Molecular mechanisms of the negative effect of insulin-like growth factor-I on growth hormone gene expression in MtT/S somatotroph cells.
Although insulin-like growth factor-I (IGF-I) is shown to have a suppressive effect on GH gene expression at the pituitary level, its molecular mechanism has not yet been clarified. To study the issue, we established a new in vitro system using MtT/S, a recently established rat somatotroph tumor cell line that retains the basic characteristics of somatotroph function. Plasmids containing the GH 5' promoter (approximately 1.75 kb or shorter)-luciferase fusion gene were transfected stably or transiently into the cells, and the effect of IGF-I on the GH promoter activity was estimated by a luciferase assay. The results showed that IGF-I inhibited GH promotor activity (more than 50% suppression) in a time- and dose-related manner. IGF-I also inhibited GH secretion. A study using deletion mutants of the GH promoter revealed that the negative effect was maintained in the shortest construct (-80 to +6), suggesting that IGF-I-related factor is acting at the region very close to the minimal promoter. Interestingly, the negative effect was completely eliminated by a PI3 kinase inhibitor wortmannin (1 microM), whereas a MAP kinase inhibitor PD98059 (20 microM) or S6 kinase inhibitor rapamycin (10 nM) did not influence the effect. Our results suggest that IGF-I suppresses GH gene expression at the transcriptional level and that the PI3 kinase-mediated signaling pathway plays a major role in the negative effect of IGF-I. We believe that our system using MtT/S cells is an excellent experimental model system for studying the cellular and molecular mechanisms of the transcriptional regulation of GH in vitro.
Niiori-Onishi
A
A
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Iwasaki
Y
Y
Mutsuga
N
N
Oiso
Y
Y
Inoue
K
K
Saito
H
H
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D019833:Genistein / Q000494:pharmacology; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics*; D006131:Growth Inhibitors / Q000494:pharmacology; D007328:Insulin / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000008:administration & dosage / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010911:Pituitary Neoplasms / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D019893:Ribosomal Protein S6 Kinases / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D005419:Flavonoids; D006131:Growth Inhibitors; D007328:Insulin; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone; D019833:Genistein; D008156:Luciferases; D019893:Ribosomal Protein S6 Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D020123:Sirolimus; D000077191:Wortmannin