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Division of Endocrinology and Metabolism, Department of Medicine, and the Center for Research in Reproduction, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Daniel J. Haisenleder, Aurbach Medical Research Building, P.O. Box 801412, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: djh2q{at}virginia.edu.
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, LHß, and FSHß transcriptional responses to GnRH or Bay K 8644+KCl (BK+KCl) pulses in vitro in the absence or presence of the Ca/CAMK II-specific inhibitor, KN-93. Gonadotropin subunit transcription was assessed by measuring primary transcripts (PTs) by quantitative RT-PCR. In time-course studies, both GnRH and BK+KCl pulses given alone increased all three subunit PTs after 6 h (2- to 4-fold). PT responses to GnRH increased over time (3- to 8-fold over basal at 24 h), although BK+KCl was ineffective after 24 h. KN-93 reduced the LHß and FSHß transcriptional responses to GnRH by 50–60% and completely suppressed the PT response. In contrast, KN-93 showed no inhibitory effects on basal transcriptional activity or LH or FSH secretion. In fact, KN-93 tended to increase basal , LHß, and FSHß PT levels and enhance LH secretory responses to GnRH. These results reveal that Ca/CAMK II plays a central role in the transmission of pulsatile GnRH signals from the plasma membrane to the rat , LHß, and FSHß subunit genes. |
Introduction |
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, LHß and FSHß gene expression at both transcriptional and the steady-state mRNA levels (4, 5, 6). More specifically, rapid- or intermediate-pulse frequencies maximally stimulate expression of the - and LHß genes, and slower-frequency pulses selectively stimulate FSHß. Alterations in GnRH pulse amplitude and frequency can also differentially regulate other gonadotrope genes including the GnRH receptor (GnRHR), follistatin, and inhibin subunits (7, 8, 9). The GnRHR is a member of the seven-transmembrane receptor family, with receptor-binding activating various GTP-binding proteins, including Gq and G11, stimulating increased phosphoinositide turnover within the plasma membrane, cytoplasmic diacylglycerol levels, and the activation of protein kinase C (PKC) (10, 11). GnRHR binding increases intracellular calcium through activation of L-type voltage-sensitive calcium channels and inositol 1,4,5-triphosphate-induced release from intracellular calcium storage pools (12, 13). GnRH has also been shown to activate other intracellular pathways, including cAMP/protein kinase A, ERK, p38, and c-Jun N-terminal kinase (JNK) (14, 15, 16, 17, 18, 19).
Although the intracellular messenger pathways that mediate GnRH pulsatile actions on gonadotropin subunit genes remain to be fully characterized, calcium has been shown to be a major component in the transduction of GnRH signals (10, 12). GnRH-induced increases in intracellular calcium stimulate a rise in gonadotropin subunit transcription or promoter activity, subunit mRNAs, and LH and FSH secretion (13, 20, 21, 22). Calcium plays an essential role in gonadotropin subunit responses to pulsatile GnRH (23), and pulsatile calcium signals (via pulses of the L-type channel activator Bay K 8644) are more effective in stimulating , LHß, and FSHß mRNAs than continuous calcium influx (23). Recently we demonstrated that the frequency of the calcium pulse signal can differentially regulate , LHß, and FSHß transcriptional activity and mRNA expression (22, 24), similar to results seen for pulsatile GnRH (6).
Although data suggest that alterations in intracellular calcium plays a role in the transmission of GnRH pulsatile signals, the downstream third-messenger pathway(s) that mediate GnRH actions on the gonadotrope remain to be fully delineated. Calcium/calmodulin-dependent kinase II (Ca/CAMK II) is an important mediator of calcium signaling in various cell types, including the pituitary (25, 26). Ca/CAMK II plays a role in cross-talk between intracellular messengers by activating adenylate cyclase, calcium ATPase, nitric oxide synthetase, calcineurin, and other enzyme systems (27). Ca/CAMK II has been shown to regulate gene expression and secretion in various tissues (25, 28). In the brain, Ca/CAMK II stimulates c-fos expression and norepinephrine and glutamate secretion (29). In the pituitary, the enzyme stimulates prolactin secretion and promoter activity (26, 30, 31).
Ca/CAMK II is a complex of subunits derived from four genes (, ß, 
, and 
), with - and ß-subunits prominent in the brain and - and -isoforms in other tissues, with each Ca/CAMK II subunit having catalytic activity (25, 28). Ca/calmodulin binding to the regulatory domain of the subunit stimulates activation of Ca/CAMK II by disinhibiting the enzyme and promoting binding of ATP and appropriate peptide substrate (28, 29). Following activation the subunit is autophosphorylated, increasing the affinity for Ca/calmodulin-CAMK II binding and extending the duration of enzyme activation state (29). Studies have shown that Ca/CAMK II autophosphorylation plays an important role in Ca/CAMK II activity (25), including recent findings that reveal that Ca/CAMK II autophosphorylation is required for hippocampal long-term potentiation and spatial learning in the mouse (32). Ca/CAMK II is transported to the nucleus, allowing the enzyme to influence the expression of specific genes by directly activating various transcription factors (28).
The aim of the present studies was to determine whether GnRH stimulates Ca/CAMK II activity in the rat pituitary and whether the enzyme system mediates , LHß, and FSHß transcriptional responses to pulsatile GnRH.
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Materials and Methods |
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In vitro perifusion studies
For each experiment, pituitaries from 36 adult female rats were pooled and dissociated in medium containing 0.35% collagenase, 0.1% hyaluronidase, and 0.01% Dnase. Following dissociation, the cell suspension was aliquoted into 12 culture wells (5–6 x 106 cells per well) containing 22-mm plastic coverslips coated with Matrigel (Becton Dickinson and Co., Bedford, MA). The cells were cultured for 48 h before beginning each experiment. The in vitro procedure and culture medium constituents have previously been described (16, 23). To allow LHß mRNA expression in response to pulsatile GnRH, testosterone [at a concentration present on proestrus, 500 pg/ml (34)] was added during the last 24 h of plating and during perifusion. After plating, the coverslips were inserted into custom-made chambers and allowed to equilibrate for 1 h before initiating treatment. The perifusion flow rate was 200 µl/min, and 100-µl pulses were administered over a 10-sec duration via Autosyringe pumps. Studies were conducted as three to four separate experiments (12 chambers per experiment), with all treatment groups represented in each experiment (three chambers/treatment per experiment). Data from each experiment were expressed as percent change vs. vehicle-pulsed controls.
GnRH pulse studies.
Chambers were given pulses of GnRH (peak chamber concentration = 200 pM, medium pulses to controls) every 60 min for 6–24 h. For some experiments, chambers were perifused with medium containing the water-soluble Ca/CAMK II-specific inhibitor KN-93 (10 µM), the inactive isoform KN-92 (10 µM), or vehicle. LH and FSH secretory responses were determined by collecting 10-min perifusate fractions over 60 min after 2 h and 22 h (for 24-h studies) of treatment. Cells were recovered 5 min after the last pulse; total RNA extracted with guanidinium thiocyanate; and , LHß, and FSHß primary transcripts (PTs) determined by quantitative RT-PCR.
Bay K 8644+KCl pulse studies.
Perifusion chambers received pulses of Bay K 8644 (BK) plus potassium chloride (BK+KCl) [peak chamber concentration = 3 µM (BK) plus 10 mM (KCl)], every 60 min for 6 or 24 h. This treatment paradigm is based on previous data showing that BK is more effective in stimulating pituitary secretion or mRNA responses in the presence of a threshold depolarization concentration of K+ in the medium (23). Control groups received vehicle pulses (0.2% ethanol/medium). Then 10 µM KN-93 (or vehicle) were added to the perifusion medium for some studies. LH and FSH secretory responses to pulse treatments were determined by collecting 10-min perifusate fractions over 60 min between 2 and 3 h of treatment. Five minutes after the final pulse, the cells were recovered; total RNA extracted; and , LHß, and FSHß PTs measured.
Ca/CAMK II immunoblot assay
Cell lysate protein (120 µg) was resolved by electrophoresis (4–20% SDS-PAGE). Protein bands were transferred to nitrocellulose filters and immunoblotted using antibodies specific to phosphorylated - and ß-Ca/CAMK II subunits (P-CAMK II, Promega Corp., Madison, WI), and phosphorylated and unphosphorylated -, ß-, -, and -subunits [total Ca/CAMK II (T-CAMK II), Santa Cruz Biotechnology, Santa Cruz, CA]. The secondary antibody was horseradish peroxidase-conjugated goat antirabbit (Upstate Biotechnology, Lake Placid, NY). Bands were detected using the Super Signal Pico West chemiluminescent system (Pierce Chemical Co., Rockford, IL), followed by autoradiography. P-CAMK II bands was quantitated by densitometry and corrected to the amount of T-CAMK II per sample. Treatment-induced responses were similar for both - and ß-Ca/CAMK II subunits. However, because the intensity of the signal for the ß-subunit was greater than that seen for -Ca/CAMK II subunit, results were quantitated using ß-Ca/CAMK II.
Quantitative RT-PCR
The , LHß, and FSHß PT concentrations were determined by quantitative RT-PCR assay, as previously described (35). The method is based on adding known amounts of a size-altered competitor RNA to a constant amount of pituitary RNA. Briefly, for each subunit, regions of intron/exon were amplified using specific oligonucleotide primers and a size-altered competitive template (CT) RNA for each gene. A four-point standard curve was generated by adding a fixed amount of pituitary RNA (100–400 ng/PCR) to an increasing amount of CT (2, 10, 50, 250 fg). The pituitary and CT RNAs were reverse transcribed, followed by 35 cycles of PCR in the presence of 32P-dCTP. The PCR products were separated by electrophoresis in 3% agarose, DNA bands were excised, and 32P-dCTP incorporation determined by scintillation counting. PT concentrations were expressed as femtomoles/100 µg pituitary RNA. Intraassay coefficients of variation are 8.9% (), 6.7% (LHß), and 5.0% (FSHß); interassay coefficients of variation are 22.2% (), 19.2% (LHß), and 14.1% (FSHß). To reduce the effect of interassay variation, all samples from each experiment were run within a single PCR assay.
RIA
LH and FSH secretory responses were measured in perifusate fractions by RIA, using reagents provided by the National Hormone and Pituitary Program. The assays were performed by the University of Virginia Center for Research in Reproduction, Ligand Assay and Analysis Core Facility. The RIA standards were NIDDK RP-3 (for LH) and RP-2 (for FSH). The assay sensitivities were 0.09 ng/tube for LH and 0.8 ng/tube for FSH. The coefficients of variation were 8.3% and 10.8% for the LH assay and 6.3% and 9.6% for FSH (intraassay and interassay, respectively). All samples from each experiment were assayed in a single RIA.
Statistical analysis
The data were analyzed by one-way ANOVA, with differences between treatment groups determined by Duncan’s multiple range test.
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Results |
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shows the Ca/CAMK II activational response to a single pulse of GnRH in GnRH-deficient rats in vivo, as determined by Ca/CAMK II autophosphorylation. The upper panel presents a representative autoradiograph of the Ca/CAMK II phosphorylation response (P-CAMK II) 2, 5, 15, and 45 min after the GnRH stimulus. As shown in the lower panel, GnRH stimulated a rapid rise in Ca/CAMK II phosphorylation, with a significant increase (84%) seen 2 min after the pulse [P < 0.05 vs. control (Con)]. Peak values were observed 5 min after the GnRH pulse (245% increase vs. Con; P < 0.05), maintained through 15 min and then declined to control levels 45 min after GnRH administration.
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, pulsatile GnRH for 6 h stimulated a 2- to 4-fold increase in , LHß, and FSHß PTs (P < 0.05 vs. Con). However, maximal increases occurred after longer treatment durations, 14 h for (7-fold increase), and 24 h for LHß (5-fold), and FSHß (8-fold). BK+KCl pulses also stimulated subunit PTs, but in contrast to GnRH, significant increases (P < 0.05 vs. Con) were only seen after 6 h of treatment. Also of note, maximal responses for each subunit PT to GnRH were greater than that seen for BK+KCl (P < 0.05).
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, GnRH stimulated increases in (3-fold), LHß, (6-fold) and FSHß (9-fold) PT (P < 0.05 vs. Con). KN-93 suppressed LHß and FSHß PT responses to GnRH by 50–60% and completely blocked the GnRH-induced increase in -PT. Of interest, gonadotropin subunit PTs in pituitary cells treated with KN-93 alone were not suppressed vs. vehicle controls and tended to be elevated, although this did not achieve significance.
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(mean ± SEM = 100 ± 6), LHß (100 ± 18), FSHß (100 ± 17); KN-92: (99 ± 3), LHß (108 ± 16), FSHß (94 ± 12)]. Pulsatile GnRH stimulated a significant increase in LHß and FSHß PTs (P < 0.05 vs. Veh controls) in both Veh-treated and KN-92-treated groups (LHß: Veh+GnRH = 451 ± 88, KN-92+GnRH = 322 ± 67; FSHß: Veh+GnRH = 343 ± 74, KN-92+GnRH = 365 ± 53). LHß and FSHß PT responses to GnRH in Veh and KN-92 treated groups were not significantly different. Of note, in this particular study a GnRH-induced increase in -PT was not seen in either Veh or KN-92 treated groups. This likely is due to the experimental duration used (24 h), which is not optimal for (as shown in Fig. 2
).
The effect of KN-93 on gonadotropin secretory responses to pulsatile GnRH after 2 h and 22 h of treatment in vitro is shown in Fig. 4. LH and FSH in KN-93 controls were not different from Veh controls and are not shown. GnRH stimulated a pulsatile LH and FSH release pattern that continued over 22 h. Although LH and FSH secretory responses to GnRH alone or in combination with KN-93 were similar, LH release to GnRH tended to be greater during the initial phase of the study (2-h time point) in chambers given KN-93. Over the course of the study, LH release remained stable in control chambers. Peak LH levels were 7- to 8-fold above pre-GnRH concentrations. The magnitude of FSH secretory responses to GnRH was lower than that seen for LH (3.5-fold vs. 8-fold), and KN-93 had no effect on FSH release. Similar to responses seen for gonadotropin subunit PT, KN-92 had no effect on basal or GnRH-stimulated LH or FSH release (data not shown).
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). Pulses of BK+KCl stimulated PT concentrations of (50%), LHß (80%), and FSHß (115%) vs. vehicle-pulsed controls (P < 0.05). However, because KN-93 alone tended to increase , LHß, and FSHß PTs vs. controls and the magnitude of responses to pulsatile BK+KCl were reduced, compared with GnRH, the effect of KN-93 on responses to BK+KCl pulses were inconclusive. Similar to results for GnRH on LH secretion, KN-93 tended to enhance the effectiveness BK+KCl on LH release but not to a significant degree (data not shown).
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Discussion |
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, LHß, and FSHß transcription. These findings are physiologically relevant because the studies used differentiated pituitary cells, a physiological treatment paradigm (i.e. pulsatile GnRH), and measured endogenous gene products. In primary pituitary cells, the Ca/CAMK II-specific inhibitor, KN-93, either completely () or partially (LHß and FSHß) suppressed gonadotropin subunit PT responses to pulsatile GnRH. This action was specific because the inactive isoform, KN-92, had no effect on GnRH-induced increases in gonadotropin subunit PTs or secretion. Of note, we recently showed that KN-93, but not KN-92, completely blocked GnRH-induced Ca/CAMK II phosphorylation in gonadotrope-derived LßT2 cells (36). Previous studies revealed that Ca/CAMK II regulates the expression of several genes in various cell types, including the pituitary (25, 28, 31). Nowakowski et al. (31) showed that Ca/CAMK II stimulates prolactin promoter activity via actions on the pituitary-specific transcription factor, Pit 1. In rat lactotrope cells or lactotrope-derived GH3 cells, Ca/CAMK II mediates TRH or potassium depolarization-induced prolactin release, through actions on L-type voltage sensitive calcium channels (26, 30). In contrast, our results suggest that Ca/CAMK II has little or no effect on gonadotropin secretion. Indeed, LH secretory responses to pulsatile GnRH or BK+KCl in the presence of KN-93 tended to be increased vs. GnRH or BK+KCl alone.
A rise in intracellular calcium is essential in the activation of Ca/CAMK II (25, 28), and recent results reveal that Ca/CAMK II is regulated by alterations in the amplitude and duration of intracellular calcium spikes (37). Based on these observations and our previous data showing that pulsatile BK+KCl stimulates , LHß, and FSHß transcription (24), we examined whether Ca/CAMK II plays a role in calcium-induced gonadotropin subunit PT responses. Unfortunately, we were not able to answer the question using the current experimental paradigm. This was due to the consistent observation that KN-93 tended to increase , LHß, and FSHß PTs, which did not allow for statistically significant changes to be seen following pulsatile BK+KCl.
Although we have previously demonstrated that BK+KCl pulses replicate most effects of pulsatile GnRH (22, 24), including the differential , LHß, and FSHß transcriptional responses to pulse frequency, some differences have been noted. As seen in the present study, the optimal treatment duration for BK+KCl and GnRH differs (6 h vs. 24 h, Fig. 2), which may reflect differences in site of action for both compounds (GnRHR vs. L-type calcium channels). One possibility is that L-type channels within the pituitary become desensitized to BK+KCl pulses over longer durations. In support, we have previously noted that LH secretory responses to BK+KCl pulses in perifused rat pituitary cells decline between 3 and 21 h of treatment to a greater extent than that seen for pulsatile GnRH (22). Also of note, the magnitude of transcriptional responses to GnRH was greater than that for BK+KCl (as shown in Fig. 2). GnRHR binding stimulates the activation of various second-messenger pathways that play a role in gonadotropin subunit gene expression, including PKC, protein kinase A, ERK, JNK, and p38 (10, 15, 16, 17, 18). Although calcium may mediate the activation of Ca/CAMK II and other third-messenger pathways, its role in activating various second-messenger systems associated with GnRHR binding is either permissive or less important than G protein-initiated activational responses (10, 12, 17, 38). Thus, although alterations in intracellular calcium may be critical to the transmission of pulse frequency signals within the gonadotrope, it is likely that maximal responses to pulsatile GnRH require cross-talk among calcium, Ca/CAMK II, and other intracellular pathways.
We and others have shown that calcium plays an important role in GnRH-induced stimulation of , LHß, and FSHß subunit gene expression, both at the steady-state mRNA and transcriptional levels in various cell models, including primary rat and mouse pituitary cells, as well as gonadotrope-derived T3 and LßT2 cells (20, 22, 24, 39). However, some results have differed, showing either no effect or selective stimulation of one but not other subunit genes (21). Recent findings in LßT4 cells reveal that calcium may play a role in the inhibitory effect of long-term continuous GnRH desensitization on rat LHß promoter activity (40). The reason(s) for divergent results remain unclear, but differences in cell model, experimental paradigms using pulsatile or continuous stimulation, and end products measured are likely contributory factors. Also, because cross-talk among intracellular messenger systems play an important role in the transduction of GnRH signals (10, 15, 38, 41), differences in interactions among messenger systems within primary cells vs. cell line models may be a factor in the differing results.
Our results showed that -PT responses to pulsatile GnRH were completely suppressed by Ca/CAMK II inactivation, suggesting that Ca/CAMK II plays an essential role in rat -transcription. These findings differ from that seen for the murine -subunit gene because one report showed that transfecting T3 cells with a Ca/CAMK II expression vector did not stimulate mouse -subunit promoter activity (42). We and others have shown that ERK also plays an important role in GnRH stimulation of the -subunit gene (16, 42, 43). Recent finding suggest that ERK stimulates -transcription by activating a member of the Ets family of transcription factors, which binds to the GnRH-responsive element of both the mouse and rat -subunit promoters (44, 45). Thus, the present results suggest that cross-talk between the Ca/CAMK II and ERK pathways may be critical to transmitting GnRH signals to the rat, but not the mouse, -subunit gene.
Both LHß and FSHß PT responses to GnRH were partially suppressed by blocking Ca/CAMK II autophosphorylation, suggesting that this intracellular pathway is only partially responsible for transmitting GnRH signals to these two gonadotropin subunit genes. Similar to , we and others have shown that ERK plays a role in GnRH actions on FSHß mRNA expression (16, 46). Results from other investigations reveal that PKC also stimulates FSHß gene expression in both the rat and sheep (39, 46). In contrast to and FSHß, evidence does not support a role for ERK in the regulation of LHß (16, 20). However, recent findings reveal that PKC and JNK may mediate GnRH action on the rat LHß promoter via effects on one or both GnRH-responsive elements (19, 47).
In summary, our findings reveal that Ca/CAMK II plays an important role in the GnRH signal transduction pathway within the rat gonadotrope. Although Ca/CAMK II mediates transcriptional responses to GnRH for all three gonadotropin subunits to some extent, the magnitude of responses differ among subunits, with Ca/CAMK II playing a greater role for than for FSH ß or LH ß. Whether Ca/CAMK II plays a critical role in modulating gonadotropin subunit responses to transient increases in intracellular calcium will require further investigation. However, these findings suggest that cross-talk between Ca/CAMK II and other intracellular messenger pathways are essential in the transmission of GnRH signals from the plasma membrane to the gonadotropin subunit genes.
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Footnotes |
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Abbreviations: BK, Bay K 8644; Ca/CAMK II, calcium/calmodulin-dependent kinase II; Con, control; CT, competitive template; GnRHR, GnRH receptor; JNK, c-Jun N-terminal kinase; P-CAMK II, phosphorylated - and ß-Ca/CAMK II subunit; PKC, protein kinase C; PT, primary transcript; T-CAMK II, total Ca/CAMK II; Veh, vehicle.
Received December 19, 2002.
Accepted for publication March 20, 2003.
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T3-1 cells: evidence for cross-talk between PKA and protein kinase C pathways. Endocrinology 138:2259–2266T3-1 cell line is mediated by protein kinase C, c-Src, and CDC42. Mol Endocrinol 12:815–824T3–1 cell line: differential roles of calcium and protein kinase C. Endocrinology 138:1673–1682-subunit promoter by gonadotropin-releasing hormone. Mol Cell Biol 15:3531–3539[Abstract]
-subunit gene expression. Recent Prog Horm Res 54:455–484
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