权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular mechanism of human growth Hormone secretagogue receptor gene transcription

人生长激素促分泌素受体基因转录的分子机制

基本信息

批准号：
12671086
负责人：
KAJI Hidesuke
金额：
$ 2.18万
依托单位：
Collage of Nursing Art & Science Hyogo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671086/
关键词：
growth hormone secretagogue ghrelin receptor gene transcription regulation pituitary cell hepatocyte GH分泌物質 GH3細胞

项目摘要

Growth hormone secretagogue (GHS) was originally designed as an artificial peptide, followed by more potent analogues and non-peptide compounds. Natural ligand for GHS receptor (GHS-R) has been recently identified as ghrelin. We have analyzed molecular mechanism of negative regulation of GHS-R gene expression in GH3 cells. TPA/Bay K8644, mimicking GHS action, caused an inhibition of GHS-R/luciferase activity (GHS-R/Luc) through -669〜640 bp upstream of translation initiation site of GHS-R gene. By electrophoretic mobility shift assay, this DNA fragment specifically bound nuclear proteins extracted from GH3 cells, which was unlike AP2 expected from the consensus element. Glucocorticoid (GC) also caused inhibition of GHS-R/Luc through -53l〜475bp upstream of GHS-R gene, where negative GC responsive element located. This DNA fragment specifically bound nuclear proteins extracted from GH3 cells, which was unlike GC receptor (GR) expected from the consensus element. Furthermore, overexpression of cyclic AMP responsive element binding protein (CBP) failed to abolish the inhibition by GC of GHS-R/Luc. These results suggested that neither GR nor CBP was involved in the negative regulation of GHS-R/Luc by GC. Next, we found that ghrelin modulated intracellular signaling of insulin action such as cellular proliferation as well as anti-insulin action such as stimulation of glycogen synthesis or inhibition of gluconeogenesis on hepatocytes H4-II-E. Therefore, GHS-R/Luc was measured in human hepatoma cells HepG2. Deletion from -1224 to -608 bp caused an increase in GHS-R/Luc, which was sustained by deletion to -445bp, suggesting that the regulatory element for basal promoter activity of GHS-R gene in HepG2 was located more proximal than that in GH3. Ghrelin failed to change GHS-R/Luc in HepG2 cells cultured in vitro. In summary, this study demonstrated the molecular mechanism of the cell specific regulation of human GHS-R gene expression.

生长激素促分泌素(GHS)最初被设计为一种人造多肽，然后是更有效的类似物和非肽化合物。GHS受体的天然配体(GHS-R)最近被鉴定为Ghrelin。我们分析了GHS-R基因在GH3细胞中表达负调控的分子机制。TPA/Bay K8644模拟GHS的作用，通过GHS-R基因翻译起始点上游-669~640个碱基，抑制GHS-R/LUC的活性。通过电泳迁移率改变分析，该DNA片段与从GH3细胞中提取的核蛋白特异性结合，这与从共识元件中预期的AP2不同。糖皮质激素(GC)也通过GHS-R基因上游-53l~475bp的负性GC反应元件对GHS-R/Luc产生抑制作用。这一DNA片段与从GH3细胞中提取的核蛋白特异性结合，不同于从共有元件中预期的GC受体(GR)。此外，过表达cAMP反应元件结合蛋白(CBP)不能取消GC对GHS-R/Luc的抑制作用。这些结果提示，GR和CBP均不参与GC对GHS-R/Luc的负性调节。接下来，我们发现Ghrelin调节细胞内胰岛素信号的作用，如细胞增殖，以及抗胰岛素作用，如刺激肝细胞H4-II-E糖原合成或抑制糖异生。因此，我们在人肝癌细胞株HepG2中检测了GHS-R/Luc。-1224~-608bp的缺失导致GHS-R/Luc的增加，并通过缺失维持到-445bp，这表明在HepG2中，GHS-R基因的基本启动子活性的调控元件比在GH3中更接近。Ghrelin不能改变体外培养的HepG2细胞的GHS-R/Luc。综上所述，本研究揭示了细胞特异性调控人GHS-R基因表达的分子机制。