Uchida K, Shiraishi M, Naito Y, Torii Y, Nakamura Y, Osawa T
Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan.
J Biol Chem. 1999 Jan 22;274(4):2234-42. doi: 10.1074/jbc.274.4.2234.
In the present study, we studied the signal transduction mechanism that is involved in the expression of c-Jun protein evident after exposure of rat liver epithelial RL34 cells to the major end product of oxidized fatty acid metabolism, 4-hydroxy-2-nonenal (HNE). HNE treatment of the cells resulted in depletion of intracellular glutathione (GSH) and in the formation of protein-bound HNE in plasma membrane. In addition, HNE strongly induced intracellular peroxide production, suggesting that HNE exerted oxidative stress on the cells. Potent expression of c-Jun occurred within 30 min of HNE treatment, which was accompanied by a time-dependent increase in activator protein-1 (AP-1) DNA binding activity. We found that HNE caused an immediate increase in tyrosine phosphorylation in RL34 cells. In addition, HNE strongly induced phosphorylation of c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases and also moderately induced phosphorylation of extracellular signal-regulated kinases. The phosphorylation of JNK was accompanied by a rapid and transient increase in JNK and p38 activities, whereas changes in the activity of extracellular signal-regulated kinase were scarcely observed. GSH depletion by L-buthionine-S, R-sulfoximine, a specific inhibitor of GSH biosynthesis, only slightly enhanced peroxide production and JNK activation, suggesting that HNE exerted these effects independent of GSH depletion. This and the findings that (i) HNE strongly induced intracellular peroxide production, (ii) HNE-induced JNK activation was inhibited by pretreatment of the cells with a thiol antioxidant, N-acetylcysteine, and (iii) H2O2 significantly activated JNK support the hypothesis that pro-oxidants play a crucial role in the HNE-induced activation of stress signaling pathways. In addition, we found that, among the inhibitors of tyrosine kinases, cyclooxygenase, and Ca2+ influx, only quercetin exerted a significant inhibitory effect on HNE-induced JNK activation. In light of the JNK-dependent induction of c-jun transcription and the AP-1-induced transcription of xenobiotic-metabolizing enzymes, these data may show a potential critical role for JNK in the induction of a cellular defense program against toxic products generated from lipid peroxidation.
在本研究中，我们探究了大鼠肝上皮RL34细胞暴露于氧化脂肪酸代谢的主要终产物4-羟基-2-壬烯醛（HNE）后，参与c-Jun蛋白表达的信号转导机制。用HNE处理细胞导致细胞内谷胱甘肽（GSH）耗竭，并在质膜中形成蛋白结合的HNE。此外，HNE强烈诱导细胞内过氧化物生成，表明HNE对细胞施加了氧化应激。HNE处理后30分钟内即出现c-Jun的强力表达，同时激活蛋白-1（AP-1）的DNA结合活性呈时间依赖性增加。我们发现HNE使RL34细胞中的酪氨酸磷酸化立即增加。此外，HNE强烈诱导c-Jun氨基末端激酶（JNK）和p38丝裂原活化蛋白激酶的磷酸化，还适度诱导细胞外信号调节激酶的磷酸化。JNK的磷酸化伴随着JNK和p38活性的快速短暂增加，而细胞外信号调节激酶活性的变化几乎未观察到。L-丁硫氨酸-S，R-亚砜亚胺（一种GSH生物合成的特异性抑制剂）使GSH耗竭，仅轻微增强过氧化物生成和JNK激活，表明HNE发挥这些作用与GSH耗竭无关。这以及以下发现：（i）HNE强烈诱导细胞内过氧化物生成，（ii）用硫醇抗氧化剂N-乙酰半胱氨酸预处理细胞可抑制HNE诱导的JNK激活，以及（iii）H2O2显著激活JNK，支持了促氧化剂在HNE诱导的应激信号通路激活中起关键作用的假说。此外，我们发现，在酪氨酸激酶、环氧化酶和Ca2+内流抑制剂中，只有槲皮素对HNE诱导的JNK激活有显著抑制作用。鉴于c-jun转录依赖JNK以及AP-1诱导异源生物代谢酶转录，这些数据可能表明JNK在诱导针对脂质过氧化产生的有毒产物的细胞防御程序中具有潜在的关键作用。
