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
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
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DOI：
10.1073/pnas.95.17.10188
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发表时间：
1998-08-18
影响因子：
11.1
通讯作者：
Giudice, LC
中科院分区：
文献类型：
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作者：
Tazuke, SI;Mazure, NM;Giudice, LC
IGFBP-1在长期、慢性缺氧和宫内生长受限的胎儿中升高。我们在体内研究了低氧对人胎儿IGFBP-1的调节作用，并在体外研究了IGFBP-1基因和蛋白的表达。用免疫放射分析法检测了足月新生儿呼吸性酸中毒(急性缺氧)、正常新生儿、呼吸性/代谢性酸中毒(重度缺氧和长期缺氧)新生儿脐动脉IGFBP-1水平(Mean+/-SEM)。正常组(n=12)和急性缺氧组(n=6)的IGFBP-1水平相似(分别为189.1±71.8vs 175.8±45.9 ng/ml，P=0.789)。但随着低氧程度的加深和时间的延长(n=19)，IGFBP-1水平显著升高(470.6±80.00 ng/ml，P=0.044)。为探讨低氧对IGFBP-1的调节作用，将人肝癌细胞株HepG2分别置于低氧(Po(2)=2%)和常氧(Po(2)=20%)中培养。在低氧条件下，IGFBP-1蛋白和mRNA分别增加8倍和12倍。低氧不影响IGFBP-3或-4的蛋白或mRNA水平。在对照细胞中检测不到的IGFBP-5和-6mRNAs在低氧条件下不被诱导，而IGFBP-3mRNA的最低表达增加了一倍。对IGFBP-1基因结构的研究揭示了第一内含子中缺氧反应元件(HRE)的三个潜在的共同序列。为了研究IGFBP-1内含子1的功能，以荧光素酶为报告基因，将IGFBP-1内含子1的一个372个碱基的片段插入到一个异源HSP70启动子的5‘端。在低氧条件下，报告基因活性最高可提高30倍。中间HRE的突变取消了报告活性对低氧的反应，表明这种HRE在IGFBP-1的低氧反应中具有功能。将HRE报告基因与组成性表达的缺氧诱导因子1共转染入HepG2细胞，可诱导4倍的报告基因活性，提示缺氧诱导因子1在低氧诱导IGFBP-1基因表达中起作用。这些数据支持这一假设，即IGFBP-1的低氧调节可能是在慢性低氧和底物可获得性有限的条件下，在人类胚胎中运行的一种机制，以限制胰岛素样生长因子介导的子宫内生长。
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 a 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 (pO(2) = 2%) and normoxia (pO(2) = 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-3 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.