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 polypeptide that forms a ternary complex with IGFs and an acid-labile subunit. The hormonal regulation of the components of this complex is highly controversial, and both IGF-I and GH have been shown to mediate the expression/synthesis of IGFBP-3. This study investigates the regulation of IGFBP-3 protein, measured by RIA and Western ligand blot, and messenger RNA (mRNA) expression, measured by Northern analysis and reverse transcriptase-PCR, in SKHEP-1 human hepatocarcinoma cells. SKHEP-1 cells significantly increased the IGFBP-3 concentrations in conditioned 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, time-course studies showed that the GH-induced increase in IGFBP-3 mRNA preceded the IGF-I-induced increase (6 h for GH-induced IGFBP-3 mRNA; 12 h for IGF-I-induced IGFBP-3 mRNA). The half-life of IGFBP-3 mRNA was evaluated after transcriptional arrest by treatment with a RNA polymerase II inhibitor (5,6-dichloro-1beta-D-ribofuranosylbenzimidazole), and was found to be 14-18 h and unaltered by GH or IGF-I treatment. The induction of IGFBP-3 by GH was not due to the indirect action of locally synthesized IGF-I, because 1) no immunoreactive IGF-I was detected in the CM of control or GH-treated cells; 2) Northern blots revealed 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-course studies showed an earlier increase in IGFBP-3 mRNA after GH treatment 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.
Gucev
Z S
ZS
Department of Pediatrics, Oregon Health Sciences University School of Medicine, Portland 97201-3042, USA.
Oh
Y
Y
Kelley
K M
KM
Labarta
J I
JI
Vorwerk
P
P
Rosenfeld
R G
RG
D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D019382:Human Growth Hormone / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000096:biosynthesis*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008113:Liver Neoplasms / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D018972:Insulin-Like Growth Factor Binding Protein 3; D012333:RNA, Messenger; D019382:Human Growth Hormone; D007334:Insulin-Like Growth Factor I