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Endocrinology Vol. 144, No. 6 2409-2416
Copyright © 2003 by The Endocrine Society

The Calcium Component of Gonadotropin-Releasing Hormone-Stimulated Luteinizing Hormone Subunit Gene Transcription Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase Type II

Daniel J. Haisenleder, Heather A. Ferris and Margaret A. Shupnik

Division of Endocrinology (D.J.H., M.A.S.), Department of Medicine and Department of Molecular Physiology and Biological Physics (H.A.F., M.A.S.), University of Virginia Health Science Center, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Division of Endocrinology, Department of Medicine, and +Department of Molecular Physiology and Biological Physics, University of Virginia Health Science Center, Charlottesville, Virginia 22908. E-mail: djh2q{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Calcium influx plays a critical role in GnRH regulation of rat LH subunit gene transcription, but the site(s) of action are undefined. We investigated the potential of GnRH acting through calcium to activate calcium/calmodulin-dependent protein kinase type II (Ca/CaMK II) in mouse gonadotrope-derived LßT2 cells. GnRH stimulated Ca/CaMK II ß subunit activity 3-fold 2 min after treatment and returned to control values by 45 min. The Ca/CaMK II response to GnRH was blocked by administration of the Ca/CaMK II-specific inhibitor, KN-93. The calcium channel activator Bay K 8644 stimulated a 3-fold increase in Ca/CaMK II activity, similar to GnRH. Blocking calcium influx with nimodipine or depleting intracellular calcium storage pools with thapsigargin each resulted in a partial suppression of GnRH-induced activation of Ca/CaMK II, and in combination, completely suppressed the Ca/CaMK II response to GnRH. KN-93 and nimodipine also suppressed {alpha}-subunit and LHß promoter responses to GnRH by 40–60%. LHß promoter constructs containing either proximal or proximal and distal GnRH-responsive regions were sensitive to inhibition. These data show for the first time that Ca/CaMK II activation plays an important role in the transmission of GnRH signals from the plasma membrane to the LH subunit genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY GONADOTROPIN secretion and gene transcription are tightly controlled by pulses of hypothalamic GnRH (1). GnRH pulses vary in frequency and amplitude with physiological state and modulate transcription of the three gonadotropin subunit genes in a frequency-dependent, subunit-specific manner (2, 3). This has prompted suggestions that differential signaling through the GnRH receptor (GnRH-R) may play a role in defining gonadotropin gene expression. The GnRH-R is a member of the seven-transmembrane receptor family. Binding to the GnRH-R activates several members of the GTP-binding protein family, including Gq and G11, resulting in the activation of phospholipase Cß, an increase in phosphoinositide turnover, and a rise in intracellular diaclyglycerol levels leading to the activation of protein kinase C (PKC; Refs.3, 4, 5, 6, 7). GnRH-R binding also stimulates a rise in intracellular calcium concentration, which is derived from inositol 1,4,5-trisphosphate-activated intracellular storage pools, as well as influx from extracellular fluid through L-type voltage-sensitive calcium channels (8). GnRH has been shown to activate various additional intracellular signaling pathways, including protein kinase A (PKA), PKC, ERK 1 and 2 (MAPK), c-Jun N-terminal kinase (JNK), and p38 (5, 9, 10, 11, 12, 13, 14, 15). Although the specific signal transduction pathways mediating GnRH-induced regulation of gonadotropin subunit transcription remain to be completely characterized, stimulation of the LH subunit genes appears to require components of the PKC, MAPK, and calcium-stimulated pathways (13, 16, 17, 18, 19).

Calcium has been implicated as a major component of the GnRH mechanism of action, stimulating both gonadotropin secretion and gene expression (13, 19, 20, 21). Calcium signaling and secretion or exocytosis have been studied in several model gonadotrope systems, including primary cultured pituitary cells as well as {alpha}T3 and LßT2 clonal gonadotrope cells (22, 23, 24). The elegant work of Hille and colleagues (23) demonstrated that acute GnRH-stimulated oscillations in intracellular calcium stimulated rhythmic exocytosis in rat gonadotropes. Studies in LßT2 cells demonstrated dose-dependent biphasic GnRH-induced calcium mobilization and calcium sensitivity of exocytosis, which were modulated by steroid treatment (3, 24). Changes in cytoplasmic calcium concentrations and calcium-stimulated secretion may not necessarily correlate with altered gene transcription. For example, changes in nuclear calcium alter gene transcription independently from changes in cytoplasmic calcium in neural cells, and specific transcription factors may respond independently to altered cytoplasmic [serum-responsive factor (SRF)] or nuclear (cAMP response element binding protein) calcium (25, 26). In our studies, we concentrated on the role of GnRH-stimulated changes in calcium that could regulate transcription of the LH subunit genes.

Recent studies using primary rat pituitary cells or LßT2 cells showed that a rise in intracellular calcium increases steady-state levels of {alpha}, LHß, and FSHß mRNAs and stimulates gene transcription (13, 27, 28, 29). Pulsatile application of a calcium channel agonist to normal gonadotrope cells regulates gonadotropin subunit transcription in a differential manner, reminiscent of GnRH frequency-dependent effects (2, 29). However, the downstream third messenger system(s) that transmit GnRH-induced calcium signals to the gonadotropin subunit genes have yet to be determined.

Calcium/calmodulin-dependent kinase II (Ca/CaMK II) is an important intracellular mediator of calcium signaling in several cell and tissue types, including the pituitary (30, 31). Ca/CaMK II is a complex of subunits derived from 4 genes ({alpha}, ß, {gamma}, and {delta}), with {alpha} and ß prominent within the brain and {gamma} and {delta} distributed throughout the body (30, 31). Each Ca/CaMK II subunit has catalytic activity. Ca/CaMK II is activated by Ca/CaM binding to the regulatory domain, disinhibiting the enzyme, and allowing it to bind ATP and peptide substrate (31, 32). Following activation, the subunit is autophosphorylated, thus increasing the affinity for CaM-CaMK binding, and phosphorylation is correlated with enzyme activity (32). Ca/CaMK II is transported to the nucleus, allowing the enzyme to directly activate various transcription factors (31). Ca/CaMK II plays a role in cross-talk between intracellular messengers by activating calcium ATPase, adenylyl cyclase, calcineurin, nitric oxide synthase, and other enzyme systems (33). Ca/CaMK II regulates secretion and gene expression in several tissues (30, 31). In the brain, Ca/CaMK II stimulates glutamate and norepinephrine release and c-fos gene expression (32). In the pituitary, the enzyme stimulates both PRL secretion and promoter activity (34, 35, 36).

The potential role for Ca/CaMK II in the GnRH-stimulated gonadotrope has not been examined. However, several GnRH-response regions on the rat LH subunit gene promoters have been defined, and transcription factors binding to these sites could be targets for Ca/CaMK II (15, 16, 17, 37, 38). The rat {alpha}- subunit GnRH responsive element (-411 to -384 bp) has two Ets factor binding motifs, and members of the Ets protein family may be modulated by calcium (37, 39). The rat LHß promoter contains two GnRH-responsive elements. The distal GnRH-responsive element (-456 to -342 bp) contains 5' and 3'-Sp1 binding sites and a CArG box element, and the proximal GnRH-responsive element (-121 to -50 bp) contains two early growth response protein 1 (Egr-1) sites, two steroidogenic factor 1 (SF-1) sites, and a pituitary homeobox-1 (Ptx-1) site (37, 40). Any or all of these proteins could be modulated by calcium (41, 42, 43).

The present study was conducted to address two questions. First, does GnRH stimulate Ca/CaMK II activity within gonadotrope cells? Second, does Ca/CaMK II help mediate the calcium component of the {alpha}-subunit and LHß transcriptional responses to GnRH (13, 17, 18, 19)? We performed these experiments in the LßT2 clonal gonadotrope cell line, which represents a homogeneous system for biochemical and transcriptional studies. These cells express functional GnRH-R and the gonadotropin subunit genes and respond to GnRH with increased calcium mobilization and stimulated gonadotropin secretion and gene transcription (18, 24, 27, 37).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transfection studies
Each experiment contained three wells per treatment group and was repeated at least five times. LßT2 cells were originally obtained from Dr. Pamela Mellon (University of California, San Diego, CA). Cells were grown in DMEM (Life Technologies, Inc., Grand Island, NY) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Approximately 5 x 105 cells were plated per 20 mm well and allowed to grow for approximately 24 h in phenol-free DMEM/5% stripped newborn calf serum before calcium phosphate transfection. Cells were transfected for 16 h with 1 µg of reporter DNA per well before initiating experimental treatments. Plasmid vectors containing the rat {alpha}-subunit promoter (-411 to +44/I77 bp, relative to the transcriptional start site) or two rat LHß promoter regions (the -617 to +44-bp region containing both distal and proximal GnRH-responsive elements, and the -245 to +44-bp region containing only the proximal response region; Ref. 37) fused to a luciferase (Luc) reporter were used for these studies. As controls for transfection efficiency and GnRH and drug responsiveness, we transfected several additional constructs including the cytomegalovirus (CMV) promoter (CMV-Luc; 0.5 µg/well) construct, or a construct (GAL-Luc; 1 µg/well) with five copies of the DNA binding site for the GAL4 protein and a minimal TATA box (PG5Luc; Promega Corp., Madison, WI) fused to Luc (8, 44). The ratio of basal expression of these constructs to LH subunit constructs gives a measure of transfection efficiency. In all studies comparing different constructs, such as {alpha}-subunit and LHß (see Fig. 5Go), various LHß constructs (see Fig. 7Go), or LH subunit constructs and control constructs (see Fig. 6Go as an example, but as done routinely), constructs were transfected in parallel in the same experiment. Following transfection, cells were washed with PBS and treated with 100 nM GnRH or vehicle for 6 h. The Ca/CaMK II water soluble inhibitor KN-93 (10 µM) and the inactive form, KN-92 (10 µM) and the L-type calcium channel antagonist nimodipine (Nimo, 500 nM) were all obtained from Calbiochem (San Diego, CA) and were added to the cells 30 min before GnRH treatment. These concentrations were shown in pilot studies to be maximally effective for transcription in this cell system on LH subunits. At the end of the treatment, cells were washed with PBS and collected in 150 µl of lysis buffer (Promega Corp., Madison, WI) and frozen overnight. Cell lysate samples were assayed in a Turner 20e luminometer (Turner Designs, Mountain View, CA) using D-luciferin (ICN Biomedicals, Aurora, OH) and then assayed for protein concentration using the protein assay reagent from Bio-Rad Laboratories, Inc. (Hercules, CA). All transfection assays are expressed as arbitrary light units per 100 µg of protein or in some studies, as indicated in the figures, also normalized to control levels. As an additional control for GnRH and inhibitor sensitivity, parallel wells were transfected with the promoterless vector used for LH subunit contructs (pLUCLINK), or with a heterologous promoter (PGL3-basic) and treated with GnRH ± the various inhibitors. In some studies, these values were used to normalize LH promoter Luc values, expressed as arbitrary light units/100 µg protein.



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  		Figure 5. Rat {alpha} and LHß promoter responses to GnRH ± KN-93, Nimo or vehicle. LßT2 cells were transfected with Luc constructs (-411 to +77 bp for {alpha}-subunit and -617 to +44 bp for LHß; see Materials and Methods for details) and later pretreated for 30 min with 10 µM KN-93, 500 nM Nimo, or vehicle, followed by 6 h of GnRH (100 nM). Each experiment is normalized to control and then expressed as mean ± SEM. Values are calculated from three to six independent experiments with three samples per group. *, P < 0.05 vs. appropriate controls; #, P < 0.05 vs. vehicle + GnRH. The increase in fold between treatment and treatment plus GnRH is shown in parentheses above each plus GnRH bar.

 


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  		Figure 7. The effect of Ca/CaMK II blockade on rat LHß promoter responses to GnRH. LHß Luc constructs (-617 to +44 bp, -245 to +44 bp) were transfected into LßT2 cells, pretreated with KN-93 or vehicle, then given GnRH for 6 h. Mean ± SEM for each group are shown. Values are calculated from seven to nine independent experiments with three samples per group. *, P < 0.05 vs. appropriate controls; #, P < 0.05 vs. vehicle plus GnRH. The fold-stimulation by GnRH is shown in parentheses above each appropriate bar.

 


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  		Figure 6. A, The -617 to +44 LHß Luc construct was transfected into LßT2 cells, treated with 10 µM KN-93 or the inactive form, KN-92, with or without 100 nM GnRH. The fold-stimulation by GnRH is shown in parentheses above each appropriate bar. B, The -617LHß Luc, CMV-Luc; C, GAL-Luc constructs were transfected into LßT2 cells and treated with 100 nM GnRH, 10 µM KN-93, or the combination. The figure is a representative graph of three independent experiments performed in triplicate. *, P < 0.05 vs. appropriate control. #, P < 0.05 vs. -617 vehicle plus GnRH.

 
CaMK II immunoblot assay
Cell lysates.
Ca/CaMK II activity was measured in parallel with transfected promoter activity. LßT2 cells were plated in DMEM/10% fetal bovine serum at a density of 4.5 x 10-6 cells per 60-mm tissue culture well. After 24 h, cells were transferred to phenol red-free DMEM/5% stripped newborn calf serum. The following day, cells were pretreated with 500 nM Nimo, 10 µM KN-93, 10 µM KN-92, 50 nM thapsigargin (Thap, an endoplasmic reticulum calcium-ATPase inhibitor), 10 µM GF109203X (GF, a pan-PKC inhibitor), 50 µM PD098059 [PD, a MAPK kinase (MEK) inhibitor] or vehicle for 30–60 min. Wells were then treated with 100 nM GnRH or 100 nM Bay K 8644 (BK, an L-type calcium channel agonist) for the duration indicated in the figure legends. After treatment cells were washed with PBS, then collected in 200 µl of 2x gel loading buffer (100 mM Tris, pH 6.8; 4% sodium dodecyl sulfate; 20% glycerol; 150 mM NaF; 1 mM sodium orthovanadate) and boiled for approximately 10 min. Protein concentration was determined by Bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL). BayK 8644, Thap, GF109203X, and PD098059 were obtained from Sigma (St. Louis, MO).

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 {alpha} and ß Ca/CaMK II subunits (P-CaMK II, Upstate Biotechnology, Inc., Lake Placid, NY) and phosphorylated and unphosphorylated {alpha}, ß, {gamma}, and {delta} CaMK II subunits (Total Ca/CaMK II [T-CaMK], Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Enzyme autophosphorylation increases the affinity of the Ca/CaMK II for calmodulin and is correlated with functional enzyme activity (30, 45). The horseradish peroxidase-conjugated goat antirabbit secondary antibody was also obtained from Upstate Biotechnology, Inc. Bands were detected using the Super Signal Pico West chemiluminescent system (Pierce Chemical Co., Rockford, IL), followed by autoradiography. P-CaMK II bands were quantified by densitometry and corrected to the amount of T-CaMK II per sample. Treatment-induced responses were similar for both {alpha} and ß Ca/CaMK II subunits. However, as the intensity of the signal for the ß Ca/CaMK II subunit was greater than that seen for {alpha} Ca/CaMK II subunit, results were quantified using ß Ca/CaMK II bands for each sample.

ERK immunoblot assay
For ERK assays, cell lysate protein (50 µg) was resolved by SDS-PAGE, transferred to nitrocellulose filters and assayed with a kit purchased from Cell Signaling Technology (Beverly, MA) as described previously (10). ERK1 and 2 phosphorlylation was measured and normalized for total ERK protein as previously described (10).

Statistical analysis
The Ca/CaMK II blot data were analyzed by one-way ANOVA, with differences between treatment groups determined by Duncan’s multiple range test. Transfection data were analyzed using ANOVA and the Tukey post hoc test. P < 0.05 was considered to be statistically significant. Statistical analysis was performed using the Prism statistics program (GraphPad Software, Inc., San Diego, CA). When data were normalized within an individual experiment, the average of the three control points was determined and set to 100%. For analysis of multiple transfection experiments, the mean value of the treatment group from one experiment was used as a single value to average results from multiple experiments and expressed as the mean ± SEM for each group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH stimulates Ca/CaMK II phosphorylation
Ca/CaMK II phosphorylation by 100 nM GnRH in LßT2 cells is shown in Fig. 1Go. GnRH stimulated a 3-fold increase within 2 min (P < 0.05 vs. vehicle controls). The Ca/CaMK II response to GnRH was maintained after 5 and 15 min (3- to 4-fold vs. controls; P < 0.05 vs. controls) and then declined after 45 min of treatment. Figure 2Go shows the Ca/CaMK II response to GnRH in the presence of the specific Ca/CaMK II inhibitor, KN-93. The upper panel of Fig. 2Go shows a representative autoradiograph of the Ca/CaMK II response to GnRH ± KN-93, and the lower panel shows the quantitation of four experiments. Five or 15 min of GnRH administration stimulated a 3-fold increase in Ca/CaMK II phosphorylation (P < 0.05 vs. controls). However, in cells pretreated for 30 min with KN-93, the stimulatory response to GnRH was completely suppressed. In contrast, pretreatment of LßT2 cells with KN-92, an inactive form of KN-93, had no inhibitory effect on the Ca/CaMK II response to a 5-min GnRH stimulus.



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  		Figure 1. Time course response of GnRH-induced phosphorylation of Ca/CaMK II in LßT2 cells. GnRH (100 nM) was given for 2, 5, 15, or 45 min [medium only to controls (Con)]. The results are presented as % change vs. Con; mean ± SEM are shown from three independent experiments, with two to three samples/group. *, P < 0.05 vs. Con.

 


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  		Figure 2. Ca/CaMK II responses to GnRH ± KN-93 (or vehicle) in LßT2 cells. The Ca/CaMK II blocker KN-93 (10 µM) or the inactive isoform KN-92 (10 µM) were given 30 min before 100 nM GnRH (5' or 15') and phosphorylated Ca/CaMK II (P-CMK) and total Ca/CaMK II (T-CMK) measured by immunoblot following treatment. Phosphorylated Ca/CaMK II results for each sample were corrected to T-CMK for the same sample. Mean ± SEM are shown from four independent experiments; *, P < 0.05 vs. controls (Con). A representative immunoblot is presented in the upper panel.

 
To determine the link between alterations in intracellular calcium and GnRH-induced phosphorylation of Ca/CaMK II, studies were conducted using compounds that either increase or decrease intracellular calcium. As shown in Fig. 3Go, the L-type calcium channel agonist BK stimulated a 3-fold increase in Ca/CaMK II activity (P < 0.05 vs. vehicle controls), similar to responses seen for GnRH. In separate studies, the BK stimulation of Ca/CaMK II phosphorylation (332% vs. untreated controls) was specifically suppressed by treatment with KN-93 (133% of control) but not by KN-92 (375% of control). GnRH-stimulated Ca/CaMK II phosphorylation could occur by increases in calcium influx or by increased release from intracellular calcium stores. Pretreating the cells with Thap, a compound that depletes intracellular inositol-releasable calcium storage pools, reduced basal and partially suppressed GnRH-induced increases in Ca/CaMK II activity. The L-type calcium channel antagonist, Nimo, also suppressed basal and partially suppressed GnRH-induced Ca/CaMK II responses. Partial suppression by either Nimo or Thap was not due to submaximal doses of either compound, as dose-response studies using concentrations up to 1.5 µM Nimo or 1000 nM Thap showed no additional effects. The combination of Thap and Nimo completely inhibited the Ca/CaMK II responses to GnRH. GnRH effects on Ca/CaMK II phosphorylation were also examined in the presence of the PKC inhibitor, GF109203X or the MEK inhibitor, PD098059, and blocking either pathway had no effect on GnRH-induced phosphorylation of Ca/CaMK II, although they are effective at blocking GnRH stimulation of ERK (our unpublished data, and Ref. 10). Because the ERK pathway is also stimulated by GnRH, and plays an important role in gonadotropin gene expression, we tested if the Ca/CaMK II inhibitor could influence ERK activation by GnRH (Fig. 4Go). Neither KN-93 nor the inactive compound KN-92 significantly suppressed GnRH-stimulated ERK phosphorylation, demonstrating the specificity of the response.



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  		Figure 3. Ca/CaMK II responses to alterations in intracellular calcium. LßT2 cells were pretreated for 60 min with 50 nM Thap, 500 nM Nimo, 10 µM GF109203X (GF), 50 µM PD098059 (PD), or vehicle (Con), followed by 5 min of treatment with 100 nM GnRH or 100 nM Bay K 8644 (BK). Mean ± SEM are shown from three independent experiments. *, P < 0.05 vs. appropriate minus-GnRH control group (Con, Thap, Nimo, GF or PD); #, P < 0.05 vs. Con + GnRH; a, P < 0.05 vs. Con.

 


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  		Figure 4. ERK1/2 phosphorylation in response to GnRH stimulation with and without KN-92 and KN-93. Additional lysate from samples used in Fig. 2Go to determine Ca/CaMK II phosphorylation were also assayed for ERK1/2 phosphorylation by immunoblot. Fifty micrograms of protein were loaded in each well. Immunoblotting was performed for phosphorylated ERK1/2 and total ERK proteins on the same blot. Phosphorylated ERK1/2 was measured and normalized for total ERKI in the same sample. Results are expressed as mean ± SEM for percent change vs. vehicle controls. Mean ± SEM are shown from four independent experiments; *, P < 0.05 vs. controls (Con) *, P < 0.05 vs. appropriate minus-GnRH control group.

 
GnRH stimulation of LH subunit promoter activity requires Ca/CaMK II
Studies were next conducted to investigate the role of Ca/CaMK II activation in the regulation of LH subunit promoter activity. As shown in Fig. 5Go, the {alpha}-subunit and LHß promoters were stimulated by GnRH in LßT2 cells (3.3-fold and 21.3-fold, respectively; P < 0.05 vs. vehicle controls), in agreement with our previous data (37). In cells pretreated with 10 µM KN-93, there was a trend toward slightly decreased basal expression for the {alpha}-subunit and the LHß promoter, but this was not statistically significant. Pretreatment with KN-93 partially suppressed both {alpha}-subunit and LHß responses to GnRH (to 1.9- and 15-fold, respectively P < 0.05 vs. vehicle + GnRH), and activity of the promoters overall were decreased. Similar inhibition of GnRH stimulation was seen in cells pretreated with Nimo (to 2.0-fold for {alpha}-subunit and 11.2-fold for LHß, P < 0.05 vs. vehicle + GnRH). These findings clearly demonstrate that both Ca/CaMK II and calcium influx play a role in {alpha}-subunit and LHß promoter activity, and partially suppress the stimulation by GnRH.

To demonstrate the specificity of the response, we tested the KN-93 and KN-92 compounds, as well as several different promoter constructs (Fig. 6Go). The LHß promoter was partially suppressed by KN-93 in the presence of GnRH but KN-92, which does not inhibit Ca/CaMK II phosphorylation, had no effect on GnRH stimulation (Fig. 6AGo). In contrast to the effects on the LHß promoter, GnRH did not stimulate expression of the CMV-Luc or GAL-Luc constructs, and KN-93 also had little effect on activity (Fig. 6Go, B and C). KN-92 had no affect on activity on any promoter in any experiment.

To determine which of the GnRH-responsive regions in the rat LHß promoter are sensitive to Ca/CaMK II, LßT2 cells transfected with two constructs were treated with GnRH ± KN-93 (Fig. 7Go). Constructs included an LHß promoter construct (-617 to +44 bp) containing both the distal (-456 to -352 bp) and the proximal GnRH-responsive regions (-121 to -50 bp), and a truncated promoter construct (-245 to +44 bp) containing only the proximal response element. The -245 bp construct is stimulated to a lesser extent by GnRH, as expected with the loss of the distal response region (37, 40). In both constructs, GnRH stimulation is partially suppressed by KN-93, approximately one third lower compared with vehicle plus GnRH. These data demonstrate that the entire LHß promoter and the proximal GnRH-response region are sensitive to Ca/CaMK II. The proximal region of the promoter contains binding sites for Egr-1, a protein whose synthesis is rapidly stimulated by GnRH (17, 46). We performed immunoblotting studies of Egr-1 protein under basal and GnRH-treated conditions, and found that the GnRH-stimulation of Egr-1 protein in our cultures was not altered by KN-93 or KN-92 (Ferris, H., unpublished data). These data suggest that at least the proximal GnRH-sensitive region of the promoter contains a Ca/CaMK II-sensitive region. Overall, these studies demonstrate that GnRH can stimulate Ca/CaMK II phosphorylation in gonadotrope cells and that at least part of the calcium component of GnRH stimulation of the LH subunit genes is mediated by Ca/CaMK II.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study is the first to show that GnRH stimulates Ca/CaMK II phosphorylation within gonadotrope cells and that this intracellular signal transduction enzyme plays an important role in the {alpha}-subunit and LHß transcriptional responses to GnRH. Ca/CaMK II activity increased rapidly with GnRH stimulation (i.e. within 2 min). This effect was completely blocked by the specific Ca/CaMK II inhibitor, KN-93, but not by KN-92, the MEK inhibitor PD098059, or the general PKC inhibitor GF109203X. Our results also showed that a rise in intracellular calcium is essential in the GnRH activation of Ca/CaMK II, with increasing calcium from both intracellular pools (suppressed by Thap) and extracellular influx (stimulated by BK and suppressed by Nimo) playing a role.

Calcium is an important mediator in the activation of downstream intracellular messenger systems, including PKA, PKC, ERK, JNK, and Ca/CaMK I, II, and IV (47, 48, 49, 50). In the present study, we investigated the link between GnRH-R activation and Ca/CaMK II. This enzyme is present within the anterior pituitary, and is stimulated by TRH binding to its seven-transmembrane, G protein-coupled receptor, the receptor superfamily in which the GnRH-R is also a member (51). Studies by Dannies and colleagues (34, 35) reveal that Ca/CaMK II mediates TRH-induced PRL secretion by activating voltage-dependent calcium channels within rat lactotrope and GH3 cells. Ca/CaMK II also stimulates PRL promoter activity, via actions on the pituitary specific transcription factor, Pit-1 (36). GnRH also binds to a pituitary seven transmembrane G protein-coupled receptor, stimulates intracellular calcium through L-type calcium channels and the release of intracellular calcium stores (8), and modulates gonadotropin gene expression via calcium (13). We thus postulated that Ca/CaMK II could be the link between GnRH calcium signaling and gonadotropin gene transcription.

The role played by calcium in the regulation of gonadotropin subunit gene expression has been a point of intensive investigation and some controversy over the past few years, with results dependent on the biological system used and the endpoint measured. Studies with primary cultures of rat pituitary cells have shown that mature mRNAs for {alpha}, LHß, and FSHß subunits are stimulated by calcium influx (21, 52). We also showed, by measurement of primary transcripts of the endogenous genes in rat pituitary cells, that pulsatile BK duplicates the differential gonadotropin subunit transcriptional response to GnRH pulse frequency (29). This current work agrees with our previous studies showing that calcium influx plays an important role in GnRH stimulation of endogenous rat {alpha}-subunit and LHß gene transcription in normal rat pituitary cells as determined by nuclear run-on assays, and in the GnRH stimulation of an LHß promoter transgene in transgenic mice (13). Thus, increased intracellular calcium and calcium influx appear to be necessary for the GnRH stimulation of LH subunit gene transcription in normal rodent gonadotrope cells.

The importance of calcium in GnRH-stimulated gene transcription has not been as clear in studies with immortalized cell lines. Transient transfection studies in the heterologous GGH3–1 cells found that rat LHß and FSHß were primarily regulated by PKC, with calcium having only a minor role, whereas calcium played a major role in stimulation of the rat {alpha}-subunit promoter (19). Investigations in LßT2 gonadotrope cells demonstrated that calcium influx was important for the induction of the endogenous LHß and c-fos genes, but that MEK activation was important for stimulation of LHß protein (27). A more recent report (18) using a slightly different cell line, the LßT4 gonadotrope cells, showed that calcium played little role in acute GnRH stimulation of the transfected rat LHß promoter but is required for long-term repression of LHß gene expression by continuous GnRH. One intriguing difference with the results in the LßT4 cell line is that the proximal rat LHß promoter construct was not stimulated by GnRH, in contrast to our results and those from other investigators using LßT2 cells (37, 40, 53, 54, 55). Proximal promoter activity was suppressed by GnRH in a calcium influx-dependent process in LßT4 cells, whereas we find that the proximal promoter is stimulated by GnRH and calcium in LßT2 cells. These transcriptional studies did not link biological responses to calcium to specific intracellular signaling molecules.

Some of the differences between results in various cell lines, and between specific cell lines and primary cultures, could be due to differential GnRH receptor coupling in different cell contexts. For example, the GnRH receptor couples to Gq/11 and Gs in primary pituitary cells, LßT2 cells and GGH3–1 cells, but not to Gs in {alpha}T3 cells (5). LßT2 cells and {alpha}T3 cells, like primary cells, demonstrate GnRH dose-dependent increases in secretion and internal calcium concentrations (3, 24). Increases in calcium concentrations are biphasic, with rapid increases within 15 sec followed by a prolonged secondary phase of GnRH-induced calcium response. However, the oscillations in internal calcium noted at low concentrations of GnRH in primary gonadotropes are not observed in the cell lines (3). Cross-talk between GnRH signal transduction pathways is likely to play a role in transcriptional responses. It is probable that cross talk between pathways may vary in differing experimental models, adding to different outcomes seen by various investigators. The LßT2 cells represent a mature gonadotrope phenotype to study regulation of the LH subunit gene promoters. Calcium-dependent stimulation of LH subunit gene transcription occurs in both primary pituitary cells and LßT2 cells (13, 19, 27, 28, 29). Furthermore, recent collaborative studies have shown that Ca/CaMK II inhibition by KN-93 also suppresses GnRH-stimulated transcription of the LHß and {alpha}-subunit genes in primary cultures of normal rat pituitary cells (56).

Overall, results from several laboratories have demonstrated that expression of the rat, mouse and human {alpha}-subunit genes are regulated by several intracellular pathways, including calcium, cAMP/PKA, PKC, and ERK (7, 13, 16, 46, 57, 58). Our current and previous data show that GnRH stimulation of the endogenous rat {alpha}-subunit gene and the transfected rat {alpha}-subunit promoter are partially dependent on increases in intracellular calcium, and partially on increased MEK/MAPK activity (13, 19). The present study shows that the calcium component of GnRH stimulation of the rat {alpha}-subunit promoter occurs through Ca/CaMK II, and that blocking the enzyme with KN-93 reduces the GnRH response. The addition of PKC or MEK blockers did not affect GnRH stimulation of Ca/CaMK II, but Nimo and Thap together eliminated the GnRH stimulation. Thus, the Ca/CaMK II action on the {alpha}-subunit promoter is independent of the PKC/ERK pathway. This does not rule out the possibility that cross-talk between these various intracellular pathways is essential for physiological regulation of {alpha}-subunit transcription by GnRH. The rat {alpha}-subunit promoter contains a binding site for the orphan receptor SF-1, but this appears to play a critical role in basal, rather than GnRH-stimulated expression (15). The GnRH-responsive region of the rat {alpha}-subunit promoter (-411 to -384 bp) contains two putative Ets protein-binding sites, and the GnRH-response element of the mouse promoter contains one such site, but these proteins have not been identified (37, 59). Promoter stimulation by Ets proteins could be modulated by MAPK and Ca/CaMK II, potentially by protein modifications (59, 60).

Previous studies demonstrated that the endogenous rat LHß promoter is sensitive to GnRH-stimulated calcium influx (13). In these investigations of Ca/CaMK II regulation of the LHß promoter, we attempted to determine if the constructs containing the entire promoter or only the proximal GnRH response element could also be calcium sensitive. The -617 to +44-bp LHß construct contains both proximal and distal GnRH responsive regions, whereas the truncated promoter (-245 to +44 bp) construct contains only the proximal GnRH responsive region. As we have previously shown, elimination of the distal response region reduces but does not eliminate the stimulatory response to GnRH (37). KN-93 partially suppressed GnRH stimulation of the -617 construct, similar to results with Nimo, suggesting that calcium influx may act via Ca/CaMK II. Addition of KN-93 to cells transfected with the truncated LHß promoter construct (-245 to +44 bp) also partially suppressed the response to GnRH, suggesting the presence of an important Ca/CaMK II responsive site(s) within the proximal promoter region.

Ca/CaMK II could influence one or several important GnRH-sensitive transcription factors on the LHß promoter or act on a separate protein site to influence GnRH stimulation, as was found for chronic GnRH suppression in LßT4 cells (18). The distal GnRH-sensitive region contains two binding sites for Sp1, and a CArG box element, which may bind SRF-like complexes (61). The proximal GnRH responsive region of the LHß promoter contains two composite SF-1/Egr-1 binding sites separated by a Ptx-1 site (17, 54, 55). SF-1, Ptx-1, and Egr-1 interact functionally to increase LHß promoter activity (54). SF-1 is a tissue specific factor and plays a role in basal, but not GnRH-induced, LHß promoter activity, and is a less likely target for Ca/CaMK II (54). Sp1 and SRF may be phosphorylated by several intracellular kinases including Ca/CaMK II, which could modify DNA binding or recruitment of coactivators (25, 42, 43). GnRH stimulation of the mouse Egr-1 promoter, which uses Sp1, Egr-1, and SRF binding sites, is partially suppressed by KN-93, suggesting that Ca/CaMK II could play a role in GnRH stimulation of this promoter and the rise in Egr-1 protein that stimulates LHß (41). Under our cell culture conditions, we did not observe a suppression of Egr-1 protein by KN-93, but this does not preclude potential protein modification by Ca/CaMK II. GnRH treatment modifies the transcriptional activity of Egr-1 (62), although the signaling pathways have not been identified. Thus, the possibilities include Ca/CaMK II modification of Sp1, SRF, or Egr-1 selectively, modification of the SF-1/Ptx-1/Egr-1 complex, or additional promoter regions binding proteins with yet to be identified Ca/CaMK II-responsive sites. Because the rat LHß promoter requires cooperation of both GnRH-responsive regions for maximal stimulation, modification of one region will influence overall activity (40, 53).

In summary, these results show that Ca/CaMK II plays an important role in the GnRH signal transduction pathway within the gonadotrope, with significant effects on both {alpha}-subunit and LHß promoters. The GnRH-induced activation of Ca/CaMK II is mediated via increases in intracellular calcium derived from cellular storage pools and influx from plasma membrane calcium channels.


   Footnotes
 
This research was supported by NICHD/NIH through a cooperative agreement (U54-HD-28934, Molecular Core) as part of the Specialized Cooperative Centers Program in Reproductive Research (to M.A.S. and D.J.H.).

Abbreviations: Ca/CaMK II, Calcium/calmodulin-dependent protein kinase type II; CMV, cytomegalovirus; Egr-1, early growth response protein 1; GnRH-R, GnRH receptor; JNK, c-Jun N-terminal kinase; Luc, luciferase; MEK, MAPK kinase; Nimo, nimodipine; PKA, protein kinase A; PKC, protein kinase C; Ptx-1, pituitary homeobox-1; SF-1, steroidogenic factor 1; SRF, serum-responsive factor; Thap, thapsigargin.

Received November 6, 2002.

Accepted for publication February 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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