Expression of the Ghrelin Axis in the Mouse: An Exon 4-Deleted Mouse Proghrelin Variant Encodes a Novel C Terminal Peptide
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内分泌学杂志 2005年第1期
Ghrelin Research Group, School of Life Sciences, Queensland University of Technology, Brisbane 4001, Australia
Address all correspondence and requests for reprints to: Penny Jeffery, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane 4001, Australia. E-mail: p.jeffery@student.qut.edu.au.
Abstract
Ghrelin, an n-octanoylated 28-amino-acid peptide capable of inducing GH secretion and food intake in humans and rats, is the endogenous ligand for the GH secretagogue receptor (GHS-R). Here we describe the expression and tissue distribution of the ghrelin/GHS-R axis in the mouse. We also report for the first time the identification of a novel mouse ghrelin mRNA variant in which there is a complete deletion of exon 4. Translation of this variant mRNA yields a protein containing ghrelin and an alternative C-terminal domain with a unique C-terminal peptide sequence. RT-PCR with primers specific for mouse ghrelin was used to demonstrate the mRNA expression of the full preproghrelin transcript and the exon 4-deleted variant in multiple mouse tissues. Real-time PCR was also employed to quantitate mRNA expression of ghrelin, the novel isoform and a previously reported ghrelin gene variant, ghrelin gene-derived transcript. We also demonstrated the tissue expression of the functional GHS-R in the mouse. Immunohistochemistry, employing antibodies raised against the mature human n-octanoylated ghrelin peptide and the putative C-terminal peptide encoded by the exon 4-deleted proghrelin variant, was used to demonstrate protein expression of ghrelin and the variant in multiple mouse tissues including stomach, kidney, and reproductive tissues. The coexpression of ghrelin and its receptor in a wide range of murine tissues suggests varied autocrine/paracrine roles for these peptides. Exon 4-deleted proghrelin, a novel mouse proghrelin isoform with a unique C-terminal peptide sequence, is also widely expressed in the mouse and thus may possess biological activity in these tissues.
Introduction
GHRELIN, A RECENTLY DISCOVERED 28-amino-acid GH-releasing peptide originally isolated from rat stomach (1), is now recognized to have a wide range of physiological roles. The in vitro and in vivo GH secretory activity of ghrelin is dependent on a unique n-octanoylation at Ser-3 (1) and is mediated by its endogenous receptor, the GH secretagogue (GHS) receptor (GHS-R), a G protein-coupled receptor (2). Two GHS-R isoforms have been identified. Type 1a is the full-length, seven-transmembrane domain functional receptor, and the type 1b isoform is a C-terminally truncated, five-transmembrane domain variant (3). The 1b isoform is thought to be nonfunctional. GHS-R signaling in the pituitary triggers a transient increase in intracellular calcium levels that results in GH release (1).
Although ghrelin is produced and expressed predominantly in the endocrine cells of the stomach (4), coexpression of ghrelin and the GHS-R 1a has been widely observed throughout rat tissues, suggesting that the ghrelin/GHS-R axis possesses diverse physiological functions. In addition to GH release, ghrelin stimulates food intake and adiposity in rats (5, 6), and the administration of ghrelin improves cardiac function in humans (7) and rats with experimentally induced chronic heart failure (8). Ghrelin is expressed in pancreatic islets, although conflicting results have been reported regarding its influence on insulin secretion (9, 10, 11). Ghrelin and the GHS-R may also have important roles in cell proliferation and the progression of hormone-dependent cancer (for review see Ref. 12).
Ghrelin has been isolated from several animal species. Human and rat ghrelin differ by only two amino acids (1), and there is a high degree of homology for the first four amino acids (GSSF) of the mature peptide across species, including the human and rat (1), mouse (13), fish (14, 15), and chicken (16). These four amino acids comprise the minimum active core of ghrelin and dictate its ability to activate the GHS-R in vitro (17). However, the remaining sequence of ghrelin may be biologically essential in vivo because truncated synthetic ghrelin molecules fail to stimulate GH secretion in rats (18).
In the mouse, preproghrelin is encoded by the ghrelin gene, which consists of four coding exons and a 19-bp noncoding first exon (13). The mature ghrelin peptide, which is cleaved from the proghrelin product, is encoded by parts of exons 2 and 3 (13). A number of ghrelin isoforms have recently been identified in rats. Des-octanoylated ghrelin lacks the Ser-3 octanoic acid modification (19) and therefore cannot activate GHS-R-expressing cells to stimulate GH secretion. Alternative splicing of the ghrelin gene is thought to produce des-Gln14-ghrelin, which is identical with ghrelin except for the loss of one glutamine residue (Gln14) and induces a similar profile of GH release in rats as does ghrelin (20).
The ghrelin gene-derived transcript (GGDT) is a murine proghrelin mRNA variant encoded by exons 1, 4, and 5 and therefore does not encode mature ghrelin. GGDT displays temporally regulated expression in mouse testis tissue and may play a role in reproduction (21). It is not known, however, whether this transcript is translated in vivo because the predicted protein sequence does not contain a signal peptide. Very recently a truncated des-Gln14-ghrelin isoform has been reported in the mouse (22).
In this paper, we report the identification and characterization of a novel mouse proghrelin isoform in which there is a complete deletion of exon 4, potentially generated by alternative splicing of the mouse ghrelin gene. We previously reported the identification of the human counterpart of this isoform in prostate cancer cell lines (23) and presented preliminary data demonstrating the expression of the mouse isoform, along with full-length preproghrelin, in mouse embryos (24). The expression of this variant has now been examined in a wide range of murine tissues at the protein and mRNA levels. Expression of other components of the ghrelin/GHS-R axis was also examined at the mRNA and protein level in these tissues.
Materials and Methods
Mice
Quackenbush Swiss mice were obtained from the Central Animal Breeding House (University of Queensland, Brisbane, Australia) and killed by cervical dislocation in accordance with Queensland University of Technology (QUT) animal ethics guidelines (QUT approval number 187/4a). Heart, liver, kidney, testis, ovary, spleen, lung, muscle, adrenal gland, seminal vesicles, brain, fat, and gastrointestinal tissues were dissected and stored in RNA later (Ambion, Austin, TX) or frozen in liquid nitrogen and stored at –80 C. RNA was extracted from the tissues using the Trizol method (Life Technologies, Rockville, MD). Tissues were also collected for immunohistochemistry and Western analysis.
Reverse transcriptase (RT) of extracted tissue RNA
The first-strand cDNA synthesis kit for RT-PCR [avian myeloma virus (AMV)] (Invitrogen, Carlsbad, CA) was used to generate cDNA from RNA extracted from mouse tissues. Total RNA from each tissue (1 μg) was incubated in a 20-μl reagent solution containing 1x reaction buffer, 5 mM MgCl2, 1 mM deoxynucleotide triphosphate mix, 3.2 μg random hexamers, 50 U RNase inhibitor, and 20 U AMV RT for 10 min at 25 C and then for 60 min at 42 C. The reaction was treated at 99 C for 5 min and 4 C for 5 min to denature the AMV RT. PCR using intron-spanning mouse ?-actin primers (Table 1) demonstrated that cDNA was present and devoid of genomic DNA contamination.
TABLE 1. RT-PCR primer sequences, exon positioning, annealing temperatures (Ta) and expected amplicon sizes
Detection of full-length ghrelin, exon 4-deleted proghrelin, and GHS-R mRNA expression in mouse tissues with RT-PCR
Transcripts for full-length mouse ghrelin (GenBank accession no. AB035701), exon 4-deleted proghrelin, and GHS-R 1a (AY056474) were detected with RT-PCR using the primers and PCR conditions listed in Table 1. Discrete expression of exon 4-deleted proghrelin mRNA was confirmed using a specific sense primer that spans the putative exon 3/5 boundary (Table 1).
PCRs contained 10x PCR buffer, 100 μM deoxynucleotide triphosphates, 1.5 mM MgCl2, 100 pM primers (Proligo, Armidale, Australia), 2 μl cDNA or water (no template-negative control), and 1 U Red Hot polymerase (Integrated Sciences, Melbourne, Australia) or 1 U platinum Taq polymerase (Roche, Stockholm, Sweden). Thermal cycling consisted of 5 min at 95 C followed by 40 cycles of 30 sec at 95 C, 30 sec at annealing temperature (Table 1), 1 min at 72 C, followed by a final 10-min incubation at 72 C on a PTC-200 thermal cycler (MJ Research, Watertown, MA). Southern analysis was performed on blotted RT-PCR products as previously described (23) using a digoxigenin-labeled oligonucleotide probe and chemiluminescent detection (Roche).
Sequencing
The RT-PCR products were purified from an agarose gel using the High Pure gel purification kit (Eppendorf, Hamburg, Germany). Sequencing was performed at the Australian Genome Research Facility (University of Queensland, Brisbane, Australia) using the 377 DN automated DNA sequencer (Applied Biosystems, Warrington, UK) and ABI Big Dye version 2 terminator reagents (Applied Biosystems).
Relative quantitation of full-length ghrelin, exon 4-deleted proghrelin, and GGDT mRNA transcripts with real-time PCR
Real-time quantitative PCR was performed using the ABI 7000 prism sequence detection system (Applied Biosystems). Intron-spanning primers were designed using Primer Express software (version 2.0, PE Applied Biosystems) and were synthesized by Proligo (Table 2). Validation experiments were performed to ensure that the amplification efficiencies of both the target and reference primer sets were equal (user bulletin 2, ABI PRISM 7000 sequence detection system; Applied Biosystems). Serial dilutions of cDNA for full-length ghrelin, exon 4-deleted proghrelin, and GGDT were used in the data validation experiments. All quantitative PCRs were performed on 5 μl cDNA in a total volume of 20 μl. Each reaction comprised 100 pmol relevant primers and 10 μl 2x SYBR green I master mix (Applied Biosystems). Quantification of ghrelin, exon 4-deleted proghrelin, and GGDT transcripts in the tissue samples was achieved by the comparative CT method (user bulletin 2, ABI PRISM 7000 sequence detection system). All samples were run in triplicate and normalized against ribosomal 18S RNA. Data are represented as fold change (with confidence intervals), compared with brain tissue (calibrator tissue).
TABLE 2. Quantitative light cycler PCR primer sequences
Antibodies and Western analysis
Polyclonal primary antibodies were raised in rabbits (Institute for Medical and Veterinary Sciences, Adelaide, Australia) against the human n-octanoylated ghrelin peptide, GSSFLSPEHQRVQQRKESKKPPAKLQPR (Mimotopes, Victoria, Australia), and the novel C-terminal peptide encoded by the exon 4-deleted proghrelin variant, RRQLTSNHGQA (Mimotopes). The antibodies were affinity purified (Mimotopes) and concentrated by ultracentrifugation with Amicon centrifugal filter units (Millipore, Watertown, MA).
Western blot analysis was undertaken to detect the expression of ghrelin, preproghrelin, proghrelin, and the exon 4-deleted proghrelin peptide in mouse stomach tissue and confirm antibody specificity. Tissue protein was extracted in buffer containing 1% Triton X-100 and a protease inhibitor cocktail tablet without EDTA (Roche). Protein (20 μg) was boiled for 5 min in 4x loading buffer [250 mM Tris-Cl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 20 mM dithiothreitol, and 0.01% bromophenol blue] and electrophoresed on 10% SDS-PAGE gels against a protein standard (Multimark, Invitrogen) and synthetic exon 4-deleted proghrelin peptide or n-octanoylated ghrelin peptide (0.1 μg) (Mimotopes). Protein was transferred to a Protran nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) for 2 h at 100 V in transfer buffer (10 mM NaHCO3, 3 mM Na2CO3, methanol) and the membrane blocked overnight at 4 C in Tris-buffered saline/0.05% Tween 20/1% BSA. The membrane was incubated with anti-exon 4-deleted proghrelin peptide primary antibody solution (1/1000 to 1/2000) or anti-ghrelin antibody (1/40 to 1/800) at room temperature for 1 h. After washing in Tris-buffered saline/Tween20, the membrane was incubated with an antirabbit secondary antibody (1/100,000) (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. Femto chemiluminescent substrate solution (Pierce, Rockford, IL) was layered onto the membrane and incubated for 5 min. The membrane was then exposed to x-ray film and developed using a Curix 60 automatic processor (Agfa-Gevaert, Morstel, Belgium).
Immunohistochemistry
Detection and localization of ghrelin, the exon 4-deleted proghrelin variant, and GHS-R 1a protein was performed using immunohistochemistry on mouse tissue specimens. Antibodies cross-reactive with the putative murine GHS-R 1b isoform are unavailable at this stage. Tissues were dissected and fixed in 4% paraformaldehyde solution for 4–6 h. The tissues were then embedded in paraffin wax, and 7-μm sections were cut on a microtome. Tissue sections were dewaxed, dehydrated, and then microwaved in 0.01 M citric acid buffer (pH 6) to facilitate antigen retrieval. Sections were incubated for 1 h with primary antibody diluted in 0.01 M PBS/1% BSA. Ghrelin antibody was diluted 1/25 to 1/100 and anti-exon 4-deleted proghrelin peptide antibody from 1/1000 to 1/3000. GHS-R 1a antibody was diluted 1/200 as described previously (25). Immunodetection was performed using the Envision plus diaminobenzamine antirabbit immunodetection kit (Dako, Kyoto, Japan) according to the manufacturer’s instructions. Negative controls included the abolition of staining by preabsorbing the primary antibody overnight at 4 C with ghrelin or the exon 4-deleted proghrelin peptide (1 mg/ml), omission of primary antibody, or the application of preimmune sera. Tissues were counterstained with hematoxylin (Sigma, St. Louis, MO).
Results
Expression of full-length preproghrelin and a novel exon 4-deleted proghrelin mRNA isoform in mouse issues
Using primers positioned in exons 2 and 5 (Fig. 1), RT-PCR identified full-length preproghrelin transcripts of the expected size (464 bp) in all tissues examined including heart, brain, and the positive control tissue, stomach (Fig. 2A). DNA sequencing confirmed the identity of this transcript. In addition, a smaller PCR product of 355 bp was also generated in all of the tissues examined (Fig. 2A). Southern analysis using an internal oligonucleotide probe for mouse ghrelin gave a positive signal, indicating that this transcript had homology with ghrelin (data not shown). DNA sequencing identified this transcript as a novel mouse proghrelin isoform with a complete lack of exon 4. Thus, alternative splicing of the mouse preproghrelin pre-mRNA can potentially produce the exon 4-deleted isoform as well as the expected full-length transcript (Fig. 1). The novel nucleotide sequence of the exon 4-deleted variant has been deposited with GenBank (accession no. AY179430).
FIG. 1. Mouse preproghrelin pre-mRNA (i) can be processed to give full-length ghrelin mRNA (ii) or splicing of ghrelin pre-mRNA (shown here with exons numbered and introns represented by plain lines) can result in the skipping of exon 4, thus generating exon 4-deleted proghrelin (iii). Positioning of the sense and antisense primers used in the RT-PCRs is indicated by arrows. The exon 4-deleted proghrelin transcript-specific sense PCR primer (dashed arrow) spans the unique exon 3/5 junction created by the lack of exon 4 and under stringent conditions detects only this variant.
FIG. 2. A, RT-PCR products showing the expression of full-length preproghrelin and exon 4-deleted proghrelin mRNA in mouse tissues derived using primers located in exon 2 (sense) and exon 5 (antisense). The upper band (464 bp) seen in all tissues corresponds to mRNA for the full-length form of ghrelin, and the lower band (355 bp) was identified through Southern analysis and sequencing as a proghrelin mRNA isoform that lacks exon 4. B, Exon 4-deleted proghrelin mRNA potentially results from the alternative splicing of mouse preproghrelin pre-mRNA. Ethidium bromide stained agarose gel of RT-PCR products using primers designed specifically to detect this isoform. Exon 4-deleted proghrelin was amplified in all mouse tissues with RT-PCR employing a sense exon 3/5 primer specific for the exon 4-deleted transcript and the exon 5 antisense primer. C, RT-PCR amplification of the housekeeping ?-actin mRNA in all mouse tissues shows a single band at 243 bp, indicating that cDNA is devoid of genomic DNA contamination.
Based on the sequencing data for the exon 4-deleted proghrelin transcript, a forward primer that spans the unique exon 3/5 boundary created by the deletion of exon 4 in this variant was designed to establish a PCR protocol for the specific amplification of this variant only (Fig. 1). Using this primer together with the exon 5 reverse primer used for the full-length PCR (Table 1), a specific band of the expected size for exon 4-deleted proghrelin (131 bp) was observed in all of the mouse tissues examined (Fig. 2B). Sequencing confirmed the transcript as being an exon 4-deleted isoform of preproghrelin. The internal housekeeping gene ?-actin was amplified in all tissues examined (Fig. 2C) and confirmed the integrity of the cDNA.
GHS-R 1a transcript expression in mouse tissues
GHS-R 1a mRNA transcripts were amplified in various mouse tissues including the adrenal gland, brain, kidney, stomach, heart, lung, and pancreas. These cDNA products were confirmed by Southern analysis using an internal probe to increase the specificity of the assay (Fig. 3) and by a 100% sequence homology with the published mouse GHS-R 1a nucleotide sequence (accession no. NM-177330).
FIG. 3. GHS-R 1a isoform expression in mouse tissues detected by Southern blot analysis after RT-PCR using intron-spanning primers. PCR products were detected using an internal digoxigenin end-tailed oligonucleotide antisense probe and chemiluminescent detection (Roche).
Relative quantitation of full-length ghrelin, exon 4-deleted proghrelin, and GGDT transcripts in mouse tissues with real-time PCR
As expected, ghrelin gene expression was found to predominate in mouse stomach tissue as indicated in Fig. 4A. Pancreas, muscle, ovary, kidney, and fat express low levels of full-length preproghrelin mRNA with negligible expression in adrenal gland and liver (data not shown). Stomach was also the primary site of exon 4-deleted proghrelin mRNA expression with markedly lower levels in brain, muscle, ovary, and kidney (Fig. 4B). Negligible amounts of transcript were detected in pancreas, fat, adrenal gland, and liver (data not shown). In agreement with Tanaka et al. (21), GGDT transcript was found to be expressed predominantly in mouse testis tissue (data not shown), even with the use of a more sensitive detection method in this present study.
FIG. 4. Quantitative RT-PCR analysis of ghrelin (A) and the novel exon 4-deleted proghrelin isoform (B) mRNA expression in various mouse tissues. Data are represented as fold change relative to expression of transcripts in brain (1.0) with 95% confidence intervals. The full-length and exon 4-deleted isoforms are predominantly expressed in the stomach. All other tissues tested express low levels of ghrelin mRNA transcript. Exon 4-deleted proghrelin mRNA was present at low levels in the brain, muscle, ovary, and kidney with expression in the fat, pancreas, and adrenal tissues undetectable in this assay.
The absence of exon 4 results in a novel truncated C-terminal peptide
The exclusion of exon 4 from mouse preproghrelin mRNA results in a frameshift that, when translated, encodes a novel C-terminal peptide (RRQLTSNHGQA) while still coding for the mature ghrelin sequence (Fig. 5A). The generation of a dibasic pair of amino acids (RR) at the N terminus of the unique peptide sequence is particularly interesting, raising the possibility that enzymatic cleavage of this peptide may occur. After taking into account conservative amino acid changes, the C-terminal peptide sequence of the mouse exon 4-deleted isoform shares significant homology with the human (exon 3-deleted) homolog (Fig. 5B).
FIG. 5. cDNA and corresponding amino acid sequence (AY179430) for the exon 4-deleted proghrelin transcript with bases numbered on the left and amino acids on the right (A). The mature ghrelin peptide is underlined and the potential n-octanoylation site indicated by solid triangle. The unique C-terminal peptide sequence created by deletion of exon 4 is in italic font with the tribasic motif (RRR) in bold italic. Homology between human exon 3-deleted proghrelin C-terminal peptide and the mouse exon 4-deleted proghrelin peptide is illustrated (B). Exact amino acid matches are highlighted.
Western blot detection and size estimation of full-length ghrelin, exon 4-deleted proghrelin, and their cleavage products in mouse stomach protein extract
Western analysis of mouse stomach tissue extracts using the antighrelin antibody detected three distinct bands at 13, 11, and 3 kDa, respectively (Fig. 6A). These correspond to the predicted sizes of the potential preproghrelin cleavage products: preproghrelin itself, proghrelin, and mature ghrelin (Fig. 6B). The exon 4-deleted equivalents of the preproghrelin cleavage products (10 and 9kDa) were also detected in mouse stomach using the anti-exon 4-deleted proghrelin antibody (Fig. 6A); however, a cleaved form of the unique C-terminal peptide was not detected at the predicted approximate size of 1 kDa (Fig. 6B). No other bands were detected for either antibody, confirming antibody specificity.
FIG. 6. A, Western blot analysis of mouse stomach protein extracts detected with either an anti-ghrelin (i) or anti-exon 4-deleted proghrelin (ii) antiserum. Full-length preproghrelin (13 kDa), proghrelin (11 kDa), and the mature ghrelin peptide (3 kDa) were detected in mouse stomach tissue using the ghrelin antiserum, but only bands corresponding to the predicted sizes of exon 4-deleted preproghrelin (10 kDa) and proghrelin (9 kDa) were detected in this tissue using the exon 4-deleted proghrelin antiserum. B, Predicted cleavage products and approximate sizes (kilodaltons) of full-length and exon 4-deleted preproghrelin. The antighrelin antibody can be predicted to detect three peptides: preproghrelin, proghrelin, and the mature ghrelin peptide. The anti-exon 4-deleted proghrelin peptide antibody will not detect mature ghrelin but should detect truncated preproghrelin, proghrelin, and the putatively cleaved unique C-terminal peptide.
Immunohistochemical localization of ghrelin, GHS-R 1a, and exon 4-deleted proghrelin peptide expression in mouse histological specimens
As expected, ghrelin protein is strongly expressed in the glands of the mouse stomach as indicated by intense ghrelin immunostaining (Fig. 7A). Expression is also evident in the pancreatic islets (Fig. 7B) and blood vessel endothelium and visceral fat of the kidney (Fig. 7C). The proximal and distal kidney tubules also stain positively for ghrelin with negligible staining in the glomeruli (Fig. 7D). Skeletal muscle tissue also expresses ghrelin at the protein level (Fig. 7E). Preabsorption of the antibody with synthetic n-octanoylated ghrelin completely inhibits immunostaining in all of the tissues tested (stomach shown here, Fig. 7F) again confirming the specificity of the antighrelin antibody. GHS-R 1a protein expression is also present in mouse stomach glands (Fig. 7G) and kidney tubules with faint immunoreactivity apparent in the kidney glomeruli (Fig. 7H). No staining was evident in the kidney tissue when the primary antibody step was omitted (Fig. 7I).
FIG. 7. Immunohistochemistry on mouse tissue specimens to detect expression of the ghrelin/GHS-R axis. A, Ghrelin immunostaining is present in stomach glandular tissue, the positive control tissue. B, Mouse pancreatic islets (PI), blood vessel endothelium (BV) (C), and visceral fat cells (F) of the kidney are immunoreactive for full-length ghrelin peptide. D, The proximal and distal tubules of the kidney also stain for ghrelin, whereas glomeruli (arrow) are nonimmunoreactive. E, Ghrelin is expressed in skeletal muscle fibers. F, Preabsorption of the ghrelin antibody with synthetic ghrelin peptide abolishes immunostaining (shown in stomach). GHS-R 1a is expressed in the stomach glands (G) and kidney tubules (H) with negligible staining in the glomeruli. I, Preabsorbed control did not show staining (shown here in kidney). Scale bars, 100 μm (A); 50 μm (B–I).
The exon 4-deleted proghrelin C-terminal peptide is highly expressed at the protein level in stomach glandular tissue with intense immunoreactivity noted in some cells (Fig. 8A). Negative controls, consisting of primary antibody omission and preabsorption of the antibody with the C-terminal peptide, were devoid of any immunoreactivity (representative preabsorption control, Fig. 8B). Corpus lutea of the mouse ovary stain strongly for the peptide (Fig. 8C). Faint staining was noted in blood vessel endothelium of the kidney (Fig. 8D), with negligible expression of the isoform noted in the seminiferous tubules of the testis (Fig. 8E) and in cardiac and skeletal muscle fibers (Fig. 8, F and G). Intense immunoreactivity was detected in mouse brain tissue in the dentate gyrus of the hippocampal formation (Fig. 8H). Kidney tubules and glomeruli also express the C-terminal peptide of exon 4-deleted proghrelin (Fig. 8I) as do the epithelial cells of mouse prostate gland (Fig. 8J).
FIG. 8. Immunohistochemistry on mouse tissue specimens for the novel exon 4-deleted proghrelin isoform. A, Exon 4-deleted proghrelin is expressed highly in stomach glands. B, Preabsorption of the antibody with the C-terminal peptide results in negative staining in the stomach. C, Corpus lutea (CL) of the mouse ovary show strong immunoreactivity for the peptide as does blood vessel endothelium (D, arrow). Seminiferous tubules (ST) of the testis (E) and cardiac (F) and skeletal muscle fibers (G) demonstrate weak staining. H, Strong immunoreactivity is evident in the dentate gyrus region of the mouse hippocampal formation. Inset, Preabsorbed control demonstrates no staining in this tissue. I, The kidney tubules express the isoform as do glandular epithelial cells (J) of the mouse prostate. Scale bar, 50 μm (C, D, F, G, H, and I); 100 μm (A, B, and J); and 150 μm (E).
Discussion
We report for the first time the expression of both mouse ghrelin and its receptor and a novel mouse proghrelin isoform designated exon 4-deleted proghrelin. This variant, along with full-length ghrelin, is expressed at the mRNA and protein level in all of the mouse tissues examined, and in particular the stomach and brain. The exon 4-deleted proghrelin protein results from the translation of an mRNA transcript that completely lacks exon 4 (which is equivalent to human exon 3). We previously identified a human homolog of this isoform, exon 3-deleted proghrelin, which is expressed in a wide range of human tissues including the prostate (23).
By performing Southern analysis under low-stringency conditions, Hosoda et al. (20) determined that ghrelin and des-gln14-ghrelin are likely to be products of a single ghrelin gene. Therefore, it is probable that the exon 4-deleted variant is also a product of this gene, produced by alternative splicing mechanisms such as exon skipping. Alternative splicing contributes significantly to protein diversity in the human and mouse, giving rise to the production of multiple, often functionally diverse proteins from a single gene. The exclusion of exon 4 from mouse preproghrelin mRNA transcript results in a cDNA frameshift and the generation of a premature stop codon. When translated, this sequence encodes a novel peptide sequence at the C terminal of proghrelin while still retaining the coding sequence for mature ghrelin. Thus, translation of the exon 4-deleted proghrelin isoform is likely to lead to the production of n-octanoylated (active) ghrelin.
We also predict that the unique C-terminal tail of the exon 4-deleted proghrelin protein is cleaved in vivo at an arginine-rich junction introduced between the 11-amino-acid C-terminal peptide and the rest of the propeptide (Fig. 5A). Alternatively, if not cleaved and secreted in vivo, this unique C-terminal tail may influence the properties and/or processing of the entire exon 4-deleted proghrelin variant.
Pemberton et al. (25) recently demonstrated the presence of full-length proghrelin C-terminal peptides in human plasma that circulate at higher concentrations than mature octanoylated ghrelin and are elevated in heart failure. This study suggests that proghrelin C-peptides may possess as-yet-unknown physiological functions and supports our hypothesis that the generation of the unique C-peptide from the exon 4-deleted variant of proghrelin may also have physiological significance.
The antibody used for the immunohistochemical studies in this paper does not distinguish between cleaved and noncleaved exon 4-deleted proghrelin; therefore, this study could not determine whether mouse tissues express intact or C-terminally cleaved exon 4-deleted proghrelin or both. Although we were not able to detect the cleaved 11-amino-acid C-terminal peptide by Western blot analysis, the relatively high degree of conservation of this sequence with the unique C-terminal peptide of the human exon 3-deleted proghrelin (12) implies functional relevance. Biological assays to test for peptide function are currently being undertaken.
Previous studies documented that ghrelin and the type 1a GHS-R are widely coexpressed in rat tissues including the heart, pancreas, adipose tissue (1), adrenal gland (26, 27), and reproductive tissues such as the testis (28) and ovary (29). This coexpression suggests autocrine/paracrine functions for ghrelin and its receptor in these tissues. To our knowledge, this study is the first to quantitate, at the mRNA level, the expression of ghrelin and the novel exon 4-deleted isoform in an extensive range of murine tissues. To date, GHS-R 1b expression has been identified only in the human and domestic pig (2) and recently in black seabream hypothalamus (30). Using primers based on a Mus musculus chromosome 3 genomic contig, we were able to identify PCR products that represented fragments of murine GHS-R 1b cDNA (data not shown); however, further sequencing and proteomic analysis is required before this sequence can be fully reported.
Exon 4-deleted proghrelin mRNA and protein are expressed at the highest levels in mouse stomach glandular tissue, which reflects the expression pattern of ghrelin. GHS-R 1a expression was also demonstrated in this tissue. Further experiments are required to determine whether the unique C-terminal peptide of the exon 4-deleted proghrelin isoform influences the orexigenic properties of ghrelin and its functional receptor, GHS-R 1a. Preliminary data also indicate immunoreactivity for the isoform in the adrenal medulla (data not shown) in which ghrelin and the GHS-R are also present (26) and are postulated to be a part of an autocrine/paracrine axis, which stimulates adrenal cell growth (27).
Our quantitative PCR studies also support previous findings that ghrelin is expressed in whole kidney (31); however, we were unable to detect significant protein expression in glomeruli, in which mRNA expression has previously been reported (31). GHS-R 1a immunoreactivity was also evident in kidney tubules along with the exon 4-deleted proghrelin isoform, and thus, this study supports the possibility that an autocrine/paracrine axis encompassing ghrelin, exon 4-deleted proghrelin, and the GHS-R 1a is present in the mouse kidney.
There is now a growing body of research investigating the physiological influence of ghrelin and the synthetic mimics of ghrelin, the GH secretagogues (GHSs), on cardiovascular function. Ghrelin binding sites have been documented in rat peripheral vascular tissue (32). The synthetic GHS hexarelin, and to a lesser extent ghrelin, protect against ischemia-induced myocardial damage in rat hearts (33), and ghrelin improves left ventricular dysfunction in rats with induced heart failure (8). The present study is the first to show ghrelin peptide immunoreactivity in mouse blood vessel endothelium. In addition, the expression of the exon 4-deleted proghrelin isoform in this tissue indicates that it may also have a role in cardiovascular physiology.
Ghrelin and the GHS-R are coexpressed in rat adipose tissue (1), and ghrelin stimulates rat preadipocyte differentiation, the preliminary step of adipogenesis (34). We have also now demonstrated the protein expression of ghrelin in visceral fat of the mouse kidney. The novel exon 4-deleted variant is also present in these fat cells, and its role in adipogenesis is perhaps worthy of further investigation.
Ghrelin and its receptor appear to have an influential role in reproduction and development. Ghrelin is expressed in the Leydig cells of rat testis and is capable of suppressing human chorionic gonadotropin and cAMP-stimulated testosterone secretion (28). Although we detected exon 4-deleted transcript in testis cDNA by RT-PCR, we were not able to demonstrate significant protein expression of the variant in the seminiferous tubules. Exon skipping, or transcription factors that influence this, is often tissue or developmentally regulated (35) and may therefore favor full-length preproghrelin mRNA transcription in the mouse testis. Expression of the GGDT isoform in testis is developmentally regulated (21), and Barreiro et al. (36) demonstrated that the GHS-R gene is expressed under the influence of varying developmental and hormonal cues (including ghrelin).
The cyclic and pregnant rodent ovary expresses ghrelin primarily in the functional corpus lutea (29), and we have demonstrated ovarian exon 4-deleted proghrelin mRNA expression and immunoreactivity in our experiments. Thus, the novel isoform may also have endocrine roles in female reproductive tissues. We previously demonstrated the expression of full-length ghrelin and the exon 4-deleted isoform in mouse embryos at varying stages of development (24). Kawamura et al. (22) also recently reported the expression of ghrelin and a des-Gln14 truncated isoform in mouse embryos, in addition to demonstrating that mouse preimplantation embryo development is inhibited after incubation with ghrelin. It is yet to be determined whether the novel proghrelin isoform reported here has a similar influence on mouse embryo preimplantation development.
As previously described, we detected ghrelin and GHS-R 1a mRNA transcripts in mouse brain together with expression of the exon 4-deleted isoform. The localization of the exon 4-deleted isoform protein expression in the dentate gyrus of the mouse hippocampal formation is particularly interesting. GHS-R mRNA has previously been reported to be highly expressed in the dentate gyrus of the rat (37). The dentate gyrus is part of a cortical structure involved in complex neural processes such as learning, emotional response, and memory. It is interesting to speculate that the ghrelin axis may be active in memory formation and function, especially considering the importance of GH and IGF-I in memory and other cognitive processes (for review see Ref. 38).
In previous studies, we reported that the in vitro proliferation of human prostate cancer cells is enhanced by exogenous ghrelin, potentially through an autocrine/paracrine mode of action because both ghrelin and its functional receptor are expressed in these cells. Exon 3-deleted human proghrelin is also expressed in prostate cancer cells and tissues (12), and we now report the peptide expression of the equivalent mouse exon 4-deleted proghrelin in the epithelium of the mouse prostate gland. Prostate epithelial cells express and secrete a wide range of growth factors important for development and maintenance of prostate gland morphology and function. The potential role of the novel exon 4-deleted proghrelin isoform in mouse prostate biology requires further in vivo investigation.
In conclusion, the findings of this comprehensive study demonstrate that the ghrelin axis is expressed in a wide range of murine tissues, suggesting much broader, and potentially tissue specific, functional roles for this recently discovered hormone and its cognate receptor, the GHS-R. A novel mRNA variant, the exon 4-deleted proghrelin transcript, is most likely to result from alternative splicing of the preproghrelin gene, and it is translated into a unique protein in many murine tissues. Exon 4-deleted proghrelin represents a new facet in the field of ghrelin research, and studies are continuing to determine its potential biological function.
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Address all correspondence and requests for reprints to: Penny Jeffery, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane 4001, Australia. E-mail: p.jeffery@student.qut.edu.au.
Abstract
Ghrelin, an n-octanoylated 28-amino-acid peptide capable of inducing GH secretion and food intake in humans and rats, is the endogenous ligand for the GH secretagogue receptor (GHS-R). Here we describe the expression and tissue distribution of the ghrelin/GHS-R axis in the mouse. We also report for the first time the identification of a novel mouse ghrelin mRNA variant in which there is a complete deletion of exon 4. Translation of this variant mRNA yields a protein containing ghrelin and an alternative C-terminal domain with a unique C-terminal peptide sequence. RT-PCR with primers specific for mouse ghrelin was used to demonstrate the mRNA expression of the full preproghrelin transcript and the exon 4-deleted variant in multiple mouse tissues. Real-time PCR was also employed to quantitate mRNA expression of ghrelin, the novel isoform and a previously reported ghrelin gene variant, ghrelin gene-derived transcript. We also demonstrated the tissue expression of the functional GHS-R in the mouse. Immunohistochemistry, employing antibodies raised against the mature human n-octanoylated ghrelin peptide and the putative C-terminal peptide encoded by the exon 4-deleted proghrelin variant, was used to demonstrate protein expression of ghrelin and the variant in multiple mouse tissues including stomach, kidney, and reproductive tissues. The coexpression of ghrelin and its receptor in a wide range of murine tissues suggests varied autocrine/paracrine roles for these peptides. Exon 4-deleted proghrelin, a novel mouse proghrelin isoform with a unique C-terminal peptide sequence, is also widely expressed in the mouse and thus may possess biological activity in these tissues.
Introduction
GHRELIN, A RECENTLY DISCOVERED 28-amino-acid GH-releasing peptide originally isolated from rat stomach (1), is now recognized to have a wide range of physiological roles. The in vitro and in vivo GH secretory activity of ghrelin is dependent on a unique n-octanoylation at Ser-3 (1) and is mediated by its endogenous receptor, the GH secretagogue (GHS) receptor (GHS-R), a G protein-coupled receptor (2). Two GHS-R isoforms have been identified. Type 1a is the full-length, seven-transmembrane domain functional receptor, and the type 1b isoform is a C-terminally truncated, five-transmembrane domain variant (3). The 1b isoform is thought to be nonfunctional. GHS-R signaling in the pituitary triggers a transient increase in intracellular calcium levels that results in GH release (1).
Although ghrelin is produced and expressed predominantly in the endocrine cells of the stomach (4), coexpression of ghrelin and the GHS-R 1a has been widely observed throughout rat tissues, suggesting that the ghrelin/GHS-R axis possesses diverse physiological functions. In addition to GH release, ghrelin stimulates food intake and adiposity in rats (5, 6), and the administration of ghrelin improves cardiac function in humans (7) and rats with experimentally induced chronic heart failure (8). Ghrelin is expressed in pancreatic islets, although conflicting results have been reported regarding its influence on insulin secretion (9, 10, 11). Ghrelin and the GHS-R may also have important roles in cell proliferation and the progression of hormone-dependent cancer (for review see Ref. 12).
Ghrelin has been isolated from several animal species. Human and rat ghrelin differ by only two amino acids (1), and there is a high degree of homology for the first four amino acids (GSSF) of the mature peptide across species, including the human and rat (1), mouse (13), fish (14, 15), and chicken (16). These four amino acids comprise the minimum active core of ghrelin and dictate its ability to activate the GHS-R in vitro (17). However, the remaining sequence of ghrelin may be biologically essential in vivo because truncated synthetic ghrelin molecules fail to stimulate GH secretion in rats (18).
In the mouse, preproghrelin is encoded by the ghrelin gene, which consists of four coding exons and a 19-bp noncoding first exon (13). The mature ghrelin peptide, which is cleaved from the proghrelin product, is encoded by parts of exons 2 and 3 (13). A number of ghrelin isoforms have recently been identified in rats. Des-octanoylated ghrelin lacks the Ser-3 octanoic acid modification (19) and therefore cannot activate GHS-R-expressing cells to stimulate GH secretion. Alternative splicing of the ghrelin gene is thought to produce des-Gln14-ghrelin, which is identical with ghrelin except for the loss of one glutamine residue (Gln14) and induces a similar profile of GH release in rats as does ghrelin (20).
The ghrelin gene-derived transcript (GGDT) is a murine proghrelin mRNA variant encoded by exons 1, 4, and 5 and therefore does not encode mature ghrelin. GGDT displays temporally regulated expression in mouse testis tissue and may play a role in reproduction (21). It is not known, however, whether this transcript is translated in vivo because the predicted protein sequence does not contain a signal peptide. Very recently a truncated des-Gln14-ghrelin isoform has been reported in the mouse (22).
In this paper, we report the identification and characterization of a novel mouse proghrelin isoform in which there is a complete deletion of exon 4, potentially generated by alternative splicing of the mouse ghrelin gene. We previously reported the identification of the human counterpart of this isoform in prostate cancer cell lines (23) and presented preliminary data demonstrating the expression of the mouse isoform, along with full-length preproghrelin, in mouse embryos (24). The expression of this variant has now been examined in a wide range of murine tissues at the protein and mRNA levels. Expression of other components of the ghrelin/GHS-R axis was also examined at the mRNA and protein level in these tissues.
Materials and Methods
Mice
Quackenbush Swiss mice were obtained from the Central Animal Breeding House (University of Queensland, Brisbane, Australia) and killed by cervical dislocation in accordance with Queensland University of Technology (QUT) animal ethics guidelines (QUT approval number 187/4a). Heart, liver, kidney, testis, ovary, spleen, lung, muscle, adrenal gland, seminal vesicles, brain, fat, and gastrointestinal tissues were dissected and stored in RNA later (Ambion, Austin, TX) or frozen in liquid nitrogen and stored at –80 C. RNA was extracted from the tissues using the Trizol method (Life Technologies, Rockville, MD). Tissues were also collected for immunohistochemistry and Western analysis.
Reverse transcriptase (RT) of extracted tissue RNA
The first-strand cDNA synthesis kit for RT-PCR [avian myeloma virus (AMV)] (Invitrogen, Carlsbad, CA) was used to generate cDNA from RNA extracted from mouse tissues. Total RNA from each tissue (1 μg) was incubated in a 20-μl reagent solution containing 1x reaction buffer, 5 mM MgCl2, 1 mM deoxynucleotide triphosphate mix, 3.2 μg random hexamers, 50 U RNase inhibitor, and 20 U AMV RT for 10 min at 25 C and then for 60 min at 42 C. The reaction was treated at 99 C for 5 min and 4 C for 5 min to denature the AMV RT. PCR using intron-spanning mouse ?-actin primers (Table 1) demonstrated that cDNA was present and devoid of genomic DNA contamination.
TABLE 1. RT-PCR primer sequences, exon positioning, annealing temperatures (Ta) and expected amplicon sizes
Detection of full-length ghrelin, exon 4-deleted proghrelin, and GHS-R mRNA expression in mouse tissues with RT-PCR
Transcripts for full-length mouse ghrelin (GenBank accession no. AB035701), exon 4-deleted proghrelin, and GHS-R 1a (AY056474) were detected with RT-PCR using the primers and PCR conditions listed in Table 1. Discrete expression of exon 4-deleted proghrelin mRNA was confirmed using a specific sense primer that spans the putative exon 3/5 boundary (Table 1).
PCRs contained 10x PCR buffer, 100 μM deoxynucleotide triphosphates, 1.5 mM MgCl2, 100 pM primers (Proligo, Armidale, Australia), 2 μl cDNA or water (no template-negative control), and 1 U Red Hot polymerase (Integrated Sciences, Melbourne, Australia) or 1 U platinum Taq polymerase (Roche, Stockholm, Sweden). Thermal cycling consisted of 5 min at 95 C followed by 40 cycles of 30 sec at 95 C, 30 sec at annealing temperature (Table 1), 1 min at 72 C, followed by a final 10-min incubation at 72 C on a PTC-200 thermal cycler (MJ Research, Watertown, MA). Southern analysis was performed on blotted RT-PCR products as previously described (23) using a digoxigenin-labeled oligonucleotide probe and chemiluminescent detection (Roche).
Sequencing
The RT-PCR products were purified from an agarose gel using the High Pure gel purification kit (Eppendorf, Hamburg, Germany). Sequencing was performed at the Australian Genome Research Facility (University of Queensland, Brisbane, Australia) using the 377 DN automated DNA sequencer (Applied Biosystems, Warrington, UK) and ABI Big Dye version 2 terminator reagents (Applied Biosystems).
Relative quantitation of full-length ghrelin, exon 4-deleted proghrelin, and GGDT mRNA transcripts with real-time PCR
Real-time quantitative PCR was performed using the ABI 7000 prism sequence detection system (Applied Biosystems). Intron-spanning primers were designed using Primer Express software (version 2.0, PE Applied Biosystems) and were synthesized by Proligo (Table 2). Validation experiments were performed to ensure that the amplification efficiencies of both the target and reference primer sets were equal (user bulletin 2, ABI PRISM 7000 sequence detection system; Applied Biosystems). Serial dilutions of cDNA for full-length ghrelin, exon 4-deleted proghrelin, and GGDT were used in the data validation experiments. All quantitative PCRs were performed on 5 μl cDNA in a total volume of 20 μl. Each reaction comprised 100 pmol relevant primers and 10 μl 2x SYBR green I master mix (Applied Biosystems). Quantification of ghrelin, exon 4-deleted proghrelin, and GGDT transcripts in the tissue samples was achieved by the comparative CT method (user bulletin 2, ABI PRISM 7000 sequence detection system). All samples were run in triplicate and normalized against ribosomal 18S RNA. Data are represented as fold change (with confidence intervals), compared with brain tissue (calibrator tissue).
TABLE 2. Quantitative light cycler PCR primer sequences
Antibodies and Western analysis
Polyclonal primary antibodies were raised in rabbits (Institute for Medical and Veterinary Sciences, Adelaide, Australia) against the human n-octanoylated ghrelin peptide, GSSFLSPEHQRVQQRKESKKPPAKLQPR (Mimotopes, Victoria, Australia), and the novel C-terminal peptide encoded by the exon 4-deleted proghrelin variant, RRQLTSNHGQA (Mimotopes). The antibodies were affinity purified (Mimotopes) and concentrated by ultracentrifugation with Amicon centrifugal filter units (Millipore, Watertown, MA).
Western blot analysis was undertaken to detect the expression of ghrelin, preproghrelin, proghrelin, and the exon 4-deleted proghrelin peptide in mouse stomach tissue and confirm antibody specificity. Tissue protein was extracted in buffer containing 1% Triton X-100 and a protease inhibitor cocktail tablet without EDTA (Roche). Protein (20 μg) was boiled for 5 min in 4x loading buffer [250 mM Tris-Cl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 20 mM dithiothreitol, and 0.01% bromophenol blue] and electrophoresed on 10% SDS-PAGE gels against a protein standard (Multimark, Invitrogen) and synthetic exon 4-deleted proghrelin peptide or n-octanoylated ghrelin peptide (0.1 μg) (Mimotopes). Protein was transferred to a Protran nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) for 2 h at 100 V in transfer buffer (10 mM NaHCO3, 3 mM Na2CO3, methanol) and the membrane blocked overnight at 4 C in Tris-buffered saline/0.05% Tween 20/1% BSA. The membrane was incubated with anti-exon 4-deleted proghrelin peptide primary antibody solution (1/1000 to 1/2000) or anti-ghrelin antibody (1/40 to 1/800) at room temperature for 1 h. After washing in Tris-buffered saline/Tween20, the membrane was incubated with an antirabbit secondary antibody (1/100,000) (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. Femto chemiluminescent substrate solution (Pierce, Rockford, IL) was layered onto the membrane and incubated for 5 min. The membrane was then exposed to x-ray film and developed using a Curix 60 automatic processor (Agfa-Gevaert, Morstel, Belgium).
Immunohistochemistry
Detection and localization of ghrelin, the exon 4-deleted proghrelin variant, and GHS-R 1a protein was performed using immunohistochemistry on mouse tissue specimens. Antibodies cross-reactive with the putative murine GHS-R 1b isoform are unavailable at this stage. Tissues were dissected and fixed in 4% paraformaldehyde solution for 4–6 h. The tissues were then embedded in paraffin wax, and 7-μm sections were cut on a microtome. Tissue sections were dewaxed, dehydrated, and then microwaved in 0.01 M citric acid buffer (pH 6) to facilitate antigen retrieval. Sections were incubated for 1 h with primary antibody diluted in 0.01 M PBS/1% BSA. Ghrelin antibody was diluted 1/25 to 1/100 and anti-exon 4-deleted proghrelin peptide antibody from 1/1000 to 1/3000. GHS-R 1a antibody was diluted 1/200 as described previously (25). Immunodetection was performed using the Envision plus diaminobenzamine antirabbit immunodetection kit (Dako, Kyoto, Japan) according to the manufacturer’s instructions. Negative controls included the abolition of staining by preabsorbing the primary antibody overnight at 4 C with ghrelin or the exon 4-deleted proghrelin peptide (1 mg/ml), omission of primary antibody, or the application of preimmune sera. Tissues were counterstained with hematoxylin (Sigma, St. Louis, MO).
Results
Expression of full-length preproghrelin and a novel exon 4-deleted proghrelin mRNA isoform in mouse issues
Using primers positioned in exons 2 and 5 (Fig. 1), RT-PCR identified full-length preproghrelin transcripts of the expected size (464 bp) in all tissues examined including heart, brain, and the positive control tissue, stomach (Fig. 2A). DNA sequencing confirmed the identity of this transcript. In addition, a smaller PCR product of 355 bp was also generated in all of the tissues examined (Fig. 2A). Southern analysis using an internal oligonucleotide probe for mouse ghrelin gave a positive signal, indicating that this transcript had homology with ghrelin (data not shown). DNA sequencing identified this transcript as a novel mouse proghrelin isoform with a complete lack of exon 4. Thus, alternative splicing of the mouse preproghrelin pre-mRNA can potentially produce the exon 4-deleted isoform as well as the expected full-length transcript (Fig. 1). The novel nucleotide sequence of the exon 4-deleted variant has been deposited with GenBank (accession no. AY179430).
FIG. 1. Mouse preproghrelin pre-mRNA (i) can be processed to give full-length ghrelin mRNA (ii) or splicing of ghrelin pre-mRNA (shown here with exons numbered and introns represented by plain lines) can result in the skipping of exon 4, thus generating exon 4-deleted proghrelin (iii). Positioning of the sense and antisense primers used in the RT-PCRs is indicated by arrows. The exon 4-deleted proghrelin transcript-specific sense PCR primer (dashed arrow) spans the unique exon 3/5 junction created by the lack of exon 4 and under stringent conditions detects only this variant.
FIG. 2. A, RT-PCR products showing the expression of full-length preproghrelin and exon 4-deleted proghrelin mRNA in mouse tissues derived using primers located in exon 2 (sense) and exon 5 (antisense). The upper band (464 bp) seen in all tissues corresponds to mRNA for the full-length form of ghrelin, and the lower band (355 bp) was identified through Southern analysis and sequencing as a proghrelin mRNA isoform that lacks exon 4. B, Exon 4-deleted proghrelin mRNA potentially results from the alternative splicing of mouse preproghrelin pre-mRNA. Ethidium bromide stained agarose gel of RT-PCR products using primers designed specifically to detect this isoform. Exon 4-deleted proghrelin was amplified in all mouse tissues with RT-PCR employing a sense exon 3/5 primer specific for the exon 4-deleted transcript and the exon 5 antisense primer. C, RT-PCR amplification of the housekeeping ?-actin mRNA in all mouse tissues shows a single band at 243 bp, indicating that cDNA is devoid of genomic DNA contamination.
Based on the sequencing data for the exon 4-deleted proghrelin transcript, a forward primer that spans the unique exon 3/5 boundary created by the deletion of exon 4 in this variant was designed to establish a PCR protocol for the specific amplification of this variant only (Fig. 1). Using this primer together with the exon 5 reverse primer used for the full-length PCR (Table 1), a specific band of the expected size for exon 4-deleted proghrelin (131 bp) was observed in all of the mouse tissues examined (Fig. 2B). Sequencing confirmed the transcript as being an exon 4-deleted isoform of preproghrelin. The internal housekeeping gene ?-actin was amplified in all tissues examined (Fig. 2C) and confirmed the integrity of the cDNA.
GHS-R 1a transcript expression in mouse tissues
GHS-R 1a mRNA transcripts were amplified in various mouse tissues including the adrenal gland, brain, kidney, stomach, heart, lung, and pancreas. These cDNA products were confirmed by Southern analysis using an internal probe to increase the specificity of the assay (Fig. 3) and by a 100% sequence homology with the published mouse GHS-R 1a nucleotide sequence (accession no. NM-177330).
FIG. 3. GHS-R 1a isoform expression in mouse tissues detected by Southern blot analysis after RT-PCR using intron-spanning primers. PCR products were detected using an internal digoxigenin end-tailed oligonucleotide antisense probe and chemiluminescent detection (Roche).
Relative quantitation of full-length ghrelin, exon 4-deleted proghrelin, and GGDT transcripts in mouse tissues with real-time PCR
As expected, ghrelin gene expression was found to predominate in mouse stomach tissue as indicated in Fig. 4A. Pancreas, muscle, ovary, kidney, and fat express low levels of full-length preproghrelin mRNA with negligible expression in adrenal gland and liver (data not shown). Stomach was also the primary site of exon 4-deleted proghrelin mRNA expression with markedly lower levels in brain, muscle, ovary, and kidney (Fig. 4B). Negligible amounts of transcript were detected in pancreas, fat, adrenal gland, and liver (data not shown). In agreement with Tanaka et al. (21), GGDT transcript was found to be expressed predominantly in mouse testis tissue (data not shown), even with the use of a more sensitive detection method in this present study.
FIG. 4. Quantitative RT-PCR analysis of ghrelin (A) and the novel exon 4-deleted proghrelin isoform (B) mRNA expression in various mouse tissues. Data are represented as fold change relative to expression of transcripts in brain (1.0) with 95% confidence intervals. The full-length and exon 4-deleted isoforms are predominantly expressed in the stomach. All other tissues tested express low levels of ghrelin mRNA transcript. Exon 4-deleted proghrelin mRNA was present at low levels in the brain, muscle, ovary, and kidney with expression in the fat, pancreas, and adrenal tissues undetectable in this assay.
The absence of exon 4 results in a novel truncated C-terminal peptide
The exclusion of exon 4 from mouse preproghrelin mRNA results in a frameshift that, when translated, encodes a novel C-terminal peptide (RRQLTSNHGQA) while still coding for the mature ghrelin sequence (Fig. 5A). The generation of a dibasic pair of amino acids (RR) at the N terminus of the unique peptide sequence is particularly interesting, raising the possibility that enzymatic cleavage of this peptide may occur. After taking into account conservative amino acid changes, the C-terminal peptide sequence of the mouse exon 4-deleted isoform shares significant homology with the human (exon 3-deleted) homolog (Fig. 5B).
FIG. 5. cDNA and corresponding amino acid sequence (AY179430) for the exon 4-deleted proghrelin transcript with bases numbered on the left and amino acids on the right (A). The mature ghrelin peptide is underlined and the potential n-octanoylation site indicated by solid triangle. The unique C-terminal peptide sequence created by deletion of exon 4 is in italic font with the tribasic motif (RRR) in bold italic. Homology between human exon 3-deleted proghrelin C-terminal peptide and the mouse exon 4-deleted proghrelin peptide is illustrated (B). Exact amino acid matches are highlighted.
Western blot detection and size estimation of full-length ghrelin, exon 4-deleted proghrelin, and their cleavage products in mouse stomach protein extract
Western analysis of mouse stomach tissue extracts using the antighrelin antibody detected three distinct bands at 13, 11, and 3 kDa, respectively (Fig. 6A). These correspond to the predicted sizes of the potential preproghrelin cleavage products: preproghrelin itself, proghrelin, and mature ghrelin (Fig. 6B). The exon 4-deleted equivalents of the preproghrelin cleavage products (10 and 9kDa) were also detected in mouse stomach using the anti-exon 4-deleted proghrelin antibody (Fig. 6A); however, a cleaved form of the unique C-terminal peptide was not detected at the predicted approximate size of 1 kDa (Fig. 6B). No other bands were detected for either antibody, confirming antibody specificity.
FIG. 6. A, Western blot analysis of mouse stomach protein extracts detected with either an anti-ghrelin (i) or anti-exon 4-deleted proghrelin (ii) antiserum. Full-length preproghrelin (13 kDa), proghrelin (11 kDa), and the mature ghrelin peptide (3 kDa) were detected in mouse stomach tissue using the ghrelin antiserum, but only bands corresponding to the predicted sizes of exon 4-deleted preproghrelin (10 kDa) and proghrelin (9 kDa) were detected in this tissue using the exon 4-deleted proghrelin antiserum. B, Predicted cleavage products and approximate sizes (kilodaltons) of full-length and exon 4-deleted preproghrelin. The antighrelin antibody can be predicted to detect three peptides: preproghrelin, proghrelin, and the mature ghrelin peptide. The anti-exon 4-deleted proghrelin peptide antibody will not detect mature ghrelin but should detect truncated preproghrelin, proghrelin, and the putatively cleaved unique C-terminal peptide.
Immunohistochemical localization of ghrelin, GHS-R 1a, and exon 4-deleted proghrelin peptide expression in mouse histological specimens
As expected, ghrelin protein is strongly expressed in the glands of the mouse stomach as indicated by intense ghrelin immunostaining (Fig. 7A). Expression is also evident in the pancreatic islets (Fig. 7B) and blood vessel endothelium and visceral fat of the kidney (Fig. 7C). The proximal and distal kidney tubules also stain positively for ghrelin with negligible staining in the glomeruli (Fig. 7D). Skeletal muscle tissue also expresses ghrelin at the protein level (Fig. 7E). Preabsorption of the antibody with synthetic n-octanoylated ghrelin completely inhibits immunostaining in all of the tissues tested (stomach shown here, Fig. 7F) again confirming the specificity of the antighrelin antibody. GHS-R 1a protein expression is also present in mouse stomach glands (Fig. 7G) and kidney tubules with faint immunoreactivity apparent in the kidney glomeruli (Fig. 7H). No staining was evident in the kidney tissue when the primary antibody step was omitted (Fig. 7I).
FIG. 7. Immunohistochemistry on mouse tissue specimens to detect expression of the ghrelin/GHS-R axis. A, Ghrelin immunostaining is present in stomach glandular tissue, the positive control tissue. B, Mouse pancreatic islets (PI), blood vessel endothelium (BV) (C), and visceral fat cells (F) of the kidney are immunoreactive for full-length ghrelin peptide. D, The proximal and distal tubules of the kidney also stain for ghrelin, whereas glomeruli (arrow) are nonimmunoreactive. E, Ghrelin is expressed in skeletal muscle fibers. F, Preabsorption of the ghrelin antibody with synthetic ghrelin peptide abolishes immunostaining (shown in stomach). GHS-R 1a is expressed in the stomach glands (G) and kidney tubules (H) with negligible staining in the glomeruli. I, Preabsorbed control did not show staining (shown here in kidney). Scale bars, 100 μm (A); 50 μm (B–I).
The exon 4-deleted proghrelin C-terminal peptide is highly expressed at the protein level in stomach glandular tissue with intense immunoreactivity noted in some cells (Fig. 8A). Negative controls, consisting of primary antibody omission and preabsorption of the antibody with the C-terminal peptide, were devoid of any immunoreactivity (representative preabsorption control, Fig. 8B). Corpus lutea of the mouse ovary stain strongly for the peptide (Fig. 8C). Faint staining was noted in blood vessel endothelium of the kidney (Fig. 8D), with negligible expression of the isoform noted in the seminiferous tubules of the testis (Fig. 8E) and in cardiac and skeletal muscle fibers (Fig. 8, F and G). Intense immunoreactivity was detected in mouse brain tissue in the dentate gyrus of the hippocampal formation (Fig. 8H). Kidney tubules and glomeruli also express the C-terminal peptide of exon 4-deleted proghrelin (Fig. 8I) as do the epithelial cells of mouse prostate gland (Fig. 8J).
FIG. 8. Immunohistochemistry on mouse tissue specimens for the novel exon 4-deleted proghrelin isoform. A, Exon 4-deleted proghrelin is expressed highly in stomach glands. B, Preabsorption of the antibody with the C-terminal peptide results in negative staining in the stomach. C, Corpus lutea (CL) of the mouse ovary show strong immunoreactivity for the peptide as does blood vessel endothelium (D, arrow). Seminiferous tubules (ST) of the testis (E) and cardiac (F) and skeletal muscle fibers (G) demonstrate weak staining. H, Strong immunoreactivity is evident in the dentate gyrus region of the mouse hippocampal formation. Inset, Preabsorbed control demonstrates no staining in this tissue. I, The kidney tubules express the isoform as do glandular epithelial cells (J) of the mouse prostate. Scale bar, 50 μm (C, D, F, G, H, and I); 100 μm (A, B, and J); and 150 μm (E).
Discussion
We report for the first time the expression of both mouse ghrelin and its receptor and a novel mouse proghrelin isoform designated exon 4-deleted proghrelin. This variant, along with full-length ghrelin, is expressed at the mRNA and protein level in all of the mouse tissues examined, and in particular the stomach and brain. The exon 4-deleted proghrelin protein results from the translation of an mRNA transcript that completely lacks exon 4 (which is equivalent to human exon 3). We previously identified a human homolog of this isoform, exon 3-deleted proghrelin, which is expressed in a wide range of human tissues including the prostate (23).
By performing Southern analysis under low-stringency conditions, Hosoda et al. (20) determined that ghrelin and des-gln14-ghrelin are likely to be products of a single ghrelin gene. Therefore, it is probable that the exon 4-deleted variant is also a product of this gene, produced by alternative splicing mechanisms such as exon skipping. Alternative splicing contributes significantly to protein diversity in the human and mouse, giving rise to the production of multiple, often functionally diverse proteins from a single gene. The exclusion of exon 4 from mouse preproghrelin mRNA transcript results in a cDNA frameshift and the generation of a premature stop codon. When translated, this sequence encodes a novel peptide sequence at the C terminal of proghrelin while still retaining the coding sequence for mature ghrelin. Thus, translation of the exon 4-deleted proghrelin isoform is likely to lead to the production of n-octanoylated (active) ghrelin.
We also predict that the unique C-terminal tail of the exon 4-deleted proghrelin protein is cleaved in vivo at an arginine-rich junction introduced between the 11-amino-acid C-terminal peptide and the rest of the propeptide (Fig. 5A). Alternatively, if not cleaved and secreted in vivo, this unique C-terminal tail may influence the properties and/or processing of the entire exon 4-deleted proghrelin variant.
Pemberton et al. (25) recently demonstrated the presence of full-length proghrelin C-terminal peptides in human plasma that circulate at higher concentrations than mature octanoylated ghrelin and are elevated in heart failure. This study suggests that proghrelin C-peptides may possess as-yet-unknown physiological functions and supports our hypothesis that the generation of the unique C-peptide from the exon 4-deleted variant of proghrelin may also have physiological significance.
The antibody used for the immunohistochemical studies in this paper does not distinguish between cleaved and noncleaved exon 4-deleted proghrelin; therefore, this study could not determine whether mouse tissues express intact or C-terminally cleaved exon 4-deleted proghrelin or both. Although we were not able to detect the cleaved 11-amino-acid C-terminal peptide by Western blot analysis, the relatively high degree of conservation of this sequence with the unique C-terminal peptide of the human exon 3-deleted proghrelin (12) implies functional relevance. Biological assays to test for peptide function are currently being undertaken.
Previous studies documented that ghrelin and the type 1a GHS-R are widely coexpressed in rat tissues including the heart, pancreas, adipose tissue (1), adrenal gland (26, 27), and reproductive tissues such as the testis (28) and ovary (29). This coexpression suggests autocrine/paracrine functions for ghrelin and its receptor in these tissues. To our knowledge, this study is the first to quantitate, at the mRNA level, the expression of ghrelin and the novel exon 4-deleted isoform in an extensive range of murine tissues. To date, GHS-R 1b expression has been identified only in the human and domestic pig (2) and recently in black seabream hypothalamus (30). Using primers based on a Mus musculus chromosome 3 genomic contig, we were able to identify PCR products that represented fragments of murine GHS-R 1b cDNA (data not shown); however, further sequencing and proteomic analysis is required before this sequence can be fully reported.
Exon 4-deleted proghrelin mRNA and protein are expressed at the highest levels in mouse stomach glandular tissue, which reflects the expression pattern of ghrelin. GHS-R 1a expression was also demonstrated in this tissue. Further experiments are required to determine whether the unique C-terminal peptide of the exon 4-deleted proghrelin isoform influences the orexigenic properties of ghrelin and its functional receptor, GHS-R 1a. Preliminary data also indicate immunoreactivity for the isoform in the adrenal medulla (data not shown) in which ghrelin and the GHS-R are also present (26) and are postulated to be a part of an autocrine/paracrine axis, which stimulates adrenal cell growth (27).
Our quantitative PCR studies also support previous findings that ghrelin is expressed in whole kidney (31); however, we were unable to detect significant protein expression in glomeruli, in which mRNA expression has previously been reported (31). GHS-R 1a immunoreactivity was also evident in kidney tubules along with the exon 4-deleted proghrelin isoform, and thus, this study supports the possibility that an autocrine/paracrine axis encompassing ghrelin, exon 4-deleted proghrelin, and the GHS-R 1a is present in the mouse kidney.
There is now a growing body of research investigating the physiological influence of ghrelin and the synthetic mimics of ghrelin, the GH secretagogues (GHSs), on cardiovascular function. Ghrelin binding sites have been documented in rat peripheral vascular tissue (32). The synthetic GHS hexarelin, and to a lesser extent ghrelin, protect against ischemia-induced myocardial damage in rat hearts (33), and ghrelin improves left ventricular dysfunction in rats with induced heart failure (8). The present study is the first to show ghrelin peptide immunoreactivity in mouse blood vessel endothelium. In addition, the expression of the exon 4-deleted proghrelin isoform in this tissue indicates that it may also have a role in cardiovascular physiology.
Ghrelin and the GHS-R are coexpressed in rat adipose tissue (1), and ghrelin stimulates rat preadipocyte differentiation, the preliminary step of adipogenesis (34). We have also now demonstrated the protein expression of ghrelin in visceral fat of the mouse kidney. The novel exon 4-deleted variant is also present in these fat cells, and its role in adipogenesis is perhaps worthy of further investigation.
Ghrelin and its receptor appear to have an influential role in reproduction and development. Ghrelin is expressed in the Leydig cells of rat testis and is capable of suppressing human chorionic gonadotropin and cAMP-stimulated testosterone secretion (28). Although we detected exon 4-deleted transcript in testis cDNA by RT-PCR, we were not able to demonstrate significant protein expression of the variant in the seminiferous tubules. Exon skipping, or transcription factors that influence this, is often tissue or developmentally regulated (35) and may therefore favor full-length preproghrelin mRNA transcription in the mouse testis. Expression of the GGDT isoform in testis is developmentally regulated (21), and Barreiro et al. (36) demonstrated that the GHS-R gene is expressed under the influence of varying developmental and hormonal cues (including ghrelin).
The cyclic and pregnant rodent ovary expresses ghrelin primarily in the functional corpus lutea (29), and we have demonstrated ovarian exon 4-deleted proghrelin mRNA expression and immunoreactivity in our experiments. Thus, the novel isoform may also have endocrine roles in female reproductive tissues. We previously demonstrated the expression of full-length ghrelin and the exon 4-deleted isoform in mouse embryos at varying stages of development (24). Kawamura et al. (22) also recently reported the expression of ghrelin and a des-Gln14 truncated isoform in mouse embryos, in addition to demonstrating that mouse preimplantation embryo development is inhibited after incubation with ghrelin. It is yet to be determined whether the novel proghrelin isoform reported here has a similar influence on mouse embryo preimplantation development.
As previously described, we detected ghrelin and GHS-R 1a mRNA transcripts in mouse brain together with expression of the exon 4-deleted isoform. The localization of the exon 4-deleted isoform protein expression in the dentate gyrus of the mouse hippocampal formation is particularly interesting. GHS-R mRNA has previously been reported to be highly expressed in the dentate gyrus of the rat (37). The dentate gyrus is part of a cortical structure involved in complex neural processes such as learning, emotional response, and memory. It is interesting to speculate that the ghrelin axis may be active in memory formation and function, especially considering the importance of GH and IGF-I in memory and other cognitive processes (for review see Ref. 38).
In previous studies, we reported that the in vitro proliferation of human prostate cancer cells is enhanced by exogenous ghrelin, potentially through an autocrine/paracrine mode of action because both ghrelin and its functional receptor are expressed in these cells. Exon 3-deleted human proghrelin is also expressed in prostate cancer cells and tissues (12), and we now report the peptide expression of the equivalent mouse exon 4-deleted proghrelin in the epithelium of the mouse prostate gland. Prostate epithelial cells express and secrete a wide range of growth factors important for development and maintenance of prostate gland morphology and function. The potential role of the novel exon 4-deleted proghrelin isoform in mouse prostate biology requires further in vivo investigation.
In conclusion, the findings of this comprehensive study demonstrate that the ghrelin axis is expressed in a wide range of murine tissues, suggesting much broader, and potentially tissue specific, functional roles for this recently discovered hormone and its cognate receptor, the GHS-R. A novel mRNA variant, the exon 4-deleted proghrelin transcript, is most likely to result from alternative splicing of the preproghrelin gene, and it is translated into a unique protein in many murine tissues. Exon 4-deleted proghrelin represents a new facet in the field of ghrelin research, and studies are continuing to determine its potential biological function.
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