Download Molecular and functional analyses of growth hormone

Comparative Biochemistry and Physiology, Part B 159 (2011) 84–91
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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).
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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).
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