Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Comparative Biochemistry and Physiology, Part B 159 (2011) 84–91 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b Molecular and functional analyses of growth hormone-releasing hormone (GHRH) from olive flounder (Paralichthys olivaceus) Bo-Hye Nam ⁎, Ji-Young Moon, Young-Ok Kim, Hee Jeong Kong, Woo-Jin Kim, Kyong-Kil Kim, Sang-Jun Lee Biotechnology Research Division, National Fisheries Research and Development Institute, 408-1, Sirang-ri, Gijang-eup, Gijang-gun, Busan 619-902, Republic of Korea a r t i c l e i n f o Article history: Received 29 October 2010 Received in revised form 21 February 2011 Accepted 24 February 2011 Available online 1 March 2011 Keywords: Growth hormone-releasing hormone Growth hormone Fish Bidirectional communication Growth Immunity a b s t r a c t Growth hormone-releasing hormone (GHRH) is an important neuroendocrine factor that stimulates the release of growth hormone (GH) from the anterior pituitary. Several nonmammalian GHRH-like peptides were reported previously to be encoded by PACAP and processed from the same transcript and prepropolypeptide. However, the true nonmammalian GHRHs in amphibian and fishes were only recently discovered. We identified and characterized the primary structure of the GHRH gene and determined its expression profiles under normal and infectious conditions in the teleost fish, Paralichthys olivaceus. The 142 amino acids of the GHRH precursor are encoded by six exons spanning 2290 bp. The flounder GHRH precursor mRNA was constitutively expressed in the brain as well as gills and ovary. Inducible expression of GHRH mRNA was observed in the gills of Edwardsiella tarda-challenged fish. Induction of GHRH mRNA was highest at 24 h post-bacterial challenge. Subsequently, the biological role of GHRH was investigated by exogenous treatment of flounder embryogenic cells (hirame natural embryonic cells, HINAE cells) and primary cultured pituitary cells with a synthetic GHRH peptide (fGHRH-28). The 10− 6 M concentration of fGHRH-28 produced intracellular cAMP in HINAE cells and induced growth hormone mRNA in both of HINAE and pituitary cells. The profiles of TNF-α mRNA expression differed from HINAE and pituitary cells after fGHRH-28 treatment. TNF-α mRNA levels elevated approximately 3-fold in HINAE cells, but decreased to one-third in pituitary cells stimulated by fGHRH-28. These results suggest that the flounder GHRH plays roles in the bidirectional communication network between growth and immunity in fish. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Growth hormone-releasing hormone (GHRH) belongs to the glucagon/secretin superfamily, which also includes glucagons, glucagons-like peptides (GLP-1 and GLP-2), pituitary adenylate cyclaseactivating polypeptide (PACAP), PACAP-related peptide (PRP), secretin, and vasoactive intestinal peptide (VIP), peptide histidine methionine (PHM) or peptide histidine isoleucine (PHI) and glucose-dependent insulinotropic peptide (GIP) (Sherwood et al., 2000). The glucagons/secretin superfamily is believed to have originated from a common ancestral gene. The VIP, PACAP, and glucagon genes encode two or three bioactive peptides, whereas the GHRH, secretin, and GIP genes encode only a single bioactive peptide in mammals. Previously, PACAP and GHRH were hypothesized to be encoded on the same gene in nonmammalian vertebrates such as fish (Parker et al., 1993) and birds (McRory and Sherwood, 1997), whereas in mammals, PACAP and GHRH are encoded by separate genes on separate chromosomes (Hosoya et al., 1992; Mayo et al., 1985). This hypothesis implies that duplication of the GHRH-like/PACAP gene took place just ⁎ Corresponding author. Tel.: + 82 51 720 2452; fax: + 82 51 720 2456. E-mail address: [email protected] (B.-H. Nam). 1096-4959/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2011.02.006 before the divergence of mammals, giving rise to the PRP/PACAP gene, via modifications of the GHRH-like sequences (Holmgren and Jensen, 2001). However, recent research on the evolution of GHRH in nonmammalian vertebrates showed that the GHRH-like peptides that encode PACAP in non-mammals are in fact the counterparts of mammalian PRPs, and the real GHRH peptides encoded in cDNA isolated from goldfish, zebrafish, and African clawed frog were identified (Lee et al., 2007). Moreover, the goldfish GHRH reportedly functions to dose-dependently activate cAMP production in receptortransfected CHO cells as well as GH release from goldfish pituitary cells (Lee et al., 2007). The primary function of GHRH is to stimulate growth hormone (GH) synthesis and release from anterior pituitary cells via specific interaction with its receptor, the GHRH receptor (GHRHR). For many years, investigators reported a connection between the nervous system and immune system, and named this relationship the “bidirectional communication network.” The bidirectional communication network is possible because the nervous and immune systems share a common biochemical language involving shared ligands and receptors, including neurotransmitters, neuropeptides, growth factors, neuroendocrine hormones, and cytokines (Kelley et al., 2007). In particular, GH, prolactin (PRL), and insulin-like growth factor (IGF) are well-known as modulators of the immune system in mammals B.-H. Nam et al. / Comparative Biochemistry and Physiology, Part B 159 (2011) 84–91 (Gala, 1991; Clark, 1997; Kooijman et al., 1996; Auernhammer and Strasburer, 1995; Velkeniers et al., 1998). PACAP and VIP, which belong to the glucagon superfamily, were also demonstrated as important modulators in the mammalian immune system (reviewed in Pozo, 2003; Brenneman, 2007; Bik et al., 2006). Recently, the roles of PACAP and VIP in the fish immune system have been investigated and reported by several groups. For example, the administration of recombinant PACAP by immersion baths increased lysozyme, nitric oxide synthase (NOS)-derived metabolites, and antioxidant defenses in African catfish fry (Carpio et al., 2008). In addition, injection of recombinant PACAP increased the NOS and total immunoglobulin M (IgM) concentration in the serum of juvenile catfish and tilapia (Lugo et al., 2010). Furthermore, we demonstrated that the prepro-VIP mRNA expression was significantly upregulated in the spleen and kidney of olive flounder after an artificial bacterial challenge with Edwardsiella tarda (Nam et al., 2009). The present work aimed to characterize a fish GHRH gene and examine its biological activity with respect to inducing GH expression, in addition to evaluating the potential function of GHRH within the teleostean immune system. 2. Materials and methods 2.1. Fish and cell culture Olive flounder (Paralichthys olivaceus; mean mass 100 g) was supplied by the Genetic and Breeding Research Center of NFRDI on Geoje Island (southern South Korea) and maintained in a circulating seawater system at 23 ± 1 °C with commercial feeding. For the artificial bacterial infection, the fish were anesthetized with MS-222 (3-aminobenzoic acid ethyl ester; Sigma, St. Louis, MO, USA) and infected with E. tarda by intraperitoneal injection of a sublethal dose (1.2 × 106 cells) suspended in phosphate-buffered saline (PBS). Tissues were collected from three fish at 0, 1, 3, 6, 12, 24, and 72 h postinjection and frozen at −80 °C for RNA extraction. HINAE olive flounder embryonic cell lines were maintained in Leibovitz L-15 medium with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco-BRL, Grand Island, NY, USA) and 1% (v/v) penicillin–streptomycin (PS; Gibco-BRL) at 20 °C. Primary pituitary cell culture was performed according to the method described by Salamat et al. (2009). Pituitary glands were removed from twelve flounders (1-year-old). The glands were rinsed several times with sterile ice-cold Eagle's minimum essential medium (MEM) with 15 mM HEPES and 9 mM bicarbonate. The collected glands were chopped into small pieces and subjected to dispersion for 2–3 h at 20 °C in MEM containing 1% collagenase H and 1% FBS. Enzymatic dissociation was followed mechanically by aspiration of the cell clusters using pipettes. The cells were harvested by centrifugation (200 g, 20 °C, 10 min) and washed twice with preincubation medium containing FBS (2%) and PS (1%) solution. Pituitary cells were resuspended and maintained at 20 °C in MEM containing 10% FBS and 1% PS solution until use. 2.2. Full-length cDNA cloning The EST clone “Gill-1-B01,” which carries an 806-bp insertion, showed significant sequence homology to the goldfish GHRH gene (GenBank accession no. DQ991243), lacking the 5′-UTR and 3′-UTR complete sequences. For the full-length cDNA cloning of GHRH, the 5′and 3′-UTRs of flounder GHRH were amplified from the first-strand cDNA of brain tissue using the rapid amplification of cDNA ends (RACE) technique with a SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA). Total RNA from the collected tissue was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The 5′- and 3′ endspecific primers of GHRH (5′-TAG AGT GGG GAG CCT GAT AGA GAC ATG-3′ for 5′-RACE or 5′-ACT GCT GAG CTG AGA TCA GAG GAC CA-3′ 85 for 3′-RACE) and a nested universal primer included in the amplification kit were used independently for amplifying the 5′and 3′-ends of GHRH cDNA. PCR products were cloned into a pGEM®T Easy Vector (Promega, Madison, WI, USA), and individual cDNA clones were sequenced using the ABI 3130 Automatic DNA Sequencer (Applied Biosystems, Foster City, CA, USA). 2.3. Cloning of the GHRH gene The olive flounder genomic BAC library (Nam et al., 2010) was screened to isolate the GHRH gene using the BAC pooling system with PCR primers specific for the GHRH coding region. PCR-based BAC library screening was carried out as previously reported (Chae et al., 2007). The obtained GHRH genomic BAC clone was purified and used for genomic structure analysis and determination of nucleotide sequence of the flounder GHRH 5′-flanking region with two specific primers for genome walking (GHRH gw-1: 5′-TCC TCT CTC ATC ATC GCT TAC T-3′ and GHRH gw-2: 5′-GCT CGA GGG ATA CAG GTT ATA T-3′). 2.4. Sequence analysis The relevant sequences were retrieved from GenBank for multiple sequence alignments using ClustalX and refined using GENETYX version 8.0 (SDC Software Development, Tokyo, Japan). A phylogenetic tree was created based on the amino acid distances between the aligned sequences using the neighbor-joining method with 1000 bootstrap replications using the MEGA software (version 4.1). A computer search for putative cis-elements was performed using the Transcription Element Search System (TESS) program (http://www. cbil.upenn.edu/cgi-bin/tess). The putative signal peptide was predicted using the SignalP 3.0 server (http://www.cbs.dtu.dk/services/ SignalP/). 2.5. Tissue distribution of GHRH Total RNA was extracted from each tissue using TRIzol reagent according to the manufacturer's instructions and treated with DNase I (Invitrogen) to destroy contaminating genomic DNA before conversion to cDNA. First-strand cDNA synthesis was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany). The tissue distribution and profile changes by bacterial challenge of GHRH mRNA expression were studied by semiquantitative RT-PCR using gene-specific primers (GHRH-ORF-F: 5′-ACT CTT CTC CAG GAC GAG CTG TG-3′ and GHRH-ORF-R: 5′-GGC AGT CTG GTC CTC TGA TCT-3′). PCR amplification cycles were as follows: 95 °C for 5 min; 30 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a final extension period at 72 °C for 10 min. We used 18S rRNA as an internal control with each primer pair (18S rRNA-F: 5′-ATG GCC GTT CTT AGT TCC TG-3′ and 18S rRNA-R: 5′-CCA CGC TGA TCC AGT CAG T-3′). 2.6. Synthesis and purification of the peptide The olive flounder GHRH peptide (fGHRH-28: HADAIFTNSYRKVLGQISARKFLQTVMG) was commercially synthesized by Peptron Inc. (Daejeon, South Korea). Briefly, the peptide was synthesized using Fmoc SPPS (solid phase peptide synthesis) with ASP48S (Peptron Inc.) and purified using reverse phase HPLC with a Vydac Everest C18 column (250 mm × 22 mm, 10 μm; Grace, Deerfield, IL). Elution was carried out with a water–acetonitrile linear gradient (3– 40% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. Molecular weights of the purified peptide were confirmed using LC/ MS (HP1100 series; Agilent, Santa Clara, CA, USA). 86 B.-H. Nam et al. / Comparative Biochemistry and Physiology, Part B 159 (2011) 84–91 2.7. Measurement of intracellular cAMP HINAE cells were seeded in 24-well plates (2.5 × 105 cells/well) and incubated for 24 h. Before treatment with various concentration of fGHRH-28, the cells were pretreated with 10− 6 M fGHRH-28 for 2 h. Subsequently, medium was removed and cells were treated with 10–6, 10− 8, and 10− 10 M fGHRH-28 for 4 h. After 4 h of treatment with peptides, cells were lysed, and cAMP was measured as described in the Format A Cyclic AMP “PLUS” Enzyme Immunoassay Kit (Enzo Life Sciences, Plymouth Meeting, PA, USA). 2.8. mRNA expression of GH and TNF-α in HINAE and primary pituitary cells treated with fGHRH-28 Treatment of cells with fGHRH-28 peptide was performed after 24 h culture. On the day of the experiment, the culture medium was changed to serum-free L-15 medium, and 10− 6 M fGHRH-28 was added to 2.5 × 105 cells/well in a 6-well culture plate in replenished incubation medium for 0, 6, 12, 24, 48, and 72 h. All experiments were performed in triplicate per time point. To detect GH and TNF-α mRNA in response to the fGHRH-28 peptide, cells from the same time-points were harvested and pooled. Total RNA extraction was performed with control and treated cells at each time point. For the detection of GHRH-induced GH (GenBank accession no. M23439) and TNF-α (GenBank accession no. AB040448) mRNA, an adapted real-time PCR technique was applied to distinguish different expression levels because differences could not be clearly detected using traditional RT-PCR. Quantitative real-time PCR was performed using the LightCycler system (Roche Diagnostics) with FastStart DNA Master SYBR Green I (Roche Diagnostics). The specific primer sets used are as follows: for flounder GH, fGH-RT-F: 5′-CCG ATC GAC AA CAC GAG AC-3′ and fGH-RT-R: 5′-GAA TCC ACC TGC TCC ATC CT-3′; for flounder TNF-α, fTNF-a RT-F: 5′-GAC TTC CGA AAA CAA GCT GGT-3′ and fTNF-a ORF-R: 5′-TCA AAG TGC AAA GAC ACC GAA-3′. Following an initial 10-min Taq activation step at 95 °C, 40 cycles of LightCycler PCR was conducted under the following cycling conditions: 95 °C for 10 s, 55 °C for 5 s, and 72 °C for 20 s with fluorescence reading. Immediately following the PCR, the machine performed a melting curve analysis by gradually (0.1 °C/s) increasing the temperature while measuring fluorescence emission intensity. The relative expression of each gene was determined by the 2−ΔΔCT method (Livak and Schmittgen, 2001), using 18S rRNA as the reference gene. All assays were performed in triplicate per cDNA sample in independent experiments. 2.9. Data analysis All experiments were performed at least three times with reproducible results. Data are presented as means ± standard deviation (SD). Values were analyzed using one-way analysis of variance (ANOVA) when comparing the control and samples at different time points, followed by Duncan's multiple comparison test. A P-value less than 0.05 was considered to indicate statistical significance. All analyses were carried out using the SAS software (ver. 9.1; SAS Institute, Cary, NC, USA). 3. Results 3.1. Sequence analysis The EST clone “Gill-1-B01” (GenBank accession no. CX283280), which carries an 806-bp insertion, showed significant sequence homology to the goldfish GHRH gene (GenBank accession no. DQ991243), lacking the 5′-UTR and 3′-UTR complete sequences. The 5′- and 3′-UTRs of flounder GHRH were amplified from the firststranded cDNA of brain tissue using 5′-RACE and 3′-RACE with primers based on the Gill-1-B01 sequence. The full-length cDNA of GHRH was 842 bp in length, consisting of a 429-bp open reading frame encoding a GHRH precursor of 142 amino acids, 149 bp of 5′UTR, and 264 bp of 3′-UTR (GenBank accession no. HQ402560; Fig. 1). To determine the genomic sequence and organization of the GHRH gene, we isolated a BAC clone containing the GHRH gene using a PCRbased BAC library screening method. The genomic DNAs of the coding region and 5′-flanking region were amplified from GHRH-BAC DNA by PCR. Sequence analysis showed that the genomic DNA sequence of the flounder GHRH gene obtained in the present study contains 2290 bp and is composed of six exons and five introns (GenBank accession no. HQ402561), which are 209, 665, 428, 89, and 99 bp in length, respectively. Typical intronic splice motifs were observed at the 5′ (GT)- and 3′(AG)-ends of each intron. The first exon represents a 5′UTR sequence of 120 bp. The second exon encodes a putative signal peptide of 19 amino acids and 9 additional amino acids. The third and fourth exons encode a cryptic peptide and GHRH peptide, respectively. The fifth exon contains 126 bp, which encodes a carboxyl terminal Fig. 1. The nucleotide and deduced amino acid sequences of GHRH precursor cDNA: signal peptide (underlined), GHRH peptide sequence (shaded), and predicted polyadenylation sequence (bold). B.-H. Nam et al. / Comparative Biochemistry and Physiology, Part B 159 (2011) 84–91 flanking peptide (C-peptide). The sixth exon directs the three amino acid sequences and contains a stop codon and a 3′-UTR (Fig. 2). Sequence analysis of the 1400-bp 5′-flanking upstream from the transcription start site of the GHRH gene revealed multiple potential cis-acting elements, including three binding sites for activating protein-1 (AP-1), two binding sites for cAMP response element binding protein (CRE-BP), and nuclear factor of activated T cells (NF-AT), as well as one site each for interleukin-6 responsive element-binding protein (IL-6 RE-BP), stimulating protein-1 (Sp-1), and TATA-binding protein (TBP) (Fig. 2). 87 3.2. Gene organization and phylogenetic analysis Although the deduced amino acid sequence of GHRH is highly conserved between teleosts and mammals, the flounder GHRH precursor includes a cryptic peptide encoded by exon 3, which is absent in mammals but exists in avian, amphibian, and fish species examined with the exception of zebrafish (Fig. 3). A phylogenic tree was constructed based on the deduced GHRH precursor amino acid sequences of reported species using the neighbor-joining method. As Fig. 2. DNA sequence of the flounder GHRH gene. Nucleotide numbering is from the proposed transcriptional start site. Underlined regions indicate consensus sequences as described in the text. B.-H. Nam et al. / Comparative Biochemistry and Physiology, Part B 159 (2011) 84–91 Human SP GHRH Mouse SP Chicken Xenopus SP CP ? SP GHRH CP SP Flounder 5,804 bp C-peptide 17,160 bp GHRH C-peptide C-peptide 7,172 bp GHRH C-peptide ? 1,945 bp GHRH SP Zebrafish C-peptide GHRH CP C-peptide 89,938 bp C-peptide 2,290 bp Fig. 3. Comparison of the gene organization of the flounder GHRH and that of other species. The lengths of the exons and introns are not proportional so that exons can be aligned between genes. Open boxes indicate untranslated regions, and closed and shaded boxes indicate the coding regions of the exons for each gene. The accession numbers of the GHRH genomic sequences used in the GenBank database are as follows: human, NC_000020; mouse, NC_000068; chicken, NC_006107; Xenopus tropicalis, Scallfold_38 in Ensembl; zebrafish, NC_007122; flounder, HQ402561. Sp, signal peptide; GHRH, growth hormone releasing hormone; CP, cryptic peptide; C-peptide, C-terminal peptide. shown in Fig. 4, the flounder GHRH precursor formed a cluster with Xenopus and other fishes. 3.3. Expression of GHRH mRNA To determine the tissue distribution of the GHRH transcript in healthy flounder, RT-PCR was performed in 12 tissues (brain, heart, gill, liver, kidney, spleen, stomach, ovary, testis, pyloric cecum, intestine, and skin). As a result, the GHRH transcript was detected in the brain, gill, and ovary of healthy flounders (Fig. 5A). To investigate the flounder immune responses through transcriptional regulation of GHRH, the expression of GHRH mRNA was analyzed by RT-PCR in gill tissues following infection with E. tarda because the flounder GHRH was originally identified from an EST analysis of gill tissue. As shown in Fig. 5B, the expression of GHRH mRNA in the gill gradually increased from 12 h post-challenge and reached its highest level at 24 h post-challenge. 3.4. Effects of GHRH treatment on HINAE and primary pituitary cells in vitro To examine the function of flounder GHRH, the intracellular cAMP production was measured in HINAE cells after treatment with fGHRH-28 at various concentrations. Prior to the experiments for intracellular cAMP production, HINAE cells were treated with 10− 6, 89 85 Human GHRH Mouse GHRH 97 Rat GHRH 92 Chicken GHRH Xenopus GHRH Flounder GHRH Goldfish GHRH 99 87 10− 8, or 10− 10 M fGHRH-28 for 4 h without any pre-treatment. However, a single treatment of fGHRH-28 did not induce cAMP production from HINAE cells (data not shown). To raise the basal level of the GHRH-receptor on HINAE cells, the cells were pretreated with 10− 6 M fGHRH-28 for 2 h. Subsequently, the medium was removed and cells were treated with 10− 6, 10− 8, and 10− 10 M fGHRH-28 for 4 h. Of interest was that fGHRH-28 could stimulate intracellular cAMP production in HINAE cells at 10− 6 M (Fig. 6). To confirm whether flounder GHRH could induce GH, real-time RT-PCR was performed to detect the expression of GH mRNAs in HINAE and pituitary cells after 10− 6 M fGHRH-28 treatment at various incubation times. GH mRNA was induced at 24 h and the highest levels were detected at 48 h after peptide treatment in both HINAE (1.97-fold) and pituitary (2.03-fold) cells (Fig. 7). Additionally, to test whether the flounder GHRH affects the immune system, the expression of TNF-α mRNA was also observed in both cells treated with 10− 6 M fGHRH-28. The expression level of TNF-α mRNA decreased slightly at the 12-h incubation time, but TNF-α mRNA was induced at 24 h, reached 2.9-fold at 48 h, then gradually decreased in HINAE cells (Fig. 8A). However, in pituitary cells, TNFα mRNA levels reduced 27% during 10− 6 M fGHRH-28 treatment (Fig. 8B). 4. Discussion Previous reports suggested that several nonmammalian GHRHlike peptides are encoded with PACAP and processed from the same transcript and prepropolypeptide. However, the previously named 3.0 ** 2.5 Dog GHRH Bovine GHRH 100 Fig. 5. mRNA expression of GHRH. A. Tissue distribution of GHRH in the normal conditioned fish. B. Transcriptional change of GHRH in gills following E. tarda challenge. Expression analysis of GHRH mRNA was performed by RT-PCR and 18S rRNA was used for the internal control. B, brain; H, heart; G, gills; L, liver; K, kidney; Sp, spleen; St, stomach; O, ovary; T, testis; P, pyloric cecum; I, intestine; and Sk, skin. cAMP induction (fold) 88 2.0 1.5 1.0 0.5 Zebrafish GHRH 0.1 Fig. 4. Phylogenetic analysis of GHRH. The tree was constructed based on amino acid sequences. The number at each node indicates the percentage of bootstrapping after 1000 replications. The accession numbers of the GHRH amino acid sequences used in the GenBank database are as follows: human, AAA52609; bovine, NP_847895; dog, XP_542987; mouse, NP_034415; rat, NP_113765; chicken, NP_001035554; Xenopus, NP_001090197; goldfish, ABJ55977; zebrafish, NP_001073561; flounder, HQ402560). 0 Cont. 10-10 10-8 10-6 GHRH concentration (M) Fig. 6. Intracellular cAMP production in HINAE cells stimulated with various concentration of fGHRH-28. Values represent the mean ± SD from three independent experiments. The asterisk indicates a statistically significant difference (double asterisk, P b 0.01). B.-H. Nam et al. / Comparative Biochemistry and Physiology, Part B 159 (2011) 84–91 A. HINAE cells 89 A. HINAE cells 3.5 3.5 ** * 3.0 mRNA expression (Fold increase) 2.5 2.0 1.5 TNF- GH mRNA expression (Fold increase) 3.0 1.0 2.0 1.5 1.0 0.5 0.5 0 2.5 0 Cont. 6 12 24 48 72 Cont. Duration of GHRH treatment 1.2 3.0 1.0 mRNA expression (Fold increase) 3.5 2.5 * * 2.0 1.5 TNF- GH mRNA expression (Fold increase) 12 24 48 72 B. Pituitary cells B. Pituitary cells 1.0 0.8 0.6 * 0.4 ** 0.2 0.5 0 6 Duration of GHRH treatment Cont. 6 12 24 48 72 Duration of GHRH treatment 0 Cont. 6 12 24 48 72 Duration of GHRH treatment Fig. 7. Regulation of GH mRNA levels by synthetic fGHRH-28. Expression profiles of GH mRNA in HINAE (A) and primary pituitary cells (B). The expression level of GH mRNA was detected at 0, 6, 12, 24, 48, and 72 h after treatment with 10–6 M of fGHRH-28 by real-time PCR. The levels of GH mRNA at each time point were quantified by expressed relative to the 18S rRNA level and then compared to that of the PBS-treated control (Cont.). Data are presented as the mean ± SD from three independent cDNA samples with three replicates at each time point. The asterisk indicates a statistically significant difference (single asterisk, P b 0.05). Fig. 8. Induction of TNF-α mRNA by synthetic fGHRH-28. Expression profiles of TNF-α mRNA in HINAE (A) and primary pituitary cells (B). The expression level was detected at 0, 6, 12, 24, 48, and 72 h after treatment with 10− 6 M of fGHRH-28 by real-time PCR. The levels of TNF-α mRNA at each time point were quantified relative to expressed 18S rRNA levels and then compared with that of the PBS-treated control (Cont.). Data are presented as the mean ± SD of three independent cDNA samples from three replicates at each time point. The asterisk indicates a statistically significant difference (single asterisk, P b 0.05; double asterisks, P b 0.01). GHRH-like peptides of nonmammalian vertebrates were unable to demonstrate GH-releasing activities. Here, we identified and characterized a GHRH gene from the teleost fish P. olivaceus and investigated the biological role of GHRH by in vitro stimulation upon induction of GH mRNA. The true nonmammalian GHRHs were discovered from chicken, African clawed frog, zebrafish, takifugu, pufferfish, rainbow trout, and flounder by in silico analysis (Lee et al., 2007). The flounder GHRH, identified by our group, is an EST clone containing a 678-bp insert, lacking 5′- and 3′-UTRs, and was deposited into the NCBI database. The flounder GHRH precursor cDNA identified in this study encodes 142 amino acids containing a 20-amino acid signal peptide. The flounder GHRH peptide shares high homology with the GHRHs of other species, especially in the bioactive core region (GHRH1–28) as shown in Table 1. The amino acid sequence of the bioactive core region of flounder is identical with goldfish and zebrafish GHRHs and also shows high homology (approximately 78.6%) with human GHRH at the amino acid level. The flounder GHRH gene spans 2290 bp and consists of six exons and five introns. The 120-bp exon I consists of the 5′-UTR of the gene, the 80-bp exon II, which encodes the signal peptide and N-terminus of a cryptic peptide, the 103-bp exon III, which encodes a cryptic peptide, the 105-bp exon IV encoding GHRH, the 126-bp exon V containing the C-terminal region of GHRH and the C-terminal peptide, and the 247-bp exon VI, which contains three amino acids, a termination codon, and the 3′-UTR of the gene (Fig. 2). However, this structural organization of the flounder GHRH gene differs from its mammalian counterparts. In general, the orthologous gene structure is conserved within different species; specifically, 86% of the orthologous gene pairs are estimated to have the same number of coding exons, 46% of which are of the same coding length, whereas only 1.5% have the same coding length but a different number of 90 B.-H. Nam et al. / Comparative Biochemistry and Physiology, Part B 159 (2011) 84–91 Table 1 The amino acid sequence identity of the GHRH core region from various species including flounder. Species Amino acid sequences Identity (%) Flounder Goldfish Zebrafish X. laevis Chicken Rat Mouse Bovine Dog Human HADAIFTNSYRKVLGQISARKFLQTVMG ............................ ............................ .V......T...F.......RY..NM.. .......DN...F............II. .......S...RI...LY...L.HEI.N .V.....TN...L.S.LY...VI.DI.N Y...............L....L..DI.N Y...............L....L..DI.S Y...............L....L..DI.S 100 100 100 75 82.1 64.3 53.6 78.6 78.6 78.6 Dots indicate identical amino acids and the alternative amino acid is indicated when the sequences differ. exons (Meyer and Durbin, 2004). Chicken, Xenopus, and flounder have a cryptic peptide encoding exons between the signal peptide and GHRH peptide encoding exons, while mammals and zebrafish do not (Fig. 3). Additionally, the chicken has two C-terminal peptides between the GHRH peptide and 3′-UTR encoding exons (Wang et al., 2007). VIP, which belongs to the same glucagon superfamily, shows a different gene structure within fish compared to mammals, as demonstrated in our previous study (Nam et al., 2009). The cases in which the exon number and organization differ may be explained by independent exon duplication or exon splitting events after two rounds of genome duplication. The tissue distribution of GHRH mRNA was determined using RTPCR. Flounder GHRH mRNA was detected in the brain as well as in the gill and the ovary (Fig. 5A). Both GHRH gene expression and GHRH immunoreactivity have also been demonstrated in the rat ovary (Bagnato et al., 1992). This implies that GHRH may play important biological roles in germ cell maturation and hormone production in the ovary. Gills are responsible for respiration as well as osmoregulation in euryhaline fish. Insulin-like growth factor-I (IGF-I) is an important regulator for hydromineral balance, and the GHRH/GH axis directly regulates IGF-I gene expression in gills during seawater acclimation (Sakamoto and McCormick, 2006). Additionally, gills trap and process antigens in fish (Zapata et al., 1994). Fish gills are the mucosal-associated lymphoid organs, along with the gut and skin. Leukocytes and plasma cells, which are present in the epithelia of the mucosal-associated lymphoid tissues, express immune-related molecules and take up antigens (Koppang et al., 1998; Press and Evensen, 1999). Therefore, the GHRH/GH/IGF-I axis in fish gills may modulate the induction of immune responses following pathogen infection. GHRH is well documented to play a critical role in stimulating pituitary GH synthesis and release both in vitro and in vivo in mammals (Müller et al., 1999). GHRH binding to the GHRH receptor results in increased GH production, mainly via the cAMP-dependent pathway (Mayo, 1992). The cAMP-dependent pathway is initiated by GHRH binding to its receptor, causing receptor conformation that activates the G-alpha subunit of the closely associated G-protein complex on the intracellular side. This results in stimulation of membrane-bound adenylate cyclase and increased intracellular cAMP. cAMP binds to and activates the regulatory subunits of protein kinase A (PKA), allowing the free catalytic subunits to translocate to the nucleus and phosphorylate the transcription factor cAMP response element-binding protein (CRE-BP). Phosphorylated CREBP, together with its coactivators, p300 and CRE-BP binding protein (CBP), enhances the transcription of GH by binding to cAMP-response elements (CREs) in the promoter region of the GH gene (Frohman and Kineman, 2002). Based on this mechanism of GH induction described above, we investigated whether the synthetic flounder GHRH (fGHRH-28) induces flounder GH mRNA in the embryonic cell line HINAE. After 24 h of 10− 6 M fGHRH-28 treatment, flounder GH mRNA induction was detected by RT-PCR. GHRH was shown to stimulate GHrelease in vivo (Wehrenberg et al., 1982) and in vitro (Brazeau et al., 1982) without affecting the release of other pituitary hormones in mammals. Additionally, GHRH stimulated GHRH mRNA expression in rat pituitary cells (Gick et al., 1984). In nonmammalian vertebrates, however, the previously named GHRH-like peptides were unable to demonstrate robust GH-releasing activities (Montero et al., 2000). Recently, a “true” GHRH cDNA was identified from nonmammalian vertebrates including fish, and was shown to activate cAMP production dose-dependently in receptor-transfected CHO cells as well as GH release from pituitary cells (Lee et al., 2007). Although the goldfish GHRH could stimulate intracellular cAMP and GH release at 1.8 × 10− 7 M and 10− 8 M, respectively, the flounder GHRH showed activities of cAMP production and GH mRNA induction at 10− 6 M (Fig. 6). These differences are a reflection of differing features of the pituitary and embryonic cells. Past research has shown that cytokines, such as TNF-α and IL-1, play important roles in pituitary function and regulation (reviewed in Haedo et al., 2009). Conversely, the pituitary hormone, GH, has been suggested to directly modulate the release of both TNF-α and IL-1 (Bozzola et al., 1998; Uronen-Hansson et al., 2003). As shown in Fig. 8, flounder TNF-α mRNA was induced in fGHRH-28-treated HINAE cells, suggesting that indirect effects of GHRH are mediated through GH release, which, in turn, induces TNF-α expression. In pituitary cells, however, TNF-α mRNA decreased after fGHRH-28 treatment, indicating that its expression might be regulated negatively by GHRH, in a direct or indirect manner. These different responses may be attributed to the distinct features of each cell type. Although this study was limited to the direct in vitro effects of GHRH, TNF-α mRNA expression, which was regulated by fGHRH-28 treatment, supports the hypothesis of bidirectional communication between neuroendocrine and immune systems through shared protein hormones and cytokines as messengers. In summary, the full-length cDNA and genomic DNA encoding the flounder GHRH precursor were identified and characterized. The GHRH mRNA was found constitutively in the whole brain as well as in the gills and the ovary. Under infectious conditions, the expression level of GHRH mRNA responded and increased time-dependently, which supports that the 5′-flanking region of the GHRH gene includes binding elements for transcription factors involved in cytokinemediated activation. A bioactivity assay with a synthetic peptide fGHRH-28 demonstrated that the expression of flounder GH mRNAs was induced in HINAE and pituitary cells by peptide treatment. Additionally, fGHRH-28 induced mRNA expression of the inflammatory cytokine TNF-α in HINAE cells, but suppressed TNF-α mRNA expression in primary cultured pituitary cells. Overall, these results demonstrate that, similar to mammals, GHRH plays roles in the immune and neuroendocrine systems of fish through bidirectional communication. Acknowledgment This work was funded by a grant from the National Fisheries Research and Development Institute (RP-2011-BT-010). References Auernhammer, C.J., Strasburer, C.J., 1995. Effects of growth hormone and insulin-like growth factor on the immune system. Eur. J. Endocrinol. 133, 635–645. Bagnato, A., Moretti, C., Onishi, J., Frajese, G., Catt, K.J., 1992. Expression of the growth hormone-releasing hormone gene and its product in the rat ovary. Endocrinology 130, 1097–1102. Bik, W., Wolinska-Witort, E., Pawlak, J., Skwarlo-Sonta, K., Chmielowska, M., Martynska, L., Baranowska-Bik, A., Baranowska, B., 2006. PACAP38 as a modulator of immune and endocrine responses during LPS-induced acute inflammation in rats. J. Neuroimmunol. 177, 76–84. Bozzola, M., De Amici, M., Zecca, M., Schimpff, R.M., Rapaport, R., 1998. Modulating effect of human growth hormone on tumour necrosis factor-α and interleukin-1β. Eur. J. Endocrinol. 138, 640–643. B.-H. Nam et al. / Comparative Biochemistry and Physiology, Part B 159 (2011) 84–91 Brazeau, P., Ling, N., Böhlen, P., Esch, F., Ying, S.Y., Guillemin, R., 1982. Growth hormone releasing factor, somatocrinin, releases pituitary growth hormone in vitro. Proc. Natl. Acad. Sci. U. S. A. 79, 7909–7913. Brenneman, D.E., 2007. Neuroprotection: a comparative view of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Peptides 28, 1720–1726. Carpio, Y., Lugo, J.M., León, K., Morales, R., Estrada, M.P., 2008. Novel function of recombinant pituitary adenylate cyclase-activating polypeptide as stimulator of innate immunity in African catfish (Clarias gariepinus) fry. Fish Shellfish Immunol. 25, 439–445. Chae, S.H., Kim, J.W., Cho, J.M., Larkin, D.M., Everts-van der Wind, A., Park, H.S., Yeo, J.S., Choi, I., 2007. Chromosomal localization of Korean cattle (Hanwoo) BAC clones via BAC end sequence analysis. Asian Australas. J. Anim. Sci. 20, 316–327. Clark, R., 1997. The somatogenic hormones and insulin-like growth factor-1: stimulators of lymphopoiesis and immune function. Endocrinol. Rev. 18, 157–179. Frohman, L.A., Kineman, R.D., 2002. Growth hormone-releasing hormone and pituitary development, hyperplasia and tumorigenesis. Trends Endocrinol. Metab. 13, 299–303. Gala, R.R., 1991. Prolactin and growth hormone in the regulation of the immune system. Proc. Soc. Exp. Biol. Med. 198, 513–526. Gick, G.G., Zeytin, F.N., Brazeau, P., Ling, N.C., Esch, F.S., Bancroft, C., 1984. Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells. Proc. Natl. Acad. Sci. U. S. A. 81, 1533–1555. Haedo, M.R., Gerez, J., Fuertes, M., Giacomini, D., Páez-Pereda, M., Labeur, M., Renner, U., Stalla, G.K., Arzt, E., 2009. Regulation of pituitary function by cytokines. Horm. Res. 72, 255–274. Holmgren, S., Jensen, J., 2001. Evolution of vertebrate neuropeptides. Brain Res. Bull. 55, 723–735. Hosoya, M., Kimura, C., Ogi, K., Ohkubo, S., Miyamoto, Y., Kugoh, H., Shimizu, M., Onda, H., Oshimura, M., Arimura, A., Fujino, M., 1992. Structure of the human pituitary adenylate cyclase activating polypeptide (PACAP) gene. Biochim. Biophys. Acta 1129, 199–206. Kelley, K.W., Weigent, D.A., Kooijman, R., 2007. Protein hormones and immunity. Brain Behav. Immun. 21, 384–392. Kooijman, R., Hooghe-Peters, E.L., Hooghe, R., 1996. Prolactin, growth hormone and insulin-like growth factor-1 in the immune system. Adv. Immunol. 63, 377–454. Koppang, E.O., Lundin, M., Press, C.McL., Rønningen, K., Lie, Ø., 1998. Differing levels of Mhc class II β chain expression in a range of tissues from vaccinated and nonvaccinated Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol. 8, 183–196. Lee, L.T.O., Siu, F.K.Y., Tam, J.K.V., Lau, I.T.Y., Wong, A.O.L., Lin, M.C.M., Vaudry, H., Chow, B.K.C., 2007. Discovery of growth hormone-releasing hormones and receptors in non-mammalian vertebrates. Proc. Natl. Acad. Sci. U. S. A. 104, 2133–2138. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real time quantitative PCR and the 2−ΔΔCt method. Methods 25, 402–408. Lugo, J.M., Carpio, Y., Oliva, A., Morales, A., Estrada, M.P., 2010. Pituitary adenylate cyclase-activating polypeptide (PACAP): a regulator of the innate and acquired immune function in juvenile fish. Fish Shellfish Immunol. 29, 513–520. Mayo, K.E., 1992. Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone. Mol. Endocrinol. 6, 1734–1744. Mayo, K.E., Cerelli, G.M., Lebo, R.V., Bruce, B.D., Rosenfeld, M.G., Evans, R.M., 1985. Gene encoding human growth hormone-releasing factor precursor: structure, sequence, and chromosomal assignment. Proc. Natl. Acad. Sci. U. S. A. 82, 63–67. 91 McRory, E., Sherwood, N.M., 1997. Two protochordate genes encode pituitary adenylate cyclase-activating poly peptide and related family members. Endocrinology 138, 2380–2390. Meyer, I.M., Durbin, R., 2004. Gene structure conservation aids similarity based gene prediction. Nucleic Acids Res. 32, 776–783. Montero, M., Yon, L., Kikuyama, S., Dufour, S., Vaudry, H., 2000. Molecular evolution of the growth hormone-releasing hormone/pituitary adenylate cyclase-activating polypeptide gene family. Functional implication in the regulation of growth hormone secretion. J. Mol. Endocrinol. 25, 157–168. Müller, E.E., Locatelli, V., Cocchi, D., 1999. Neuroendocrine control of growth hormone secretion. Physiol. Rev. 79, 511–607. Nam, B.H., Kim, Y.O., Kong, H.J., Kim, W.J., Lee, S.J., Choi, T.J., 2009. Identification and characterization of the prepro-vasoactive intestinal peptide gene from the teleost Paralichthys olivaceus. Vet. Immunol. Immunopathol. 127, 249–258. Nam, B.H., Moon, J.Y., Kim, Y.O., Kong, H.J., Kim, W.J., Lee, S.J., Kim, K.K., 2010. Multiple β-defensin isoforms identified in early developmental stages of the teleost Paralichthys olivaceus. Fish Shellfish Immunol. 28, 267–274. Parker, D.B., Coe, I.R., Dixon, G.H., Sherwood, N.M., 1993. Two salmon neuropeptides encoded by one brain cDNA are structurally related to members of the glucagons superfamily. Eur. J. Biochem. 215, 439–448. Pozo, D., 2003. VIP- and PACAP-mediated immunomodulation as prospective therapeutic tools. Trends Mol. Med. 9, 211–217. Press, C.McL., Evensen, Ø., 1999. The morphology of the immune system in teleost fishes. Fish Shellfish Immunol. 9, 309–318. Sakamoto, T., McCormick, S.D., 2006. Prolactin and growth hormone in fish osmoregulation. Gen. Comp. Endocrinol. 147, 24–30. Salamat, N., Alboghobeish, N., Hashemitabar, M., Mesbah, M., Ahangarpour, A., 2009. Pituitary primary cell culture of common carp (Cyprinus Carpio) and evaluation of its secretion effect on endocrine activity of incubated ovarian follicles. Iran. J. Vet. Res. 10, 61–65. Sherwood, N.M., Krueckl, S.L., McRory, J.E., 2000. The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagons superfamily. Endocrinol. Rev. 21, 619–670. Uronen-Hansson, H., Allen, M.L., Lichtarowicz-Krynska, E., Aynsley-Green, A., Cole, T.J., Höidén-Guthenberg, I., Fryklund, L., Klein, N., 2003. Growth hormone enhances proinflammatory cytokine production by monocytes in whole blood. Growth Horm. IGF Res. 13, 282–286. Velkeniers, B., Dogusan, Z., Naessens, F., Hooghe, R., Hooghe-Peters, E.L., 1998. Prolactin, growth hormone and the immune system in humans. Cell. Mol. Life Sci. 54, 1102–1108. Wang, Y., Li, J., Wang, C.Y., Kwok, A.H.Y., Leung, F.C., 2007. Identification of the endogenous ligands for chicken growth hormone-releasing hormone (GHRH) receptor: evidence for a separate gene encoding GHRH in submammalian vertebrates. Endocrinology 148, 2405–2416. Wehrenberg, W.B., Ling, N., Brazeau, P., Esch, F., Böhlen, P., Baird, A., Ying, S., Guillemin, R., 1982. Somatocrinin, growth hormone releasing factor, stimulates secretion of growth hormone in anesthetized rats. Biochem. Biophys. Res. Commun. 109, 382–387. Zapata, A.G., Chibá, A., Varas, A., 1994. Cells and tissues of the immune system of fish. In: Iwama, G., Nakanishi, T. (Eds.), The Fish Immune System: Organism, Pathogen, and Environment. Academic Press, San Diego, pp. 1–62.