Activation of Stress Signaling Pathways by the End Product of Lipid Peroxidation

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 proteinbound 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 timedependent 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 signalregulated 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 Ca 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. Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids and has been implicated in the pathogenesis of numerous diseases including atherosclerosis, diabetes, cancer, and rheumatoid arthritis as well as in drug-associated toxicity, postischemic reoxygenation injury, and aging (1). The peroxidative breakdown of polyunsaturated fatty acids has also been implicated in the pathogenesis of many types of liver injury and especially in the hepatic damage induced by several toxic substances. Among these are the haloalkanes, carbon tetrachloride, trichlorobromomethane, chloroform, dibromoethane, and halothane; in addition, paracetamol, bromobenzene, iron, bipyridyl compounds, allyl alcohol, and in some instances ethanol have been shown to stimulate lipid peroxidation (2). There is increasing evidence that aldehydes generated endogenously during the process of lipid peroxidation are causally involved in most of the pathophysiological effects associated with oxidative stress in cells and tissues (3). Compared with free radicals, lipid peroxidation-derived aldehydes are generally stable and can diffuse within or even escape from the cell and attack targets far from the site of the original free radicalinitiated event, therefore suggesting that they are not only end products and remnants of lipid peroxidation processes but also may act as mediators for the primary free radicals that initiated lipid peroxidation. Among the lipid peroxidation-derived aldehydes, 4-hydroxy-2-nonenal (HNE) that can be produced from arachidonic acid, linoleic acid, or their hydroperoxides in relatively large amounts at concentrations of 10 mM to 5 mM in response to oxidative insults is believed to be largely responsible for the cytopathological effects observed during oxidative stress in vivo (Scheme 1) (3). HNE exhibits a wide range of biological activities including inhibition of protein and DNA synthesis, inactivation of enzymes, stimulation of phospholipase C, reduction of gap-junction communication, and stimulation of neutrophil migration (3). In addition, HNE modulates the expression of various genes, including c-myc and globin genes (4), procollagen type I (5) and aldolase reductase (6) genes, c-myb (7), and transforming growth factor b1 gene (8). HNE is a potent alkylating agent that reacts with a variety of