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Department of Obstetrics and Gynecology, University of British Columbia (K.W.C., S.K.K., P.C.K.L.), Vancouver, Canada V6H 3V5; and Department of Zoology, University of Hong Kong (E.S.W.N., B.K.C.C.), Hong Kong, China
Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H30–4490 Oak Street, British Columbia Women’s Hospital, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1) cells with a
2297-bp human GnRHR promoter-luciferase construct (p2300-LucF). A
dose- and time-dependent decrease in human GnRHR promoter activity
was observed after GnRHa treatment. An average 71% decrease in
promoter activity was observed after 24-h treatment with 0.1
µM GnRHa, which was blocked by cotreatment of the
GnRH antagonist, antide. This effect was mimicked by phorbol
12-myristate 13-acetate (TPA) administration. In addition, the
GnRHa- and TPA-mediated decrease in the human GnRHR promoter
activity was reversed by a specific protein kinase C (PKC) inhibitor,
GF109203X, or depletion of PKC by TPA pretreatment. These findings
indicate that the activation of the PKC pathway is important in
regulating the human GnRHR gene expression.
By progressive 5'-deletion studies, we have identified a 248-bp DNA
fragment (-1018 to -771, relative to the translation start site) at
the 5'-flanking region of the human GnRHR gene that is responsible for
the GnRHa-mediated down-regulation of human GnRHR promoter activity.
Analysis of this sequence reveals the existence of two putative
activating protein-1 (AP-1) sites with 87% homology to the
consensus sequence
(5'-TGAG/CTC/AA-3'),
located at -1000 to -994 (5'-TTAGACA-3', in complementary
orientation) and -943 to -937 (5'-TGAATAA-3'). Using competitive
gel mobility shift assays, AP-1 binding was observed within this 248-bp
region. Site-directed mutation of the putative AP-1-binding site
located at -1000 to -994 abolished the GnRHa-induced inhibition.
Further competitive GMSA and supershift experiments confirmed the
identity of AP-1 binding in this region. By the use of Western blot
analysis, a significant increase in c-Jun (100%; P
< 0.05) and c-Fos (50%; P < 0.05) protein levels
was observed after GnRHa treatment in T3–1 cells. In addition, our
data suggested that a change in AP-1 composition, particularly c-Fos,
was important in mediating GnRHa-induced inhibition of human GnRHR gene
expression.
We conclude that activation of the PKC pathway by GnRH is important in controlling human GnRHR gene expression. In addition, the putative AP-1-binding site located at -1000 to -994 of the human GnRHR 5'-flanking region has been functionally identified to be involved in mediating this down-regulatory effect.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells (16). Others
have reported no change in GnRHR mRNA levels in T3–1 cells
(17) and in the rat (18). To understand the
molecular mechanism involved in transcriptional regulation of
GnRHR gene expression, the 5'-flanking region of the GnRHR gene was
isolated from mouse (19, 20). Progressive 5'-deletions in
the mouse GnRHR gene promoter revealed that the binding sites for
steroidogenic factor-1 (SF-1), activating protein-1 (AP-1), and a novel
element referred to as the GnRHR-activating sequence appear to be
responsible for regulating cell-specific expression of the mouse
GnRHR gene (21).
Using T3–1, the homologous up-regulation of mouse GnRHR
gene (mGnRHR) by GnRH was studied (22, 23). A significant
increase in luciferase activity was observed after GnRH agonist (GnRHa)
treatment for 4 h. Progressive deletion and mutation studies have
identified a putative AP-1-binding site that responds to the
GnRH-induced stimulation of luciferase activity (22, 23).
This GnRHa-induced increase in mGnRHR promoter activity was mimicked by
phorbol ester treatment. Furthermore, pretreatment with a specific
protein kinase C (PKC) inhibitor blocked the GnRH- and phorbol
12-myristate 13-acetate (TPA)-induced increase luciferase activity,
suggesting the involvement of PKC in regulating the expression of
mGnRHR gene (22, 23). The depletion of PKC by
pretreatment with 10 nM TPA for 20 h prevented
the increase in mGnRHR promoter activity caused by GnRH, and TPA
treatment further supported the role of PKC (22).
Clinically, GnRH analogs have proven to be efficacious in treating a
wide variety of gonadal hormone-dependent disorders, such as
endometriosis, precocious puberty, and polycystic ovarian syndrome
(24). In addition, GnRH agonists have been used
extensively in assisted reproductive technologies (24, 25). The clinical applications of GnRH agonists are based
primarily on the decrease in gonadotropin release as a result of GnRH
receptor down-regulation by continuous GnRH agonist administration
(26, 27). To date, the molecular mechanisms underlying the
transcriptional regulation of the human GnRHR gene after continuous
GnRH treatment remain poorly understood. As the first step in
understanding the possible transcriptional regulation of the human
GnRHR gene by GnRH, a 2297-bp 5'-flanking region of the human GnRHR
gene (28) was functionally characterized by luciferase
reporter gene assays in a pituitary gonadotrope-derived T3–1
cell line.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cell line,
provided by Dr. P. L. Mellon (Department of Reproductive Medicine,
University of California, San Diego, CA), were maintained in DMEM (with
4.5 mg/ml glucose; Life Technologies, Inc., Burlington,
Canada) supplemented with 10% FBS (HyClone Laboratories, Inc., Logan, UT). Cultures were maintained at 37 C in a
humidified atmosphere of 5% CO2 in air.
Cells were passaged when they reached about 90% confluence using a
trypsin/EDTA solution (0.05% trypsin/0.53 mM EDTA).
Preparation of human GnRHR promoter-luciferase constructs
A 2.3-kb 5'-flanking region of human GnRHR-luciferase construct
(p2300-LucF) was prepared as previously described (28).
Progressive 5'-deletion constructs were prepared using various
restriction endonucleases or exonuclease III/SI nuclease digestion
(Pharmacia Biotech, Piscataway, NJ). The 5'-deleted human
GnRHR promoter clones Nde-HLuc, Sac-HLuc,
Pst-HLuc, Spe-HLuc, and p167-Luc were prepared by
digesting p2300-LucF with NdeI, SacI,
PstI, SpeI, and HincII. Deletion clone
p577-Luc was prepared by exonuclease III/SI nuclease digestion. The
positive clones were identified by restriction mapping and DNA sequence
analysis using a T7 DNA sequencing kit (Pharmacia Biotech). Plasmid DNA for transfection studies was prepared
using QIAGEN Plasmid Maxi Kits (QIAGEN,
Chatsworth, CA) following the manufacturer’s suggested procedure. The
concentration and integrity of DNA were determined by measuring
absorbance at 260 nm and by agarose gel electrophoresis, respectively.
Purified plasmid DNA was then dissolved in 0.1 x TE (1
mM Tris-Cl, pH 7.5, and 0.1
mM EDTA) to a final concentration of 1
µg/µl.
Site-directed mutagenesis
Mutations were introduced by a three-step PCR mutagenesis method
as described previously (29), using mutagenic primers
mAP-1(-1000) and mAP-1(-943) and universal primers UP-T3F, UP, and
T7R (see Table 1
for complete sequence
information). Mutation was confirmed by sequence analysis after the
mutagenesis reaction.
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Pharmacological treatments
Pharmacological reagents, including a GnRH agonist,
D-(Ala6)GnRH (GnRHa), a GnRH
antagonist (antide), and TPA, were purchased from Sigma-Aldrich Corp. (Oakville, Ontario, Canada). The PKC inhibitor
bisindolymaleimide I (GF109203X) was obtained from
Calbiochem (La Jolla, CA). In time-course experiments,
treatments of the transfected cells were initiated at the same time
with the indicated agents, and the treated cells were harvested at each
time point of interest. In experiments in which the effects of GnRHa,
antide, and TPA on GnRHR-Luc activity were studied, the cells were
treated with the corresponding drug for 24 h before luciferase and
ß-galactosidase activities were measured.
Gel mobility shift assay (GMSA)
Oligodeoxynucleotides corresponding to the putative AP-1 element
(hG-AP-1) at the human GnRHR 5'-flanking region (-1004 to -988),
mutated hG-AP-1a, mutated hG-Ap-1b, and their complements were
synthesized by the Oligonucleotide Synthesis Laboratory (University of
British Columbia, Vancouver, Canada) and annealed to form an
18-bp double-stranded oligodeoxyribonucleotides (Table 1
).
Consensus AP-1, mutated AP-1, and progesterone response element
(PRE) oligonucleotide DNA; c-Fos antibody (catalog no. sc-52X),
c-Jun/AP-1 antibody (catalog no. sc-45X); GATA-2 antibody (catalog no.
sc-276X); cAMP response element-binding protein (CREB-1) antibody
(catalog no. sc-186X); and progesterone receptor (PR) antibody (catalog
no. sc-539X) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Probes for GMSA were end radiolabeled
with [32P]ATP by T4 polynucleotide kinase
(Life Technologies, Inc.) and separated from
unincorporated radionucleotides by passage over a Sephadex G-50 or G-25
column. Nuclear extracts was prepared from GnRHa-treated T3–1 cells
according to the method described previously (30). Protein
concentrations were determined by a modified Bradford assay
(Bio-Rad Laboratories, Inc., Hercules, CA). GMSAs were
carried out in 20 µl containing 20 mM HEPES (pH 7.5), 20
mM KCl, 20 mM NaCl, 1.5 mM
MgCl2, 1 mM dithiothreitol, 1
mM EDTA, 10% glycerol, 2 µg poly(dI-dC), 5 µg nuclear
proteins, 2 mg/ml BSA, and radiolabeled probe.
For the competition assays, the unlabeled DNA was added simultaneously with the labeled probe. Antibodies used in supershift experiments were added to the nuclear extract at room temperature for 1 h before the addition of labeled probe. The binding mixture was incubated at room temperature for 20 min and separated in 4–8% polyacrylamide gel containing 1 x TBE (Tris-borate-EDTA: 0.09 M Tris-borate and 2 mM EDTA, pH 8.0). Before loading of samples, the gel was prerun for 90 min at 100 V at 4 C. Electrophoresis was carried out at 30 mA at 4 C. The gel was then dried under vacuum and exposed to x-ray film (X-OMAT AR film, Eastman Kodak Co., Rochester, NY) at -70 C.
Western blot analysis
For Western blot analysis, GnRHa-treated T3–1 cells
were incubated in 75 µl cell lysis RIPA [containing 1 x PBS
(pH 7.4), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10
µg/ml phenylmethylsulfonylfluoride, 30 µg/ml aprotinin, and 10
µg/ml leupeptin] for 15 min on ice. The cellular debris was removed
by centrifugation, and the protein concentration in the cell lysates
was determined using a modified Bradford assay (Bio-Rad Laboratories, Inc.). Aliquots (35 µg) were taken from the
total cell lysates and subjected to SDS-PAGE under reducing conditions.
The separated proteins were then electrophoretically transferred onto
nitrocellular paper (Hybond-C, Amersham Pharmacia Biotech,
Morgan, Canada). The membranes were blocked with 5% (wt/vol) nonfat
milk in Tris-buffered saline containing 20 mM Tris-Cl (pH
8.0), 140 mM NaCl, and 0.05% (wt/vol) Tween-20 for at
least 1 h before the addition of c-Jun or c-Fos antibodies in a
final concentration of 0.2 µg/ml. Monoclonal antibodies against PKC
isoforms (anti-PKC mAB kit, catalogue no. S85080) were purchased
from Transduction Laboratory, Inc. (BD Biosciences, Mississauga,
Canada). The antibodies used for PKC isoforms were in the
following dilutions: 1:1000 for PKC, 1:250 for PKCß, 1:500
for PKC
, and 1:1000 for PKC
. All antibody incubation and
washing were performed in Tris-buffered saline with 0.05% Tween-20.
The Amersham Pharmacia Biotech enhanced
chemiluminescence system (ECL) was used for detection. Membranes
were visualized by exposure to Kodak X-OMAT film. The
radioautograms were then scanned and quantified with Scion
Image-Released ß 3b software (Scion Corp., Bethesda, MD).
Data analysis
For transfection assay, data are shown as the mean ±
SD of triplicate assays in at least three independent
experiments. For Western blot analysis, data were obtained from three
independent experiments. All data were analyzed by one-way ANOVA
followed by Tukey’s multiple comparison test or t test,
using the computer software PRISM (Version 2, GraphPad Software, Inc., GraphPad Software, Inc., San Diego, CA). Data
were considered significantly different from each other at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 pituitary tumor cell line, which expresses high
levels of GnRHR, as an experimental model for our transient
transfection assays. Our preliminary studies showed that a 24-h
transfection with 8 µl lipofectin resulted in the highest
transfection efficiency. As a result, all transfections hereinafter
were preformed accordingly. To determine the effects of GnRHa, antide,
and TPA on the promoter activity of the human GnRHR 5'-flanking region,
p2300-LucF was transiently transfected into T3–1 cells and treated
with the corresponding pharmacological agent before measurement of
luciferase activity. A dose- and time-dependent decrease in human GnRHR
promoter activity was observed after GnRHa treatment (Fig. 1). A longer posttransfection incubation
resulted in increased basal luciferase activity. A significant decrease
in luciferase activity (31%; P < 0.001 vs.
control) was observed after 6 h of 0.1-µM
GnRHa treatment, and maximum inhibition (71%; P <
0.001) was reached after 24 h of 0.1-µM
GnRHa treatment (Fig. 1A). In addition, this inhibitory effect was
observed when treating the p2300-LucF-transfected cells with a
concentration as low as 0.1 nM GnRHa for 24
h (Fig. 1B). To investigate whether the activation of GnRHR is
essential for inhibiting the human GnRHR promoter activity, a GnRH
antagonist, antide, was used to treat the p2300-LucF-transfected
T3–1 cells for 24 h, alone or in combination with GnRHa. No
significant change in human GnRHR promoter activity was observed after
antide treatment alone, and the magnitude of inhibition by GnRHa was
reduced from 55% to 20% (P < 0.001) and was
completely blocked in the presence of 0.1 and 10
µM antide, respectively (Fig. 2). These results suggest that the
activation of GnRHR by GnRHa is important and results in activation of
an intracellular mechanisms that subsequently inhibits human GnRHR gene
expression in the pituitary cells. Similar to the results obtained with
GnRHa treatment, a dose- and time-dependent inhibition of human GnRHR
promoter activity was observed after TPA treatment (Fig. 3). A significant decrease (30%;
P < 0.01) in luciferase activity was observed after
6 h of 10 µM TPA treatment, and the
degrees of inhibition increased with time in culture (Fig. 3A). The
inhibitory effect of GnRHa on GnRHR promoter activity was mimicked by
the administration of TPA, suggesting that the PKC pathway is involved
in regulating human GnRHR gene expression in pituitary gonadotropes at
the transcriptional level.
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T3–1
cells were transiently transfected with p2300-LucF and treated with
vehicle, 1 or 5 µM GF109203X, 0.1 µM GnRHa,
10 µM TPA, or 0.1 µM GnRHa or 10
µM TPA plus GF109203X (Fig. 4). Administration of GF109203X alone did
not affect GnRHR promoter activity. However, in the presence of 1
µM GF109203X, the inhibitory effects of GnRHa and TPA on
human GnRHR promoter activity were reduced from 62.5% to 30%
(P < 0.01) and from 57% to 30% (P <
0.05), respectively (Fig. 4, A and B). These inhibitory effects were
completely reversed by 5 µM GF109203X. These
results indicate the participation of the PKC pathway in the control of
human GnRHR gene expression at the transcriptional level after GnRHa
treatment. The role of PKC in regulating transcriptional activation of
the GnRHR gene was further examined by depleting PKC by pretreatment
with 0.1 µM TPA during transfection (Fig. 5). The PKC-depleted transfected T3–1
cells were then treated with vehicle, 0.1 µM
GnRHa, or 10 µM TPA for 24 h. Using
Western blot analysis (Fig. 5A), pretreatment of T3–1 cells with
TPA resulted in significant decreases in PKC (75%;
P < 0.001), PKCß (60%; P < 0.001),
PKC (80%; P < 0.001), and PKC (40%;
P < 0.001) levels, with no significant decrease in
basal luciferase activity (Fig. 5B). However, TPA-induced inhibition of
GnRHR promoter activity was completely blocked in PKC-depleted cells.
Similarly, TPA pretreatment resulted in a significant reduction of
GnRH-induced inhibition of GnRHR promoter activity (Fig. 5B). These
results indicate that the PKC pathway is important in regulating at
least in part human GnRHR gene expression at the transcriptional
level.
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T3–1 cells. All deletion clones were functionally
active in T3–1 cell, with a minimal 7-fold increase in luciferase
activity compared with pGL2-Basic, except p167-HLuc (Fig. 6). A deletion in the human GnRHR
5'-flanking sequences up to the PstI site (1018 bases away
from the translation start site) did not affect its responsiveness to
GnRHa inhibition. However, further deletion to the SpeI site
(771 bases away from the translation start site) abolished the
GnRHa-induced inhibitory effects on luciferase activity (Fig. 6). These
results suggest that this 248-bp DNA fragment located between
PstI and SpeI sites in the human GnRHR
5'-flanking region is responsible for the GnRH-mediated down-regulation
of human GnRHR gene expression in T3–1 cells.
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T3–1 cells after GnRHa treatment. In a preliminary study
DNA-protein complexes were formed after 5 min of incubation, and
prolonged incubation for 30 min resulted in decreasing signals, as the
complexes were too big to be separated in 4% polyacrylamide gel (data
not shown). As a result, a 10-min incubation time was chosen in this
particular study. As shown in Fig. 7, there were three DNA-protein complexes formed with the 248-bp DNA
fragments. The specificity of these complexes was confirmed by the
addition of both specific (unlabeled 248-bp probe; -1018 to -771) and
nonspecific (unlabeled DNA fragment; -2300 to -2000, relative to
translation start site) DNA competitors (Fig. 7, lanes 2 and 3).
Furthermore, the competition with increasing concentrations of
unlabeled AP-1 consensus element displaced the binding of two
sequence-specific protein complexes (Fig. 7, bands 1 and 2), suggesting
the presence of AP-1-binding sites within this region.
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). The mutated
constructs were transiently transfected into T3–1 cells and treated
with 0.1 µM GnRHa for 24 h. Site-directed mutation
of the putative AP-1-binding site located at -943 to -937 did not
alter GnRHa-induced inhibition of GnRHR promoter activity. However,
mutation of the AP-1-binding site at -1000 to -994 abolished the
GnRHa-induced inhibition (Fig. 8). These effects were again mimicked by
TPA treatment (data not shown).
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T3–1 cells after GnRHa treatment, two DNA-protein complexes were formed
(bands 1 and 2) using nuclear extract isolated from GnRHa-treated
T3–1 cells. These bands were eliminated by incubation with
increasing competitor DNA fragment containing a consensus AP-1 site or
unlabeled probe, but not with a competitor containing mutated AP-1
site, mutated hG-AP-1 site, or nonrelated sequence (PRE; Fig. 9).
Furthermore, the addition of anti-c-Jun or anti-c-Fos antibodies
supershifted these complexes, supporting the binding of c-Jun and c-Fos
to the hG-AP-1 (Fig. 10).
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T3–1 cells. A
100% increase (P < 0.05) in c-Jun protein was
observed after 3 h of 0.1 µM GnRHa
treatment and was maintained up to 24 h (Fig. 11A). Similarly, a significant increase
in c-Fos levels (50%; P < 0.05) was observed after
GnRHa treatment (Fig. 11B). The GnRHa-mediated regulation of AP-1
protein production was further examined by gel mobility assay using
hG-AP-1. Interestingly, differential AP-1 binding was observed. Only
one DNA-protein complex was formed with the nuclear extract isolated
from non-GnRHa-treated T3–1 cells, whereas two DNA-protein
complexes were formed with the GnRHa-treated nuclear extract (Fig. 12A). The increase in DNA-protein
complex intensity implied that more AP-1-like protein was available
after GnRHa treatment. In addition, the DNA-protein complex formed
with nuclear extract isolated from non-GnRHa-treated cells was
supershifted only by anti-c-Jun, not by anti-c-Fos, antibodies (Fig. 12B). Similar to those shown in Fig. 10, the DNA-protein complexes
formed by nuclear extract isolated from GnRHa-treated T3–1 cells
were supershifted by both c-Jun and c-Fos antibodies (Fig. 12C).
These results suggest that a different composition of AP-1 complex,
particularly with c-Fos, may bind to the hG-AP-1 site after GnRHa
treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells. As no human pituitary gonadotrope cells were available,
we employed mouse gonadotrope-derived T3–1 cells as an experimental
model in the present study. Our results suggest that one potential
mechanism for down-regulation of the human GnRHR is by decreasing
its gene expression, as continuous administration of GnRH resulted in
the reduction of human GnRHR promoter activity. Prolonged incubation
(24 h) of the transfected T3–1 cells with a GnRH agonist inhibits
human GnRHR promoter activity in a dose-dependent manner. In addition,
our results indicate that activation of GnRHR by GnRH is essential for
the regulation of GnRHR gene activity, as no reduction in luciferase
activity was observed after antide treatment alone, and the inhibitory
effect of GnRHa on human GnRHR promoter activity was reversed in the
presence of the GnRH antagonist.
Interestingly, no stimulatory effect on human GnRHR promoter activity
by GnRHa treatment was observed as in the mouse gene. In these study an
increase in mGnRHR promoter activity occurred in the transfected
T3–1 cells after 4 h of 0.1-µM GnRH treatment
(22, 23). Further analysis of the time course of the GnRH
response revealed a higher effect at 6 h of treatment
(22). Although the basal luciferase activity was increased
by prolonged transfection, it reduced the responsiveness of the
transfected cells to GnRH treatment (23). We did not
observe any increase in human GnRHR promoter activity after 4 and
6 h of 0.1-µM GnRHa treatment. Instead, a
significant inhibition of human GnRHR promoter activity was obtained
after 6 h of 0.1-µM GnRHa treatment. This difference
is possibly due to the different experimental conditions in the present
study compared to the mouse studies: 1) transient transfection was
carried out by lipofectin in contrast to the calcium precipitation; 2)
the incubation time for the transfection reaction was 24 h in our
study but 30 min (22) or 4 h (23) in the
mouse studies; 3) a mouse gonadotrope-derived cell-line was used in
this study to examine regulation of the human gene, so it is possible
that a species-specific regulator(s) required to control the increase
in expression of human GnRHR gene is not present in T3–1 cells; or
4) a different regulatory mechanism was used in controlling the human
gene vs. the rodent gene.
It is well known that agonist occupancy of GnRHR results in a
Gq/11-mediated activation of phospholipase Cß
(PLCß), with consequent generation of diacylglycerol and inositol
phosphates, which, in turn, activates PKC and elevates cytosolic
Ca2+, respectively (36, 37). We have
shown in the present study that activation of the PKC pathway is
involved in controlling human GnRHR gene expression at the
transcriptional level. The inhibition of human GnRHR promoter activity
by GnRHa can be mimicked by TPA administration. Our data from the
experiments using the specific PKC inhibitor, GF109203X, further
confirmed that GnRH acts through a PKC-dependent pathway in regulating
transcription of the human GnRHR gene. The role of PKC in regulating
transcriptional activation of the GnRHR gene was further demonstrated
by depleting PKC by pretreatment with 0.1 µM TPA. In
agreement with previous studies (38), we observed a
significant decrease in PKC, -ß, -, and - isoforms after PKC
depletion. In addition, depletion of endogenous PKC eliminated the
TPA-induced inhibition of GnRHR promoter activity, further supporting
the role of PKC. However, the GnRHa-induced inhibition of GnRHR
promoter activity cannot be abolished completely after depletion of PKC
by TPA pretreatment, suggesting that an additional mechanism might be
involved. It has been recently shown that the desensitizing effect of
GnRH remained unchanged despite the presence of PKC inhibitor or
down-regulation of PKC by TPA pretreatment in T3–1 cells
(38). Apart from PLCß, phospholipase A2 (PLA) and
phospholipase D (PLD) are also activated by GnRH, which resulted in the
production of arachidonic acid and phosphatidic acid (PA), respectively
(38, 39, 40, 41). PA can be converted to diacylglycerol, by a
specific PA phosphohydrolase, which activates
Ca2+-independent PKC isoforms such as novel PKC
(42). In addition, arachidonic, oleic, linoleic, and
linolenic acids (derived from PA via PLA) were found to be capable of
supporting the activation of specific PKC isoforms (43).
Recent studies have demonstrated that GnRH activates and regulates
various PKC isoforms in T3–1 cells (38, 44, 45, 46). Of
particular interest, GnRH activated PKC
(46), which is
insensitive to either GnRH or TPA down-regulation (38).
Perhaps the stimulation of this TPA-insensitive PKC isoform after GnRHR
activation is also involved in regulating the expression of the human
GnRHR gene.
The mechanism(s) underlying GnRHR homologous desensitization
remains unclear and might be different from other G protein-coupled
receptors (47) due to the lack of C-terminal tail and
parts of the third intracellular loop (48). As GnRH
exposure to gonadotropes resulted in a decrease in GnRHR number and
mRNA levels, modification of GnRHR synthesis has been proposed as a
mechanism of GnRHR homologous desensitization. Thus, a decrease in
GnRHR promoter activity may be a mechanism of GnRHR desensitization.
This is the first demonstration of GnRH down-regulation of the
human GnRHR promoter activity. Using deletion, mutation, and gel
mobility shift studies, a putative AP-1-binding site located between
-1000 and -994 was identified as being responsible for this
inhibitory effect. Interestingly, AP-1 has been shown to be important
in regulating gonadotrope-specific expression of mouse GnRHR and to
mediate the GnRH-induced increase in mGnRHR promoter activity, as
mutation of the AP-1 site resulted in a significant loss of promoter
activity as well as GnRH responsiveness (21, 22, 23). However,
we did not observe any significant loss of promoter activity after
mutating the AP-1 site located at -1000 to -994, suggesting that this
AP-1 site may not regulate basal human GnRHR gene expression. It has
been further supported by the observation that the deletion of this
AP-1 site form Pst-Hluc to Spe-Hluc did not
affect basal promoter activity. It has been demonstrated in the rodent
GnRH gene that a 1-bp difference from the consensus AP-1-binding
element in the human GnRH gene resulted in a loss of DNA-protein
binding (49). Indeed, there are two nucleotide differences
between the identified AP-1-binding site in mouse (TGACTCA) and human
(TGTCTAA) GnRHR genes, suggesting that a different regulatory mechanism
may be used in controlling GnRHR gene expression in rodent and human.
Nevertheless, a recent study demonstrated that the inhibition of rat
GnRH gene expression at the transcriptional level by phorbol ester may
be mediated by AP-1, as deletion of a putative AP-1-binding site
abolished this TPA inhibition (50). Similarly,
overexpression of c-Jun and c-Fos in rat pituitary cells significantly
reduced gonadotropin -subunit promoter activity (51).
These studies support the potential role of AP-1 in down-regulation of
gene expression.
Activation of GnRHR in primary cultures of rat pituitary gonadotropes
and T3–1 cells by GnRH caused an increase in mRNA levels of c-Jun,
c-Fos and JunB (52). The GnRHa-induced expression of these
genes was mimicked by activation of PKC by phorbol ester. In addition,
depletion of cellular PKC by prior treatment of TPA reduced GnRH- and
TPA-induced expression of these genes, further supporting the role of
PKC in mediating the GnRH stimulatory effect (52). In
agreement with these studies, a significant increase in c-Fos and c-Jun
protein levels was observed after GnRHa treatment in the present study
by Western blot analysis. Using nuclear extracts from T3–1 cells,
with or without GnRHa treatment, a differential binding of AP-1 was
observed in hG-AP-1. Our data suggested that the possible mechanism of
GnRHa-mediated inhibition in human GnRHR promoter activity may be
the result of a change in AP-1 composition, as there was no retarded
migration of the AP-1 complex caused by c-Fos antibody in
non-GnRHa-treated nuclear extract. Furthermore, a second
DNA-protein complex, which can be retarded by both c-Jun and c-Fos
antibodies, was only observed in GnRHa-treated nuclear extract. These
results indicate that c-Fos may play an important role in mediating
GnRHa-induced inhibition of human GnRHR gene expression. Several
studies have demonstrated the negative transcriptional regulatory
action of c-Fos (53, 54). A dose-dependent decrease in rat
GnRH promoter activity (53) and clusterin promoter
activity (54) was observed after cotransfection of
increasing c-Fos expression vector. Furthermore, this inhibitory effect
was reversed in the presence of mutant c-Fos expression vector
(53, 54). It is worth noting that AP-1 is a family of
nuclear transcription factors composed of either homodimeric Jun or
heterodimeric Fos-Jun complexes that interact with the AP-1-binding
site to regulate gene expression at the transcriptional level. The Jun
family includes c-Jun, JunB, and JunD, whereas Fos gene family members
include c-Fos, FosB, Fra-1, and Fra-2. The differential expression
among these gene products could certainly affect the composition of
AP-1 and their actions (55). In the present study we only
studied the possible role of c-Jun and c-Fos in mediating GnRHa-induced
down-regulation of GnRHR gene expression. Studies of the involvement of
other AP-1 proteins are currently underway.
In summary, we demonstrated a decrease in human GnRHR
promoter activity after GnRHa treatment in T3–1 cells. Activation
of GnRHR and the PKC pathway is important for transcriptional
down-regulation of the human GnRHR gene. In addition, a putative
AP-1-binding site within the human GnRHR 5'-flanking region (-1000 to
-994) has been functionally identified to be involved in the molecular
mechanism of this down-regulation.
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Footnotes |
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2 Recipient of a studentship from the British Columbia Research
Institute for Children’s and Women’s Health.
3 Career investigator with the British Columbia Research Institute
for Children’s and Women’s Health.
Received January 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 and HEK-293 cells expressing the GnRH
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