Osada Mayuko, Imaoka Susumu, Sugimoto Toshikado, Hiroi Toyoko, Funae Yoshihiko
Department of Chemical Biology, Osaka City University Medical School, 1-4-3 Asahimatchi, Abeno-ku, Osaka 545-8585, Japan.
J Biol Chem. 2002 Jun 28;277(26):23367-73. doi: 10.1074/jbc.M112413200. Epub 2002 Apr 23.
Hypoxia induces a group of physiologically important genes that include erythropoietin (EPO) and vascular endothelial growth factor (VEGF). Hypoxia-inducible factor 1 (HIF-1) was identified as a hypoxia-activated transcription factor; however, the molecular mechanisms that underlie hypoxia signal transduction in mammalian cells remain undefined. In this study, we found that a flavoprotein, NADPH-P450 reductase (NPR), could regulate the induction of EPO mRNA under hypoxic conditions. Hypoxic EPO mRNA induction in Hep3B cells was inhibited by diphenyleneiodonium chloride, which is an inhibitor of NADPH-dependent enzymes. NPR antisense cDNA was transfected into Hep3B cells, and NPR-deficient hepatocyte cells (NPR(-) cells) were established. NPR(-) cells lacked EPO induction under hypoxia, and HIF-1alpha in NPR(-) cells did not respond to either transcriptional activation or translocation to the nucleus based on electrophoretic mobility shift assays and reporter gene assay including hypoxia response element. In contrast, NPR overexpression in Hep3B cells enhanced the DNA binding activity of HIF-1alpha by luciferase reporter gene assay. A study with HeLa S3 cells produced the same results. Furthermore, anti-NPR IgG inhibited EPO induction. EPO induction inhibited by diphenyleneiodonium chloride was recovered by bovine serum albumin-NADPH (a covalent binding complex of bovine serum albumin and NADPH) as well as NADPH. These results suggested that NPR located at the plasma membrane regulates EPO expression in hypoxia, including HIF-1 activation and translocation. We further studied the expression of NPR and VEGF mRNAs in human tumor tissues and found that the NPR mRNA levels were correlated with the VEGF mRNA levels, suggesting that NPR might be an important factor in the hypoxic induction of genes such as VEGF in vivo.
缺氧可诱导一组具有重要生理意义的基因，其中包括促红细胞生成素（EPO）和血管内皮生长因子（VEGF）。缺氧诱导因子1（HIF-1）被鉴定为一种缺氧激活的转录因子；然而，哺乳动物细胞中缺氧信号转导的分子机制仍不明确。在本研究中，我们发现一种黄素蛋白，即NADPH-P450还原酶（NPR），可在缺氧条件下调节EPO mRNA的诱导。氯化二苯碘鎓（一种NADPH依赖性酶的抑制剂）可抑制Hep3B细胞中缺氧诱导的EPO mRNA表达。将NPR反义cDNA转染到Hep3B细胞中，建立了NPR缺陷型肝细胞（NPR(-)细胞）。NPR(-)细胞在缺氧条件下缺乏EPO诱导，基于电泳迁移率变动分析和包括缺氧反应元件的报告基因分析，NPR(-)细胞中的HIF-1α对转录激活或转位至细胞核均无反应。相反，通过荧光素酶报告基因分析，Hep3B细胞中NPR的过表达增强了HIF-1α的DNA结合活性。对HeLa S3细胞的研究也得到了相同的结果。此外，抗NPR IgG抑制EPO诱导。氯化二苯碘鎓抑制的EPO诱导可通过牛血清白蛋白-NADPH（牛血清白蛋白与NADPH的共价结合复合物）以及NADPH恢复。这些结果表明，位于质膜的NPR在缺氧时调节EPO表达，包括HIF-1的激活和转位。我们进一步研究了NPR和VEGF mRNA在人肿瘤组织中的表达，发现NPR mRNA水平与VEGF mRNA水平相关，提示NPR可能是体内缺氧诱导VEGF等基因的重要因素。
