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Division of Endocrinology, Departments of Medicine and Microbiology (M.E.C., S.J.P.), University of Virginia Health Science Center, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Dr. Daniel J. Haisenleder, Division of Endocrinology, Department of Medicine, University of Virginia Health Science Center, 5041 MR-4 Building, Charlottesville, Virginia 22908. E-mail:djh2q{at}virginia.edu
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Abstract |
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The role of MAPK activation on gonadotrope (
, LHß, FSHß, and
GnRH receptor) gene expression was determined in vitro.
Preliminary studies demonstrated that the MAPK inhibitor, PD-098059 (50
µM), completely blocked GnRH-induced increases in MAPK
activity in adult male pituitary cells. Further studies revealed that
PD-098059 blocked gonadotrope messenger RNA (mRNA) responses to
pulsatile GnRH (100 pg/ml, 60-min interval, 24-h duration) in a
selective manner, with , FSHß, and GnRH receptor (but not LHß)
mRNA responses being suppressed. These results show that a pulsatile
GnRH signal is required to maintain MAPK activation for durations of
longer than 2 h, and that slower frequency pulses are more
effective. Further, MAPK plays a crucial role in , FSHß, and GnRH
receptor mRNA responses to pulsatile GnRH. Thus, divergent MAPK
responses to alterations in GnRH signal pattern may be one mechanism
involved in differential regulation of gonadotrope gene expression.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, LHß, and FSHß), GnRH-R, and follistatin
(1, 2, 3, 4, 5). To some degree, the effect of altering the GnRH pulse pattern
on gonadotropin subunit gene expression is a direct action at the
transcriptional level (6). In contrast to the effects of pulsatile
GnRH, a continuous signal pattern desensitizes secretory responses to
GnRH (7) and, with the exception of the -subunit, is also
ineffective in stimulating a rise in the expression of gonadotrope
genes (8, 9, 10). The GnRH-R is a member of the seven-transmembrane receptor family, with receptor binding activating two specific GTP-binding proteins, Gq and G11 (11). GnRH-R occupation rapidly stimulates an increase in phosphoinositide turnover and a rise in intracellular diaclyglycerol levels, which results in the activation of protein kinase C (PKC) (12). GnRH-R activation also stimulates an increase in intracellular calcium (Ca2+) concentrations (13) as well as a rise in intracellular cAMP levels (14). In recent years, we have investigated the intracellular mechanisms involved in the transduction of pulsatile vs. continuous GnRH signals from the plasma membrane to the nucleus, focusing on the second messenger systems that are activated by GnRH (15, 16, 17). The results from these studies suggest that, as has been shown for GnRH, intermittent increases in specific second messenger systems may play an important role in the transduction of signals from the plasma membrane to the cytoplasm in the gonadotrope. However, the critical downstream site(s) in this signal transduction pathway (i.e. specific protein kinases and nuclear regulatory proteins) remain to be characterized.
Recent studies have shown that mitogen-activated protein kinase (MAPK)
is located in primary rat pituitary cells as well as in
pituitary-derived cell lines, including the gonadotrope-derived T-3
cell and the lactotrope-derived GH3 cell (18, 19, 20, 21, 22). MAPK is
a serine/threonine kinase that is activated in response to a variety of
stimuli, including tyrosine kinase, G protein-coupled receptors, and
Ca2+ influx (23). Two prominent isoforms of MAPK (also
called extracellular signal-regulated kinase; ERK) have been
identified, a 44-kDa isoform (ERK-1) and a 42-kDa isoform (ERK-2)
(24). MAPK has been shown to play a role in the regulation of gene
expression in various systems, either by activating nuclear
transcription factors directly or by phosphorylating downstream
cytoplasmic kinases (25). Studies by Mitchell et al.
(18) demonstrated that GnRH stimulates an increase in MAPK activity in
female rat pituitary cells in vitro. Their results suggested
that MAPK may play a role in the regulation of LH secretion and that
GnRH-induced activation of gonadotrope MAPK may be mediated by PKC.
More recent studies by Roberson et al. (21) indicate that
MAPK may play an important role in basal as well as GnRH-induced
stimulation of -subunit transcription.
The present study was conducted to determine whether MAPK plays a
physiological role in the differential gonadotrope messenger RNA (mRNA)
responses to alterations in GnRH treatment mode (pulsatile
vs. continuous; pulse frequency). This issue was addressed
by asking the following questions. 1) Is MAPK activity regulated by
alterations in GnRH signal pattern? 2) Does MAPK play a role in
GnRH-induced actions on gonadotrope (, LHß, FSHß, and GnRH-R)
gene expression? This later question was addressed by determining
whether the stimulatory effect of pulsatile GnRH on gonadotrope mRNAs
can be suppressed by administering a MAPK inhibitor. The inhibitor
chosen for these studies was the recently identified compound,
PD-098059 (PD), which has been shown to be a relatively specific
inhibitor of the MAPK activator, MAPK kinase (MEK) (26).
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Materials and Methods |
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Exp 1: GnRH dose response: Twenty-four hours after surgery and before treatment (as described above), rats received a single bolus of GnRH (30, 100, or 300 ng) or BSA-saline and were killed 4 min later.
Exp 2: time-course response to a single GnRH pulse: Animals were given a single bolus of GnRH (50 ng) and were killed 4, 10, or 30 min later. Controls were killed 4 min after receiving a single bolus of BSA-saline.
Exp 3: time-course responses to pulsatile or continuous
GnRH: Rats received GnRH pulses (50 ng, 10-sec duration, 60-min
interval) or a continuous infusion (25 ng/min) for 1, 2, 4, or 8
h. This infusion dose was selected to provide sustained levels of
circulating GnRH that are similar to peak levels obtained after a
single 50-ng pulse (i.e. 
200 pg/ml) (28). The total GnRH
doses for the 8-h groups were 450 ng (pulsatile treatment) and 12 µg
(continuous infusion). Controls received pulsatile or continuous
BSA-saline for 8 h.
Exp 4: effect of GnRH pulse interval: GnRH pulses (50 ng) were administered every 30, 60, or 120 min for 8 h, with a total GnRH dose of 850 ng (30-min pulse group), 450 ng (60-min group), or 250 ng (120-min group). Controls received 60-min BSA-saline pulses.
In vitro studies: cell model. Adult male pituitaries were collected, pooled, and dissociated in DMEM (without phenol red) containing 0.35% collagenase, 0.01% deoxyribonuclease, and 0.1% hylauronidase, as previously described (15, 16, 17). The cells were plated for 48 h to allow attachment to plastic coverslips (22 mm in diameter) that were coated with Matrigel (Collaborative Biomedical Products, Bedford, MA). Plating medium contained 10% FCS, 5% horse serum, penicillin (100 U/ml), streptomycin (100 µg/ml), gentomicin (36 µg/ml), nystatin (240 U/ml), insulin (2 ng/ml), and transferrin (10 µg/ml). The medium also contained dopamine (500 nM) with 100 µM ascorbic acid to inhibit lactotrope-derived MAPK activity (27) and T (2.5 ng/ml) to allow LHß mRNA expression in response to pulsatile GnRH (29). Experiments were initiated by either inserting the coverslips into fresh wells that contained treatment compounds (static culture studies) or inserting them into perfusion chambers (GnRH pulse studies), as previously described (16). Perifusion medium contained DMEM (without phenol red), penicillin (100 U/ml), streptomycin (100 µg/ml), nystatin (240 U/ml), insulin (2 ng/ml), and transferrin (10 µg/ml). Perifusion medium did not contain serum.
Exp 1: effect of PD on GnRH-stimulated activation of MAPK: Coverslips were inserted into culture wells containing PD (50 µM; Calbiochem, La Jolla, CA) or vehicle (0.1% dimethylsulfoxide-plating medium). One hour later, the coverslips were transferred to fresh wells containing the same treatment (PD or vehicle) with or without GnRH (1 ng/ml; medium only to controls). Cells were recovered 5 or 60 min later (GnRH treatment duration, 5 or 60 min).
Exp 2: to determine gonadotrope recovery from 24 h of PD treatment: Coverslips were transferred into wells containing either PD (50 µM) or vehicle. Twenty-four hours later, the cells were washed and inserted into fresh wells containing GnRH (1 ng/ml), pituitary adenylate cyclase-activating polypeptide (PACAP; 45 ng/ml; Penninsula Lab, Belmont, CA), or medium only. At specific time points (4, 8, and 24 h), the coverslips were transferred to fresh wells containing the same treatment (i.e. GnRH, PACAP, or medium). Culture medium from each time point were collected and frozen (-20 C) for LH measurement.
Exp 3: effect of PD on GnRH-induced increases in , LHß,
FSHß, and GnRH-R mRNAs and gonadotropin secretion: Fifty male
rat pituitaries were pooled, dissociated, and plated on Matrigel-coated
coverslips. Forty-eight hours later, the coverslips were inserted into
chambers and perifused with medium containing either PD (50
µM) or vehicle. One hour later, chambers from both groups
received either GnRH pulses (peak chamber concentration, 100 pg/ml;
60-min interval) or pulses of perifusion medium alone for 24 h.
Perifusate fractions (10 min) were collected for 60 min after 2 and
22 h of treatment to assess LH and FSH secretory responses. After
completion of the experiment, the cells were recovered, and total RNA
was extracted with phenol for mRNA determination. , LHß, FSHß,
and GnRH-R mRNAs were measured by dot blot hybridization, as previously
described (15, 34). mRNA concentrations were expressed as femtomoles of
complementary DNA bound per 100 µg pituitary DNA and were presented
as the percent increase vs. the concentrations in
medium-pulsed controls.
MAPK assay
Tissue recovery. After each experiment, whole pituitaries or
cultured cells were removed and homogenized in a lysis buffer
containing leupeptin, aprotinin, mycrocystin,
p-nitrophenylphosphate, and sodium vanadate (30). The
homogenate was centrifuged at 10,000 x g for 5 min,
and the supernatant was recovered. An aliquot was removed for protein
determination, and the remaining sample was diluted 1:1 in a protein
denaturing sample buffer (containing 4% SDS, 10% ß-mercaptoethanol,
and 20% glycerol), boiled for 3 min, then stored at -70 C until
assayed.
Immunoblot assay. Activation of MAPK occurs by phosphorylation of specific tyrosine and threonine residues. Phospho-specific MAPK antibodies have been raised against these phosphorylated sites and were used to measure MAPK activation by Western blot. This assay provides a sensitive, specific, and simple method to determine alterations in MAPK activation. Compared with other MAPK activation assays [i.e. immunoprecipitation/substrate assay (30), in-gel kinase renaturation assay (31)], we found this method to be more sensitive and consistent (32).
Whole cell lysate protein (20–50 µg) was resolved via electrophoresis through a 12% SDS-polyacrylamide gel. Protein bands were transferred to nitrocellulose filters and immunoblotted using antibodies specific to phosphorylated p44/42 MAPK (P-MAPK) and total p44/42 MAPK (T-MAPK; antibodies provided by New England Biolabs, Beverly, MA). These antibodies recognize the p44 and p42 isoforms to a similar degree. The secondary antibody was horseradish peroxidase-conjugated antirabbit (Pierce Chemical Co., Rockford, IL). MAPK bands were identified by phosphorylated and nonphosphorylated MAPK control proteins (p42 and ERK-2; New England Biolabs) run on each gel. Bands were detected using the ECL chemiluminescent system (Amersham, Arlington Heights, IL), followed by autoradiography. Pituitary P-MAPK was quantitated by densitometry (NIH Image 1.61) and corrected to the amount of T-MAPK per sample. Preliminary data revealed that ERK-1 and -2 were activated by GnRH to a similar degree. However, as the intensity of the signal for ERK-2 was greater than that for ERK-1, results were quantitated using ERK-2 bands for each sample.
RIA
LH and FSH were determined in culture medium and perifusate
samples by RIA, using reagents provided by the National Hormone and
Pituitary Program. The RIA standards were NIDDK RP-3 for LH and RP-2
for FSH. The assay sensitivities were 0.09 ng/tube (LH) and 0.8 ng/tube
(FSH). The coefficients of variation were 8.3% and 10.8% for the LH
assay and 6.3% and 9.6% for the FSH assay (intra- and interassay,
respectively).
Statistical analysis
Most data were analyzed by one-way ANOVA, with differences
between treatment groups determined by Duncan’s multiple range test.
Treatment-induced changes in perifusate LH and FSH were determined by
two-way ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T-3 cells (20), in which a GnRH agonist
stimulated ERK-1 to a greater extent than ERK-2. The upper
panel of Fig. 1
shows the
autoradiograph of the ERK-2 response 4 min after a single pulse of
GnRH. The results reveal that GnRH stimulated an increase in pituitary
MAPK activity in a dose-responsive manner (P < 0.05
vs. controls), with a 2-fold rise after a 30-ng pulse, a
4-fold rise after a 100-ng pulse, and an 8-fold increase after a 300-ng
dose.
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, GnRH stimulated a rapid, transient increase in pituitary MAPK activity
(maximal within 4 min; P < 0.05 vs.
controls). Although MAPK activity decreased over time, it remained
elevated 30 min after the GnRH pulse (4-fold; P <
0.05). Of note, the magnitude of the response to GnRH (8-fold increase,
4 min after the pulse) was similar to that of the response to the
higher (300 ng) pulse dose in Fig. 1
.
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) that pulsatile GnRH stimulated a
2- to 4-fold increase in MAPK activity (P < 0.05
vs. controls) that was maintained over the 8-h study
duration. In contrast, continuous GnRH only stimulated a rise in MAPK
activity (2- to 3-fold vs. controls; P <
0.05) for the initial 2 h of infusion.
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, similar increases in MAPK were
seen after 30- or 60-min pulses (2 fold; P < 0.05
vs. controls). However, slower (120-min) interval pulses
were more effective in increasing MAPK activity than faster pulses
(P < 0.05 vs. 30- or 60-min pulse
groups).
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, LHß, FSHß, and GnRH-R) mRNA expression. The
first study was conducted to demonstrate that pretreating pituitary
cells in static culture with the MAPK inhibitor, PD (50
µM), can suppress GnRH-induced increases in MAPK
activity. The GnRH dose used (1 ng/ml) was determined from preliminary
studies showing that this dose produced maximal stimulatory effects on
MAPK (data not shown), similar to previous findings (18, 20). Figure 5 shows that GnRH increased MAPK activity
within 5 min (4-fold; P < 0.05 vs.
controls), which was maintained to a lesser degree after 60 min.
Pretreating the cells with PD not only inhibited basal MAPK activity
(by 65%), but completely suppressed the MAPK response to GnRH.
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). Of importance, no significant
difference in basal or stimulated LH secretion was seen between cells
pretreated with PD or vehicle. This suggests that PD, over the dose and
duration given, does not affect cell viability, as determined by
secretory function.
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shows the effect of PD on GnRH-induced
increases in gonadotrope mRNAs. The GnRH treatment paradigm used (100
pg/ml pulses, 60-min interval, 24-h duration) was based on previous
perifusion data showing that this protocol increases all four
gonadotrope mRNAs under investigation (16, 34). Pulsatile GnRH
stimulated an increase (P < 0.05) in (91%
increase vs. controls), LHß (52% increase), FSHß (51%
increase), and GnRH-R (56% increase) mRNAs. The addition of PD to the
perifusion medium completely suppressed the , FSHß, and GnRH-R
mRNA responses to pulsatile GnRH. Of note, PD had no effect on the
GnRH-induced increase in LHß mRNA. These results suggest that MAPK
plays a critical role in GnRH stimulation of , FSHß, and GnRH-R
(but not LHß) gene expression.
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. Pulsatile
GnRH increased LH and FSH release in both vehicle- and PD-treated
groups (P < 0.05 vs. controls). Although
perifusate gonadotropin responses to GnRH tended to be lower after 2
and 22 h in PD-treated chambers, a significant reduction was only
seen for FSH [total release over the 60-min sampling duration (2 and
22 h; P < 0.05 vs. the vehicle-GnRH
group, as determined by two-way ANOVA)]. Thus, both mRNA data
presented in Fig. 6 and hormone secretion (Fig. 7) suggest that MAPK
plays a greater role in GnRH regulation of FSH than LH.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T-3 cells
(18, 19, 20, 21). The present investigation is the first to examine
GnRH-induced effects on MAPK in vivo and to assess the role
of alterations in the pattern of GnRH signals in pituitary MAPK
responses. Specifically, we report that MAPK responsiveness to GnRH is
maintained for at least 8 h if the peptide is presented in a
pulsatile manner, but that continuous GnRH only stimulates a short term
(2-h) rise in MAPK activation. Further, the frequency of GnRH pulse
signals can influence pituitary MAPK responses, with slower pulses
(120-min interval) being optimal. To investigate the physiological
implications of these results, in vitro studies were
conducted to determine whether blocking GnRH-induced activation of MAPK
(using PD) (26) influences gonadotrope mRNA responses to pulsatile
GnRH. The results showed that suppression of GnRH-induced activation of
MEK/MAPK blocked gonadotrope mRNA responses in a selective manner. As
presented in Fig. 6, PD completely inhibited the rise in , FSHß,
and GnRH-R mRNA in response to pulsatile GnRH, but the LHß response
was not altered. These results strongly suggest that the MEK/MAPK
pathway plays a critical role in the GnRH signal transduction pathway
for , FSHß, and GnRH-R gene expression. The data also suggest that
GnRH stimulation of LHß uses a distinct and different intracellular
pathway.
A recent investigation by Reiss et al. (20) revealed that
PKC plays a critical role, and that tyrosine kinase and
Ca2+ also participate in GnRH-induced activation of MAPK in
T-3 cells. We and others have shown that both Ca2+ and
PKC pathways regulate gonadotrope gene expression in primary pituitary
cells (16, 17, 34, 35). More specifically, increasing intracellular
Ca2+ stimulates all four gonadotrope mRNAs studied (16, 34). In contrast, the actions of PKC appear to be more selective, with
PKC activation increasing and LHß mRNAs, but not FSHß (17, 35).
The effects of PKC on rat GnRH-R mRNA have yet to be determined. Taken
together, these results suggest that GnRH regulation of LHß mRNA
expression uses Ca2+ or PKC downstream pathways that are
separate from the MAPK system. At present, the findings that
GnRH-induced increases in FSHß mRNA are mediated via MAPK activation
are difficult to explain in view of data showing that PKC activation
[via phorbol 12-myristate 13-acetate (PMA) or diacylglycerol pulses]
does not stimulate FSHß mRNA expression (17). As GnRH has been shown
to activate various PKC isoforms (36), perhaps stimulation of a
PMA-insensitive PKC isoform regulates MAPK activation in the
gonadotrope (37). Alternatively, cross-talk between other signal
transduction pathways (i.e. tyrosine kinase and
Ca2+) may play a greater role in MAPK activation in rat
pituitary cells than is suggested by studies in T-3 cells (20). A
further possibility is that PMA selectively suppresses FSHß mRNA
expression at a site downstream from MAPK activation, canceling out any
stimulatory effects.
We and others have shown that in rats, rapid GnRH pulse signals (or a
continuous infusion) stimulate mRNA expression, rapid to medium
pulse frequencies (15- to 30-min intervals) are optimal for LHß, and
slower pulses (>60-min intervals) maximally stimulate FSHß (3, 6, 10). For GnRH-R mRNA, in vivo results in GnRH-deficient rats
reveal that maximal increases are seen after slower (240-min interval)
pulses (5). In light of these findings, the present data showing that
longer interval pulses are optimal for MAPK activation are of interest
and may explain the link between slower pulse frequencies and
regulation of FSHß/GnRH-R mRNAs. Although GnRH has been shown to
stimulate pituitary MAPK activity in the female rat (18, 32), it
remains to be determined whether the female responds to alterations in
GnRH pulse frequency in a manner similar to that seen in the male or
whether gonadal steroids play a role. The MAPK response to a single
pulse peaked at 4 min and was declining after 10 and 30 min, but
remained elevated compared with that in controls at the 30 min point.
Although not measured in this study, it is likely that during the
120-min period after the pulse, MAPK activity would return to basal
levels. This suggests that transient, high amplitude increases in MAPK
activity may be optimal to maintain maximal activation of downstream
components of the signal transduction pathway for the FSHß and GnRH-R
genes.
The present data confirm GnRH activation of gene expression through
the MAPK pathway (19, 21). As noted above, mRNA expression is
selectively stimulated by fast frequency pulses or continuous GnRH
signals. This could suggest that the observed short term increases in
MAPK activation in response to continuous GnRH are sufficient to
stimulate downstream mediators of GnRH action on transcriptional
activity. Recent studies by Robinson et al. (21) have shown
that MAPK phosphorylates ELK-1, a member of the Ets family of
transcription factors (38, 39). ELK-1 has been shown to bind to the
GnRH-responsive element located within the mouse -subunit gene
promoter region, and GnRH increases ELK-1 promoter activity in T-3
cells (21). However, whether ELK-1 or another Ets protein binds to this
region in the rat remains to be determined.
Previous studies have suggested that MAPK may play a role in GnRH regulation of LH secretion in the rat (18). In the present investigation, we were unable to confirm those findings. However, differences in animal models (cyclic females vs. T-treated males) and experimental paradigm are possible explanations for the differing results. More specifically, in that earlier report (18), data suggest that MAPK plays a selective role in one specific aspect of GnRH-induced LH release (i.e. the augmentation of LH secretory responsiveness to GnRH, the priming effect). This issue was not addressed in the present study. In contrast to LH, a significant reduction in FSH secretion to pulsatile GnRH in the presence of MAPK inhibition was observed. Whether the suppression of FSH secretion is a direct action on FSH release pathways or via indirect intracellular regulatory mechanisms (i.e. reduction in FSHß mRNA expression/subunit synthesis) remains to be determined.
For the assessment of pulsatile vs. continuous GnRH signals,
we chose a GnRH infusion dose to maintain serum GnRH similar to peak
concentrations after a 50-ng pulse (200 pg/ml) (28). This required
administration of a higher total dose of GnRH over 8 h compared
with that in the pulse-treated group; however, in another experiment
(32), we used a lower GnRH infusion dose (1.7 ng/min) in
vivo and obtained similar results (i.e. MAPK returned
to baseline within 4 h). The intracellular mechanism(s) involved
in the decrease in MAPK responsiveness to continuous GnRH remain
uncertain. Studies in T-3 cells showed that continuous GnRH
maintains an increase in MAPK activity for at least 60 min, but that
the rise in MEK is transient (returning to baseline within 10 min)
(20). Perhaps MEK is a critical site involved in desensitization of the
MAPK pathway to continuous GnRH. Other studies reveal that T-3
cells contain at least two MAPK-specific phosphatases [MAPK
phosphatase-1 (MKP-1) and MKP-2] (21), which suppress MAPK activity by
selectively dephosphorylating tyrosine and threonine residues (40).
GnRH has been shown to stimulate MKP-2 mRNA levels in this cell model,
and overexpression of MKP-2 can suppress GnRH-induced stimulation of
-subunit promoter activity in T-3 cells (21). In light of this
observation, it is possible that continuous GnRH is more effective than
a pulsatile signal pattern in stimulating a rise in MKP-2 gene
expression.
In conclusion, these data show that a pulsatile GnRH signal is required
to maintain MAPK activation for durations longer than 2 h and that
slower frequency pulses are more effective in maintaining maximal
increases in MAPK activity. Further, MAPK plays a critical role in GnRH
stimulation of , FSHß, and GnRH-R gene expression, but not that of
LHß. Thus, alterations in MAPK activity may be a physiologically
important component in the signal transduction pathway within the
gonadotrope and may play a role in the differential regulation of
gonadotrope gene expression.
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Acknowledgments |
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Footnotes |
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Received December 10, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit gene expression. Endocrinology 131:3027–3033
T-3 cell line: differential roles of calcium and PKC. Endocrinology 138:1673–1682-subunit promoter
by GnRH. Mol Cell Biol 15:3531–3539[Abstract]
T-3 cells. J
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A. Chamson-Reig, E. M. Sorianello, P. N. Catalano, M. O. Fernandez, O. P. Pignataro, C. Libertun, and V. A. R. Lux-Lantos Gonadotropin-Releasing Hormone Signaling Pathways in an Experimental Ovarian Tumor Endocrinology, July 1, 2003; 144(7): 2957 - 2966. [Abstract] [Full Text] [PDF]
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D. J. Haisenleder, H. A. Ferris, and M. A. Shupnik The Calcium Component of Gonadotropin-Releasing Hormone-Stimulated Luteinizing Hormone Subunit Gene Transcription Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase Type II Endocrinology, June 1, 2003; 144(6): 2409 - 2416. [Abstract] [Full Text] [PDF]
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D. Harris, D. Chuderland, D. Bonfil, S. Kraus, R. Seger, and Z. Naor Extracellular Signal-Regulated Kinase and c-Src, But Not Jun N-Terminal Kinase, Are Involved in Basal and Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone {alpha}-Subunit Promoter Endocrinology, February 1, 2003; 144(2): 612 - 622. [Abstract] [Full Text] [PDF]
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K.-C. Choi, C.-J. Tai, C.-R. Tzeng, N. Auersperg, and P. C.K. Leung Adenosine Triphosphate Activates Mitogen-Activated Protein Kinase in Pre-Neoplastic and Neoplastic Ovarian Surface Epithelial Cells Biol Reprod, January 1, 2003; 68(1): 309 - 315. [Abstract] [Full Text] [PDF]
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V. V. Vasilyev, M. A. Lawson, D. Dipaolo, N. J. G. Webster, and P. L. Mellon Different Signaling Pathways Control Acute Induction versus Long-Term Repression of LH{beta} Transcription by GnRH Endocrinology, September 1, 2002; 143(9): 3414 - 3426. [Abstract] [Full Text] [PDF]
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F.E.M. Rebers, G.A.M. Hassing, W. van Dijk, E. van Straaten, H.J.Th. Goos, and R.W. Schulz Gonadotropin-Releasing Hormone Does Not Directly Stimulate Luteinizing Hormone Biosynthesis in Male African Catfish Biol Reprod, June 1, 2002; 66(6): 1604 - 1611. [Abstract] [Full Text] [PDF]
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 K.-C. Choi, S. K. Kang, C.-J. Tai, N. Auersperg, and P. C. K. Leung Follicle-Stimulating Hormone Activates Mitogen-Activated Protein Kinase in Preneoplastic and Neoplastic Ovarian Surface Epithelial Cells J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2245 - 2253. [Abstract] [Full Text] [PDF]
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V. V. Vasilyev, F. Pernasetti, S. B. Rosenberg, M. J. Barsoum, D. A. Austin, N. J. G. Webster, and P. L. Mellon Transcriptional Activation of the Ovine Follicle-Stimulating Hormone-{beta} Gene by Gonadotropin- Releasing Hormone Involves Multiple Signal Transduction Pathways Endocrinology, May 1, 2002; 143(5): 1651 - 1659. [Abstract] [Full Text] [PDF]
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F. Liu, D. A. Austin, P. L. Mellon, J. M. Olefsky, and N. J. G. Webster GnRH Activates ERK1/2 Leading to the Induction of c-fos and LH{beta} Protein Expression in L{beta}T2 Cells Mol. Endocrinol., March 1, 2002; 16(3): 419 - 434. [Abstract] [Full Text] [PDF]
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D.J. Haisenleder, L.J. Workman, L.L. Burger, K.W. Aylor, A.C. Dalkin, and J.C. Marshall Gonadotropin Subunit Transcriptional Responses to Calcium Signals in the Rat: Evidence for Regulation by Pulse Frequency Biol Reprod, December 1, 2001; 65(6): 1789 - 1793. [Abstract] [Full Text] [PDF]
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G. Maya-Nunez and P. Michael Conn Cyclic Adenosine 3',5'-Monophosphate (cAMP) and cAMP Responsive Element-Binding Protein Are Involved in the Transcriptional Regulation of Gonadotropin-Releasing Hormone (GnRH) Receptor by GnRH and Mitogen-Activated Protein Kinase Signal Transduction Pathway in GGH3 Cells Biol Reprod, August 1, 2001; 65(2): 561 - 567. [Abstract] [Full Text] [PDF]
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H.-J. Huang, J. Sebastian, B. D. Strahl, J. C. Wu, and W. L. Miller Transcriptional Regulation of the Ovine Follicle-Stimulating Hormone-{beta} Gene by Activin and Gonadotropin-Releasing Hormone (GnRH): Involvement of Two Proximal Activator Protein-1 Sites for GnRH Stimulation Endocrinology, June 1, 2001; 142(6): 2267 - 2274. [Abstract] [Full Text] [PDF]
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S. K. Kang, C.-J. Tai, P. S. Nathwani, K.-C. Choi, and P. C. K. Leung Stimulation of Mitogen-Activated Protein Kinase by Gonadotropin-Releasing Hormone in Human Granulosa-Luteal Cells Endocrinology, February 1, 2001; 142(2): 671 - 679. [Abstract] [Full Text] [PDF]
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A. C. Dalkin, L. L. Burger, K. W. Aylor, D. J. Haisenleder, L. J. Workman, S. Cho, and J. C. Marshall Regulation of Gonadotropin Subunit Gene Transcription by Gonadotropin-Releasing Hormone: Measurement of Primary Transcript Ribonucleic Acids by Quantitative Reverse Transcription-Polymerase Chain Reaction Assays Endocrinology, January 1, 2001; 142(1): 139 - 146. [Abstract] [Full Text] [PDF]
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J. Weck, A. C. Anderson, S. Jenkins, P. C. Fallest, and M. A. Shupnik Divergent and Composite Gonadotropin-Releasing Hormone-Responsive Elements in the Rat Luteinizing Hormone Subunit Genes Mol. Endocrinol., April 1, 2000; 14(4): 472 - 485. [Abstract] [Full Text]
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X.-b. Han and P. M. Conn The Role of Protein Kinases A and C Pathways in the Regulation of Mitogen-Activated Protein Kinase Activation in Response to Gonadotropin-Releasing Hormone Receptor Activation Endocrinology, May 1, 1999; 140(5): 2241 - 2251. [Abstract] [Full Text]
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B. R. White, D. L. Duval, J. M. Mulvaney, M. S. Roberson, and C. M. Clay Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene Is Partially Mediated by Protein Kinase C Activation of an Activator Protein-1 Element Mol. Endocrinol., April 1, 1999; 13(4): 566 - 577. [Abstract] [Full Text]
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