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.

生长马促分析（GHS）最初被设计为人造肽，其次是更多的潜在类似物和非肽化合物。 GHS受体（GHS-R）的天然配体最近已被鉴定为Ghrelin。我们已经分析了GH3细胞中GHS-R基因表达负调控的分子机制。 TPA/BAY K8644，模仿GHS的作用，通过-669至640 bp的GHS-R基因的上游抑制GHS-R/荧光素酶活性（GHS-R/LUC）的抑制作用。通过电泳迁移率转移测定法，该DNA片段特异性结合了从GH3细胞中提取的核蛋白，这与从共识元件中预期的AP2不同。糖皮质激素（GC）还导致GHS-R基因上游的-53L至475bp抑制GHS-R/LUC，其中负GC响应元件位于其中。该DNA片段特异性结合的核蛋白从GH3细胞中提取，这与从共识元件中预期的GC受体（GR）不同。此外，环状AMP响应元件结合蛋白（CBP）的过表达未能消除GC对GHS-R/LUC的抑制作用。这些结果表明，GR和CBP均未参与GC对GHS-R/LUC的负调控。接下来，我们发现，生长素素调节了胰岛素作用的细胞内信号传导，例如细胞增殖以及抗胰岛素作用，例如刺激糖原合成或抑制糖节生成对肝细胞H4-II-E的抑制。因此，在人肝癌细胞HEPG2中测量了GHS-R/LUC。从-1224到-608 bp的删除导致GHS -R/LUC的增加，这是由于缺失到-445bp所维持的，这表明HEPG2中GHS -R基因基本启动子活性的调节元素比GH3中的近端更近端。在体外培养的HEPG2细胞中，生长素素无法改变GHS-R/LUC。总而言之，这项研究证明了人GHS-R基因表达的细胞特异性调节的分子机制。