Hypoxia Stimulates Insulin-Like Growth Factor Binding Protein 1 (IGFBP-1) Gene Expression in HepG2 Cells: A Possible Model for IGFBP-1 Expression in Fetal Hypoxia

• Published in: Proceedings of the National Academy of Sciences - PNAS 1998-08, Vol.95 (17), p.10188-10193
• Main Authors: Tazuke, Salli I., Mazure, Natalie M., Sugawara, Junichi, Carland, Grace, Faessen, Gerry H., Suen, Lii-Feng, Irwin, Juan C., Powell, David R., Giaccia, Amato J., Giudice, Linda C.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological Sciences; Cell Hypoxia - genetics; Cell Line; Consensus Sequence; DNA Primers - genetics; Embryonic and Fetal Development - genetics; Embryonic and Fetal Development - physiology; Female; Fetal Blood - metabolism; Fetal growth retardation; Fetal Growth Retardation - etiology; Fetal Growth Retardation - genetics; Fetal Growth Retardation - physiopathology; Fetal hypoxia; Fetal Hypoxia - complications; Fetal Hypoxia - genetics; Fetal Hypoxia - physiopathology; Fetus; Fetuses; Gene Expression; Genes; Genes, Reporter; Hep G2 cells; Humans; Hypoxia; Infant, Newborn; Insulin-Like Growth Factor Binding Protein 1 - blood; Insulin-Like Growth Factor Binding Protein 1 - genetics; Insulin-Like Growth Factor Binding Protein 1 - physiology; Introns; Medical research; Messenger RNA; Mice; Models, Biological; Plasmids; Pregnancy; Proteins; Rats; Reporter genes; RNA, Messenger - genetics; RNA, Messenger - metabolism; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.17.10188

IGFBP-1 is elevated in fetuses with long-term, chronic hypoxia and intrauterine growth restriction. We investigated the hypothesis that hypoxia regulates IGFBP-1 in the human fetus in vivo and IGFBP-1 gene expression and protein in vitro. Umbilical artery IGFBP-1 levels (mean ± SEM) from term babies with respiratory acidosis (acute hypoxia), normal babies, and those with mixed respiratory/metabolic acidosis (more profound and prolonged hypoxia) were measured using an immunoradiometric assay. IGFBP-1 levels were similar in normal (n = 12) and acutely hypoxic (n = 6) babies (189.1 ± 71.8 vs. 175.8 ± 45.9 ng /ml, respectively, P = 0.789). However, with more profound and prolonged hypoxia (n = 19), IGFBP-1 levels were markedly elevated (470.6 ± 80.0 ng/ml, P = 0.044). To investigate IGFBP-1 regulation by hypoxia in vitro, HepG2 cells were incubated under hypoxia (pO2= 2%) and normoxia (pO2= 20%). IGFBP-1 protein and mRNA increased 8- and 12-fold, respectively, under hypoxic conditions. Hypoxia did not affect protein or mRNA levels of IGFBP-2 or -4. IGFBP-5 and -6 mRNAs, undetectable in control cells, were not induced by hypoxia, whereas minimally expressed IGFBP-3 mRNA increased twofold. Investigation into IGFBP-1 gene structure revealed three potential consensus sequences for the hypoxia response element (HRE) in the first intron. To investigate functionality, a 372-bp fragment of IGFBP-1 intron 1, containing putative HREs, was placed 5′to a heterologous hsp70 promoter in a plasmid using luciferase as a reporter gene. Under hypoxia, reporter gene activity increased up to 30-fold. Mutations in the middle HRE abolished reporter activity in response to hypoxia, suggesting that this HRE is functional in the IGFBP-1 hypoxia response. Cotransfection of HRE reporter genes with a constitutively expressing hypoxia-inducible factor 1 plasmid in HepG2 cells resulted in a fourfold induction of reporter activity, suggesting a role for hypoxia-inducible factor 1 in hypoxia induction of IGFBP-1 gene expression. These data support the hypothesis that hypoxia regulation of IGFBP-1 may be a mechanism operating in the human fetus to restrict insulin-like growth factor-mediated growth in utero under conditions of chronic hypoxia and limited substrate availability.