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Endocrinology Vol. 148, No. 5 2405-2416
Copyright © 2007 by The Endocrine Society

Identification of the Endogenous Ligands for Chicken Growth Hormone-Releasing Hormone (GHRH) Receptor: Evidence for a Separate Gene Encoding GHRH in Submammalian Vertebrates

Yajun Wang, Juan Li, Crystal Ying Wang, Amy Ho Yan Kwok and Frederick C. Leung

Department of Zoology, The University of Hong Kong, Hong Kong, China

Address all correspondence and requests for reprints to: Dr. Frederick C. Leung, Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong, China. E-mail: fcleung{at}hkucc.hku.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is generally believed that hypothalamic GHRH activates GHRH receptor (GHRHR) to stimulate GH synthesis and release in the pituitary of mammals. However, the identity of the endogenous ligand of GHRHR is still unresolved in submammalian vertebrates including birds. In this study, we have successfully identified the chicken GHRH (cGHRH) gene, which consists of seven exons including two exons (exons 4 and 5) coding for the predicted mature GHRH peptide of 47 amino acids. Interestingly, the differential usage of splice donor sites at exon 6 results in the generation of two prepro-GHRHs (172 and 188 amino acids in length) with different C-terminal tails. Similar to mammals, cGHRH was detected to be predominantly expressed in the hypothalamus by RT-PCR assay. Using the pGL3-CRE-luciferase reporter system, we further demonstrated that both the synthetic cGHRH peptides (cGHRH1–47 and cGHRH1–31) and conditioned medium from CHO cells expressing cGHRH could strongly induce luciferase activity via activation of cGHRHR, indicating that cGHRH could bind cGHRHR and activate downstream cAMP-protein kinase A signaling pathway. Using the same system, cGHRH-like peptide was also shown to be capable of activating cGHRHR in vitro. As in chicken, a conserved GHRH gene was identified in the genomes of lower vertebrate species including zebrafish, fugu, tetraodon, and Xenopus by synteny analysis. Collectively, our data suggest that GHRH, perhaps together with GHRH-like peptide (chicken/carp-like), may function as the authentic endogenous ligands of GHRHR in chicken as well as in other lower vertebrate species.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY GH SYNTHESIS and release are regulated by two main hypothalamic peptides: GHRH and somatostatin in mammals (1, 2, 3). GHRH belongs to the glucagon superfamily, which also includes pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, and vasoactive intestinal polypeptide (VIP) (4). Mammalian GHRH is a polypeptide of 42–44 amino acid residues and encoded by a gene of five exons (5, 6, 7, 8, 9). Compelling evidence demonstrated that GHRH could strongly stimulate pituitary GH synthesis and release both in vivo and in vitro (6, 10, 11). The biological actions of GHRH are mediated by GHRH receptor (GHRHR), a member of the G protein-coupled receptor family, whose activation could increase intracellular cAMP levels and activate the protein kinase A (PKA) signaling pathway (12, 13, 14, 15). Mutations in either GHRH gene or GHRHR gene would cause pituitary hypoplasia and serum GH deficiency in mouse and human (12, 16, 17, 18, 19, 20, 21), further emphasizing the pivotal role of GHRH in controlling GH synthesis and secretion, as well as somatotroph proliferation in the pituitary of mammals.

In contrast to studies reported in mammals, the physiological role of GHRH in pituitary GH release is controversial in lower vertebrates (22). In teleost fish, human GHRH or fish GHRH-like peptide has only modest or weak effects on GH release in cultured pituitary cells of several species including rainbow trout (23), tilapia (24), and sockeye salmon (25). In European eels (Anguilla anguilla), one of the most primitive teleost fish, human GHRH is totally devoid of an effect on GH secretion in cultured pituitary cells (26). In contrast to the minimal response to GHRH, PACAP has been demonstrated to act as a GH-releasing factor in pituitaries of grass carp (27, 28), sockeye salmon (25), and European eel (26, 29). Similar stimulatory effects of PACAP have also been reported in amphibians (30). All the evidence points to the possibility that PACAP, instead of GHRH, may play a dominant role in GH release in the pituitary of lower vertebrates, especially in teleost fishes (22). Using a cDNA cloning strategy, the sequences of GHRH-like peptide have been determined in a number of lower vertebrate species including zebrafish (31, 32, 33), sockeye salmon (25), catfish (34), and frog (35, 36). Interestingly, GHRH-like peptide and PACAP are encoded by a single gene, whereas GHRH and PACAP are encoded by two distinct genes in mammals (4, 22). The distinct organization of GHRH and PACAP between mammals and lower vertebrates seems to partially explain the difference in pituitary responsiveness to GHRH and PACAP.

Although the same precursor encompasses GHRH-like peptide and PACAP in chickens as reported in teleosts and amphibians (4, 37), we and others have clearly demonstrated that human GHRH, and not PACAP, play a dominant role in chicken GH release both in vivo and in vitro (38, 39, 40, 41, 42, 43, 44, 45, 46). Despite the fact that PACAP can increase intracellular cAMP level and GH release in cultured chicken pituitary cells, its effect on GH release is much less potent than human GHRH (47). Surprisingly, iv administration of synthetic chicken (c) GHRH-like peptide has little effect on GH release even at a high dosage (44). Consistent with this finding, synthesized cGHRH-like peptide also has little potency at cGHRHR activation (48). In view of these findings, there arises several fundamental issues that need to be addressed: 1) whether cGHRH-like peptide represents the authentic ligand of cGHRHR; 2) whether a new GHRH with high potency in stimulating pituitary GH secretion exists in the chicken and other lower vertebrate species; and 3) how GHRH gene and GHRH-PACAP gene have evolved in the course of vertebrate evolution. Answering these questions would help us to reevaluate the physiological roles of GHRH in the hypothalamic-pituitary axis of lower vertebrates.

In the present study, we isolated the full-length cDNAs coding for chicken GHRH. Using a luciferase reporter system, we further demonstrated that cGHRH could activate cGHRHR with high potency (EC50 < 0.1 nM). Together with the information collected from genome database of model vertebrate animals (http://www.ensembl.org/), our studies for the first time indicate that a conserved GHRH gene exists in submammalian vertebrates including birds, amphibians, and teleosts, and most likely, the identified GHRH is one of the endogenous ligands of GHRHR in submammalian vertebrate species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and hormones
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), and restriction enzymes were obtained from Amersham Biosciences (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) unless stated otherwise. Human pancreatic GHRH (hpGHRH1–40) and ovine pituitary adenylate cyclase-activating polypeptide (oPACAP38) were purchased from Bachem (Torrance, CA). cGHRH1–31 and cGHRH1–47 with free carboxyl termini were synthesized using solid-phase Fmoc chemistry (GL Biochem, Shanghai, China). The purity of synthesized cGHRHs is greater than 95% (analyzed by HPLC), and their structure was verified by mass spectrometry (GL Biochem). cGHRH1–47, cGHRH1–31, hpGHRH1–40, and oPACAP38 were first dissolved in distilled water and then diluted to the desired concentrations with medium before use.

Animals
Adult chickens used in all experiments are of a local chicken strain (Shek-ki) of Hong Kong and was kindly provided by Kadoorie Agricultural Research Centre (Hong Kong). All experiments were performed under license from the Government of the Hong Kong Special Administrative Region and endorsed by the Animal Experimentation Ethics Committee of The University of Hong Kong.

Total RNA isolation
Adult chickens were decapitated and different tissues including small intestine, kidney, liver, lung, muscle, ovary, pituitary, spleen, testis, and brain were collected for total RNA extraction. Total RNA was extracted from chicken tissues with Tri-reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions and dissolved in diethylpyrocarbonate-treated H2O.

Cloning of the full-length cDNAs for GHRH from chicken brain
Two gene-specific primers were designed based on an unannotated cDNA sequence deposited in GenBank (accession no. BX929984, ChEST294a6) (Table 1Go). Two micrograms of total RNA from adult chicken brain were reverse transcribed into single-strand cDNA with Moloney murine leukemia virus reverse transcriptase (Promega Corp., Madison, WI) followed by PCR amplification using high-fidelity Taq DNA polymerase (Roche Diagnostics, Basel, Switzerland). The PCR amplification (35 cycles) was performed using a cycle profile of 30 sec at 95 C, 30 sec at 56 C, and 60 sec at 72 C, followed by a 15-min extension at 72 C. The PCR products were cloned into pBluescript SK (+/–) (Stratagene, La Jolla, CA). The cloned GHRH cDNAs were sequenced with the Bigdye terminator cycle sequencing, version 3.0, ready reaction kit (PerkinElmer, Foster City, CA) and analyzed on Prism 3100 genetic analyzer (PerkinElmer). Each form (short and long form) of GHRH full-length cDNAs was finally determined by sequencing three independent clones.


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  		TABLE 1. Primers used1

 
RT-PCR
Reverse transcription was performed at 42 C for 2 h in a total volume of 10 µl consisting of 2 µg total RNA from different tissues, 1x single-strand buffer, 0.5 mM each deoxynucleotide triphosphate, 0.5 µg oligodeoxythymide, and 100 U Moloney murine leukemia virus reverse transcriptase (Promega). All negative controls were carried out under the same condition without reverse transcriptase added in the 10 µl of reaction mix.

PCR was carried out in a total volume of 20 µl consisting of 1x PCR buffer, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2, 0.2 µM each primer, and 0.5 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA) on the PTC-225 Peltier thermal cycler (MJ Research Inc., Waltham, MA). To evaluate the relative mRNA levels of target genes, the optimal cycle numbers were first determined according to our previously established semiquantitative RT-PCR methods (49, 50, 51, 52). For ß-actin gene, 23 cycles of 30 sec at 95 C, 30 sec at 58 C, and 60 sec at 72 C were used followed by 5 min extension at 72 C. For GHRH gene, 31 cycles of 30 sec at 95 C, 30 sec at 58 C, and 60 sec at 72 C were used followed by 5 min extension at 72 C. For GHRH-PACAP gene, 33 cycles were used based on our previous report (53). The primers used for GHRH gene, GHRH-PACAP gene, and ß-actin gene were listed in Table 1Go. The PCR products were visualized on a UV-transilluminator (Bio-Rad Laboratories, Inc., Hercules, CA) after electrophoresis on 2% agarose gel containing ethidium bromide.

Establishment of a system to monitor the biological activities of cGHRH, cGHRH-like peptide, and cPACAP
Experiment 1: cloning of cGHRHR and chicken PACAP type I receptor (PAC1-R) into pcDNA3.1(+) expression vector.
Based on the cDNA sequences of cGHRHR (53) and PACAP type I receptor [cPAC1-R, short form, 471 amino acids (a.a.)] (54), two pairs of gene-specific primers flanked by restriction enzyme recognition sites were designed to amplify the complete open reading frame (ORF) regions of chicken GHRHR and PAC1-R with high-fidelity Taq DNA polymerase (Roche Diagnostics) (Table 1Go). The amplified PCR products were first cloned into pBluescript SK (+/–) vector (Stratagene) and subjected to sequencing analysis (PerkinElmer). Then the inserts of interest were released by restriction enzyme digestions and subcloned into pcDNA3.1(+) expression vector (Invitrogen).

Experiment 2: cell culture and cotransfection of cGHRHR (or cPAC1-R) expression plasmid and pGL3-CRE-luciferase reporter construct into Chinese hamster ovary (CHO) cells.
In this experiment, pGL3-CRE-luciferase reporter construct, in which the expression of luciferase gene is driven by a promoter containing multiple cAMP-response elements (CRE), was first constructed by cloning the promoter region of pCRE-SEAP vector (CLONTECH, Palo Alto, CA) into the promoterless pGL3-Basic vector (Promega). CHO cells were cultured in DMEM supplemented with 10% (vol/vol) fetal bovine serum (HyClone, Logan, UT), 100 U/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies, Inc., Grand Island, NY) in a 90-cm culture dish (NUNC, Rochester, NY) and incubated at 37 C with 5% CO2. Then CHO cells were plated in a 6-well plate at a density of 3 x 105 cells/well 1 d before transfection. A mixture containing 700 ng of pGL3-CRE-luciferase reporter construct, 200 ng of pcDNA3.1 construct containing either cGHRHR or cPAC1-R (or empty vector), and 6 µl of DOSPER liposomal transfection reagent (Roche Diagnostics) was prepared in 50 µl of PBS solution and transfection was performed according to the manufacturer’s instructions when cells were 70% confluent. After 24 h of culture, CHO cells were trypsinized and cultured in a 96-well plate at a density of 2 x 104 cells/well at 37 C for 24 h before hormone treatment.

Experiment 3: examination of hormone specificity of hpGHRH1–40 and oPACAP38 on the activation of cGHRHR and cPAC1-R.
The hpGHRH1–40 and oPACAP38 were diluted by serum-free DMEM to the desired concentrations, respectively. After removal of the DMEM from 96-well plate, 50 or 60 µl of hormone-containing medium or hormone-free medium (used as control) were added and CHO cells were incubated for an additional 6 h at 37 C with 5% CO2 before being harvested for luciferase assay. After removal of the culture medium, CHO cells were lysed by adding 50 µl of 1x passive lysis buffer (Promega) per well, and the luciferase activity of 15 µl of cellular lysates was determined using luciferase assay reagent (Promega).

Transient expression of cGHRH(s), cGHRH(l), cGHRH(d), and cGHRH(c) in CHO cells and conditioned medium collection
Five gene-specific primers flanked by restriction sites (KpnI or EcoRI) at their 5'-ends were designed to prepare four expression plasmids, named cGHRH(s) (encoding the short cGHRH precursor, 172 a.a.), cGHRH(l) (encoding the long cGHRH precursor, 188 a.a.), cGHRH(d) (containing the signal peptide, cryptic peptide and putative GHRH peptide, 121 a.a.), and cGHRH(c) (including signal peptide and cryptic peptide only, 72 a.a.), respectively (see Table 1Go and Fig. 6AGo). The PCR was first performed with high fidelity Taq DNA polymerase (Roche) by using cloned GHRH plasmids as templates. Then the PCR products were cloned into pBluescript SK(+/–) vector (Stratagene) and sequenced by ABI3100 genetic analyzer (PerkinElmer). The inserts with correct DNA sequences were released by KpnI and EcoRI restriction enzyme digestions and ligated into pcDNA3.1(+) expression vector (Invitrogen). The transfection procedure is slightly different from the above experiments. In brief, five mixtures containing 3 µg of different pcDNA3.1(+) constructs and 6 µl of DOSPER liposomal transfection reagent (Roche) were prepared in 50 µl of PBS solution, and transfection was carried out according to the manufacturer’s instruction when CHO cells were 70% confluent in a 6-well plate. Six hours after transfection, the medium was replaced by fresh medium supplemented with 10% fetal bovine serum and CHO cells were incubated at 37 C for an additional 18 h. Then the medium was replaced by 1 ml of serum-free DMEM. The serum-free conditioned medium was collected after 36 h of culture at 37 C with 5% CO2. To avoid any contamination from cells or cell debris, the conditioned medium was centrifuged at 3000 rpm for 3 min and the supernatant was aliquoted and stored at –80 C before measurement of biological activity.


Figure 6
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  		FIG. 6. A, Schematic representation of constructing the expression plasmids encoding GHRH precursor of different length. B–E, Effects of conditioned medium (0–25 µl added, total volume 50 µl/well) from CHO cells with transient expression of different cGHRH expression plasmids [B, cGHRH(l); C, cGHRH(s); D, cGHRH(d); and E, cGHRH(c)] on basal luciferase activities of CHO cells cotransfected with cGHRHR (or cPAC1-R) and pGL3-CRE-luciferase reporter constructs. Each data point represents mean ± SEM of three replicates. Serum-free medium (50 µl added) was used as the control. SP, Signal peptide; CP, cryptic peptide; C-peptide, carboxyl terminal flanking peptide. *, P < 0.001 vs. control.

 
Transient expression of cGHRH-PACAP(w), cGHRH-PACAP(d), and cGHRH-PACAP(c) in chicken DF-1 cells and conditioned medium collection
Using the same approach as mentioned above, three sets of gene-specific primers were designed to prepare three pcDNA3.1 expression plasmids: named cGHRH-PACAP(w) (encoding the GHRH-PACAP precursor, 172 a.a.), cGHRH-PACAP(d) (containing signal peptide, cryptic peptide and GHRH-like peptide only, 125 a.a.), and cGHRH-PACAP(c) (containing signal peptide and cryptic peptide only, 77 a.a.) respectively (see Table 1Go and Fig. 8AGo). An immortalized chicken embryo fibroblast cell line DF-1 (cultured in high glucose DMEM) was chosen for transient expression of various cGHRH-PACAP constructs because the conditioned medium collected from CHO cells had low activity (perhaps due to the low expression level of recombinant peptides in CHO cells) on the activation of cGHRHR (or cPAC1-R). Transfection and conditioned medium collection were performed in the same way mentioned above.


Figure 8
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  		FIG. 8. A, Schematic representation of constructing the expression plasmids encoding GHRH-PACAP precursor of different length. SP, Signal peptide; CP, cryptic peptide. B–D, Effects of conditioned medium (0–30 µl added, total volume 60 µl/well) from DF-1 cells with transient expression of different cGHRH-PACAP expression plasmids [B, cGHRH-PACAP(w); C, cGHRH-PACAP(d); and D, cGHRH-PACAP(c)] on basal luciferase activities of CHO cells cotransfected with GHRHR (or PAC1-R) expression plasmid and pGL3-CRE-luciferase reporter construct. Serum-free medium (60 µl added) was used as the control. Each data point represents mean ± SEM of three replicates. # and *, P < 0.001 vs. respective controls.

 
Data analysis
The luciferase activities in each treatment group were expressed as relative fold increase as compared with the control group (without hormone treatment). The data were analyzed by one-way ANOVA followed by Dunnett’s test using GraphPad Prism 4 (GraphPad Software, San Diego, CA). To validate our results, all experiments were repeated at least three times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of the full-length cDNAs encoding cGHRH
Based on an unannotated cDNA sequence deposited in GenBank (accession no. BX929984), we successfully cloned the full-length cDNA coding for GHRH from adult chicken brain. It is 704 bp in length and encodes a GHRH precursor of 172 amino acids (GenBank accession no. DQ465018). Comparison of GHRH cDNA sequence with the chicken genome database (http://www.ensembl.org/Gallus_gallus) revealed that cGHRH gene spans more than 6 kb on chromosome 20 and consists of 7 exons (Fig. 1Go). The predicted mature GHRH peptide is 47 amino acid residues in length (encoded by exons 4 and 5) and shares high amino acid sequence identity with that of human (50%), rat (41%), and zebrafish (69%) (accession no. DQ832172) (Fig. 2AGo). Within GHRH1–47 region, the N terminus (cGHRH1–27) is of the highest sequence identity, whereas the carboxyl termini diversify significantly among species (Fig. 2AGo). Unlike GHRH precursors of human, rat, and zebrafish, cGHRH precursor has a unique long carboxyl terminal flanking peptide (49 a.a.) and a cryptic peptide encoded by exon 3, which is absent in human, rat, and zebrafish GHRH precursors (Fig. 2AGo).


Figure 1
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  		FIG. 1. A, Amplification of cGHRH full-length cDNAs from brain by RT-PCR. –, RT without reverse transcriptase; +, RT with reverse transcriptase. B, Genomic structure of cGHRH gene. Seven exons are labeled with e1, e2, e3, e4, e5, e6, and e7, respectively. The short and long prepro-GHRHs are designated as cGHRH-s and cGHRH-l, respectively. SP, Signal peptide; CP, cryptic peptide; C-peptide, carboxyl terminal flanking peptide. Arrow (B) indicates the location of an alternative splice donor site at exon 6 (e6).

 

Figure 2
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  		FIG. 2. A, Comparison of GHRH precursors of chicken (cGHRH-s and cGHRH-l) with that of zebrafish (zfGHRH: DQ832172), rat (rGHRH: M73486), and human (hGHRH: X00094). The putative cryptic peptide is underlined and the deduced or identified GHRH peptides are shaded. A putative amidation site (G28) is boxed. B, Comparison of the deduced amino acid sequence of cGHRH precursor with that of cGHRH-PACAP precursor (AY956323). The predicted GHRH and C-peptide are boxed. GHRH-like peptide and PACAP38 are boxed and shaded. Dots (A and B) indicate the amino acid residues identical with those of chicken prepro-GHRH (cGHRH-s). C-peptide, Carboxyl terminal flanking peptide.

 
Although the GHRH gene may have evolved from a duplicated GHRH-PACAP gene, the putative cGHRH1–47 is only 46% identical with cGHRH-like peptide with the highest sequence identity (70%) noted in the N-terminal 23 amino acid residues (37, 53), and little or no homology was found in the remaining parts of the two precursors (Fig. 2BGo).

Interestingly, a minor PCR band (667 bp) with faint intensity was also observed when RT-PCR assay was performed (Fig. 1AGo). Sequencing analysis revealed that it is resulted from the alternative usage of a splice donor site located at 3' end of exon 6, which leads to a deletion of 37 bp including the stop codon on exon 6 (Fig. 1BGo). Thus, this short cDNA fragment using a new stop codon on exon 7 encodes a larger prepro-GHRH (188 a.a.) with an altered carboxyl terminal tail (GenBank accession no. DQ465017) (Fig. 2AGo). Consistent with our finding, the unannotated cDNA sequence (BX929984) also encodes an identical precursor of 188 amino acid residues although sequence variation was noted in the 5' untranslated region, confirming the presence of alternative mRNA splicing in cGHRH gene. In this study, the short and long GHRH precursors are designated as cGHRH-s and cGHRH-l, respectively.

Expression of GHRH gene and GHRH-PACAP gene in adult chicken brain
To demonstrate the hypothalamic origin of this novel cGHRH, spatial distribution of GHRH mRNA in adult chicken brain was examined by RT-PCR assay. As shown in Fig. 3Go, GHRH mRNA was detected to be predominantly expressed in the hypothalamus and weakly expressed in the midbrain and hindbrain, clearly indicating that this polypeptide is produced mainly by the hypothalamus (Fig. 3Go). Moreover, when specific primers detecting both the long and short GHRH precursor mRNAs were used, similar expression pattern was noted (Table 1Go and Fig. 3Go). In contrast, cGHRH-PACAP mRNA is widely expressed in all regions of adult chicken brain investigated, including telencephalon, cerebellum, midbrain, hindbrain, and hypothalamus (Fig. 3Go).


Figure 3
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  		FIG. 3. Expression of GHRH gene and GHRH-PACAP gene in adult chicken brain regions including telencephalon (Te), cerebellum (Cb), midbrain (Mb), hindbrain (Hb), and hypothalamus (Hp). Gel pictures A and B show the specific amplification of N-terminal region and variable C-terminal regions of GHRH precursors, respectively. Only very faint minor band corresponding to cGHRH-l was observed in gel picture B. Numbers in brackets indicate the PCR cycles used (all negative controls are not shown).

 
Tissue distribution of GHRH mRNA expression in chicken tissues
To examine the extrahypothalamic sources of cGHRH, we also investigated the expression of cGHRH in other nine adult chicken tissues including small intestine, kidney, liver, lung, muscle, ovary, pituitary, spleen, and testis using RT-PCR assay. The expression of cGHRH was low but detectable in small intestine, kidney, ovary, pituitary, and spleen using lower PCR cycle (31 cycles) (Fig. 4Go). However, using higher PCR cycle number (35 cycles), the expression of cGHRH, predominantly the cGHRH-s, was detected in all tissues except muscle (Fig. 4Go). Considering the low or no expression of cGHRHR in extrapituitary tissues (48, 53, 55), the physiological relevance of extrahypothalamic GHRH remains to be determined.


Figure 4
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  		FIG. 4. Expression of GHRH gene in chicken extrahypothalamic tissues including small intestine (In), kidney (Ki), liver (Li), lung (Lu), muscle (Mu), ovary (Ov), pituitary (Pi), spleen (Sp), and testis (Te). Gel images A and B indicate the specific amplification of cGHRH N terminus, and gel image C shows the specific amplification of variable cGHRH C-termini. Numbers in brackets indicate the PCR cycle numbers used (all negative controls are not shown).

 
Validating the system for monitoring the biological activities of cGHRH, cGHRH-like peptide, and cPACAP
To test the biological activities of cGHRH, cGHRH-like peptide, and cPACAP from conditioned medium, a system monitoring hormone specificity on cGHRHR activation was first established. In view of the fact that the GHRH gene is likely to be originated from a duplication of GHRH-PACAP gene (4, 22, 56), cPAC1-R was also introduced in the present study to monitor whether peptides released from the short or long cGHRH precursor could activate cPAC1-R. As shown in Fig. 5Go, human GHRH (10–12 to 10–6 M) could induce luciferase activities in a dose-dependent manner via activation of cGHRHR (EC50 0.106 nM) but not cPAC1-R (Fig. 5AGo), whereas oPACAP38 could increase basal luciferase activities via cPAC1-R, even at a low dosage (EC50 0.867 nM) (Fig. 5BGo). Interestingly, PACAP could also activate cGHRHR at higher dosages (≥10–8 M, EC50 37.7 nM) (Fig. 5BGo), coincided with the report by Toogood et al. (48). Most important of all, neither hpGHRH1–40 nor oPACAP38 could increase basal luciferase activity in cultured CHO cells without transfected cGHRHR and cPAC1-R expression plasmids, indicating the binding specificity of hormone to receptor(s) (Fig. 5Go). Thus, this system was used to evaluate the biological activities of cGHRH, cGHRH-like peptide, and cPACAP from conditioned medium.


Figure 5
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  		FIG. 5. Hormone specificity of hpGHRH (10–12 to 10–6 M) (A) and oPACAP38 (10–12 to 10–7 M) (B) on the activation of cGHRHR and PACAP type I receptor (PAC1-R) monitored by a system of cotransfection of pGL3-CRE-luciferase reporter construct and GHRHR (or PAC1-R) expression plasmid in CHO cells. Each data point represents mean ± SEM of three replicates (cotransfection of pcDNA3.1 empty vector and pGL3-CRE-luciferase reporter construct was used as an internal control).

 
Activation of cGHRHR by conditioned medium collected from CHO cells with transient expression of cGHRH(s), cGHRH(l), and cGHRH(d) constructs
To examine whether cGHRH could activate cGHRHR specifically, transient expression of four cGHRH constructs [cGHRH(s), cGHRH(l), cGHRH(d), and cGHRH(c)] in CHO cells was performed in this study (Fig. 6Go). As expected, only the conditioned medium collected from CHO cells with transient expression of constructs containing cGHRH region [cGHRH(s), cGHRH(l), and cGHRH(d)] could increase luciferase activity in a dose-dependent manner via activation of cGHRHR (not cPAC1-R) (Fig. 6Go, B–D). In contrast, cGHRHR could not be activated by conditioned medium collected from CHO cells transfected with construct encoding only the cryptic peptide [cGHRH(c)] under the same condition (Fig. 6EGo). Similar results were obtained by using conditioned medium from cultured DF-1 cells (data not shown). These results suggest that cGHRH is a potential ligand of cGHRHR.

Activation of cGHRHR by synthetic cGHRH1–31 and cGHRH1–47 peptides
To further evaluate the potency of cGHRH at cGHRHR activation, two cGHRH peptides of different length were synthesized. One is the predicted long cGHRH1–47 peptide. The other is a short cGHRH1–31 peptide containing the N-terminal 31 amino acid residues conserved between chicken and zebrafish (Fig. 2AGo). As shown in Fig. 7Go, both synthetic cGHRH1–47 and cGHRH1–31 could increase luciferase activity via activation of cGHRHR with high potency (cGHRH1–47, EC50 0.053 nM; cGHRH1–31, EC50 0.087 nM) (Fig. 7Go). This finding strongly suggests that cGHRH is a potent ligand of cGHRHR.


Figure 7
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  		FIG. 7. Activation of cGHRHR and cPAC1-R by synthetic cGHRH peptides (10–12 to 10–6 M, 6 h). A and B, Dose-dependent effects of synthetic cGHRH1–47 (A) and GHRH1–31 (B) on basal luciferase activities of CHO cells cotransfected with pGL3-CRE-luciferase reporter construct and cGHRHR (or PAC1-R) expression plasmid. Each data point represents mean ± SEM of three replicates (cotransfection of pcDNA3.1 empty vector and pGL3-CRE-luciferase reporter construct was used as an internal control).

 
In contrast to hpGHRH1–40, cGHRH peptide, either the long or short form, could activate cPAC1-R at higher dosages (≥10–7 M, 1000-fold less potent than oPACAP38) (Figs. 5Go and 7Go). This experiment was repeated more than three times, and similar patterns were noted, suggesting the low-affinity binding of cGHRH to cPAC1-R and a close evolutionary relationship between GHRH and PACAP (4, 22).

Activation of cGHRHR by cGHRH-like peptide
It has been reported that the synthesized cGHRH-like peptide (or carp GHRH-like peptide) shows little detectable binding to either cGHRHR or hGHRHR in vitro (48), suggesting that cGHRH-like peptide may not function as a ligand of cGHRHR. To test this possibility, three GHRH-PACAP expression plasmids were used to transfect chicken DF-1 cells and conditioned medium was collected. As shown in Fig. 8Go, the conditioned medium from transient expression of the construct cGHRH-PACAP(d) encoding GHRH-like peptide could increase luciferase activity in a dose-dependent manner via specific activation of cGHRHR (Fig. 8CGo). In contrast, the conditioned medium from transient expression of construct cGHRH-PACAP(c) encoding the cryptic peptide had no effect on cGHRHR activation (Fig. 8DGo), also supporting the specific action of cGHRH-like peptide on cGHRHR activation. Interestingly, the conditioned medium from transient expression of the construct encoding complete ORF region of GHRH-PACAP seemed to induce luciferase activity via activation of cPAC1-R only (Fig. 8BGo). We repeated these experiments five times, and similar results were obtained.

Evolutionary evidence for a separate gene encoding GHRH in submammalian vertebrate species
Two separate genes were shown to encode GHRH and GHRH-PACAP in chickens (37), leading us to speculate that chicken GHRH gene and GHRH-PACAP gene may be orthologous to mammalian GHRH gene and PACAP gene, respectively. To test this hypothesis, we blasted genome database of human (Homo sapiens), Xenopus (Xenopus tropicalis), zebrafish (Danio rerio), tetraodon (Tetraodon nigroviridis), and fugu (Takifugu rubripes) using chicken GHRH and GHRH-PACAP cDNAs as references (http://www.ensembl.org). As a result, the orthologous regions containing GHRH gene have been identified in all genomes examined (Fig. 9AGo), and the GHRH1–31 peptides of different species (deduced from the identified conserved exon) are highly conserved (Fig. 10AGo). Furthermore, the existence of GHRH gene in lower vertebrates was evidenced by cloning a full-length cDNA of GHRH gene from zebrafish brain in our laboratory (accession no. DQ832172; 1017 bp, 112 a.a.) (Fig. 2AGo), confirming the expression of GHRH gene in lower vertebrate species (Figs. 2AGo and 9AGo). In contrast, cGHRH-PACAP gene and mammalian PACAP gene are localized on another conserved synteny, which could also be found in teleosts and amphibians, indicating that they are of the same origin (Figs. 9BGo and 11Go).


Figure 9
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  		FIG. 9. Identification of two conserved syntenies containing GHRH gene and GHRH-PACAP (or PRP-PACAP) gene, respectively, in vertebrate species including chicken, human, Xenopus, and zebrafish. A, GHRH, RPN2, MANBAL, and Src genes are located on a conserved synteny. B, GHRH-PACAP, clusterin-like 1 (CLUL1), thymidylate synthetase (TYMS), and v-yes-1 Yamaguchi sarcoma viral oncogene (YES1) genes are located on another conserved synteny. A duplication of GHRH-PACAP gene (GHRH-PACAP1 and GHRH-PACAP2) is identified in zebrafish. Chr., Chromosome.

 

Figure 10
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  		FIG. 10. A, Alignment of amino acid sequences of the partial GHRH peptides from various species including chicken (cGHRH: DQ465018), Xenopus (xGHRH: CX961070), zebrafish (zGHRH: DQ832172), fugu (fGHRH: scaffold_79), tetraodon (tGHRH: CAF96902), and human (hGHRH: X00094). B, Alignment of amino acid sequences of GHRH-like peptides (or PRP in human) from different species including chicken (cGHRH-L: AY956323), carp (cpGHRH-L), salmon (sGHRH-L: CAA51705), zebrafish (zGHRH-L1: AF217251; zGHRH-L2: AF329730), tetraodon (tGHRH-L1: CAG10213; tGHRH-L2: CAG12289), Xenopus (xGHRH-L: scaffold_420), and human (hPRP: NP_001108). Numbers in brackets indicate sequence identity of chicken GHRH (A) or GHRH-Like peptide (B) with that of other species. Dots represent the amino acid residues identical with those of cGHRH or cGHRH-like peptide. The predicted C-terminal amidation (G28) sites in A are boxed.

 

Figure 11
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  		FIG. 11. Proposed evolutionary history of GHRH gene and GHRH-PACAP gene in vertebrate species. The GHRH gene had already appeared in the last common ancestor of tetrapods and teleosts. After emergence of the mammalian lineage, GHRH gene and GHRH-PACAP gene have undergone an accelerated evolutionary event. The whole-genome duplication (WGD) occurred in teleost lineage resulted in the duplicated GHRH-PACAP genes (GHRH-PACAP1 and GHRH-PACAP2) in teleost species. Presence of an additional copy of GHRH gene in teleosts remains questionable. CP, Cryptic peptide; GHRH-L, GHRH-like peptide; C-peptide, carboxyl terminal flanking peptide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well documented that GHRH plays a critical role in stimulating pituitary GH synthesis and release both in vitro and in vivo in mammals (1). However, whether a GHRH gene exists in submammalian vertebrate species is still an open question (4, 22). In the present study, two chicken prepro-GHRH precursors with distinct C-terminal tails have been identified and both of them display biological activities on cGHRHR activation. Our studies also provide direct evidence that a functional GHRH is expressed predominantly in the chicken hypothalamus.

cGHRH peptide shares high homology with mammalian GHRHs, especially in the bioactive core region (GHRH1–29) (Fig. 2AGo). However, the chicken GHRH gene differs from its mammalian counterparts. In chickens, the GHRH gene consists of seven exons, whereas five exons have been noted in mammals (5, 7), and the predicted chicken prepro-GHRHs are much longer (172 and 188 a.a.) than mammalian prepro-GHRHs (103–108 a.a.). Unlike that in mammals, a cryptic peptide is still present in chicken GHRH precursors (Fig. 2AGo) (4). In addition, no homology could be noted in the carboxyl terminal flanking peptides between human, zebrafish, and chicken, suggesting that C-terminal flanking peptides may not have a similar physiological role in chickens as proposed in mammals (57, 58, 59, 60). In this study, two cGHRH mRNA species have been identified. The multiple splice variants coexpressed in hypothalamus and extrahypothalamic tissues have also been reported in mammals (5, 9, 61). However, the splicing sites are not conserved between chicken and mammals. All the difference indicates that mammalian GHRH gene must have undergone an accelerated evolutionary process after its divergence from a common ancestor (Figs. 2Go and 11Go). Meanwhile, the obvious difference in GHRH gene between chicken and mammals raises an interesting question regarding whether cGHRH is a functional cGHRHR ligand.

The supporting evidence from our present study first comes from the restricted spatial expression pattern of cGHRH in adult chicken brain. cGHRH mRNA is detected to be expressed predominantly in the hypothalamus. This is consistent with findings in mammals that GHRH is mainly localized in the arcuate nuclei of hypothalamus using immunohistochemistry and in situ hybridization (62, 63). In contrast, cGHRH-PACAP mRNA was detected to be expressed in all regions of the brain examined including hypothalamus. The ubiquitous expression of cGHRH-PACAP mRNA in the brain also concurs with previous findings in chickens (64). The differential mRNA expression of the two genes strongly hints that GHRH gene and GHRH-PACAP gene may have distinct functions in chicken brain. At least, the abundant expression of cGHRH mRNA in the hypothalamus, together with the predominant expression of cGHRHR in the pituitary (48, 53, 55), provides spatial evidence that cGHRH is likely to be a hypophysiotropic factor in the regulation of pituitary functions.

Another proof to support the functionality of cGHRH comes from the transient expression of cGHRH in CHO cells. Only the conditioned medium from CHO cells transfected with constructs encoding GHRH region could activate cGHRHR (Fig. 6Go, B–D) and thus increase luciferase activity in a dose-dependent manner, suggesting that cGHRH, as in mammals, could activate the intracellular cAMP-PKA signaling pathway (13). It also strongly suggests that cGHRH could function as the endogenous ligand of cGHRHR (Fig. 6Go, B–D). At present, we do not know how many amino acid residues are present in the mature chicken GHRH. But the predicted GHRH peptide is flanked by monobasic (R74) and dibasic residues (R122R123) at its N and C terminus, respectively, which are the putative proteolytic cleavage sites for mature peptide release (4). Thus, it is conceivable that cGHRH may have 47 amino acid residues with a free carboxyl terminus (named cGHRH1–47). This speculation is directly supported by our study that the synthetic cGHRH1–47 peptide is of the highest potency at cGHRHR activation (EC50 0.053 nM) (Fig. 7AGo). Because the carboxyl-terminally truncated cGHRH peptide (cGHRH1–31) still displays high potency at cGHRHR activation comparable with that of cGHRH1–47 (Fig. 7Go), whether the short form of cGHRH exists in vivo remains to be determined. Interestingly, a conserved consensus amidation site (G28), which also exists in the precursors of other glucagon family members such as PACAP and VIP (4), has been identified within the cGHRH1–47 region (Figs. 2AGo and 10AGo). Therefore, whether an amidation would occur at this specific location, thereby generating a short amidated form of cGHRH (cGHRH1–27-NH2) in vivo, would be of considerable interest to be investigated (Figs. 2AGo and 10AGo).

Finally, the evolutionary evidence supports that chicken GHRH gene is orthologous to the mammalian GHRH gene. It was originally hypothesized that mammalian GHRH gene originated from a duplicated GHRH-PACAP gene just before the emergence of mammals (4, 22). In this study, we have found that GHRH gene is located on a conserved synteny of all vertebrate species examined, including teleosts and amphibians (Fig. 9AGo), revealing that a separate GHRH gene had already existed in the last common ancestor of tetrapods and teleosts (Figs. 9Go and 11Go). Moreover, partial or whole GHRH regions, especially the bioactive core of GHRH, have been identified in zebrafish, tetraodon, fugu, and Xenopus. Interestingly, the cGHRH1–31 is 84% identical with the predicted teleost GHRH1–31 but is only 61% identical with human GHRH1–31 (Fig. 10AGo). The high homology between chicken and fish strongly suggests that teleost and cGHRH may play a conserved role in the pituitary. Despite accumulated evidence suggesting that PACAP, instead of GHRH, appears to act as a major hypophysiotropic GH-releasing factor in teleosts (22, 27, 28), it should be noted that cGHRHR could be activated by oPACAP38 at higher dosages (≥10–8 M, EC50 37.7 nM) (Fig. 5BGo) (48), pointing out the possibility that the GH-releasing effect of PACAP, particularly in vitro, may be partially mediated by pituitary GHRHR in the lower vertebrate species including chicken (Figs. 5BGo and 12Go) (48). Undoubtedly, the identification of the conserved GHRH gene in lower vertebrate species provides us an opportunity to reevaluate the roles of GHRH and PACAP and their receptors in the pituitary.


Figure 12
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  		FIG. 12. Proposed actions of GHRH, GHRH-like peptide (GHRH-L), and PACAP in chicken pituitary. Both GHRH and GHRH-L can activate GHRHR and thus are supposed to be the endogenous ligands of GHRHR responsible for GH synthesis and release in somatotroph. The GH-releasing action of PACAP in lower vertebrate species including chicken is likely to be mediated by PAC1-R (or other PACAP receptors) or GHRHR or both. The physiological relevance of GHRH-induced PAC1-R activation remains to be clarified.

 
In agreement with the concept of a separate gene encoding GHRH in lower vertebrates, the GHRH-PACAP gene is located on another conserved synteny of all vertebrate species investigated (Fig. 9BGo). Interestingly, two copies of the GHRH-PACAP gene have been identified in teleosts (32, 33), providing a possibility that cGHRH might have evolved from one of them. However, two copies of the GHRH-PACAP gene and its neighboring genes including clusterin-like 1, thymidylate synthetase, and v-yes-1 Yamaguchi sarcoma viral oncogene were identified on two chromosomes of zebrafish (chromosomes 2 and 7) (Fig. 9BGo), clearly showing that two copies of the GHRH-PACAP gene have resulted from a whole-genome duplication event that occurred in the teleost lineage (Figs. 9Go and 11Go) (65), as proposed in our previous study (32).

The identification of GHRH gene in lower vertebrates also questions the authentic identity of GHRH-like peptide in submammalian species. Is it a GHRH-like peptide with GHRH-like activity or a PACAP-related peptide (PRP) without known functions? In this study, we have demonstrated that cGHRH-like peptide is likely to be a ligand of cGHRHR. However, the biological activity of cGHRH-like molecule detected in conditioned medium largely depends on the type of construct tested. The conditioned medium from cultured DF-1 cells with transient expression of GHRH-like peptide could activate cGHRHR but not cPAC1-R (Fig. 8CGo). In contrast, the conditioned medium from DF-1 cells with transient expression of construct encoding complete ORF region of GHRH-PACAP could activate cPAC1-R but not cGHRHR (Fig. 8BGo). This finding is quite interesting because both cGHRH-like peptide and cPACAP are supposed to be released from the same precursor at a theoretical ratio of 1:1 and then activate both cGHRHR and cPAC1-R to a certain extent. Because the recombinant cGHRH-like peptide still displays an effect on cGHRHR activation; thus, the inability of recombinant GHRH-PACAP(w) on cGHRHR activation is unlikely to be due to the instability of processed GHRH-like peptide under our experimental condition. One possible explanation is that tissue-specific posttranslational processing may be involved. In mammals, the precursor of glucagon gene, also a member of secretin/VIP/PACAP family, encompasses three bioactive peptides (glucagon, glucagon-like peptide-1, and glucagon-like peptide-2) (4, 13), and the list of functional peptides produced depends on a tissue-specific processing event (13). If this is the case for cGHRH-PACAP, it would be of considerable importance to determine how cGHRH-PACAP precursor is processed in the hypothalamus and extrahypothalamic tissues, a question yet to be answered in all lower vertebrates. Recently Toogood et al. (48) demonstrated that GHRH-like peptide has little potency in increasing intracellular cAMP level via activation of cGHRHR (48). In this study, we clearly demonstrated that cGHRH-like peptide could activate cGHRHR with high reproducibility. The discrepancy between the two studies may be due to different approaches used (48). In agreement with our findings, chicken GHRH-like peptide is 76% identical with carp or salmon GHRH-like peptide (also named GHRH-L1 in this study) of biological activity (66, 67, 68), but is only 47–67% identical with the other type of teleost GHRH-like peptide (named GHRH-L2 in this study) with no effect on GHRHR activation (Fig. 11BGo) (68). The high degree of conservation between cGHRH-like peptide and carp GHRH-like peptide, together with evidence showing the biological activities of chicken and carp GHRH-like peptides (66), suggests that GHRH-like peptide (chicken/carp-like) may have important biological actions, at least in part, mediated by GHRHR in chicken and teleost fish (Fig. 12Go).

In summary, the full-length cDNAs encoding chicken GHRH have been cloned in the present study. RT-PCR assay revealed that cGHRH mRNA is predominantly expressed in the hypothalamus and functional studies confirmed that cGHRH could activate cGHRHR with high potency (EC50 < 0.1 nM). The activation of cGHRHR by cGHRH-like peptide was also observed. Moreover, the conserved GHRH gene was identified in genomes of Xenopus, zebrafish, fugu, and tetraodon. Evidence presented here demonstrates that GHRH, perhaps together with GHRH-like peptide (chicken/carp-like), function as the endogenous ligands of GHRHR in the chicken, and may act similarly in other lower vertebrate species (Fig. 12Go).


   Footnotes
 
This work was supported by the Research Grant Council of the Hong Kong Government (HKU7345/03M).

The cDNA sequences encoding chicken GHRH and zebrafish GHRH have been submitted to the DNA Data Base of Japan/European Molecular Biology Laboratory/GenBank databases under accession no. DQ465018, DQ465017, and DQ832172.

Disclosure Statement: Y.W., J.L., C.Y.W., A.H.Y.K., and F.C.L. have nothing to disclose.

First Published Online February 1, 2007

Abbreviations: a.a., Amino acid(s); c, chicken; CHO, Chinese hamster ovary; CRE, cAMP response element; GHRH-L, GHRH-like peptide; GHRHR, GHRH receptor; hpGHRH, human pancreatic GHRH; l, long form; oPACAP, ovine pituitary adenylate cyclase-activating polypeptide; ORF, open reading frame; PACAP, pituitary adenylate cyclase-activating polypeptide; PRP, PACAP-related peptide; PAC1-R, PACAP type I receptor; PKA, protein kinase A; s, short form; VIP, vasoactive intestinal polypeptide.

Received July 27, 2006.

Accepted for publication January 24, 2007.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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