GnRH signaling pathways regulate differentially the tilapia gonadotropin subunit genes

Molecular and Cellular Endocrinology 189 (2002) 125– 134 www.elsevier.com/locate/mce

GnRH signaling pathways regulate differentially the tilapia gonadotropin subunit genes G. Gur a, D. Bonfil b, H. Safarian a, Z. Naor b, Z. Yaron a,c,* a Department of Zoology, Tel-A6i6 Uni6ersity, Tel A6i6 69978, Israel Department of Biochemistry, Tel-A6i6 Uni6ersity, Tel A6i6 69978, Israel c The Norman and Rose Lederer Chair of Experimental Biology, Tel-A6i6 Uni6ersity, Tel A6i6 69978, Israel b

Received 13 July 2001; accepted 30 October 2001

Abstract Exposure of tilapia pituitary cells in culture to salmon gonadotropin-releasing hormone (sGnRH; 0.01– 100 nM) elevated the phosphorylated extracellular signal-regulated kinase (pERK) levels. sGnRH also elevated the a, FSHb and LHb subunit mRNA levels. The phorbol ester, 1-O-tetradecanoyl phorbol-13-acetate (TPA; 12.5 nM) increased pERK levels, whereas protein kinase C (PKC) depletion or inhibition by GF109203X (GF; 0.01– 10 mM) suppressed GnRH-activated ERKs. GF too abated the GnRH-induced a and LHb mRNA levels, but had no effect on those of FSHb. Forskolin (0.001– 100 mM) activated ERK, while inhibition of protein kinase A (PKA) by H89 (0.01–10 mM) suppressed pERK levels and all GnRH-stimulated gonadotropin subunit transcripts. Exposure of cells to the mitogen-activated protein kinase kinase (MAPK kinase; MEK) inhibitor (PD98059; PD 10, 25 and 50 mM) completely blocked GnRH-induced increase in ERKs activation. Furthermore, PD suppressed the a and LHb mRNA responses to GnRH, but had no effect on FSHb mRNA levels. It is suggested that in tilapia the differential regulation of gonadotropin subunit gene expression by GnRH results from a divergent recruitment of signal transduction pathways, activated upon GnRH binding; PKC-ERK cascade is involved in elevating a and LHb mRNAs, whereas induction of FSHb transcript is ERK-independent and is under direct cAMP-PKA regulation or through other MAPK cascades. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tilapia; GnRH; PKC; PKA; MAPK; a; LHb; FSHb

1. Introduction Gonadotropin-releasing hormone (GnRH) is the main hypothalamic regulator of the reproductive system in teleost fish as in mammals. Upon binding to its G protein-coupled receptors (GPCRs) in the pituitary gonadotrophs, GnRH elicits multiple signaling events leading to the synthesis and release of FSH and LH (Naor et al., 1998). In tilapia too, GnRH binding to its membrane receptor induces signaling steps that include Ca2 + influx and mobilization from intracellular sources (Levavi-Sivan and Yaron, 1989). Phospholipase C

* Corresponding author. Tel.: +972-3-640-9398; fax: + 972-3-6409403. E-mail address: [email protected] (Z. Yaron).

(PLC) activated by GnRH stimulates phosphoinositide turnover, resulting in the formation of inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG) and subsequently the activation of protein kinase C (PKC). Phospholipase A2 and arachidonic acid were also found as components of the GnRH second messenger system (Yaron and Levavi-Sivan, 1990). Furthermore, exposure of tilapia pituitary cells in perifusion and in static culture to GnRH also resulted in increased cAMP levels. It was suggested, therefore, that the cAMPprotein kinase A (PKA) pathway acts as an additional signaling cascade in tilapia (Levavi-Sivan and Yaron, 1992; Yaron et al., 2001). Whereas the intracellular messenger cascades mediating the GnRH stimulation of gonadotropin release in fish are becoming clearly mapped, those involved in GnRH regulation of gonadotropin subunit gene expres-

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sion remain scantily understood. Previously, GnRH was found to stimulate LHb subunit transcript via the PKC and cAMP-PKA pathways, and elevation of cAMP was found to stabilize the transcript (Melamed et al., 1996). In addition, GnRH-induced expression of glycoprotein hormone a (a) subunit gene was reported to involve both transduction pathways (Gur et al., 2001), while preliminary results have indicated the involvement of only PKA in that of FSHb. Nevertheless, little is known in fish about the molecular events downstream of GnRH-activation of PKC and cAMP-PKA. Like receptor tyrosine kinases (RTKs), some GPCRs were found to further convey the extracellular signal by a set of sequentially activated cytosolic protein kinases collectively known as the MAPK cascades (Gutkind, 2000). In mammalian cell lines, MAPK pathways are activated by GPCRs via mechanisms involving PKC, PKA, the G-proteins bg subunits, and by various potential sites of activation such as Ras and Raf-1 (Seger and Krebs, 1995). GnRH was found to activate various MAPK cascades in mammals i.e. extracellular signalregulated kinase (ERK; Roberson et al., 1995; Sundaresan et al., 1996; Reiss et al., 1997), Jun N-terminal kinase (JNK; Levi et al., 1998) and P38 MAPK (Roberson et al., 1999; Naor et al., 2000; Cheng and Leung, 2000; Haisenleder et al., 1998). Of particular interest is ERK (p42, p44), which is involved in gene expression of the a (Roberson et al., 1995; Naor et al., 2000; Haisenleder et al., 1998) and FSHb subunits (Haisenleder et al., 1998), while the JNK cascade is necessary to elicit the rat LHb promoter activity in LbT2 cells (Yokoi et al., 2000). The present work aimed to clarify the role of GnRH transduction pathways in regulating gene expression of gonadotropin subunits in tilapia, and to shed light on the conservation of these pathways along vertebrate evolution.

2. Materials and methods

2.1. Fish Experiments were carried out on primary cultures of pituitary cells from immature (BW 35– 110 g, GSI of 0.08 90.01, n=29) or early maturing (BW 40– 108 g, GSI of 0.127 9 0.002– 0.19 90.03, n =30) male tilapia hybrids (Oreochromis niloticus×O. aureus). Fish were collected from local farms and housed in the laboratory in 600-l tanks under natural photoperiod and ample aeration. In 21–30 specimens from each batch of donor fish, the gonado-somatic-index (GSI) was determined as a parameter reflecting the reproductive stage. The research procedures were approved by the Tel-Aviv University Committee for Animal Care and Use.

2.2. Culture of dispersed pituitary cells Primary cultures of pituitary cells were prepared as described previously (Levavi-Sivan and Yaron, 1992). Briefly, pituitary cells were collectively dispersed, counted, and then plated at 3×106 cells per well in 3 ml medium or 1× 106 cells per well in 4 ml medium [M199, 10% fetal calf serum (FCS), 10 mM HEPES, 1% antibiotics (Pen-Strep-Nystatin suspension); Biological Industries, Bet Ha’emek, Israel], and cultured for 4 days at 28 °C under 5% CO2. For MAPK determination, at the end of the third day the cells were serum-starved for 18 h in M-199 containing 0.1% FCS, after which they were challenged with the test substances. For measurement of the gonadotropin subunit mRNA steady-state levels, cells were incubated for 4 days and then challenged with GnRH for 24 h, with the inhibitors given 15 or 30 min prior to hormone administration. Salmon gonadotropin-releasing hormone ([Trp7, Leu8]-GnRH; sGnRH), the PKC inhibitor, GF 109203X (GF), and the phorbol ester, 1-O-tetradecanoyl phorbol-13-acetate (TPA) were all purchased from Sigma (St. Louis, MO, USA). The MEK inhibitor, 2%-amino-3%-methoxyflavone (PD98059; PD) and the PKA inhibitor, H89, were purchased from Biomol Research Laboratories (Plymouth Meeting, OA, USA). sGnRH was dissolved directly in the medium, whereas GF, H89, TPA and PD were dissolved first in dimethylsulfoxide. The final concentration of the solvent in the medium did not exceed 0.1% and lacked any visible effect on the cells.

2.3. MAPK (ERK1 -2) acti6ity At the end of the incubation period, the cells were rinsed in ice-cold phosphate buffer saline (PBS) and then with buffer A [50 mM b-glycerophosphate, (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM sodium orthovanadate]. Subsequently, the cells were harvested in ice-cold buffer H [50 mM b-glycerophosphate, (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate, 1 mM benzamidine, 10 mg/ml aprotinin, 10 mg/ml leupeptin and 2 mg/ml pepstatin A]. Cell lysates were disrupted by two 7 s sonications (50 W) on ice, followed by centrifugation at 20 000× g (4 °C; 15 min). The supernatant was assayed for protein content (Bradford reagent, Bio-Rad, UK). Aliquots of cell supernatant containing cytosolic protein (10 mg) were resolved via electrophoresis through a denaturing 10% PAGE-SDS gel (ratio acrylamide to bisacrylamide 29:1) and electrotransferred to nitrocellulose membrane at 4 °C and 100 mV in transfer buffer (50 mM glycine and 50 mM Tris–HCl buffer, pH 8.8). The membranes were then blocked for 60 min

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in 5% dried low fat milk (w/v) and 0.05% Tween-20 in TBS (20 mM Tris, pH 8.3 and 150 mM NaCl). For detection of phosphorylated (active) extracellular signal-regulated kinase (pERK; p44/p42 ERK, corresponding to ERK1/2), membranes were probed with monoclonal mouse anti-activated-MAPK (diphosphorylated ERK-1&2; Sigma, 1: 10 000 v/v). For detection of general ERK, membranes were probed with rabbit anti-MAPK (ERK-1&2; Sigma, 1:10 000 v/v). After intensive washing, the signals were visualized using horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG, respectively (both at 1:10 000 v/v), and the enhanced chemiluminescence system (Biological Industries). Signals were analyzed by scanning densitometry using TINA (PC BAS) program. The active ERK values were normalized with the values of the general ERK levels in the same sample.

2.4. RNA extraction, blotting and hybridization For measurements of the gonadotropin subunits mRNA steady-state levels, cells were incubated (1× 106 cells per well) for 4 days and then challenged with sGnRH for 24 h, alone or with the inhibitors. RNA was extracted from each well using guanidinium thiocyanate–phenol:chloroform method (Chomczynski and Sacchi, 1987) as modified by Melamed et al. (1996). Briefly, the cells were lysed in the well with 360 ml guanidinium thiocyanate (4 M). They were then homogenized and 36 ml sodium acetate (2 M, pH 4) was added. Nucleic acids were extracted in phenol: chloroform (1:1 v/v) and then precipitated with isopropanol (30 min, −20 °C). The precipitate was then rinsed in 0.5 ml ethanol (70%). Samples were dissolved in diethyl pyrocarbonatetreated water (45.2% v/v), 37% formaldehyde (4.8% v/v) and formamide (50% v/v), and were loaded onto nylon membranes (Nytran N; Schleicher and Schuell, Dassell, Germany) using slot-blot apparatus of the same manufacturer. After prehybridization of the membrane [1% sodium dodecyl sulphate (SDS), 10% dextran sulfate, 5.8% NaCl and 100 mg/ml calf thymus DNA, Sigma; 65 °C, 4 h], RNA was hybridized overnight with cDNA probes, labeled with 32P dCTP using Megaprime DNA labeling system (Amersham, UK). The tilapia FSHb and LHb subunit cDNA sequences have been described by Rosenfeld et al. (1997) and that of the a subunit by Gur et al. (2001). Subunit mRNA values were normalized against those of 18S ribosomal RNA (rRNA) in the same sample. Rinsing procedures were according to the manufacturer’s recommendations. The membranes were exposed to an imaging plate of a phosphoimager (BAS 1000, Fuji), and the data were analyzed using the TINA (PC BAS) program.

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2.5. Statistical analysis Statistical analysis was done between control and treatment groups using one-way analysis of variance (ANOVA) followed by an A posteriori Bonferroni ttest, which simultaneously compares means of all examined groups. Three independent experiments were carried out, each in triplicate, and the results are presented as means of the three experiments.

3. Results

3.1. Effect of sGnRH on ERK acti6ation and GtH mRNA le6els Dispersed pituitary cells of immature tilapia (BW 35–110 g, GSI 0.089 0.01, n= 29) were exposed to increasing doses of sGnRH (0.01–100 nM) for 15 min, after which proteins were extracted and immunoblotted with the respective antibodies. A dose-dependent elevation was seen for both pERKs, reaching a peak at 10 nM. At the higher dose, a slight decrease was noticed (Fig. 1A). Cells from the same batch were exposed to identical doses of sGnRH in order to check the peptide effect on the expression of gonadotropin subunit genes. sGnRH elevated the mRNA of a subunit up to 2.8-fold at 1–10 nM, but was lower at 100 nM, albeit remaining above basal levels (Fig. 1B). LHb and FSHb mRNA levels increased dose-dependently after exposure to sGnRH, reaching a peak of 2.8- and 2.4-fold at 1–10 nM, respectively, and declined at the higher dose (Fig. 1C and D).

3.2. Role of PKC 3.2.1. Effect of PKC acti6ation on ERK induction by GnRH Cells from early maturing male tilapia (BW 46–108 g, GSI 0.12790.015, n= 30) were exposed to an acute activation of PKC by TPA (12.5 nM) for 15 min, which resulted in a 7-fold increase in pERK levels above the control (Fig. 2). Addition of sGnRH (10 nM) did not result in any further increase. PKC depletion by TPA (12.5 nM for 16 h) resulted in a 50% decrease in pERK levels as compared with the 15 min exposure. sGnRH had no effect when added after 16 h exposure to the phorbol ester, although this treatment suppressed pERK levels by 30% compared with sGnRH alone (Fig. 2). 3.2.2. Effect of PKC inhibition on GnRH-induced ERK and ele6ation of GtH mRNAs Exposure of dispersed pituitary cells from the same

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Fig. 1. sGnRH effect on (A) pERK levels and (B –D) gonadotropin subunit mRNA levels. Pituitary cells of immature tilapia (BW 35 – 110 g, GSI 0.0890.01, n = 29) were stimulated with sGnRH (0.01 –100 nM) for 15 min (A) or 24 h (B – D). (A) pERK isoform levels are expressed as ratio to the level of general ERKs in the same sample. Statistical analysis was done separately for each pERK isoform. (B – D) a, FSHb and LHb mRNA levels. Results are expressed as percentage of the level in untreated cells. Mean 9S.E.M., n = 3. Means designated by the same letter are not significantly different (P\0.05).

group of fish to the selective PKC inhibitor, GF, given 15 min prior to sGnRH administration (10 nM), resulted in a gradual decrease in GnRH-induced ERK activation, totally abating the hormone effect at 10 mM GF (Fig. 3A). In parallel experiments from the same batch of cells, sGnRH elevated the mRNA of a, LHb and FSHb by 2.7, 2.8 and 2-fold of the control, respectively. GF suppressed the GnRH-induced elevation of a mRNA as from 0.01 mM, decreasing it to below the basal level (Fig. 3B). The PKC inhibitor also suppressed the elevation of LHb transcript in a dose-dependent manner (Fig. 3D). However, FSHb mRNA levels were not affected by any of the GF concentrations (Fig. 3C). It should be noted that GF, when given alone, also decreased the basal level of the a transcript (Fig. 3B), while no such effect was seen for that of LHb or FSHb (Fig. 3C and D).

3.3.2. Effect of PKA inhibition on GnRH-induced ERK and ele6ation of GtH mRNAs Dispersed pituitary cells from the same group of fish were exposed to the PKA inhibitor, H89, 15 min prior to GnRH administration (10 nM). The presence of H89 resulted in gradual suppression of GnRH-induced

3.3. Role of PKA 3.3.1. The effect of PKA acti6ation on MAPK induction Exposure of cells from early maturing male tilapia (BW 38 –107 g, GSI 0.199 0.03, n =30) to increasing doses of forskolin (0.001– 100 mM) for 15 min resulted in a dose-dependent elevation in both pERKs, reaching a peak at 10 mM forskolin, while a slight decrease was noted at a higher dose (Fig. 4).

Fig. 2. Effect of acute and prolonged treatment with TPA on ERK activation by sGnRH. Cells from early maturing male tilapia (BW 46 – 108 g, GSI 0.127 90.015, n =30) were pretreated with TPA (12.5 nM) for 15 min or 16 h. sGnRH (10 nM) was then added to the medium for 15 min. Mean 9S.E.M., n =3. pERK isoform levels are expressed as ratio to the level of general ERKs in the same sample. Statistical analysis was done separately for each pERK isoform. Means designated by the same letter are not significantly different (P \0.05).

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Fig. 3. Effect of PKC inhibition on (A) pERK levels and (B – D) gonadotropin subunit mRNA levels. Cells from early maturing male fish (BW 46–108 g, GSI 0.127 90.015, n= 30) were exposed to GF109203X (GF; 0.01 – 10 mM) 15 min before the addition of sGnRH (10 nM) for 15 min (A) or 24 h (B – D). (A) pERK isoform levels are expressed as ratio to the level of general ERKs in the same sample. Statistical analysis was done separately for each pERK isoform. (B –D) a, FSHb and LHb mRNA levels. Results are expressed as percentage of the level in untreated cells. Mean9 S.E.M., n = 3. Means designated by the same letter are not significantly different (P \0.05).

pERK levels, reaching the control level at 10 mM H89 (Fig. 5A). Parallel experiments from the same batch of cells showed an elevation of all three GtH subunit mRNAs in response to GnRH. Administration of H89 abated the GnRH-induced a subunit mRNA levels as from the lowest H89 dose, although at 0.1– 1 mM the inhibition was less obvious (Fig. 5B). GnRH-elevated LHb and FSHb transcript levels were reduced dose-dependently after H89 administration (Fig. 5C and D).

4. Discussion The present study provides evidence for the differential regulation of the gonadotropin subunit genes by GnRH in tilapia. This is achieved mainly by divergent signal transduction pathways participating in the mediation of GnRH effects on each of the GtH subunits.

3.4. Role of ERK Pituitary cells from early maturing fish (BW 40–100 g, GSI of 0.169 0.02, n =21) were exposed to the MEK inhibitor, PD (10– 50 mM) alone or 30 min prior to the administration of sGnRH (10 nM). PD suppressed the GnRH-induced pERK levels, gradually decreasing both pERKs to the basal levels at 50 mM (Fig. 6A). In parallel experiments from the same batch of cells, sGnRH elevated the mRNA of a, LHb and FSHb. PD suppressed the GnRH-induced elevation of the a transcript (Fig. 6B). sGnRH-stimulated increase in LHb mRNA levels was also suppressed dose-dependently by PD, decreasing it to even below the basal level (Fig. 6D). Nevertheless, FSHb mRNA level was not affected by any concentration of the MEK inhibitor (Fig. 6C).

Fig. 4. Effect of forskolin on ERK activation. Cells from early maturing male tilapia (BW 38 – 107 g, GSI 0.19 9 0.03, n =30) were exposed to forskolin (0.001 – 100 mM) for 15 min. Mean 9 S.E.M., n =3. pERK isoform levels are expressed as ratio to the level of general ERKs in the same sample. Statistical analysis was done separately for each pERK isoform. Means designated by the same letter are not significantly different (P\0.05).

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Fig. 5. Effect of PKA inhibition on (A) pERK levels and (B –D) gonadotropin subunit mRNA levels. Cells from early maturing male fish (BW 38–107 g, GSI 0.19 9 0.03, n=30) were exposed to H89 (0.01 – 10 mM) 15 min before addition of sGnRH (10 nM) for 15 min (A) or 24 h (B –D). (A) pERK isoform levels are expressed as ratio to the level of general ERKs in the same sample. Statistical analysis was done separately for each pERK isoform. (B– D) a, FSHb and LHb mRNA levels. Results are expressed as percentage of the level in untreated cells. Mean 9S.E.M., n= 3. Means designated by the same letter are not significantly different (P\ 0.05).

It should be noted that the experiments described here were carried out on dispersed pituitary cells containing all cell types in the gland. GnRH binding sites were found in the gonadotrophs, somatotrophs, lactotrophs and somatolactin (SL) cells in fish (Stefano et al., 1999; Vissio et al., 1999), which could theoretically have contributed to the experimental results. Although GnRH can affect release of the gonadotropins, growth hormone (GH), prolactin (PRL) and SL from their respective cells in fish pituitary (Melamed et al., 1996; Weber et al., 1997; Kakizawa et al., 1997), it lacks any effect on the GH mRNA levels in tilapia, while increasing those of LHb (Melamed et al., 1996) and those of FSHb and a transcripts (present study). Furthermore, while GnRH does not affect the mRNA levels of the PRL and GH in sockeye salmon (Oncorhynchus nerka), it can increase those of SL but only in males (Taniyama et al., 2000). Admittedly, the fact that selective inhibition of PKA, PKC and ERK affects the gonadotropin subunit mRNAs does not exclude the possibility that SL cells could have contributed to the pERK levels determined in this study. However, due to their scarcity in the pituitary of tilapia (Mousa and Mousa, 1999) as compared with the gonadotrophs (Melamed et al., 1998), this contribution is likely to be only marginal. GnRH plays a central role in the regulation of gonadotropin synthesis and release in mammals as

well as fish. In the present study, sGnRH led to a 2.8 increase in LHb and a subunit mRNA levels and a 2.4- fold increase in that of FSHb. These results are in line with those reported in maturing male striped bass (Morone saxatilis) in which the response of a and LHb mRNA levels to GnRH implantation was higher than that of FSHb (Hassin et al., 1998). Similarly, implanting GnRHa in vitellogenic homing sockeye salmon resulted in increased a and LHb mRNA levels but not that of FSHb (Kitahashi et al., 1998). Somewhat contradictory results were obtained in primary culture of pituitary cells of 2-year-old coho salmon (Oncorhynchus kisutch) in which sGnRH (1 and 100 nM) increased the levels of the two a subunits (a1 and a2) and FSHb mRNAs but not that of LHb (Dickey and Swanson, 2000). It was assumed that the donor fish had been at a stage too early for LH production, which normally appears only when fish approach spawning (Yaron et al., 2001). In this context, it should be noted that although the pituitary cells used in the present study were taken from fish at two reproductive phases (immature or early maturing), the general response to sGnRH, TPA or forskolin was similar (present study and unpublished results). This allowed combining the results obtained from the fish at these particular phases into a single scheme. The stimulation of gonadotropin biosynthesis and secretion by GnRH in mammals is dependent on the

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pulsatile nature of GnRH delivery, either in vivo (e.g. Haisenleder et al., 1991) or in vitro (Kaiser et al., 1997). Similarly, in goldfish and common carp, continuous treatment with either sGnRH or chicken GnRH-II results in rapid attenuation of GtH release and a significant reduction in the pituitary content of GnRH highaffinity binding sites (Habibi, 1991; Murthy and Peter, 1994; Lin et al., 1994). However, in tilapia sGnRH (0.1 –100 nM) given for 24 h elevated GnRH receptor mRNA levels dose-dependently, with no obvious desensitization (Safarian et al., 2001), as well as those of a, LHb and FSHb subunit mRNAs (present results). It is, therefore, reasonable to assume that in this fish, sGnRH at 0.1–100 nM is effective in elevating mRNA levels even when given continuously, differing from the situation in mammalian (Haisenleder et al., 1991; Shupnik, 1990) and goldfish gonadotrophs. In the present study, acute treatment with TPA elevated the pERK levels, an effect that was not further augmented by GnRH. The non-additivity of sGnRH and TPA effects on pERK levels indicates that both are conveyed through the same pathway, namely through activation of PKC. This is further corroborated by the inability of sGnRH to stimulate pERKs level after PKC depletion by continuous exposure to the phorbol ester. Furthermore, the specific PKC inhibitor, GF109203X decreased GnRH-induced ERK activation, and a high dose of GF completely abated GnRH effect,

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and even reduced pERK to below basal levels. Taken together, these results demonstrate that PKC is involved in the GnRH-induced ERK activation. PKC involvement in the GnRH-induced MAPK activation was also reported in the aT3-1 cell line, in which it was found to be necessary and sufficient for mediating GnRH effect (Sundaresan et al., 1996; Reiss et al., 1997; Sim et al., 1995). PKC inhibition by GF resulted in a decrease in the GnRH-induced a and LHb subunit mRNA levels, while none of the GF doses had any effect on that of FSHb. This indicates that PKC mediates the GnRH-induced a and LHb but not FSHb gene expression. Differential regulation of a and LHb versus the FSHb subunit mRNAs was also found in rat pituitary cells, where the GnRH pulse frequency, which stimulates the a and LHb subunits differs from that stimulating FSHb mRNA (Kaiser et al., 1997). Controversy exists as to the nature of the signaling systems involved in this differential regulation of gonadotropin subunit gene expression in mammalian cells. Weck et al. (1998) suggested that the rat a-subunit promoter is activated by PKC/MAPK, while the LHb subunit is mainly regulated by Ca2 + in a PKC-independent manner. However, Saunders et al. (1998) reported that the asubunit promoter is activated by Ca2 + while LHb subunit is PKC-dependent and Ca2 + -independent. Similarly, Call and Wolfe (1999) reported that the PKC/

Fig. 6. Effect of MAPK kinase (MEK) inhibition on (A) pERK isoform levels and (B – D) gonadotropin subunit mRNA levels after exposure to sGnRH. Pituitary cells from early maturing fish (BW 40 –100 g, GSI 0.16 90.02, n =21) were exposed to PD98059 (PD; 10 – 50 mM) for 30 min with or without subsequent stimulation by sGnRH (10 nM) for 15 min (A) or 24 h (B – D). (A) pERK isoform levels are expressed as ratio to the level of general ERKs in the same sample. Statistical analysis was done separately for each pERK isoform. (B – D) a, FSHb and LHb mRNA levels. Results are expressed as percentage of the level in untreated cells. Mean 9S.E.M., n = 3. Means designated by the same letter are not significantly different (P \0.05).

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MAPK system is involved in GnRH-induced LHb gene expression in a Ca2 + -independent manner. This controversy might be due to different species or different regions of the promoter used. Taken together with the present results, it appears that GnRH regulation of the gonadotropin subunit gene expression in fish, as in mammals, is conveyed differentially via signaling cascades specific for each subunit. In its general lines, such differential regulation appears to be conserved throughout vertebrate evolution from fish to mammals. The differential regulation of gonadotropin subunit genes by GnRH in tilapia was also demonstrated when the MAPK kinase (MEK) inhibitor, PD, was given to the pituitary cell culture. The fact that PD reduced both pERKs and GnRH-stimulated a and LHb mRNA levels indicates that ERK is an integral part in the mediation of GnRH effect on the expression of these subunit genes in the fish. Furthermore, as PD was not able to reduce the GnRH-stimulated FSHb mRNA levels, ERK is unlikely to be involved in mediating the transcription of this subunit gene. In contrast, PD at a similar concentration (50 mM) was able to block the mRNA response of FSHb and a to GnRH given in pulses to pituitary cells of male rats (Haisenleder et al., 1998), possibly reflecting species differences. It may be concluded that in tilapia the PKC-ERK cascade mediates the GnRH-stimulated a and LHb subunit gene expression, while having no effect on that of FSHb. The present data demonstrate that increased cAMP levels by forskolin elevate pERK levels, indicating the co-involvement of PKA and PKC in the MAPK cascade activation. Moreover, as the PKA inhibitor, H89, decreased the GnRH-induced pERK isoform level, it is suggested that the cAMP-PKA pathway also participates in the GnRH-induced ERK activation. In the GGH3 cells too, activation of ERK1/2 involves both PKC and PKA (Han and Conn, 1999). Similarly, in the wt28 cells (a GH3 cell line stably transfected with a wild type murine GnRH receptor) raising cAMP levels by forskolin strongly increases ERK phosphorylation (Johnson et al., 2000). It may be concluded that GnRH can activate the ERK cascade in tilapia too via the cAMP-PKA pathway. The fact that inhibition of PKA by H89 resulted in suppression of the GnRH-stimulated increase in all GtH subunits implicates cAMP-PKA cascade in their gene expression. Elevated cAMP can affect LHb mRNA by stabilization of the transcript in tilapia (Melamed et al., 1996), in the rat pituitary (Ishizaka et al., 1993), as well as in a and hCGb subunits mRNA of human choriocarcinoma cells (Fuh et al., 1989). It should be noted, however, that both H89 and GF at 0.01 mM had only a marginal inhibitory effect on ERK activation while considerably suppressing the a subunit mRNA level. Therefore, we cannot currently exclude the possibility that these drugs have more distal effects on

the cascade leading to expression of the a subunit gene. Considering the fact that LHb subunit mRNA levels were suppressed dose-dependently in parallel to those of ERK, this would indicate the involvement of an additional transduction element(s), which differentiate the regulation of a from the LHb subunits. Although elevation of intracellular cAMP level by forskolin in tilapia does result in an increase in both pERK and FSHb mRNA, and the inhibition of PKA decreases both of them, inhibition of MEK was not able to reduce the GnRH-induced FSHb transcript. This would suggest that the effect of cAMP-PKA on ERK and its effect on FSHb gene are uncoupled. We conclude, therefore, that PKA involvement in FSHb gene expression is not mediated via ERK. In this context, a study of the FSHb gene of tilapia (tFSHb) revealed several putative sequences of cis-acting motifs in its 5% flanking region (5% FR). Among these motifs, a cAMP response element (CRE) was detected together with multiple AP1 sites known as the recognition site for Fos and Jun (Rosenfeld et al., 2001). Functional analysis of the tFSHb gene was carried out using a series of luciferase fusion constructs (tFSHbLUC) containing various lengths of the 5%FR. The analysis revealed that the CRE motif, which is positioned at − 1062 bp upstream from the start site, may play a central role in its expression by regulating the tFSHb gene in a positive or a negative manner (Rosenfeld et al., 2001). Transcriptional regulation stimulated by cAMP signaling pathway is known to be mediated by a family of cAMP-responsive nuclear factors, which may act as activators or repressors employing either the cAMP-response-element-binding-protein (CREB) or the cAMPresponsive modulatory inducible cAMP early repressor (CREM/ICER), respectively (Sassone-Corsi, 1998). It is also possible that MAPK cascades other than ERK (i.e. JNK, P38 and BMK) are involved in GnRH-induced FSHb gene expression, similar to the situation in LbT2 cells where GnRH differentially activates the ERK and JNK cascades, with JNK being essential for rat LHb promoter activity (Yokoi et al., 2000). In conclusion, the results of this study show that, although GnRH stimulates the expression of all three GtH subunit genes, its signal in tilapia is transduced differentially, in a manner distinct from that in mammals. The transcription of a and LHb appears to be mediated through the PKC-ERK and PKA-ERK cascades, whereas the effect on FSHb mRNA is via the cAMPPKA system, without involving ERK.

Acknowledgements The authors wish to thank A. Gissis and HaMaapil Hatchery for the supply and maintenance of the fish, and Naomi Paz for critical reading of the manuscript.

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