gonadotropin-Releasing Hormone-Stimulated Luteinizing Hormone Subunit Gene Transcription Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase Type II Endocrinology, June 1, 2003; 144(6): 2409 - 2416. [Abstract] [Full Text] [PDF]
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Y. Okada, Y. Fujii, J. P. Moore Jr., and S. J. Winters Androgen Receptors in Gonadotrophs in Pituitary Cultures from Adult Male Monkeys and Rats Endocrinology, January 1, 2003; 144(1): 267 - 273. [Abstract] [Full Text] [PDF]
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D. A. Schreihofer, D. F. Rowe, E. F. Rissman, E. M. Scordalakes, J.-a. Gustafsson, and M. A. Shupnik Estrogen Receptor-{alpha} (ER{alpha}), But Not ER{beta}, Modulates Estrogen Stimulation of the ER{alpha}-Truncated Variant, TERP-1 Endocrinology, November 1, 2002; 143(11): 4196 - 4202. [Abstract] [Full Text] [PDF]
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M. Abdelrahim, I. Samudio, R. Smith III, R. Burghardt, and S. Safe Small Inhibitory RNA Duplexes for Sp1 mRNA Block Basal and Estrogen-induced Gene Expression and Cell Cycle Progression in MCF-7 Breast Cancer Cells J. Biol. Chem., August 2, 2002; 277(32): 28815 - 28822. [Abstract] [Full Text] [PDF]
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J. E. Pawlowski, J. R. Ertel, M. P. Allen, M. Xu, C. Butler, E. M. Wilson, and M. E. Wierman Liganded Androgen Receptor Interaction with beta -Catenin. NUCLEAR CO-LOCALIZATION AND MODULATION OF TRANSCRIPTIONAL ACTIVITY IN NEURONAL CELLS J. Biol. Chem., May 31, 2002; 277(23): 20702 - 20710. [Abstract] [Full Text] [PDF]
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