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Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622
Address all correspondence and requests for reprints to: Dr. William L. Miller, Department of Biochemistry, Box 7622, North Carolina State University, Raleigh, North Carolina 27695-7622.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß heterodimeric glycoprotein, the formation of which is
regulated primarily by expression of its ß-subunit. Recent studies on
transcriptional regulation of the ovine FSH ß-subunit gene (oFSHß)
have defined two functional activating protein-1 (AP-1) enhancers in
the proximal promoter (located at -120 and -83 bp) that are probably
physiologically important for FSHß expression. As GnRH is a major
regulator of FSHß expression and is also known to stimulate the
synthesis of Jun and Fos family members (AP-1), we investigated the
possibility that oFSHß transcription may be regulated by GnRH through
AP-1. Here we report the use of an in vitro cell system
involving transient transfection of GnRH receptors (GnRHR) into HeLa
cells to define regulatory elements involved in GnRH-mediated induction
of oFSHß. This system was used to show that expression of luciferase
constructs containing either the -4741/+759 region of the oFSHß gene
(-4741oFSHß-Luc) or the -846/+44 region of the human gene
(-Luc; a positive control) was stimulated 3.1 ± 0.3- and
7.7 ± 1.9-fold, respectively, by 100 nM GnRH. Another
luciferase expression plasmid containing the Rous sarcoma virus
promoter (a negative control) showed no response to GnRH. Similar
results with these constructs were obtained in COS-7 cells. Studies
with progressive 5'-deletion constructs and site-specific mutations
demonstrated that this stimulation was dependent on each AP-1 site in
the proximal promoter of oFSHß. Gel shift assays demonstrated the
ability of GnRHR in HeLa cells to increase AP-1 binding activity.
Responses in the HeLa cell system were dependent on GnRH
(ED50 = 0.5 nM) and GnRHR, which was identified
by photoaffinity labeling. In addition, GnRHR-expressing HeLa cells
exhibited a normal GnRH-dependent mobilization of intracellular
calcium. Finally, as protein kinase C (PKC) is a known target of GnRH
action in gonadotropes, the role of PKC in transcriptional regulation
of oFSHß and -subunit genes by GnRH in HeLa cells was
investigated. Although 12-O-tetradecanoyl 13-acetate
induction of -Luc and -215oFSHß-Luc could be completely blocked
in a dose-dependent manner by the specific PKC inhibitor
bisindolylmaleimide I, only 57–65% of the GnRH-mediated stimulation
of these promoters was blocked, demonstrating the involvement of PKC as
well as other signaling systems in GnRH induction. These data define a
molecular action of GnRH on oFSHß gene transcription that involves
two proximal AP-1 enhancer elements and PKC activation. Furthermore,
these studies establish the usefulness of HeLa and COS-7 cells to
investigate specific aspects of GnRH action on gonadotropin subunit
gene expression, as similar signaling pathways and transcription
factors that are activated by GnRH in gonadotropes (such as PKC,
mitogen-activated protein kinase, Ca2+, and AP-1) exist in
these cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß heterodimeric
glycoprotein hormone necessary for folliculogenesis and the female
reproductive cycle. It is produced, along with LH, in gonadotropes of
the anterior pituitary. The -subunit in FSH is also common to LH,
but the biological activity of each hormone is defined by its unique
ß-subunit (1). As expression of the ß-subunit for FSH is the
primary rate-limiting step in overall FSH production, those factors
controlling FSHß synthesis are the key regulators of gonadal and
reproductive function.
One primary regulator of FSHß expression in vivo is GnRH.
This decapeptide hormone is released from the hypothalamus and binds a
specific G protein-coupled receptor (GnRHR) on plasma membranes of
gonadotropes to activate intracellular signals that lead to
gonadotropin secretion and increased transcription of all gonadotropin
subunits (1, 2, 3). Evidence is now emerging that GnRH induces and/or
activates components of the AP-1 transcriptional complex, which usually
consists of Jun/Jun homodimers or Jun/Fos heterodimers that bind target
genes to increase their expression. It has been shown that GnRH
administration to cultured rat pituitary or immortal T3–1
gonadotrope cells causes a rapid increase in the messenger RNAs (mRNAs)
for the protooncogenes c-jun, junB, and
c-fos (4). Additionally, pulsatile administration of GnRH to
ovariectomized hypogonadotropic (GnRH-deficient) lambs was shown to
increase pituitary mRNAs for c-jun and c-fos (5).
This same study demonstrated that pituitary levels of c-jun
mRNA increased 2- to 3-fold just before the preovulatory LH surge,
strongly suggesting that dynamic changes in activating protein-1 (AP-1)
transcriptional complexes were occurring in gonadotropes throughout the
reproductive cycle. Finally, it has been shown that GnRH administration
to perifused rat pituitary cells activates protein kinase C (PKC), and
PKC is a known activator of c-jun (6, 7). These data, taken
together, imply an important role for AP-1 transcriptional complexes in
gonadotropes and, further, suggest the possibility that GnRH action on
gonadotropin subunit gene expression may include mechanisms involving
AP-1.
Recent studies with the ovine FSHß gene (oFSHß) uncovered two functionally linked AP-1 sites in the proximal promoter, lending strength to a hypothesis that FSHß gene transcription in vivo is regulated at least in part through Jun and Fos family members (8). Additionally, these studies provided the first evidence that the proteins for Jun and Fos family members are directly expressed in a majority of gonadotropes in vivo, demonstrating that AP-1 is present to potentially act on the oFSHß gene. Working on the hypothesis that GnRH is an important regulator of FSHß, and that AP-1 may be a component of GnRH action on FSHß gene transcription, we engineered an in vitro cell assay system to analyze regulatory elements involved in GnRH action on the FSHß gene. In this report we use such a system to demonstrate that transcriptional activation of the oFSHß gene by GnRH in HeLa cells involves each of the previously characterized AP-1 sites in the oFSHß gene and, further, show that this stimulation partially involves PKC activation. HeLa cells were used as a model system because there are no FSHß-producing cell lines available and because all transformed pituitary cell lines known to date poorly express oFSHß promoter/luciferase constructs. These data are the first to characterize a mechanism of action of GnRH at the level of FSHß promoter sequence and provide a direct physiological role for GnRH-induced AP-1 transcriptional complexes in gonadotropes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit promoter or the Rous sarcoma virus (RSV) promoter (-Luc
and RSV-Luc, respectively) (9) were provided by Dr. J. Larry Jameson
(Department of Medicine, Northwestern University, Chicago, IL). The
cytomegalovirus (CMV) mouse GnRHR expression plasmid (10) was a gift
from Dr. Stuart C. Sealfon (Mt. Sinai School of Medicine, New York,
NY). The RSV-ß-galactosidase expression construct (pRSVßgal), an
internal control for these studies, has been described previously
(8).
Cell culture and transient transfection
HeLa and COS-7 cells were obtained from the American Type
Culture Collection (Manassas, VA). T3–1 cells were provided
by Dr. Pamela L. Mellon (University of California-San Diego, La Jolla,
CA). All transformed cells in this study were grown at 37 C in DMEM
(Life Technologies, Grand Island, NY) containing 10% FBS
(HyClone Laboratories, Inc., Logan, UT) under 95% air-5%
CO2. Cells used for transfection were grown in
150-cm2 flasks until they were confluent and then were
replated in six-multiwell plates (diameter, 35 mm/well) at a
concentration of 300,000 cells/well the day before transfection. Unless
otherwise indicated, HeLa cells were transiently transfected in
triplicate with 8 µg reporter construct, 1 µg GnRHR, and 10 µg
pRSVßgal (total volume, 0.5 ml) using the calcium phosphate method
(11). COS-7 cells were transfected in the same way as HeLa cells,
except that 6 µg reporter construct and 4 µg pRSVßgal were used.
Eighteen hours after the start of transfection, the precipitates were
removed and replaced with low serum medium (0.5% FBS). Six hours
later, cells were treated with either 100 nM GnRH
(Sigma Chemical Co., St. Louis, MO) or 10 nM
12-O-tetradecanoylphorbol 13-acetate (TPA;
Calbiochem-Novabiochem International, La Jolla, CA). For
transfection studies involving the PKC inhibitor bisindolylmaleimide I
(BIM; also known as GF 109203X; Calbiochem-Novabiochem
International), HeLa cells were pretreated with the indicated
concentration of BIM 15 min before the addition of GnRH or TPA. Cells
were harvested for determination of luciferase and ß-galactosidase
activities 12 h after GnRH or TPA treatment. Luciferase and
ß-galactosidase activity assays from transient transfections were
performed as previously described (8).
Normalization and statistical analysis of the transient
transfection data
Activity in the internal control ß-galactosidase expression
construct, pRSVßgal, was used to normalize the transfection
efficiency in HeLa or COS-7 cell culture studies. The transfected gene
for ß-galactosidase typically showed 5- to 10-fold higher activity
than the endogenous ß-galactosidase gene in all transfections.
Variation in transfection efficiency was normalized by dividing the
ß-galactosidase values directly into the luciferase values from
control and GnRH- or TPA-treated cultures. Expression of the pRSVßgal
construct under GnRH treatment was enhanced 1.5- to 1.8-fold in these
experiments. Because the expressions of RSV-Luc, -84oFSHß-Luc, and
-120/-83 mut FSHß-Luc were increased to the same degree as that of
pRSVßgal, this slight enhancement was judged to be a nonspecific
effect caused by GnRH. Thus, although the negative control RSV-Luc and
deletion/mutation oFSHß-Luc constructs listed above were normalized
to about 1.0 (no stimulation), a typical 5.0- to 6.0-fold increase in
-215oFSHß-Luc expression was normalized to 3.2- to 3.8-fold.
To combine data from independent experiments, the basal expression for each construct in each experiment was averaged and then assigned a relative value of 1 (see figures). Induction ratios (fold induction) were obtained by dividing the average value for the GnRH-induced or TPA-induced reporter by the average basal expression of the same reporter. Differences between means obtained for basal expression, stimulated GnRH or TPA expression, and induction ratios of all luciferase expression constructs were determined using ANOVA followed by Tukey’s multiple range test for individual comparisons (12).
Measuring intracellular calcium
([Ca2+]i)
HeLa cells were transiently transfected as described above,
using 1 µg GnRHR and 18 µg Bluescript or Bluescript alone (19
µg). Eighteen hours after transfection, HeLa cells as well as
T3–1 cells were plated into four-well Nunc chamber slides
(Fisher Scientific International, Inc., Pittsburgh, PA;
catalog no. 1256517) and cultured 24 h before treatment with 100
nM GnRH. According to protocols provided by Molecular
Probes, Inc., cells were loaded with 4 µM
indo-1/AM, washed in HBSS (1 mM Ca2+), and
mounted on an ACAS 570 Interactive Laser Cytometer (Meridian
Instruments, Inc., Ann Arbor, MI). Two-dimensional spatial fluorescent
10-sec image scans were collected before and after GnRH treatment.
Intracellular calcium concentrations ([Ca2+]i) were
determined using quantitative ratiometric fluorescence analysis.
The total number of lines in each graph corresponds to the total number
of cells in the field of analysis. Each line in the graph represents
the change in [Ca2+]i within a single cell.
Photoaffinity labeling of GnRHR
HeLa cells were transiently transfected as described above using
1 µg GnRHR and 18 µg Bluescript or Bluescript alone (19 µg).
Transfected HeLa and T3–1 cells used for photoaffinity labeling
studies were harvested and stored at -80 C before analysis. HeLa cells
(3 x 106) or T3–1 cells (9 x
106; equal cell pellet sizes) were incubated with 100,000
cpm
[125I]GnRH-N-hydroxysuccinimidyl-4-azido-benzoate
(13) at 4 C for 4 h and then exposed to a mercury lamp for
5 min to covalently link the radiolabeled ligand to the GnRHR.
Radiolabeled GnRHR was fractionated using 10% SDS-PAGE. Gels were
fixed, dried, and visualized by a PhosphorImager (model 445 SI,
Molecular Dynamics, Inc., Sunnyvale, CA).
Electrophoretic mobility shift analysis
To analyze induction of nuclear proteins by GnRH, HeLa cells
were stably transfected with the GnRHR expression plasmid. Twenty
micrograms of BglII-linearized CMV-GnRHR were transfected as
described above, and HeLa cells expressing CMV-GnRHR were selected
using G418 (Mediatech, Herndon, VA). Individual HeLa cell colonies were
isolated, expanded, and analyzed for GnRHR expression by assaying for
GnRH induction of transiently transfected -215oFSHß-Luc.
Micropreparations of nuclear protein extracts from stably transfected
HeLa (HeLa-Rec) or T3–1 cells were prepared as described by Andrews
and Faller (14), using 1 x 106 HeLa-Rec or 3 x
106 T3–1 cells. Before harvesting, cells were incubated
for 12 h in low serum medium and then were treated with or without
100 nM GnRH for 1 h. Binding reactions with HeLa-Rec
or T3–1 nuclear extract were performed by mixing 8 µg nuclear
protein with binding buffer [final concentrations, 1 mM
MgCl2, 2 mM EDTA, 2 mM
dithiothreitol, 25 mM NaCl, 10 mM Tris-Cl (pH
7.5), and 4% glycerol], 0.75 µg poly(dI-dC) (Boehringer Mannheim,
Indianapolis, IN), and 0.4 ng (13,000–14,000 cpm) end-labeled AP-1
consensus (Promega Corp., Madison, WI) at room temperature
in 20-µl reaction volumes. All binding reactions (including those
with competitor oligonucleotides or antibodies) were carried out
simultaneously. For supershift assays, 0.15 µg anti-Fos rabbit
polyclonal antibody (provided by Dr. M. J. Iadarola, NIH,
Bethesda, MD) (15), 0.5 µg anti-JunD rabbit polyclonal antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 0.5
µg nonspecific rabbit IgG (Sigma) were preincubated in the binding
reactions for 15 min at room temperature before the addition of
radiolabeled probe. The anti-Fos antibody is directed to residues
128–152 of human c-Fos and recognizes all of the Fos family members
(c-Fos, FosB, Fra-1, and Fra-2). The affinity-purified rabbit anti-JunD
polyclonal antibody was directed against residues 329–341 of mouse
JunD and was specific for JunD only (confirmed by Santa Cruz
Biotechnology, Inc. using Western blot analysis). For
competition studies, unlabeled AP-1 or SP-1 competitor oligonucleotides
(Promega Corp.) were preincubated in binding reactions at
a 75-fold molar excess for 15 min before the addition of radiolabeled
probe. After the addition of probe, all binding reactions were
incubated for an additional 25 min at room temperature before being
loaded onto 5.6% nondenaturing polyacrylamide gels (1.5 mm), which
were run at 200 V for 1.5 h at 4 C in 0.5 x TBE
(0.089 M Tris; 0.089 M borate; 0.002
M EDTA). Gels were fixed, dried, and visualized by a
PhosphorImager (model 445 SI, Molecular Dynamics,
Inc.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1, GH3,
RC-4B/C, and GT-1 cells, efficiently expresses oFSHß
promoter/luciferase expression constructs. To investigate the ability
of GnRHR to activate oFSHß gene transcription, increasing amounts of
mouse GnRHR expression plasmid were cotransfected along with a
luciferase expression construct containing the -215/+759 region of the
oFSHß gene (-215oFSHß-Luc) into HeLa cells that were subsequently
treated with 100 nM GnRH for 12 h. As shown in Fig. 1A
, the promoter activity of
-215oFSHß-Luc increased with increasing amounts of cotransfected
GnRHR. A maximal response of 3.8 ± 0.8-fold was obtained with 1
µg GnRHR and remained constant with up to 4 µg GnRHR (3.5 ±
0.5-fold induction). Similar induction of -215oFSHß-Luc by GnRH was
observed as early as 6 h and as late as 18 h (data not
shown). Stimulation by GnRH was dependent upon cotransfection of GnRHR.
The ED50 of GnRH action was determined from a dose-response
study using 1 µg GnRHR. As Fig. 1B shows, increasing amounts of GnRH
resulted in a dose-dependent increase in -215oFSHß-Luc promoter
activity with an ED50 of 0.5 nM. It is notable
that this response is in the range of normal GnRHR action in
gonadotropes (4, 16).
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, addition of 100 nM GnRH to HeLa cells
transiently expressing GnRHR resulted in mobilization of intracellular
calcium similar to that observed in T3–1 cells. More than three
independent experiments showed similar results with calcium responses
in T3–1 and GnRH-responsive HeLa cells typically ranging from
0.2–3.5 µM after GnRH treatment. The level of calcium
mobilized in GnRH-responsive HeLa cells is similar to that reported for
ovine pituitary gonadotropes (17). As only a small population of HeLa
cells actually became transfected with GnRHR using the
CaPO4 method (15–30%), only a limited number of cells
were expected to be responsive to GnRH. HeLa cells transfected only
with control plasmid DNA (Bluescript) lacked the ability to mobilize
intracellular calcium in response to GnRH. Ionomycin, a calcium
ionophore that permits calcium to flow freely into cells, was used as a
positive control in each study to confirm the integrity of the calcium
detection system (data not shown).
To explore the physical nature of GnRHR expressed in HeLa cells, GnRHR
from transiently transfected HeLa cells were radiolabeled by
photoaffinity labeling methods using
[125I]azidobenzoyl-GnRH agonist
([125I]azidobenzoyl-GnRH-A; see Materials and
Methods). Mouse GnRHR from T3–1 cells was radiolabeled as a
positive control, and SDS-PAGE studies revealed the presence of a band
corresponding to authentic GnRHR at approximately 46 kDa (Fig. 1D, lane
1). The photoaffinity labeling of
[125I]azidobenzoyl-GnRH-A to GnRHR in T3–1 cells was
specific, as the addition of 100 nM GnRH-A (unlabeled
competitor) to the binding reaction prevented formation of the
radiolabeled band (Fig. 1D, lane 2). HeLa cells transiently transfected
with GnRHR contained a radiolabeled band with an apparent molecular
mass of 63 kDa (Fig. 1D, lane 4). This band was specific, as 100
nM GnRH-A prevented the photoaffinity labeling with
[125I]azidobenzoyl-GnRH-A (Fig. 1D, lane 5). Without the
cotransfection of GnRHR, no radiolabeled bands were observed (Fig. 1D, lane 3). To address whether the higher molecular mass form of GnRHR in
HeLa cells was due to differences in glycosylation, we used the
recombinant glycosidase peptide-N-glycosidase F (Oxford
GlycoSciences, Inc., Bedford, MA) to cleave all carbohydrates from the
GnRHR and found that deglycosylated GnRHR from either transfected HeLa
or T3–1 cells exhibited a single major band at 28 kDa (data not
shown). It should be noted that other studies have shown a similar
apparent molecular mass of 57–65 kDa for mouse GnRHR expressed in
COS-1 cells and that this receptor binds GnRH with wild-type affinity
(18).
GnRH induces -4741oFSHß-Luc and human -Luc in transformed
cell types
The -subunit gene is known to be GnRH responsive through
several characterized promoter elements (reviewed in Refs. 19, 20).
To determine whether GnRH-responsive HeLa cells would
support stimulation of the human -subunit gene in addition to a
4.7-kb oFSHß promoter construct, luciferase expression constructs
containing either the -846/+44 region of the human -subunit gene
(-Luc) or the -4741/+759 region of the oFSHß gene
(-4741oFSHß-Luc) were assayed for GnRH stimulation as described
above. As a negative control, a luciferase expression construct
containing the RSV promoter (RSV-Luc) was also assayed. As shown in
Fig. 2, both -4741oFSHß-Luc and
-Luc were stimulated (3.1 ± 0.3- and 7.7 ± 1.9-fold,
respectively) by 100 nM GnRH, whereas RSV-Luc expression
was not changed. Similar results with these constructs were observed in
COS-7 cells (Fig. 2), indicating that this regulation is not restricted
to HeLa cells. It should be noted that the level of stimulation caused
by GnRH in these gonadotropin subunit promoters in HeLa and COS-7 cells
are within the ranges observed by others for these genes both in
vivo and in vitro (2, 3, 21, 22). Several other cell
lines tested for GnRH stimulation (JAR, T47-D, and NIH 3T3), however,
did not result in significant levels of -4741oFSHß-Luc and -Luc
stimulation (data not shown), suggesting that either factors required
for GnRH induction are absent from these cell types or that GnRHR is
not efficiently expressed.
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, constructs containing
deletions from -4741 to -215 bp were stimulated between 2.8- to
3.2-fold by 100 nM GnRH. An additional deletion to -84 bp
resulted in a complete loss of GnRH responsiveness, demonstrating that
sequences between -215 and -84 bp are involved in the GnRH-mediated
induction of oFSHß-Luc. Basal expression of all deletion constructs
was statistically equivalent (P > 0.05) and did not
vary by more than 15,000–30,000 relative light units/100 µl (1/10th
of the total sample) between individual experiments.
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, mutations in either site significantly
reduced (P < 0.05) the level of GnRH activation of
-215oFSHß-Luc from 3.2-fold to about 2-fold (50% reduction).
Luciferase expression of a construct containing mutations at both sites
(-83/-120 mutFSHß-Luc) was not enhanced by GnRH, demonstrating that
both sites are essential for GnRH induction. To determine whether any
up-stream regions (>215 bp) in the oFSHß gene possessed functional
elements that might be contributing to the GnRH induction, the -120
and -83 sites were mutated in -4741oFSHß-Luc by site-directed
mutagenesis and assayed for GnRH responsiveness as described. The
results of these studies paralleled those observed with the
-215oFSHß-Luc mutation constructs, indicating that the two AP-1
sites in the oFSHß gene are the only GnRH-responsive elements that
can be detected in HeLa cells (data not shown). Basal level expression
of all constructs containing wild-type or mutated oFSHß sequences did
not vary significantly between individual experiments.
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T3–1 and
GnRHR-expressing HeLa cellsT3–1 cells were prepared and analyzed along with the HeLa-Rec
nuclear extracts. As shown in Fig. 5, incubation of [32P]conAP-1 with nuclear proteins from
control or GnRH-treated cells resulted in the formation of distinct
DNA/ protein complexes (lanes 2, 3, 9, and 10) that were not
present in the control lane (lane 1). Comparison of the control
vs. GnRH-treated bands (lanes 2 vs. 3 and lanes 9
vs. 10) revealed a 4.5-fold increase in AP-1 binding in
T3–1 nuclear extracts and a 2.2-fold increase in AP-1 binding in
HeLa-Rec nuclear extracts. Three independent experiments showed similar
results, with increases in AP-1 binding ranging from 2- to 3-fold in
HeLa-Rec nuclear extract after 1 h of GnRH treatment. Similar
increases in AP-1-associated DNA binding were also observed when either
the -120 or -83 oFSHß AP-1 sites were radiolabeled and analyzed by
gel shift analysis using the extracts described above (data not shown).
Increases in [32P]conAP-1 binding in GnRH-treated
HeLa-Rec were seen as early as 30 min after GnRH treatment and lasted
as long as 4 h before returning to control levels (data not
shown). The [32P]conAP-1 shift observed with these
extracts was specific, as competition with a 75-fold molar excess of
unlabeled AP-1, but not a SP-1 competitor oligonucleotide, prevented
the formation of DNA-protein complexes (compare lanes 4 vs.
5 and lanes 11 vs. 12). Additional Western blot
analyses employing an anti-Fos antibody showed that the increase in
AP-1 binding in both T3–1 and GnRHR-expressing HeLa cells
paralleled increases in the proteins for various Fos family members,
indicating that the increased AP-1 binding activity is due at least in
part to induction of AP-1 family members (data not shown).
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(lanes 6, 7,
13, and 14), the anti-Fos and anti-JunD antibodies were each able to
supershift the DNA-protein complexes formed under GnRH-treated T3–1
and HeLa-Rec cells, whereas nonspecific rabbit IgG could not (lanes 8
and 15). The Fos antibody, in addition to supershifting, showed the
ability to prevent the formation of DNA-protein complexes (Fig. 5, lanes 7 and 14). Additional supershift experiments showed that the
DNA-protein complexes formed from control extracts also contained JunD
and Fos proteins (data not shown).
Transcriptional activation of oFSHß-Luc and -Luc is mediated
in part through PKC
The role of PKC in GnRH-induced activation of -215oFSHß-Luc and
-Luc in HeLa cells was investigated using a highly specific
inhibitor of PKC activation, BIM (also known as GF 109203X). As a
positive control of PKC inhibition, varying concentrations of BIM (from
1 nM to 2 µM) were administered to HeLa cells
transfected with -215oFSHß-Luc or -Luc before the addition of 10
nM TPA for 12 h. As shown in Fig. 6, A and B, increasing concentrations of
BIM completely blocked a TPA-mediated PKC response (in a dose-dependent
fashion) for both the oFSHß and -subunit genes. Under similar
experimental conditions, GnRH induction of either -Luc or
-215oFSHß-Luc was reduced by 57% and 65%, respectively, by BIM
(Fig. 6, C and D), suggesting that GnRH is targeting PKC in addition to
other signaling pathways to stimulate gonadotropin subunit gene
transcription in HeLa cells. Similar results were obtained when another
highly specific and structurally different PKC inhibitor, chelerythrine
chloride, was tested (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether GnRH regulation of oFSHß gene transcription involves the two oFSHß AP-1 sites, a GnRH-responsive HeLa cell system was established through transient expression of mouse GnRHR along with oFSHß promoter/luciferase constructs. These cells responded to GnRH by inducing oFSHß-Luc expression 2.8- to 3.8-fold when the constructs contained both AP-1 sites. Site-directed mutation of either the -120 or -83 sites resulted in a significant reduction of GnRH stimulation (50%), and a double mutation abolished all GnRH responsiveness. These results demonstrated that each site independently promotes GnRH- stimulated transcription.
Although the data presented here show that both sites function
independently under GnRH, previous data demonstrated that each site is
required for c-Jun/c-Fos or TPA induction (8). It is unclear why the
mechanism of GnRH regulation differs from that of AP-1 regulation, but
several theories are suggested. First, it is known that GnRH induces
certain Jun/Fos family members, so it may be that specific combinations
of Jun and Fos induced by GnRH function at each site independently (4).
Binding studies show, for instance, that each site has specific
capacities to bind different AP-1 family members (our
unpublished data). Further, certain combinations of Jun and Fos family
members possess distinct and separate affinities for an AP-1 consensus,
and these combinations possess unique biological activities different
from those of c-Jun/c-Fos (23, 24). A second hypothesis to explain the
differences between AP-1 and GnRH regulation may be found in the
ability of GnRH to induce non-AP-1 proteins that cooperate with Jun or
Fos to bind and trans-activate oFSHß through each site. In
support of this, data show that the -120 and -83 sites bind proteins
other than AP-1 (8). It may be that these proteins, in conjunction with
Jun or Fos, bind to and activate transcription through each site.
Finally, it is possible that additional GnRH-responsive
elements/regions exist between sequences -215 and +759, allowing each
site to cooperate with a yet unknown factor. Theoretical support for
this comes from the concept that a composite region consisting of
separate and distinct elements participates in mediating normal GnRH
regulation of the -subunit promoter (reviewed in Refs. 19, 20).
Thus, it is possible that other elements in the oFSHß gene may also
be involved in mediating the observed GnRH regulation. Studies are
underway to determine which, if any, of the theories described above
are correct.
The -120 AP-1 site is conserved in the FSHß genes of all species analyzed to date (sheep, cow, pig, human, rat, mouse, and rabbit). This suggests a universal involvement of the -120 site in GnRH induction of most, if not all, mammals. For the -83 site to be considered part of the normal GnRH response in vivo, one would predict that it should also be a common feature of all FSHß genes, but the -83 site is conserved only among the ovine, bovine, and porcine species. In addition, oligonucleotides coding for the human, rabbit, and rat -83 sites show no ability to compete for AP-1 proteins (our unpublished results). As the rat FSHß gene has been shown to be transcriptionally activated by GnRH, and to similar or higher levels than those reported here, these data suggest that other sites, in addition to the -120 site, contribute to the GnRH responsiveness in these FSHß genes (2, 21).
Given that the -120 and -83 sites appear to be important for GnRH
regulation of oFSHß gene transcription, a comparison of these
elements was made to those identified as being involved in GnRH
regulation of other GnRH-responsive genes. Comparison of the -83 AP-1
site to these elements reveals a striking sequence similarity between
the -83 site and the pituitary glycoprotein hormone basal element
(PGBE) located in the mouse -subunit promoter at -342 bp (-83:
5'-CTTACTAAT-3'; PGBE:
5'-taCTTAgCTAATta-3'; lower case
letters in the PGBE represent sequence mismatches to oFSHß
sequences, and underlined letters highlight regions of high
homology) (22). The PGBE acts as a basal enhancer and is reported to
bind members of the LIM homeodomain family of transcription factors,
including LH-2 and mLim-3 (22, 25). These factors are thought to bind
to the PGBE and cooperate with other GnRH-responsive elements in the
-subunit promoter to promote GnRH induction. Mutating the PGBE
severely disrupts GnRH responsiveness as well as LH-2 binding (22, 25).
As gel shift studies with the -83 AP-1 site using HeLa nuclear extract
demonstrate the ability of the -83 AP-1 site to bind proteins other
than AP-1, it may also be that the -83 site of oFSHß binds LIM
homeodomain transcription factors. The ability of the -83 site in the
oFSHß gene to bind to and be trans-activated by LH-2 or
mLim-3 awaits confirmation.
A comparison of the -120 and -83 AP-1 sites in oFSHß can be made to
the tandem cAMP response elements (CREs) located in the human
-subunit gene, which appear to play a role in GnRH-regulated
-subunit gene transcription (Jameson, J. L., personal
communication). Consensus CREs differ by only one nucleotide from
consensus AP-1 enhancer elements (CRE: TGACGTCA; AP-1:
TGAC/GTCA) and it has been demonstrated that
both elements can be regulated by Jun/Fos as well as CRE-binding
factors (24). Because GnRH action in gonadotropes is known to induce
AP-1 transcriptional complexes, such regulation by GnRH at the tandem
CREs may involve Jun and/or Fos family members. In support of this,
studies in our laboratory show that human -Luc is stimulated 5-fold
by c-Jun/c-Fos transcriptional complexes in HeLa cells (unpublished
data). Therefore, AP-1 may be a common regulatory mechanism used by
GnRH to stimulate the - and FSH ß-subunit genes.
Studies with GnRH-responsive HeLa and COS-7 cells showed that GnRH
could stimulate the promoter activities of human -Luc and
-4741oFSHß-Luc (but not RSV-Luc) to physiologically relevant levels
(approximately 7- and 3-fold, respectively). These data suggest that
HeLa and COS-7 cells contain the required components necessary for
proper GnRH signaling to these gonadotropin subunit genes. This is
probably due to the fact that these cell lines possess many of the
major signaling pathways and some of the transcription factors known to
be important for GnRH regulation of -subunit gene transcription,
including PKC, mitogen-activated protein kinase members ERK1 and ERK2,
as well as Ets family members (26, 27, 28, 29, 30). In addition, it is well
established that HeLa and COS-7 cells contain PKC-inducible AP-1
transcription complexes, which appear to be important for FSHß
transcription in gonadotropes. Thus, it is reasonable to conclude that
the observed GnRH-induced regulation found in HeLa and COS-7 cells is
probably physiologically relevant.
PKC inhibition studies demonstrated the involvement of PKC in
GnRH-mediated induction of both human -Luc and -215oFSHß-Luc in
HeLa cells (57–65% of the GnRH-mediated stimulation of these
promoters was blocked by BIM). Because these same studies showed that a
TPA-induced PKC response can be completely blocked by BIM, these data
indicate that GnRH action in HeLa cells involves PKC as well as
additional signaling mechanisms. It is notable that others have
observed PKC involvement in GnRH-mediated -Luc expression in
T3–1 cells (31). It is not clear what additional signaling
mechanisms are involved in this GnRH induction, although it may include
mechanisms involving Ca2+/calmodulin, as an antagonist of
calmodulin activity (W-7) completely blocked the GnRH response to
oFSHß-Luc (data not shown).
Given that GnRHR-expressing HeLa cells represent a new cell system to
study the effects of GnRH, studies were performed to characterize their
physical and functional nature. Cotransfection studies revealed the
ability of GnRHR to activate -215oFSHß-Luc transcription in both a
dose- and receptor-dependent manner, suggesting normal GnRHR function
in these cells. Further investigations to determine whether GnRHR was
targeting gonadotrope-specific signaling pathways in HeLa cells showed
that GnRHR was capable of mobilizing intracellular calcium similar to
that observed in T3–1 and ovine gonadotrope cells. In addition, gel
shift studies demonstrated the ability of GnRHR in HeLa cells to
activate/induce components of AP-1 as is normally seen in T3–1
cells, albeit to a lesser extent. These studies, taken together,
suggest that GnRHR is not only expressed at physiologically relevant
levels in these HeLa cells, but is capable of signaling through
pathways that are similar or common to those used by gonadotropes.
Through photoaffinity labeling studies it was determined that mouse
GnRHR expressed in HeLa cells was higher in molecular mass compared
with endogenous mouse GnRHR expressed in T3–1 cells. Subsequent
glycosidase studies, however, revealed that this higher molecular mass
was due to differences in glycosylation between these receptors (data
not shown). Differences in the glycosylation patterns observed are
probably due to the differences between HeLa cells and gonadotropes in
their protein secretion and maturation pathways. Nevertheless,
additional glycoslyation on GnRHR has been shown to not alter normal
GnRH binding and, further, appears to not alter its normal function
(18).
Regulation of gonadotropin subunit expression by GnRH in vivo normally requires pulsatile administration of GnRH from the hypothalamus, whereas continuous administration of GnRH is known to prevent stimulation of the LHß and FSHß genes. The studies presented here as well as other studies involving cotransfection of GnRHR into GH3 cells (21) did not require pulsatile administration of GnRH for FSHß-Luc stimulation. This may be due in part to the fact that the GnRHR expression plasmid is driven by a CMV promoter, which continuously produces GnRHR and is not down-regulated by GnRH. Additionally, it has been shown in rat pituitary cell cultures that continuous GnRH treatment dramatically increases the level of follistatin, which would block the essential function of activin for FSHß expression (32). The GnRH-responsive HeLa cells are not likely to produce follistatin under GnRH treatment and may not even produce activin. In some aspects, the HeLa cell system seems to be especially valuable in studying FSHß regulation, as it can be engineered to respond to only one hormone without interference of other pathways normally induced by that same hormone in gonadotrope cells.
In summary, we have used a GnRHR-expressing HeLa cell system to study
the transcriptional regulation of the oFSHß gene and have shown that
GnRH-mediated induction of oFSHß gene transcription involves two AP-1
enhancers and PKC activation. In addition, these studies demonstrate
the usefulness of GnRHR-expressing HeLa cells to study the effects of
GnRH on gonadotropin subunit gene expression, as GnRHR signaling in
HeLa cells leads to normal mobilization of intracellular calcium,
activation of PKC, activation/induction of AP-1, and stimulation of
and FSHß promoter activities. The roles of these AP-1 sites in GnRH
regulation of oFSHß gene transcription in vivo are
currently under investigation using transgenic mouse models.
|
Acknowledgments |
|---|
and RSV luciferase expression constructs, Dr. Stuart C.
Sealfon for the mouse GnRHR expression plasmid, Dr. Michael J. Iadorola
for generously providing the Fos antibody for the studies, Dr. William
L. Flowers for performing statistical analyses, and Dr. Jean Harry at
the NIEHS (Research Triangle Park, NC) for providing access to the
Meridian ACAS 570 Interactive Laser Cytometer. |
Footnotes |
|---|
2 Present address: Division of Reproductive Toxicology, U.S.
Environmental Protection Agency, Research Triangle Park, North Carolina
27711.
Received June 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit promoter. Mol Endocrinol 6:1767–1773[Abstract]
-subunit gene. J Biol
Chem 268:3903–3910-subunit promoter by a
LIM-homeodomain transcription factor. Mol Cell Biol 14:2985–2993-promoter by
gonadotropin-releasing hormone. Mol Cell Biol 15:3531–3539[Abstract]
promoter in
transfected T3 gonadotrope cells. Endocrinology 134:568–573[Abstract]
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