Somatostain (SS) is known to inhibit growth hormone (GH) and prolactin (PRL) secretion. Somatolactin (SL) is a member of the GH/PRL family, but its regulation by goldfish brain somatostatin-28 (gbSS-28) has not been examined. To this end, the structural identity of goldfish SLα was established by 5′/3′-rapid amplification of cDNA ends. As revealed by in situ hybridization and immunohistochemical staining, the expression of SL isoforms was detected in pituitary cells located in the neurointermediate lobe (NIL). The transcripts of goldfish SS receptor 5a (Sst5a) but not Sst1b, Sst2, or Sst3a were detected in the goldfish NIL cells by RT-PCR. In goldfish pituitary cells, gbSS-28 not only had an inhibitory effect on basal SLα and SLβ mRNA levels but also could abolish insulin-like growth factor-stimulated SL gene expression. In primary cultures of goldfish NIL cells, gbSS-28 reduced forskolin-stimulated total cAMP production. With the use of a pharmacological approach, the adenylate cyclase (AC)/cAMP and phospholipase C (PLC)/inositol trisphosphate (IP3)/protein kinase C (PKC) cascades were shown to be involved in gbSS-28-inhibited SLα mRNA expression. Similar postreceptor signaling cascades were also observed for gbSS-28-reduced SLβ mRNA expression, except that PKC coupling to PLC was not involved. These results provide evidence that gbSS-28 can inhibit SLα and SLβ gene expression at the goldfish pituitary level via Sst5 through differential coupling of AC/cAMP and PLC/IP3/PKC cascades.
- goldfish pituitary cells
- signaling pathways
- goldfish brain somatostatin-28
somatostatin (SS) is a multifunctional and multimember family of peptides. The evolution of the SS family appears to result from a series of gene duplication events (31). In goldfish, multiple SS mRNAs encoded three distinct SS isoforms: SS-14, goldfish brain SS-28 (gbSS-28), and [Pro2]SS-14 (13). SS has a critical role in exerting inhibitory effects on endocrine and exocrine secretions through binding to specific G protein-coupled receptors (39). Five subtypes of SS receptor (Sst1–5) have been identified in mammalian species. In fish, four Sst subtypes have been characterized (homologous to mammalian Sst1, -2, -3, and -5), some of which possess multiple isoforms depending on species (31). In goldfish, eight Ssts (Sst1a, -1b, -2, -3a, -3b, -5a, -5b, and -5c) have been identified, and multiple Ssts are expressed in pituitary cells, with Sst2, Sst5a, and Sst5b mRNAs being the most abundant (24–27), which is also consistent with mammalian counterparts. The diversity of Sst subtypes may have contributed to the pleiotropic cellular functions of SS.
It has been demonstrated that Sst2 and Sst5 are involved in inhibition of growth hormone (GH) release, primarily by affecting the timing and amplitude of GH secretion (30), which may indicate “rebound” GH secretion after SS priming and withdrawal in both the rat and human (23, 38). Despite its potent inhibitory actions on GH release, inhibition of hormone synthesis has not been proven conclusively. Some studies showed a decrease in somatotroph GH mRNA levels (1, 40), whereas others showed no change (15), probably reflecting rebound GH synthesis after cessation of SS treatment. In fish species, SS-14 does not alter steady-state GH mRNA levels in tilapia (29) and rainbow trout (42). Besides inhibiting GH secretion, SS has been found to suppress not only PRL secretion in both in vivo and in vitro studies (4), but also PRL gene expression in primary rat pituitary cells (15). The binding of SS to its G protein-coupled receptors leads to regulation of multiple postreceptor signaling events, including, but not limited to, adenylate cyclase (AC), phospholipase C (PLC), various ion channels and exchangers, mitogen-activated protein kinases, and several other kinases and phosphatases (7). In orange-spotted grouper pituitary cells, SS inhibition of GH release is mediated by both the PLC/inositol trisphosphate (IP3)/protein kinase C (PKC) and the AC/cAMP/protein kinase A (PKA) pathways (41). All of the three distinct SS isoforms (SS-14, gbSS-28, and [Pro2]SS-14, identified in goldfish) could reduce GH secretion with overlapping yet distinct signaling pathways (8, 45, 46). Also, SS can inhibit PRL release by blocking a Ca2+- or cAMP-mediated mechanism from the organ-cultured rostral pars distalis of the tilapia (14).
Somatolactin (SL) is the latest member of the GH/PRL family. Because SL could not be identified in tetrapods and might have been lost during the evolution of land vertebrates (21), SL is a pituitary hormone unique to fish species. Two SL isoforms, SLα and SLβ, have been identified, and phylogenetic analysis reveals that they are paralogs that arose by genome duplication in bony fish (47). Biochemical and physiological studies have demonstrated that SL may be involved in the regulation of reproduction (19), water-mineral balance (28), body pigmentation (48), steroidogenesis (36), lipid metabolism (12), and immune responses (5). Similar to other pituitary hormones, SL expression is also under the control of hypothalamic regulators [e.g., gonadotropin-releasing hormone, melanin-concentrating hormone (6), pituitary adenylate cyclase-activating polypeptide (17), dopamine and serotonin (20)], and feedback signals from the periphery [e.g., sex steroid (34), insulin-like growth factors (IGFs; see Ref. 18), and leptin (35)]. However, little is known of whether SS affects SL gene expression. Given that 1) gbSS-28 (preprosomatostatin II) mRNA was detected in dispersed goldfish pituitary cells (46) and 2) in sculpin, the green molly, and the catfish, somatostatin-immunoreactive fibers were found to approach the pars intermediate and to end among the SL cells (3), it is plausible that the SS peptides participate in the neuroendocrine, as well as paracrine and/or autocrine, regulation of fish pituitary physiology. These findings have prompted us to speculate that SS may play a role in SL regulation at the pituitary level. In goldfish, SLβ had been previously reported (10). In our present studies, the other SL isoform, namely SLα, was cloned by 5′/3′-rapid amplification of cDNA ends (5′/3′-RACE), and their spatial distribution in the goldfish pituitary has been characterized by in situ hybridization and immunohistochemical staining. With the use of primary cultures of goldfish pituitary cells as a model, the pituitary actions of gbSS-28 and postreceptor signaling mechanisms for SLα and SLβ gene expression were investigated. In this study, we have demonstrated for the first time that gbSS-28 can act at the pituitary level via Sst5 to regulate SLα and SLβ gene expression through differential coupling of AC/cAMP and PLC/IP3/PKC cascades.
MATERIALS AND METHODS
Goldfish (Carassius auratus) with body weight ranging from 35 to 45 g were acquired from a local supplier and maintained in a 100-liter aquariium at 20 ± 2°C under a 12:12-h dark-light photoperiod for at least 7 days before tissue sampling and pituitary cell preparation. To minimize the effects of sex steroids on the action of SL, goldfish at late stages of gonadal regression (gonadosomatic index was ≤0.1%) were used in the present study. Given that sexually regressed goldfish do not exhibit sexual dimorphism of external features, goldfish of mixed sexes were routinely used for pituitary cell preparation. During the process of tissue sampling, the fish were anesthetized by immersion in 0.05% MS222 (Sigma, St. Louis, MO) before cervical transection and pituitary collection according to the procedures approved by the Committee of Animal Use for Research of the University of Hong Kong and the Animal Ethics Committee of Sichuan University.
The gbSS-28 was synthesized by GL Biochem (Shanghai) using the standard procedures for solid-phase peptide synthesis. As revealed by HPLC analysis, the purity of gbSS-28 was ≥95%, and the homogeneity of the peptide was further confirmed by mass spectrometry. Human IGF-I and IGF-II were obtained from Sigma. IGFs and a synthetic peptide were dissolved in double-distilled deionized water and stored frozen in small aliquots at −80°C as 0.1 mM stocks. 3-Isobutyl-1-methylxanthine (IBMX), CPT-cAMP, forskolin, U-73122, 2-aminoethoxydiphenyl borate (2-APB), and GF-109203 were obtained from Calbiochem (San Diego, CA). Similar to the peptide hormones, these pharmacological agents were prepared as 10-mM frozen stocks in dimethyl sulfoxide (DMSO). Stock solutions of test substances were diluted with prewarmed (28°C) culture medium to appropriate concentrations 15 min before drug treatment. The final dilutions of DMSO were <0.1% and had no effects on SL gene expression in goldfish pituitary cells.
Molecular cloning of goldfish SLα.
Total RNA was extracted from the pituitary of goldfish using TRIzol (Invitrogen, Grand Island, NY) and reverse transcribed using a SuperScript II First-Strand cDNA Synthesis kit (Invitrogen). Using primers designed based on the conserved regions of SLα open reading frame (ORF) reported in grass carp and zebrafish, nested PCR was performed, and a partial fragment of goldfish SLα cDNA was isolated. On the basis of the sequences obtained, gene-specific primers were designed, and 5′/3′-RACE was conducted using a GeneRacer kit (Invitrogen). After size-fractionation, PCR products of appropriate sizes were purified from agarose gel and subcloned into pGEM-T Easy vector (Promega, Madison, WI) for DNA sequencing using a BigDye Sequencing kit (Applied Biosystems, Foster City, CA). The full-length cDNA for goldfish SLα was then compiled from their 5′- and 3′-sequences and analyzed with MacVector version 9.5.2 software (Oxford Molecular, Madison, WI). After that, phylogenetic analysis of goldfish SLα and SLβ was also conducted using the neighbor-joining method with MEGA 4.0 programs. For structural comparison of the two goldfish SL isoforms, the three-dimensional (3-D) models of goldfish SLα and SLβ were deduced using the knowledge-based modeling program ProMod II provided by the SWISS-MODEL Server (www.expasy.org/swissmod).
Tissue distribution of SLα and SLβ by RT-PCR.
Tissue distribution of SLα and SLβ expression was examined by using RT-PCR. Briefly, total RNA (10 μg) was isolated from selected tissue and brain areas of the goldfish using TRIzol, digested with RNase-free DNase I to remove genomic DNA contamination, and reversely transcribed using SuperScript II (Invitrogen). The RT sample obtained was used as the template for PCR using the primers specific for goldfish SLα (SLα forward primer: 5′-ATATGTTTGTCCCGTACCCTCT-3′ and SLα reverse primer: 5′-TTTATCAGACACCCACTTGGTC-3′) and SLβ (SLβ forward primer: 5′-AGGGACCATGTGTTCTCCTAAA-3′ and SLβ reverse primer: 5′-AGAACCAGTATACCCTGCTCCA-3′). In PCR assay system for SLα and SLβ, the PCR cycle number was fixed at 30 cycles with 30 s at 94°C for denaturing, 30 s at 56°C for annealing, and 30 s at 72°C for primer extension. After that, PCR products obtained were resolved in 1% gel, visualized by staining with ethidium bromide staining, and transblotted on a positively charged nylon membrane. Afterward, digoxigenin (DIG)-labeled probes for goldfish SLα and SLβ were prepared for PCR Southern blot using a PCR DIG Probe Synthesis Kit (Roche, Mannheim, Germany). To confirm the authenticity of PCR products, Southern blot was conducted using the DIG-labeled cDNA probes for goldfish SLα and SLβ. RT-PCR for β-actin was also performed to serve as an internal control in this study. To further characterize the transcripts of goldfish SLα and SLβ expressed in the pituitary, Northern blot was also performed using total RNA isolated from the goldfish pituitary as described previously (17). After size-fractionation in 1% agarose gel containing 1× MOPS and 1% formaldehyde, the RNA sample was transblotted on a nylon membrane, ultraviolently cross-linked, prehybridized for >2 h with blocking solution, and hybridized overnight at 42°C with the DIG-labeled probes for SLα and SLβ. On the following day, hybridization signals were revealed using a DIG chemiluminescent detection kit (Roche) and visualized in an IC440 CF Image Station (Eastman Kodak, New Haven, CT). To ensure the absence of RNA degradation in our samples, parallel blotting of β-actin mRNA was also conducted in these experiments.
Western blot of SL immunoreactivity.
In our recent studies, the grass carp SL antisera is specific for each isoform and also does not cross-react with GH and PRL (18). Given that the sequence homology of grass carp SLα and SLβ compared with their goldfish counterparts is quite high (85% for SLα and 86% for SLβ), it is likely that the antisera for grass carp SLα and SLβ can also be used in goldfish. To confirm the specificity of these antisera in goldfish, Western blot was performed with goldfish pituitary cell lysate and condition medium after 24-h incubation. Briefly, primary cultures of pituitary cells were prepared from goldfish and seeded in 24-well culture plates at a density of 1.3 × 106 cells·ml−1·well−1. After overnight incubation to allow for recovery of membrane receptors, drug treatment was initiated by replacing the old medium with M199 medium (43). After that, culture medium was collected for monitoring SL release while pituitary cells were lysed in RIPA buffer (50 mM Tris·HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 0.25% sodium deoxycholate) supplemented with a cocktail of protease inhibitors and phosphatase inhibitors (Roche). These protein samples were resolved by SDS-PAGE, electroblotted on nitrocellulose membrane, and subjected to Western blot using the antisera for SLα (1:1,000,000) and SLβ (1:5,000). After that, signal development was performed using Immobilon Western Chemiluminescent Reagent (Millipore, Billerica, MA). For antiserum preabsorption on SL immunoreactivities, Western blot of goldfish pituitary lysate was performed with SLα and SLβ antisera incubated for 15–18 h at 4°C with the respective isoform of recombinant SL (0.1 μg/ml).
N-glycosidase F assay.
To evaluate the glycosylation status of goldfish SL, N-gylcosidase F assay was performed according to the method described previously (18). Briefly, cell lysate (for SL content) and culture medium (for SL released) were harvested from goldfish pituitary cells after static incubation for 24 h. These protein samples were digested with N-glycosidase F (1 U/reaction; Calbiochem) for 3 h at 37°C and subjected to SDS-PAGE followed by Western blot using the antiserum for SLα and SLβ, respectively.
In situ hybridization and immunohistochemical staining of pituitary SLα and SLβ.
Pituitaries were freshly excised from goldfish, fixed in 4% paraformaldehyde, and embedded in paraffin wax according to standard procedures (17). Pituitary sections of 5 μm in thickness were prepared and mounted on slides precoated with 2% 3-aminopropyltriethoxy silane (Sigma). For in situ hybridization, pituitary sections were dewaxed with xylene, rehydrated with decreasing levels of ethanol, and postfixed with 4% paraformaldehyde. After that, the sections were digested with proteinase K, washed, and incubated at 37°C for 10 min with hybridization solution (4× SSC, 1× Denhardt's solution, 10% dextran sulfate, 10 mM DTT, 1 mg/ml calf thymus DNA, 500 μg/ml yeast tRNA, 5 μg/ml polydeoxyadenylic acid, and 100 μg/ml polyadenylic acid) containing 40% formamide. DIG-labeled antisense riboprobes for goldfish SLα and SLβ prepared by in vitro transcription were added, and the pituitary sections were incubated at 55°C overnight in a humidified chamber. On the following day, posthybridization washing was performed in decreasing dilutions of SSC solution at 42°C. After the clearance of unbound riboprobes by RNase A (Invitrogen) digestion, signal development was conducted with anti-DIG antibody (1:500; Roche) using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate as the substrates. In these experiments, hybridization with the sense strands of the SLα and SLβ riboprobes was used as the negative control. To confirm the expression pattern of SLα and SLβ at the protein level, immunohistochemical staining was also performed in goldfish pituitary sections using a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. In this study, antisera for SLα and SLβ was used at 1:10,000 dilutions, and signal detection of immunostaining was based on the avidin-biotin-peroxidase complex method using diaminobenzidine (0.05%) as the substrate.
RT-PCR of Sst subtypes in goldfish neurointermediate lobe cells.
The neurointermediate lobe (NIL) of individual pituitaries was isolated by manual dissection under a stereomicroscope. Total RNA was isolated using TRIzol and reversely transcribed with SuperScript II. RT samples obtained were then subjected to RT-PCR using primers specific for goldfish Sst1b (GenBank no. AF097727), Sst2 (GenBank no. AF139597), Sst3a (GenBank no. AF311307), and Sst5a (GenBank no. AF252879). In these experiments, RT-PCR of β-actin mRNA was used as an internal control.
Real-time PCR measurement of SLα and SLβ mRNA.
To shed light on gbSS-28 regulation of SL gene expression, SLα and SLβ mRNA expression in goldfish pituitary cells was monitored by real-time PCR after gbSS-28 treatment. Primary cultures of pituitary cells were prepared from goldfish and seeded in 24-well culture plates at a density of 1.3 × 106 cells·ml−1·well−1. After overnight incubation to allow for recovery of membrane receptors, drug treatment was initiated by replacing the old medium with M199 medium (43). After drug treatment, total RNA (6 μg) was isolated from the cell culture using TRIzol, digested with DNase I to remove genomic DNA contamination, and reversely transcribed with SuperScript II. The RT samples obtained were then subjected to quantitative PCR using a Bio-Rad real-time PCR System. PCR reactions were conducted with a Fast-Plus EvaGreen qPCR master mix (Biotium) using the primers specific for goldfish SLα (SLα forward primer: 5′-ATATGTTTGTCCCGTACCCTCT-3′ and SLα reverse primer: 5′-TTTATCAGACACCCACTTGGTC-3′) and SLβ (SLβ forward primer: 5′-AGGGACCATGTGTTCTCCTAAA-3′ and SLβ reverse primer: 5′-AGAACCAGTATACCCTGCTCCA-3′). On the basis of our validation, these primers were highly specific and did not “cross-amplify” the transcript of each isoform. In our assay system, the PCR cycle number was fixed at 30 cycles with 30 s at 94°C for denaturing, 30 s at 56°C for annealing, and 30 s at 72°C for primer extension. During the reactions, the fluorescence signals for the respective PCR products were captured at 86°C for both goldfish SLα and SLβ transcripts. For data calibration, serial dilutions of plasmid DNA containing the respective ORF of goldfish SLα and SLβ were used as the standards for these real-time PCR assays. As an internal control for data normalization, parallel real-time PCR for β-actin mRNA was also conducted as described previously (43).
Measurement of cAMP production.
The NIL cells were seeded at a density of ∼1.5 × 106 cells·2 ml−1·dish−1 in poly-d-lysine-precoated 35-mm dishes and cultured overnight at 28°C as previously described (16). On the following day, culture medium was replaced with 0.9 ml HEPES-buffered Hanks' balanced salt solution with 0.1% BSA and 0.1 mM IBMX. IBMX, the inhibitor for phosphodiesterases, was included to prevent cAMP degradation in NIL cells. Drug treatment was initiated with various combinations of drugs at appropriate concentrations, and the cells were allowed to incubate at 28°C for 30 min. After that, culture medium was harvested for the measurement of cAMP release, whereas cellular cAMP content was extracted from NIL cells with 1 ml PBS. These cAMP samples were quantified by using a cAMP ELISA kit (Wuhan EIAab Science). In these studies, total cAMP production was defined as the sum of cellular cAMP content and the amount of cAMP released in the culture medium.
Data transformation and statistics.
For real-time PCR of SLα and SLβ transcripts, standard curves with a dynamic range of 105 and correlation coefficient of ≥0.95 were used for data calibration. SLα and SLβ mRNA expressions were quantified in terms of femtomole target transcript detected per million cells. Because no major changes in β-actin mRNA were noted in our studies, the raw data of SLα and SLβ mRNA expression were simply transformed as a percentage of the mean value in the control group without drug treatment (referred to as “%Ctrl”). Data presented (as means ± SE) are the results pooled from four separate experiments and were analyzed using ANOVA followed by Fisher's least-significant difference test. Differences between groups were considered to be significant at P < 0.05.
Molecular cloning of goldfish SLα cDNA.
With the use of 5′/3′-RACE, the full-length cDNA for SLα was isolated from the goldfish pituitary. The SLα cDNA is 1,404 bp in size with five putative polyadenylation signals and two long structures of “g” repeats in the 3′-untranslated region and an ORF of 705 bp encoding a 235-amino acid (AA) precursor for goldfish SLα (Fig. 1A). The SLα precursor is composed of a 24-AA signal peptide followed by a 211-AA mature protein with a deduced molecular mass of 26.8 kDa. Seven conserved Cys residues and an N-linked glycosylation site can be located in the mature protein of goldfish SLα. As shown in Fig. 1B, sequence alignment at the protein level also reveals that the AA sequence of goldfish SLα is highly homologous to that reported in grass carp (85%), zebrafish (78%), medaka (77%), and salmon (74%). Phylogenetic analysis based on nucleotide sequence has confirmed that the newly cloned goldfish SLα cDNA can be clustered within the clade of the SLα family and is closely related to SLα carp species, e.g., grass carp and zebrafish (Fig. 2A). In silico protein modeling also confirms that the 3-D structures of goldfish SLα and SLβ are highly comparable with a central core of four α-helices arranged in an “up-up-down-down” topography (Fig. 2B).
Tissue distribution of SLα and SLβ expression.
To characterize the tissue expression profile of SLα and SLβ, Northern blot analysis was performed in RNA samples prepared from various goldfish tissues. However, hybridization signals were detected in the goldfish pituitary (Fig. 3A) but not in other tissues (data not shown). At the pituitary level, single transcripts of SLα and SLβ with the size of 2.0 and 1.6 kb, respectively, could be detected. Because the hybridization signal for SLβ required a longer exposure and was found to be much weaker than that of SLα, it would be reasonable to assume that SLα is the dominant form of SL expressed in the goldfish pituitary. To further examine the possibility of extrapituitary expression of SLα and SLβ in goldfish, a more sensitive method, namely, RT-PCR, was used for SLα and SLβ mRNA detection in selected tissues. In accordance with the results of Northern blot, the pituitary was confirmed to be the tissue with the highest levels of SLα and SLβ mRNA expression. Except for the testis, lower levels of SLα transcript were detected in the brain, liver, gills, heart, intestine, fat, kidney, and ovary (Fig. 3B). In contrast to the wide range of tissue expression for SLα, apart from the high level of transcript expression in the pituitary, low levels of SLβ gene expression could be noted only in the brain and liver but not in other tissues examined (Fig. 3D). Because SLα and SLβ gene expressions were located in the brain, the spatial distribution of the two SL isoforms in different goldfish brain areas was also examined. In this case, SLα transcript was found to be expressed at relatively high levels in the olfactory bulb and hypothalamus, low levels in the telencephalon, medulla oblongata, and spinal cord, but not in the optic tectum and cerebellum (Fig. 3C). In contrast, goldfish SLβ was restricted to the hypothalamus (Fig. 3E). Given that the PCR signals for β-actin expression were consistently detected in all the tissues, the lack of PCR signals for SLα and SLβ caused by RNA degradation during sample processing was highly unlikely.
SLα and SLβ immunoreactivities in goldfish pituitary.
To characterize the protein expression of SLα and SLβ in the goldfish pituitary, the antisera raised against grass carp SLα and SLβ were used in Western blot analysis of protein lysate prepared form goldfish pituitary cells. As shown in Fig. 4A, two protein bands of 28 and 30 kDa in size were recognized by SLα antiserum, whereas only a single protein band of 26 kDa in size could be detected by SLβ antiserum. In these studies, no Western blot signals could be detected with the preimmunized serum for the respective antiserum. Furthermore, serial dilutions of the two antisera resulted in a gradual reduction of the respective signals for their target proteins. In parallel experiments, using recombinant protein of carp SLα and SLβ as the standard, the two antisera were confirmed to be highly specific for each isoform. In this case, the 28- and 30-kDa protein bands for SLα and 26-kDa protein band for SLβ were not only detected in pituitary cell lysate but also in culture medium, suggesting that the respective forms of SL could be secreted from goldfish pituitary cells (Fig. 4B). The Western blot signals for goldfish SLα and SLβ were further confirmed to be highly specific, since preabsorption of the two antisera for 15–18 h at 4°C with the recombinant proteins of carp SLα (1 μg/ml) and SLβ (1 μg/ml) could totally abolish the signals for SLα and SLβ immunoreactivities (Fig. 4C).
Because 1) an N-linked glycosylation site could be located in the coding sequence of goldfish SLα and SLβ and 2) SL is known to be expressed in periodic acid-Schiff-positive cells in the pars intermedia of the fish pituitary (33), the two SL isoforms expressed in the goldfish pituitary are suspected to be N-linked glycosylated. N-glycosidase digestion was performed in protein lysate and conditioned medium prepared from goldfish pituitary cells. Treatment with N-glycosidase F resulted in disappearance of the 30-kDa but not the 28-kDa protein band for SLα, implying that the 30-kDa SLα was a N-linked glycoprotein. Similar to the 28-kDa protein band for SLα, the 26-kDa protein band for SLβ was also not affected by N-glycosidase F treatment (Fig. 4D).
Spatial distribution of SLα and SLβ cells in the goldfish pituitary.
To establish the anatomical distribution of SL cells within the goldfish pituitary, in situ hybridization was performed in goldfish pituitary sections using DIG-labeled antisense riboprobes for goldfish SLα and SLβ. As revealed in Fig. 5A, hybridization signals for SLα and SLβ transcripts were detected in the pituitary cells located in the NIL region. At a high magnification, the hybridization signal for SLα and SLβ transcripts was located only in the cytoplasm but not the nucleus of the target cells. Among the two SL cell populations, SLα cells appeared to be the dominant form and distributed widely throughout the NIL region. However, SLβ cells were of a smaller population size, and their distributions were restricted to the area of the NIL adjacent to the boundary of the proximal pars distalis (PPD).
To further confirm the regional distribution of the two SL isoforms at the goldfish pituitary level, immunohistochemical staining of goldfish pituitary sections was conducted using the antisera for carp SLα and SLβ (Fig. 5B). Consistent with the findings based on the in situ hybridization, the cell population with SLα immunoreactivity was found to be the major form of the SL. Although both SLα and SLβ immunostaining signals could be also recognized in the PPD by immunohistochemical staining, the cause for different expression patterns of two isoforms between the mRNA and protein levels is unclear. Furthermore, no immunostaining signals could be noted in the goldfish pituitary section by replacing the SLα and SLβ antisera with the respective preimmunized serum (data not shown), confirming that the antisera used in the present study were specific for the respective ligand.
Expression profiles of Sst subtypes in the goldfish NIL cells.
To investigate the expression profiles of Sst subtypes in the goldfish NIL cells, the mRNA expression patterns of Sst1b, Sst2, Sst3a, and Sst5a were determined by RT-PCR. Target PCR fragment for Sst5a transcript were detected in NIL cells, whereas Sst1b, Sst2, and Sst3a transcripts could not be detected in parallel experiments (Fig. 6). Because the PCR signal was also detected in RT sample prepared from mixed populations of pituitary cells but not in RNA samples obtained from whole pituitary and NIL cells without reverse transcription, the possibility of “false positive” for Sst subtypes expression caused by genomic DNA contamination is unlikely.
gbSS-28 inhibition of SLα and SLβ mRNA expression.
To investigate the mechanisms for gbSS-28 inhibition of SL gene expression, the effects of gbSS-28 treatment on SLα and SLβ gene expression were examined in goldfish pituitary cells. As shown in Fig. 7A, gbSS-28 (100 nM) could noticeably inhibit SLα and SLβ mRNA expression in a time-related manner, and the maximal inhibition on SLα and SLβ mRNA expression was observed at 48 h. In parallel experiments, a 48-h incubation of pituitary cells with increasing concentrations (0.1–100 nM) of gbSS-28 resulted in a concentration-related decrease in SLα and SLβ mRNA levels (Fig. 7B). The minimal effective dose for gbSS-28 to inhibit SLα and SLβ mRNA expression could be noted at 1 nM, whereas the maximal responses were noted in the 10 to 100 nM dose range.
More recently, IGF-I and IGF-II were found to increase SL gene expression in a dose-dependent manner using primary culture of carp pituitary cells (18). To shed light on the functional interactions between gbSS-28 and IGFs in regulating SL gene expression at the pituitary level, IGF-stimulated SLα and SLβ gene expression was tested in the presence or absence of gbSS-28 (100 nM). In this case, both IGF-I (10 nM) and IGF-II (10 nM) were effective in stimulating SLα and SLβ mRNA expression; however, this stimulatory effect could be abolished in the presence of gbSS-28 treatment (Fig. 7, C and D).
Signal transduction mechanisms for gbSS-28-inhibited SL gene expression.
Given that SS is known to inhibit cAMP synthesis via activation of AC activity at the pituitary level in fish (44), the effects of gbSS-28 on cAMP production were tested in NIL cells prepared from the goldfish pituitary. As shown in Fig. 8A, forskolin was effective in elevating total cAMP production. In contrast, basal levels and forskolin-induced increases in cAMP production were significantly suppressed by treatment with 100 nM gbSS-28 using primary cultured goldfish NIL cells. To further evaluate the functional role of the cAMP-dependent pathway in gbSS-28-inhibited SL gene expression, mixed populations of goldfish pituitary cells were exposed to gbSS-28 (100 nM) in the presence of the AC activator forskolin (1 μM) and cAMP analog CPT-cAMP (100 μM). In this case, application of the forskolin (1 μM) and CPT-cAMP (100 μM) increased SLα and SLβ gene expression. The addition of gbSS-28 (100 nM) significantly inhibited not only basal SL gene expression but also forskolin- and CPT-cAMP-stimulated SL gene expression (Fig. 8, B and C). In mammals, Ssts are also known to couple with the PLC/IP3/PKC pathway (7). The involvement of the PLC/IP3/PKC-dependent pathway in gbSS-28 inhibition of SL gene expression is suspected. To test the hypothesis, the inhibitory effects of gbSS-28 (100 nM) were examined in the presence of the PLC inhibitor U-73122 (5 μM) and IP3 receptor inhibitor 2-APB (10 μM). In these experiments, gbSS-28 suppression of SLα and SLβ mRNA expression could be prevented by these pharmacological inhibitors (Fig. 9, A and B). In parallel experiments, the inhibitory effects of gbSS-28 on SL gene expression were tested in the presence of the PKC inhibitor GF-109203 (10 μM). In this case, the inhibition on SLα mRNA expression induced by gbSS-28 was totally abolished by cotreatment with the PKC inhibitor (Fig. 9C). Similar treatment with the PKC inhibitor GF-109203, however, did not alter gbSS-28 inhibition of SLβ gene expression in goldfish pituitary cells (Fig. 9D).
To date, two isoforms of SL, namely SLα and SLβ, have been identified in fish species, e.g., zebrafish (47) and grass carp (17), but only the SLβ isoform has been reported in goldfish (10). To elucidate the structure of SLα in goldfish, 5′/3′-RACE was conducted to pull out the full-length cDNA of goldfish SLα. As revealed by phylogenetic analysis, the newly cloned cDNA could be clustered within the clade of SLα but not SLβ gene family. Although its sequence homology at the protein level is relatively low when compared with that of goldfish SLβ (47%), goldfish SLα is highly homologous to the SLα isoforms reported in other fish species (74–85%). Similar to the 3-D structure of goldfish SLβ, the newly cloned SLα also exhibits the four-helical core structure arranged in an up-up-down-down topography that is typical for the members of the class I cytokines, including GH and PRL (9). Similar to our recent studies in grass carp (17), transcript expression of SLα and SLβ could be noted in extrapituitary tissues and different brain areas of the goldfish, but the highest levels of gene expression for the two isoforms were detected in the pituitary, consistent with the functional roles of SL as a pituitary hormone in fish. At the pituitary level, as revealed by in situ hybridization and immunohistochemical staining, although SLα and SLβ were expressed in pituitary cells located in the NIL region, we could not rule out the possibility that SLβ was produced by a subpopulation of SLα cells and vice versa. Given that 1) SLα “positive cells” represent the major cell population within the goldfish NIL, 2) the transcript expression of SLα in the goldfish pituitary was found to be much higher than that of SLβ, and 3) SLα is widely expressed in different extrapituitary tissues and brain areas in fish [e.g., grass carp (17) and goldfish], it would be logical to assume that SLα but not SLβ is the major form of SL with a functional role in goldfish.
Recently, we raised specific antisera against carp SLα and SLβ (18). In our present studies, these antisera were confirmed to be specific for respective ligands in goldfish since 1) specific signal of target protein could be detected with the respective antiserum but not by the preimmunized serum and 2) these specific signals of immunostaining could be reduced gradually with serial dilution of antiserum. With the use of these antisera coupled with an N-glycosidase F assay, both glycosylated (30 kDa) and nonglycosylated (28 kDa) forms of SLα and a nonglycosylated form of SLβ (26 kDa) were detected in the cell lysate and conditioned medium prepared from goldfish pituitary cells. These results suggest that 1) the two SL isoforms are produced and released from goldfish pituitary cells and 2) posttranslational modification by an N-linked glycosylation has occurred during the process of SLα but not SLβ synthesis. Although an N-linked glycosylation site could be identified in the coding sequence of goldfish SLα, the nonglycosylated form of SLα appears to be the major product in goldfish pituitary cell culture. SLs with different levels of glycosylation have also been reported in other fish species, e.g., in gilthead seabream (2), Atlantic cod (37), and grass carp (17), but the functional role of SL's glycosylation has not been elucidated.
Sst subtypes have been cloned in goldfish and confirmed to be expressed at the pituitary level (31). Identification of multiple types of Sst raises a question whether a given physiological response is selective for one type of receptors or whether multiple types of receptors are involved. In CCL39 cells stably expressing the gfSst5a, gbSS-28 and mammalian SS-28 analog bind gfSst5a with higher affinity than SS-14 and [Pro2]SS-14 (26). In our present study, all four subtypes of Sst1b, Sst2, Sst3a, and Sst5a were recognized to be expressed in goldfish pituitary, whereas only Sst5a transcripts were located in NIL cells, suggesting Sst5 is a predominant subtype in goldfish NIL cells and involved in the direct regulation of SL gene expression. This idea is supported by our in vitro studies with goldfish pituitary cells in which gbSS-28 was found to reduce SLα and SLβ mRNA expression in a time- and concentration-related manner. In goldfish pituitary cells, short-term incubation with gbSS-28 did not alter SL gene expression, but inhibition of mRNA levels for the respective isoforms was noted by prolonging the drug treatment to 24–48 h. The “delayed” responses for SL transcript expression might be the result of the late onset of gene transcription at the pituitary cell level. In a recent study with grass carp, both IGF-I and IGF-II were effective in stimulating SLα and SLβ gene expression in a dose-dependent manner (18). In our present studies, both IGF-I and IGF-II could also increase goldfish SLα and SLβ mRNA levels, but these stimulatory actions were blocked by cotreatment with gbSS-28, suggesting SLα and SLβ gene expressions are coordinately regulated in goldfish pituitary cells by gbSS-28 and IGFs. In goldfish, gbSS-28 mRNA was identified within preparations of dispersed pituitary cells (46), implying that gbSS-28 may also be produced locally at the level of the pituitary. Our results suggest that gbSS-28 via Sst5a activation can act directly at the pituitary level to inhibit SL gene expression in goldfish. Our findings also raise the possibility that SS may be produced locally in the fish pituitary and serve as an autocrine/paracrine regulator.
Molecular inhibitory mechanisms of SS in teleosts are mainly derived from its action on GH secretion at the pituitary level. However, the signal transduction mechanisms responsible for SL gene expression are largely unknown. In CCL39 cells stably expressing the gfSst5a, gbSS-28 binding Sst5a are coupled to Gi/o protein (26, 32). In NIL cells prepared from the goldfish pituitary, gbSS-28 treatment could inhibit total cAMP production, and the ability of forskolin to increase cAMP production was attenuated by gbSS-28. Our present findings are also in line with the previous reports that treatment with gbSS-28 decreased basal cAMP levels from primary cultures of dispersed goldfish pituitary cells (44). In parallel experiments with mixed populations of goldfish pituitary cells, gbSS-28 was effective in abolishing the SLα and SLβ responses to CPT-cAMP and forskolin. These results, taken together, indicate gbSS-28 interferes with cAMP-dependent mechanisms to inhibit SL gene expression in goldfish pituitary cells. Apart from cAMP-dependent mechanisms, differential coupling with the PLC/IP3/PKC pathway was also observed for SLα and SLβ gene expression. In this case, PLC inhibitor U-73122, IP3 receptor inhibitor 2-APB, and PKC inhibitor GF-109203 were effective in blocking gbSS-28-inhibited SLα mRNA expression. These results indicate that the PLC/IP3/PKC pathway is involved in gbSS-28 inhibition of SLα gene expression. Despite the fact that the reduction in SLβ mRNA levels caused by gbSS-28 could be negated by inhibiting PLC and IP3 receptors, this inhibitory action was not affected by PKC blockade, suggesting that the PKC component may be uncoupled from PLC-dependent mechanisms leading to SLβ gene expression. Because PKC isoforms are expressed in the goldfish pituitary in a cell type-specific manner (22), the differential regulation of the SL isoforms may result from different components of PKC isoforms expressed in NIL cells. Also, given that IP3 receptors are Ca2+ channels responsible for intracellular Ca2+ concentration ([Ca2+]i) release from IP3-sensitive Ca2+ stores (11), we should not rule out the possibility that [Ca2+]i mobilization may contribute to gbSS-28-inhibited SL gene expression.
In summary, we have cloned SLα cDNA in goldfish and confirmed that the two isoforms of SL, namely, SLα and SLβ, could be detected in separate cell populations of the NIL region. Using goldfish pituitary cells as a model, we provide evidence for the first time that gbSS-28 can act at the pituitary level to inhibit SLα and SLβ gene expression via Sst5 receptors. The inhibitory action of gbSS-28 was mediated by overlapping and yet distinct signaling pathways (Fig. 10, A and B). Furthermore, our report also provides novel information on the signal transduction for the SS neuroendocrine system in basal vertebrates. Further investigations on the role of gbSS-28 in regulating SL secretion are clearly warranted.
The project was supported by grants from the Natural Science Foundation of China (31302165), the Scientific Research Foundation for Young Teachers (Grant No. 2013SCU11021, Sichuan University) to Q. Jiang, and GRF grants (RGC, HK) to A. O. L. Wong.
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: Q.J. and A.O.W. conception and design of research; Q.J. performed experiments; Q.J. analyzed data; Q.J. and A.O.W. interpreted results of experiments; Q.J. prepared figures; Q.J. and A.O.W. drafted manuscript; Q.J. edited and revised manuscript; Q.J. and A.O.W. approved final version of manuscript.
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