Regulation of human growth hormone gene expression by insulin-like growth factor I in transfected cells

Insulin-like growth factor I (IGF-I), a target hormone for growth hormone (GH) action, has been shown to regulate rat GH gene transcription. We further investigated its direct action on the human GH gene transfected in human choriocarcinoma cells (JEG3) which possess abundant IGF-I receptors. The 2.6 kilobase (kb) hGH gene (EcoRI fragment) in pUC18 (phGH) was transfected into cells by calcium-phosphate-dimethyl sulfoxide shock. The cells were subsequently treated for 72 h, when GH gene expression was measured. 8-Bromo-cAMP (Br-cAMP, 2.5 mM) and hydrocortisone (100 nM), respectively, stimulated GH secretion in transfected cells by 50% over unstimulated control cells, while combined treatment with these two agents caused a 340% increase of GH secretion as measured by radioimmunoassay. IGF-I (6.5 nM) did not suppress the basal level of Gh secretion but did inhibit the stimulated GH secretion by over 50%. Immunoprecipitation analysis showed that 8-Br-cAMP + hydrocortisone markedly increased newly synthesized 22-kDa [35S]GH. This de novo GH synthesis was inhibited by IGF-I. Northern gel analysis of poly(A) RNA showed that 8-Br-cAMP + hydrocortisone induced a 1.0-kb mRNA species as detected with 32P-labeled hGH-cDNA. IGF-I suppressed the GH mRNA induced by cAMP + hydrocortisone as well as suppressing a larger 2.2-kb GH mRNA precursor. The results show that the transfected human GH gene was expressed and regulated in homologous cells. The 2.6-kb hGH fragment therefore contains non-tissue-specific cis-acting regulatory elements responsive to cAMP, hydrocortisone, and IGF-I. Specific IGF-I-responsive GH DNA sequences may therefore reside in the 0.5-kb 5'-flanking region, or in an intron, as described for hydrocortisone.