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Endocrinology, doi:10.1210/en.2006-1473
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Endocrinology Vol. 148, No. 3 1099-1107
Copyright © 2007 by The Endocrine Society

Gonadotropin-Releasing Hormone Couples to 3',5'-Cyclic Adenosine-5'-Monophosphate Pathway through Novel Protein Kinase C{delta} and -{epsilon} in LßT2 Gonadotrope Cells

Sigolène Larivière, Ghislaine Garrel, Violaine Simon, Jae-Won Soh, Jean-Noël Laverrière, Raymond Counis and Joëlle Cohen-Tannoudji

Unité Mixte de Recherche Centre National de la Recherche Scientifique 7079 (S.L., G.G., J.-N.L., R.C., J.C.-T.), Physiologie and Physiopathologie, Université Pierre and Marie Curie-Paris 6, 75252 Paris, France; Department of Pharmacology and Experimental Therapeutics (V.S.), Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112; and Biomedical Research Center for Signal Transduction Networks (J.-W.S.), Inha University Incheon, 402-751 Incheon, Korea

Address all correspondence and requests for reprints to: Prof. J. Cohen-Tannoudji, UMR CNRS 7079 Université Pierre and Marie Curie-Paris 6, Case 256, 4 Place Jussieu, 75252 Paris cedex 05, France. E-mail: joelle.cohen-tannoudji{at}snv.jussieu.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although the cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LßT2 gonadotrope cells (maximal increase, 2.5-fold; EC50, 0.30 nM), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca2+ independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, indicating that the GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with Gö6976 did not prevent GnRH-induced cAMP production, whereas down-regulation of novel PKC{delta}, -{epsilon}, and -{theta} by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKC{delta} or -{epsilon} but not PKC{theta} impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKC{delta} and -{epsilon} are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LßT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms, i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP, suggesting that in vivo, the cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NEUROPEPTIDE GnRH is a key regulator of the mammalian reproductive system. This neuropeptide is synthesized in the hypothalamus and released into the hypophyseal portal circulation in a pulsatile manner. It then acts in the anterior pituitary gland via a G protein-coupled receptor located in the membrane of gonadotrope cells to differentially control both the synthesis and release of gonadotropin hormones LH and FSH (1, 2). Binding of GnRH to its receptor initiates a wide array of signaling events. The GnRH receptor (GnRH-R) is mainly coupled to phospholipase Cß via G{alpha}q/11, leading to a rapid increase in diacylglycerol and inositol triphosphate, which propagate signaling cascades that account for many of the biological effects of GnRH. Inositol triphosphate mobilizes Ca2+ from intracellular stores that, together with GnRH-stimulated Ca2+ influx, regulates acute gonadotropin release. Elevation of intracellular Ca2+ also activates the NO synthase (NOS I) cascade (NOS I/NO/soluble guanylate cyclase) resulting in a rapid increase of cGMP (3, 4, 5). GnRH-induced diacylglycerol activates protein kinase C (PKC) isoforms that mediate, notably, activation by GnRH of all MAPK cascades and regulation of gonadotropin subunits gene expression (6, 7). PKC-dependent ERK activation was recently demonstrated to be mediated by transactivation of the epithelial growth factor receptor via matrix metalloproteases 2 and 9 in the gonadotrope {alpha}T3-1 and LßT2 cell lines (8). To date, several PKC isoforms have been characterized in gonadotrope cells (9, 10, 11). These isoforms are members of the three known families of PKC : the conventional isoforms that are activated by Ca2+ and phorbol esters, the novel isoforms that are Ca2+-insensitive and the atypical isoforms that are both Ca2+- and phorbol esters-insensitive. Most of them have been identified as targets of GnRH, however, except for PKC{delta} which was recently reported to mediate GnRH activation of ERK in LßT2 cells (12), the respective contribution of the different PKC isoforms in the action of GnRH is not yet determined.

It has been clearly established that the cAMP/cAMP-dependent protein kinase (PKA) pathway is essential for gonadotrope function. Indeed, cAMP analogs mimic most of the effects of GnRH as they enhance the release of newly synthesized LH and the expression of several key genes including LHß and {alpha} subunits as well as GnRH-R and NOS I (13, 14, 15). However, the involvement of cAMP in the mechanism of action of GnRH is still debated. An early observation of Borgeat et al. (16) showed that GnRH stimulates cAMP accumulation in rat hemipituitaries, and it was shortly afterward confirmed by others (17). Since then, several studies performed on dispersed rat pituitary cell culture and, later, in the {alpha}T3-1 gonadotrope cell line were unable to substantiate any GnRH-induced cAMP production (18, 19, 20). More recently, the ability of GnRH to induce cAMP was documented in the more differentiated gonadotrope cell line LßT2 (21).

Furthermore, the potential mechanisms linking GnRH-R activation and cAMP production are still controversial. Using palmitoylation as a measure of G proteins activation, GnRH-R was reported to couple to Gs{alpha} in rat pituitary cells (22), and this was confirmed in LßT2 cells using cell-permeable peptides that uncouple Gs{alpha} from receptor activation (21). In contrast with these observations and based on photolabeling of the receptor-activated G proteins in {alpha}T3-1 gonadotrope cells, Grosse et al. (23) argued for the exclusive coupling of GnRH-R to Gq/11. These authors postulated that GnRH-dependent Ca2+ elevation may mediate cAMP production via activation of Ca2+/calmodulin-sensitive adenylyl cyclase (AC) isoforms, independently from Gs. An increase in cAMP concentrations may indeed result from multiple informational inputs because the nine known AC isoforms integrate various signals to differentially produce cAMP. In addition to stimulation through Gs{alpha}, AC1, AC3, and AC8 can be activated by the Ca2+-calmodulin complex, whereas AC2, AC5, and AC7 are targets for PKC phosphorylation and activation (24, 25). Rapid GnRH-induced intrasignaling events, by cross-reacting with AC, may thus indirectly regulate cAMP levels in gonadotrope cells.

The present study was conducted to analyze the coupling mechanisms linking GnRH-R to the cAMP pathway in gonadotrope cells. We demonstrate that GnRH increases cAMP levels in LßT2 gonadotrope cells with an atypical time course and efficiency. Using different pharmacological strategies, we present evidence for a PKC-mediated activation of AC and demonstrate using dominant-negative (DN) mutants of selective PKC isoforms that GnRH acts through novel PKC{delta} and -{epsilon}. Accordingly, we have characterized two PKC-sensitive AC isoforms that are likely targets of GnRH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[2,8-3H]Adenine (22.8 Ci/mmol) was purchased from Perkin-Elmer (Courtaboeuf, France). AG50 Dowex W-4X was from Bio-Rad Laboratories (Marne la Coquette, France). Aluminum oxide 90 active neutral and bisindolylmaleimide I (GF109203X) were from VWR International (Strasbourg, France). GnRH and GnRH agonist [D-Trp6]GnRH were from Sigma (Saint-Quentin Fallavier, France). GnRH antagonist (antarelix) was a generous gift from Dr. R. Deghenghi (Europeptides, Rueil-Malmaison, France). The 38-amino-acid form of pituitary AC-activating polypeptide (PACAP38) was from Calbiochem (VWR International). Antibodies against AC5/6 (sc 590), AC7 (sc 1966), PKC{delta} (sc 937), PKC{theta} (sc 212), and PKC{zeta} (sc 216) were supplied by Santa Cruz Biotechnology (Tebu, France). Antibodies against PKC{alpha} (4334), PKCß2 (3203), and PKC{epsilon} (8458) and all other drugs and products were from Sigma.

Cell culture
Pituitary gonadotrope cell line LßT2 generated by Pamela Mellon (26, 27) was grown in DMEM (Sigma) supplemented with high glucose (4.5 g/liter) containing 10% fetal calf serum (PAA Laboratories, Les Mureaux, France), penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were passaged weekly and incubated at 37 C in a humidified atmosphere with 5% CO2.

Determination of cAMP accumulation
LßT2 cells were plated at a density of 1 x 106 cells per well and grown to 80% confluence in 12-well plates. To determine the intracellular content of cAMP, LßT2 cells were first loaded overnight with 2 µCi [3H]adenine per well in serum-free DMEM. Before stimulation with GnRH or other agonists, cells were preincubated 30 min in the presence of 250 µM 3-isobutyl-1-methylxanthine (IBMX) in serum-free DMEM containing 20 mM HEPES. Stimulations were then performed for various periods of time with GnRH (100 nM) or the GnRH agonist [D-Trp6]GnRH (100 nM), the phorbol ester phorbol 12-myristate 13-acetate (PMA, 50 nM), forskolin (10 µM), or PACAP38, as described in text. When tested, pharmacological PKC inhibitors or GnRH antagonist (antarelix, 10 µM) were added during the preincubation period before the addition of GnRH agonist. After stimulation, the medium was removed and cells were lysed for 10 min in 1 ml ice-cold lysis solution containing 2.5% perchloric acid and 3.5% cAMP. [3H]cAMP-containing supernatants were neutralized with 0.1 ml 4.2 M KOH. The intracellular cAMP produced was then separated from other labeled nucleotides by two successive chromatographies on Dowex and alumina columns, and the radioactivity was quantitated using an LKB/Wallac liquid scintillation counter as previously described (28).

RNA extraction and RT-PCR amplification
Total RNA was extracted from gonadotrope cell lines LßT2 and {alpha}T3-1 as well as from Swiss mouse brain with the Tri-Insta-Pure reagent (Eurogentec SA, Seraing, Belgium). After RT of total RNA (1 µg) with Superscript II (Invitrogen Life Technologies, Cergy Pontoise, France), PCR amplifications were performed in a final volume of 50 µl with 1 µl of the previously obtained cDNA, 2 U Taq DNA polymerase (Eurobio, Les Ulis, France), 10 mM dNTPs, 1.5 mM MgCl2, and 0.5 µM of each specific intron-spanning primer, as follows: forward 5'-GGG AAG ATT AGT ACC ACG GAT-3' and reverse 5'-AGG AGA AGC CAA GGA TGG ACG-3' for the 334-bp mouse AC2 (29); forward 5'-ACC ATT GTG CCC CAC TCC CTG TT-3' and reverse 5'-TCG TCG CCC AGG CTG TAG TTG AA-3' for the 338-bp mouse AC5 (30); and forward 5'-CTG TCT GTG GAA GAA GAA GT-3' and reverse 5'-AAT CAC TCC AGC AAT CAC AGG C-3' for the 535-bp mouse AC7. Cycling conditions consisted of 28–35 cycles of denaturation for 30 sec at 95 C, annealing for 30 sec at 58 C, and extension for 30 sec at 72 C. Amplicons were electrophoresed on a 1.5% agarose gel, purified using the Jetsorb kit (Genomed, Q-BIOgene, Illkirch, France), and sequenced (Genome Express, Grenoble, France).

Immunocytochemical detection of PKC-sensitive AC isoforms
LßT2 cells (3 x 105) were plated in poly-L-lysine-coated chambers of SUPCELL8 slides (CML, Nemours, France) in DMEM containing 10% fetal calf serum. After 24 h culture, cells were washed with cold PBS and fixed in 4% paraformaldehyde for 20 min. After washings with PBS, cells were blocked in PBS containing pig gelatin (2 g/liter) and 0.2% Triton X-100 for 1 h and then incubated overnight at 4 C with anti-AC type 5/6 or 7 antibody (dilution 1:100 and 1:50, respectively, in PBS containing 5% BSA and 0.1% Triton X-100). Cells were then treated for 1 h at room temperature with the appropriate Alexa Fluor 488 secondary antibody; AC5/6 was revealed with goat antirabbit Ig (A-11070, 1:1000) and AC7 with donkey antigoat Ig (A-11055, 1:800), both purchased from Molecular Probes (Invitrogen Life Technologies). The slides were finally mounted with Mowiol 4-88 (Calbiochem, VWR International) and observed under an epifluorescence microscope (Leica, Reuil-Malmaison, France).

Negative controls were performed by adsorption of AC antibodies with an excess of appropriate peptide antigens (overnight incubation at 4 C) supplied by Santa Cruz.

Protein extraction and Western blot analysis
LßT2 cells were grown to 80% confluence in 12-well plates, washed three times with cold PBS, and lysed in RIPA buffer [150 mM NaCl, 25 mM Tris-HCl (pH 7.4), 1 mM EGTA, 0.25% sodium deoxycholate, 1% Igepal CA-630] supplemented with protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). The cell suspension was homogenized by five passages through a 21-gauge needle, and the insoluble material was removed by centrifugation at 15,000 x g for 20 min at 4 C. Protein concentration was determined by the Bio-Rad protein assay, using BSA as a standard, and samples were frozen at –80 C until use. For immunoblot analysis, protein extracts (20 µg) were separated by SDS-PAGE using 10% polyacrylamide separating gel in a Mini-Protean-3 apparatus (Bio-Rad). Protein molecular weight markers (Bio-Rad) were coelectrophoresed. After electrotransfer onto nitrocellulose membrane (0.2 µm, PROTRAN; Schleicher & Schuell, Darmstadt, Germany), membranes were blocked with 6% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20. PKC isoforms were immunodetected using specific antibodies directed against each isoform (dilutions 1:20,000, 1:100, 1:6000, 1:15,000, 1:5000, and 1:200 for PKC{alpha}, -ß2, -{delta}, -{epsilon}, -{zeta}, and -{theta}, respectively) and the enhanced chemiluminescent detection system (Amersham Pharmacia Biotech, Les Ulis, France). Blots were exposed to Kodak XAR-5 films (Eastman Kodak Co., Rochester, NY). Quantification of PKC expression was determined using densitometric scanning and image analysis software (NIH Image, version 1.62).

Transfection of LßT2 with the cAMP-sensitive reporter plasmid {Delta}MMTV-Luc and DN mutants of PKC
Expression vectors for DN mutants of PKC isoforms {alpha}, {delta}, {epsilon}, and {theta} were generated as described (31). All constructs are in the mammalian expression vector pHACE, which provides a hemagglutinin epitope and encodes a full-length PKC with a single-point mutation that abolishes the ATP-binding ability (K368R for PKC{alpha}, K376R for PKC{delta}, K437R for PKC{epsilon}, and K409R for PKC{theta}). Transfections were performed using the LipofectAMINE-2000 assay (Invitrogen Life Technologies) according to the manufacturer’s recommendations. Triplicate samples of LßT2 cells were plated in 24-well plates and grown to 80–90% confluence for 24 h before transfection. Cells were then cotransfected with 150 ng of the cAMP-sensitive reporter plasmid {Delta}MMTV-Firefly Luciferase (wtCRE) containing several copies of the canonical cAMP-responsive element and, when appropriate, with 50 ng of one of the DN constructs. A combination of DN PKC{delta} and PKC{epsilon} (25 ng/well of each) was also tested. Transfection with equal amounts of pcDNA3 empty vector was used as control. Cotransfection of cells with pTK-renilla luciferase plasmid (30 ng) (Promega Corp., Charbonnières, France) was used as an internal control of transfection efficacy. Transfection efficacy was not significantly different between the different DN constructs (TK-renilla luciferase activity was, respectively, 81 ± 9, 102 ± 5, 88 ± 10, and 93 ± 10% of pcDNA3 for DN-PKC{alpha}, -{delta}, -{epsilon}, and -{theta} transfected cells). Cells were grown overnight in 2% fetal calf serum DMEM and then stimulated or not (basal conditions) by 100 nM GnRH agonist in the presence of 250 µM IBMX during 6 h. Cells extracts were prepared, and luciferase activity was measured using the dual-luciferase reporter assay system (Promega).

Statistical analysis
All given values are the mean ± SEM of at least three separate experiments, typically with three replicates for each experimental group. Statistical differences were first determined by one-way ANOVA followed by Dunnett’s t test for multiple comparisons. Individual pairwise comparisons were performed using Student’s t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH activates the cAMP pathway in LßT2 cells
In the first series of experiments, the potential coupling of GnRH to the AC/cAMP pathway was examined by incubating LßT2 gonadotrope cells for increasing periods of time (5 min to 20 h) with the GnRH agonist, [D-Trp6]GnRH (100 nM) in the presence of 250 µM IBMX and measuring the intracellular cAMP accumulation. As illustrated in the time-course (Fig. 1AGo) and concentration-dependence (Fig. 1BGo) experiments, GnRH activates the cAMP pathway in LßT2 cells. As shown in Fig. 1AGo, intracellular cAMP accumulation was 172 ± 10% of basal at 30 min and reached near maximal at 2 h. A maximal 254 ± 27% of basal was observed after 4 h, and cAMP levels remained elevated during the entire examined period (20 h). No significant increase in cAMP levels was detected after short stimulations (5 or 15 min) with GnRH. Increasing GnRH concentrations from 10–11 to 10–7 M resulted in a significant and dose-dependent increase of intracellular cAMP levels (Fig. 1BGo). Under a 2-h stimulation, the maximal response (215 ± 4% over basal) was attained at 10–8 M, and the deduced EC50 value was 0.30 ± 0.07 nM. Preincubation of cells with the GnRH antagonist antarelix completely abolished cAMP accumulation, demonstrating the specificity of the GnRH effect. Conversely, no significant increase in cAMP levels was measurable in {alpha}T3-1 gonadotrope cells even with maximal GnRH concentration (data not shown), indicating that GnRH-induced cAMP accumulation is dependent on the cell context.


Figure 1
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  		FIG. 1. Characteristics of GnRH-induced cAMP accumulation in the LßT2 gonadotrope cell line. LßT2 cells were grown in 12-well plates and loaded overnight with 2 µCi/well [2,8-3H]adenine in serum-free DMEM. Cells were stimulated with the indicated drugs after a 30-min preincubation period in the presence of the phosphodiesterase inhibitor IBMX (250 µM), and the intracellular [3H]cAMP produced was determined by chromatography as described in Materials and Methods. Results are expressed as the percentage over basal. The average basal cAMP production ranged from 21–35 fmol/well. A, Time course of GnRH-induced cAMP accumulation. LßT2 cells were incubated with (bullet) or without ({circ}) 100 nM GnRH agonist for increasing periods of time (5 min to 20 h), and the intracellular cAMP accumulation was determined. Data shown are mean ± SEM of three to six experiments. *, P ≤ 0.05; **, P ≤ 0.01 compared with basal. B, Concentration dependence of GnRH-induced cAMP accumulation. LßT2 cells were stimulated for 2 h with increasing concentrations (0–100 nM) of GnRH agonist. The apparent affinity (EC50) and the maximal velocity (Vmax) were determined as described (49 ). Some cells were pretreated for 30 min with the GnRH antagonist antarelix ( Figure 1, 10 µM). Data shown are mean ± SEM of three to eight experiments. **, P ≤ 0.01 compared with unstimulated cells. C, Differential efficiency of GnRH agonist and PACAP38 to induce cAMP accumulation. LßT2 cells were stimulated for increasing times (5–120 min) by either the GnRH agonist (100 nM) or PACAP38 (100 nM), and the intracellular cAMP produced was assayed. Data are expressed in percentage of the respective maximal response and are shown as mean ± SEM of two to three experiments.

 
The GnRH-induced cAMP production was compared with the neuropeptide PACAP, known to be efficiently coupled to the cAMP pathway (32). PACAP strongly stimulates cAMP production with a maximal stimulation of 120-fold over basal. As shown in Fig. 1CGo, half-maximal effect was attained as early as after 5 min, whereas no measurable increase of cAMP production was induced by GnRH at the same time. Maximal stimulation was observed at 30 min for PACAP (Fig. 1CGo) and only at 4 h for GnRH (Fig. 1AGo). To examine whether the observed latency may be due to the synthesis of an intermediate protein, cells were treated with cycloheximide. However, this treatment did not affect the cAMP increase in response to GnRH, demonstrating the lack of requirement for protein neosynthesis in this process (173 ± 24 vs. 188 ± 20% over basal with or without cycloheximide, respectively, data not shown).

PKC activity but not Ca2+ is required for GnRH-induced cAMP production in LßT2 cells
The observed lag in the cAMP response to GnRH may reflect indirect mechanisms of AC activation. The activity of several AC isoforms can be increased by Ca2+ or via phosphorylation by PKC. Because GnRH-R activation leads to an immediate increase in cytosolic calcium as well as a rapid activation of PKC, we investigated whether these events might mediate AC activation by GnRH. Elevation of cytosolic Ca2+ with the Ca2+-selective ionophore A23187 did not affect the levels of intracellular basal cAMP (Table 1Go), suggesting that Ca2+-activated AC isoforms are not predominant in LßT2 cells. Furthermore, reduction of GnRH-induced Ca2+ stores mobilization by incubating LßT2 cells in a Ca2+-free medium (250 mM EGTA) and thapsigargin (2 µM), known to inhibit refilling of the reticulum Ca2+ pool (33), had no significant effect on the GnRH-induced cAMP accumulation (Table 1Go).


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  		TABLE 1. GnRH-induced cAMP accumulation is calcium independent in LßT2 cells

 
We next evaluated the possible involvement of PKC. As illustrated in Fig. 2AGo, stimulation of LßT2 cells with the phorbol ester PMA, known to directly activate most PKC, led to a significant and time-dependent increase in intracellular cAMP concentration. Levels were significantly raised after a 30-min lag period and then attained a plateau after 120 min of stimulation. No significant difference was observed between PMA and GnRH at each time. Moreover, costimulation of cells with GnRH and PMA did not lead to any additional significant increase of cAMP levels compared with PMA alone, suggesting that PKC mediated the GnRH effect. In agreement with such a hypothesis, treatment of cells with the broad-spectrum PKC inhibitor bisindolylmaleimide I, GF109203X (2 µM), for 2 h caused a 58% reduction in GnRH-induced cAMP accumulation (Fig. 2BGo). Treatment of cells with Gö6976 (1 µM), which inhibits conventional PKC and PKCµ (34), had no influence on GnRH-induced cAMP increase (Fig. 2BGo), suggesting a major involvement of novel and/or atypical isoforms.


Figure 2
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  		FIG. 2. GnRH-induced cAMP accumulation mainly depends on PKC activation in LßT2 cells. A, Effect of the PKC activator PMA. LßT2 cells were stimulated (gray bar) or not (basal, white bar) for increasing periods of time (5 min to 4 h) with the phorbol ester PMA (50 nM). GnRH agonist-induced cAMP accumulation is also shown (black bar). Inset, Effect of a costimulation with PMA and GnRH agonist for 30 min (hatched column). Data, expressed as a percentage over basal at each time, are mean ± SEM of 4 to 7 experiments. *, P ≤ 0.05; **, P ≤ 0.01 compared with basal. B, Effect of selective pharmacological PKC inhibitors. LßT2 cells were pretreated or not (white bar) for 30 min with GF109203X (2 µM) or Gö6976 (1 µM) and then exposed to 100 nM GnRH agonist for the next 2 h. Data are mean ± SEM of four to seven experiments and are expressed as a percentage of maximal response to GnRH agonist. GF109203X did not significantly affect basal cAMP production (94 ± 5% of control), whereas a slight reduction was observed with Gö6976 (86 ± 4% of control). This difference was taken into account in data calculation. **, P ≤ 0.01 compared with cells that were not pretreated.

 
Novel PKC isoforms {delta} and {epsilon} mediate GnRH-induced cAMP accumulation in LßT2 cells
To further discriminate which isoform(s) might be involved in GnRH-induced cAMP production, we then evaluated the consequences of selective down-regulation of PKC isoforms on GnRH-induced cAMP accumulation. As illustrated in Fig. 3Go, inset, overnight pretreatment of LßT2 with PMA (1 µM) or GnRH (100 nM) induced a drastic and differential decrease in PKC isoforms in agreement with previously described data (11, 35). PMA efficiently down-regulated both conventional and novel PKC, i.e. PKC{alpha}, -ß2, -{delta}, -{epsilon}, and -{theta}, respectively, by 91 ± 2, 92 ± 2, 71 ± 7, 62 ± 7, and 93 ± 2%. Conversely, the same treatment with GnRH down-regulated only the novel PKC{delta}, -{epsilon}, and -{theta} isoforms by 37, 43, and 60%, respectively. Neither treatment affected the amount of PKC{zeta}. As illustrated in Fig. 3Go, these long-term treatments with PMA or GnRH both induced significant reduction of the cAMP response to GnRH by 37 ± 3 and 48 ± 4%, respectively. Altogether, these experiments suggest that GnRH-induced cAMP production does not require conventional or atypical PKC but rather novel isoforms {delta}, {epsilon}, and/or {theta}.


Figure 3
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  		FIG. 3. Effect of selective PKC isoforms down-regulation on GnRH-induced cAMP accumulation. Selective down-regulation of PKC isoforms was performed by incubating LßT2 cells overnight with either 1 µM PMA (gray bar) or 100 nM GnRH agonist (black bar). Cells were then stimulated with 100 nM GnRH agonist for 2 h, and cAMP was assayed. Data are mean ± SEM of three to five experiments and are expressed as a percentage of maximal response to GnRH agonist. **, P ≤ 0.01 compared with GnRH agonist response. Insets, Immunoblot analysis of the PKC isoforms under each treatment. Western blots of LßT2 cells lysates were submitted to specific antibodies directed against PKC{alpha}, -ß2, -{delta}, -{epsilon}, -{zeta}, and -{theta} as described in Materials and Methods. The immunoreactive bands shown are representative of three to four independent experiments.

 
To assess whether one or several of the expressed novel PKC isoforms was involved, we used a DN mutant strategy to specifically inactivate one of these endogenous PKC in LßT2 cells. To this end, LßT2 cells were cotransfected with DN mutant expression vectors and the cAMP-sensitive reporter plasmid containing the {Delta}MMTV-Luc(wtCRE) promoter (36). The contribution of the PKC isoforms was evaluated by their ability to affect GnRH-induced promoter activity. As illustrated in Fig. 4Go, GnRH induced a 2.7 ± 0.2-fold increase over basal of Luc activity in control pcDNA3-cotransfected cells. In agreement with our pharmacological data, inhibition of conventional PKC{alpha} with DN PKC{alpha} did not affect the GnRH activation of the cAMP-sensitive promoter. Transfection with even higher amounts of DN PKC{alpha}, i.e. up to 150 ng, did not lead to any significant effect (data not shown). No effect of DN PKC{theta} construct was observed suggesting the noninvolvement of this novel PKC isoform despite its being a target of GnRH as evidenced by down-regulation experiments. Conversely, transfection of cells with DN PKC{delta} or DN PKC{epsilon} efficiently decreased GnRH-induced Luc activity by 71 ± 11 and 49 ± 7%, respectively. Cotransfection of both DN PKC{delta} and DN PKC{epsilon} did not lead to any additional inhibition of GnRH-induced promoter activity. None of the constructs had any significant influence on basal luciferase activity. Expression of each DN PKC isoform was confirmed by Western blot analysis with a hemagglutinin antibody, and cotransfection of cells with TK-renilla luciferase expression vector was used to assess transfection efficiency (data not shown). Taken together, these experiments provide evidence that PKC{delta} and -{epsilon} constitute the two major endogenous PKC isoforms involved in the stimulation of the cAMP pathway by GnRH in LßT2 cells.


Figure 4
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  		FIG. 4. GnRH-stimulated transcriptional activity of the cAMP-dependent CRE-MMTV promoter in LßT2 cells: effect of DN PKC. LßT2 cells were transiently cotransfected with the cAMP-sensitive reporter {Delta}MMTV-Luc plasmid (150 ng/well) and the empty vector pcDNA3 (50 ng/well) or PKC DN constructs (PKC{alpha}-DN, PKC{delta}-DN, PKC{epsilon}-DN, or PKC{theta}-DN; 50 ng/well). A combination of both PKC{delta}- and PKC{epsilon}-DN (25 ng/well of each) was also tested. Cells were then stimulated or not (basal) with 100 nM GnRH agonist for 6 h, and luciferase activity was measured from cell lysates and expressed as a percentage over its respective basal. None of the constructs significantly affected basal luciferase activities (83 ± 8, 122 ± 13, 94 ± 12, and 115 ± 4% for DN PKC{alpha}, PKC{delta}, PKC{epsilon}, and PKC{theta}, respectively). Data shown are mean ± SEM and are representative of three to eight independent experiments in which each assay was done in triplicate. a, P ≤ 0.01 compared with GnRH agonist response of pcDNA3-cotransfected cells. **, P ≤ 0.01; *, P ≤ 0.05 compared with its respective basal.

 
PKC-sensitive AC5 and AC7 isoforms, but not AC2, are expressed in LßT2 cells
Because these data argue for the involvement of PKC in the GnRH effect and the ability of PKC to directly phosphorylate and activate some AC isoforms has been reported, we evaluated whether preexposure of LßT2 cells to GnRH may potentiate cAMP response to forskolin. To this end, cells were pretreated with GnRH for 2 h. Figure 5Go shows that GnRH pretreatment enhanced by 29 ± 8% forskolin-induced cAMP accumulation in LßT2 cells, suggesting that AC intrinsic activity has been modified by previous exposure to GnRH.


Figure 5
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  		FIG. 5. Effect of GnRH pretreatment on forskolin-stimulated cAMP accumulation in LßT2 cells. LßT2 cells were pretreated or not for 2 h with 100 nM GnRH agonist in the presence of 250 µM IBMX. The medium was removed, and forskolin (10 µM) was then added for 5 min. Intracellular cAMP accumulation was assayed and expressed as a percentage of basal in cells that were not pretreated. Data are expressed as mean ± SEM of four experiments. **, P ≤ 0.01 compared with basal in cells that were not pretreated; a, P ≤ 0.05 compared with forskolin-induced cAMP production in cells that were not pretreated.

 
To further address the likely targets of PKC, we next characterized the PKC-sensitive AC isoforms, i.e. AC2, AC5, and AC7, in LßT2 cells. Expression of AC isoforms was first determined by RT-PCR using intron-spanning primers designed to specifically amplify each PKC-sensitive isoform. Mouse brain, known to express all the AC isoforms, was used as an internal positive control. As illustrated in Fig. 6Go, single PCR products of the expected size were detected for each AC isoform in mouse brain. AC5 and AC7 transcripts were both detected in LßT2 as well as in {alpha}T3-1 cells, whereas no product was obtained for AC2 in either gonadotrope cell line. Expression of AC5 and AC7 was further evaluated using immunocytochemical staining with specific antibodies. In LßT2 cells, both the AC5/6 and AC7 antibodies produced a clear labeling that was more abundantly localized at the membrane region (Fig. 7Go, Aa and Ba). As shown in Fig. 7Go, Ab and Bb, the specific labeling was abrogated after AC5/6 or AC7 peptide adsorption. In {alpha}T3-1 cells, a staining was obtained with the AC5/6 antibody that was also suppressed by the appropriate peptide adsorption (Fig. 7Go, Ca and Cb), but no significant labeling was observed with the AC7 antibody (Fig. 7DGoa).


Figure 6
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  		FIG. 6. RT-PCR analysis of PKC-sensitive AC isoforms in LßT2 cells. Total RNA was extracted from LßT2 cells, {alpha}T3-1 cells, and mouse brain (positive control) and reverse-transcribed into cDNA. PCR amplification of AC isoforms 2, 5, and 7 was performed as described in Materials and Methods. PCR performed on reverse-transcriptase-deleted assay product as negative controls generated no band (not shown). A 100-bp DNA ladder was used as a size marker. The data shown are representative of three different experiments.

 

Figure 7
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  		FIG. 7. Immunocytochemical identification of PKC-sensitive AC isoforms in gonadotrope cell lines. LßT2 and {alpha}T3-1 cells were plated in SUPCELL8 slides and cultured for 24 h before fixation with paraformaldehyde. Cells were then subjected to immunocytochemical staining using antibodies directed against AC isoforms 5/6 (AC5/6) and 7 (AC7) as described in Materials and Methods (a). Negative controls were performed by adsorption of each AC antibody with an excess of appropriate peptide antigen (b). A and B, AC5/6 and AC7 detection in LßT2 cells, respectively; C and D, AC5/6 and AC7 detection in {alpha}T3-1 cells, respectively. Note the absence of signal for AC7 in {alpha}T3-1 cells. Scale bar, 50 µm.

 
GnRH-induced cAMP formation required sustained stimulation of GnRH-R
Because GnRH is secreted in a pulsatile fashion, leading to intermittent activation of its receptor, we next compared the effects on cAMP accumulation of pulsatile vs. continuous administration of GnRH. LßT2 cells were exposed to three 5-min pulses of GnRH (100 nM) delivered over a 4-h period. This pulse regimen was not able to induce cAMP accumulation (Fig. 8Go) in contrast to prolonged exposure of cells during the same period of time.


Figure 8
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  		FIG. 8. cAMP accumulation in response to pulsatile vs. continuous stimulation of LßT2 cells by GnRH. After a 30-min preincubation period in serum-free DMEM with 250 µm IBMX, LßT2 cells were either exposed to 4 h incubation with 100 nM GnRH (continuous stimulation) or submitted to three periods of 5 min stimulation by GnRH over the same period of time (pulsatile stimulation). After each short stimulation, GnRH-containing medium was removed; cells were carefully rinsed and then left in control medium during the interpulse intervals. Intracellular cAMP accumulation was expressed as a percentage of cAMP produced in respective basal. Data are mean ± SEM of three separate experiments. **, P ≤ 0.01 compared with basal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although cAMP has been clearly involved in the regulation of gonadotrope function, the ability of GnRH to induce cAMP accumulation in gonadotrope cells as well as the mechanisms coupling GnRH-R to cAMP remain a matter of controversy. Our study clearly shows that GnRH is functionally linked to the cAMP pathway in LßT2 gonadotrope cells and provides evidence for an unusual coupling between GnRH-R and the cAMP pathway requiring the activation of novel PKC{delta} and -{epsilon}.

Using cAMP biochemical assays, we established that GnRH-R activation specifically increases the intracellular cAMP levels in a time- and concentration-dependent manner, with EC50 and maximal stimulation values in agreement with those reported in hemipituitaries (16). The coupling of GnRH with the cAMP pathway was further demonstrated by the ability of the neuropeptide to induce the cAMP-sensitive {Delta}MMTV-Luc(wtCRE) promoter activity. These results confirmed and further extended the previous observation of Liu et al. (21).

GnRH-induced rises of cAMP levels were observed in the presence of a potent phosphodiesterase inhibitor and, therefore, are assumed to reflect activation of AC rather than inhibition of cAMP degradation. Surprisingly, the generation of cAMP in response to GnRH required a longer time to develop compared with PACAP, which induced a rapid and robust increase in intracellular cAMP levels. This characteristic prompted us to evaluate whether some indirect, Gs{alpha}-independent mechanisms may contribute to the GnRH coupling to the cAMP pathway.

Because GnRH rapidly increases the cytosolic Ca2+ in gonadotrope cells, we suspected that GnRH effect on cAMP might reflect Ca2+/calmodulin-mediated AC activation. This hypothesis was ruled out, first because cAMP accumulation was not mimicked by Ca2+ entry with A23187 and also because acute depletion of cytosolic Ca2+ with EGTA and thapsigargin did not prevent GnRH-induced cAMP production. In addition to increasing cytosolic Ca2+ in gonadotrope cells, GnRH also rapidly activates PKC, which have been implicated in the cross-talk between phospholipase C and AC pathways in many cell lines. Here, we demonstrated that PKC mediates, at least partly, the stimulation of cAMP pathway by GnRH in LßT2 cells. Indeed, PMA was able to increase intracellular cAMP levels, and the effect of GnRH was markedly reduced by the PKC inhibitor bisindolylmaleimide I. The fact that PMA and GnRH effects were not additive further confirms that PKC mediate GnRH action. Because the PKC inhibitor only partly blocked the GnRH effect, additional mechanisms of coupling may occur, probably involving Gs{alpha} as demonstrated by Liu et al. (21) using a cell-permeable Gs inhibitory peptide. Surprisingly, these authors reported that such a peptide completely blocked the GnRH-induced cAMP accumulation. One possible explanation for this apparent discrepancy with our results might be that the inhibitory peptide also disrupted GnRH coupling with Gq. In agreement with this hypothesis, a DN mutant of Gs was reported to block signaling from the calcitonin receptor to both Gs and Gq (37).

LßT2 cells express several PKC isoforms, {alpha}, ß2, {delta}, {epsilon}, {theta}, and {zeta}, belonging to the three known PKC classes (11, 12). Although there is growing evidence that each isoform may subserve distinct cellular activities, their respective contributions in the GnRH signaling network remain obscure in gonadotrope cells. To discriminate which PKC isoform(s) might be involved in activation of the cAMP pathway, we used pharmacological inhibitors and chronic GnRH or PMA treatments to impair the activity and expression of selected isoforms, respectively. The involvement of conventional PKC{alpha} and -ß2 was eliminated because Gö6976, an inhibitor of conventional PKC (34), had no effect on GnRH-induced cAMP accumulation. The involvement of atypical PKC was also rejected because long-term treatment of cells with GnRH or PMA reduced the GnRH-induced rise in cAMP levels without altering the expression of PKC{zeta}. Novel PKC that are inhibited by bisindolylmaleimide I and are the only isoforms significantly down-regulated by GnRH are the likely candidates mediating GnRH activation of the cAMP pathway. To discriminate between the different novel isoforms, we next used selective inhibition of endogenous PKC by expressing DN, catalytically inactive forms of PKC in LßT2 cells. These DN mutants have previously been shown to be powerful inhibitors of a targeted PKC isoform in several cell lines by competing with the endogenous PKC for cofactors, substrates, and cellular binding sites (31, 38, 39). We took advantage of the coexpression of a cAMP-sensitive reporter gene to evaluate the impact of DN mutants and demonstrated that PKC{delta} and -{epsilon} couple GnRH-R to the cAMP pathway in LßT2 cells. Interestingly, both isoforms seem to be indifferently recruited by GnRH-R because both DN significantly impaired GnRH signaling. The involvement of Ca2+-insensitive novel PKC is in agreement with our observation that Ca2+ did not affect the GnRH effect. PKC{delta} was recently reported to couple GnRH to the ERK cascade (12). This, together with our results, suggests that PKC{delta} mediate coupling of GnRH-R to different signaling pathways in LßT2 cells.

This proposed new mechanism of coupling between the GnRH-R and cAMP pathway is only possible provided that PKC-sensitive AC isoforms are expressed in LßT2 cells. To date, AC2, AC5, and AC7 have been shown to be directly phosphorylated by PKC (39, 40, 41). We characterized AC5 and AC7 in {alpha}T3-1 and LßT2 cells. In contrast, as demonstrated by RT-PCR analysis, AC2 appears not to be expressed in any gonadotrope cell lines. To our knowledge, this is the first identification of AC isoforms in gonadotrope cells. The AC7 isoform was unambiguously identified both at its transcript and protein levels, whereas expression of AC5 protein remains to be confirmed because the antibody used does not discriminate between AC5 and AC6 isoforms. We postulate that AC5 and/or AC7 may be phosphorylated by GnRH-activated PKC in LßT2 cells. The fact that GnRH pretreatment of LßT2 cells potentiated AC activity, as illustrated by the increased cAMP response to forskolin stimulation, is in agreement with such a hypothesis. In {alpha}T3-1 cells, we characterized only AC7 transcript but not the protein form, indicating that {alpha}T3-1 and LßT2 gonadotrope cell lines express a different set of PKC-sensitive AC. This may contribute to the observed inability of these cells, known to be less differentiated than LßT2 cells (26), to produce detectable amounts of cAMP in response to GnRH.

Intriguingly, the coupling of GnRH-R to the cAMP pathway appears dependent on the characteristics of GnRH stimulation. Intermittent stimulation of cells with GnRH, known to stimulate Ca2+ release in pituitary gonadotropes as well as ERK pathway in LßT2 cells (1, 42) was ineffective in inducing cAMP. We previously reported that the NOS I gene was induced under prolonged stimulation (24–48 h) of rats in vivo with the long-lasting GnRH agonist, Decapeptyl, contrasting with the inhibition of LHß and FSHß expression as well as gonadotropin release (5). Similarly, the {alpha}-subunit gene was reported to be activated under sustained receptor activation (43, 44). This showed that GnRH signaling can be maintained in gonadotrope cells under conditions that induce desensitization for most G protein-coupled receptors. The peculiar structure of mammalian GnRH-R, i.e. lack of the cytoplasmic tail that protects it from rapid agonist-induced internalization, is the likely explanation of this phenomenon (45, 46, 47). Interestingly, we demonstrated that both {alpha}-subunit and NOS I genes are induced by GnRH via the cAMP pathway (13, 48). Based on these observations and our present study, we speculate that GnRH-R may switch to the cAMP pathway under high GnRH stimulation frequency, as observed for example during the preovulatory surge, and initiate the activation of a new set of targets in gonadotrope cells.


   Acknowledgments
 
We are grateful to Dr. Pamela Mellon, University of California, San Diego (San Diego, CA) for providing the LßT2 cell line. We also thank Dr. Dietmar Spengler, Max-Planck Institute of Psychiatry (Munich, Germany), and Dr. Michelle Breuiller-Fouché, Institut National de la Santé et de la Recherche Médicale, U427 (Paris, France) for their kind gift of {Delta}MMTV-Luc(wtCRE) plasmid and PKC antibodies, respectively.


   Footnotes
 
This work was supported by grants from the Centre National de la Recherche Scientifique and Université Pierre et Marie Curie (Paris, France). S.L. is a recipient of a fellowship from the Ministère de la Recherche et de l’Education Nationale.

A preliminary report of this work was presented at the 88th Annual Meeting of The Endocrine Society, Boston, Massachusetts, 2006.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 21, 2006

Abbreviations: AC, Adenylyl cyclase; DN, dominant negative; GnRH-R, GnRH receptor; IBMX, 3-isobutyl-1-methylxanthine; NOS, NO synthase; PACAP, pituitary adenylyl cyclase-activating polypeptide; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C.

Received November 3, 2006.

Accepted for publication December 11, 2006.


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 Introduction
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 Results
 Discussion
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