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Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Ursula B. Kaiser, G. W. Thorn Research Building, Room 1009, Brigham and Women’s Hospital, 20 Shattuck Street, Boston, Massachusetts 02115. E-mail: kaiser{at}rascal.med.harvard.edu
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Abstract |
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-, LHß-, and FSHß-gene expression. Previous studies indicate
that the GnRH receptor couples to G proteins of the Gq/11
family, with phosphoinositide turnover and its resultant increase in
intracellular calcium concentration and protein kinase C (PKC)
activation, to stimulate secretion of LH and FSH. However, the
molecular mechanisms by which GnRH mediates its transcriptional effects
remain largely unknown. We used GH3 cells, constitutively
expressing the rat GnRH receptor (GGH3-1' cells) and
transiently transfected with a luciferase reporter gene controlled by
either the , LHß, or FSHß gene regulatory region (LUC,
LHßLUC, and FSHßLUC, respectively), to examine the roles of several
signal transduction pathways in the GnRH-mediated stimulation of
gonadotropin subunit gene expression. Activation of PKC by phorbol,
12-myristate, 13-acetate resulted in an increase in the luciferase
activity of all three gonadotropin subunit gene reporter constructs.
Phorbol, 12-myristate, 13-acetate had a greater stimulatory effect,
relative to the maximal stimulation with GnRH, for the ß-subunit
genes than for the -subunit gene. Depletion of PKC, or inhibition of
PKC by GF109203X, demonstrated that PKC-dependent pathways play a
larger role in the GnRH-mediated transcriptional control of the LHß-
and FSHß-genes than the -subunit gene. In contrast, an L-type
calcium channel agonist, Bay K 8644, was able to stimulate LUC but
not LHßLUC or FSHßLUC. Nimodipine, an L-type calcium channel
antagonist, had a larger inhibitory effect on the GnRH response of
LUC, relative to LHßLUC or FSHßLUC. We conclude from these
results that the differential regulation of gonadotropin subunit gene
expression by GnRH is caused, in part, by differential use of signal
transduction pathways, activated upon GnRH binding. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit and a specific ß-subunit, produced in the gonadotropes of
the anterior pituitary gland (1). Both the biosynthesis and the
secretion of the gonadotropins are under the regulation of a
hypothalamic secretagogue, GnRH, which is released in a pulsatile
fashion. The actions of GnRH on gonadotropes are exquisitely sensitive
to the pulse frequency. Exogenous GnRH given in a pulsatile fashion
will stimulate the biosynthesis and secretion of LH and FSH, whereas
sustained exposure to GnRH leads to suppression of gonadotropin
biosynthesis and secretion (2, 3, 4, 5, 6, 7).
There is a growing body of evidence for the role of certain
intracellular messenger molecules in the control of LH and FSH release
by GnRH. The GnRH receptor (GnRHR) couples to a member(s) of the
Gq/11 family of heterotrimeric G proteins to effect
gonadotropin secretion (8). As anticipated by coupling to
Gq/11, GnRH binding to the GnRHR results in a rapid
hydrolysis of phosphatidylinositol 4,5-bisphosphate and generation of
inositol 1,4,5-triphosphate (IP3) and diacylglycerol (9).
Further, GnRH induces a rise in intracellular calcium concentration
(10). GnRH can evoke a translocation of protein kinase C (PKC) from the
cytosol to the membrane (11). Investigations of GnRH action have
identified additional potential mediators of GnRH-activated signal
transduction. Some studies suggest that the GnRHR may be coupled to a
cholera toxin-sensitive G protein (12, 13, 14). Consistent with this
observation, a GnRH agonist can elicit the production of cAMP (15).
Stimulation of T3–1 cells with GnRH results in the phosphorylation
and activation of two isoforms of mitogen-activated protein kinase
(16, 17, 18). Interestingly, pertussis toxin blocked GnRH-induced
mitogen-activated protein kinase activation, suggesting that this
signaling pathway is coupled to the pertussis toxin-sensitive
Gi or Go pathway. These data provide evidence
for Gs and Gi/Go-mediated signal
transduction by GnRHR in addition to Gq/11-mediated signal
transduction.
Once established that GnRH can stimulate PKC activity and cause an increase in the intracellular calcium concentration, investigators began to examine the possible roles of those second messengers in GnRH-mediated LH and FSH secretion. Most studies have concluded that PKC plays little role in the regulated exocytosis of LH and FSH (11, 19). Calcium has been conclusively shown to play a major role in mediating GnRH-induced gonadotropin release (9, 10, 20). Studies have shown that calcium ionophores and calcium channel agonists can stimulate gonadotropin release. The stimulatory actions of GnRH on LH and FSH secretion can be inhibited by calcium channel antagonists and culture in calcium-free medium.
Whereas the intracellular messenger cascades mediating the stimulation
of gonadotropin secretion by GnRH are becoming clearly mapped, those
signaling pathways that are activated by GnRH and specifically regulate
the expression of the gonadotropin subunit genes remain poorly
understood. Preliminary studies performed in cultures of primary rat
pituitary cells demonstrated that activation of PKC can increase the
levels of gonadotropin subunit messenger RNAs (mRNAs), and, conversely,
depletion of PKC by phorbol ester treatment can blunt the stimulation
of LHß-gene expression by GnRH (21, 22). Two reports have used
calcium channel agonists and calcium-free culture medium to demonstrate
the importance of extracellular calcium in the activity of the
-subunit gene promoter (22, 23). Nonetheless, it remains unclear
which are the primary signal transduction pathways that are used by
GnRH to modulate gonadotropin subunit gene transcription, and,
moreover, what is the signal transduction basis for the GnRH pulse
frequency-dependent difference in subunit gene expression.
A systematic approach to identifying mechanisms of hormonal regulation
of gonadotropin subunit gene expression has been hampered by the lack
of an available cell line that expresses the -, LHß-, and
FSHß-genes in a regulated manner. Primary anterior pituitary cells
have the disadvantage of being a heterogeneous cell population in which
gonadotropes constitute only 6–15% of the secretory cells in the
anterior pituitaries of normal adult animals (24). In our studies, we
have used GH3 cells as a model for the analysis of
transcriptional regulation of the gonadotropin subunit genes.
GH3 cells are a well-characterized rat pituitary
somatolactotropic cell line (25, 26). We have demonstrated previously
that GH3 cells, stably transfected with the rat GnRHR
complementary DNA (GGH3-1' cells), bind and respond to
GnRH. Cotransfection with the 5'-flanking region of the -, LHß-,
or FSHß-subunit gene, fused to a luciferase reporter, results in the
expression of luciferase and a stimulation of luciferase activity in
response to GnRH. Characterization of this cell model has demonstrated
many similarities in the GnRH response, compared with that in primary
pituitary cells, including the intracellular signal transduction
pathways activated; the degree of stimulation of -, LHß-, and
FSHß-gene promoter activities; and differential regulation of the
gonadotropin subunit gene promoter activities by GnRH (27, 28, 29).
GH3 cells thus seem to be a useful model for the study of
the regulation of the gonadotropin subunit genes by GnRH.
The goal of the present studies is to elucidate the signal transduction
pathways involved in mediating the differential effects of GnRH on
gonadotropin subunit gene expression. We demonstrate that there are
distinct signaling cascades responsible for the GnRH stimulation of
transcriptional activity in the - vs. the ß-subunit
genes. The regulation of the -subunit gene promoter by GnRH is
primarily through a signaling pathway dependent on a rise in
intracellular calcium concentration. In contrast, the GnRH-mediated
expression of the LHß- and FSHß-subunit gene occurs primarily via a
PKC-dependent pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reporter plasmids and expression vectors
The reporter constructs that we used contain the 5'-flanking
regions of the human (-846/0: with position -1 assigned to the
nucleotide immediately 5' to the transcriptional start site), rat LHß
(-791/+5), and rat FSHß (-2000/+1709) genes fused to the luciferase
reporter gene (LUC, LHßLUC, FSHßLUC, respectively), as
previously described (29, 30). The LUC plasmid was a generous gift
from Dr. J. Larry Jameson. An expression vector expressing
ß-galactosidase driven by the Rous sarcoma virus (RSV) promoter
(RSV-ßGal) was used as an internal standard and control.
Cell culture and transfection
GH3 cells, stably transfected with an expression
vector containing the rat GnRHR complementary DNA sequence
(GGH3-1' cells) (29), were maintained in monolayer culture
in DMEM supplemented with 600 µg/ml Geneticin (Gibco BRL, Grand
Island, NY), 10% (vol/vol) heat-inactivated FBS, and
penicillin/streptomycin at 37 C in humidified 5% CO2-95%
air. For transient transfection studies, GGH3-1' cells were
cultured to 50–70% confluence and transfected by electroporation. In
each experiment, approximately 5 x 106 cells were
suspended in Dulbecco’s PBS plus 5 mM glucose containing
the DNA to be transfected. The cells received a single electrical pulse
of 240 V from a total capacitance of 1000 µF, using an Invitrogen
Electroporator II apparatus (Invitrogen, San Diego, CA). After
electroporation, cells were resuspended in serum-containing medium and
plated in 9.62-cm2 wells. Cells were analyzed at either 24
or 48 h after transfection. For 48-h incubations, medium was
replaced 24 h after transfection. Cells were treated with hormone,
pharmacologic agent, or vehicle for 6 h immediately before
harvesting. These conditions have been tested and optimized previously
to give maximal levels of basal expression and GnRH stimulation (28, 29). Dose-response studies were performed for all agents to optimize
the dose used to treat cells for both specificity and transcriptional
response. Cells were harvested in lysis buffer [125 mM
Tris (pH 7.6), 0.5% (vol/vol) Triton X-100]. Supernatants were
collected by centrifugation at 14,000 x g for 15 min
at 4 C. Luciferase activity was measured using an LB 953 Autolumat
(EC&G Berthold, Nashua, NH), by standard protocols, as previously
described (28). Luciferase activity was normalized for expression of
RSV-ßGal. ß-galactosidase activity was assayed colorimetrically by
standard protocols, as previously described (28).
Statistical analysis
Transfections were performed in triplicate and repeated at least
three times. Data in each experiment were normalized to the level of
luciferase activity of LUC, LHßLUC, or FSHßLUC when treated with
the appropriate vehicle. Data were then combined across experiments to
give a mean ± SEM for control and treated samples.
Data were analyzed by Student’s t test for independent
samples when appropriate. A value of P < 0.05 was
considered statistically significant.
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Results |
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LUC, LHßLUC, or FSHßLUC. The cells
were treated with vehicle, PMA, GnRHAg, or a combination of PMA and
GnRHAg for the final 6 h before harvesting. Maximally effective
doses of GnRHAg and PMA were used to ensure complete activation of
their respective downstream signal transduction cascades. The cells
were harvested and luciferase activity measured 48 h after
transfection (Fig. 1
). Treatment with PMA
alone resulted in a significantly increased level of luciferase
activity, relative to vehicle, for each reporter construct (LUC,
2.02 ± 0.07-fold; LHßLUC, 2.80 ± 0.16-fold; FSHßLUC,
1.71 ± 0.17-fold; P < 0.001 for all reporter
constructs, compared with treatment with vehicle alone). However,
relative to the maximal subunit gene promoter activity elicited by
treatment with GnRHAg (LUC, 6.38 ± 0.29-fold; LHßLUC,
5.14 ± 0.42-fold; FSHßLUC, 3.04 ± 0.14-fold), the
response to PMA treatment seemed to be greater for the ß-subunits
than for the -subunit. For LHßLUC and FSHßLUC, the magnitude of
the response to PMA treatment was approximately 50% of that seen with
GnRHAg stimulation. In contrast, the PMA response of LUC was less
than one-third of the GnRHAg stimulation of LUC. There was no
further stimulation of activity of any of the gonadotropin subunit gene
reporter constructs upon costimulation with GnRHAg and PMA. These data
suggest that GnRH-mediated regulation of gonadotropin subunit gene
expression may act, in part, through a PKC-dependent pathway.
Autonomous activation of this pathway, which alone can stimulate
subunit gene expression, does not augment the maximal transcriptional
activity of the gonadotropin subunit gene promoters elicited by GnRH.
Further, this PKC-dependent pathway has a larger stimulatory effect on
the transcriptional activity of the gonadotropin ß-subunit genes than
on the -subunit gene.
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LUC, LHßLUC, or FSHßLUC and treated with vehicle,
8BrcAMP, GnRHAg, or a combination of 8BrcAMP and GnRHAg (Fig. 2). The LUC reporter plasmid responded
to stimulation by 8BrcAMP with a 7.59 ± 0.78-fold increase in
luciferase activity, relative to basal (P < 0.005). In
contrast, each of the reporter constructs containing the regulatory
regions of the ß-subunit genes was stimulated significantly by
8BrcAMP (LHßLUC, 1.41 ± 0.1-fold; FSHßLUC, 1.29 ±
0.08-fold; P < 0.005) but by less than 1.5-fold,
relative to basal levels of expression. Treatment of transfected cells
with both GnRHAg and 8BrcAMP resulted in luciferase activity
significantly greater than that observed with treatment by GnRHAg alone
for LUC and LHßLUC (LUC, GnRHAg + 8BrcAMP: 20.59 ±
1.42-fold vs. GnRHAg alone: 9.2 ± 0.4-fold,
P < 0.0001; LHßLUC, 6.22 ± 0.2-fold
vs. 5.0 ± 0.12-fold, P < 0.0001). The
increase in FSHßLUC activity seen upon treatment with both GnRHAg and
8BrcAMP, however, failed to reach statistical significance, relative to
treatment by GnRHAg alone, likely because of the small magnitude of the
effect of 8BrcAMP (FSHßLUC, GnRHAg + 8BrcAMP: 3.86 ± 0.43-fold
vs. GnRHAg alone: 3.11 ± 0.35-fold; P
= NS). Consistent with previous studies in rat primary pituitary cells
(12, 14), these data suggest that the gonadotropin subunit gene
promoters are responsive to cAMP-dependent pathways, albeit to a much
greater extent for the -subunit gene than the ß-subunit genes. In
addition, these data imply that GnRH-mediated regulation of
gonadotropin subunit gene transcription is not via a cAMP-dependent
pathway, because activation of cAMP-dependent pathways in the presence
of a maximal transcriptional stimulation by GnRH resulted in a further
increase in the level of gene expression and luciferase activity.
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- vs. the ß-subunit genes. To explore more fully the
role of PKC, we sought to determine the response to GnRH in the absence
of active PKC. Short-term treatment of intact cells with PMA results in
an activation of PKC. However, longer-term treatment of cells with PMA
results in a specific catalytic and immunological depletion of
PMA-sensitive isoforms of PKC (11, 21). GGH3-1' cells were
transiently transfected with either LUC, LHßLUC, or FSHßLUC,
treated with PMA for 24 h, and then challenged with vehicle, PMA,
or GnRHAg for the final 6 h before harvesting (Fig. 3). The 24-h pretreatment with PMA
abolished all transcriptional responses to the final 6-h challenge with
PMA, as expected. The LUC reporter construct maintained a 3.57
± 0.36-fold response to GnRHAg, even in the absence of active PKC
(P < 0.0005 vs. PMA-pretreated cells
challenged with vehicle for the final 6 h). This is in contrast to
the reporter plasmids containing the regulatory regions of the
gonadotropin ß-subunits. Both the LHßLUC and the FSHßLUC
constructs had statistically significant responses to treatment with
GnRHAg in the absence of active PKC (LHßLUC, 1.89 ± 0.09-fold;
FSHßLUC, 1.78 ± 0.17-fold, P < 0.0005 for each
vs. the PKC-depleted, vehicle-challenged cells). However,
these responses are greatly blunted when compared with the response to
GnRHAg seen in the presence of active PKC, as observed in Figs. 1
and 2. These data support the idea that a PKC-dependent pathway is critical
to the GnRH modulation of gonadotropin subunit gene expression.
Further, the relative degree to which the GnRH responses are blunted in
the absence of active PKC supports a greater role for a PKC-dependent
pathway in the transcriptional regulation of the gonadotropin
ß-subunit genes than in the -subunit gene.
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LUC, LHßLUC, or FSHßLUC. The cells were treated with
vehicle, GF109203X, stimulant, or both (Fig. 4). GF109203X alone had no effect on the
basal level of expression of any of the gonadotropin subunit gene
reporter constructs. The specificity of GF109203X in inhibiting only
PKC-dependent pathways was shown by a nearly complete blockade of
stimulated luciferase activity upon treatment with both GF109203X and
PMA, whereas GF109203X had no significant inhibitory effect when given
to transfected cells in combination with 8BrcAMP. Consistent with the
ability of PMA (and thus, a PKC-dependent pathway) to stimulate some
level of transactivation of all three gonadotropin subunit gene
reporter constructs, GF109203X blunted the transcriptional response of
each of the reporter constructs to 100 nM GnRHAg (LUC,
GnRHAg + GF109203X: 7.03 ± 0.59-fold vs. GnRHAg alone:
9.55 ± 0.8-fold; LHßLUC, 8.88 ± 0.62-fold vs.
15.09 ± 1.06-fold; FSHßLUC, 4.81 ± 0.28-fold
vs. 6.66 ± 0.59-fold; P < 0.02 for
all reporter constructs). The degree of inhibition was consistent with
the apparent relative importance of a PKC-dependent pathway in the
GnRH-mediated regulation of expression of each of the three subunit
genes. More specifically, GF109203X caused a larger degree of
inhibition of the maximal GnRH response for the LHß-subunit gene than
for the -subunit gene. The inhibitory effect of GF109203X on the
GnRH response of the FSHß-gene is intermediate, relative to the
effects on LHß and .
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LUC, LHßLUC, or FSHßLUC. The
cells were treated with vehicle, Bay K 8644, GnRHAg, or a combination
of Bay K 8644 and GnRHAg (Fig. 5).
Treatment with Bay K 8644 alone resulted in a significantly increased
expression (1.86 ± 0.12-fold, P < 0.0001) of the
LUC reporter construct, relative to vehicle alone. This is in
contrast to the lack of stimulation by Bay K 8644 of either LHßLUC or
FSHßLUC (LHßLUC, 0.95 ± 0.06-fold; FSHßLUC, 1.06 ±
0.08-fold; P = NS). When cells transfected with LUC
were treated with a combination of GnRHAg and Bay K 8644, there was a
greater stimulation of luciferase expression, relative to treatment
with GnRHAg alone (GnRHAg + Bay K 8644: 12.91 ± 0.71-fold
vs. GnRH alone: 7.87 ± 0.42-fold, P <
0.005). However, neither LHßLUC nor FSHßLUC had a further increase
in luciferase expression upon costimulation with GnRHAg and Bay K 8644,
when compared with treatment with GnRHAg alone (LHßLUC, GnRHAg + Bay
K 8644: 2.82 ± 0.07-fold vs. GnRHAg alone: 3.29
± 0.12-fold; FSHßLUC, 2.31 ± 0.22-fold vs.
2.03 ± 0.14-fold). Treatment of transfected cells with Bay K 8644
for a brief time (30 min), rather than the full 6 h during which
the GnRHAg is present, yielded similar results (data not shown). These
data suggest that calcium has a role in the GnRH-mediated regulation of
-subunit gene expression. In contrast, the lack of stimulation by
Bay K 8644 and the lack of any augmentation of the GnRH response
suggests that calcium plays little role in the GnRH-mediated regulation
of gonadotropin ß-subunit gene expression.
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LUC, LHßLUC, or
FSHßLUC. The cells were treated with vehicle, nimodipine, GnRHAg, or
a combination of nimodipine and GnRHAg (Fig. 6). Nimodipine significantly blunted the
response of the -subunit gene promoter to GnRHAg (GnRHAg +
nimodipine: 3.5 ± 0.15-fold vs. GnRHAg alone:
7.81 ± 0.21-fold, P < 0.05). The inhibition
caused by nimodipine of LUC expression was 55%. Although the
blunting of the response to GnRHAg by nimodipine did reach statistical
significance for the LHßLUC reporter construct (GnRHAg + nimodipine:
3.78 ± 0.32-fold vs. GnRHAg alone: 4.91 ±
0.33-fold, P < 0.05), the inhibitory effect was less
for each of the ß-subunit gene reporter constructs than for the
-subunit gene promoter reporter plasmid (LHßLUC, 23% inhibition;
FSHßLUC, 32% inhibition). Similar results were seen in parallel
experiments using the calcium channel antagonist verapamil (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunits,
particularly intracellular calcium fluxes, can effect exocytosis of
stored LH and FSH. Our present data provide evidence that these same
second-messenger systems, PKC and calcium, are responsible, in part,
for mediating the transcriptional regulation of the gonadotropin
subunit genes by GnRH. Strikingly, however, there is a dichotomous
relationship between the signaling cascades that mediate GnRH
stimulation of the -subunit gene and those that mediate the GnRH
regulation of the gonadotropin ß-subunit genes. Calcium-dependent
signal transduction pathways play a greater role in GnRH-mediated
stimulation of -subunit gene expression, whereas PKC-dependent
pathways are of greater importance in transducing a signal for
transcriptional stimulation from activated GnRHR to the LHß- and
FSHß-genes.
We have shown that all three gonadotropin subunit gene promoters are
responsive to PKC-dependent pathways. The absence of any further
augmentation of transcriptional activity of gonadotropin subunit gene
promoter reporter constructs in the presence of both GnRH and PMA,
compared with GnRH alone, provides evidence that the pathway activated
by PMA is also being maximally activated by GnRH stimulation. Data from
PKC-depleted cells and from the experiments using the specific PKC
inhibitor, GF109203X, confirm that GnRH acts through a PKC-dependent
pathway to regulate the expression of the gonadotropin subunit genes.
These data are in agreement with the findings of Schoderbek et
al. (32), who demonstrated that PMA responsiveness of the mouse
-subunit gene promoter colocalizes with GnRH responsiveness.
However, though the current data support a role for PKC-dependent
pathways in the transcriptional response of the -subunit gene to
GnRH, this role is quantitatively less than that stimulated by an
increased concentration of cytosolic calcium. The mechanism for the
decrease in maximal stimulated luciferase activity, noted for the
-subunit gene promoter with cotreatment with GnRH and PMA, is not
clear. It is possible that PMA may activate an isoform of PKC that has
either an inhibitory effect on a stimulatory pathway activated by GnRH
binding or an independent inhibitory effect on the -subunit gene
promoter. A second possibility is that maximal stimulation by both PMA
and GnRH results in dose-dependent down-regulation of PKC within the
6-h time frame of our experimental paradigm. The degree of stimulated
transactivation of the luciferase reporter plasmids achieved in the
series of experiments employing GF109203X was quantitatively greater
than that measured for any other set of experiments. These experiments
used a transfection paradigm in which there was only 24 h between
transfection and harvest. The qualitative relationship among the
various pharmacologic stimulants for the level of luciferase activity
remained the same as the 48-h paradigm.
An increase in intracellular calcium concentration can elicit a
transcriptional response from the LUC construct. This is in contrast
to the inability of a calcium channel agonist to stimulate either the
LHß- or the FSHß-gene promoter. The involvement of the
calcium-mediated transcriptional response of the -subunit gene in
pathways activated by GnRH is demonstrated by the ability of the
dihydropyridine calcium channel antagonist to inhibit partially the
GnRH response. Both primary pituitary gonadotropes and GH3
cells have L-type calcium channels. It has been demonstrated previously
that GnRH-induced depolarization of primary pituitary gonadotropes
results in the activation of voltage-sensitive calcium channels of the
L-type (33). The synergistic effect of Bay K 8644 and GnRH stimulation
of the -subunit gene promoter might be caused by the spatial and
temporal regulation of calcium waves mediated by each of these
agonists. Calcium entry likely will occur in close spatial proximity to
those GnRHRs that have bound ligand. In contrast, the calcium channel
agonist, Bay K 8644, will indiscriminately activate all L-type calcium
channels in a diffuse manner over the entire surface area of the cell.
Thus, Bay K 8644 will evoke a larger and more sustained calcium influx
than GnRH. This supraphysiologic increase in cytosolic calcium may be
able to stimulate further the calcium-dependent pathway that is
activated by GnRH.
Though the calcium channel agonist was unable to stimulate a
transcriptional response in either of the gonadotropin ß-subunit gene
promoter reporter constructs, the calcium channel antagonist did cause
a slight decrease in the level of GnRH-mediated expression. This
combination of results suggests that, although calcium alone cannot
stimulate the LHß- or FSHß-gene promoters, it can augment the
activity of another signaling component that is activated by GnRH and
is important for ß-subunit gene transcription. One candidate for such
a signaling pathway is PKC. Complete activation of PKC requires not
only diacylglycerol stimulation but also calcium binding (34). This is
supported by the ability of a calcium channel agonist to augment the
PMA response, and of nimodipine to partially inhibit the PMA-stimulated
expression, of LUC, LHßLUC, and FSHßLUC (data not shown). Thus,
it seems that calcium serves to augment the PKC-dependent pathway
response in the gonadotropin ß-subunits but independently mediates
GnRH transcriptional stimulation of the -subunit gene promoter.
Though examining the role of extracellular calcium, our present studies
do not address the role of the intracellular calcium stores. Holdstock
et al. (23) have demonstrated in T3–1 cells that
depletion of intracellular calcium stores with thapsigargin has no
effect on GnRH-stimulated -promoter activity. Similarly, we have
noted that thapsigargin has no effect on either basal or
GnRH-stimulated activity of LUC, LHßLUC, or FSHßLUC in
GGH3-1' cells (data not shown).
Based on our current data, we hypothesize that one of the mechanisms by
which the expression of the gonadotropin subunit genes is
differentially regulated by GnRH is a selective use of either the PKC-
or the calcium-dependent pathways activated by this hormone binding to
its receptor. Some speculation as to the consistency of our findings
with the physiological, GnRH pulse frequency-based regulation of the
gonadotropin genes can be offered. Continuous administration of GnRH
results in the down-regulation of the LHß and FSHß mRNA levels. PKC
plays the dominant role in mediating the GnRH effect on LHß- and
FSHß-subunit gene expression. Continued stimulation of PKC leads to
catalytic depletion of this enzyme. This may be one of the mechanisms
resulting in the desensitization of GnRH signal transduction pathways
that we have previously described (28). Continuous stimulation by GnRH,
then, would result in the removal of a major signaling component from
the gonadotrope and, thus, in the inability for GnRH to regulate LHß-
and FSHß-gene expression. The -subunit gene is much less
stringently regulated by this pulse frequency. Even in the presence of
continuous GnRH stimulation, the -subunit gene continues to be
expressed (35, 36, 37). This can be reconciled by the current data that
influx of calcium into the cytosol plays the major role in mediating
GnRH regulation of -gene expression. There is a vast abundance of
extracellular calcium, relative to cytosolic calcium. Thus, as long as
this substantial concentration gradient remains in place, GnRH may be
able to elicit a flux of calcium, down its concentration gradient and
into the gonadotrope. Relative to PKC desensitization, calcium stores
can be loaded and unloaded more rapidly.
Further investigations are needed to characterize the full complement
of signaling molecules involved in transmitting a signal from GnRH
binding, at the cell surface, into the nucleus. Moreover, the question
remains as to how increasing GnRH pulse frequencies can lead to
up-regulation of both -and LHß-subunit gene expression concomitant
with a specific down-regulation of FSHß-gene expression. Our
GGH3-1' cell line seems well suited to dissect the answers
to these questions. The current transfection paradigm represents the
response to a single pulse of GnRH. Though GnRH is actually present for
6 h before harvesting, this represents the length of time needed
for the transcription and subsequent translation of the luciferase
reporter gene and intracellular accumulation of the luciferase enzyme.
Thus, our data are consistent with previous studies that demonstrate an
increase in gonadotropin subunit gene mRNA upon pulsatile GnRH
stimulation (6, 7). However, some degree of caution must always be
taken when using a heterologous cell line. Our current data will
require confirmation of physiologic importance in a primary gonadotrope
cell population. It is possible that there may be gonadotrope-specific
factors necessary to recapitulate completely GnRH-mediated signaling.
Nevertheless, we have shown previously that the regulation of the
gonadotropin subunit promoter activities by GnRH in this cell line
closely reflects the regulation observed in primary pituitary cells
(29). We have demonstrated that differential regulation by GnRH of the
gonadotropin subunit genes is caused, in part, by the selective use by
the gonadotropin subunit gene promoters of the signal transduction
pathways activated upon ligand binding to the GnRHR.
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Acknowledgments |
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Footnotes |
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Received October 21, 1997.
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References |
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-subunit promoter
by gonadotropin-releasing hormone. Mol Cell Biol 15:3531–3539[Abstract]
-subunit gene expression and secretion in
T3–1 gonadotropes. Mol Endocrinol 10:1308–1317-subunit gene. J Biol
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X. Lin and P. M. Conn Transcriptional Activation of Gonadotropin-Releasing Hormone (GnRH) Receptor Gene by GnRH: Involvement of Multiple Signal Transduction Pathways Endocrinology, January 1, 1999; 140(1): 358 - 364. [Abstract] [Full Text]
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) or 1 µM
GF109203X (
), stimulus (
), or both (
) for 6 h immediately
before harvesting. GF109203X or its vehicle (dimethyl sulfoxide) was
applied 30 min before stimulus. Stimuli used were: 100 nM
GnRHAg, 100 ng/ml PMA, and 1 mM 8BrcAMP. Levels of
luciferase activity are internally standardized, as in Fig. 1
