Genomic structure and characterization of the 5'-flanking region of the human ghrelin gene.
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 site-directed 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.
Kanamoto
Naotetsu
N
Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan.
Akamizu
Takashi
T
Tagami
Tetsuya
T
Hataya
Yuji
Y
Moriyama
Kenji
K
Takaya
Kazuhiko
K
Hosoda
Hiroshi
H
Kojima
Masayasu
M
Kangawa
Kenji
K
Nakao
Kazuwa
K
D024506:5' Flanking Region / Q000235:genetics*; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D054439:Ghrelin; D006801:Humans; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D036361:Peptide Hormones / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D015533:Transcriptional Activation / Q000235:genetics; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D054439:Ghrelin; D036361:Peptide Hormones; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors