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Department of Animal and Avian Sciences (T.E.P., L.E.E., A.F., J.L.S., I.B.) and Molecular and Cell Biology Program (T.E.P., L.E.E.), University of Maryland, College Park, Maryland 20742
Address all correspondence and requests for reprints to: Dr. Tom E. Porter, Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland 20742.
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Introduction |
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In this report, we identify the chicken GHRH-R gene and characterize the regulation of pituitary GHRH-R mRNA expression. Through random sequencing of a chicken cDNA library constructed from pituitary, hypothalamic, and pineal gland RNA, we identified two cDNA clones whose predicted amino acid sequence was similar to mammalian GHRH-Rs. After sequencing of these clones, we located the GHRH-R gene within the chicken genome. Comparisons of this region of the chicken genome with mammalian GHRH-R amino acid sequences allowed us to predict the chicken GHRH-R gene and cDNA sequences. Expression of each exon and of the full-length mRNA was confirmed by RT-PCR. Effects of corticosterone (CORT) GHRH, ghrelin, pituitary adenylate cyclase-activating peptide (PACAP), SRIF, and TRH on pituitary cell GH and GHRH-R gene expression in vitro were determined by quantitative real-time PCR.
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Materials and Methods |
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Bioinformatics and sequence comparisons
DNA sequence assembly and alignments were conducted using Vector NTI Advance 9.0 software (Invitrogen, Carlsbad, CA). Sequence retrieval (Ndjinn), prediction (SIXFRAME), alignment (CLUSTALW), and comparison (BOXSHADE; DRAWTREE; RPBLAST) tools were used within Biology Workbench maintained by the San Diego Super Computer Center (http://workbench.sdsc.edu/). Comparisons of chicken GHRH-R cDNA sequences with the chicken genome sequence were performed through the Ensemble database (http://www.ensembl.org/Multi/blastview?species=Gallus_gallus). Analysis for transcription factor binding sites within the 5'-flanking region of the chicken GHRH-R gene was accomplished using the vertebrate TRANSFAC transcription factor matrix within TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html) and TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess33/) and a threshold matrix score of 85.0. Intron/exon boundaries were predicted by comparing the GHRH-R mRNA sequence with the chicken genome and assuming all introns begin and end with GT and AG, respectively.
Pituitary cell culture and RNA extraction
All use of animals in this study was approved by the Institutional Animal Care and Use Committee of the University of Maryland. Fertile chicken eggs of the Ross broiler strain were obtained from Allens Hatchery (Seaford, DE) and incubated at 37.5 C and 60% relative humidity. Animals were provided feed and water ad libitum after hatching. Anterior pituitary glands were dissected from juvenile male chickens 32 d after hatching and dissociated into single cells using a combination of trypsin digestion and mechanical trituration as described previously (23, 24). Resulting pituitary cells were plated in 12-well plates and cultured (2.5 x 106 cells/ml) for 18 h in a 1:1 mixture of DMEM and Nutrient Mixture F-12 (DMEM/F-12; Invitrogen) supplemented with 10% heat-inactivated horse serum and antibiotics. Cultures were maintained at 37.5 C in a humidified atmosphere. After the 18-h preculture, medium was replaced with serum-free DMEM/F-12 (supplemented with 0.1% BSA and antibiotics) containing hormone treatments. Medium and treatments were replaced every 24 h. Cells were recovered after 4 and 72 h of hormone treatment by mild trypsin digestion (0.25 mg/ml) in MEM (Invitrogen) followed by aspiration and centrifugation. Harvested cells were snap frozen in liquid nitrogen. RNA was extracted from snap-frozen pituitaries and cultured cells using RNeasy minikits, according to the manufacturer’s protocol (QIAGEN, Valencia, CA). Human GHRH, ghrelin, PACAP, SRIF, and TRH were purchased from Phoenix Pharmaceuticals (Belmont, CA), and used at 1 x 10–8 M. Human peptides were used because, with the exception of PACAP, they all have been previously shown to regulate GH secretion in the chicken (6, 7, 9, 25). CORT was purchased from Sigma Chemical Co. (St. Louis, MO) and tested at 1 x 10–8 M. Three replicate experiments were performed.
GHRH binding and cross-linking
Binding assays of GHRH to pituitary membrane proteins were performed based on published procedures (15) with minor modifications. Briefly, pituitary cells from embryonic d (e) 16 chick embryos were homogenized on ice with a Dounce homogenizer in 50 mM Tris-HCl, 5 mM MgCl2, 2 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). This age was chosen because we had previously reported that e16 somatotrophs respond to human GHRH (6). Membranes were collected at 4000 x g for 10 min and resuspended in binding buffer (25 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.1 mM PMSF, 0.1 mg/ml leupeptin, 1 mg/ml bacitracin, and 0.1% BSA). The microbicinchoninic acid protein assay (Pierce, Rockford, IL) was used to calculate protein content, and 75 µg of membrane protein were incubated with 100 pM [125I-Tyr10] human (h)GHRH-(1–44) (Amersham Biosciences, Piscataway, NJ) for 1 h at room temperature. Binding reactions were terminated by microcentrifugation for 5 min at 4 C. Nonspecific binding was estimated by inclusion of 1 µM hGHRH(1–44).
For estimation of the relative molecular weight of the chicken GHRH-R protein, pooled e16 chicken pituitaries were placed in ice-cold assay buffer [50 mM Tris, 5 mM MgCl2, 2 mM EDTA, 0.42% BSA, 0.1 mM PMSF, 0.1 mg/ml leupeptin (pH 7.4)] and mechanically homogenized using a Dounce homogenizer. The protein content was estimated using the microbicinchoninic acid protein assay (Pierce), and the desired concentration was obtained with addition of assay buffer. Then 1.5 ml of this preparation (400 µg protein) was incubated in 12 x 75-mm glass culture tubes with 500 pM [125I-Tyr10] hGHRH-(1–44). The assay tubes were incubated at room temperature for 90 min, and then the reactions were transferred to siliconized 2-ml microcentrifuge tubes and centrifuged for 5 min at 12,000 x g. The supernatant was aspirated, and the membrane pellets were washed twice with ice-cold assay buffer. Subsequently the membrane pellets were resuspended in 25 mM HEPES-KOH buffer (pH 8.0), containing 10 mM of the cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) alone or in combination with 5 mM N-hydroxy-succinimide and incubated at 23 C for 30 min. To stop the reaction, glycine was added to a final concentration of 50 mM, and the incubation was continued for 15 min before centrifugation (12,000 x g, 5 min, 4 C). The pellets were solubilized in 50 µl Laemmli buffer, boiled for 5 min, subjected to SDS-PAGE, and transferred to polyvinyl difluoride membranes (Bio-Rad Laboratories, Hercules, CA). Autoradiograms were generated by exposing polyvinyl difluoride membranes to X-OMAT AR films (Kodak, Rochester, NY).
Analysis of mRNA levels by quantitative real-time RT-PCR (qRT-PCR)
Levels of GH, GHRH-R, glyceraldehyde-phosphodehydrogenase (GAPDH) and ß-actin (ACTB) mRNA and 18S ribosomal RNA were determined by qRT-PCR. Total RNA (1 µg) from each sample was reverse transcribed using either an oligo(dt) primer or random hexamers and Superscript III reverse transcriptase (Invitrogen). The reverse transcribed (RT) reactions were then diluted 3-fold for quantification of all mRNA levels and 40-fold for quantification of 18S ribosomal RNA. The diluted RT samples (2 µl) were then analyzed using the QuantiTect SYBR Green PCR kit (QIAGEN) and a Bio-Rad iCycler. Primers used for quantification of GH (GH_3prime_forward and GH_3prime_reverse), GHRH-R (GHRHR11-fwd and GHRHR12-rev), GAPDH (GAPDH_forward and GAPDH_reverse), and ACTB (ACTBfwd and ACTBrev) mRNA and 18S ribosomal RNA (18S forward and 18S reverse) are provided in Table 1
. Cycling parameters were 40 cycles of 94 C for 15 sec and 60 C for 1 min. Results for each sample were corrected for any potential background genomic DNA contamination using the formula Ct(no RT control) – Ct(sample), with cycle threshold (Ct) being the cycle number at which fluorescence surpassed background (determined during the first 10 cycles of amplification). Levels of each mRNA were then normalized to levels of GAPDH mRNA by subtracting the corrected Ct for GAPDH for each sample from the corrected Ct values for each of the other genes (
Ct). Relative expression levels were then calculated by transforming the log-based Ct values into a linear scale using the formula 2Ct and dividing the transformed data by the mean basal value for each time point.
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Results |
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Based on the cDNA sequences encoding for each of the predicted exons, oligonucleotide primers were designed for amplification and sequencing of the chicken GHRH-R mRNA. All primers used are described in Table 1
. Pituitary RNA was reverse transcribed using the Anchored-dT-one-V primer. PCR products were purified and sequenced, and the resulting sequences were assembled into a contig sequence for chicken GHRH-R mRNA. Determination of the 3' end of the mRNA sequence was accomplished by performing 3' rapid amplification of cDNA ends using the following primers: GHRHR_exon6_sense and Anchored-dT-one-V. Primer GHRH-R-5'sense was designed to be upstream of the predicted start codon, and this primer was used in combination with primers GHRHR_exon5_antisense and GHRHR_exon9_antisense to amplify and determine the sequence of the 5' end of the chicken GHRH-R mRNA. A summary of the sequences assembled in determining the chicken GHRH-R mRNA sequence is provided in Fig. 1. The final sequence for the chicken GHRH-R mRNA resulted from a minimum of three sequencing reactions for any region (
3-fold coverage). Primers GHRH-R-5' sense and cGHRH-R-3'-AS were used to amplify and verify expression of the full-length cDNA (Fig. 2).
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. The contribution of the 13 exons to the GHRH-R mRNA is presented in Fig. 1.
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). Similarly, amplification from exon 11 to exon 13 detected only a product devoid of introns (Fig. 2D). In all cases the size of the product agreed with that predicted from our final GHRH-R mRNA sequence. These results suggest that splice variants containing exons 1 and 13, which encode for the beginning and end of the protein, are rare. During the preparation of this report, two nucleic acid sequences nearly identical with our chicken GHRH-R sequence were submitted to GenBank (accession no. DQ114791 and DQ230840). Both sequences contain an extended 3' untranslated region that was not found in our 3' rapid amplification of cDNA ends determination of the chicken GHRH-R mRNA sequence. In addition, none of the 5' untranslated region determined in the present study was included in DQ230840. Within the overlapping region of the three sequences, which included all of the protein-coding region, only a single nucleotide was different between our sequence (DQ284530) and DQ230840, and this is in the 3' untranslated region. DQ114791 contained seven nucleotide differences between our sequence and DQ284530. Although six of these were within the protein-coding region, none of them altered the predicted amino acid sequence. As a result, the predicted amino acid sequences of the three chicken GHRH-R mRNA sequences submitted to GenBank were identical. Reports describing these other sequences have not been published.
The nucleic acid sequence of the chicken GHRH-R mRNA determined by sequencing of RT-PCR products in the present study was used to predict the chicken GHRH-R amino acid sequence. The cDNA encodes for a 419-amino acid protein, with a predicted molecular mass of 48.9 kDa. All 13 exons contribute to the open reading frame encoding for the GHRH-R protein. The predicted amino acid sequence for chicken GHRH-R was then compared with those for rat, human, pig, sheep, cow, and pufferfish GHRH-R (Fig. 2). As expected, the mammalian sequences were more closely related to one another, with the pufferfish GHRH-R sequence being most distantly related. The chicken GHRH-R sequence was more closely related to the mammalian sequences and was located at the expected phylogenetic branch point between the pufferfish and the mammals. Comparison of the chicken GHRH-R amino acid sequence with the Pfam database using the RPBLAST procedure revealed that it is similar to the secretin family of seven transmembrane domain receptors (pfam00002.11). The locations of the seven transmembrane domains predicted in the chicken GHRH-R protein are shown in Fig. 2, along with two potential sites for N-linked glycosylation in the extracellular domains. The GHRH-R sequence also contains four conserved cysteines found in a family of hormone receptors (pfam02793.11), and these cysteines probably form disulfide bridges and may form the GHRH ligand-binding domain. This domain is located in the amino-terminal, extracellular region of the sequence.
Estimation of GHRH-R relative molecular weight
To estimate the relative molecular weight of the native receptor for GHRH, plasma membrane proteins from chicken anterior pituitary glands were incubated with 125I-hGHRH and then cross-linked. The preparations were then separated in SDS-PAGE gels and subjected to autoradiaography. Results are presented in Fig. 3. Two GHRH-binding proteins were identified. The larger GHRH-binding protein was approximately 72 ± 3 kDa, and the smaller protein was approximately 45 ± 3 kDa. The larger was observed using cross-linking with EDAC alone or in combination with N-hydroxy-succinimide. However, the smaller was found only with cross-linking with EDAC in combination with N-hydroxy-succinimide. In a single experiment, deglycosylation of the protein preparation with glycosidase F slightly reduced the relative molecular weights of the bands, indicating that they are glycoproteins. However, glycosidase F did not reduce the size of the upper band to that of the smaller band (data not shown). These proteins likely represent the chicken GHRH receptor(s) because binding of 125I-hGHRH to pituitary membrane proteins was displaceable with nonlabeled GHRH. The EC50 for inhibition of 125I-hGHGH binding by GHRH was less than 1 nM, suggesting the presence of high-affinity receptors for GHRH.
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. GHRH-R mRNA was readily detected in the pituitary samples. Faint bands were also seen in the hypothalamic and total brain samples, but not in the other tissues. In addition to the Pit-1 sites, 21 glucocorticoid response element (GRE) half-sites and two cAMP response elements were found within the 2-kb sequence, suggesting control by hypophysiotropic hormones and glucocorticoids. To determine the effects of GHRH and other hormones on pituitary GH and GHRH-R mRNA levels, anterior pituitary cells were isolated and cultured for 4 and 72 h in the absence and presence of CORT, GHRH, ghrelin, PACAP, SRIF, and TRH. Results are summarized in Fig. 4. GHRH-R mRNA levels were increased and decreased by GHRH (P < 0.05) and SRIF (P < 0.01), respectively, at 4 h. These effects were modest at best. In contrast, treatment with CORT for 72 h dramatically reduced levels of GHRH-R mRNA by about 80% (P < 0.01). The most dramatic effect on GHRH-R gene expression was found with CORT, and suppression of GHRH-R mRNA levels by CORT was also observed when mRNA levels were normalized to levels of 18S RNA or not normalized to any internal control (data not shown). CORT affected GH mRNA levels in an opposite manner to its regulation of GHRH-R. CORT increased GH mRNA levels more than 2-fold at 4 and 72 h (P < 0.01). Similarly, GHRH, ghrelin, PACAP, SRIF, and TRH also increased GH mRNA levels at 4 h (P < 0.01). These effects were gone by 72 h. No effects on ACTB expression were found.
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Discussion |
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The predicted molecular mass of the chicken GHRH-R protein was 48.9 kDa, and this was confirmed experimentally. Binding of GHRH to pituitary membranes indicated the presence of a single high-affinity receptor with an affinity constant of less than 1 nM; 1 nM hGHRH displaced more than 50% of the 125I-hGHRH bound to chicken pituitary membranes. Similar affinity was reported for the rat GHRH-R (26). Interestingly, GHRH bound to a protein(s) that migrated with two relative molecular masses. The smaller of the two was approximately 45 kDa, consistent with the molecular mass predicted by the open reading frame of the mRNA sequence determined in the present study. The second band with a higher relative molecular mass of approximately 72 kDa may either represent the product of a second unknown GHRH receptor gene, a splice variant of the same GHRH-R gene, or the same GHRH-R protein modified to increase its relative migration in SDS-PAGE. Although two sites for N-linked glycosylation were found in the GHRH-R amino acid sequence, treatment with glycosidase did not reduce mobility of the higher molecular mass band to that of the lower. Alternatively, the presence of long hydrophobic stretches in the seven transmembrane domains may reduce mobility in SDS-PAGE. Similar findings have been made in mammals. The relative molecular mass of the bovine and rat GHRH-R was reported to be about 75 kDa (27), even though the molecular masses predicted by their mRNA sequences are approximately 47 kDa. However, in another report, binding proteins of 72, 50, 30, and 26 kDa were reported for the rat (28). Analysis of the chicken GHRH-R amino acid sequence indicated the presence of seven transmembrane domains and a hormone-binding domain conserved among species in the N-terminal extracellular domain of the protein. The N-terminal domain and the extracellular loops between the seven transmembrane domains are needed for GHRH binding to the human GHRH-R (29). In all, 12 cysteine residues in the GHRH-R sequences were conserved among all species compared.
Analysis of the 5'-flanking region of the chicken GHRH-R gene revealed the presence of numerous putative transcription factor response elements, including four Pit-1 sites, two cAMP response elements, and 21 potential GRE half-sites. Expression of GHRH-R in the pituitary in mammals is dependent on the pituitary-specific transcription factor Pit-1 (30), and the presence of Pit-1 sites in the chicken GHRH-R promoter suggests a similar dependence on Pit-1 for expression. This is supported by our finding that GHRH-R mRNA was highly expressed in pituitary samples but was not readily detectable in hypothalamic, brain, lung, adrenal, ovarian, or pineal gland samples. Although the function of the putative response elements was not confirmed experimentally in the present study, experiments were performed to characterize effects of various hormones on pituitary cell expression of GHRH-R mRNA. Consistent with the presence of many putative GRE half-sites in the 5'-flanking region of the GHRH-R gene, we found that CORT treatment for 72 h dramatically decreased GHRH-R mRNA levels by 80%. This finding supports a substantial role for glucocorticoids in regulating pituitary responsiveness to GHRH. Although eighteen potential GREs were reported in the rat GHRH-R 5'-flanking region (16), the opposite effect of glucocorticoids has been observed. In rats and humans, glucocorticoids appear to increase GHRH-R gene expression in the pituitary. GHRH-R mRNA levels in cultured rat pituitary cells were increased by glucocorticoids (32, 33, 34, 35), and glucocorticoids increased activity of the rat and human GHRH-R promoters in transient transfections of reporter constructs (17, 36). Glucocorticoids also increase pituitary GHRH-R mRNA levels in rats in vivo, and adrenalectomy decreases pituitary GHRH-R gene expression (37). Glucocorticoids have also been reported to increase GHRH binding to pituitary membranes in the rat (26, 38). In contrast, no effect of glucocorticoids on pituitary cell GHRH-R mRNA levels were found in the pig (18). Our current findings indicate that glucocorticoids have an opposite effect on GHRH-R gene expression in the pituitary of birds than in mammals.
In contrast to the dramatic effect of CORT on GHRH-R mRNA levels, the other treatments had modest or no effect on GHRH-R gene expression. We evaluated effects of short-term and extended treatment with GHRH, ghrelin, PACAP, SRIF, and TRH. Treatments were replenished every 24 h. However, no clear effects of any of these hypophysiotropic hormones on GHRH-R mRNA levels were found. Stimulatory effects of GHRH and inhibitory effects of SRIF were noted, but these were modest. This was surprising because clear effects of homologous and heterologous hormones on GHRH-R mRNA levels have been reported in mammals. Effects of GHRH on GHRH-R mRNA in pituitary cells in vitro in mammals are species and time dependent. GHRH decreased GHRH-R mRNA levels at 4 h in the rat and pig (18, 19, 20) but increased GHRH-R mRNA at 72 h in the rat (19). In contrast, GHRH increased GHRH-R mRNA levels at 2 h in sheep (39). Our current results indicate that the regulation of GHRH-R mRNA in chickens does not include major roles for GHRH itself, ghrelin, PACAP, SRIF, or TRH. However, these hormones were not without effect in our study. All were found to increase levels of GH mRNA at 4 h. We were not surprised to find stimulatory effects of GHRH, ghrelin, PACAP, or TRH because all are known to stimulate GH secretion. However, we were surprised to find that treatment with SRIF, which decreases GH secretion in chickens as in mammals, increased GH mRNA levels at 4 h. A potential reason for this effect is not clear. All effects of GHRH, ghrelin, PACAP, SRIF, and TRH on GH mRNA levels were gone by 72 h of treatment, possibly due to down-regulation of their receptors or signal transduction cascades. In contrast, CORT stimulated GH mRNA expression at 4 and 72 h. We noted stimulatory effects of CORT on GH mRNA levels in chicken embryos in previous reports (40, 41), and it has been known for some time that glucocorticoids stimulate pituitary expression of GH mRNA in mammals (31).
In conclusion, a number of findings support that our cDNA sequence represents the chicken GHRH-R. First, the nucleic acid and predicted amino acid sequences were highly homologous to GHRH-R in mammals. Second, the structure of the predicted protein with its hormone-binding domain and seven transmembrane regions is consistent with other GHRH-Rs. Third, GHRH binding to chicken pituitary membranes was displaceable and demonstrated the presence of a protein with a molecular weight similar to that predicted by the cDNA. Fourth, the structure of the chicken GHRH-R gene, with its 13 exons spanning 16 kb, was similar to that of mammals. The rat GHRH-R gene is approximately 15 kb in size and consists of 14 exons (16). Thus, we conclude that we have identified the chicken GHRH-R mRNA and gene. This report represents the first study of GHRH-R gene expression in any avian species, and our findings indicate that the regulation of pituitary GHRH-R mRNA levels in birds is distinctly different from that in mammals. Whereas all hormones tested increased GH mRNA levels, only GHRH, SRIF, and CORT were found to alter levels of GHRH-R mRNA. SRIF induced a modest decrease in levels of GHRH-R mRNA, and this may be one mechanism by which SRIF suppresses GHRH stimulation of GH secretion. The dramatic suppression of GHRH-R mRNA levels by CORT suggests that levels of adrenal glucocorticoids play a significant role in defining pituitary GH responses to GHRH in the chicken.
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Footnotes |
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T.P., L.E., A.F., J.S., and I.B. have nothing to declare.
First Published Online February 9, 2006
Abbreviations: ACTB, ß-Actin; CORT, corticosterone; e, embryonic day; Ct, cycle threshold; EDAC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GAPDH, glyceraldehyde-phosphodehydrogenase; GHRH-R, GHRH receptor; GRE, glucocorticoid response element; h, human; PACAP, pituitary adenylate cyclase-activating peptide; PMSF, phenylmethylsulfonyl fluoride; qRT-PCR, quantitative real-time RT-PCR; RT, reverse transcription; SRIF, somatostatin.
Received December 5, 2005.
Accepted for publication February 1, 2006.
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