Oxygen-Dependent Modulation of Insulin-Like Growth Factor Binding Protein Biosynthesis in Primary Cultures of Rat Hepatocytes

• Published in: Endocrinology (Philadelphia) 2005-12, Vol.146 (12), p.5433-5443
• Main Authors: Scharf, Jens-Gerd, Unterman, Terry G, Kietzmann, Thomas
• Format: Article
• Language: English
• Subjects: Actinomycin; Animals; Base Sequence; Biological and medical sciences; Biosynthesis; Cell Hypoxia - physiology; Cells, Cultured; Deferoxamine; Deferoxamine - pharmacology; Destabilization; Expression vectors; Fundamental and applied biological sciences. Psychology; Gene expression; Growth factors; Hepatocytes; Hepatocytes - drug effects; Hepatocytes - metabolism; Hydrogen peroxide; Hydrogen Peroxide - pharmacology; Hypoxia; Hypoxia-inducible factor 1a; Insulin-Like Growth Factor Binding Protein 1 - biosynthesis; Insulin-Like Growth Factor Binding Protein 1 - genetics; Insulin-Like Growth Factor Binding Protein 1 - metabolism; Insulin-Like Growth Factor Binding Protein 4 - biosynthesis; Insulin-Like Growth Factor Binding Protein 4 - genetics; Insulin-Like Growth Factor Binding Protein 4 - metabolism; Insulin-like growth factor-binding protein 1; Insulin-like growth factor-binding protein 4; Insulin-like growth factors; Iron; Isoenzymes - pharmacology; Liver; Male; Oxidants - pharmacology; Oxygen; Oxygen - blood; Oxygen - metabolism; Oxygen tension; Procollagen-Proline Dioxygenase - pharmacology; Promoter Regions, Genetic; Protein biosynthesis; Proteins; Rats; Rats, Wistar; Reactive oxygen species; Reporter gene; RNA, Messenger - biosynthesis; RNA, Messenger - metabolism; Siderophores - pharmacology; Time Factors; Transcription factors; Transcription initiation; Transfection; Veins; Vertebrates: endocrinology
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2005-0948

Higher levels of IGF-binding protein 1 (IGFBP-1) mRNA are expressed in the less aerobic perivenous zone of the liver. Because gradients in oxygen tension (pO2) may contribute to zonated gene expression, the influence of arterial and venous pO2 on IGFBP-1 biosynthesis was studied in primary cultures of rat hepatocytes. Maximal IGFBP-1 mRNA and protein levels were observed under venous pO2, whereas less than 30% of maximal levels were observed under arterial pO2. In contrast, the expression of IGFBP-4 was greatest under arterial pO2, indicating that this effect of hypoxia on IGFBP-1 gene expression is specific. The response to hypoxia appears to involve reactive oxygen species, because treatment with H2O2 results in a dose-dependent decrease of IGFBP-1 mRNA levels under venous pO2, whereas IGFBP-1 mRNA expression under arterial pO2 was not affected. Inhibition of the hypoxia-dependent IGFBP-1 mRNA induction by actinomycin D indicates that this effect is mediated at the level of gene transcription, and inhibition of IGFBP-1 mRNA by the iron chelator desferrioxamine under both venous and arterial pO2 suggested the involvement of hypoxia-inducible transcription factors (HIF). Transfection experiments demonstrated that especially HIF-3α and HIF-2α, and to a lesser extent HIF-1α, contribute to the induction of IGFBP-1 mRNA expression in isolated hepatocytes, whereas experiments with vectors for the HIF prolyl hydroxylases (PHD) indicated a major role of PHD-2 in destabilization of HIFs, attenuating the induction of IGFBP-1 under venous pO2. Reporter gene studies indicate that hypoxia stimulates IGFBP-1 expression through a putative HIF response element located approximately 250 bp upstream from the transcription initiation site. Together, these results support the concept that iron, radical oxygen species, and the HIF-2 and -3 as well as the PHD pathways play important roles in mediating effects of hypoxia on IGFBP-1 gene expression in the liver.