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 (growth factorygene expressionyfetusynewborn)

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 6 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 5 12) and acutely hypoxic (n 5 6) babies (189.1 6 71.8 vs. 175.8 6 45.9 ng /ml, respectively, P 5 0.789). However, with more profound and prolonged hypoxia (n 5 19), IGFBP-1 levels were markedly elevated (470.6 6 80.0 ng /ml, P 5 0.044). To investigate IGFBP-1 regulation by hypoxia in vitro, HepG2 cells were incubated under hypoxia (pO2 5 2%) and normoxia (pO2 5 20%). IGFBP-1 protein and mRNA increased 8and 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. Fetal growth is a complex process influenced by genetic, nutritional, environmental, placental, and maternal factors, each of which can have profound effects on fetal outcome (1). In humans intrauterine growth restriction (IUGR, or birthweight ,10th percentile for gestational age) is a leading cause of severe fetal and neonatal morbidity and mortality. Decreased placental perfusion and placental and fetal hypoxia are leading causes of IUGR (1). The role of oxygen in fetal growth is also underscored by observations that IUGR accompanies pregnancies complicated by decreased maternal oxygen-carrying capacity and fetal anemia and hypoxia. In addition to oxygen, growth factors and particularly the fetal and maternal insulin-like growth factor (IGF) systems are important regulators of fetal growth (2, 3). IGF-I and IGF-II gene knockout studies have demonstrated that homozygous mouse pups are born with marked IUGR (4). A naturally occurring deletion of exons 4 and 5 of the human IGF-I gene results in severe intrauterine and postnatal growth restriction (5). In humans there is also a direct correlation between IGF-I concentrations in cord blood and fetal size and birthweight (6). IGFBP-1 regulates minute-to-minute bioavailability of IGFs in the circulation and inhibits IGF actions (7). In humans and animals there is a striking inverse correlation between maternal and fetal circulating levels of IGFBP-1 and fetal size, and IGFBP-1 levels are elevated up to 20-fold in fetuses with marked IUGR (6, 8–17). Recent evidence suggests that hypoxia and the IGF system are interrelated. Animal models of uteroplacental insufficiency (caused by uterine artery ligation or maternal hypoxia) result in fetal hypoxia and IUGR, with markedly elevated circulating levels of IGFBP-1 and overexpression of IGFBP-1 mRNA in the fetal liver, the primary source of circulating IGFBP-1 (11, 12, 18–21). These studies suggest that IGFBP-1 may be an important regulator of fetal growth by regulating IGF bioavailability. IGFBP-1 gene regulation is complex, with multiple regulatory elements in its promoter that act independently or coordinately to regulate expression (7, 22). The specific stimuli for elevated IGFBP-1 in the fetal circulation in pregnancies complicated by IUGR and hypoxia have not been determined, although stress (glucocorticoids) and hypoinsulinemia have been implicated (2). Hypoxia may itself be a stimulus for elevated IGFBP-1 levels observed in these babies. Hypoxic regulation of gene expression involves binding of a nuclear transcription factor, hypoxiainducible factor 1 (HIF-1), to a hypoxia response element (HRE) (23, 24). In the current study, we report markedly elevated levels of IGFBP-1 in babies with intrauterine hypoxia, the presence of a functional HRE in intron 1 of the IGFBP-1 gene, evidence in vitro that IGFBP-1 mRNA and protein are induced in HepG2 cells by hypoxia, that this increase is mediated directly through an HRE located in the first intron of the hIGFBP-1 gene, and that HIF-1 is likely involved in hypoxia regulation of IGFBP-1 gene expression. We propose that transcriptional regulation of IGFBP-1 by hypoxia is a mechanism to explain the observed increased The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9510188-6$2.00y0 PNAS is available online at www.pnas.org. Abbreviations: IUGR, intrauterine growth restriction; IGF, insulinlike growth factor; HRE, hypoxia response element; HIF-1, hypoxiainducible factor 1; VEGF, vascular endothelial growth factor. §To whom reprint requests should be addressed at: Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, CA 94305-5317. e-mail: [email protected].