Wenger R H, Rolfs A, Marti H H, Bauer C, Gassmann M
Institute of Physiology, University of Zürich-Irchel, Switzerland.
J Biol Chem. 1995 Nov 17;270(46):27865-70. doi: 10.1074/jbc.270.46.27865.
Transcriptional regulation of gene expression by hypoxia is an important, but yet only marginally characterized mechanism by which organisms adapt to low oxygen concentrations. The human hepatoma cell line HepG2 is a widely used model for studying hypoxic induction of the hematopoietic growth factor erythropoietin. In an attempt to identify additional genes expressed in HepG2 cells during hypoxia, we differentially screened a cDNA library derived from hypoxic (1% O2) HepG2 cells using probes isolated from either normoxic (21% O2) or hypoxic cells. Two genes were identified, one encoding aldolase, a member of the glycolytic enzymes, and the other encoding alpha 1-antitrypsin which belongs to the family of the acute phase (AP) responsive proteins. Whereas hypoxic induction of glycolytic enzymes is well established, oxygen-dependent regulation of AP genes has not been reported so far. AP proteins are liver-derived plasma proteins whose production during inflammation is either up-regulated (positive AP reactants) or down-regulated (negative AP reactants). In the present study, we demonstrate that on the mRNA level hypoxic stimulation of HepG2 cells led to (i) an induction of the positive AP reactants alpha 1-antitrypsin, alpha 1-antichymotrypsin, complement C3, haptoglobin, and alpha 1-acid glycoprotein; (ii) a down-regulation of the negative AP reactant albumin; (iii) an up-regulation of the negative AP reactant transferrin; and (iv) unchanged levels of the positive AP reactants alpha- and beta-fibrinogen as well as hemopexin. Cycloheximide inhibited hypoxic up-regulation of AP mRNAs demonstrating that de novo protein synthesis is required for hypoxic induction. Nuclear run-on assays indicate that the hypoxic increase in AP mRNAs is mainly due to transcriptional regulation. The hypoxic response was compared to AP stimulation by interleukin 6. The results suggest that the adaptive response to hypoxia overlaps with, but is not identical with, the AP response mediated by interleukin 6.
缺氧对基因表达的转录调控是生物体适应低氧浓度的一种重要但尚未完全明确的机制。人肝癌细胞系HepG2是研究造血生长因子促红细胞生成素缺氧诱导的常用模型。为了鉴定缺氧时HepG2细胞中表达的其他基因，我们用从常氧（21% O₂）或缺氧细胞中分离的探针，对源自缺氧（1% O₂）HepG2细胞的cDNA文库进行了差异筛选。鉴定出两个基因，一个编码醛缩酶（糖酵解酶家族成员），另一个编码α1-抗胰蛋白酶（属于急性期反应蛋白家族）。虽然糖酵解酶的缺氧诱导已得到充分证实，但急性期基因的氧依赖性调控迄今尚未见报道。急性期反应蛋白是肝脏来源的血浆蛋白，其在炎症期间的产生要么上调（阳性急性期反应物），要么下调（阴性急性期反应物）。在本研究中，我们证明在mRNA水平上，HepG2细胞的缺氧刺激导致：（i）阳性急性期反应物α1-抗胰蛋白酶、α1-抗糜蛋白酶、补体C3、触珠蛋白和α1-酸性糖蛋白的诱导；（ii）阴性急性期反应物白蛋白的下调；（iii）阴性急性期反应物转铁蛋白的上调；（iv）阳性急性期反应物α-和β-纤维蛋白原以及血红素结合蛋白水平不变。放线菌酮抑制缺氧时急性期反应蛋白mRNA的上调，表明缺氧诱导需要从头合成蛋白质。核转录分析表明，急性期反应蛋白mRNA的缺氧增加主要是由于转录调控。将缺氧反应与白细胞介素6引起的急性期反应进行了比较。结果表明，对缺氧的适应性反应与白细胞介素6介导的急性期反应重叠，但并不相同。
