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Endocrinology Vol. 145, No. 9 4144-4153
Copyright © 2004 by The Endocrine Society

Genomic Structure and Characterization of the 5'-Flanking Region of the Human Ghrelin Gene

Naotetsu Kanamoto, Takashi Akamizu, Tetsuya Tagami, Yuji Hataya, Kenji Moriyama, Kazuhiko Takaya, Hiroshi Hosoda, Masayasu Kojima, Kenji Kangawa and Kazuwa Nakao

Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine (N.K., T.A., Y.H., K.M., K.N.), Kyoto 606-8507, Japan; Ghrelin Research Project, Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto University School of Medicine (T.A., K.T., H.H., K.K.), Kyoto 606-8507, Japan; Clinical Research Institute, Center for Endocrine and Metabolic Diseases, Kyoto National Hospital (T.T.), Kyoto 612-8555, Japan; Department of Biochemistry, National Cardiovascular Center Research Institute (H.H., M.K., K.K.), Osaka 565-8565, Japan; and Institute of Life Science, Kurume University (M.K.), Fukuoka 839-0861, Japan

Address all correspondence and requests for reprints to: Dr. Takashi Akamizu, Ghrelin Research Project, Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto University School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: akataka{at}kuhp.kyoto-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ghrelin, an endogenous ligand for the GH secretagogue receptor, induces GH secretion, food intake, and positive energy balance. Although ghrelin exhibits a variety of hormonal actions, the mechanisms regulating ghrelin expression and secretion remain unclear. To understand regulation of human ghrelin gene expression, we examined the genomic structure of approximately 5,000 bp of the 5'-flanking region of the human ghrelin gene. We performed rapid amplification of cDNA ends to estimate transcriptional start sites, indicating that there are two transcriptional initiation sites within the human ghrelin gene. Both transcripts were equally expressed in the human stomach, whereas the longer transcript was mainly expressed in a human medullary thyroid carcinoma (TT) cell line. Functional analysis using promoter-reporter constructs containing the 5'-flanking region of the gene indicated that the sequence residing within the –349 to –193 region is necessary for human ghrelin promoter function in TT cells. Within this region existed several consensus sequences for a number of transactivating regulatory proteins, including an E-box site. Destruction of this site decreased to 40% of the promoter activity. The upstream region of the promoter has two additional putative E-box sites, and sitedirected mutagenesis suggested that these are also involved in promoter activation. Electrophoretic mobility shift assays demonstrated that the upstream stimulatory factor specifically bound to these E-box elements. These results suggest a potential role for upstream stimulatory factor transcription factors in the regulation of human ghrelin expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VARIOUS PEPTIDE HORMONES produced by endocrine cells of the digestive tract control important physiological functions, including growth and repair of gut epithelium, motility of the gut wall, gastric emptying, glycemia, and exocrine pancreatic secretion (1). Ghrelin, an endogenous ligand for the GH secretagogue receptor, is a 28-amino acid peptide discovered in the stomach (2). Ghrelin is produced in X/A-like cells, whose functions have yet to be clarified (3). In addition to the stomach, ghrelin is expressed in the arcuate nucleus of the hypothalamus, pituitary, pancreas, kidney, placenta, and testes (2, 4, 5, 6, 7, 8, 9). Ghrelin exerts a variety of actions, including GH secretion, food intake, vagal control of gastric function, cardiovascular effects, and control of energy balance (2, 10, 11, 12, 13, 14, 15, 16, 17, 18). Plasma ghrelin levels are elevated by fasting and suppressed after feeding. Ghrelin secretion is also suppressed by somatostatin (19, 20, 21). It remains controversial, however, whether plasma insulin or glucose affects plasma ghrelin levels (21, 22, 23) and whether ghrelin expression is altered by leptin (24, 25). Thus, the regulatory mechanisms governing ghrelin expression and secretion remain to be clarified.

We recently reported significant production of ghrelin by a human medullary thyroid carcinoma (hMTC) cell line, TT cells (26). The TT cell line is the most suitable model system developed to date for human thyroid parafollicular C cells, the origin of hMTC (27, 28, 29, 30). TT cells were found to produce a variety of hormones, some of which are products of gastrointestinal endocrine and/or neuroendocrine cells. Thus, the TT cell line is an excellent tool to study the regulation of ghrelin gene expression.

To understand the regulation of human ghrelin gene expression, we identified the transcriptional initiation site, examined the promoter activity of 5 kb in the 5'-flanking region of the human ghrelin gene, and characterized proteins binding to the regulatory elements within this region. Transcription factors of the basic/helix-loop-helix (bHLH) family specifically bind to the putative E-box within the human ghrelin promoter to participate in the regulation of human ghrelin gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The hMTC cell line, TT cells (29, 30), were cultured as described previously (26). The human hepatoma cell line, HepG2 cells, obtained from American Type Culture Collection (Manassas, VA), were cultured in DMEM (Invitrogen Life Technologies, Carlsbad, CA) with 4 mM L-glutamine and 2.1 g/liter sodium bicarbonate supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD) at 37 C in a humidified atmosphere containing 5% CO2.

RNA isolation and rapid amplification of cDNA ends (RACE)
Polyadenylated RNA was extracted from TT cells using a FastTrack 2.0 kit (Invitrogen Life Technologies). Human stomach polyadenylated RNA was purchased from BD Clontech (Palo Alto, CA). The transcriptional start of the human ghrelin gene was estimated by 5'-RACE using a 5'-Full RACE Core Set (Takara Shuzo Co., Shiga, Japan) according to the manufacturer’s instructions (31, 32). Polyadenylated RNA from TT cells was reverse transcribed, then subjected to nested PCR. PCR products were subcloned into the pGEM-T vector (Promega Corp., Madison, WI) and sequenced using a BigDye Terminator cycle sequencing kit FS and 3100 genetic analyzer (Applied Biosystems, Foster, CA).

RT-PCR
cDNA was synthesized using a First-Strand cDNA Synthesis Kit (Amersham Biosciences, Little Chalfont, UK) and 1 µg polyadenylated RNA according to the manufacturer’s instructions. Resulting cDNAs were subjected to PCR using the sense and antisense primers specified in Table 1Go. All primers were designed to recognize separate exons to eliminate the possibility of DNA contamination.


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  		TABLE 1. List of primers for RT-PCR

 
Quantitative PCR
Quantitative PCR was performed as described previously (4, 33). Primers and probes are designated in Table 2Go and Fig 3AGo. Probes were labeled with fluorescent (6-carboxy-fluorescein) and nonfluorescent quencher (minor groove binder) dyes. cDNAs (1 ng) were amplified using the following conditions: 50 C for 2 min, 95 C for 10 min, and 40 cycles of 95 C for 15 sec and 60 C for 1 min, then continuous incubation at 25 C. Quantitative PCRs were performed, recorded, and analyzed by using the an ABI7700 PRISM Sequence Detection System (Applied Biosystems).


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  		TABLE 2. List of primers and probes for quantitative PCR

 


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  		FIG. 3. Quantitative PCR analysis of the human ghrelin gene transcripts containing 5'-untranslated region. A, Schematic diagram representing the positions of primers and probes used for quantitative PCR (sequences are shown in Table 2Go). B, Ghrelin expression levels in the human stomach and TT cells. Polyadenylated RNA from the human stomach and TT cells were reverse transcribed and subjected to quantitative PCR. Ghrelin expression was normalized to glyceraldehyde-3-phosphate dehydrogenase expression in each sample. {square}, Transcript-A; {blacksquare}, Transcript-B. The data represent the mean ± SE for triplicate samples. *, Statistically significant differences (P < 0.01) measured by Student-Newman-Keuls tests.

 
Cloning of the 5'-flanking region of the human ghrelin gene
The PCR primers used were based on the sequence data of a bacterial artificial chromosome (CITB-187P1, GenBank accession no. AC008116 mapped to 3p26–25) (34): sense, 5'-GGGGAGAGAGGGTCTCCAGGCAGCTGCCTC-3' [nucleotide (nt) 47534–47563]; and antisense, 5'-AAGAATAGAAGTGGGGAAATGAAAGCATTC-3' (nt 52531–52560). Genomic sequences from human leukocytes was used as a template. PCR products were subcloned into pCR II-TOPO vector (Invitrogen Life Technologies) to obtain the 5027-bp (–5059 to –33, the translation start site was set at +1) fragment of the 5'-flanking region of the human ghrelin gene. The sequences were confirmed as described above.

Plasmid construction
To analyze the function of the 5'-flanking region of the human ghrelin gene, we generated various deletion mutants of the ghrelin promoter by PCR. The MluI and the HindIII or XhoI site overhang the oligonucleotides used as sense and antisense primers, respectively. PCR products were subcloned into the MluI-HindIII site of a reporter plasmid, pGL3-basic vector [Promega Corp.; –4110, –2109, –1509, –1109, –780, –349, and –192/–33 GHRE-luciferase (Luc) and –2109, –1509, –1109, and –780/–490 GHRE-Luc] or the MluI-XhoI site of pGL3-promoter vector (Promega) (–2109, –1509, –1109, and –780/–490 GHRE-simian virus 40 Luc), to create a fusion with the Luc gene. The correct orientation of these deletion mutant constructs was confirmed by sequencing.

Mutations were created using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer’s instruction. –1509/–33 GHRE-Luc was used as the template for PCR amplification. For mutagenesis, each sequence of E-boxes was replaced as follows: CAGGTG to tcGGTG for E1, CACCTG to tcCCTG for E2; and CATCTG to tcTCTG for E3 (the mutated bases are in lower case). Mutated construct was isolated from each reaction and verified by sequencing.

The putative transcription factor binding sites on the 5'-flanking region of the human ghrelin gene were identified by computational analysis using the DNASIS (Hitachi Software Engineering Co., Tokyo, Japan) and TFSEARCH databases (www.cbrc.jp/research/db/TFSEARCHJ.html), based on the TRANSFAC databases (35).

Transient transfection and Luc assay
TT and HepG2 cells were plated at 5–8 x 105 and 1 x 105 cells/well in 12-well tissue culture plates (Corning Inc., Corning, NY), respectively. Cells were maintained in 1 ml antibiotic-free medium for 1 d before transfection. Transient transfections were performed using Lipofectamine Plus reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. Transfections included 800 ng experimental reporter gene and 80 ng pRL-thymidine kinase (TK), which contained the cDNA encoding Renilla Luc (Promega Corp.) as an internal control for transfection efficiency. After transfection, cells were grown in antibiotic-free medium and harvested after 48 h. Luc activities were determined using the Dual-Luciferase Reporter Assay System (Promega Corp.), and luminescence was measured with a Wallac 1420 ARVOsx multilabel reader (PerkinElmer Life Sciences, Tokyo, Japan). Firefly Luc activity was normalized to Renilla Luc activities in each well. Each experiment was performed at least three times with triplicate samples.

Preparation of cell extracts and EMSA
Nuclear extracts were prepared from TT cells using a nuclear extract kit (Active Motif, Carlsbad, CA), according to the manufacturer’s instruction. EMSAs were conducted using a LightShift chemiluminescent EMSA kit (Pierce Chemical Co., Rockford, IL) with slight modifications of the original manufacturer’s instruction. The double-strand oligonucleotide probe was end labeled using a biotin 3' end DNA labeling kit (Pierce Chemical Co.). Biotinylated probe (200 fmol) was incubated with approximately 10 µg nuclear protein and 0.5 µg poly(dI-dC) in the presence or absence of competing oligonucleotide in 10x binding buffer (containing 100 mM Tris, 500 mM KCl, and 10 mM dithiothreitol, pH 7.5). After 30-min incubation at room temperature, DNA-protein complexes were separated by electrophoresis on a 6% DNA retardation gel (Invitrogen Life Technologies) at 4 C in 0.5x Tris-borate, EDTA buffer (containing 89 mM Tris-borate and 2 mM EDTA, pH 8.0). For supershift assay experiments, binding reactions were incubated for 45 min at room temperature with antibodies before the addition of labeled probes. The antibodies used in supershift assay experiments were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). After electrophoresis, samples were transferred onto nylon membranes (Amersham Biosciences) and fixed by UV irradiation. Biotinylated DNA was detected using a Fujix Lumino-image analyzer (LAS-1000, Fuji Photo Film Co., Ltd., Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of the transcription initiation site
To identify the transcriptional initiation site within the human ghrelin gene, we performed 5'-RACE using polyadenylated RNA isolated from TT cells. In TT cells, two distinct clones were obtained with a 30-bp difference in length (Fig. 1AGo). Sequencing of these PCR products indicated the presence of two different transcriptional initiation sites, one located at –80 and the other at –555 (each transcript was named Transcript-A and Transcript-B, respectively) (Fig. 1Go B). These results indicated that the short first exon, which is only 20 bp in length, is present in the noncoding region of the human ghrelin gene.



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  		FIG. 1. 5'-RACE of the human ghrelin gene. A, Polyadenylated RNA isolated from TT cells was reverse transcribed as described in Materials and Methods. The resultant cDNAs were subjected to 5'-RACE. The secondary products obtained by nested PCR using TT cell cDNAs contained two band species, as visualized by agarose gel electrophoresis (arrows). M, Molecular size marker. B, Sequence and genomic location of the 5'-flanking region of the human ghrelin gene. Bold and fine bars indicate exons and introns, respectively. As shown in (a) and (b), the human ghrelin gene has two transcriptional initiation sites (named Transcript-A and Transcript-B, respectively). The nucleotide sequences indicated are within the 5'-untranslated region.

 
The presence of these two transcripts was confirmed by RT-PCR analysis of TT cells and human stomach RNA. PCR products of the expected sizes were detected by agarose gel electrophoresis (Fig. 2BGo), suggesting that both transcripts were present in TT cells and human stomach.



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  		FIG. 2. RT-PCR analysis of the human ghrelin gene transcripts containing 5'-untranslated region. A, Schematic diagram representing the positions of primers used for RT-PCR. The sequences of the sense primers, from (1 )–(4 ), are shown in Table 1Go. B, Electrophoretic analysis of RT-PCR products using polyadenylated RNA from TT cells and the human stomach. The number in the parentheses corresponds to the sense primer used in the reaction. Glyceraldehyde-3-phosphate dehydrogenase (G) was used as a positive control for PCRs.

 
Tissue specificity of transcription in the human ghrelin gene
To examine how these two transcripts were expressed in different tissues, we performed quantitative PCR. In the human stomach, both transcripts were equally expressed, whereas in TT cells Transcript-B was expressed at much lower levels (Fig. 3BGo). These results suggest tissue specificity in ghrelin gene expression. The finding that the total amount of transcripts isolated from the human stomach was greater than that isolated from TT cells was compatible with previous reports of ghrelin contents estimated by Northern blotting or RIA (3, 26, 36).

Genomic analysis of the 5'-flanking region of the human ghrelin gene
The 5059-bp fragment contained a partial sequence of the 5'-flanking region of the human ghrelin gene (Fig. 4Go). No typical GC or CAAT box was identified. A TATA box-like sequence, a TATATAA element, was identified 24 bp upstream (–585 to –579) of the transcriptional initiation site of Transcript-B. A comparison of the 5'-flanking sequence of human ghrelin with the initiator consensus sequence Py-Py-A-N-A/T-Py-Py identified putative initiator elements located at positions –557 to –551 and –82 to –76, in agreement with the transcriptional initiation sites estimated by 5'-RACE.



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  		FIG. 4. The nt sequences of the 5'-flanking region of the human ghrelin gene. The translational start site was set at +1. The 5' ends of the 5'-RACE products are denoted by asterisks. The sequences shown in bold letters indicate Transcript-A. The sequences enclosed by an open box indicate Transcript-B. Dotted-lined sequences indicate TATA box-like or initiator-like sequences. Underlined sequences indicate putative binding sites for transcription factors.

 
Functional analysis of the 5'-flanking region of the human ghrelin gene
To identify the regulatory regions important for expression of the human ghrelin gene, a series of 5' and/or 3' deletion constructs of the promoter were subcloned into the pGL3-Basic vector, then cotransfected with pRL-TK into TT cells (Fig. 5AGo). Figure 5BGo exhibits the effects of these deletions on Luc reporter activity in TT cells. Luc activity increased by deleting the region from –4110 to –1509 and decreased after deletion of –1509 to –1109. Although Luc activity was restored by additional deletion of –780 to –349, it was markedly decreased by deletion of –349 to –192. To elucidate the significance of the transcription factor binding sites between –193 and –349, the 3' truncations (to –490 bp) of some of the 5' deletion series were also created. As shown in the middle part of the Fig. 5BGo, all promoter activity was lost. However, because this deletion also removes the alternative transcription start site at –80 bp (i.e. Transcript-A), and essentially all the ghrelin transcripts in the TT cells used in the transfection assay originate here (Fig. 3BGo), we next examined the promoter activity using heterologous promoter. A series of 3' deletion constructs of the promoter were subcloned into the pGL3-promoter vector (in the bottom part of Fig. 5AGo), then cotransfected into TT cells. The Luc activity was restored by the simian virus 40 promoter and the pattern of the activity was similar to the corresponding native constructs, indicating that the initiator/transcription start site here is mainly active in this cell type. These results suggest the presence of activating sequences within the –1509 to –1110 and –349 to –193 regions.



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  		FIG. 5. The human ghrelin promoter activity. A, Schematic diagram representing deletions of the human ghrelin gene introduced upstream of the Luc gene, containing variable of 5' and/or 3' ends. Exon 1 is represented by vertical bars. B, Each construct was transiently cotransfected with pRL-TK into TT cells. Promoter activity, after normalization to Renilla Luc activity, was expressed as a percentage of the activity of promoterless pGL3-basic. The data represent the mean ± SE for triplicate samples. Similar results were obtained in all independent experiments. C, Cell specificity of human ghrelin promoter activity. –1509/–33 GHRE-Luc or –349/–33 GHRE-Luc were transiently transfected into TT and HepG2 cells. Promoter activity was normalized to Renilla Luc activity, then expressed as a percentage of the activity of promoterless pGL3-basic. The data represent the mean ± SE for triplicate samples. Similar results were obtained in all independent experiments.

 
To examine the cell specificity of human ghrelin promoter function, –1509/–33 GHRE-Luc or –349/–33 GHRE-Luc were transiently transfected into TT and HepG2 cells. The Luc activity of both constructs was very low in HepG2 cells in comparison with TT cells (Fig 5CGo). These results indicate that these promoter/regulatory regions contain elements essential for cell-specific expression.

A computational analysis of the –349 to –193 regions upstream of the human ghrelin gene revealed the presence of several consensus binding sequences for a number of transactivating regulatory proteins (Fig. 4Go). We predicted a consensus E-box motif at –236 to –231. Sequence analysis identified additional putative cis-acting elements for several transcription factors, including two myeloid zinc finger protein-1 sites (–330 to –323, and –274 to –267) and sites for NF-{kappa}B (–270 to –261), activator protein-2 (–263 to –254), stimulating protein 1(–262 to –253), and GATA (–255 to –246).

Activation of transcription by E-box
To elucidate the role of the E-box (E1) in the –349 to –193 region necessary for human ghrelin promoter activity in TT cells, we examined the activation of gene expression using site-directed mutagenesis. The putative E-box sequence of E1 site CAGGTG was mutated to TCGGTG using –1509/–33 GHRE-Luc as a template (Fig. 6AGo). The Luc activity exhibited considerably reduced activity (60% of –1509/–33 GHRE-Luc levels; Fig. 6BGo). Because progressive 5' deletion analysis of upstream of the promoter showed less, but a significant, decrease in activity, and the additional putative E-box sites were detected at –1157 to –1152 (E2) and –1429 to –1424 (E3) among other transcription factors (Fig. 4Go), we next examined the effects of these two distal E-box elements. As shown in Fig. 6BGo, sequential disruption of the sites decreased Luc activity in an additive manner. Mutations of both distal E-box sites decreased promoter activity by 50%. These results indicated that both proximal and distal E-boxes within the human ghrelin promoter are required for the enhancer activity.



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  		FIG. 6. Activation of the human ghrelin promoter activity by E-boxes. A, Schematic diagram representing wild-type and site-specific mutations of the human ghrelin promoter, introduced into the upstream region of the Luc gene. A cross represents the site-specific mutation of the putative E-box. B, Each construct was transiently cotransfected with pRL-TK into TT cells. Promoter activity was normalized to Renilla Luc activity, then expressed as a percentage of the activity of –1509/–33 GHRE-Luc. The data represent the mean ± SE for triplicate samples. Similar results were obtained in all independent experiments.

 
Putative E-boxes in the human ghrelin promoter bind upstream stimulatory factor 1 (USF1)/USF2
As transcription factors of the bHLH family are important regulators of cell fate determination and differentiation in enteroendocrine cells (37), we examined the role of bHLH transcription factors in the regulation of human ghrelin gene expression. Computational analysis predicted that these putative E-box sites bind the bHLH-leucine zipper (LZ) transcription factor USF1. To determine the partner nuclear proteins for these elements in TT cells, oligonucleotides containing the sequences were used for EMSA. We first examined the E1 sequences. Utilization of the oligonucleotide (Fig. 7Go A, WT E1) generated several DNA/protein complexes (Fig. 7Go B, lane 2). The formation of two of these complexes is specific, because they were competed by a 25-fold excess of WT E1, but not by a mutant oligonucleotide MUT E1 (Fig. 7BGo, lanes 3–6).



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  		FIG. 7. Specific binding of the transcription factor USF to the –236 to –231 E-box element (E1) in the human ghrelin promoter using nuclear proteins from TT cells. A, Sequences of the double-stranded oligonucleotides used in EMSA. WT E1, Wild-type human ghrelin sequence identical to the –249 to –220 region, containing the E-box at –236 to –231. MUT E1 contains a mutated E-box (the E-box element is underlined; the mutated base pairs are indicated by italic bold letters). B, Oligonucleotide WT E1 was used as the probe for EMSA either without competitor (lane 2) or in the presence of 25- and 50-fold molar excesses of unlabeled WT E1 (lanes 3 and 4, respectively) and MUT E1 (lanes 5 and 6, respectively) oligonucleotides. The specific complex formed from TT cell nuclear extract and WT E1 is indicated by an arrowhead. Supershift assay experiments were performed using 1 µl (200 µg/0.1 ml) antibody against USF1 (U1; lane 7), USF2 (U2; lane 8), or E47 (E47; lane 9) and 5 µl (200 µg/0.5 ml) normal rabbit IgG (C; lane 10).

 
To examine the binding of E1 to USF, antibodies against USF1 and USF2 were used for supershift assay experiments. Addition of antibody against USF1 resulted in the complete disappearance of the two complexes (Fig. 7BGo, lane 7). Addition of antibody against USF2 also completely dissociated them (Fig. 7BGo, lane 8). Antibody against E47, which recognizes a similar E-box sequence, or normal rabbit IgG did not disrupt them (Fig. 7BGo, lanes 9 and 10). These results suggest that the putative E-box specifically binds USF1/USF2; the complexes probably contain a USF1/USF2 heterodimer. Similarly, we next examined the E2 and E3 sequences. In EMSA using oligonucleotides containing the E2 and E3 sequences, one of DNA/protein complexes proved to be specific using specific competitors and antibodies, respectively (Fig. 8Go). These results suggest a potential role for USF transcription factors in the regulation of human ghrelin gene expression.



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  		FIG. 8. Specific binding of the transcription factor USF to the distal E-box element (E2 and E3) in the human ghrelin promoter. Sequences of the double-stranded oligonucleotides used in the EMSA are shown on the top of each figure. A, WT E2 represents wild-type human ghrelin sequence identical to the –1169 to –1140 region, containing the E-box at –1157 to –1152. MUT E2 contains a mutated E-box (the E-box element is underlined; the mutated base pairs are indicated by italic bold letters). B, WT E3 represents wild-type human ghrelin sequence identical to the –1441 to –1412 region, containing the E-box at –1429 to –1424. MUT E3 contains a mutated E-box. Representative EMSAs were shown on the bottom of each figure. Oligonucleotide WT E2 or E3 was used as the probe for EMSA either without competitor (lane 2) or in the presence of 25- and 50-fold molar excesses of unlabeled WT E2 or E3 (lanes 3 and 4, respectively) and MUT E2 or E3 (lanes 5 and 6, respectively) oligonucleotides. The specific complex formed from TT cell nuclear extract and WT E2 or E3 is indicated by an arrowhead. Supershift assay experiments were performed using 1 µl (200 µg/0.1 ml) antibody against USF1 (U1; lane 7), USF2 (U2; lane 8), or E47 (E47; lane 9) and 5 µl (200 µg/0.5 ml) normal rabbit IgG (C; lane 10).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we analyzed the 5'-flanking region of the human ghrelin gene to identify a set of protein-DNA interactions within the human ghrelin promoter. By 5'-RACE using polyadenylated RNA isolated from TT cells, we identified two transcription initiation sites, located at –80 (Transcript-A) and –555 (Transcript-B). This analysis indicated the presence of a short noncoding first exon, only 20 bp in length, within the human ghrelin gene presented at the latter transcript (Transcript-B). Tanaka et al. (38) reported that the mouse ghrelin gene contains five exons and four introns, with a similar first exon of 19 bp, and they postulated a possible function for a TATATAA sequence within the mouse ghrelin promoter. They predicted that the presence of a short noncoding first exon is a feature common to mammalian ghrelin genes, as an upstream region in the reported human ghrelin sequence shows high sequence homology to the exon 1 region of mouse ghrelin (38). In contrast, Kishimoto et al. (39) reported recently that the 5' end of cDNA of the human ghrelin gene was –32 bp from the translation start site as a major transcription start site, suggesting the absence of such a noncoding exon. However, the cDNA library they used was commercially based, and there is a possibility that it may not necessarily express a full-length cDNA. Although the expression levels were different, both longer transcripts (Transcripts A and B) were actually expressed in TT cells and stomach in our study. Taken together, there are at least two transcription initiation sites, one of which contains a short first exon, and the promoter usage may be regulated in a cell-specific manner.

Our functional analysis of the 5'-flanking region of the human ghrelin gene demonstrates that Luc activity exhibited the biphasic changes (Fig. 5BGo) different from those reported by Kishimoto et al. (39). This discrepancy probably results from differences in the cells used for transfections. Ghrelin-producing X/A-like cells (3), primarily found in the oxyntic gland and infrequently in the pyloric gland and small intestine, represent a major endocrine cell population in the human oxyntic gland. These cells possess round, compact, and electron-dense neurosecretory granules (40, 41). The ECC10 cell line (42), derived from a human gastric carcinoid tumor, had characteristic neurosecretory granules, well to moderately developed microvilli, and demosomal junctions. Neuron-specific enolase and carcinoembryonic antigen, however, are not detected in these cells. In addition, no amines or peptide hormones could be detected in ECC10 cells by immunocytochemical and cytochemical studies, indicating that ECC10 cells belong to the variant type that is used in the classification of small cell lung carcinoma cell lines. The ECC10 cell line is supposed to arise via neoplastic neometaplasia from adenocarcinoma cells to endocrine cells. The TT cell line possesses a considerable number of secretory granules, a well developed rough endoplasmic reticulum, and a prominent Golgi apparatus. In addition to calcitonin (CT), TT cells produce a variety of hormones, namely CT gene-related peptide, ACTH, neurotensin, enkephalin, PTH-related peptide, gastrin-releasing peptide, serotonin, synaptophysin, neuron-specific enolase, calbindin, tyrosine hydroxylase, and chromogranin A, all of which are expressed in a wide variety of endocrine and neuroendocrine tissues (43, 44, 45). Additional marker proteins have been detected in the cytosol (carcinoembryonic antigen) and as part of the cytoskeleton ({alpha}-tubulin and cytokeratin). However, the fact that TT cells expressed Transcript-B, which contains a noncoding short exon, at lower levels than Transcript-A and functional analysis of the human ghrelin promoter using TT cells (Fig. 5BGo) support the idea that the transcriptional initiation site located at –80 is more important for transcriptional regulation of ghrelin in TT cells. These results also suggest that a TATA box-like sequence, TATATAA, located at –585 does not function significantly, at least in TT cells, similar to the previous report using ECC10 cells (39). Although the stomach equally expressed both transcripts, there are no functional stomach cell lines to determine whether the TATATAA element located at the 5'-flanking region of the human ghrelin gene may function in the tissue.

In both invertebrates and vertebrates, transcription factors of the bHLH family regulate cell fate determination and differentiation in a variety of cell types (46, 47). In the digestive tract, endocrine cell development is also regulated by members of the bHLH transcription factor family. The member of the neurogenin family, neurogenin 3, is transiently expressed in endocrine progenitors during digestive tract development. This molecule controls endocrine cell fate specifications in multipotent endodermal progenitors of the digestive tract (37, 48, 49). In the mouse intestine, loss of Math1 leads to depletion of secretory cell lineages, including enteroendocrine cells (50). BETA2/NeuroD controls terminal differentiation of the enteroendocrine secretin-producing cells through a coordination of secretin gene transcription with cell cycle arrest (51, 52, 53).

USF proteins, members of the bHLH-LZ family of transcription factors, were first identified by their role in the regulation of adenovirus major late promoter transcription (54, 55). USF proteins contain 43-kDa (USF1) and 44-kDa (USF2) polypeptides, encoded by separate genes (56, 57). USF proteins primarily bind as dimers to consensus sequences containing the CACGTG motif termed an E-box (54, 57, 58). USF proteins are ubiquitously expressed, although different ratios of USF homo- and heterodimers are found in different cell types (59). Although the biological functions of USF are poorly understood, these proteins regulate the expression of a wide variety of genes, including CT/CT gene-related peptide (60), TGFß2 (61), and Pdx-1, a critical player in pancreatic development (62, 63). USF proteins have also been reported to bind other sequences, including CGCGTG (64), CCCGTG (65), CAGCTG (60, 66), CACCTG (67), and CACATG (68, 69). By EMSA, we demonstrated that USF proteins also bind the CAGGTG and CATCTG motifs within the human ghrelin promoter. Thus, USF proteins are capable of binding to a variety of E-box motifs to regulate gene expression.

The data presented here indicate that putative E-boxes in the human ghrelin promoter specifically bind the USF1/USF2 heterodimer. USF proteins play a potential role in the regulation of human ghrelin expression. As the transcriptional activity was not completely lost upon mutations of the E-box sites (Fig. 6Go), other transcription factors may participate, together with USF proteins, to exert full activity of the promoter. Indeed, the LZ domains of several members of the bHLH-LZ family participate in various interactions with additional transcription factors (70, 71). USF proteins can interact with transcription factor for RNA polymerase II D when bound to the TATA box motif (54). Thus, it is possible that human ghrelin promoter activity is regulated by modulation of these essential protein-protein interactions. We are investigating the possibility of other putative cis-acting elements for several transcription factors functioning in this pathway.

In summary, the bHLH-LZ transcription factors USF1 and USF2 specifically bind to E-boxes in the human ghrelin promoter as a heterodimer, probably playing a role in the regulation of human ghrelin expression. Although the molecular mechanism of human ghrelin transcriptional regulation remains unclear, the present study contributes greatly to our understanding of the controls governing ghrelin secretion.


   Acknowledgments
 
We thank Miss Hitomi Hiratani for excellent technical assistance, and Miss Maki Kouchi for excellent secretarial work.


   Footnotes
 
This work was supported in part by grants-in-aids from the Ministry of Education, Science, Culture, Sports, and Technology of Japan; the Ministry of Health, Labor, and Welfare of Japan; and the Foundation for Growth Science.

Abbreviations: bHLH, Basic/helix-loop-helix; CT, calcitonin; hMTC, human medullary thyroid carcinoma; Luc, luciferase; LZ, leucine zipper; NF-{kappa}B, nuclear factor {kappa}B; nt, nucleotide; RACE, rapid amplification of cDNA ends; TK, thymidine kinase; USF, upstream stimulatory factor.

Received December 18, 2003.

Accepted for publication May 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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