Gonadotropin-Releasing Hormone Pulses Are ... - Semantic Scholar

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society

Vol. 138, No. 7 Printed in U.S.A.

Gonadotropin-Releasing Hormone Pulses Are Required to Maintain Activation of Mitogen-Activated Protein Kinase: Role in Stimulation of Gonadotrope Gene Expression* DANIEL J. HAISENLEDER, MICHAEL E. COX, SARAH J. PARSONS, JOHN C. MARSHALL

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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 ABSTRACT The present study examined the effect of alterations in GnRH signal pattern (pulsatile vs. continuous; pulse frequency) on mitogenactivated protein kinase (MAPK) activity and whether MAPK plays a role in regulating gonadotrope gene expression. Pituitary MAPK activity was measured by immunoblot, using a phospho-specific MAPK antibody, corrected to the amount of total MAPK per sample. In vivo studies were conducted in adult castrate testosterone-replaced male rats (to suppress endogenous GnRH). Animals received pulsatile or continuous GnRH (or BSA-saline for controls) via jugular cannulas. Initial studies revealed that pulsatile GnRH stimulated a dose-dependent rise in MAPK activity (30 ng, 2-fold increase; 100 ng, 4-fold; 300 ng, 8-fold) 4 min after the pulse. The effect of pulsatile vs. continuous GnRH was examined by administering 50-ng pulses (60-min interval) or a continuous infusion (25 ng/min) for 1, 2, 4, or 8 h. Pulsatile GnRH stimulated a 2- to 4-fold rise in MAPK activity (P , 0.05 vs. controls) that was maintained over the 8-h duration. In contrast, continuous GnRH only increased MAPK activity (2- to 3-fold; P , 0.05 vs. controls) for 2 h, with MAPK activity returning to baseline at later time points. The effect of GnRH pulse frequency on MAPK activation was determined by giving GnRH pulses (50 ng) at

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E AND other investigators have shown that alterations in GnRH pulse pattern can differentially regulate LH and FSH secretory activity, GnRH receptor (GnRH-R) number, as well as the expression of several gonadotrope-specific genes, including the gonadotropin subunits (a, LHb, and FSHb), GnRH-R, and follistatin (1–5). 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 a-subunit, is also ineffective in stimulating a rise in the expression of gonadotrope genes (8 –10). Received December 10, 1997. 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:[email protected]. * This work was supported by USPHS Grants HD-11489 and HD33039 (to J.C.M.) and DK-44199 and CA-40041 (to S.J.P.), Jeffress Memorial Trust Grant J-257 (to D.J.H.), and the University of Virginia Center for Cellular and Molecular Studies in Reproduction (NIH Grant P30-HD-28934).

30-, 60-, or 120-min intervals for 8 h. Maximal increases (3-fold vs. controls; P , 0.05) were seen after 120-min pulses, with faster (30- to 60-min interval) pulses stimulating 2-fold increases in MAPK activity (P , 0.05 vs. controls and 120-min GnRH pulse group). The role of MAPK activation on gonadotrope (a, LHb, FSHb, and GnRH receptor) gene expression was determined in vitro. Preliminary studies demonstrated that the MAPK inhibitor, PD-098059 (50 mM), 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 a, FSHb, and GnRH receptor (but not LHb) 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 a, FSHb, 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. (Endocrinology 139: 3104 –3111, 1998)

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 (Ca21) 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 (15–17). 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

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as in pituitary-derived cell lines, including the gonadotropederived a T-3 cell and the lactotrope-derived GH3 cell (18 – 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 Ca21 influx (23). Two prominent isoforms of MAPK (also called extracellular signalregulated 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 a-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 GnRHinduced actions on gonadotrope (a, LHb, FSHb, 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, PD098059 (PD), which has been shown to be a relatively specific inhibitor of the MAPK activator, MAPK kinase (MEK) (26). Materials and Methods Experimental protocol In vivo studies: animal model. During preliminary studies, rats were given bromocriptine (to inhibit lactotrope-derived MAPK activity; 1.2 mg, sc) (27) and were compared with animals that received vehicle injections. The data revealed that basal pituitary MAPK activity was reduced in bromocriptine-treated animals, resulting in an increase in the magnitude of MAPK responses to GnRH and improved consistency of treatment effects between experiments. Two days before the experiment, adult male rats were castrated, testosterone (T) implants were placed sc (serum T, 2– 4 ng/ml) to suppress endogenous GnRH, and jugular cannulas were inserted. The following day animals received bromocriptine. Twenty-four hours later, the animals received GnRH either as a single bolus or in a pulsatile or continuous manner (BSA-saline pulses or continuous infusion to controls) and were killed 4 min after completing the treatment protocol (with the exception of Exp 2). All animal procedures were approved by the University of Virginia animal research committee. 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

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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 mg (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 (15–17). 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 mg/ml), gentomicin (36 mg/ml), nystatin (240 U/ml), insulin (2 ng/ml), and transferrin (10 mg/ml). The medium also contained dopamine (500 nm) with 100 mm ascorbic acid to inhibit lactotrope-derived MAPK activity (27) and T (2.5 ng/ml) to allow LHb 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 mg/ml), nystatin (240 U/ml), insulin (2 ng/ml), and transferrin (10 mg/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 mm; 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 mm) or vehicle. Twenty-four hours later, the cells were washed and inserted into fresh wells containing GnRH (1 ng/ml), pituitary adenylate cyclaseactivating 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 (220 C) for LH measurement. Exp 3: effect of PD on GnRH-induced increases in a, LHb, FSHb, and GnRH-R mRNAs and gonadotropin secretion: Fifty male rat pituitaries were pooled, dissociated, and plated on Matrigel-coated coverslips. Fortyeight hours later, the coverslips were inserted into chambers and perifused with medium containing either PD (50 mm) 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. a, LHb, FSHb, 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 mg 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 3 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% b-mercaptoethanol, and 20% glycerol), boiled for 3 min, then stored at 270 C until assayed. Immunoblot assay. Activation of MAPK occurs by phosphorylation of specific tyrosine and threonine residues. Phospho-specific MAPK anti-

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bodies 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 mg) 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 (TMAPK; 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.

Results

Preliminary studies used the immunoprecipitation/substrate assay and in-gel kinase renaturation assay (32) and showed that GnRH stimulated activation of both MAPK isoforms (ERK-1 and -2) to a similar degree. These results in primary pituitary cells differ from data in gonadotropederived a 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 doseresponsive 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. As we have previously shown that maximal increases in gonadotropin subunit mRNA responses vs. those in vehiclepulsed controls are seen with GnRH pulse amplitudes less than 100 ng/pulse in adult male rats (3), a 50-ng pulse dose was used for subsequent in vivo studies. As shown in Fig. 2, 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 in-

FIG. 1. MAPK responses to alterations in the GnRH pulse dose. Male rats received a single pulse of GnRH (30, 100, or 300 ng) or BSA-saline (Con), and pituitaries were collected 4 min later. Activated P-MAPK and T-MAPK were measured by immunoblot. The upper panel shows the autoradiograph of p42 (ERK-2) bands for P-MAPK and T-MAPK. The lower panel shows alterations in P-MAPK levels, corrected to T-MAPK for each sample. The data are presented as the percent increase (mean 6 SD) vs. controls (n 5 3/group, except for the 100-ng dose, in which n 5 2). *, P , 0.05 vs. controls.

crease, 4 min after the pulse) was similar to that of the response to the higher (300 ng) pulse dose in Fig. 1. To determine whether a pulsatile signal pattern is required to maintain MAPK responsiveness to GnRH over longer durations, groups of animals were given pulses of GnRH every 60 min and compared with animals that received a continuous GnRH infusion for 1– 8 h. The results reveal (Fig. 3) 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. As it was established that a pulsatile signal was required to maintain MAPK responsiveness to GnRH, further studies were conducted to determine whether MAPK is also sensitive to alterations in pulse frequency. GnRH pulses were given every 30, 60, or 120 min for 8 h, and MAPK activity was measured 4 min after the last pulse. As shown in Fig. 4, similar increases in MAPK were seen after 30- or 60-min pulses (2 fold; P , 0.05 vs. controls). However, slower (120min) interval pulses were more effective in increasing MAPK activity than faster pulses (P , 0.05 vs. 30- or 60-min pulse groups). In light of the in vivo findings described above, which showed that MAPK activity is regulated by alterations in GnRH signal pattern, further studies were conducted to determine whether GnRH-induced increases in MAPK activa-


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FIG. 2. Time-course effects of a single pulse of GnRH on pituitary MAPK activity. Groups of male rats received a single GnRH pulse (50 ng) and were killed 4, 10, or 30 min later [controls (C) were killed 4 min after a single pulse of BSA-saline], and pituitary MAPK activity was measured (see Fig. 1 for details). The mean 6 SEM are shown (n 5 4/group). *, P , 0.05 vs. controls.

tion play a role in the regulation of gonadotrope (a, LHb, FSHb, 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 mm), 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. To determine whether longer durations of PD treatment might compromise cell viability and confound data relating to putative inhibitory actions on gonadotrope mRNA expression, cells were cultured with 50 mm PD (or vehicle) in the medium. Twenty-four hours later, the cells were washed and put into fresh medium containing GnRH (1 ng/ml), PACAP (45 ng/ml), or medium only. Results revealed that both GnRH (which uses Ca21/PKC pathways) (12, 13) and PACAP (Ca21/cAMP) (33) stimulated a significant increase (2- to 11-fold; P , 0.05 vs. medium-vehicle controls) in LH secretion over the subsequent 24 h (Table 1). 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. In preliminary studies we determined that PD did not alter basal gonadotrope mRNA concentrations or gonadotropin secretion over 24 h in perifused pituitary cells (data not shown). Figure 6 shows the effect of PD on GnRH-induced increases in gonadotrope mRNAs. The GnRH treatment par-

FIG. 3. MAPK responses to pulsatile or continuous GnRH after 1, 2, 4, or 8 h. GnRH was administered to adult male rats either in a pulsatile (50 ng/pulse, 60-min interval) or continuous manner [25 ng/min; this 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). Controls (C) received BSA-saline pulses or a continuous infusion for 8 h. The mean 6 SEM are shown (n 5 3/group, except in the pulsatile control group, in which n 5 4). *, P , 0.05 vs. controls.

adigm 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 a (91% increase vs. controls), LHb (52% increase), FSHb (51% increase), and GnRH-R (56% increase) mRNAs. The addition of PD to the perifusion medium completely suppressed the a, FSHb, and GnRH-R mRNA responses to pulsatile GnRH. Of note, PD had no effect on the GnRH-induced increase in LHb mRNA. These results suggest that MAPK plays a critical role in GnRH stimulation of a, FSHb, and GnRH-R (but not LHb) gene expression. Perifusate samples (10-min fractions collected for a period of 60 min) were obtained during the initial (second to third hour of treatment) and later (22–23 h of treatment) portions of the perifusion study, and results are shown in Fig. 7. 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

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TABLE 1. LH secretory responses in pituitary cells in vitro to GnRH (1 ng/ml) or PACAP (45 ng/ml) after 24 h of pretreatment with PD (50 mM) or vehicle

Medium 1 vehicle Medium 1 PD GnRH 1 vehicle GnRH 1 PD PACAP 1 vehicle PACAP 1 PD

4h

8h

24 h

100 6 10 111 6 5 192 6 5a 212 6 9a 242 6 54a 187 6 13a

100 6 19 150 6 13 289 6 40a 291 6 41a 577 6 140a 388 6 49a

100 6 5 142 6 21 274 6 32a 257 6 42a 1071 6 83a 1131 6 12a

Results are presented as the percent change vs. medium plus vehicle control values for the same timepoint (see Materials and Methods for experimental details). The mean 6 SEM are shown (n 5 3/group). Control LH levels were 33 6 3 ng/ml (4 h), 15 6 3 (8 h), and 14 6 1 (24 h). a P , 0.05 vs. medium plus vehicle controls.

FIG. 4. The effect of GnRH pulse frequency on pituitary MAPK activity. Groups of male rats received GnRH pulses (50 ng) at 30-, 60-, or 120-min intervals for 8 h. Controls (Con) received BSA-saline pulses. n 5 3/group, except in the control group, in which n 5 4. *, P , 0.05 vs. controls; **, P , 0.05 vs. 30- and 60-min GnRH groups.

FIG. 5. PD suppression of GnRH-induced increases in MAPK activity in vitro. Cultured male pituitary cells were treated with PD (or vehicle) for 60 min before initiation of GnRH treatment (1 ng/ml, for 5 or 60 min). n 5 4/group. *, P , 0.05 vs. appropriate vehicle-treated group; **, P , 0.05 vs. controls.

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. Discussion

Recent studies have shown that GnRH stimulates the activation of MAPK in primary pituitary cells or gonadotropederived a T-3 cells (18 –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 a, FSHb, and GnRH-R mRNA in response to pulsatile GnRH, but the LHb response was not altered. These results strongly suggest that the MEK/MAPK pathway plays a critical role in the GnRH signal transduction pathway for a, FSHb, and GnRH-R gene expression. The data also suggest that GnRH stimulation of LHb 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 Ca21 also participate in GnRH-induced activation of MAPK in a T-3 cells. We and others have shown that both Ca21 and PKC pathways regulate gonadotrope gene expression in primary pituitary cells (16, 17, 34, 35). More specifically, increasing intracellular Ca21 stimulates all four gonadotrope mRNAs studied (16, 34). In contrast, the actions of PKC appear to be more selective, with PKC activation increasing a and LHb mRNAs, but not FSHb (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 LHb mRNA expression uses Ca21 or PKC downstream pathways that are separate from the MAPK system. At present, the findings that GnRH-induced increases in FSHb mRNA are mediated via MAPK activation are difficult to explain in view of data showing that PKC activation [via phorbol 12-myristate 13acetate (PMA) or diacylglycerol pulses] does not stimulate FSHb 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 Ca21) may play a greater role in MAPK activation in rat pituitary cells than is suggested by studies in a T-3 cells (20). A further possibility is that PMA selectively suppresses FSHb mRNA


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FIG. 6. Gonadotrope mRNA responses to pulsatile GnRH (peak chamber concentration, 100 pg/ml) given every 60 min for 24 h in the presence of PD or vehicle in perifused male pituitary cells (n 5 4/group). The data are presented as the percent change vs. medium-pulsed controls (Con). Control mRNA levels were 1.38 6 0.12 fmol bound/100 mg pituitary DNA (a), 0.68 6 0.07 (LHb), 0.84 6 0.08 (FSHb), and 0.17 6 0.01 (GnRH-R). *, P , 0.05 vs. controls.

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 a mRNA expression, rapid to medium pulse frequencies (15- to 30-min intervals) are optimal for LHb, and slower pulses (.60-min intervals) maximally stimulate FSHb (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 FSHb/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 FSHb and GnRH-R genes. The present data confirm GnRH activation of a gene expression through the MAPK pathway (19, 21). As noted above, a 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 a 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 a-subunit gene promoter region, and GnRH increases ELK-1 promoter activity in a 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

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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 a 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 a 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 a-subunit promoter activity in a 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 a, FSHb, and GnRH-R gene expression, but not that of LHb. 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. Acknowledgments

FIG. 7. LH and FSH secretory responses to pulsatile GnRH with or without PD (data from experiment described in Fig. 6). Perifusate samples (10 min/sample) were collected for a period of 60 min at two time points during the study (2 and 22 h of treatment). The arrows indicate the timing of the pulse. The mean 6 SEM are shown. Perifusate LH and FSH levels were significantly greater in GnRH-treated cells (with or without PD) than those in vehicle-pulsed controls (Con) after 2 and 22 h of treatment (P , 0.05). Perifusate FSH levels in the GnRH plus PD group were significantly reduced compared with those in the GnRH alone group (P , 0.05) after 2 and 22 h (as determined by two-way ANOVA).

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 FSHb 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

The authors thank the University of Virginia Center for Cellular and Molecular Studies in Reproduction for providing iodinated LH and FSH, and the National Hormone and Pituitary Program for providing rat LH and FSH RIA reagents and standard preparations.

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