Marquis J C, Demple B
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115-6021, USA.
Cancer Res. 1998 Aug 1;58(15):3435-40.
NO is a biologically generated free radical that serves diverse roles in mammalian cell signaling and immune-mediated cell killing. Because mammalian cells might be exposed to varying levels of NO, we tested for possible defense genes and proteins induced upon treatment of cells with sublethal fluxes of pure NO. Two-dimensional gel analysis was performed for human embryonic lung fibroblasts (IMR-90) exposed for 90 min to pure NO at approximately 280 nM/s, which revealed the reproducible induction of at least 12 proteins. Among these, a prominent polypeptide had Mr approximately 32,000, similar to the well-known oxidative stress protein heme oxygenase-1 (HO-1). Northern blot analysis of IMR-90 and HeLa cells demonstrated the NO-mediated induction of HO-1 mRNA up to 70-fold over the levels in untreated cells. HO-1 induction depended on the NO dose and subsequent expression time and was maximal 3-5 h after a 1-h exposure to NO at a constant flux of approximately 280 nM/s. The mRNA encoding a tyrosine/threonine phosphatase (CL100/MKP-1) was also NO inducible (approximately 20 fold), whereas there was no increase in expression of the mRNA encoding manganese-containing superoxide dismutase. Induction of HO-1 mRNA was independent of the guanylate cyclase signaling pathway; addition of the analogue 8-bromo-cyclic GMP did not induce the HO-1 transcript, and the soluble guanylate cyclase inhibitor LY-83583 did not block HO-1 induction by NO in IMR-90 cells. Luciferase reporter constructs containing up to 4.7 kb of DNA upstream of the HO-1 transcription start site showed < or = 2.5-fold induction in IMR-90 or HeLa cells exposed to NO. However, HO-1 mRNA was dramatically stabilized after exposure of IMR-90 cells to NO. Even a transient NO exposure produced elevated levels of HO-1 protein for > or = 10 h, whereas continuous low-level NO treatment (35 nM/s) maintained elevated HO-1 mRNA expression for > or = 8 h. These results reveal a complex mammalian response to NO that involves a new level of posttranscriptional control in response to this radical.
一氧化氮（NO）是一种生物生成的自由基，在哺乳动物细胞信号传导和免疫介导的细胞杀伤中发挥多种作用。由于哺乳动物细胞可能会暴露于不同水平的NO中，我们测试了用亚致死通量的纯NO处理细胞后诱导产生的可能的防御基因和蛋白质。对人胚肺成纤维细胞（IMR-90）进行二维凝胶分析，这些细胞在约280 nM/s的纯NO中暴露90分钟，结果显示至少有12种蛋白质可重复性诱导产生。其中，一种突出的多肽分子量约为32,000，类似于著名的氧化应激蛋白血红素加氧酶-1（HO-1）。对IMR-90和HeLa细胞进行Northern印迹分析表明，NO介导的HO-1 mRNA诱导水平比未处理细胞中的水平高出70倍。HO-1的诱导取决于NO剂量和随后的表达时间，在以约280 nM/s的恒定通量暴露于NO 1小时后3 - 5小时达到最大值。编码酪氨酸/苏氨酸磷酸酶（CL100/MKP-1）的mRNA也可被NO诱导（约20倍），而编码含锰超氧化物歧化酶的mRNA表达没有增加。HO-1 mRNA的诱导独立于鸟苷酸环化酶信号通路；添加类似物8-溴环鸟苷酸不会诱导HO-1转录本，可溶性鸟苷酸环化酶抑制剂LY-83583也不会阻断IMR-90细胞中NO对HO-1的诱导。含有HO-1转录起始位点上游高达4.7 kb DNA的荧光素酶报告构建体在暴露于NO的IMR-90或HeLa细胞中显示出≤2.5倍的诱导。然而，IMR-90细胞暴露于NO后，HO-1 mRNA显著稳定。即使短暂暴露于NO，HO-1蛋白水平也会在≥10小时内升高，而持续低水平的NO处理（35 nM/s）会使HO-1 mRNA表达在≥8小时内维持升高。这些结果揭示了哺乳动物对NO的复杂反应，其中涉及对这种自由基的转录后控制的新水平。
