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.