RAPID ACTIVATION OF RAT INSULIN-LIKE GROWTH-FACTOR-I GENE-TRANSCRIPTION BY GROWTH-HORMONE REVEALS NO CHANGES IN DEOXYRIBONUCLEIC-ACID PROTEIN INTERACTIONS WITHIN THE 2ND PROMOTER

Insulin-like growth factor-I (IGF-I) is a highly conserved 70-residue circulating peptide that mediates many of the systemic growth-promotin g effects of GH. This laboratory has found previously that GH rapidly stimulates hepatic IGF-I transcription in hypophysectomized (hyper) ra ts by activating promoter 1, the major rat IGF-I gene promoter. In thi s study, the hormonal regulation of IGF-I expression through promoter 2, a minor promoter in most tissues but active in the liver, was inves tigated. Through use of a sensitive RNase protection assay, GH was sho wn to rapidly induce the accumulation of correctly initiated transcrip ts directed by this promoter in hepatic nuclei. Using in vitro DNase-I footprinting, six DNA-protein interactions were identified within pro moter 2 with hepatic nuclear extracts from juvenile male hyper rats gi ven a single ip injection of GH or saline 60 min before death. These D NA-protein-binding complexes also were investigated for specificity an d for regulation by GH by gel mobility shift assays. All DNA-protein i nteractions were detected in hepatic nuclear protein extracts from hyp er rats and did not change within 15-120 min after GH treatment. These results thus identify and characterize a series of constitutive nucle ar protein-binding sites within the second rat IGF-I promoter that may be involved in mediating its transcriptional activity.