Sogawa K, Numayama-Tsuruta K, Ema M, Abe M, Abe H, Fujii-Kuriyama Y
Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-77, Japan.
Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7368-73. doi: 10.1073/pnas.95.13.7368.
Nitric oxide (NO) is known to have various biologic and pathophysiologic effects on organisms. The molecular mechanisms by which NO exerts harmful effects are unknown, although various O2 radicals and ions that result from reactivity of NO are presumed to be involved. Here we report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-L-glutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 microM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 = 6.6 microM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 = 2.5 microM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1alpha or HIF-1alpha-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1alpha to a DNA-binding form.
已知一氧化氮（NO）对生物体具有多种生物学和病理生理学作用。尽管推测NO反应产生的各种氧自由基和离子参与其中，但NO发挥有害作用的分子机制尚不清楚。在此我们报告，缺氧时由转录因子缺氧诱导因子1（HIF-1）控制的适应性细胞反应受到NO的抑制。在缺氧培养的人肝癌细胞系Hep3B细胞中，促红细胞生成素和糖酵解醛缩酶A mRNA的诱导分别被自发释放NO的硝普钠（SNP）完全和部分抑制。构建了一个携带与荧光素酶结构基因相连的四个缺氧反应元件序列的报告质粒，并将其转染到Hep3B细胞中。在缺氧条件下可诱导表达的荧光素酶活性在加入SNP和其他两种结构不同的NO供体S-亚硝基-L-谷胱甘肽和3-吗啉代亚磺酰亚胺后受到抑制，IC50值分别为7.8、211和490 microM。在Neuro 2A和HeLa细胞中也观察到SNP的抑制作用，表明这种抑制不是细胞类型特异性的。由HIF-1控制的血管内皮生长因子启动子活性也受到SNP的抑制（IC50 = 6.6 microM）。加入钴离子（这种处理模拟缺氧）所产生的诱导也受到SNP的抑制（IC50 = 2.5 microM）。在缺氧条件下共转染HIF-1α或HIF-1α样因子的效应质粒所表达的增强的荧光素酶活性也受到NO供体的抑制。我们还表明，这种抑制是通过阻断HIF-1α向DNA结合形式的激活步骤来实现的。
