Differential Gonadotropin-Releasing Hormone Stimulation of Rat Luteinizing Hormone Subunit Gene Transcription by Calcium Influx and Mitogen-Activated Protein Kinase-Signaling Pathways
Jennifer Weck,
Patricia C. Fallest,
Leslie K. Pitt and
Margaret A. Shupnik
Department of Molecular Physiology and Biological Physics
(J.W., M.A.S.) Department of Medicine Division of Endocrinology
and The National Science Foundation Center for Biological Timing
(P.C.F., L.K.P., M.A.S.) University of Virginia, Charlottesville,
Virginia 22908
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ABSTRACT
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Gonadotropin secretion and gene expression are
differentially regulated by hypothalamic GnRH pulses by unknown
mechanisms. GnRH stimulates calcium influx through L-type voltage-gated
channels and activates phospholipase C, leading to increased protein
kinase C (PKC) and mitogen-activated protein kinase activity. We found
differential contributions of these pathways to GnRH-stimulated rat LH
subunit transcription in pituitary gonadotropes and cell lines.
Endogenous transcription of the
- and LHß-subunits in rat
pituitary cells was stimulated by GnRH. Independent PKC activation by
phorbol myristate acid stimulated only the
-subunit gene. In
contrast, an L-channel antagonist (nimodipine) inhibited only LHß
stimulation by GnRH, and an L-channel agonist (BayK 8644) stimulated
only basal LHß transcription. GnRH induction of a rat
-subunit
promoter construct in
T3 cells was unaffected by nimodipine or
elimination of external calcium, while both treatments eliminated the
LHß response. Application of a mitogen-activated kinase kinase (MEK)
inhibitor (PD098059) decreased basal and GnRH-stimulated
-subunit
promoter activity and had no effect on LHß promoter activity. In
pituitary cells from mice bearing an LHß promoter-luciferase reporter
transgene, GnRH stimulation was inhibited by nimodipine but not by
PD098059. Thus, GnRH induction and basal control of the
-subunit
gene seem to occur through the PKC/mitogen-activated protein kinase
pathway, while induction of the LHß gene is dependent on calcium
influx. Differential signaling from the same receptor may be a
mechanism for preferential regulation of transcription.
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INTRODUCTION
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The hypothalamic peptide GnRH is released into pituitary portal
vessels in intermittent pulses, the frequency and amplitude of which
vary physiologically (1, 2, 3, 4). Pulsatile GnRH release is necessary for
reproductive function, as continuous infusion of the peptide results in
infertility. GnRH acts on anterior pituitary gonadotropes to stimulate
production and secretion of the gonadotropins LH and FSH. These
proteins are composed of two noncovalently linked subunits, the
(common to TSH, FSH, and LH) and a ß-subunit specific to each
protein. Each ß-subunit as well as the common
is encoded by
different genes on separate chromosomes. LH stimulates gametogenesis
and gonadal steroid production necessary for fertility. Transcriptional
activation of gonadotropin subunit genes is differentially regulated by
GnRH pulse frequency in vivo and in vitro (5, 6).
Transcription from the rat
-subunit gene is stimulated by pulses of
intermediate or short intervals, or by constant GnRH, whereas
activation of the rat LHß gene occurs only with
intermediate-frequency (30 min), high-amplitude pulses. Given this
differential regulation, some mechanistic differences must exist.
The mechanisms by which GnRH causes such differential control within
gonadotropes are unknown but may include activation of different
transcription factors for each gene or preferential sensitivity
to distinct second messenger pathways. These mechanisms are not
mutually exclusive. Transfection experiments in cultured pituitary
cells and cell lines and studies involving transgenic mice have
identified several cis-acting elements responsible for
expression of the human, bovine, and mouse
-subunit promoters
(7, 8, 9, 10, 11). Less is known about LHß promoter regulation. However,
transfection studies in primary cultures, heterologous cell lines, and
transgenic mice expressing either rat or bovine LHß reporters
illustrate activation of both LHß transgene and endogenous gene
expression by GnRH, and this activation is attenuated in
vivo by increased steroid levels or by a GnRH antagonist (12, 13, 14, 15, 16).
GnRH stimulation of gonadotropes activates two primary signaling
pathways, calcium and protein kinase C (PKC) (17, 18, 19, 20). Receptor
activation induces a biphasic increase in internal calcium, with an
initial spike dependent on internal calcium stores, and a sustained
plateau that is dependent on increased Ca++ influx through
L-type channels (21, 22). GnRH binding also activates G proteins
(Gq
and G11
) that stimulate phospholipase
C ß activity to generate inositol triphosphate and diacylgycerol.
Activation of this pathway results in PKC and thus mitogen-activated
protein kinase (MAPK) stimulation (23, 24, 25, 26, 27).
We sought to determine the mechanisms by which the rat
and LHß
genes are regulated by GnRH. In the current study we examined rat LH
subunit promoter expression in normal pituitaries, transfected cell
lines, and pituitaries from mice bearing a rat LHß-
promoter-luciferase transgene. By manipulating the two major pathways
activated in response to GnRH, the influx of Ca++ and the
activation of MAPK via PKC, we illustrate stimulation of MAPK activates
the rat
-subunit gene preferentially, while Ca++ influx
is more important in activating the LHß gene.
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RESULTS
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Transcriptional Analysis of the Endogenous Genes
Transcription rates of endogenous rat
and LHß genes were
measured by nuclear run-off assays in isolated rat pituitary cells
treated with GnRH and various pharmacological agents (Fig. 1
). Both genes were stimulated by GnRH
(
, 5-fold, and LHß, 9-fold) but differ in their sensitivity to
modulators of GnRH-signaling pathways. To determine the contribution of
GnRH-stimulated increase in calcium influx through L-channels, Bay
K8644, a specific L-channel agonist, and nimodipine, an L-channel
antagonist, were used. Bay K only slightly increased
gene mRNA
synthesis (1.5-fold) but stimulated LHß gene transcription by 7- to
9-fold, equal to the stimulation seen with GnRH. Nimodipine decreased
GnRH stimulation of the
gene only partially, but had a marked
effect on the LHß response, decreasing GnRH-mediated stimulation from
9- to 3-fold. The effects of PKC activation were assessed by
stimulating the pathway with the phorbol ester phorbol myristate acid
(PMA). PMA stimulated
-subunit mRNA synthesis 4-fold, but did not
significantly increase transcription of the LHß gene. Therefore,
although GnRH stimulation of pituitary gonadotropes activates both the
Ca++- and PKC-regulated second messenger systems, these
pathways do not affect rat
-subunit and LHß gene transcription
identically. The
-subunit gene is much more susceptible to
stimulation by the PKC pathway, and increases in Ca++
influx preferentially stimulate LHß gene transcription.

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Figure 1. Transcription Rates of Endogenous LH Subunit Genes
in Isolated Rat Pituitaries
mRNA synthesis from endogenous and LHß genes was measured in
response to different treatments. Dispersed pituitary cells were
treated for 30 min with GnRH (10 nM), the PKC activator PMA
(100 nM), the L-type calcium channel agonist BayK8644
(BayK, 50 nM), or the L-type calcium channel antagonist
nimodipine (100 nM). Nuclei were then isolated and
transcription rates measured. Values are expressed in parts per million
(ppm) and represent mean ± SEM for three or four
samples per group. * indicates P < 0.05 and **
P < 0.01 vs. control.
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Transfection Studies
Transfection data were supported by studies in which the
homologous rat LHß promoter from -617 to +41 was stimulated by GnRH
when transfected into RC4B cells (5.17 ± 3.17 arbitrary light
units (ALU)/100 µg protein vs. 1.0 ± 0.26 ALU/100
µg protein for control). Nimodipine eliminated GnRH-induced promoter
activity (0.96 ± 0.02 ALU/100 µg protein) as in endogenous gene
transcription experiments. To investigate both subunit genes in a
gonadotrope line with well characterized signaling pathways,
transfections were performed in
T3 cells. Luciferase constructs
containing the homologous
promoter (
LUC) or the GnRH-responsive
portion of the LHß gene fused to a heterologous nonresponsive
thymidine kinase (tk) minimal promoter were used (LHßtkLUC). The
LHßtkLUC construct consistently responded to GnRH in both RC4B and
T3 cell lines while expression of a tkLUC control was unchanged by
GnRH or any other treatment.
In
T3 cells, GnRH stimulated both
LUC and LHßtkLUC reporter
activity (Fig. 2
). Chelation of external
calcium with EGTA did not alter the
LUC response to GnRH. However,
the same amount of EGTA in the media decreased GnRH stimulation of
LHßtkLUC to basal levels. EGTA alone had no effect on either
construct. GnRH stimulation of
LUC was slightly decreased with
nimodipine, the specific L-channel antagonist (Fig. 2
). In contrast,
application of nimodipine eliminated GnRH activation of LHßtkLUC,
indicating that calcium influx through L-type voltage-gated channels is
necessary for GnRH stimulation of the LHß promoter region.
The contribution of GnRH-induced protein kinase C activation to the
transcriptional response was also examined. As PKC stimulation of the
mouse and human
-subunit genes in primary cultures and
T3
cells has been shown to result in MAPK activation, we examined the
sensitivity of the rat
and LHß gene GnRH-responsive regions to
this pathway (25, 26). Transfected cells were treated with GnRH in the
absence or presence of the specific MAPK kinase (MEK) inhibitor
PD098059. Basal and GnRH-stimulated levels of
LUC were both
decreased in the presence of this inhibitor. While GnRH stimulated the
reporter with the inhibitor present, the extent of this stimulation was
less than in the absence of the inhibitor. PD098059 did not
significantly alter basal activity of LHßtkLUC, and GnRH treatment
stimulated reporter activity (Fig. 3
).
These data illustrate that while rat
promoter expression requires
activation of the MAPK pathway for basal and stimulated expression, the
rat LHß gene is regulated differently by MAPK. Inhibition of the
MAPK pathway had no effect on the LHßtkLUC reporter, indicating this
pathway is not necessary for rat LHß activation.
Transgenic Mouse Studies
To examine the homologous rat LHß promoter in a more
physiological setting, we measured luciferase activity in pituitaries
from transgenic female mice containing 2 kb of the rat LHß
5'-flanking region fused to a luciferase reporter. The response of this
LHß-LUC transgene to GnRH, antide (a GnRH antagonist) and gonadal
steroids in vivo has been well documented (15). Isolated
pituitary cells were stimulated with GnRH or treated with nimodipine or
PD098059 plus GnRH (Fig. 4
). Nimodipine
eliminated GnRH stimulation of the LHß reporter, while PD did not
effect GnRH induction. These results agree with transcription data from
the endogenous gene (Fig. 1
) and with transfection data (Figs. 2
and 3
), illustrating the relative importance of Ca++ influx in
activating the LHß gene. Similar results were observed with
pituitaries from both males and females, and inhibitors did not affect
basal promoter activity (data not shown).

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Figure 4. LHß Promoter Stimulation in Transgenic Mice
Isolated pituitary cells from male transgenic mice bearing the
LHß-luciferase transgene were treated for 4 h with GnRH (10
nM), PD098059 (50 µM), or nimodipine (100
nM). *, P < 0.05 vs.
control for six samples per group.
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DISCUSSION
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Pulsatile release of hypothalamic GnRH is necessary for pituitary
LH synthesis and secretion in a variety of species (1, 2, 3, 4). Its
importance in rodents is illustrated by the hypogonadal mouse, where
lack of pulsatile GnRH results in loss of LH production and therefore
inadequate gonadal development (28). Replacement of pulsatile GnRH in
these mice restores production of LH and gonadal steroids. Messenger
RNA levels and gene transcription of all three gonadotropin subunits
(
, FSHß, and LHß) are differentially modulated by specific GnRH
pulse frequencies in vivo and in vitro (5, 6).
Our results demonstrate the rat
-subunit and LHß gene
GnRH-responsive regions can also be differentially activated by
Ca++ influx and the PKC/MAPK pathways. Stimulation of the
rat LHß gene requires pulsatile GnRH in vitro, while the
-subunit gene can be stimulated by pulsatile or constant GnRH. GnRH
pulses also increase GnRH receptor levels, thus amplifying the signal
(20). One mechanism to favor LHß vs.
-subunit gene
transcription, therefore, could be due to increased receptor levels
caused by pulsatile GnRH. This is supported by studies in a
somatomammotrope cell line stably transfected with the GnRH receptor
and transiently transfected with human
, rat LHß, and rat FSHß
promoter-reporter constructs. GnRH receptor density correlates with
preferential gonadotrope subunit promoter activation, with high density
favoring GnRH stimulation of the LHß gene (29). However,
T3 cells
do not increase the level of GnRH receptor mRNA in response to GnRH
(30). Additionally, stimulation of both LH subunit reporters occurred
with the same GnRH application, so that any increase in receptor number
would be identical in both cases. The different stimulation of the rat
LH subunits by different second messengers here indicates that
mechanisms other than change in receptor number are also involved. Our
studies on endogenous gene transcription, expression of GnRH-responsive
areas in transfected cells, and LHß promoter activity in transgenic
mice indicate that differential sensitivity to second messenger
pathways exists in a physiological context.
The requirement for MAPK activation for GnRH stimulation of the rat
promoter agrees with data using the mouse and human
-subunit
promoters (25, 26). GnRH activation of the mouse
-subunit promoter
was attenuated by overexpression of a kinase-defective MAPK or
overexpression of MAPK phosphatase 2 in
T3 cells (25). The
transcription factors binding the GnRH-responsive region of the mouse
gene have not been described although one has been postulated to be an
Ets family protein. Members of this family have been demonstrated to be
phosphorylated and activated by MAPK (31, 32). The DNA-binding domains
of two family members, Ets-2 and ER81, contact a GGAA sequence in the
mouse GnRH response element, and cotransfection with a dominant
negative Ets-2 reduces GnRH stimulation. Others demonstrated that both
PKC depletion and cotransfection of dominant negative MAPK (ERK1 and
ERK2) suppress basal and GnRH-stimulated human
-subunit promoter
activity (26, 27). The human and mouse
-subunit genes contain an Ets
domain protein-binding site in the GnRH-responsive regions, whereas the
rat gene contains two, between bases -411 and
-375.1 Thus, all the
-subunit genes
contain potential sites for binding proteins directly activated by
MAPK. Several studies illustrate the dependence of human
-subunit
promoter expression on both calcium influx and PKC activation in
T3
cells (33, 34). In our experiments, GnRH-induced rat
-subunit
promoter activity was only slightly affected by nimodipine or by
chelation of external calcium by EGTA, indicating that external calcium
influx has less influence on the rat
than the human
-subunit
promoter when studied in the same cell context. As promoter sequence
homology between species ranges from 7190%, some differences in DNA
sequence within the GnRH-sensitive region or other promoter areas could
account for observed variations in the transcriptional responses to
calcium vs. MEK-stimulated pathways.
The differences in transcriptional responses of the rat LH subunit
genes are reflected in promoter sequences (12, 41). No Ets
family-binding sites were identified in the rat LHß GnRH-responsive
region between -617 and -245, perhaps explaining the relative lack of
sensitivity to the MEK/MAPK pathway. Steroidogenic factor-1 (SF-1)
sites are found in the
-subunit, GnRH receptor, and rat and bovine
LHß subunit promoters and are critical for basal activity of these
genes in vitro and in vivo (35, 36, 37). However,
these SF-1 sites are not contained in the GnRH-responsive portion of
the rat LHß reporter used in transfection studies. Others have also
suggested that these SF-1 sites are not primarily responsible for GnRH
stimulation of this gene (36). Our studies using transgenes,
transfection constructs, and endogenous LHß gene activity show
similar sensitivity to pharmacological agents and suggest that calcium
influx plays a major role in GnRH stimulation of this gene.
Calcium-sensitive activation is not regulated by a single transcription
factor, and several types of transcription factor-binding sites and
different transcription factors can contribute to stimulation by this
pathway (38, 39). Cross-talk between signaling pathways has also been
noted in gonadotropes and other cell types; therefore, cooperation
between PKC stimulation and calcium influx may also occur, and this
could result in LHß stimulation (17, 38, 39, 40).
The results of these studies in a variety of experimental systems
demonstrate that the rat LH subunit genes are differentially stimulated
by the two signaling pathways activated by the GnRH receptor. The
relative concentrations of molecules contributing to these pathways are
unknown but could vary with GnRH pulses. Even a single GnRH pulse, for
example, induces internal Ca++ oscillations in
gonadotropes, suggesting complicated dynamics involving multiple
cellular compartments (41). The GnRH receptor can also couple to
multiple G proteins, enabling preferential associations under different
GnRH concentrations and regimens to occur (23, 42). Differential
signaling pathways may also be activated by the same receptor, as both
the
- and ß
-subunits of an individual G protein can affect
second messenger pathways (43, 44, 45). These results suggest a
mechanism by which separate genes can be preferentially modulated by a
single ligand in a physiologically relevant manner.
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MATERIALS AND METHODS
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Transcription Run-Off Assays
Dispersed pituitary cells from male CD-1 rats (200250 g,
Charles River Laboratories, Wilmington, MA) were treated for 30 min
with 10-8 M GnRH, the PKC activator PMA
(10-7 M), the L-type Ca++ channel
agonist Bay K8644 (5 x 10-7 M)
(Calbiochem, San Diego, CA), or the L-type Ca++ channel
antagonist nimodipine (10-7 M) (46, 47).
Nimodipine was added 15 min before GnRH treatment. Transcription rates
of the endogenous
and LHß genes were analyzed in isolated nuclei
incubated with [
32P]uridine triphosphate as previously
described (47, 48). Values are expressed in parts per million and are
the mean ± SEM for three or four independent samples
per group. Statistical significance was assessed using multiple
comparisons among group means by Tukeys wholly significant difference
procedure.
Vector Preparation
The rat
promoter from -411 to +77 bp relative to the
transcriptional start site (see footnote 1) was generated from genomic
DNA via PCR using primers specific to the rat
sequence and
conserved regions of the human and mouse
-subunit genes (10, 11, 49). DNA was sequenced by the Sanger method (50) and subcloned into a
luciferase reporter vector. For the rat LHß gene, two constructs were
used. The homologous promoter construct, containing -617 to +41 bp
relative to the transcriptional start site, was fused to the luciferase
reporter and used in RC4B cells. A construct containing the
GnRH-responsive region (-617 to -245 bp) of the rat LHß promoter
fused to the tk minimal promoter and cloned into the luciferase
reporter vector was used in
T3 cells. The LHß gene region from
-245 to -100 bp was unresponsive to GnRH in RC4B or
T3 cells (data
not shown), and the tk promoter was unaffected by any treatment
used.
Cell Cultures and Transfections
RC4B cells express PRL and the gonadotropins, have functional
GnRH receptors, and can express both the
and LHß genes (51). RC4B
(8 x 106 cells) were incubated with 80 µg of the
LHß promoter construct in a total volume of 0.8 ml Dulbeccos PBS,
transfected by electroporation at 320 mV and 960 µFarads (Bio-Rad,
Richmond, CA), and divided into eight 60-mm wells.
T3 cells are a
clonal gonadotropin line that expresses the
-subunit but not LHß
or FSHß. These cells contain GnRH receptors, and the response to GnRH
has been well characterized (20, 21, 22, 23, 24, 25).
T3 cells were grown in DMEM
with 2 mM L-glutamine, 10% FBS, 100 U/ml
penicillin, and 100 µg/ml streptomycin. Cells (1 x
106/60-mm well) were plated 3648 h before
CaPO4 transfection with 5 µg of the
or LHß reporter
per well. Cells were transfected for 16 h, washed, and treated.
For all studies, 10-7 M GnRH or
10-7 M of the des-gly agonist (GnRHa), 2
mM EGTA, 5 x 10-7 M PMA,
and/or 10-7 M nimodipine were applied for
6 h, with nimodipine added 15 min before GnRH. In some experiments
50 µM PD098059 (Calbiochem, San Diego, CA), a specific
MEK inhibitor, was applied 30 min before GnRH (52). Inhibition of MEK
activity by PD098059 was verified in parallel studies by measuring MAPK
phosphorylation with an antibody specific to the tyr-phosphorylated
form (New England BioLabs, Beverly, MA). After treatments, cells were
washed, collected in 250 µl lysis buffer (Promega, Madison WI),
vortexed, spun for 1 min, and assayed in a Turner 20-e luminometer
(Turner Designs, Mountain View, CA). Cell lysates were also used to
determine protein concentrations using the colorimetric Bio-Rad protein
assay system. Results are expressed as ALUs per 100 µg protein. All
data were normalized and compared with untreated controls equal to 1.0.
Data represent the averages of three to eight separate experiments and
were analyzed using Students t test comparing treatment
groups to respective controls.
Transgenic Mice
Mice expressing a -2.0 kb to +41 bp LHß promoter-luciferase
reporter transgene were previously described (15). These animals
express LHß promoter activity specifically in the pituitary, and this
activity is regulated in vivo by gonadectomy, steroids, and
a GnRH antagonist. Pituitaries from transgenic female mice over 5 weeks
old were removed and treated as previously described (15, 48). Cells
were then treated with 10-7 M GnRH, or GnRH in
addition to 10-7 M nimodipine, or 50
µM PD098059. Nimodipine and PD were given 30 min before
4-h stimulation by GnRH. Pituitaries were processed and luciferase
activity was assayed as described (15). Statistical significance was
assessed using multiple comparisons by Tukeys significant difference
procedure.
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ACKNOWLEDGMENTS
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We thank Drs. Nancy Cooke and Jean Rica of the University of
Pennsylvania Transgenic Mouse Core Facility, who performed DNA
injections to establish founder mouse lines. We also thank Drs. Derek
Schreihofer, Suzanne Moenter, and Martin Straume for helpful
discussions and careful reading of the manuscript.
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FOOTNOTES
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Address requests for reprints to: Margaret A. Shupnik, Box 578 Health Science Center, University of Virginia, Charlottesville, Virginia 22908. e-mail: mas3x@virginia.edu.
This work was supported by NIH Grants R01 HD25719 (to M.A.S.) and NRSA
F32MH10152 (to P.C.F.), the Center for Cellular and Molecular Research
in Reproduction (P30 HD28934), and the National Science Foundation
Center for Biological Timing (DIR 890162) at the University of
Virginia.
1 Rat glycoprotein hormone
-subunit sequence
submitted to GenBank. Accession number AF016702. 
Received for publication October 24, 1997.
Revision received November 20, 1997.
Accepted for publication November 24, 1997.
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