| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
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.
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