From the Division of Maternal-Fetal Medicine, Department of Obstetrics & Gynecology and Division of Genetics, Department of Medicine, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The response of the pituitary gonadotrope to
gonadotropin-releasing hormone (GnRH) correlates directly with the
concentration of GnRH receptors (GnRHR) on the cell surface, which is
mediated in part at the level of GnRHR gene expression. Several
hormones have been implicated in this regulation, most notably GnRH
itself. Despite these observations and the central role that GnRH is
known to play in reproductive development and function, the molecular mechanism(s) by which GnRH regulates transcription of the GnRHR gene
has not been well elucidated. Previous studies in this laboratory have
identified and partially characterized the promoter region of the mouse
GnRHR gene and demonstrated that the regulatory elements for
tissue-specific expression as well as for GnRH regulation are present
within the 1.2-kilobase 5'-flanking sequence. By using deletion and
mutational analysis as well as functional transfection studies in the
murine gonadotrope-derived The hypothalamic decapeptide, gonadotropin-releasing hormone
(GnRH),1 plays a pivotal role
in regulating mammalian reproductive development and function.
Pituitary gonadotropes, which make up 8-15% of all cells in the
anterior pituitary gland (1), express cell surface, G protein-coupled
receptors specific for GnRH (2, 3). Activation of this receptor by GnRH
stimulates intracellular signal transduction pathways to increase the
synthesis and release of the pituitary gonadotropins, luteinizing
hormone (lutropin; LH) and follicle-stimulating hormone (follitropin;
FSH) (4, 5). These hormones then enter the systemic circulation to
regulate gonadal function, including steroid hormone synthesis and gametogenesis.
The biosynthesis and secretion of LH and FSH by pituitary gonadotropes
are tightly regulated as evidenced by predictable and reproducible
changes in circulating levels during the menstrual cycle. This
regulation is dependent primarily on GnRH pulse amplitude and
frequency, which varies with physiological state, with the rat estrous
and human menstrual cycle, and with puberty, and the menopause. The
response of pituitary gonadotropes to GnRH correlates directly with the
concentration of GnRH receptors (GnRHR) on the cell surface, which are,
in turn, regulated by a number of hormonal factors, most notably GnRH
itself (6-9). The highest concentration of GnRHR in the pituitary
gland is associated with a GnRH pulse frequency of 30 min and results
in the optimum synthesis and release of LH. Lower concentrations of
GnRHR are seen with GnRH pulse frequencies of 2 h and correlate
with optimum synthesis and release of FSH (8-10). Continuous exposure
to high concentrations of GnRH results in down-regulation of GnRHR
mRNA (11). The difference in the concentration of GnRHR between
high and low frequency GnRH pulses is 2-3-fold (8, 12). The difference
in the concentration of GnRHR appears to be mediated at least in part
at the level of GnRHR gene expression (6). GnRH regulation of GnRHR
mRNA is well documented in rat pituitary cells (11). However, the cellular mechanism(s) by which GnRH regulates transcription of this
and other genes has not been intensively investigated. This study
was designed to identify and characterize the critical
cis-DNA element(s) and cognate trans-factors that
mediate the regulation of mouse GnRHR (mGnRHR) gene expression by GnRH.
Previous studies in this laboratory have identified and partially
characterized the promoter region of the mGnRHR gene and demonstrated
that the regulatory elements for tissue-specific expression as well as
for GnRH regulation are present within a 1.2-kilobase (kb) 5'-flanking
region of the mGnRHR gene (designated Materials--
Des-Gly10[D-Ala6]-GnRH-ethylamide
(GnRH agonist (GnRHAg)), phorbol 12-myristate 13-acetate (PMA),
forskolin, and 8-bromo-cAMP were obtained from Sigma. GF-109203X
(bisindolylmaleimide I or 2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(1H-indol-3-yl)-maleimide), a selective inhibitor of PKC, was purchased from Alexis Biochemicals (San Diego, CA). SQ 22536 (9-[tetrahydro-2'-furyl] adenine), a selective inhibitor of adenylate cyclase, was obtained from Calbiochem. Anti-Fos and anti-Jun polyclonal antibodies were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA). Full-length human c-JUN
protein was obtained from Promega (Madison, WI). All oligonucleotides were prepared by Life Technologies, Inc. Reporter Plasmids and Expression Vectors--
A fusion construct
was prepared by ligation of the 1.2-kb 5'-flanking region of the mGnRHR
gene (designated
The GH50-pXP2 construct was prepared by subcloning the rat
growth hormone gene minimal promoter (GH(
Oligonucleotides corresponding to sense and antisense strands of the
regions Cell Culture and Transient Transfection--
Northern Blot Analysis--
To investigate the effect of GnRHAg
stimulation on GnRHR mRNA, Preparation of Nuclear Extracts--
Electrophoretic Mobility Shift Assay (EMSA)--
Probe was
prepared for EMSA by digestion of the pXP2 plasmid containing the
Characterization of the Signal Transduction Pathway Involved in
the GnRHAg Stimulation of the mGnRHR Gene--
To identify the second
messenger pathway(s) involved in the GnRHAg stimulation of the mGnRHR
gene, Statistical Analysis--
Transfections were performed in
triplicate and repeated multiple times. Data in each experiment were
normalized to the basal level of activity of either pXP2 or
GH50-pXP2 (designated 1-fold). Data were then combined
across experiments. Results were expressed as mean ± S.E. for
basal and GnRHAg-stimulated activities for each construct, and fold
stimulation in response to GnRHAg was calculated. One-way analysis of
variance (ANOVA) followed by post hoc comparisons with
Fisher's protected least significant difference test was used to
assess whether changes in GnRH responsiveness among different GnRHR
promoter-luciferase reporter constructs were significant. Significant
differences were designated as p < 0.05. When
appropriate, data were analyzed by the Student's t test for
independent samples.
Northern Blot Analysis--
A selected Northern blot of RNA
extracted from Optimization of the GnRHAg Response--
To optimize conditions
for GnRHAg responsiveness, Identification of Two GnRH Response Elements (GnRH-REs) in the
mGnRHR Gene Promoter--
Transfection of
To determine whether the putative element(s) in this region is not only
necessary but also sufficient to mediate a GnRHAg response,
PCR-generated fragments of the region
PCR-generated fragments of the region
To identify and characterize further the GnRH-RE(s) within the region
To investigate further the importance of SURG-2 and SURG-1 in the
GnRHAg-stimulated response of the mGnRHR gene, single or multiple
copies of these regions were synthesized by PCR, either alone or in
combination, placed upstream of the GH50 minimal promoter in GH50-pXP2, and transfected into Identification and Characterization of trans-Factors by
EMSA--
Using nuclear extracts from
Since SURG-2 containing the AP-1-binding site had been shown to be
critical for GnRH responsiveness in the mGnRHR gene (above), anti-Fos
and anti-Jun antibodies (Santa Cruz Biotechnology) were used in
antibody-supershift EMSA experiments to identify and characterize further the trans-factors present within the protein-DNA
complex. Supershift of the lower band with an anti-Fos antibody
suggests the presence of a Fos protein within the complex (Fig.
8A). Similarly, incubation
with an anti-Jun blocking antibody resulted in diminution in binding of
the lower band, suggesting the presence of a Jun protein within the
complex (Fig. 8B). However, this effect was moderate at best
and required large amounts of anti-Jun antibody (8 µl/lane as
compared with only 2 µl/lane for positive control). Positive control
for the anti-Jun blocking antibody included EMSA with purified human
c-JUN protein (Promega) using the consensus AP-1-binding site (Santa
Cruz Biotechnology) as probe (Fig. 8B). These data suggest
that the lower band on EMSA is a complex with a member(s) of the
Jun/Fos heterodimer superfamily. This is consistent with our
transfection data suggesting that SURG-2 at position GnRHAg Stimulation of the mGnRHR Gene Promoter Is Mediated via
PKC--
A dose-response curve for PMA was used to standardize all
subsequent experiments at a final concentration of 100 ng/ml PMA for
4 h (data not shown).
Similar experiments were carried out to investigate the role of the PKA
signal transduction pathway in the response of mGnRHR gene to GnRH.
Exposure to forskolin alone (25 µM) did not stimulate luciferase activity, and the addition of forskolin did not influence GnRHAg-stimulated luciferase activity in either
GH50/ The maintenance of normal reproductive function in all vertebrate
species is dependent on the regulation of LH and FSH synthesis and
release by pituitary gonadotropes. Although the synthesis and
intermittent release of the pituitary gonadotropins are affected by a
number of endocrine, paracrine, and autocrine factors, the most
important influence appears to be that of GnRH (6-9). In this study,
we have defined the dimeric GnRH-RE within the 1.2-kb 5'-flanking
sequence of the mGnRHR gene, and we have demonstrated that the AP-1
complex plays a central role in conferring GnRH responsiveness to the
mGnRHR gene.
We have used The responsiveness of pituitary gonadotropes to GnRH correlates
directly with changes in GnRHR concentrations. It has been suggested
that the concentration of GnRHR on the cell surface is mediated in
turn, at least in part, at the level of gene expression (6, 12). Data
from Northern blot analyses presented above (Fig. 1), which demonstrate
a significant increase in GnRHR mRNA in response to GnRHAg
stimulation which was maximal at 4 h, would support this
conclusion. These findings are consistent with previous reports in
primary monolayer cultures of rat pituitary cells in which GnRHR
mRNA levels were significantly increased by pulses of GnRH (10 nM, 5 min/pulse) at all pulse frequencies tested over a
24-h period (12). In contrast, Alarid and Mellon (20) found no change
in GnRHR mRNA levels in The mGnRHR gene has been isolated, and its major TSS has been
identified (17, 22, 23). A 1.2-kb 5'-flanking region of the mGnRHR gene
has been characterized and shown to be active in transfection studies
(13). This region has also been used in transgenic mice to show that it
is sufficient to mediate gonadotrope-specific expression in
vivo (24). Preliminary studies on the 5'-flanking putative
promoter region of the mouse, human, and sheep GnRHR genes reveal
complex organization with multiple TSS that are occasionally associated
with TATA boxes (13, 25). In the mGnRHR gene, the major TSS was shown
to be located 62 nucleotides upstream of the translational start site
by primer extension and ribonuclease protection analysis of Earlier studies identified a putative gonadotrope-specific element
(5'-TGTCCTTG-3') at position +48/+55 of the mGnRHR gene and suggested
that this element may be important in conferring GnRH responsiveness
(13). This sequence was first described in the human Using nuclear extracts from Serum is a highly effective stimulus of primary response genes,
including fos and jun. Endogenous steady-state
levels of Fos and Jun in The expression of the GnRHR, The mechanism(s) by which a common and ubiquitous
cis-regulatory element, such as the AP-1-binding site, is
able to regulate differentially both GnRHR and FSH The single copy GnRHR gene is well conserved between the species, as is
its putative promoter sequence. Indeed, there appears to be 69-71%
homology of the entire 1.2-kb 5'-flanking region among the mouse (13),
rat (44), human (45, 46), and sheep genes (47). The sequence homology
of SURG-1 and SURG-2 between various species (Fig.
10) shows a relatively high concordance
between sequences in the mouse, rat, and human. The sheep GnRHR gene
promoter is poorly characterized but does not appear to contain a
consensus AP-1-binding site (47). It is likely that different
mechanisms are involved in the GnRH-mediated activation of the GnRHR
gene in different species. For example, the 5'-flanking region of the human GnRHR gene is far more complex than that of the other species (45, 46, 48). It is larger (~2.3 kb), contains multiple TSS, and
numerous putative cis-regulatory sequences have been identified by sequence homology, including thyroid hormone-RE, glucocorticoid/progesterone-RE, cAMP-RE, PEA-3, AP-1, AP-2, and Pit-1
sites (46, 48).
T3-1 cell line, we have localized GnRH
responsiveness of the mouse GnRHR gene to two DNA sequences at
276/
269 (designated Sequence Underlying Responsiveness to GnRH-2 (SURG-2), which
contains the consensus sequence for the activating protein-1-binding
site) and
292/
285 (a novel element designated SURG-1), and
demonstrated that this response is mediated via protein kinase C. By
using the electrophoretic mobility shift assay, we further demonstrate
that a member(s) of the Fos/Jun heterodimer superfamily is responsible
in part for the DNA-protein complexes formed on SURG-2, using
T3-1
nuclear extracts. These data define a bipartite GnRH response element in the mouse GnRHR 5'-flanking sequence and suggest that the activating protein-1 complex plays a central role in conferring GnRH
responsiveness to the murine GnRHR gene.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1164/+62 relative to the major
transcriptional start site (TSS)) (13). By using deletion and
mutational analysis as well as functional transfection studies in the
murine gonadotrope-derived
T3-1 cell line, we have localized GnRH
responsiveness of the mGnRHR gene to two distinct DNA elements that
appear to be both necessary and sufficient to mediate a full GnRH
response. The first and critical element (5'-TATGAGTC-3'), designated
the Sequence Underlying Responsiveness to GnRH-2 (SURG-2), lies at
position
276/
269 and contains the consensus sequence for the
canonical 12-O-tetradecanoylphorbol-13-acetate response
element, also known as the activating protein-1 (AP-1)-binding site.
The second element (5'-GCTAATTG-3'), designated SURG-1, lies at
position
292/
285 and appears to be a novel enhancer element. The
importance of these two elements in mediating GnRH responsiveness of
the mGnRHR gene was confirmed by demonstrating that both SURG-1 and
SURG-2 are capable independently of conferring activity on a
heterologous minimal promoter but that both elements are required for a
full response. Our data further suggest that the response of the mGnRHR
gene promoter to GnRH is mediated via the protein kinase C (PKC), and
not protein kinase A (PKA), signal transduction pathway. By using the
electrophoretic mobility shift assay, we further demonstrate that a
member(s) of the Fos/Jun heterodimer superfamily is responsible in part
for the DNA-protein complexes formed on SURG-2, using
T3-1 nuclear
extracts, and that such proteins are rapidly induced by GnRH
stimulation. We propose therefore that GnRH-stimulated activity of the
mGnRHR gene is regulated by two distinct elements within the GnRHR gene promoter and that the key component of this mechanism involves the AP-1
protein complex that activates transcription in a cell-specific fashion.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
T3-1 cells were generously donated by Dr. Pamela Mellon (University of California, San Diego).
1164/+62) into the luciferase reporter plasmid,
pXP2, as described previously (13). The nucleotide sequence of the
mGnRHR gene promoter used in these studies is based on previous work in
this laboratory (13), with
1 assigned to the nucleotide immediately
5' of the major TSS. 5'-Deletions of the 1.2-kb GnRHR gene promoter
were synthesized by polymerase chain reaction (PCR) using selected sense/antisense primers with the full-length construct as a template and incorporating HindIII/XhoI restriction enzyme
sites at the ends. All primers included sufficient 5'- and 3'-flanking
sequence (~18 base pairs (bp)) to ensure specific annealing. The
resultant PCR products encoding the desired 5'-deletion constructs
(
765/+62,
387/+62,
365/+62,
341/+62,
300/+62,
232/+62,
117/+62, and
38/+62) were then each digested with
HindIII and XhoI restriction enzymes and inserted
into the HindIII/XhoI polylinker restriction sites upstream of the luciferase reporter in pXP2. An expression vector
expressing
-galactosidase driven by the Rous sarcoma virus promoter
(RSV-
-galactosidase) was used as an internal standard and control.
50/+1),
designated GH50) into the BglII restriction site
in pXP2. Dideoxy sequencing was used to confirm the orientation and
sequence of the GH50 insert. PCR-generated fragments of the
region
387/
220 of the mGnRHR gene promoter were synthesized using
selected sense/antisense primers with the full-length construct as a
template and incorporating HindIII/XhoI
restriction enzyme sites at the ends. The constructs were designated
GH50/
387/
220 (wild type), GH50/
308/
220,
GH50/
267/
220, GH50/
387/
264,
GH50/
341/
264, GH50/
308/
264, and
GH50/
387/
308. A series of scanner-linker mutations of
the region
308/
264 of the mGnRHR gene incorporating
HindIII/XhoI restriction enzyme sites at the ends
were generated by synthesizing sense and antisense oligonucleotides
with an overlap of ~15 bp, self-annealing the oligonucleotides, and
reconstituting the DNA double strands using Sequenase 2.0 (United
States Biochemical Corp.). By serially replacing 8-bp segments of the
wild type sequence with the NotI restriction enzyme site
starting at the 5' end, five separate mutants of the region
308/
264
of the mGnRHR gene promoter were created and designated regions A-E
(Fig. 5A). The NotI restriction enzyme site
(5'-GCGG
CCGC-3') is novel to the mGnRHR gene, and previous transfection experiments have shown no effect of this sequence on GnRH
stimulation (14). Digestion with the restriction enzyme, NotI, and subsequent gel electrophoresis confirmed the
presence of the NotI-containing insert. A point mutation of
the AP-1-binding site at position
269 (C269T) in the wild type
308/
264 construct was similarly generated. These constructs were
then digested with HindIII and XhoI restriction
enzymes and subcloned into HindIII/XhoI polylinker restriction sites upstream of the rat growth hormone gene
minimal promoter in GH50-pXP2 to generate wild type
expression vector (designated GH50/
308/
264), a single
point mutation (designated GH50/region E/mut), and five
NotI mutants (designated GH50/region A,
GH50/region B, GH50/region C,
GH50/region D, and GH50/region E, according to
the location of the NotI restriction enzyme site (Fig.
5A)).
276/
269 (SURG-2) and
292/
285 (SURG-1) were synthesized
incorporating XhoI, HindIII, and/or
BamHI restriction enzyme sites at the ends. Single or
multiple copies of SURG-1 and/or SURG-2 were synthesized by
self-annealing complementary oligonucleotide strands, digesting out the
inserts using restriction endonucleases, and placing them upstream of
the rat growth hormone gene minimal promoter in GH50-pXP2.
The resultant constructs were designated GH50/SURG-1,
GH50/SURG-2,
GH50/SURG-2/NotI/SURG-2, and
GH50/SURG-2/NotI/SURG-1. Similarly, selected
oligonucleotides were used to create mutations of SURG-2 (a C269T point
mutation, designated
1164/+62/SURG-2mut) and SURG-1 (a
NotI replacement of SURG-1, designated
1164/+62/SURG-1mut)
in the context of the full-length
1164/+62 promoter region. The
identity of all reporter constructs were confirmed by sequencing using
the dideoxynucleotide chain termination method.
T3-1 (mouse
gonadotrope) and CV-1 (African green monkey kidney fibroblast) cells
were maintained in monolayer culture in high and low glucose
Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.),
respectively, and supplemented with 10% (v/v) fetal bovine serum (Life
Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml
streptomycin sulfate (Life Technologies, Inc.) at 37 °C in
humidified 5% CO2/95% air. For transient transfection studies, cells were divided into six-well tissue culture plates and
cultured overnight in DMEM in the absence of serum or antibiotics. Under these conditions, cells were 40-50% confluent. Cells were then
transfected by calcium phosphate co-precipitation, as described previously (15). Briefly, cells were incubated with the calcium phosphate-DNA precipitates for 4 h in media containing 10% (v/v) fetal bovine serum. In each experiment, test vector was standardized at
4 µg of DNA per well. An expression vector expressing
RSV-
-galactosidase (1 µg/well) was co-transfected in all
experiments and used as an internal standard. Following a 4-h
transfection, cells were washed once at room temperature with
phosphate-buffered saline (pH 7.4). Thereafter, cells were treated with
100 nM GnRHAg or vehicle in serum-containing DMEM (2 ml/well) for 4 h immediately prior to harvest. These conditions
were selected after optimization analysis to give maximal levels of
expression and GnRHAg stimulation. Following the final incubation, the
medium was aspirated, and cells were washed once with ice-cold
phosphate-buffered saline. Cells were lysed in the wells by addition of
200 µl of lysis buffer (125 mM Tris (pH 7.6), 0.5% (v/v)
Triton X-100). Cellular debris was removed from lysate by
microcentrifugation at 14,000 × g for 10 min at
4 °C. Supernatants were assayed immediately for luciferase and
-galactosidase activity by standard protocols. Briefly, luciferase activity was determined by adding 120 µl of cell lysate to 200 µl
of luciferin substrate (Promega) and measuring luminescence with a
LB-953 Autolumat (Berthold, Nashua, NH) luminometer set for a 30-s
integration with no delay.
-Galactosidase activity was determined by
adding 50 µl of cell lysate to 297 µl of substrate (0.1 M Na2HPO4 buffer (pH 7.3), 0.013 M 2-nitrophenyl-
-D-galactopyranoside, 0.1%
(v/v) 1.0 M MgCl2, 0.35% (v/v)
-mercaptoethanol), incubating overnight at 37 °C, and measuring
colorimetrically at 410 nm in a Beckman DU640 spectrophotometer
(Beckman, Fullerton, CA) after the addition of 100 µl of 1.0 M sodium carbonate. Luciferase activity was normalized to
expression of RSV-
-galactosidase.
T3-1 cells were treated with 100 nM GnRHAg or vehicle for varying time intervals (1, 2, 4, or 8 h), and total RNA was extracted from cells using the Qiagen
"RNEasy" RNA extraction kit (Qiagen, Santa Clarita, CA). Total RNA
(10 µg/lane) was separated by electrophoresis on a denaturing 1.2%
agarose gel containing 6.7% formaldehyde prior to capillary transfer
onto sheets of Nytran (Schleicher & Schuell) immobilization membrane.
Northern blot analysis was performed under high stringency conditions
using a [32P]UTP-labeled antisense riboprobe (5 × 105 cpm/ml) prepared from the coding region (+173/+1153) of
mGnRHR cDNA using T7 RNA polymerase (New England Biolabs, Inc.,
Beverly, MA). Washed blots were exposed to Kodak X-Omat/AR film at
70 °C for 8-38 h. Rat cyclophilin antisense riboprobe was used as an internal standard. The intensity of the individual RNA bands was
quantified in a PhosphorImager (Molecular Dynamics, Sunnyvale, CA)
according to the protocol outlined by the manufacturers. Measurements were standardized for cyclophilin mRNA.
T3-1 cells were grown to
20, 40, 60, and 80% confluence and treated with 100 nM
GnRHAg or vehicle for varying time intervals (1 or 4 h).
Thereafter, cells were harvested, and nuclear extracts were prepared by
the method of Andrews and Faller (16).
308/
220 fragment of the mGnRHR gene promoter with
HindIII and XhoI, followed by 5'-end-labeling
with [
-32P]ATP by T4 polynucleotide kinase (New
England Biolabs). Constructs were then purified using a Qiagen
nucleotide removal kit. The binding reaction for EMSA was performed by
incubating 50,000 cpm of DNA probe with 10 µg of nuclear extract and
1 µg of salmon sperm DNA in reaction buffer (20 mM HEPES
(pH 7.9), 60 mM KCl, 5 mM MgCl2, 10 mM phenylmethylsulfonyl fluoride, 10 mM
dithiothreitol, 1 mg/ml bovine serum albumin, and 5% (v/v) glycerol)
for 30 min at 4 °C. For competition studies, excess unlabeled DNA
was added 5 min prior to the addition of probe. Protein-DNA complexes
were resolved on 4% low ionic strength non-denaturing polyacrylamide gel electrophoresis in 0.5× Tris borate/EDTA buffer (45 mM
Tris-HCl (pH 8.0), 45 mM boric acid, 1 mM
EDTA). Gels were then dried for 1 h and subjected to
autoradiography for 24-48 h. Antibody supershift experiments were
performed using an anti-Fos antibody (Santa Cruz Biotechnology) which
recognizes all members of the Fos oncoprotein family. Similar
experiments were carried out using an anti-Jun blocking antibody (Santa
Cruz Biotechnology) raised against the common DNA binding domain of all
members of the Jun family. Antibody, either anti-Fos (1 µl), anti-Jun
(1, 2, 4, or 8 µl), or both, was added to the EMSA reaction samples
after 30 min and incubated at 4 °C for an additional 2 h prior
to gel electrophoresis. When indicated, the intensity of the individual
protein bands was quantified in a PhosphorImager (Molecular Dynamics).
Measurements were standardized to background intensity.
T3-1 cells were transfected with GH50-pXP2,
GH50/
308/
264 (wild type), GH50/region C
(SURG-1 mutant), or GH50/region E (SURG-2 mutant) as
described, and response to GnRHAg stimulation was measured in the
presence or absence of selective agonists/antagonists. To investigate
the role of the PKC signal transduction pathway, transfected cells were
stimulated for 4 h with PMA (100 ng/ml), GnRHAg (100 nM), or both. Final concentrations of GnRHAg (Fig.
2A) and PMA (data not shown) were chosen to give maximal
stimulation at 4 h. Similar experiments were carried out in the
presence or absence of 1 µM GF-109203X (Sigma), a
simplified derivative of staurosporine that acts as a competitive
inhibitor for the ATP-binding site of PKC. This agent is selective for
PKC isoforms
,
1,
2,
,
, and
as compared with PKA, phosphorylase kinase, various tyrosine kinases, and PKC isoform
. In the latter experiments, cells were incubated with antagonist or vehicle for 30 min immediately prior to as well as
during the 4-h period of stimulation. Reagents were initially dissolved
in dimethyl sulfoxide (Me2SO) and subsequently diluted in
culture medium to give a final concentration of 0.01% (v/v) Me2SO in each experiment. To investigate the role of the
PKA signal transduction pathway, transfected cells were stimulated for
4 h with forskolin (25 µM), GnRHAg (100 nM), or both. Identical studies were carried out using a
second PKA agonist, 8-bromo-cAMP (1 mM). The effect of 1 µM SQ 22536 (Sigma), a selective inhibitor of PKA
activity, was investigated in selected experiments.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
T3-1 cells stimulated with GnRHAg (100 nM) or vehicle for varying time intervals (1, 2, 4, or
8 h) and hybridized with mGnRHR antisense riboprobe is shown in
Fig. 1A. The size of the major
mGnRHR mRNA was 4.5 kb as previously reported (17) and that of
cyclophilin mRNA was 0.8 kb. The intensities of individual bands
were quantified in a PhosphorImager and corrected for total RNA content
by using cyclophilin mRNA levels. Results (expressed as percent of
time 0) demonstrate a significant increase in GnRHR mRNA in
response to GnRHAg stimulation that was maximal at 4 h (1.84 ± 0.2-fold stimulation; p < 0.01) but decreased
thereafter (Fig. 1B). These data are consistent with
previous reports indicating that GnRH is capable of regulating GnRHR
gene expression, both in the short term (up-regulation) and in the long
term (down-regulation) (6, 11). The following study was then designed
to identify and characterize the critical cis-DNA element(s)
and cognate trans-factors responsible for this regulation in
T3-1 cells.
View larger version (20K):
[in a new window]
Fig. 1.
Northern blot analysis. A, a
Northern blot analysis of RNA extracted from T3-1 cells treated with
100 nM GnRHAg or vehicle for varying time intervals (1, 2, 4, or 8 h) and hybridized with mGnRHR antisense riboprobe is
shown. B, graphical representation of Northern blot
analyses. The intensity of individual bands was quantified using a
PhosphorImager and corrected for cyclophilin mRNA. Results are
expressed as percent of time 0. *, p < 0.01 compared
with time 0.
T3-1 cells were transiently transfected
with the full-length 1.2-kb 5'-flanking region of the mGnRHR gene
(
1164/+62) in pXP2 luciferase vector for 4 h and then subjected
to GnRHAg stimulation for varying times (2, 4, or 20 h) and with
varying concentrations of GnRHAg (0, 10, 100, or 500 nM).
On review of the GnRHAg dose- (Fig.
2A) and time-response curves
(Fig. 2B) for
T3-1 cells, GnRHAg stimulation was optimum
at a concentration of 100 nM for 4 h. A longer
transfection time resulted in increased basal expression of luciferase
activity and a significantly diminished response to GnRHAg (Fig.
2A). Optimum response to GnRHAg stimulation was also seen in
T3-1 cells grown to 40-50% confluence and cultured overnight in
high glucose DMEM in the absence of serum or antibiotics (data not
shown). Cells grown to
80% confluence or cells cultured overnight in
medium containing serum and antibiotics showed a marked reduction in GnRHAg responsiveness (data not shown). CV-1 cells transiently transfected with
1164/+62 also demonstrated a modest increase in
basal luciferase activity with time but showed no response to GnRHAg
(Fig. 2C), suggesting that the GnRH response is
cell-specific.
View larger version (20K):
[in a new window]
Fig. 2.
Characterization and optimization of the GnRH
response. A, GnRHAg dose-response curve. T3-1 cells
were transfected for 4 h with the 1.2-kb 5'-flanking region of the
mGnRHR gene (
1164/+62), followed by 4 h treatment with
increasing concentrations of GnRHAg (0, 10, 100, and 500 nM). Results are mean ± S.E. from multiple
experiments. *, p < 0.0002 compared with no GnRHAg.
, p = 0.012 compared with 10 nM GnRHAg.
B, transfection time course for
T3-1 cells.
T3-1 cells
were transfected with pXP2 or
1164/+62 for 2, 4, or 20 h and
treated with GnRHAg (100 nM for 4 h). Measurements are
expressed as luciferase/
-galactosidase. Results are mean ± S.E. from multiple experiments. Fold stimulation to GnRHAg is shown.
**, p < 0.001 compared with no GnRHAg. C,
transfection time course for CV-1 cells. CV-1 cells were transfected
with pXP2 or
1164/+62 for 2, 4, or 20 h, and response to GnRHAg
stimulation was measured. Results are mean ± S.E. from multiple
experiments. Fold stimulation to GnRHAg is shown.
T3-1 cells with
1164/+62 resulted in a 9.1 ± 1.3-fold increase in luciferase
activity in response to GnRHAg stimulation as compared with vector
alone (p < 0.0001; ANOVA) (Fig.
3). Transfection with serial 5'-deletion
mutants of the full-length construct demonstrated a significant
decrease in GnRHAg-stimulated luciferase activity between the
constructs
765/+62,
387/+62,
365/+62,
341/+62,
300/+62 (in
which fold stimulation was not different from each other nor from
1162/+62 but were all significantly different from pXP2 vector alone
(p < 0.01; ANOVA)) and the remaining constructs
232/+62,
117/+62, and
38/+62 (in which fold stimulation was not
significantly different from each other nor from pXP2 vector alone)
(Fig. 3). Despite a 2.4 ± 0.4-fold and 2.3 ± 0.4-fold stimulation in the
232/+62 and
117/+62 constructs, respectively, these measurements were not significantly different from pXP2 alone
(p = 0.56 and p = 0.57, respectively;
ANOVA; n = 13 separate experiments). Basal luciferase
activity was not significantly different between the various constructs
(data not shown). These data suggest the presence of an element(s)
within the region
300/
232 of the mGnRHR gene promoter which is
necessary for GnRH responsiveness.
View larger version (16K):
[in a new window]
Fig. 3.
Effect of 5'-deletions of the mGnRHR gene
promoter on GnRHAg-stimulated luciferase activity in T3-1
cells.
T3-1 cells were transfected for 4 h with pXP2, the
full-length promoter (
1164/+62), or one of a series of 5'-deletions
of the mGnRHR gene promoter (
765/+62,
387/+62,
365/+62
341/+62,
300/+62,
232/+62,
117/+62,
38/+62), followed by GnRHAg
stimulation (100 nM for 4 h). Measurements are
expressed as fold stimulation of luciferase activity by GnRHAg. Results
are mean ± S.E. from multiple experiments. *, p < 0.04 compared with the fold stimulation of pXP2,
232/+62,
117/+62, and
38/+62 by GnRHAg. NS = not
significant compared with pXP2.
387/
220 were placed in
control of a heterologous promoter, and response to GnRHAg stimulation
was measured. A number of heterologous minimal promoters were tested,
including the human
-subunit gene promoters (
(
99/+1) or
(
77/+1)), the rat growth hormone promoter (GH50), and the herpes simplex virus
thymidine kinase promoters (PT(
109/+1) or
PT(
81/+1)). The GH50 minimal promoter was
found to be most appropriate for our system (data not shown) and was
therefore used in all subsequent experiments. GH50-pXP2
alone did not increase luciferase activity in response to GnRHAg
stimulation, but incorporation of selected mGnRHR constructs upstream
of GH50 allows for a 10-12-fold response to GnRHAg (Fig.
4).
View larger version (14K):
[in a new window]
Fig. 4.
Identification of GnRH-RE(s) within the
mGnRHR gene promoter. PCR-generated fragments of the region
387/
220 of the mGnRHR gene promoter were placed in control of the
heterologous minimal promoter, GH50, and transfected into
T3-1 cells. Measurements are expressed as fold stimulation of
luciferase activity by GnRHAg (100 nM for 4 h).
Results are mean ± S.E. from multiple experiments. *,
p < 0.0001 compared with pXP2 (positive control).
,
p < 0.0001 compared with GH50-pXP2.
NS, not significant compared with
GH50-pXP2.
387/
220 of the mGnRHR gene
promoter were placed in control of the GH50 minimal
promoter, transfected into
T3-1 cells, and stimulated with GnRHAg.
Wild type sequence (GH50/
387/
220) showed only a
2.5 ± 0.6-fold response to GnRHAg stimulation that was not
statistically different from GH50-pXP2 alone (Fig. 4).
5'-Deletion from
387/
220 to either
308/
220 or
267/
220 did
not significantly affect GnRHAg-stimulated luciferase activity
(2.8 ± 1.2-fold and 1.1 ± 0.2-fold, respectively). However,
a 3'-deletion from
387/
220 to
387/
264 resulted in a 10.3 ± 1.7-fold increased response to GnRHAg (p < 0.0001 compared with GH50-pXP2), suggesting the presence of a
putative repressor element in the region
264/
220. Further
5'-deletion studies localized the primary GnRH-RE(s) to the 44-bp
region
308/
264 of the mGnRHR gene promoter (Fig. 4). The magnitude
of the GnRH response to
308/
264 (10.4 ± 1.3-fold) was similar
to that observed with the full-length mGnRHR promoter,
1164/+62
(10.4 ± 2.3-fold), which had been included as a positive control.
Thus, region
308/
264 is sufficient for GnRH responsiveness.
308/
264 of the mGnRHR gene promoter, five scanner-linker mutants of
this region were synthesized as detailed above (Fig. 5A), placed upstream of the
GH50 minimal promoter in GH50-pXP2, and
transfected into
T3-1 cells. Wild type expression vector (GH50/
308/
264) resulted in a 7.8 ± 1.2-fold
increase in luciferase activity in response to GnRHAg stimulation,
which was similar to that measured in the scanner-linker mutant
constructs for regions A, B, and D (Fig. 5B). However,
transfection with the mutant construct of region E
(GH50/region E, SURG-2mut), in which the putative AP-1-binding site had been mutated, completely abrogated the GnRHAg response (p < 0.0001 compared with wild type, but not
significant compared with GH50-pXP2; ANOVA). Similarly, a
single point mutation of the AP-1-binding site at position
269
(GH50/region E/mut) completely eliminated the GnRHAg
response, suggesting that region E at position
276/
269 of the
mGnRHR gene promoter is critical for GnRH responsiveness. Transfection
with the mutant construct of region C (GH50/region C,
SURG-1mut) resulted in a 3.6 ± 0.4-fold increase in luciferase
activity, which was significantly different from both
GH50-pXP2 alone and from the wild type mutant,
GH50/
308/
264 (p = 0.041 and
p = 0.0003, respectively; ANOVA) (Fig. 5B).
Once again, basal luciferase activity was not significantly different between the various constructs (data not shown). These data suggest that region E (SURG-2), which contains the consensus sequence for the
AP-1-binding site, is critical for GnRH responsiveness of the mGnRHR
gene and that region C (SURG-1) contains an enhancer element which may
be necessary for the full GnRH response.
View larger version (25K):
[in a new window]
Fig. 5.
Identification of two distinct GnRH-REs in
the mGnRHR gene promoter. A, five scanner-linker
mutations of the region 308/
264 of the mGnRHR gene promoter were
synthesized by insertion of the 8-bp NotI restriction enzyme
site (5'-GCGG
CCGC-3') as described. These are illustrated above. The
location of the single point mutation (C269T) of the AP-1-binding site
(designated GH50/region E/mut) is also shown. B,
scanner-linker mutations synthesized above were inserted upstream of
the GH50 minimal promoter in GH50-pXP2 and
transfected into
T3-1 cells. Measurements are expressed as fold
stimulation of luciferase activity by GnRHAg (100 nM for
4 h). Results are mean ± S.E. from multiple experiments. *,
p < 0.0001 compared with GH50-pXP2.
,
p = 0.041 compared with GH50-pXP2, and
p = 0.0003 compared with the wild type sequence
(GH50/
308/
264). NS = not significant
compared with GH50-pXP2.
T3-1 cells as
described. Both GH50/SURG-2 and GH50/SURG-1
demonstrated a significant increase in luciferase activity in response
to GnRHAg stimulation as compared with GH50 (8.8 ± 2.7-fold and 2.9 ± 0.6-fold, respectively; p < 0.001), and this response appeared to be additive so that the activity
of the reconstituted GH50/SURG-2/NotI/SURG-1
construct was similar to that seen with the full-length mGnRHR
promoter,
1164/+62 (13.7 ± 3.0-fold and 13.6 ± 3.0-fold,
respectively) (Fig. 6A).
GH50/SURG-2/NotI/SURG-2 gave a 72.7 ± 26.5-fold response to GnRHAg stimulation, lending further evidence to
the importance of SURG-2 in GnRH responsiveness in the mGnRHR gene.
Similar studies using the full-length mGnRHR gene promoter containing a
C269T point mutation of SURG-2 (
1164/+62/SURG-2mut) completely
abrogated the response to GnRHAg stimulation (Fig. 6B),
confirming that SURG-2 is critical for GnRH responsiveness. Mutation of
SURG-1 in the full-length construct (
1164/+62/SURG-1mut), on the
other hand, significantly diminished but did not abrogate the response (6.8 ± 0.9-fold as compared with 14.6 ± 1.7-fold response
seen with
1164/+62; p < 0.001 (Fig. 6B)).
These data provide further evidence in support of the hypothesis that
SURG-2 is a critical element for GnRH responsiveness of the mGnRHR gene
but that the inclusion of SURG-1 is necessary for optimal response to
GnRH.
View larger version (18K):
[in a new window]
Fig. 6.
Confirming the importance of regions SURG-1
and SURG-2 on GnRHAg-stimulated luciferase activity in T3-1
cells. A, GH50-linked constructs containing
single or multiple copies of SURG-2 (region E, AP-1-binding site)
and/or SURG-1 (region C) were synthesized as detailed above and
transfected into
T3-1 cells. Measurements are expressed as fold
stimulation of luciferase activity by GnRHAg (100 nM for
4 h). Results are mean ± S.E. from multiple experiments. *,
p < 0.0001 compared with pXP2 (positive control). **,
p < 0.001 compared with all other reactions. #,
p < 0.0001 compared with all other reactions.
,
p < 0.001 compared with GH50,
GH50/SURG-1, and
GH50/SURG-2/Not-1/SURG-2. B,
similar experiments were carried out using the full-length (1.2 kb)
mGnRHR gene promoter containing mutations of SURG-2
(
1164/+64/SURG-2mut) or SURG-1 (
1164/+64/SURG-1mut). *,
p < 0.0001 compared with pXP2 (positive control). #,
p < 0.02 compared with
1164/+62 and
1164/+62/SURG-1mut but not significant compared with pXP2.
,
p < 0.01 compared with all other reactions.
T3-1 cells and
32P-end-labeled
308/
220 of the mGnRHR gene promoter as
probe, two distinct protein-DNA bands could be identified on EMSA that
were not present with probe alone (Fig.
7). Nuclear extracts from cells grown to
approximately 40% confluence appeared to give optimum binding as
compared with nuclear extracts derived from cells grown to
approximately 20, 60, or 80% confluence (Fig. 7). These results are in
keeping with data from transfection studies suggesting that cells grown
to >40-50% confluence showed a marked reduction in GnRHAg
responsiveness (data not shown). Further EMSA experiments were
therefore standardized to nuclear extracts from
T3-1 cells grown to
40-50% confluence. GnRHAg stimulation (100 nM for 4 h) of
T3-1 cells prior to preparation of nuclear extract increased
the intensity of the lower band by 1.9 ± 0.4-fold
(p < 0.05; Student's t test), suggesting
the presence of a specific, GnRH-responsive protein within the complex
(Fig. 7). A shorter GnRHAg stimulus (100 nM for 1 h)
appeared to give similar results (1.8 ± 0.5-fold (data not
shown)). The intensity of the upper band did not change significantly
with GnRHAg stimulation.
View larger version (91K):
[in a new window]
Fig. 7.
Identification of a GnRH-responsive
trans-factor by EMSA. T3-1 cells were grown to 20, 40, 60, and 80% confluence and treated with GnRHAg (100 nM) or vehicle for 4 h prior to preparation of nuclear
extract. Using
T3-1 nuclear extracts and the
308/
220
PCR-generated fragment of the mGnRHR gene promoter as probe, EMSA
identified two distinct protein-DNA complex bands that were not present
in probe alone (designated by arrows). Nuclear extracts from
cells grown to approximately 40% confluence appeared to give optimum
binding as compared with nuclear extract derived from cells grown to
20, 60, or 80% confluence. Stimulation of
T3-1 cells by GnRHAg
prior to preparation of nuclear extract increased the intensity of the
lower band by 1.9 ± 0.4-fold as measured by a
PhosphorImager.
276/
269 of the
mGnRHR gene promoter, which contains the AP-1-binding site, is critical
for GnRH responsiveness. Exactly which of the Jun and Fos family
members make up this heterodimer complex has yet to be determined. We
are also currently investigating the identity of the protein(s)
responsible for the upper band seen on EMSA.
View larger version (41K):
[in a new window]
Fig. 8.
Identification and characterization of
trans-factors binding to critical cis-DNA
elements in the mGnRHR gene promoter by EMSA. A, using
nuclear extracts prepared from T3-1 cells and the
308/
220
fragment of the mGnRHR gene promoter as probe, the two protein-DNA
complex bands could again be identified by EMSA. Control was probe
alone. An increase in intensity of the lower band was again seen with
GnRHAg stimulation. Supershift of the lower band with anti-Fos antibody
(Santa Cruz Biotechnology) suggests the presence of a Fos protein
within this DNA-protein complex (see arrows). B,
similar experiments were carried out using an anti-Jun blocking
antibody (Santa Cruz Biotechnology). Results demonstrate a moderate but
significant diminution in binding of the lower band suggesting the
presence of Jun protein within the complex (see small
arrow). To determine the specificity of the anti-Jun antibody,
EMSA experiments were carried out using purified c-JUN protein
(Promega) and the consensus AP-1-binding site (5'-TGAGTCA-3') as probe.
Control was probe alone. Addition of excess anti-Jun antibody resulted
in significant diminishment in the intensity of the lower band (see
large arrow). Addition of excess anti-Fos antibody had no
effect on binding (data not shown).
T3-1 cells were transfected with
GH50-pXP2, GH50/
308/
264 (wild type),
GH50/region C (SURG-1mut), or GH50/region E
(SURG-2mut) and stimulated for 4 h with PMA (100 ng/ml), GnRHAg (100 nM), or both. In the wild type sequence, PMA
stimulation resulted in a 15.2 ± 3.3-fold increase in luciferase
activity, which was not significantly different from the 9.6 ± 2.1-fold increase seen with GnRHAg stimulation (p = 0.4; Student's t test). However, simultaneous stimulation
with both PMA and GnRHAg gave an additive effect resulting in a
25.2 ± 1.1-fold increase in luciferase activity
(p = 0.02 and p = 0.001 compared with
PMA alone and GnRHAg alone, respectively; ANOVA) (Fig.
9A). The addition of the
selective PKC antagonist, GF-109203X (1 µM; Sigma),
resulted in complete abrogation of the response to PMA, to GnRHAg, and to both PMA and GnRHAg to a level similar to that seen in
GH50-pXP2 vector alone (Fig. 9A). Similar
results were seen with SURG-1mut, although the magnitude of the
response to both GnRHAg and PMA was significantly diminished.
SURG-2mut, on the other hand, failed to demonstrate an increase in
luciferase activity in response to either PMA or GnRHAg (Fig.
9A). Taken together, these data suggest that both PMA and
GnRHAg stimulation of the mGnRHR gene promoter are mediated through
SURG-2.
View larger version (36K):
[in a new window]
Fig. 9.
Fig. 9. Role of selected signal transduction
pathways in GnRHAg stimulation of the mGnRHR gene. A,
to investigate the effect of PKC agonists and antagonists on
GnRHAg-stimulated luciferase activity, T3-1 cells were transfected
with GH50, wild type sequence
(GH50/
308/
264), or mutant constructs for regions C
(SURG-1mut) or E (SURG-2mut) as described and stimulated for 4 h
with PMA (100 ng/ml), GnRHAg (100 nM), or both.
Measurements are expressed as fold stimulation of luciferase activity.
Results are mean ± S.E. from multiple experiments. *,
p < 0.04 compared with fold stimulation in the
presence of GF-109203X. **, p < 0.0001 compared with
fold stimulation in the presence of GF-109203X.
, p < 0.05 compared with fold stimulation in the presence of GF-109203X.
#, p < 0.01 compared with PMA- or GnRHAg-stimulated
luciferase activity in wild type construct
(GH50/
308/
264). NS = not significant.
B, similar experiments were performed to investigate the
role of the PKA signal transduction pathway in GnRH stimulation of the
mGnRHR gene.
T3-1 cells were transfected as detailed above and
stimulated for 4 h with forskolin (25 µM), GnRHAg
(100 nM), or both. *, p < 0.001 compared
with all other reactions.
, p < 0.04 compared with
GH50-pXP2, SURG-1mut + forskolin ± SQ 22536. NS = not significant.
308/
264 (wild type), SURG-1mut, or SURG-2mut (Fig.
9B). Once again, the magnitude of the response seen with
SURG-1mut was significantly lower than that seen with the wild type
construct. Similarly, the absence of GnRHAg-stimulated luciferase
activity with SURG-2mut was consistent with previous experiments.
Furthermore, incubation with the selective PKA antagonist, SQ 22536 (1 µM; Sigma), had no effect on GnRHAg-stimulated luciferase
activity (Fig. 9B). Identical results were observed using
8-bromo-cAMP (1 mM; data not shown).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
T3-1 cells, a well characterized mouse pituitary
gonadotrope cell line, as a model for the analysis of
cis-regulatory elements in the mGnRHR gene. This cell line,
obtained by targeted tumorigenesis in the mouse pituitary with the SV40
large T antigen driven by the human glycoprotein hormone
-subunit
promoter (18), has been used to study many aspects of gonadotrope
physiology. A number of studies have shown that
T3-1 cells
constitutively express GnRHR and are capable of binding and responding
to exogenous GnRH (13, 18). Characterization of this cell model has
demonstrated many similarities in the GnRH response compared with that
in mouse primary pituitary cells, including the specific intracellular signal transduction pathways activated, the degree of stimulation of
the gonadotropin subunit promoter activities, and the presence of
differential regulation of GnRHR and
-subunit gene promoter activities by GnRH (4, 14, 19).
T3-1 cells thus appear to be a
useful model for the study of the regulation of expression of the GnRHR
gene by GnRH.
T3-1 cells in response to continuous exposure to GnRHAg for 1-24 h, and Mason et al. (21)
demonstrated a time- and dose-dependent decrease in the
level of GnRHR mRNA in
T3-1 cells in response to GnRH or GnRHAg.
The disparity among these results may be related to cell culture
conditions and the timing of GnRH stimulation. In our hands, optimal
response of mGnRHR promoter-transfected
T3-1 cells to GnRHAg
stimulation was seen after 4 h transfection and 4 h GnRHAg
stimulation. A longer transfection time resulted in increased basal
expression of luciferase activity but a significantly diminished
response to GnRHAg (Fig. 2B).
T3-1
gonadotrope mRNA (13). Functional analysis by transient
transfection of
T3-1 cells with the 1.2-kb 5'-flanking region of the
mGnRHR gene (
1164/+62 [Figs. 2 and 3]) confirmed previous
observations (13, 25) that this region contains an element(s) that is
necessary for tissue-specific basal expression as well as for GnRH
responsiveness. By using deletion and mutational analysis in
T3-1
cells, Duval et al. (26) recently identified a tripartite
enhancer that appears to be responsible for regulating cell-specific
basal expression of the GnRHR gene. Individual elements of
this putative enhancer include binding sites for steroidogenic factor-1
(SF-1), AP-1, and a novel element designated GnRH receptor activating
sequence. Although a number of hormones, including GnRH (7-9),
estradiol (9, 27), and activin A (28), either alone or in combination,
are known to affect transcriptional activation of the mGnRHR gene,
neither the cis-regulatory element(s) nor their cognate
binding protein(s) has previously been identified. In this study,
transfection of
T3-1 cells with serial 5'-deletion mutants of the
1.2-kb 5'-flanking region of the mGnRHR gene (
1164/+62) demonstrated
the existence of an element(s) within the region
300/
232 of the
mGnRHR gene promoter that is necessary for GnRH responsiveness (Fig.
3). Further studies show that the 44-bp region
308/
264 is also
sufficient to mediate GnRH responsiveness in the mGnRHR gene (Fig. 4).
Transfection studies using a series of scanner-linker mutations of this
region further localized GnRH responsiveness to two distinct GnRH-REs, designated SURG-1 and SURG-2 (Fig. 5B). SURG-2
(5'-TATGAGTC-3') lies at position
276/
269 and contains the
consensus sequence for the AP-1-binding site. The AP-1-binding site is
a ubiquitous DNA consensus motif (5'-TGAGTCA-3') that binds a family of
dimeric transcription factors, known collectively as AP-1, which are
composed of Jun, Fos, and/or ATF (activating transcription factor)
subunits. SURG-1 (5'-GCTAATTG-3') lies at position
292/
285 and
appears to be a novel element. The relative importance of SURG-1 and
SURG-2 in mediating GnRH responsiveness of the mGnRHR gene was
confirmed by demonstrating that these two elements, either alone or in
concert, are capable of conferring activity on an heterologous minimal promoter, GH50, in
T3-1 cells (Fig. 6A). In
addition, a point mutation of SURG-2 (C269T) in the full-length 1.2-kb
5'-flanking region of the mGnRHR gene completely abrogated the
GnRHAg-stimulated response, whereas mutation of SURG-1 diminished but
did not completely abolish this stimulation (Fig. 6B). These
data suggest that SURG-2 (AP-1-binding site) is critical to GnRH
responsiveness in the mGnRHR gene, whereas SURG-1 acts as an enhancer
element to facilitate full GnRH response. The putative repressor
element in the region
264/
220 of the mGnRHR gene has not been
further characterized.
-subunit gene
as an element that binds the nuclear orphan receptor, SF-1 (29), and
appears to be important for gonadotrope-specific expression of the
-
(30) and LH
-subunit genes (31). It has also been shown to be
important in regulating cell-specific basal expression of the mGnRHR
gene (26). Our studies, however, suggest that the putative
gonadotrope-specific element is not involved in GnRHAg responsiveness
of the mGnRHR gene. Functional transfection studies using serial
5'-deletion constructs of the 1.2-kb putative mGnRHR gene promoter
(
1164/+62) showed that downstream constructs (
232/+62,
117/+62,
and
38/+62) did not significantly stimulate luciferase activity in
response to GnRHAg (Fig. 3). Results from mutational studies in the
full-length
1164/+62 construct (Fig. 6B) further confirm
these observations. These data are in keeping with observations made in
SF-1 knock-out mice. Targeted disruption of the murine
ftz-F1 gene encoding SF-1 results in adrenal and gonadal
hypoplasia (32). Such SF-1 knock-out mice exhibit malformations of the
ventromedial hypothalamus as well as selective deficiency of GnRHR,
LH
, and FSH
mRNA in the pituitary (32). However, treatment
with GnRH results in partial restoration of gonadotropin subunit gene
expression as well as detectable levels of mGnRHR mRNA (32). Taken
together, these results along with our data suggest that SF-1 is not a
critical element for mGnRHR gene expression.
T3-1 cells, with or without GnRHAg
stimulation, two distinct protein-DNA bands were identified on EMSA.
Nuclear extracts from cells grown to approximately 40% confluence
appeared to give optimum binding. These results are in keeping with
data from transfection studies suggesting that cells grown to 40-50%
confluence showed optimal response to GnRHAg stimulation. The lower
protein-complex band on EMSA, but not the upper band, appeared to be
GnRH-responsive. Using antibody blocking and supershift EMSA
experiments, we have demonstrated that the lower band represents a
complex containing a member(s) of the Jun/Fos heterodimer superfamily,
also known as the AP-1 protein complex (Fig. 8). These data are
consistent with the functional transfection studies demonstrating that
the AP-1-binding site (SURG-2) is critical for GnRH responsiveness of
the mGnRHR gene. Anti-Fos supershift EMSA experiments resulted in a
supershift of the entire lower band. Anti-Jun blocking EMSA, on the
other hand, resulted in only a modest inhibition in binding. It is
possible that Jun may not itself bind to cis-regulatory
elements and that AP-1 stimulation of mGnRHR gene transcription may be
mediated by Jun interaction with another protein(s), perhaps Fos, that directly binds to the proximal mGnRHR promoter. A similar observation was made by Bruder et al. (33) investigating the role of
AP-1 in the repression of GnRH gene transcription in GT1-7 neuronal cells. Exactly which of the Jun and Fos family members make up this
heterodimer complex has yet to be determined. The identity of the
DNA-protein complex responsible for the upper band seen on EMSA has yet
to be characterized but may represent a trans-factor binding
to the SURG-1 cis-regulatory element.
T3-1 cells cultured in the presence of 10%
fetal calf serum are relatively high (34). However, levels of
c-fos, c-jun, as well as junB mRNA
have been shown to decrease progressively over a 6-h period in
T3-1
cells cultured in serum-free medium. The final steady-state mRNA
level of all three of these trans-factors after 6 h was
around 3-5% of that observed in cells cultured in the presence of
10% fetal calf serum (34). In transcription studies detailed above,
optimal response to GnRHAg stimulation was observed in
T3-1 cells
cultured overnight in serum-free medium (data not shown). By having
subsequently identified a member of the Jun/Fos heterodimer superfamily
as the critical element in GnRH responsiveness of the mGnRHR gene, we
hypothesize that an overnight incubation in serum-free medium might
decrease basal (endogenous) levels of Jun and Fos, thereby allowing for
an enhanced response to GnRHAg.
-, FSH
-, and LH
-subunit genes are
all strictly regulated in pituitary gonadotropes by GnRH and other
hormones. Separate and independent manipulation of each of these
related genes in a single cell type requires a complex regulatory
system. The individual components of this regulatory system are poorly
defined but probably include activation of distinct signal transduction
pathways, the presence of specific cis-regulatory elements
in the promoter regions of each of these genes, and the incorporation
of different trans-factors and/or coactivators/corepressors which differentially regulate gene transcription. The GnRH-RE(s) in
each of the gonadotropin subunit genes have been partially characterized. In the LH
gene, two putative Sp1-binding sites in the
proximal promoter region appear to play an important role in conferring
GnRH responsiveness (35), and two transcription factors, SF-1 and early
growth response-1 (Egr-1), are known to be involved in tissue-specific
expression of this gene (31, 36). There is no consensus Sp1-binding
site in the mGnRHR gene. Analysis of the
-subunit promoter in
T3-1 cells and in transgenic mice (14, 37) have led to the
identification of multiple cis-regulatory elements that
appear to be important for tissue-specific basal expression of the
-subunit gene. Such elements include a binding site for a LIM
homeodomain protein (38), several canonical E boxes (39), the
ACT
element that binds members of the GATA binding factor family (40), and
the gonadotrope-specific element that binds the SF-1 transcription
factor (29). A binding site for Ets factor (a family of transcription
factors that have been implicated in mediating transcriptional
responses to mitogen-activated protein kinase activation) has also been
identified in the
-subunit gene promoter (41). The precise
GnRH-RE(s) in the common
-subunit gene, however, has not been
characterized. This may be due in part to the observation that the
common
-subunit gene is less well regulated by GnRH as compared with
the GnRHR, LH
-, and FSH
-subunit genes (10). Recent studies in the
ovine FSH
gene have identified two AP-1 enhancers in the proximal
promoter that appear important for tissue-specific basal FSH
expression in vivo (42). These same AP-1 elements also
appear to mediate GnRH-stimulated transcription of the ovine FSH
gene in primary cultures of ovine pituitary cells (43). There are no
confirmed AP-1 consensus sequences in either common
- or
LH
-subunit gene promoters.
-subunit genes
within the pituitary gonadotrope cell remains unclear. A number of
potential mechanisms exist. Specific homo- and heterodimer members of
the AP-1 family, for example, may differentially regulate target genes
through a common cis-regulatory element. Alternatively, the
same AP-1 trans-factor may interact with different protein
kinases and/or transcriptional coactivators/corepressors to affect
distinct biological functions. Our data suggest another possibility,
namely the incorporation of one or more secondary
cis-elements, such as SURG-1 for the GnRHR gene (above).
Exactly which of the AP-1 family members binds to the GnRHR and
FSH
-subunit promoter regions are not known. There is, however,
evidence to suggest that different signal transduction pathways may be
involved in the regulation of these two genes. Although GnRHAg
stimulation of both FSH
-subunit (43) and GnRHR genes (above) appears
to be mediated through PKC, inhibition of PKC activity completely
blocked GnRHAg-mediated stimulation of the GnRHR gene (above) but only
partially blocked that of the FSH
-subunit gene (43).
View larger version (21K):
[in a new window]
Fig. 10.
Sequence homology in SURG-1 and SURG-2
regions of the GnRHR gene between the species. There is a high
degree of sequence homology in the regions of interest between the
mouse and the rat (numbered from the major TSS). There is no AP-1
consensus sequence in the sheep (47). In the human, there is a
consensus AP-1 site in the region 1515/
1509 of the GnRHR gene
promoter (numbered from the major translational start site) as well as
11 regions with 6/8 (76%) sequence homology with SURG-1 (the closest
region is shown).
The intracellular signal transduction pathways within pituitary
gonadotropes, which are involved in regulating gonadotropin subunit and
GnRHR gene transcription, are still not clearly described but likely
include phosphoinositides, calcium, and cAMP as second messengers
and/or mitogen-activated protein kinase cascades (see Ref. 2 for
review). GnRH induction and basal regulation of the -subunit gene
seems to occur through the PKC/mitogen-activated protein kinase
pathway, whereas induction of the LH
gene is dependent on calcium
influx (49). In this study, PMA stimulation of luciferase activity in
T3-1 cells transfected with
308/
264 of the mGnRHR gene was
similar to that seen with GnRHAg (Fig. 9A). The addition of
GF-109203X (Sigma), a inhibitor selective for the PKC isoforms
,
1,
2,
,
, and
, resulted in
complete abrogation of the response to either PMA or GnRHAg.
Simultaneous stimulation with optimal doses of both PMA and GnRHAg
resulted in an additive effect as compared with each agonist alone,
which was similarly completely blocked by GF-109203X. These data
suggest that both PMA and GnRHAg stimulation of the mGnRHR gene are
mediated via PKC. The additive effect of PMA on GnRHAg stimulation
implies either that these two agents act through different PKC isoforms
or that they act synergistically through the same PKC pathway. Whatever
the mechanism, it is clear from the transfection data presented above
that both agents act at least in part through the SURG-2 consensus
sequence. Similar experiments were carried out using forskolin and
8-bromo-cAMP to investigate the role of the PKA signal transduction
pathway in the GnRHAg response. Neither agonist was able to stimulate luciferase activity in
T3-1 cells transfected with
308/
264 nor
were they able to influence GnRHAg-stimulated luciferase activity in
such cells. Similarly, the addition of SQ 22351 (Sigma), a competitive
inhibitor of adenylate cyclase, had no effect on GnRHAg stimulation
(Fig. 9B). These findings were not unexpected given that no
cAMP-response element-like sequence has been identified in the mGnRHR
gene (13, 25), although there may be cAMP-response element-like
elements in the rat and human GnRHR genes (44, 45). Taken together,
these data suggest that the response of the mGnRHR gene to GnRHAg
stimulation is mediated via PKC and not PKA. These observations are
consistent with a number of previous reports suggesting that PKC and
its activators increase GnRH binding activity in pituitary gonadotropes
(50, 51) but in contrast with other studies in which phorbol esters did
not affect levels of GnRHR mRNA in
T3-1 cells, whereas forskolin
decreased GnRHR mRNA (20). Whether this discrepancy can be
explained on the basis of post-transcriptional modification has yet to
be determined. Although GnRH is known to induce levels of cAMP in
gonadotropes both in vitro and in vivo (52, 53),
GnRHAg stimulation of gonadotropin secretion appears to be independent
of changes in cAMP (54). These data do not exclude the possibility that
second messengers such as calcium and/or mitogen-activated protein
kinase cascades may be involved in this response downstream of PKC.
Indeed, the observations that both GnRH and PMA induce rapid increases in mRNA levels for primary response genes (including jun
and fos) with a peak response at around 30 min (34), whereas
maximal response of the mGnRHR gene to GnRHAg stimulation is achieved at around 4 h (above), suggest that a more complex intracellular signal transduction pathway may be involved.
While this paper was being completed, Lin and Conn (55) reported on
transcriptional activation of the mGnRHR gene by GnRH and cAMP in
GGH3 cells (GH3 cells stably expressing GnRHR).
By using the same full-length mGnRHR promoter construct as that
detailed above (13), the authors localized the major putative
GnRH-RE(s) to the region 331/
255 (relative to the major TSS) of the
mGnRHR gene promoter, which is in keeping with our data. In our
studies, response to GnRHAg stimulation (100 nM for 4 h) ranged from 10- to 12-fold (Fig. 2-3) as compared with a 2-fold
response to GnRHAg stimulation (Buserelin; 100 nM for
6 h) reported by Lin and Conn (55). This discrepancy may be
accounted for by the use of different cell lines (
T3-1 cells and
GGH3 cells, respectively) but is more likely due to
differences in cell culture conditions. As demonstrated above (Fig.
2B), optimal response to GnRHAg stimulation was seen after
4 h transfection. Longer transfection times were associated with
higher basal luciferase activity but a marked reduction in GnRHAg
responsiveness. In the study by Lin and Conn (55), cells were
transfected for 24 h. By using serial 5'-deletion constructs of
the full-length (12 kb) mGnRHR gene promoter in transfection studies,
the authors demonstrated a 1.5-2-fold response to both GnRHAg and
dibutyryl-cAMP in the
255/+62 construct, which they suggested was
statistically significant. Comparable studies detailed above (Fig. 3)
demonstrated a similar 2.3-2.4-fold response to GnRHAg stimulation in
the
232/+62 and
117/+62 constructs, but these measurements were not
statistically different from pXP2 alone.
In summary, we have used deletion and mutational analysis as well as
functional transfection studies in the murine gonadotrope-derived T3-1 cell line to localized GnRH responsiveness of the mGnRHR gene
to two DNA sequences at
276/
269 (SURG-2, the AP-1 consensus binding
site) and
292/
285 (a novel element designated SURG-1), and we
demonstrated that this response is mediated via PKC. By using EMSA, we
further demonstrate that a member(s) of the Fos/Jun heterodimer
superfamily is responsible for the DNA-protein complexes formed using
T3-1 nuclear extracts. These data define the dimeric GnRH-RE in the
mGnRHR gene promoter and suggest that the AP-1 complex plays a central
role in conferring GnRH responsiveness to the mGnRHR gene.
![]() |
ACKNOWLEDGEMENTS |
---|
Dr. Constance Albarracin is thanked for
mGnRHR promoter constructs (1164/+62,
765/+62,
365/+62,
232/+62, and
117/+62). Drs. Ursula Kaiser and Lisa Halvorson are
thanked for their help and advice.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant R01 HD19938 (to W. W. C.) and by the Reproductive Scientist Development Program through National Institutes of Health Grant 5K12HD00849 and the Association of Professors of Gynecology and Obstetrics (to E. R. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Association of Professors of Gynecology and Obstetrics-National
Institutes of Child Health and Development Fellow of the Reproductive Scientist Development Program. To whom correspondence and reprint requests should be addressed: G. W. Thorn Research Bldg., Rm. 1013, Brigham & Women's Hospital, 20 Shattuck St., Boston, MA 02115. Tel.:
617-732-5857; Fax: 617-732-5123; E-mail:
ernorwitz{at}bics.bwh.harvard.edu.
The abbreviations used are: GnRH, gonadotropin-releasing hormone; AP-1, activating protein-1; LH, luteinizing hormone; FSH, follicle-stimulating hormone; GnRHR, GnRH receptor; GnRHAg, GnRH agonist; EMSA, electrophoretic mobility shift assay; mGnRHR, mouse GnRHR; PKA, protein kinase A; PKC, protein kinase C; RSV, Rous sarcoma virus; SF-1, steroidogenic factor-1; PMA, phorbol 12-myristate 13-acetate; ANOVA, analysis of variance; RE, response elements; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; kb, kilobase; bp, base pair(s); mut, mutant; TSS, transcriptional start site.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|