Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus and stimulates the synthesis and secretion of gonadotropin hormones. In addition, GnRH also stimulates the production and secretion of growth hormone (GH) in some fish species and in humans with certain clinical disorders. In the goldfish pituitary, GH secretion and gene expression are regulated by two endogenous forms of GnRH known as salmon GnRH and chicken GnRH-II. It is well established that PKC mediates GnRH-stimulated GH secretion in the goldfish pituitary. In contrast, the signal transduction of GnRH-induced GH gene expression has not been elucidated in any model system. In this study, we demonstrate, for the first time, the presence of novel and atypical PKC isoforms in the pituitary of a fish. Moreover, our results indicate that conventional PKCα is present selectively in GH-producing cells. Treatment of primary cultures of dispersed goldfish pituitary cells with PKC activators (phorbol ester or diacylglycerol analog) did not affect basal or GnRH-induced GH mRNA levels, and two different inhibitors of PKC (calphostin C and GF109203X) did not reduce the effects of GnRH on GH gene expression. Together, these results suggest that, in contrast to secretion, conventional and novel PKCs are not involved in GnRH-stimulated increases in GH mRNA levels in the goldfish pituitary. Instead, PD98059 inhibited GnRH-induced GH gene expression, suggesting that the ERK signaling pathway is involved. The results presented here provide novel insights into the functional specificity of GnRH-induced signaling and the regulation of GH gene expression.
- protein kinase C isoforms
- mitogen-activated protein kinase
- extracellular regulated kinase
growth hormone (gh), prolactin, somatolactin, and placental lactogens compose a family of polypeptide hormones with structural similarities and overlapping biological properties. This family of hormones is thought to have evolved from a common ancestral gene, probably GH, through duplication and subsequent divergence (10, 14, 29). Synthesized and released by somatotropes, GH is a single-chain polypeptide that has a wide range of physiological effects, including the stimulation of skeletal, soft tissue, and overall body growth in all vertebrates. GH also affects the metabolism of carbohydrates, lipids, proteins, and minerals, either directly or indirectly by stimulating the local production of insulin-like growth factors (7, 20, 50). In addition to its many growth and metabolic effects, GH also has many interactions with the reproductive axis, including among others, the potentiation of gonadotropin hormone (GtH) effects on gonadal steroidogenesis (12, 38).
Gonadotropin-releasing hormone (GnRH) is best known for its regulation of the synthesis and secretion of pituitary GtHs. To date, a total of 16 forms of GnRH have been identified in various vertebrate and protochordate species, largely through the isolation and sequencing of peptides (1, 49). Recently, in silico analysis identified seven novel decapeptide GnRHs in tunicates (2). A 12-amino acid GnRH-like peptide has also been isolated from octopus (24). Most vertebrate species express at least two molecular variants of GnRH, including one form, chicken (c)GnRH-II (also known as GnRH-II), that is conserved from bony fish to man. In particular, the goldfish brain and pituitary contain salmon (s)GnRH and cGnRH-II (32, 64, 65). In goldfish, GnRH receptors are present on somatotropes, and GnRH stimulates GH release and mRNA levels (13, 18, 33, 43, 44). Although not universally observed (e.g., African catfish; Ref. 6), GnRH-binding sites and GnRH involvement in GH secretion has been reported in several other fish species, including tilapia (47, 48, 53), common carp (39, 40), rainbow trout (38), and pejerrey (58). GnRH has also been shown to stimulate GH mRNA levels in common carp (39) and masu salmon (5) but not sockeye salmon (59) or tilapia (48). The ability of GnRH to regulate somatotropes is not unique to fish species. GnRH-binding sites have been detected on rat somatotropes, and GnRH stimulates GH secretion from rat hemipituitaries (4, 11). In addition, GnRH receptor immunoreactivity has been detected on somatotropes in normal human pituitary cells (37), and GnRH can stimulate GH release in humans with various clinical disorders (44, 51).
In mammals, GnRH signal transduction in gonadotropes involves activation of PKC and ERK (34). The PKC family comprises 10 isoforms grouped into three distinct classes: conventional (α, γ, βI, and βII), novel (δ, ε, η, and θ), and atypical (ζ and ι/λ) PKC isoforms. Conventional PKCs are activated by phorbol esters/diacylglycerol (DAG), Ca2+ and phosphatidylserine. In contrast, novel PKCs are Ca2+ insensitive, and atypical PKCs are Ca2+ and DAG/phorbol ester insensitive (52). MAPK pathways are structurally organized as kinase cascades and are involved in the regulation of many cellular functions, including embryogenesis, cell differentiation, cell proliferation, and cell death. The kinase components of the ERK pathway include an ERK kinase kinase (Raf), which phosphorylates and activates an ERK kinase (MEK), which, in turn, phosphorylates and activates ERK (54). Although only investigated in goldfish and tilapia model systems, the signal transduction of GnRH-induced GH release is known to involve PKC (9, 48). In contrast, the signaling pathways that mediate GnRH-stimulated GH gene expression have not been elucidated in any model system, mammalian or nonmammalian. Although members of the growth hormone-releasing hormone (GHRH)/pituitary adenylate cyclase-activating polypeptide (PACAP) gene family use PKA-mediated signaling as a common mechanism for the regulation of GH production and secretion (41, 45, 46, 51), the involvement of PKA in mediating GnRH actions on GH mRNA levels in our system is not likely, as studies have shown that sGnRH and cGnRH-II do not stimulate cAMP production in goldfish pituitary cells (8). Thus the objective of this study was to test the hypothesis that the PKC and ERK signaling pathways are involved in the stimulation of GH mRNA levels by native forms of GnRH in goldfish pituitary cells.
MATERIALS AND METHODS
[Trp7,Leu8] GnRH (sGnRH) and [His5,Trp7,Tyr8] GnRH (cGnRH-II) from American Peptide (Sunnyvale, CA) were solubilized in 0.1 M acetic acid and stored at −20°C. Calphostin C (Cal C), GF109203X (GF), TPA, and PD98059 (PD) were purchased from Calbiochem (La Jolla, CA), dissolved in DMSO and stored at −20°C, 4°C, −20°C, and −20°C, respectively; 1,2-dioctanoyl-sn-glycerol (DiC8), also purchased from Calbiochem, was dissolved in DMSO just before use. 4-α-TPA was purchased from Sigma (Oakville, Ontario, Canada), dissolved in DMSO, and stored at −20°C. DMSO at a final concentration of <0.1% had no effect on GH subunit mRNA levels. Common carp GH (0.56 kb) and 18s rRNA (0.56 kb) cDNA fragments were cloned in this laboratory (23, 43). Antigenic peptides and antibodies directed against PKCβ (polyclonal; Invitrogen, Burlington, Ontario, Canada), PKCδ (polyclonal; Invitrogen), PKCη (polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), PKCθ (monoclonal; Santa Cruz Biotechnology) and PKCζ (polyclonal; Invitrogen) were a kind gift from Dr. M. P. Walsh (University of Calgary, Calgary, AB, Canada). Polyclonal anti-PKCα and γ antibodies and their respective antigenic peptides were purchased from Santa Cruz Biotechnology. The anti-carp GH (caGH) antibody was a kind gift from Dr. R. E. Peter (University of Alberta, Edmonton, Alberta, Canada).
Animals and cell preparation.
Male and female goldfish, Carassius auratus, ranging from 8 to 12 cm in length were purchased from Aquatic Imports (Calgary, Alberta, Canada) and used throughout the yearly reproductive cycle. The fish were maintained in semirecirculating tanks at 17°C on a 16:8-h light-dark photoperiod for acclimation before the experiments and fed a commercial fish diet. Goldfish were anesthetized and killed in accordance with the principles and guidelines of the Canadian Council on Animal Care. Pituitaries were dispersed using a modified trypsin-DNase protocol described by Klausen and colleagues (33). Briefly, pituitaries were placed in dispersion medium (medium 199 with Hanks’ salts, 25 mM HEPES, 2.2 g/l sodium bicarbonate, 0.3% BSA, 100 000 U/l penicillin, 100 mg/l streptomycin, pH 7.2), diced into fragments, and treated sequentially with trypsin (25 000 U/ml), trypsin inhibitor (25 000 U/ml), DNase II (0.01 mg/ml), 2 mM EGTA, and 1 mM EGTA. The fragments were dispersed by gentle trituration in calcium-free HBSS with 25 mM HEPES, 2.2 g/l sodium bicarbonate, 0.3% BSA, 100 000 U/l penicillin, 100 mg/l streptomycin (pH 7.2). Cell yield and viability were determined using trypan blue exclusion, and the cells were resuspended in culture medium (medium 199 with Earle’s salts 25 mM HEPES, 2.2 g/l sodium bicarbonate, 100,000 U/l penicillin, 100 mg/l streptomycin, pH 7.2). The cells were plated in culture medium for 2 h at 28°C, 5% CO2 and saturated humidity, after which horse serum was added to a final concentration of 1%. Depending on the experiment, the cells were allowed to recover for 24 or 72 h with a medium change at 48 h.
GH mRNA experiments.
Cells were prepared as described above and plated on 6- or 24-well Primaria plates (VWR, Edmonton, Alberta, Canada) at a density of 0.75–1.25 million cells/ml of medium. Unless otherwise indicated, cells were treated continuously with stimuli for 12 h. For studies involving the inhibition of PKC or MEK, inhibitors were added 30 min before the addition of stimuli. Continuous treatment for 12 h with sGnRH or cGnRH-II has previously been shown to be effective in stimulating GH mRNA levels in primary cultures of dispersed goldfish pituitary cells (33).
Northern blot analysis of goldfish GH mRNA levels has been previously validated and described (22, 33, 43). Briefly, total RNA was extracted from the cells using Trizol reagent (Invitrogen), and its purity was determined from ratios of the sample absorbances at 260 and 280 nm (A260:A280). Three to five micrograms of RNA was fractionated on 1.4% agarose/formaldehyde gels and blotted in the presence of 20× SSPE (3 M sodium chloride, 0.23 M sodium phosphate, 20 mM EDTA, pH 7.4) onto Hybond-XL membranes (Amersham Biosciences, Baie d'Urfé, Quebec, Canada). RNA was fixed to the membranes by baking at 80°C for 2 h. Purified cDNA fragments (GH and 18s rRNA) were labeled by the random primer method using the T7 Quickprime kit (Amersham Biosciences) and [α-32P]dCTP (3,000 Ci/mmol; Perkin Elmer Life Sciences, Woodbridge, Ontario, Canada). Membranes were prehybridized for 1 h and hybridized for 2 h at 60°C in rapid hybridization buffer (Amersham Biosciences) with the specific probe of interest. The membranes were then washed in a series of increasing stringency washes up to 0.1× SSC (15 mM sodium chloride, 1.5 mM sodium citrate, pH 7.0) in the presence of 0.1% SDS and exposed to Kodak X-Omat blue XB-1 film (Perkin Elmer Life Sciences). Additional hybridizations on the same membranes were carried out after stripping with repeated washings in boiling 0.1% SDS. 18s rRNA was used as an internal standard for normalizing mRNA levels. Autoradiograms were scanned and quantified with a computerized densitometry program (Image 1.62, National Institutes of Health, Bethesda, MD). GH mRNA levels were expressed with respect to 18s rRNA levels for the same sample and given as a percentage change with respect to a time-matched control (means ± SE, where control shows 0% change with respect to itself). Densitometry readings of mRNA and rRNA were all within the linear portion of the detection range of the system. The results from a minimum of three independent experiments performed on separate cell cultures were pooled, log-transformed, and analyzed by a one-way ANOVA. If the ANOVA analysis showed that a significant difference existed among the groups, the Student-Newman-Keuls test for multiple comparisons of means was performed to identify the treatment groups that were different from one another. Means were considered statistically different if P < 0.05 and are indicated by different letters.
Western blot analysis of PKC isoforms.
Goldfish pituitary, rat (Sprague-Dawley) brain and rat muscle tissue homogenates were prepared in protein extraction buffer (20 mM Tris, 2 mM EDTA, 2 mM EGTA, 0.1% SDS, 50 μg/ml leupeptin, 0.5 mM PMSF, pH 7.6), centrifuged at 10,000 g for 10 min, and the supernatants were collected. The protein concentration of each lysate was quantified using the bicinchoninic acid assay (MJS Biolynx, Brockville, Ontario, Canada) with BSA as a standard. Goldfish pituitary (10–40 μg of protein), rat brain (8–40 μg of protein), and rat muscle (50 μg of protein) samples were fractionated on 10% SDS-polyacrylamide gels (Mini-Protean II system, Bio-Rad Laboratories, Mississauga, Ontario, Canada). Fractionated protein extracts were transferred to methanol prewetted polyvinylidene difluoride membrane (Immun-blot, 0.2 μm, Bio-Rad Laboratories) by electroblotting (Mini-Protean II system). Depending on the antibody being used, membranes were blocked overnight at 4°C in 1× TBS (10 mM Tris·HCl, 150 mM sodium chloride, pH 7.4) with either 1% BSA or 5% nonfat dried milk (NFDM). After blocking, the membranes were washed three times (10 min each) at room temperature in 1× TBS with 0.05% Tween-20 and either 0.1% BSA or 1% NFDM. To detect the various isoforms of PKC, membranes were incubated for 2 h at room temperature with the appropriate primary antibody. After another three washes, the membranes were incubated with the appropriate secondary antibody [enhanced chemiluminescence (ECL) kit, Amersham Biosciences] for 45 min at room temperature. After a final set of washes, the ECL kit reagents were applied, and the membranes were exposed to Kodak X-Omat blue XB-1 film. Where available, the specificity of the antibodies used was examined by preabsorption with the antigenic peptide.
Goldfish pituitary glands were freshly removed and bisected at the midsagittal plane. The tissues were immersed in Boun's fixative for 1 day at 4°C and processed for conventional paraffin embedding. Serial and mirror sections, 5 μm in thickness, were mounted on silane-coated slides. Wet heat-induced antigen retrieval was performed in a microwave oven (550 W, 6 min). Immunostaining of GH and PKCα was performed by the avidin-biotinylated enzyme complex (ABC) method (21) using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). After antigen retrieval, sections were treated with 3% hydrogen peroxide (H2O2) in methanol for 5 min to block the activity of endogenous peroxidase. After blocking of nonspecific binding with 1.5% normal goat serum in 1× PBS (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM sodium chloride, pH 7.4) for 60 min, the sections were incubated with rabbit polyclonal anti-caGH and -PKCα antibodies for 16 h at 4°C. The next day the sections were incubated with biotinylated goat anti-rabbit IgG for 30 min and with ABC peroxidase reagent for 30 min. The reaction products were visualized as brown staining in 0.016% 3,3′-diaminobenzidine tetrahydrochloride in Tris·HCl buffer (0.05 M, pH 7.6) with 0.005% H2O2. All of the steps, with the exception of the incubation with normal goat serum, were followed by three washes with 1× PBS for 5 min each. The specificity of caGH and PKCα immunostaining was confirmed by omission of the primary antibody or replacement with nonimmune rabbit serum at approximately the same IgG concentration.
Detection of PKC isoforms in the goldfish pituitary.
In a previous study, immunoreactive PKC was detected in goldfish pituitary cells using a monoclonal antibody recognizing the α- and β-isoforms of PKC (25). To further investigate this question, the complement of PKC isoforms expressed in the goldfish pituitary was examined by Western blot analysis. Goldfish pituitary, rat brain, and rat muscle homogenates were subjected to SDS-PAGE and immunoblotted with antibodies targeting various conventional, novel, and atypical isoforms of PKC, including, α, β, γ, δ, η, θ, and ζ (Fig. 1). The rat tissue samples were used as positive controls and, when available, competing antigenic peptide confirmed the specificity of immunoreactivity. Immunoreactive bands of appropriate molecular mass were detected for PKCα, -β, -γ, -δ, -η and -ζ in rat brain homogenate and for PKCθ in rat muscle. PKC immunoreactivity was detected in goldfish pituitary homogenate with antibodies directed against PKCα, -δ, -θ, and -ζ. The immunoreactive band detected in goldfish pituitary homogenate by the anti-PKCθ antibody has a slightly larger molecular mass than the corresponding band in rat muscle homogenate.
Immunohistochemical analysis was performed to examine the possible expression of PKCα in goldfish pituitary somatotropes. In goldfish, GH cells are known to be distributed in the pars distalis region along the midsagittal plane (27). In serial and mirror sections prepared along the midsagittal plane of the goldfish pars distalis, a positive reaction for PKCα was identified in the cytoplasm of GH-positive cells (Fig. 2).
The effects of PKC inhibitors on GnRH-induced GH mRNA levels.
Having demonstrated the presence of conventional PKCα in goldfish somatotropes and novel PKC(s) in the goldfish pituitary, inhibitors of PKC (Cal C and GF) were used to assess its role in GnRH-stimulated GH gene expression. Cal C targets the DAG/phorbol ester-binding site in the regulatory domain and is a potent and specific inhibitor of PKC (IC50, 5 × 10−8 M) (35). The effect of Cal C on GnRH-induced GH gene expression was examined in primary cultures of goldfish pituitary cells after 24 (A) or 72 (B) h of recovery (Fig. 3). The rationale for using different recovery times relates to the stimulatory effects of PKC inhibitors on GtH subunit mRNA levels in the same experiments (Klausen C, Severson DL, Chang JP, and Habibi HR, unpublished observations). Briefly, the goldfish pars distalis is innervated by hypothalamic neurons (28). Trace amounts of neurotransmitter substances such as dopamine, which is known to inhibit GtH and to stimulate GH release, can be detected in goldfish pituitary cell cultures shortly after preparation. However, levels of dopamine became undetectable after 72 h of culture. Thus, to minimize the possible compounding effects of the remnants of active neurosubstances, such as dopamine, some experiments are performed after 72 h, as well as 24 h, of recovery from dispersion. GH mRNA levels were determined after 12 h of continuous treatment with varying doses of sGnRH or cGnRH-II (10−9 M, 10−8 M, or 10−7 M) in the absence or presence of Cal C (10−7 M) (24 h of recovery, Fig. 3A). GH mRNA levels were significantly elevated by all three concentrations of sGnRH, whereas only treatment with 10−7 M cGnRH-II generated significant increases. Treatment with 10−7 M Cal C alone did not affect basal GH mRNA levels, and GnRH-induced increases were not affected by coincubation with Cal C.
The dose-related effects of Cal C were examined in studies where GH mRNA levels were quantified after 12 h of treatment with sGnRH or cGnRH-II (10−7 M) in the absence or presence of Cal C (10−8 M, 10−7 M or 10−6 M) (72 h of recovery, Fig. 3B). Cal C did not significantly alter GH mRNA levels at any of the concentrations tested, and its presence did not affect GnRH-induced responses.
The dose-related effects of a second PKC inhibitor, GF, on GnRH-stimulated GH mRNA levels were examined after 24 h of recovery. GF targets the ATP-binding site in the catalytic domain and is a potent and selective inhibitor of PKC (IC50 for various isoforms, 10−8 to 2 × 10−7 M) (61). Cells were treated continuously for 12 h with sGnRH or cGnRH-II in the absence or presence of GF (10−8 M, 10−7 M or 10−6 M) (Fig. 4). Treatment with varying doses of GF alone did not result in any changes in GH mRNA levels; however, pretreatment with GF significantly enhanced the responses to sGnRH and cGnRH-II.
The effects of PKC activation on basal and GnRH-induced GH mRNA levels.
The phorbol ester, TPA, was used to study the effect of PKC activation on GH mRNA levels. Treatment with varying concentrations of TPA for 2 or 12 h did not alter GH mRNA levels after either 24 or 72 h of recovery (Table 1). The modified 2-h (followed by 10 h in normal medium) treatment protocol was used to control for depletion of PKC by prolonged treatment with TPA, an effect that has previously been demonstrated in goldfish pituitary cells by Western blot analysis (25). Application of an inactive phorbol ester, 4-α-TPA, did not result in any changes in GH mRNA levels (Table 1).
Coincubation of GnRH with either TPA or a cell-permeable and PKC-activating DAG analog, DiC8, was performed to further investigate the role of PKC in the signal transduction of GnRH action (72 h of recovery, Fig. 5). Similar to results from Table 1, all doses of TPA tested did not affect basal GH mRNA levels. GnRH-induced increases in GH mRNA levels were also unaffected by cotreatment with TPA. Similarly, treatment with DiC8 alone did not result in any changes in GH mRNA levels, and its presence did not alter GnRH-induced increases.
Unlike experiments with GnRH (Fig. 4), GF does not enhance the effects of PKC activators. Coincubation of GF (10−6 M) with either TPA or DiC8 did not alter GH mRNA levels in cells that were allowed to recover for 72 h (Fig. 6).
The effects of a MEK inhibitor on GnRH-induced GH mRNA levels.
Cotreatment of the MEK inhibitor PD with TPA or GnRH was performed to investigate the possible involvement of MEK-dependent ERK activation in the regulation of GH mRNA levels. Goldfish pituitary cells were allowed to recover for 72 h and then treated continuously for 12 h with TPA (10−8 M), sGnRH (10−7 M), or cGnRH-II (10−7 M) in the absence or presence of PD (10−5 M) (Fig. 7). Application of PD alone did not alter basal GH mRNA levels. Treatment with TPA, either in the absence or presence of PD, did not alter GH mRNA levels. sGnRH- and cGnRH-II-induced increases in GH mRNA levels were significantly reduced in cells pretreated with PD.
Profile of PKC isoforms in goldfish pituitary cells.
Of the conventional isoforms examined in this study (α, β, and γ), only PKCα was detected in the goldfish pituitary. This is in agreement with an earlier study showing immunoreactivity in goldfish pituitary cell homogenates using an antibody recognizing the α- and β-isoforms of PKC (25). PKCα and -β immunoreactivity mRNA has been detected in the rat pituitary (16, 36, 42), in the gonadotrope-derived LβT2 and αT3–1 cell lines (36, 57, 62), in rat pituitary GH4C1 cells (31, 55), and in the corticotropic AtT-20/D16-V cell line (42). PKCγ was not detected in the goldfish pituitary (present study), the rat pituitary, GH4C1 cells, AtT-20/D16-V cells, or αT3–1 cells, in line with the assertion that PKCγ is expressed specifically in central nervous tissue (30). The results of this study appear to indicate that immunoreactive PKCβ is not present in the goldfish pituitary; however, it remains possible that the antibody used in this study, raised against mammalian PKCβ, failed to cross-react with goldfish PKCβ.
We also demonstrate, for the first time, the presence of novel PKC isoforms in the goldfish pituitary. Immunoreactive PKCδ, but not -η, was detected in goldfish pituitary homogenate. PKCθ immunoreactivity was also detected; however, the molecular mass of the immunoreactive band differed slightly from that detected in rat muscle. Differences between the two species could be responsible for subtle variations in size. The antigenic peptide for the anti-PKCθ antibody used in this study was unavailable for use in preabsorption control experiments, and further studies will be required to confirm the presence of PKCθ in the goldfish pituitary. Rat pituitary and LβT2 cells have been shown to contain PKCδ, -ε, and -θ, but not -η, immunoreactivities (36, 42, 62). Immunoreactive PKCδ and -ε, and mRNA for PKCδ, -ε and -η, but not -θ, have been detected in GH4C1 cells (3, 31, 55). AtT-20/D16-V cells were shown to express PKCε and -θ, but not -δ or -η (42). The presence of mRNA for PKCδ in αT3–1 cells has been reported (19); however, two studies have failed to detect PKCδ immunoreactivity in these cells (36, 42). PKCε and -θ, but not -η, immunoreactivities have also been identified in αT3–1 cells (42). The present study did not examine the expression of PKCε; however, its presence in all the pituitary cell types listed above suggests that it is likely to be present in the goldfish pituitary. On the other hand, the apparent lack of PKCη expression in the goldfish pituitary appears to be consistent with reports of its absence from the aforementioned cell types, with the notable exception of GH4C1 cells. Together, these results indicate that the goldfish pituitary expresses a profile of novel PKC isoforms that is similar to other pituitary cell types.
This study also demonstrates the presence of atypical PKCζ in the goldfish pituitary. Immunoreactive PKCζ has been detected in the rat pituitary (36, 42), GH4C1 cells (31, 55), AtT-20/D16-V cells (42), and αT3–1 cells (36, 42). PKCζ is not expressed in LβT2 cells although PKCλ, another atypical isoform, has been detected (62).
The presence of immunoreactivity representing different PKC isoforms in the goldfish pituitary cell population begs the question of whether all pituitary cell types contain the same complement of PKCs. Interestingly, in this study, we show that PKCα immunoreactivity is present only in GH-staining goldfish pituitary cells (somatotropes). Moreover, gonadotropes are known to be the other goldfish pituitary cell type present alongside the somatotropes in the area of the pars distalis shown in Fig. 2 (27). This suggests that PKCα is selectively present in somatotropes but not gonadotropes in goldfish. In mammals, PKCθ was detected in the gonadotrope-derived αT3–1 and LβT2 cell lines (42, 62), but not in the somatolactotrope-derived GH4C1 cell line (3). In contrast, PKCη was detected in GH4C1 cells, but not in αT3–1 or LβT2 cells. These observations would appear to support the hypothesis that different pituitary cell types express distinct profiles of PKC isoforms. In this context, future studies in goldfish examining the profile of PKC isoforms expressed by distinct secretory cell types, including somatotropes, will be of great interest.
Conventional and novel PKCs are not involved in GnRH-induced increases in GH mRNA levels.
Goldfish pituitary cells were pretreated with PKC inhibitors (Cal C or GF) to test the hypothesis that PKC is involved in GnRH-induced increases in GH mRNA levels. Increases in GH mRNA levels induced by sGnRH or cGnRH-II were not reduced by pretreatment with Cal C or GF, indicating that GnRH-mediated effects are not likely to involve conventional and novel PKCs. Further support for this hypothesis is derived from experiments aimed at mimicking the effects of GnRH with PKC activators. Treatment of goldfish pituitary cells with TPA (2 or 12 h) or DiC8 did not alter GH mRNA levels, indicating that activation of PKC is not coupled to changes in GH gene expression, at least at the time point examined in these studies. In contrast, both TPA and DiC8 increased GtH subunit mRNA levels in the same experiments (Klausen C, Severson DL, Chang JP, and Habibi HR, unpublished observations). Unfortunately, the role of atypical PKC isoforms in the regulation of basal and GnRH-induced GH mRNA levels could not be determined with the pharmacological tools employed in this study. Having demonstrated the presence of atypical PKCζ in the goldfish pituitary, we cannot completely rule out atypical PKC-mediated signaling. Indeed, all of the pituitary cell types discussed earlier possess at least one atypical PKC, and GnRH has been shown to stimulate the translocation of PKCζ to the membrane in αT3–1 cells (36).
Given the above results and the observation that PKC activators did not affect GH mRNA levels in tilapia (48), it is tempting to conclude that PKC is not involved in the regulation of GH gene expression in the goldfish pituitary. However, this study only examined the involvement of PKC at a previously established time point and conditions associated with GnRH-induced increases in GH mRNA levels (12 h; see Ref. 33) and does not preclude the involvement of PKC in the regulation of GH gene expression by other neuroendocrine factors under different conditions. Recently, it has been proposed that growth hormone-releasing hexapeptide-6 (GHRP-6) stimulates GH release from porcine somatotropes and increases pituitary-specific transcription factor (Pit-1) promoter activity in a largely PKC-dependent fashion (15, 17). Although GH mRNA levels have not been examined, it is tempting to speculate that the regulation of GH gene expression by certain GH secretagogues may involve PKC since the expression of the GH gene in somatotropes is dependent on Pit-1 (60).
ERK mediates GnRH-induced GH gene expression.
The results of this study clearly show that GnRH-stimulated increases in GH mRNA levels in goldfish pituitary cells are mediated by MEK and thus presumably ERK activation. Our results are the first to demonstrate the involvement of the ERK pathway in GnRH-induced GH gene expression, in any model system. Other GH secretagogues, such as GHRH and PACAP, have previously been shown to induce ERK activation in rat pituitary cells and GH3 cells, respectively (56, 63). However, the significance of this activation in terms of stimulated GH gene expression has not been investigated. Interestingly, GHRP-6-induced Pit-1 promoter activity was reduced in the presence of PD, suggesting a possible role for ERKs in GHRP-6-stimulated GH gene expression (15).
Signaling pathways regulating GH gene expression and secretion are not necessarily similar.
Although a number of studies have demonstrated GnRH effects on GH secretion, there have been only two model systems in which the signaling has been investigated. It is well established that PKC and Ca2+, but not arachidonic acid or PKA, are involved in sGnRH- and cGnRH-II-stimulated GH secretion in goldfish pituitary cells (9, 34). Similarly, PKC, but not PKA, has been implicated in sGnRH-induced GH release from dispersed cultures of tilapia pituitary cells (48). Interestingly, treatment with sGnRH or TPA did not increase GH mRNA levels in the same experiments, suggesting that GnRH and PKC do not regulate GH gene expression in the tilapia pituitary. In this context, this study is the first to elucidate some of the signaling involved in GnRH-mediated increases in GH gene expression, in any model system. Our results indicate that conventional and novel PKCs are not likely to be involved in GnRH-stimulated GH gene expression, suggesting that the signal transduction pathways mediating GnRH effects on GH gene expression are different from those regulating GH secretion in the goldfish pituitary. Indeed, previous studies in goldfish examining the regulation of GH secretion, storage, and gene expression by multiple intracellular Ca2+ stores appear to support the hypothesis that secretion and gene expression can be independently controlled by function-specific signaling (26).
In summary, our results are the first to demonstrate the presence of immunoreactive novel and atypical PKCs in the pituitary of a fish. Moreover, our results suggest that among the pituitary cell types present along the midsaggital plane of the pars distalis, conventional PKCα is expressed only in somatotropes. In addition, the postreceptor mechanisms mediating sGnRH- and cGnRH-II-induced GH gene expression are also examined for the first time, in any model system. Treatment of primary cultures of dispersed goldfish pituitary cells with PKC activators did not affect basal or GnRH-induced GH mRNA levels, and two different inhibitors of PKC (Cal C and GF) did not reduce the effects of GnRH on GH gene expression. Together, these results suggest that, in contrast to secretion, conventional and novel PKCs are not involved in GnRH-stimulated increases in GH mRNA levels in goldfish pituitary cells. Instead, the ability of the MEK inhibitor PD to reduce GnRH-induced GH gene expression suggests that the effects of GnRH are mediated by the ERK signaling pathway. The results presented here provide novel insights into the functional specificity of GnRH-induced signaling and the regulation of GH gene expression.
This work was supported by grants from the Natural Sciences and Engineering Research Council to H. R. Habibi. (RGP 156910) and J. P. Chang (RGP 0121399). Studentship support for C. Klausen from the Alberta Heritage Foundation for Medical Research is gratefully acknowledged.
We thank Dr. D. A. Syme and Dr. R. L. Walker (University of Calgary, Calgary, Alberta, Canada) for the rat tissue samples used in this study.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 the American Physiological Society