Gonadotropin-Releasing Hormone Pulse Frequency-Dependent Activation of Extracellular Signal-Regulated Kinase Pathways in Perifused LT2 Cells
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《内分泌学杂志》
Division of Endocrinology, Diabetes
Hypertension (H.K., G.Y.B., K.-Y.K., S.X., U.B.K.), Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
Department of Obstetrics and Gynecology (H.K.), Shimane University School of Medicine, Izumo, Shimane 693-8501 Japan
Department of Animal and Poultry Science (G.Y.B.), University of Guelph, Guelph, Ontario, Canada N1G 2W1
Department of Occupational Therapy (K.-Y.K.), Inje University, Gimhae 621-749, South Korea
Abstract
The pattern of GnRH release is associated with differential synthesis and release of LH and FSH. Using a perifusion system, we previously reported that stimulation of the LT2 cell line with varying GnRH pulse frequencies resulted in differential stimulation of LH and FSH gene transcription, analogous to previous observations in primary gonadotropes. In the present study, we investigated the patterns of MAPK activation by GnRH and the role of MAPK in mediating the frequency-dependent effects. In static culture, ERK activation in LT2 cells stimulated with continuous GnRH (10 nM) was maximal by 10 min and persisted for up to 6 h, with a return to basal levels by 20 h. In contrast, stimulation with continuous GnRH (10 nM) in perifused cells resulted in a more sustained activation of ERK. To investigate the effects of GnRH pulse frequency on ERK activation, perifused LT2 cells were stimulated with pulsatile GnRH at a frequency of one pulse every 30 min or one pulse every 2 h for 20 h (10 nM, 5 min/pulse). After the final GnRH pulse, cells were lysed at frequent intervals and levels of ERK phosphorylation were measured. Under high-frequency conditions, ERK activation was maximal 10 min after the GnRH pulse and returned to baseline levels by 20 min. In contrast, under lower GnRH pulse frequency conditions, ERK activation occurred more rapidly and activation was more sustained, with a slower rate of ERK dephosphorylation. These changes resulted in different levels of nuclear phosphorylated ERK. Blockade of ERK activation abolished GnRH-dependent activation of LH and FSH transcription at both high and low pulse frequencies. These results demonstrate that in perifused LT2 cells, distinct patterns of ERK activation/inactivation are regulated by GnRH pulse frequency, and the difference in ERK activation may be important for GnRH pulse frequency-dependent differential stimulation of LH and FSH gene expression.
Introduction
GnRH, A HYPOTHALAMIC decapeptide, is released into the hypophyseal portal vascular system in a pulsatile manner to serve as a major regulator of the reproductive system by stimulating the synthesis and release of the gonadotropins LH and FSH from anterior pituitary gonadotropes (1). In vivo, the patterns of GnRH pulses vary physiologically as a function of hormonal status and reproductive cycle stage (2, 3). Changes in the frequency of GnRH pulse signals have been shown to differentially regulate gonadotropin subunit gene expression and LH and FSH release (4, 5, 6). In rat and primate models, one pulse per hour of GnRH sustains gonadotropin synthesis and secretion. More rapid frequencies of GnRH pulses increase the secretion of LH, whereas slower frequencies result in a decline in LH secretion but a rise in FSH secretion (7). LH gene expression is maximally stimulated by a GnRH pulse interval of 30 min. In contrast, FSH gene expression is optimally stimulated by a slower GnRH pulse frequency, every 2 h (4, 5, 6, 8). Alterations of other gonadotropin genes, including GnRH receptor (GnRHR), follistatin, and inhibin subunits, have been also observed in response to varying GnRH pulse frequencies (6, 9, 10).
The GnRHR is a member of the seven-transmembrane G protein-coupled receptor family, which upon binding GnRH stimulates an increase in phosphoinositide turnover and in diacylglycerol levels, both of which ultimately lead to increased intracellular Ca2+ concentrations and activation of protein kinase C (11, 12). As a result, GnRH activates members of the MAPK family, including ERK (13, 14), c-Jun kinase (JNK) (14, 15), and p38 MAPK (14, 16) as well as cAMP/protein kinase A (17) and calcium/calmodulin-dependent kinase pathways (18). Previous studies have demonstrated the involvement of ERK pathways in GnRH-induced -subunit (13, 19, 20), LH (14, 21, 22), and FSH (23) gene expression in pituitary cells. JNK has also been reported to be involved in LH (15, 21) and FSH expression (24). In addition, p38 MAPK has also been reported to regulate FSH (24). However, most of these studies have focused on the effects of continuous GnRH in static culture.
Few studies have addressed signaling evoked by the more physiological pulsatile GnRH stimulus. Haisenleder et al. (18, 25, 26, 27) demonstrated a role for calcium and calcium/calmodulin-dependent kinase in the induction of gonadotropin gene transcription by pulsatile GnRH, and a role for ERK activation has also been suggested in FSH- and LH-subunit gene expression induced by pulsatile GnRH (28). However, the intracellular signal transduction pathways that mediate the frequency-dependent differential effects of pulsatile GnRH on gonadotropin subunit gene expression have not been fully elucidated.
The development of immortalized murine pituitary gonadotrope-derived cell models such as T3-1 and LT2 cells has facilitated the study of the signal-transduction pathways activated by GnRHR (29, 30). In particular, the LT2 cell line expresses the -, LH-, and FSH-subunits as well as the GnRHR and synthesizes and releases LH and FSH in response to GnRH stimulation. In the present study, using LT2 cells exposed to different frequencies of pulsatile GnRH in perifusion, we have determined the kinetic profile of MAPK activation and investigated the involvement of the ERK pathway in GnRH pulse frequency-dependent regulation of gonadotropin gene expression.
Materials and Methods
Cell culture and perifusion system
The perifusion system used for our study was designed and validated in our laboratory and has been described previously (31). Briefly, LT2 cells were plated in perifusion chambers mounted on glass slides (32) previously coated with Matrigel (Becton Dickinson and Co. Labware, Bedford, MA) and then incubated for 24 h in static culture in high-glucose DMEM containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 C in a humidified atmosphere of 5% CO2 in air. The chambers were then mounted in the perifusion system and continuously perifused with high-glucose DMEM containing 1% heat-inactivated FBS and 1% penicillin-streptomycin at a constant flow rate of 0.25 ml/min. During perifusion, cells were treated with either medium alone, continuous GnRH, or pulsatile GnRH at varying frequencies (one pulse every 30 min or one pulse every 2 h). Up to 12 chambers were run simultaneously. GnRH pulses were delivered by a set of peristaltic pumps controlled by a time controller (Chrontrol XT; Chrontrol Corp., San Diego, CA). A concentration of 10–8 M GnRH and pulse duration of 5 min were chosen, based on previous studies (31, 32). Cells were perifused for a total of 20 h, and GnRH stimulation was performed as indicated.
In static culture experiments, LT2 cells were plated in 35-mm tissue culture dishes and incubated in high-glucose DMEM containing 10% heat-inactivated FBS and 1% penicillin-streptomycin at 37 C in a humidified atmosphere of 5% CO2 in air (33). After 24 h, culture medium was changed to high-glucose DMEM containing 1% heat-inactivated FBS and 1% penicillin-streptomycin and incubated for an additional 20 h without (control) or with 10–8 M GnRH for the indicated times.
All cell culture reagents were supplied by Life Technologies, Inc. (Gaithersburg, MD), and GnRH was supplied by Sigma Chemical Co. (St. Louis, MO).
Experimental paradigms
The experimental paradigms used for these studies are depicted in Fig. 1.
Continuous GnRH.
In static culture studies, LT2 cells were plated in six-well plates. GnRH (10 nM) was added to the culture dish for the indicated times (10 min to 20 h) before cell harvest (Fig. 1A). In perifusion studies, LT2 cells were plated in chambers, mounted in the perifusion system as described above, and perifused continuously for 20 h. GnRH (10 nM) was added to the perifusion medium for the indicated times (10 min to 20 h) before cell harvest (Fig. 1A).
Pulsatile GnRH.
LT2 cells were plated in perifusion chambers and mounted in the perifusion system as described above. Cells were stimulated with GnRH (10 nM) at a frequency of one pulse every 30 min (defined as high-frequency pulse rate) or one pulse every 2 h (defined as low-frequency pulse rate) for 20 h. These pulse frequencies were chosen based on previous studies indicating that these frequencies were optimal for LH gene expression and LH secretion and FSH gene expression and FSH secretion, respectively (4, 5, 6, 7, 31). For reference samples, LT2 cells were maintained in the perifusion chambers and perifused with medium only for 20 h, followed by stimulation with a single pulse of GnRH (10 nM). Cells were harvested every 5–10 min for up to 50 min after the pulse of GnRH occurring at 20 h (Fig. 1B).
Western blot analysis
After chambers were disconnected, cells were rinsed twice with PBS and then lysed on ice with RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 30 mg/ml aprotinin, and 1 mM sodium orthovanadate and sonicated for 20 sec followed by centrifugation at 14,000 x g at 4 C. Alternatively, where indicated, nuclear extracts were prepared as previously described (34, 35). Protein content was measured in the cell lysate or nuclear extract, and 20 μg denatured protein per well was separated on a 10% SDS-PAGE gel according to standard protocols. Proteins were transferred onto polyvinylidene fluoride membranes (Immobilon-P; Millipore Corp., Bedford, MA), which were blocked overnight at 4 C in Blotto [Tris-buffered saline (TBS) with 4.5% milk]. Membranes were incubated with anti-phosphorylated ERK (anti-P-ERK) antibody or anti-early growth response protein-1 (anti-Egr-1) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in Blotto overnight at 4 C and washed three times for 10 min each with TBS/0.05% Tween 20 and once for 10 min with TBS. A subsequent incubation with a monoclonal horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) was carried out for 1 h at room temperature in Blotto, and the appropriate additional washes were performed. After chemiluminescence detection (Pierce Chemical Co., Rockford, IL), membranes were exposed onto x-ray film (Kodak, Rochester, NY). After strip washing (Restore buffer; Pierce), membranes were probed with anti-ERK antibody or anti-upstream stimulating factor-1 (USF-1) antibody (Santa Cruz Biotechnology) overnight at 4 C, followed by incubation with horseradish peroxidase-conjugated secondary antibody as described above. Films were analyzed by densitometry, and the intensities of P-ERK bands were normalized to those of total ERK to correct for protein loading in the case of cellular lysates, whereas the intensities of P-ERK and Egr-1 bands were normalized to those of USF-1 to correct for protein loading in the case of nuclear extracts. Corrected results were expressed as fold of unstimulated control samples. Each experiment was repeated at least three times.
Plasmids and transfections
The reporter constructs used were generated by fusing –797/+5 of rat LH gene (LHLuc) and –2000/+698 of rat FSH gene (FSHLuc) to the firefly luciferase cDNA in pXP2, as previously described (36). LT2 cells were transiently transfected by electroporation (36) with 2 μg/chamber of reporter construct (LHLuc or FSHLuc) and 1 μg/chamber of an SV40--galactosidase expression vector, and plated in Matrigel-coated perifusion chambers. After 24 h of static culture at 37 C in a humidified atmosphere, chambers were mounted in the perifusion system and perifused as described.
Luciferase assays
At the end of each perifusion experiment with transfected cells, chambers were disconnected and cells were rinsed twice with ice-cold PBS and lysed with 125 mM Tris-HCl/0.5% Triton. Cell debris was pelleted by centrifugation at 14,000 x g for 10 min at 4 C, and luciferase and -galactosidase activities were measured in the supernatants as previously described (36). Luciferase activity was normalized to -galactosidase activity to correct for transfection efficiency and cell number, and the results are expressed as fold increase compared with the unstimulated control groups.
MAPK kinase (MEK) inhibitor studies
To study the effects of ERK pathway inhibition, the MEK inhibitor U0126 was used (37). First, to confirm the inhibitory effects of U0126 on pulsatile GnRH-stimulated ERK phosphorylation, cells were stimulated with pulsatile GnRH in perifusion as described above, in the presence or absence of 5 μM U0126 for 20 h. Ten minutes after the last GnRH pulse, cell chambers were disconnected and samples were assayed for ERK phosphorylation by Western blot analysis. For luciferase experiments, LT2 cells cotransfected with LHLuc or FSHLuc and SV40--galactosidase were cultured in the perifusion chambers and stimulated by high frequency (one pulse every 30 min) or low frequency (one pulse every 2 h) pulsatile GnRH in the presence or absence of U0126 for 20 h, after which cells were lysed and luciferase and -galactosidase assays were performed.
Statistical analysis
All studies were performed in at least three independent experiments, each performed with triplicate (in luciferase assays) or duplicate (in Western blot analyses) samples in each experimental group. Results from the independent experiments were analyzed by two-way ANOVA on repeated measurement followed by a Tukey-Kramer multiple-comparison post hoc test using Instat 3.0 (GraphPad Software, Inc., San Diego, CA). The curves in Fig. 4D were generated as cubic spline curves using Prism Software (GraphPad). The results in Fig. 6 were analyzed by Student’s t test to compare the effects of the two GnRH pulse frequencies.
Results
Activation of ERK by continuous GnRH
Previous studies have shown that GnRH can activate both ERK isoforms (ERK1 and ERK2), but little is known about the pattern of activation in response to different paradigms of GnRH exposure. We first compared the pattern of ERK activation in LT2 cells in response to continuous GnRH stimulation in static culture and in perifusion. LT2 cells were cultured under static conditions, and 10 nM GnRH was added directly to the cell culture dish for the indicated times (10 min to 20 h) before cell harvest and collection of cell lysates. ERK activation, determined by Western blot analysis, was significantly increased, by 3.7 ± 1.2-fold (P < 0.01), after 10 min of GnRH stimulation, compared with unstimulated cells (Fig. 2A). Increased ERK phosphorylation was sustained for 6 h but returned to baseline levels by 20 h. In comparison, when perifused LT2 cells were exposed to continuous GnRH for similar time intervals, ERK phosphorylation was increased to a similar degree as in static culture (4.9 ± 1.1-fold) (Fig. 2B). However, activation was more sustained and remained significantly increased after 20 h of GnRH stimulation, the longest time point examined (3.6 ± 0.9-fold; P < 0.01).
Activation of p38 MAPK by continuous GnRH
ERK was activated in response to continuous GnRH in both static and perifused culture conditions. Next, we examined whether GnRH treatment in static or perifused culture systems activated JNK or p38, additional members of the MAPK family. Previous studies have shown that GnRH can activate both JNK and p38 MAPK in LT2 cells in static culture (15, 24), but the effects of GnRH stimulation in a perifusion system remain unknown. In perifused LT2 cells, p38 MAPK phosphorylation was increased in response to continuous GnRH stimulation, with a significant increase (2.5 ± 0.6-fold) first noted after 30 min (Fig. 3). Levels of phosphorylated p38 continued to increase, reaching a maximum after 2 h of GnRH stimulation. High levels of activation were sustained through 20 h, the longest time point tested. In contrast, in static culture, we did not observe an increase in p38 MAPK phosphorylation by continuous GnRH. Furthermore, no significant activation of JNK was observed in response to continuous GnRH stimulation at any time point studied, in either static or in perifused culture conditions (data not shown).
Activation of ERK by pulsatile GnRH in perifused LT2 cells
Continuous GnRH resulted in an increase in ERK phosphorylation within 10 min that was sustained for at least 6 h in both static and perifused LT2 cells. Because GnRH is released from the hypothalamus in a pulsatile manner in vivo, exposure of the cells to pulsatile GnRH should reflect more closely physiological conditions than does continuous GnRH. Therefore, we next determined ERK activation in response to pulsatile GnRH in perifused LT2 cells. Cells were stimulated with pulsatile GnRH (10 nM, 5 min per pulse), at a frequency of one pulse every 30 min or one pulse every 2 h for 20 h. These pulse frequencies were chosen based on previous studies indicating that these frequencies were optimal for LH gene expression and LH secretion and FSH gene expression and FSH secretion, respectively (4, 5, 6, 7, 31). Cells were collected every 5 or 10 min after the pulse of GnRH administered at 20 h, and ERK phosphorylation was measured by Western blot analysis.
Interestingly, the patterns of ERK activation in response to pulsatile GnRH stimulation were different compared with those observed in response to continuous GnRH. After a pulse of GnRH, ERK phosphorylation was rapidly increased (within 5–10 min) but was not sustained as it was in response to continuous GnRH (Fig. 4). After exposure to pulsatile GnRH at high frequency (one pulse every 30 min), ERK phosphorylation increased to a maximum of 2.6 ± 0.3-fold at 10 min, and then levels rapidly decreased, returning to baseline levels within 20–30 min (Fig. 4, A and D). A similar pattern of ERK activation was seen in response to the subsequent pulse of GnRH. In contrast, a different pattern of ERK activation was observed in response to pulsatile GnRH at low frequency (one pulse every 2 h), with more rapid phosphorylation of ERK, reaching a maximum of 2.8 ± 0.3-fold by 5 min after the GnRH pulse (Fig. 4, B and D). Moreover, this increase was more sustained, remaining elevated until the 20-min time point and then slowly decreasing, returning to baseline levels by 40–50 min. As a result, 30 min after the GnRH pulse, ERK phosphorylation was still significantly increased (1.5 ± 0.2-fold) in cells treated with the slow GnRH pulse frequency, compared with levels 30 min after a pulse of GnRH in cells exposed to the high GnRH pulse frequency (Fig. 4D). These experiments were replicated at least three times for each pulse frequency, with duplicate samples for each data point in each experiment, with similar results.
For comparison, LT2 cells were perifused for 20 h with medium only, followed by stimulation with a single pulse of GnRH. Under these conditions, ERK phosphorylation was increased transiently by GnRH, with a maximal level increased by 1.9 ± 0.1-fold after 10 min, and levels of P-ERK returned to baseline within 30 min. Interestingly, the amplitude of this increase in ERK phosphorylation was less than that observed in cells treated with pulsatile GnRH.
As the patterns of ERK phosphorylation in response to pulsatile GnRH administered at high and low frequencies were distinct (Fig. 4), we directly compared levels of ERK phosphorylation in cells previously stimulated with high- vs. low-frequency pulses at each time point after a pulse of GnRH. After 20 h of perifusion, no difference in basal ERK activity (i.e. immediately before the 20-h pulse of GnRH) was observed between cells stimulated with high or low GnRH pulse frequencies (Fig. 5A). By 5 min after the final GnRH pulse, ERK phosphorylation was significantly higher in cells previously exposed to low-frequency GnRH pulses (every 2 h) compared with cells treated with the high GnRH pulse frequency (Fig. 5B). Although stimulation of ERK phosphorylation was more rapid in response to low-frequency GnRH pulses, the maximal levels of ERK phosphorylation, reached after 10 min, were not different between the two pulse frequencies (Fig. 5C). By 15 min after the GnRH pulse, levels of ERK phosphorylation were again significantly higher in the cells exposed to low-frequency GnRH pulses (every 2 h) compared with cells treated with high-frequency GnRH pulses (every 30 min).
To determine whether the changes in the patterns of ERK activation in response to pulsatile GnRH administered at high and low frequencies result in differences in levels of activated ERK in the nucleus, we measured the levels of nuclear P-ERK in response to the varying GnRH pulse frequencies (Fig. 6A). We measured levels of nuclear P-ERK by Western blot analysis 15 min after the last GnRH pulse. We selected this time point because levels of total cellular P-ERK were different in response to the different GnRH pulse frequencies at this time point. The level of nuclear P-ERK was significantly greater (P < 0.05) after exposure of cells to low-frequency GnRH pulses (every 2 h; 8.1 ± 1.2-fold) compared with cells exposed to high-frequency GnRH pulses (every 30 min; 3.2 ± 0.5-fold).
We also investigated regulation of nuclear Egr-1 levels by pulsatile GnRH (Fig. 6B). Egr-1, a member of the immediate early gene family (38), is known to be induced by GnRH and to play a central role in the induction of LH gene transcription by pulsatile GnRH (39, 40, 41). We compared the levels of nuclear Egr-1 induced after administration of high and low frequencies of pulsatile GnRH. Interestingly, Egr-1 levels were significantly higher (P < 0.05) in response to the higher GnRH pulse frequency (every 30 min; 81.0 ± 12.5-fold) compared with the lower frequency (every 2 h; 27.1 ± 6.5-fold). This pattern of Egr-1 induction is consistent with the pattern of frequency-dependent LH gene regulation, but is in contrast to the absolute levels of nuclear P-ERK measured.
Taken together, these data indicate that ERK activation occurred more rapidly and inactivation occurred more slowly after the slower frequency of GnRH pulses, resulting in a more sustained pattern of ERK activation, in contrast to a more transient pattern of ERK activation in response to the higher GnRH pulse frequency. These differences in ERK activation patterns resulted in higher levels of nuclear P-ERK accumulation in response to the slower frequency of GnRH pulses.
Effects of a MEK inhibitor on GnRH-stimulated ERK phosphorylation
The effects of a MEK inhibitor, U0126, on ERK phosphorylation in response to pulsatile GnRH were examined. LT2 cells were perifused and exposed to high- and low-frequency GnRH pulses for 20 h in the presence or absence of 5 μM U0126. Samples were collected 10 min after the final pulse of GnRH (shown in our studies to be the time of peak ERK phosphorylation) and assayed for levels of ERK phosphorylation. Activation of ERK by GnRH was almost completely eliminated in the presence of U0126 at both GnRH pulse frequencies (Fig. 7). Basal levels of ERK phosphorylation were also reduced in the presence of U0126, suggesting some intrinsic level of ERK activation in perifused LT2 cells.
Effects of a MEK inhibitor on stimulation of LH and FSH gene transcription by pulsatile GnRH
Our results show that the pattern of ERK activation/inactivation in response to pulsatile GnRH was distinct and specific for each GnRH pulse frequency. These findings parallel our previous report of the frequency-dependent pattern of stimulation of LH and FSH gene transcription by pulsatile GnRH (31). Previous studies have demonstrated that MEK inhibition can inhibit GnRH-induced LH and FSH promoter activities in static culture (21, 23). However, the effects of ERK inhibition on the stimulation of LH and FSH promoter activities by varying frequencies of pulsatile GnRH have not been studied. We therefore evaluated the effects of the MEK inhibitor, U0126, on frequency-dependent activation of LH- and FSH-subunit gene transcription by pulsatile GnRH. LT2 cells were transfected with LHLuc or FSHLuc, maintained in static culture for 24 h to allow cell adherence to the culture chambers, and then perifused for 20 h, treated with or without GnRH pulses every 30 min or every 2 h in the presence or absence of 5 μM U0126, a concentration shown to be effective in preventing ERK phosphorylation (37) (Fig. 7). In the absence of U0126, LHLuc activity was stimulated to a greater extent at the higher GnRH pulse frequency (Fig. 8A, left), whereas FSHLuc activity was preferentially stimulated at the lower GnRH pulse frequency (Fig 8B, left), consistent with our previous report (31). In the presence of 5 μM U0126, however, stimulation of LHLuc activity by pulsatile GnRH was markedly reduced at both pulse frequencies, and pulse frequency dependence was no longer evident (Fig. 8A, right). Similarly, in the presence of U0126, stimulation of FSHLuc activity by pulsatile GnRH was also markedly reduced at both pulse frequencies and pulse frequency dependence of FSHLuc was also abrogated (Fig. 8B, right). These results suggest that the MEK inhibitor U0126 inhibits GnRH-induced activation of both LH and FSH gene transcription at both pulse frequencies tested.
Discussion
The gonadotropins, LH and FSH, play an integral role in regulated hormonal output to achieve normal sexual development and gonadal function. Prolonged exposure of the pituitary to GnRH desensitizes the gonadotropes, resulting in decreased LH and FSH secretion (42). In contrast, pulsatile GnRH induces follicular development and ovulation by increasing LH and FSH levels (43). Furthermore, it is well established that the frequency of GnRH pulses changes during the menstrual cycle. In the late follicular phase, the GnRH pulse frequency increases to culminate in the preovulatory LH surge, whereas it slows in the late luteal and early follicular phase, correlating with higher FSH levels, which permit follicle development (3, 44). The purpose of this study was to determine the effects of different patterns of GnRH stimulation on activation of signal transduction pathways in gonadotropes to provide insight into the differential regulation of LH and FSH by GnRH. In these studies, we have used the gonadotrope-derived LT2 cell line as a model for gonadotropes. These cells have been shown to possess many characteristics of mature gonadotropes, including expression of -subunit, LH, FSH, and GnRHR (30, 33, 45, 46) and frequency-dependent differential regulation of LH and FSH gene expression by pulsatile GnRH (31).
Several studies have been performed in recent years to evaluate the contribution of various signal transduction pathways to the regulation of -subunit, LH, and FSH gene expression by GnRH, and the importance of the MAPK families was emphasized in a number of these reports. Three members of the MAPK family, ERK, JNK, and p38 MAPK have been shown to be activated by GnRH in LT2 cells (14, 24). ERK activation has been shown to be necessary for induction of -subunit, LH, and FSH gene expression (13, 14, 19, 21, 22), JNK for LH and FSH (15, 21, 24), and p38 MAPK for FSH (24). These data were obtained in experiments in which cells were stimulated with GnRH followed by assays for kinase activity, studies of the effects of specific inhibitors on GnRH-stimulated -subunit, LH, and FSH gene expression, and the use dominant negative forms of kinases. However, these experiments were performed in static culture; that is, cells were stimulated by continuous exposure to GnRH added to cell cultures for various time intervals. Our objective was to compare the activation of MAPK signaling pathways by different patterns of GnRH stimulation and, in particular, by varying frequencies of pulsatile GnRH.
In previous studies, stimulation of ERK phosphorylation by continuous GnRH in LT2 cells in static culture was shown to peak within 10 min and then gradually decline (14, 21). However, ERK phosphorylation had not yet returned to baseline levels by 2 h, the longest time point examined. In our study, continuous GnRH increased ERK phosphorylation rapidly by 10 min, similar to the previous results, but interestingly, this activation persisted up to 6 h (Fig. 2A). Moreover, exposure of the cells to continuous GnRH in the perifusion system resulted in persistent increases in ERK phosphorylation for up to 20 h, the longest time point examined. The reasons for this lack of desensitization are not known. We previously observed similar effects on -, LH-, and FSH-subunit gene promoter activity; all three gonadotropin subunit genes were stimulated by continuous GnRH in our perifusion system (31). These results suggest that desensitization of GnRH receptor and/or postreceptor signaling pathways that has been observed in primary pituitary gonadotropes does not occur in the same way in LT2 cells, particularly in perifusion. Consistent with our observations, Liu et al. (47) reported that ERK activation in LT2 cells was still detectable after up to 24 h of GnRH treatment in static culture but did report loss of GnRH responsiveness after 48 h of continuous GnRH exposure. ERK is inactivated by dephosphorylation by members of a unique family of dual-specificity protein phosphatases, the MAPK phosphatases (MKPs), which therefore serve as important intracellular negative regulators of MAPK signaling cascades. It has been previously shown that MKP-2 is induced by GnRH in both primary gonadotropes and in T3-1 cells (48). We have similarly observed induction of MKP-2 by GnRH in LT2 cells (data not shown), suggesting that the pathway for ERK inactivation may be intact in LT2 cells and that the persistent elevations in levels of P-ERK may be the result of ongoing stimulation of ERK activation. We can also speculate that GnRH-stimulated factors may be secreted by the cells and may contribute to autocrine/paracrine desensitization in static culture. These factors would be removed by the constant flow of medium in the perifusion system, thereby allowing continued activation of ERK.
In our studies, neither JNK nor p38 MAPK were activated by continuous GnRH in static culture. These results are in contrast to previous reports (14, 24), in which modest and delayed (relative to ERK) activation of JNK and p38 MAPK were observed in response to continuous GnRH. Nonetheless, we did observe increases in phosphorylation of p38 MAPK by continuous GnRH in perifusion. These increases were slow and sustained, with maximal levels of activation not reached until after 2 h of GnRH stimulation and persisting until 20 h, the longest time point examined. These results again indicate a lack of desensitization to continuous GnRH stimulation in the perifusion system.
The activation pattern of ERK in response to pulsatile GnRH was distinct from that of continuous GnRH. Pulsatile GnRH stimulated ERK phosphorylation rapidly, but this activation was decreased much more rapidly after each GnRH pulse, compared with the more sustained activation by continuous GnRH. As a result, levels of phosphorylation returned to baseline before the subsequent GnRH pulse. These results suggest that ERK is activated in response to GnRH occupation of its receptor, and because this receptor signaling is not prolonged, it is likely that GnRH binding to its receptor has only transient effects on downstream signaling events. Because GnRH release occurs in a pulsatile fashion in vivo, it is clear that GnRH-induced ERK activation is not continuous in pituitary gonadotropes.
To our knowledge, this is the first report demonstrating different patterns of ERK activation in response to different frequencies of pulsatile GnRH. We and others have previously reported differential regulation of LH and FSH gene expression by different GnRH pulse frequencies (4, 5, 6, 10), and we have demonstrated that LH gene promoter activity is stimulated to a greater extent at higher GnRH pulse frequencies, whereas the FSH gene is preferentially stimulated at lower pulse frequencies in LT2 cells (31). Our results are consistent with those of Haisenleder et al. (28), who showed induction of pituitary MAPK activity by pulsatile GnRH in vivo in an adult castrated, testosterone-replaced male rat model. The lack of any significant differences in basal levels of ERK phosphorylation between GnRH pulses in the two pulse frequencies studied indicates that changes in basal ERK activity are not involved in the determination of the frequency-dependent regulation of gonadotropin gene expression. The maximal activation of ERK was also not different between pulse frequencies, indicating that neither basal ERK activity nor absolute levels of ERK phosphorylation dictate the specificity for pulse frequency-dependent gonadotropin gene expression in LT2 cells. As shown in Fig. 4, the kinetics of ERK phosphorylation in response to low and high pulse frequencies were distinct from each other. Low-frequency GnRH pulses led to more rapid and sustained phosphorylation of ERK, whereas high-frequency pulses showed a more transient pattern of ERK phosphorylation. The distinct frequency-dependent patterns of activation of ERK by pulsatile GnRH raise the possibility that these distinct patterns dictate the specificity of frequency-dependent activation of LH and FSH gene expression. Indeed, inhibition of ERK activation by the MEK inhibitor U0126 markedly decreased GnRH-induced activation of both LH and FSH gene transcription by pulsatile GnRH at both high (every 30 min) and low (every 2 h) GnRH pulse frequencies (Fig. 7), confirming the importance of ERK activation as a mediator of GnRH-induced activation of both LH and FSH gene transcription at both pulse frequencies tested. These results suggest that GnRH-induced ERK activation plays an important role in stimulation of both LH and FSH gene expression by pulsatile GnRH and support our hypothesis that the kinetic pattern of ERK activation may be an important mediator of GnRH pulse frequency-dependent regulation of gonadotropin gene expression.
The observation that different patterns of ERK activation can have differential cellular effects is not unprecedented. For example, in PC12 cells, epidermal growth factor transiently stimulates ERK activation and provokes cellular proliferation. In contrast, nerve growth factor leads to sustained ERK activation and induces neuronal differentiation (49). Similarly, blockade of rapid vs. prolonged ERK activation has differential effects on insulin-induced gene expression (50).
The importance of calcium signals in dictating frequency-dependent gonadotropin gene expression has also been reported previously (18, 26, 27). Similar to GnRH, high-frequency pulsatile calcium signals have been shown to increase LH gene expression to a greater extent compared with low-frequency calcium pulses, whereas FSH gene expression was increased by low-frequency calcium pulses (26, 27). Given the evidence that calcium signaling itself regulates ERK phosphorylation (51, 52), it is possible that calcium pulse frequency-dependent gonadotropin gene expression may be mediated by differences in ERK activation patterns. On the other hand, others have shown that GnRH-induced ERK activation in static culture is only partially dependent on calcium influx, through non-L-type calcium channels (14). The precise role of calcium signaling in mediating GnRH pulse frequency-dependent ERK activation remains to be determined.
We have not directly addressed the issue of whether it is the duration or periodicity of ERK pulses that are causal in terms of the differential effects on LH and FSH gene transcription. Nonetheless, to begin to evaluate the effects of the different patterns of ERK activation on gene transcription, the level of nuclear P-ERK localization in response to the varying GnRH pulse frequencies was measured (Fig. 6). The level of nuclear P-ERK was greater after a pulse of GnRH in the cells exposed to low GnRH pulse frequency (every 2 h) than in the cells exposed to high-frequency GnRH pulses (every 30 min). This finding is consistent with the frequency-dependent change in the pattern/kinetics of ERK activation such that total cellular P-ERK was increased for a longer period of time after the slower GnRH pulse frequency, allowing more P-ERK translocation to the nucleus even though the maximal P-ERK concentrations were similar at both pulse frequencies. We speculate that the more sustained activation of ERK leads to higher levels of nuclear P-ERK, which in turn results in activation of transcription factors that lead to increases in FSH gene transcription. These transcription factors have not yet been fully characterized in the case of the rat FSH gene promoter studied here, although data suggest that an activator protein-1 site is important for GnRH responsiveness of the murine FSH gene (53). In contrast, the important role of Egr-1 in mediating GnRH stimulation of rat LH gene promoter activity has been well characterized (39, 40, 41). Interestingly, Egr-1 levels were higher in response to the higher GnRH pulse frequency (every 30 min) compared with the lower frequency (every 2 h). This pattern of Egr-1 induction is consistent with the pattern of frequency-dependent LH gene regulation, i.e. greater at the higher GnRH pulse frequencies, but is in contrast to the levels of nuclear P-ERK. These data suggest that it may be the kinetic pattern of P-ERK activation rather than the absolute levels that determines the levels of Egr-1 induction. Alternatively, the higher frequency of ERK activation after the higher GnRH pulse frequency may play a greater role in the stimulation of Egr-1 and hence LH transcription than the absolute levels of P-ERK or the pattern of P-ERK induction. Finally, the possibility that other or additional signaling pathways lead to the induction of Egr-1 in response to pulsatile GnRH needs to be considered as well.
In summary, in the present study, we have shown that the pattern of GnRH administration results in different patterns of ERK activation in LT2 cells, with distinct responses to continuous GnRH in static culture, continuous GnRH in perifusion, and pulsatile GnRH. Moreover, the pattern of ERK activation by pulsatile GnRH demonstrated frequency dependence, with a more sustained pattern of activation in response to a slower pulse frequency compared with a more transient activation pattern in response to a higher GnRH pulse frequency. The potential significance of these distinct frequency-dependent patterns of ERK activation in dictating the differential stimulation of gonadotropin gene expression by varying frequencies of pulsatile GnRH is supported by the abrogation of GnRH-stimulated LH and FSH gene transcription in the presence of an inhibitor of ERK activation at both high and low frequencies of GnRH pulses.
Acknowledgments
We thank Dr. Pamela Mellon for generously providing LT2 cells.
Footnotes
This work was supported by National Institute of Child Health and Human Development/National Institutes of Health (NIH) through cooperative agreement U54 HD28138 as part of the Specialized Cooperative Centers Program in Reproduction Research (to U.B.K.), NIH R01 HD33001 (to U.B.K.), NIH T32 DK 007529-20 (to S.X.), and the Lalor Foundation (to G.Y.B.).
First Published Online September 1, 2005
Abbreviations: Egr-1, Early growth response protein-1; FBS, fetal bovine serum; GnRHR, GnRH receptor; JNK, c-Jun kinase; MEK, MAPK kinase; MKP, MAPK phosphatase; P-ERK, phosphorylated ERK; TBS, Tris-buffered saline.
Accepted for publication August 22, 2005.
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Hypertension (H.K., G.Y.B., K.-Y.K., S.X., U.B.K.), Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
Department of Obstetrics and Gynecology (H.K.), Shimane University School of Medicine, Izumo, Shimane 693-8501 Japan
Department of Animal and Poultry Science (G.Y.B.), University of Guelph, Guelph, Ontario, Canada N1G 2W1
Department of Occupational Therapy (K.-Y.K.), Inje University, Gimhae 621-749, South Korea
Abstract
The pattern of GnRH release is associated with differential synthesis and release of LH and FSH. Using a perifusion system, we previously reported that stimulation of the LT2 cell line with varying GnRH pulse frequencies resulted in differential stimulation of LH and FSH gene transcription, analogous to previous observations in primary gonadotropes. In the present study, we investigated the patterns of MAPK activation by GnRH and the role of MAPK in mediating the frequency-dependent effects. In static culture, ERK activation in LT2 cells stimulated with continuous GnRH (10 nM) was maximal by 10 min and persisted for up to 6 h, with a return to basal levels by 20 h. In contrast, stimulation with continuous GnRH (10 nM) in perifused cells resulted in a more sustained activation of ERK. To investigate the effects of GnRH pulse frequency on ERK activation, perifused LT2 cells were stimulated with pulsatile GnRH at a frequency of one pulse every 30 min or one pulse every 2 h for 20 h (10 nM, 5 min/pulse). After the final GnRH pulse, cells were lysed at frequent intervals and levels of ERK phosphorylation were measured. Under high-frequency conditions, ERK activation was maximal 10 min after the GnRH pulse and returned to baseline levels by 20 min. In contrast, under lower GnRH pulse frequency conditions, ERK activation occurred more rapidly and activation was more sustained, with a slower rate of ERK dephosphorylation. These changes resulted in different levels of nuclear phosphorylated ERK. Blockade of ERK activation abolished GnRH-dependent activation of LH and FSH transcription at both high and low pulse frequencies. These results demonstrate that in perifused LT2 cells, distinct patterns of ERK activation/inactivation are regulated by GnRH pulse frequency, and the difference in ERK activation may be important for GnRH pulse frequency-dependent differential stimulation of LH and FSH gene expression.
Introduction
GnRH, A HYPOTHALAMIC decapeptide, is released into the hypophyseal portal vascular system in a pulsatile manner to serve as a major regulator of the reproductive system by stimulating the synthesis and release of the gonadotropins LH and FSH from anterior pituitary gonadotropes (1). In vivo, the patterns of GnRH pulses vary physiologically as a function of hormonal status and reproductive cycle stage (2, 3). Changes in the frequency of GnRH pulse signals have been shown to differentially regulate gonadotropin subunit gene expression and LH and FSH release (4, 5, 6). In rat and primate models, one pulse per hour of GnRH sustains gonadotropin synthesis and secretion. More rapid frequencies of GnRH pulses increase the secretion of LH, whereas slower frequencies result in a decline in LH secretion but a rise in FSH secretion (7). LH gene expression is maximally stimulated by a GnRH pulse interval of 30 min. In contrast, FSH gene expression is optimally stimulated by a slower GnRH pulse frequency, every 2 h (4, 5, 6, 8). Alterations of other gonadotropin genes, including GnRH receptor (GnRHR), follistatin, and inhibin subunits, have been also observed in response to varying GnRH pulse frequencies (6, 9, 10).
The GnRHR is a member of the seven-transmembrane G protein-coupled receptor family, which upon binding GnRH stimulates an increase in phosphoinositide turnover and in diacylglycerol levels, both of which ultimately lead to increased intracellular Ca2+ concentrations and activation of protein kinase C (11, 12). As a result, GnRH activates members of the MAPK family, including ERK (13, 14), c-Jun kinase (JNK) (14, 15), and p38 MAPK (14, 16) as well as cAMP/protein kinase A (17) and calcium/calmodulin-dependent kinase pathways (18). Previous studies have demonstrated the involvement of ERK pathways in GnRH-induced -subunit (13, 19, 20), LH (14, 21, 22), and FSH (23) gene expression in pituitary cells. JNK has also been reported to be involved in LH (15, 21) and FSH expression (24). In addition, p38 MAPK has also been reported to regulate FSH (24). However, most of these studies have focused on the effects of continuous GnRH in static culture.
Few studies have addressed signaling evoked by the more physiological pulsatile GnRH stimulus. Haisenleder et al. (18, 25, 26, 27) demonstrated a role for calcium and calcium/calmodulin-dependent kinase in the induction of gonadotropin gene transcription by pulsatile GnRH, and a role for ERK activation has also been suggested in FSH- and LH-subunit gene expression induced by pulsatile GnRH (28). However, the intracellular signal transduction pathways that mediate the frequency-dependent differential effects of pulsatile GnRH on gonadotropin subunit gene expression have not been fully elucidated.
The development of immortalized murine pituitary gonadotrope-derived cell models such as T3-1 and LT2 cells has facilitated the study of the signal-transduction pathways activated by GnRHR (29, 30). In particular, the LT2 cell line expresses the -, LH-, and FSH-subunits as well as the GnRHR and synthesizes and releases LH and FSH in response to GnRH stimulation. In the present study, using LT2 cells exposed to different frequencies of pulsatile GnRH in perifusion, we have determined the kinetic profile of MAPK activation and investigated the involvement of the ERK pathway in GnRH pulse frequency-dependent regulation of gonadotropin gene expression.
Materials and Methods
Cell culture and perifusion system
The perifusion system used for our study was designed and validated in our laboratory and has been described previously (31). Briefly, LT2 cells were plated in perifusion chambers mounted on glass slides (32) previously coated with Matrigel (Becton Dickinson and Co. Labware, Bedford, MA) and then incubated for 24 h in static culture in high-glucose DMEM containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 C in a humidified atmosphere of 5% CO2 in air. The chambers were then mounted in the perifusion system and continuously perifused with high-glucose DMEM containing 1% heat-inactivated FBS and 1% penicillin-streptomycin at a constant flow rate of 0.25 ml/min. During perifusion, cells were treated with either medium alone, continuous GnRH, or pulsatile GnRH at varying frequencies (one pulse every 30 min or one pulse every 2 h). Up to 12 chambers were run simultaneously. GnRH pulses were delivered by a set of peristaltic pumps controlled by a time controller (Chrontrol XT; Chrontrol Corp., San Diego, CA). A concentration of 10–8 M GnRH and pulse duration of 5 min were chosen, based on previous studies (31, 32). Cells were perifused for a total of 20 h, and GnRH stimulation was performed as indicated.
In static culture experiments, LT2 cells were plated in 35-mm tissue culture dishes and incubated in high-glucose DMEM containing 10% heat-inactivated FBS and 1% penicillin-streptomycin at 37 C in a humidified atmosphere of 5% CO2 in air (33). After 24 h, culture medium was changed to high-glucose DMEM containing 1% heat-inactivated FBS and 1% penicillin-streptomycin and incubated for an additional 20 h without (control) or with 10–8 M GnRH for the indicated times.
All cell culture reagents were supplied by Life Technologies, Inc. (Gaithersburg, MD), and GnRH was supplied by Sigma Chemical Co. (St. Louis, MO).
Experimental paradigms
The experimental paradigms used for these studies are depicted in Fig. 1.
Continuous GnRH.
In static culture studies, LT2 cells were plated in six-well plates. GnRH (10 nM) was added to the culture dish for the indicated times (10 min to 20 h) before cell harvest (Fig. 1A). In perifusion studies, LT2 cells were plated in chambers, mounted in the perifusion system as described above, and perifused continuously for 20 h. GnRH (10 nM) was added to the perifusion medium for the indicated times (10 min to 20 h) before cell harvest (Fig. 1A).
Pulsatile GnRH.
LT2 cells were plated in perifusion chambers and mounted in the perifusion system as described above. Cells were stimulated with GnRH (10 nM) at a frequency of one pulse every 30 min (defined as high-frequency pulse rate) or one pulse every 2 h (defined as low-frequency pulse rate) for 20 h. These pulse frequencies were chosen based on previous studies indicating that these frequencies were optimal for LH gene expression and LH secretion and FSH gene expression and FSH secretion, respectively (4, 5, 6, 7, 31). For reference samples, LT2 cells were maintained in the perifusion chambers and perifused with medium only for 20 h, followed by stimulation with a single pulse of GnRH (10 nM). Cells were harvested every 5–10 min for up to 50 min after the pulse of GnRH occurring at 20 h (Fig. 1B).
Western blot analysis
After chambers were disconnected, cells were rinsed twice with PBS and then lysed on ice with RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 30 mg/ml aprotinin, and 1 mM sodium orthovanadate and sonicated for 20 sec followed by centrifugation at 14,000 x g at 4 C. Alternatively, where indicated, nuclear extracts were prepared as previously described (34, 35). Protein content was measured in the cell lysate or nuclear extract, and 20 μg denatured protein per well was separated on a 10% SDS-PAGE gel according to standard protocols. Proteins were transferred onto polyvinylidene fluoride membranes (Immobilon-P; Millipore Corp., Bedford, MA), which were blocked overnight at 4 C in Blotto [Tris-buffered saline (TBS) with 4.5% milk]. Membranes were incubated with anti-phosphorylated ERK (anti-P-ERK) antibody or anti-early growth response protein-1 (anti-Egr-1) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in Blotto overnight at 4 C and washed three times for 10 min each with TBS/0.05% Tween 20 and once for 10 min with TBS. A subsequent incubation with a monoclonal horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) was carried out for 1 h at room temperature in Blotto, and the appropriate additional washes were performed. After chemiluminescence detection (Pierce Chemical Co., Rockford, IL), membranes were exposed onto x-ray film (Kodak, Rochester, NY). After strip washing (Restore buffer; Pierce), membranes were probed with anti-ERK antibody or anti-upstream stimulating factor-1 (USF-1) antibody (Santa Cruz Biotechnology) overnight at 4 C, followed by incubation with horseradish peroxidase-conjugated secondary antibody as described above. Films were analyzed by densitometry, and the intensities of P-ERK bands were normalized to those of total ERK to correct for protein loading in the case of cellular lysates, whereas the intensities of P-ERK and Egr-1 bands were normalized to those of USF-1 to correct for protein loading in the case of nuclear extracts. Corrected results were expressed as fold of unstimulated control samples. Each experiment was repeated at least three times.
Plasmids and transfections
The reporter constructs used were generated by fusing –797/+5 of rat LH gene (LHLuc) and –2000/+698 of rat FSH gene (FSHLuc) to the firefly luciferase cDNA in pXP2, as previously described (36). LT2 cells were transiently transfected by electroporation (36) with 2 μg/chamber of reporter construct (LHLuc or FSHLuc) and 1 μg/chamber of an SV40--galactosidase expression vector, and plated in Matrigel-coated perifusion chambers. After 24 h of static culture at 37 C in a humidified atmosphere, chambers were mounted in the perifusion system and perifused as described.
Luciferase assays
At the end of each perifusion experiment with transfected cells, chambers were disconnected and cells were rinsed twice with ice-cold PBS and lysed with 125 mM Tris-HCl/0.5% Triton. Cell debris was pelleted by centrifugation at 14,000 x g for 10 min at 4 C, and luciferase and -galactosidase activities were measured in the supernatants as previously described (36). Luciferase activity was normalized to -galactosidase activity to correct for transfection efficiency and cell number, and the results are expressed as fold increase compared with the unstimulated control groups.
MAPK kinase (MEK) inhibitor studies
To study the effects of ERK pathway inhibition, the MEK inhibitor U0126 was used (37). First, to confirm the inhibitory effects of U0126 on pulsatile GnRH-stimulated ERK phosphorylation, cells were stimulated with pulsatile GnRH in perifusion as described above, in the presence or absence of 5 μM U0126 for 20 h. Ten minutes after the last GnRH pulse, cell chambers were disconnected and samples were assayed for ERK phosphorylation by Western blot analysis. For luciferase experiments, LT2 cells cotransfected with LHLuc or FSHLuc and SV40--galactosidase were cultured in the perifusion chambers and stimulated by high frequency (one pulse every 30 min) or low frequency (one pulse every 2 h) pulsatile GnRH in the presence or absence of U0126 for 20 h, after which cells were lysed and luciferase and -galactosidase assays were performed.
Statistical analysis
All studies were performed in at least three independent experiments, each performed with triplicate (in luciferase assays) or duplicate (in Western blot analyses) samples in each experimental group. Results from the independent experiments were analyzed by two-way ANOVA on repeated measurement followed by a Tukey-Kramer multiple-comparison post hoc test using Instat 3.0 (GraphPad Software, Inc., San Diego, CA). The curves in Fig. 4D were generated as cubic spline curves using Prism Software (GraphPad). The results in Fig. 6 were analyzed by Student’s t test to compare the effects of the two GnRH pulse frequencies.
Results
Activation of ERK by continuous GnRH
Previous studies have shown that GnRH can activate both ERK isoforms (ERK1 and ERK2), but little is known about the pattern of activation in response to different paradigms of GnRH exposure. We first compared the pattern of ERK activation in LT2 cells in response to continuous GnRH stimulation in static culture and in perifusion. LT2 cells were cultured under static conditions, and 10 nM GnRH was added directly to the cell culture dish for the indicated times (10 min to 20 h) before cell harvest and collection of cell lysates. ERK activation, determined by Western blot analysis, was significantly increased, by 3.7 ± 1.2-fold (P < 0.01), after 10 min of GnRH stimulation, compared with unstimulated cells (Fig. 2A). Increased ERK phosphorylation was sustained for 6 h but returned to baseline levels by 20 h. In comparison, when perifused LT2 cells were exposed to continuous GnRH for similar time intervals, ERK phosphorylation was increased to a similar degree as in static culture (4.9 ± 1.1-fold) (Fig. 2B). However, activation was more sustained and remained significantly increased after 20 h of GnRH stimulation, the longest time point examined (3.6 ± 0.9-fold; P < 0.01).
Activation of p38 MAPK by continuous GnRH
ERK was activated in response to continuous GnRH in both static and perifused culture conditions. Next, we examined whether GnRH treatment in static or perifused culture systems activated JNK or p38, additional members of the MAPK family. Previous studies have shown that GnRH can activate both JNK and p38 MAPK in LT2 cells in static culture (15, 24), but the effects of GnRH stimulation in a perifusion system remain unknown. In perifused LT2 cells, p38 MAPK phosphorylation was increased in response to continuous GnRH stimulation, with a significant increase (2.5 ± 0.6-fold) first noted after 30 min (Fig. 3). Levels of phosphorylated p38 continued to increase, reaching a maximum after 2 h of GnRH stimulation. High levels of activation were sustained through 20 h, the longest time point tested. In contrast, in static culture, we did not observe an increase in p38 MAPK phosphorylation by continuous GnRH. Furthermore, no significant activation of JNK was observed in response to continuous GnRH stimulation at any time point studied, in either static or in perifused culture conditions (data not shown).
Activation of ERK by pulsatile GnRH in perifused LT2 cells
Continuous GnRH resulted in an increase in ERK phosphorylation within 10 min that was sustained for at least 6 h in both static and perifused LT2 cells. Because GnRH is released from the hypothalamus in a pulsatile manner in vivo, exposure of the cells to pulsatile GnRH should reflect more closely physiological conditions than does continuous GnRH. Therefore, we next determined ERK activation in response to pulsatile GnRH in perifused LT2 cells. Cells were stimulated with pulsatile GnRH (10 nM, 5 min per pulse), at a frequency of one pulse every 30 min or one pulse every 2 h for 20 h. These pulse frequencies were chosen based on previous studies indicating that these frequencies were optimal for LH gene expression and LH secretion and FSH gene expression and FSH secretion, respectively (4, 5, 6, 7, 31). Cells were collected every 5 or 10 min after the pulse of GnRH administered at 20 h, and ERK phosphorylation was measured by Western blot analysis.
Interestingly, the patterns of ERK activation in response to pulsatile GnRH stimulation were different compared with those observed in response to continuous GnRH. After a pulse of GnRH, ERK phosphorylation was rapidly increased (within 5–10 min) but was not sustained as it was in response to continuous GnRH (Fig. 4). After exposure to pulsatile GnRH at high frequency (one pulse every 30 min), ERK phosphorylation increased to a maximum of 2.6 ± 0.3-fold at 10 min, and then levels rapidly decreased, returning to baseline levels within 20–30 min (Fig. 4, A and D). A similar pattern of ERK activation was seen in response to the subsequent pulse of GnRH. In contrast, a different pattern of ERK activation was observed in response to pulsatile GnRH at low frequency (one pulse every 2 h), with more rapid phosphorylation of ERK, reaching a maximum of 2.8 ± 0.3-fold by 5 min after the GnRH pulse (Fig. 4, B and D). Moreover, this increase was more sustained, remaining elevated until the 20-min time point and then slowly decreasing, returning to baseline levels by 40–50 min. As a result, 30 min after the GnRH pulse, ERK phosphorylation was still significantly increased (1.5 ± 0.2-fold) in cells treated with the slow GnRH pulse frequency, compared with levels 30 min after a pulse of GnRH in cells exposed to the high GnRH pulse frequency (Fig. 4D). These experiments were replicated at least three times for each pulse frequency, with duplicate samples for each data point in each experiment, with similar results.
For comparison, LT2 cells were perifused for 20 h with medium only, followed by stimulation with a single pulse of GnRH. Under these conditions, ERK phosphorylation was increased transiently by GnRH, with a maximal level increased by 1.9 ± 0.1-fold after 10 min, and levels of P-ERK returned to baseline within 30 min. Interestingly, the amplitude of this increase in ERK phosphorylation was less than that observed in cells treated with pulsatile GnRH.
As the patterns of ERK phosphorylation in response to pulsatile GnRH administered at high and low frequencies were distinct (Fig. 4), we directly compared levels of ERK phosphorylation in cells previously stimulated with high- vs. low-frequency pulses at each time point after a pulse of GnRH. After 20 h of perifusion, no difference in basal ERK activity (i.e. immediately before the 20-h pulse of GnRH) was observed between cells stimulated with high or low GnRH pulse frequencies (Fig. 5A). By 5 min after the final GnRH pulse, ERK phosphorylation was significantly higher in cells previously exposed to low-frequency GnRH pulses (every 2 h) compared with cells treated with the high GnRH pulse frequency (Fig. 5B). Although stimulation of ERK phosphorylation was more rapid in response to low-frequency GnRH pulses, the maximal levels of ERK phosphorylation, reached after 10 min, were not different between the two pulse frequencies (Fig. 5C). By 15 min after the GnRH pulse, levels of ERK phosphorylation were again significantly higher in the cells exposed to low-frequency GnRH pulses (every 2 h) compared with cells treated with high-frequency GnRH pulses (every 30 min).
To determine whether the changes in the patterns of ERK activation in response to pulsatile GnRH administered at high and low frequencies result in differences in levels of activated ERK in the nucleus, we measured the levels of nuclear P-ERK in response to the varying GnRH pulse frequencies (Fig. 6A). We measured levels of nuclear P-ERK by Western blot analysis 15 min after the last GnRH pulse. We selected this time point because levels of total cellular P-ERK were different in response to the different GnRH pulse frequencies at this time point. The level of nuclear P-ERK was significantly greater (P < 0.05) after exposure of cells to low-frequency GnRH pulses (every 2 h; 8.1 ± 1.2-fold) compared with cells exposed to high-frequency GnRH pulses (every 30 min; 3.2 ± 0.5-fold).
We also investigated regulation of nuclear Egr-1 levels by pulsatile GnRH (Fig. 6B). Egr-1, a member of the immediate early gene family (38), is known to be induced by GnRH and to play a central role in the induction of LH gene transcription by pulsatile GnRH (39, 40, 41). We compared the levels of nuclear Egr-1 induced after administration of high and low frequencies of pulsatile GnRH. Interestingly, Egr-1 levels were significantly higher (P < 0.05) in response to the higher GnRH pulse frequency (every 30 min; 81.0 ± 12.5-fold) compared with the lower frequency (every 2 h; 27.1 ± 6.5-fold). This pattern of Egr-1 induction is consistent with the pattern of frequency-dependent LH gene regulation, but is in contrast to the absolute levels of nuclear P-ERK measured.
Taken together, these data indicate that ERK activation occurred more rapidly and inactivation occurred more slowly after the slower frequency of GnRH pulses, resulting in a more sustained pattern of ERK activation, in contrast to a more transient pattern of ERK activation in response to the higher GnRH pulse frequency. These differences in ERK activation patterns resulted in higher levels of nuclear P-ERK accumulation in response to the slower frequency of GnRH pulses.
Effects of a MEK inhibitor on GnRH-stimulated ERK phosphorylation
The effects of a MEK inhibitor, U0126, on ERK phosphorylation in response to pulsatile GnRH were examined. LT2 cells were perifused and exposed to high- and low-frequency GnRH pulses for 20 h in the presence or absence of 5 μM U0126. Samples were collected 10 min after the final pulse of GnRH (shown in our studies to be the time of peak ERK phosphorylation) and assayed for levels of ERK phosphorylation. Activation of ERK by GnRH was almost completely eliminated in the presence of U0126 at both GnRH pulse frequencies (Fig. 7). Basal levels of ERK phosphorylation were also reduced in the presence of U0126, suggesting some intrinsic level of ERK activation in perifused LT2 cells.
Effects of a MEK inhibitor on stimulation of LH and FSH gene transcription by pulsatile GnRH
Our results show that the pattern of ERK activation/inactivation in response to pulsatile GnRH was distinct and specific for each GnRH pulse frequency. These findings parallel our previous report of the frequency-dependent pattern of stimulation of LH and FSH gene transcription by pulsatile GnRH (31). Previous studies have demonstrated that MEK inhibition can inhibit GnRH-induced LH and FSH promoter activities in static culture (21, 23). However, the effects of ERK inhibition on the stimulation of LH and FSH promoter activities by varying frequencies of pulsatile GnRH have not been studied. We therefore evaluated the effects of the MEK inhibitor, U0126, on frequency-dependent activation of LH- and FSH-subunit gene transcription by pulsatile GnRH. LT2 cells were transfected with LHLuc or FSHLuc, maintained in static culture for 24 h to allow cell adherence to the culture chambers, and then perifused for 20 h, treated with or without GnRH pulses every 30 min or every 2 h in the presence or absence of 5 μM U0126, a concentration shown to be effective in preventing ERK phosphorylation (37) (Fig. 7). In the absence of U0126, LHLuc activity was stimulated to a greater extent at the higher GnRH pulse frequency (Fig. 8A, left), whereas FSHLuc activity was preferentially stimulated at the lower GnRH pulse frequency (Fig 8B, left), consistent with our previous report (31). In the presence of 5 μM U0126, however, stimulation of LHLuc activity by pulsatile GnRH was markedly reduced at both pulse frequencies, and pulse frequency dependence was no longer evident (Fig. 8A, right). Similarly, in the presence of U0126, stimulation of FSHLuc activity by pulsatile GnRH was also markedly reduced at both pulse frequencies and pulse frequency dependence of FSHLuc was also abrogated (Fig. 8B, right). These results suggest that the MEK inhibitor U0126 inhibits GnRH-induced activation of both LH and FSH gene transcription at both pulse frequencies tested.
Discussion
The gonadotropins, LH and FSH, play an integral role in regulated hormonal output to achieve normal sexual development and gonadal function. Prolonged exposure of the pituitary to GnRH desensitizes the gonadotropes, resulting in decreased LH and FSH secretion (42). In contrast, pulsatile GnRH induces follicular development and ovulation by increasing LH and FSH levels (43). Furthermore, it is well established that the frequency of GnRH pulses changes during the menstrual cycle. In the late follicular phase, the GnRH pulse frequency increases to culminate in the preovulatory LH surge, whereas it slows in the late luteal and early follicular phase, correlating with higher FSH levels, which permit follicle development (3, 44). The purpose of this study was to determine the effects of different patterns of GnRH stimulation on activation of signal transduction pathways in gonadotropes to provide insight into the differential regulation of LH and FSH by GnRH. In these studies, we have used the gonadotrope-derived LT2 cell line as a model for gonadotropes. These cells have been shown to possess many characteristics of mature gonadotropes, including expression of -subunit, LH, FSH, and GnRHR (30, 33, 45, 46) and frequency-dependent differential regulation of LH and FSH gene expression by pulsatile GnRH (31).
Several studies have been performed in recent years to evaluate the contribution of various signal transduction pathways to the regulation of -subunit, LH, and FSH gene expression by GnRH, and the importance of the MAPK families was emphasized in a number of these reports. Three members of the MAPK family, ERK, JNK, and p38 MAPK have been shown to be activated by GnRH in LT2 cells (14, 24). ERK activation has been shown to be necessary for induction of -subunit, LH, and FSH gene expression (13, 14, 19, 21, 22), JNK for LH and FSH (15, 21, 24), and p38 MAPK for FSH (24). These data were obtained in experiments in which cells were stimulated with GnRH followed by assays for kinase activity, studies of the effects of specific inhibitors on GnRH-stimulated -subunit, LH, and FSH gene expression, and the use dominant negative forms of kinases. However, these experiments were performed in static culture; that is, cells were stimulated by continuous exposure to GnRH added to cell cultures for various time intervals. Our objective was to compare the activation of MAPK signaling pathways by different patterns of GnRH stimulation and, in particular, by varying frequencies of pulsatile GnRH.
In previous studies, stimulation of ERK phosphorylation by continuous GnRH in LT2 cells in static culture was shown to peak within 10 min and then gradually decline (14, 21). However, ERK phosphorylation had not yet returned to baseline levels by 2 h, the longest time point examined. In our study, continuous GnRH increased ERK phosphorylation rapidly by 10 min, similar to the previous results, but interestingly, this activation persisted up to 6 h (Fig. 2A). Moreover, exposure of the cells to continuous GnRH in the perifusion system resulted in persistent increases in ERK phosphorylation for up to 20 h, the longest time point examined. The reasons for this lack of desensitization are not known. We previously observed similar effects on -, LH-, and FSH-subunit gene promoter activity; all three gonadotropin subunit genes were stimulated by continuous GnRH in our perifusion system (31). These results suggest that desensitization of GnRH receptor and/or postreceptor signaling pathways that has been observed in primary pituitary gonadotropes does not occur in the same way in LT2 cells, particularly in perifusion. Consistent with our observations, Liu et al. (47) reported that ERK activation in LT2 cells was still detectable after up to 24 h of GnRH treatment in static culture but did report loss of GnRH responsiveness after 48 h of continuous GnRH exposure. ERK is inactivated by dephosphorylation by members of a unique family of dual-specificity protein phosphatases, the MAPK phosphatases (MKPs), which therefore serve as important intracellular negative regulators of MAPK signaling cascades. It has been previously shown that MKP-2 is induced by GnRH in both primary gonadotropes and in T3-1 cells (48). We have similarly observed induction of MKP-2 by GnRH in LT2 cells (data not shown), suggesting that the pathway for ERK inactivation may be intact in LT2 cells and that the persistent elevations in levels of P-ERK may be the result of ongoing stimulation of ERK activation. We can also speculate that GnRH-stimulated factors may be secreted by the cells and may contribute to autocrine/paracrine desensitization in static culture. These factors would be removed by the constant flow of medium in the perifusion system, thereby allowing continued activation of ERK.
In our studies, neither JNK nor p38 MAPK were activated by continuous GnRH in static culture. These results are in contrast to previous reports (14, 24), in which modest and delayed (relative to ERK) activation of JNK and p38 MAPK were observed in response to continuous GnRH. Nonetheless, we did observe increases in phosphorylation of p38 MAPK by continuous GnRH in perifusion. These increases were slow and sustained, with maximal levels of activation not reached until after 2 h of GnRH stimulation and persisting until 20 h, the longest time point examined. These results again indicate a lack of desensitization to continuous GnRH stimulation in the perifusion system.
The activation pattern of ERK in response to pulsatile GnRH was distinct from that of continuous GnRH. Pulsatile GnRH stimulated ERK phosphorylation rapidly, but this activation was decreased much more rapidly after each GnRH pulse, compared with the more sustained activation by continuous GnRH. As a result, levels of phosphorylation returned to baseline before the subsequent GnRH pulse. These results suggest that ERK is activated in response to GnRH occupation of its receptor, and because this receptor signaling is not prolonged, it is likely that GnRH binding to its receptor has only transient effects on downstream signaling events. Because GnRH release occurs in a pulsatile fashion in vivo, it is clear that GnRH-induced ERK activation is not continuous in pituitary gonadotropes.
To our knowledge, this is the first report demonstrating different patterns of ERK activation in response to different frequencies of pulsatile GnRH. We and others have previously reported differential regulation of LH and FSH gene expression by different GnRH pulse frequencies (4, 5, 6, 10), and we have demonstrated that LH gene promoter activity is stimulated to a greater extent at higher GnRH pulse frequencies, whereas the FSH gene is preferentially stimulated at lower pulse frequencies in LT2 cells (31). Our results are consistent with those of Haisenleder et al. (28), who showed induction of pituitary MAPK activity by pulsatile GnRH in vivo in an adult castrated, testosterone-replaced male rat model. The lack of any significant differences in basal levels of ERK phosphorylation between GnRH pulses in the two pulse frequencies studied indicates that changes in basal ERK activity are not involved in the determination of the frequency-dependent regulation of gonadotropin gene expression. The maximal activation of ERK was also not different between pulse frequencies, indicating that neither basal ERK activity nor absolute levels of ERK phosphorylation dictate the specificity for pulse frequency-dependent gonadotropin gene expression in LT2 cells. As shown in Fig. 4, the kinetics of ERK phosphorylation in response to low and high pulse frequencies were distinct from each other. Low-frequency GnRH pulses led to more rapid and sustained phosphorylation of ERK, whereas high-frequency pulses showed a more transient pattern of ERK phosphorylation. The distinct frequency-dependent patterns of activation of ERK by pulsatile GnRH raise the possibility that these distinct patterns dictate the specificity of frequency-dependent activation of LH and FSH gene expression. Indeed, inhibition of ERK activation by the MEK inhibitor U0126 markedly decreased GnRH-induced activation of both LH and FSH gene transcription by pulsatile GnRH at both high (every 30 min) and low (every 2 h) GnRH pulse frequencies (Fig. 7), confirming the importance of ERK activation as a mediator of GnRH-induced activation of both LH and FSH gene transcription at both pulse frequencies tested. These results suggest that GnRH-induced ERK activation plays an important role in stimulation of both LH and FSH gene expression by pulsatile GnRH and support our hypothesis that the kinetic pattern of ERK activation may be an important mediator of GnRH pulse frequency-dependent regulation of gonadotropin gene expression.
The observation that different patterns of ERK activation can have differential cellular effects is not unprecedented. For example, in PC12 cells, epidermal growth factor transiently stimulates ERK activation and provokes cellular proliferation. In contrast, nerve growth factor leads to sustained ERK activation and induces neuronal differentiation (49). Similarly, blockade of rapid vs. prolonged ERK activation has differential effects on insulin-induced gene expression (50).
The importance of calcium signals in dictating frequency-dependent gonadotropin gene expression has also been reported previously (18, 26, 27). Similar to GnRH, high-frequency pulsatile calcium signals have been shown to increase LH gene expression to a greater extent compared with low-frequency calcium pulses, whereas FSH gene expression was increased by low-frequency calcium pulses (26, 27). Given the evidence that calcium signaling itself regulates ERK phosphorylation (51, 52), it is possible that calcium pulse frequency-dependent gonadotropin gene expression may be mediated by differences in ERK activation patterns. On the other hand, others have shown that GnRH-induced ERK activation in static culture is only partially dependent on calcium influx, through non-L-type calcium channels (14). The precise role of calcium signaling in mediating GnRH pulse frequency-dependent ERK activation remains to be determined.
We have not directly addressed the issue of whether it is the duration or periodicity of ERK pulses that are causal in terms of the differential effects on LH and FSH gene transcription. Nonetheless, to begin to evaluate the effects of the different patterns of ERK activation on gene transcription, the level of nuclear P-ERK localization in response to the varying GnRH pulse frequencies was measured (Fig. 6). The level of nuclear P-ERK was greater after a pulse of GnRH in the cells exposed to low GnRH pulse frequency (every 2 h) than in the cells exposed to high-frequency GnRH pulses (every 30 min). This finding is consistent with the frequency-dependent change in the pattern/kinetics of ERK activation such that total cellular P-ERK was increased for a longer period of time after the slower GnRH pulse frequency, allowing more P-ERK translocation to the nucleus even though the maximal P-ERK concentrations were similar at both pulse frequencies. We speculate that the more sustained activation of ERK leads to higher levels of nuclear P-ERK, which in turn results in activation of transcription factors that lead to increases in FSH gene transcription. These transcription factors have not yet been fully characterized in the case of the rat FSH gene promoter studied here, although data suggest that an activator protein-1 site is important for GnRH responsiveness of the murine FSH gene (53). In contrast, the important role of Egr-1 in mediating GnRH stimulation of rat LH gene promoter activity has been well characterized (39, 40, 41). Interestingly, Egr-1 levels were higher in response to the higher GnRH pulse frequency (every 30 min) compared with the lower frequency (every 2 h). This pattern of Egr-1 induction is consistent with the pattern of frequency-dependent LH gene regulation, i.e. greater at the higher GnRH pulse frequencies, but is in contrast to the levels of nuclear P-ERK. These data suggest that it may be the kinetic pattern of P-ERK activation rather than the absolute levels that determines the levels of Egr-1 induction. Alternatively, the higher frequency of ERK activation after the higher GnRH pulse frequency may play a greater role in the stimulation of Egr-1 and hence LH transcription than the absolute levels of P-ERK or the pattern of P-ERK induction. Finally, the possibility that other or additional signaling pathways lead to the induction of Egr-1 in response to pulsatile GnRH needs to be considered as well.
In summary, in the present study, we have shown that the pattern of GnRH administration results in different patterns of ERK activation in LT2 cells, with distinct responses to continuous GnRH in static culture, continuous GnRH in perifusion, and pulsatile GnRH. Moreover, the pattern of ERK activation by pulsatile GnRH demonstrated frequency dependence, with a more sustained pattern of activation in response to a slower pulse frequency compared with a more transient activation pattern in response to a higher GnRH pulse frequency. The potential significance of these distinct frequency-dependent patterns of ERK activation in dictating the differential stimulation of gonadotropin gene expression by varying frequencies of pulsatile GnRH is supported by the abrogation of GnRH-stimulated LH and FSH gene transcription in the presence of an inhibitor of ERK activation at both high and low frequencies of GnRH pulses.
Acknowledgments
We thank Dr. Pamela Mellon for generously providing LT2 cells.
Footnotes
This work was supported by National Institute of Child Health and Human Development/National Institutes of Health (NIH) through cooperative agreement U54 HD28138 as part of the Specialized Cooperative Centers Program in Reproduction Research (to U.B.K.), NIH R01 HD33001 (to U.B.K.), NIH T32 DK 007529-20 (to S.X.), and the Lalor Foundation (to G.Y.B.).
First Published Online September 1, 2005
Abbreviations: Egr-1, Early growth response protein-1; FBS, fetal bovine serum; GnRHR, GnRH receptor; JNK, c-Jun kinase; MEK, MAPK kinase; MKP, MAPK phosphatase; P-ERK, phosphorylated ERK; TBS, Tris-buffered saline.
Accepted for publication August 22, 2005.
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