Molecular mechanism of human growth Hormone secretagogue receptor gene transcription

生长激素促分泌素（GHS）最初被设计为人工肽，随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GH 3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基因翻译起始位点上游-669 ~640 bp处抑制GHS-R/萤光素酶活性（GHS-R/Luc）。通过电泳迁移率变动分析，该DNA片段特异性地结合从GH 3细胞提取的核蛋白，这与预期的来自共有元件的AP 2不同。糖皮质激素（GC）也可通过GHS-R基因上游-53 ~475bp处负性GC反应元件抑制GHS-R/Luc。该DNA片段特异性地结合从GH 3细胞提取的核蛋白，这与从共有元件预期的GC受体（GR）不同。此外，过表达cAMP反应元件结合蛋白（CBP）不能消除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的负调控。接下来，我们发现ghrelin调节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，例如刺激肝细胞H4-II-E上的糖原合成或抑制肝细胞H4-II-E上的糖原生成。因此，在人肝癌细胞HepG 2中测量GHS-R/Luc。从-1224到-608 bp的缺失导致GHS-R/Luc增加，并通过缺失到-445 bp来维持，这表明GHS-R基因的基础启动子活性调控元件在HepG 2中比在GH 3中位于更近的位置。Ghrelin对体外培养的HepG 2细胞GHS-R/Luc无明显影响。总之，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。

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.