EVIDENCE FOR INSULIN-LIKE GROWTH-FACTOR (IGF)-INDEPENDENT TRANSCRIPTIONAL REGULATION OF IGF BINDING PROTEIN-3 BY GROWTH-HORMONE IN SKHEP-1 HUMAN HEPATOCARCINOMA CELLS

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is a poly peptide that forms a ternary complex with IGFs and an acid-labile subu nit. The hormonal regulation of the components of this complex is high ly controversial, and both IGF-I and GH have been shown to mediate the expression/synthesis of IGFBP-3. This study investigates the regulati on of IGFBP-3 protein, measured by RIA and Western ligand blot, and me ssenger RNA (mRNA) expression, measured by Northern analysis and rever se transcriptase-PCR, in SKHEP-1 human hepatocarcinoma cells. SKHEP-1 cells significantly increased the IGFBP-3 concentrations in conditione d medium (CM) when treated with GH (0.1-10 ng/ml), IGF-I (1-100 ng/ml) , or Des(1-3)-IGF-I (1-100 ng/ml) in a dose-dependent manner (>3-fold) . The increase in IGFBP-3 protein concentrations in CM was accompanied by a corresponding increase in IGFBP-3 mRNA levels. Interestingly, ti me-course studies showed that the GH-induced increase in IGFBP-3 mRNA preceded the TGF-I-induced increase (6 h for GH-induccd IGFBP-3 mRNA; 12 h for IGF-I-induced IGFBP-3 mRNA). The half-life of IGFBP-3 mRNA wa s evaluated after transcriptional arrest by treatment with a RNA polym erase II inhibitor (5,6-dichloro-1 beta-D-ribofuranosylbenzimidazole). and was round to be 14-18 h and unaltered by GH or IGF-I treatment. T he induction of IGFBP-3 by GH was not due to the indirect action of lo cally synthesized IGF-I, because 1) no immunoreactive IGF-I was detect ed in the CM of control or GH-treated cells; 2) Northern blots reveale d no IGF-I mRNA expression in SKHEP-1 cells; 3) reverse transcriptase- PCR did not detect any expression of the IGF-I gene; and 4) time-cours e studies showed an earlier increase in IGFBP-3 mRNA after GH treatmen t than after IGF-I treatment. Thus, the results obtained in this study are consistent with an IGF-I-independent. regulation of IGFBP-3 gene expression by GH.