Activation of Stress Signaling Pathways by the End Product of Lipid Peroxidation
4-HYDROXY-2-NONENAL IS A POTENTIAL INDUCER OF INTRACELLULAR PEROXIDE PRODUCTION*

Koji UchidaDagger , Mihoko Shiraishi, Yuko Naito, Yasuyoshi Torii, Yoshimasa Nakamura§, and Toshihiko Osawa

From the Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, and the § Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

    ABSTRACT
Top
Abstract
Introduction
References

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.

    INTRODUCTION
Top
Abstract
Introduction
References

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 radical-initiated 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)1 that can be produced from arachidonic acid, linoleic acid, or their hydroperoxides in relatively large amounts at concentrations of 10 µM 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 beta 1 gene (8). HNE is a potent alkylating agent that reacts with a variety of nucleophilic sites in DNA and protein, generating various types of adducts (3), leading to the assumption that they cause a specific cellular stress response. This program may include transcriptional induction of proto-oncogenes, including c-jun. In fact, it has been demonstrated that the lipid hydroperoxide, a precursor of HNE, triggers cellular signaling pathways, leading to activation of activator protein-1 (AP-1) (9).


View larger version (11K):
[in this window]
[in a new window]
 
Scheme 1.   A proposed mechanism for the formation of HNE during peroxidation of arachidonate. Like other omega -6-polyunsaturated fatty acids, arachidonate is susceptible to free radical reaction at bis-allyl sites, e.g. C-8, to form a free radical intermediate, which further reacts with molecular oxygen to generate hydroperoxide intermediates. After intramolecular cyclization followed by oxidative C-C cleavages, HNE is produced. R, CH3(CH2)4-. R', -(CH2)3-COOH.

c-Jun is a member of a multiprotein family that has been implicated in a number of signal transduction pathways associated with cellular growth, differentiation, neuronal excitation, and cellular stress (10-14) and is thought to be required for cellular defense against toxicity (15). Induction of c-jun in response to genotoxic agents and cellular stress is mediated by two AP-1-like sites in its promoter that are recognized by c-Jun homodimers (16, 17), activation transcription factor-2 (ATF-2) homodimers, or, preferentially, c-Jun/ATF-2 heterodimers (18, 19). The transcriptional activity of these binding factors is regulated by protein kinases related to the mitogen-activated protein (MAP) kinase superfamily. To date, at least three different subtypes of MAP kinases are known (20-22). These are in turn activated by distinct upstream dual specificity kinases, thus revealing the existence of protein kinase modules that can be independently and simultaneously activated. Whereas mitogens and growth factors lead to activation of protein kinase cascades resulting in activation of extracellular signal-regulated kinase (ERK) family MAP kinases, many forms of cellular stress preferentially trigger two related signaling pathways (23-27). These center on two MAP kinases or Jun N-terminal kinases (JNKs) and p38, also termed stress-activated protein kinase and reactivating kinase, respectively. Targets of stress-activated protein kinase and JNK include several transcription factors such as c-Jun, JunD, ATF-2, and Elk-1, which become activated after exposure to cellular stresses (28-31).

In this study, we find that HNE strongly induces c-Jun expression in rat liver epithelial RL34 cells. To fully understand the signaling mechanism that triggered this response, the phosphorylation events that may lead to the activation of AP-1 transcription factors are investigated. The study reported here demonstrates that the most cytotoxic aldehyde, HNE, is a potential inducer of a signaling pathway that involves the JNK. The identification of the proto-oncoprotein AP-1 as a nuclear target of HNE signaling may provide new clues to the mutagenic and genotoxic potential of this aldehyde.

    EXPERIMENTAL PROCEDURES

Materials-- HNE was prepared by acid treatment (1 mM HCl) of HNE diethylacetal synthesized according to the procedure of De Montarby et al. (32), followed by purification with a reversed-phase high protein liquid chromatography. N-Acetylcysteine (NAC), L-buthionine-S,R-sulfoximine (BSO), herbimycin, and genistein were obtained from Sigma. AP-1, AP-2, and AP-3 consensus double-stranded oligonucleotides were obtained from Stratagene (La Jolla, CA). Indomethacin, aspirin, and quercetin were kindly provided by Dr. Y. Morimitsu of Nagoya University Graduate School of Bioagricultural Sciences.

Cell Culture-- RL34 cells were obtained from the Japanese Cancer Research Resources Bank (33). The cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal bovine serum, penicillin (100 units/ml), streptomycin (100 µg/ml), L-glutamine (588 µg/ml), and 0.16% NaHCO3 at 37 °C in an atmosphere of 95% air and 5% CO2. Cells postconfluency were exposed to the lipid peroxidation products in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum.

Glutathione Assay-- Measurement of GSH in the cells was performed fluorometrically according to the method of Hissin and Hilf (34). In brief, the cells incubated with HNE or NAC were washed twice with PBS (pH 7.0) and extracted with the 25% (w/v) metaphosphoric acid solution containing 5 mM EDTA. After ultracentrifugation (105,000 × g, 30 min), 1.8 ml of 0.1 M phosphate solution (pH 8.0) containing 5 mM EDTA and 100 µl of the o-phthalaldehyde solution (1 mg/ml) were added to the resulting supernatant (100 µl), and then the fluorescence intensity at 420 nm was determined with activation at 350 nm.

Intracellular Peroxide Production-- Cells were incubated with 50 µM 2',7'- dichlorofluorescin diacetate (DCF-DA) (dissolved in Me2SO) for 30 min at 37 °C and then treated with different agents for an additional 30 min at 37 °C. In the case of BSO, cells were treated with BSO for 6 h at 37 °C and then incubated with DCF-DA for an additional 30 min at 37  °C. After chilling on ice, cells were washed with ice-cold PBS, scraped from the plate, and resuspended at 1 × 106 cells/ml in PBS containing 10 mM EDTA. The fluorescence intensities of 2',7'- dichlorofluorescein formed by the reaction of DCF-DA with peroxides of more than 10,000 viable cells from each sample were analyzed by a flow cytometer (CytoACE 150, JASCO, Tokyo, Japan) with excitation and emission settings of 488 and 525 nm, respectively. Prior to data collection, propidium iodide was added to the sample for gating out dead cells. Experiments were repeated at least twice with similar results. The data are expressed as one representative histogram.

Western Blotting-- For detection of phosphotyrosine, whole cell lysates (200 µg of protein) were prepared in lysis buffer (1% Nonidet P-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1 mM Na3VO4, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 0.15 unit/ml aprotinin). Phosphotyrosine was monitored by SDS-PAGE and immunoblotting of 40 µg of whole cell extracts with the anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY). For detection of c-Jun, nuclear proteins were prepared as described previously (35). c-Jun was detected by SDS-PAGE and immunoblotting of 10 µg of nuclear extracts with the anti-c-Jun monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The activation of MAP kinases (JNK, p38, and ERK) was monitored by SDS-PAGE and immunoblotting of 40 µg of whole cell extracts with highly specific anti-phospho-(Thr-183/Tyr-185) JNK, anti-phospho-(Thr-180/Tyr-182) p38, or anti-phospho-(Tyr-204) ERK MAP kinase antibodies (New England Biolabs, Inc., Beverly, MA). These antibodies are directed against synthetic phosphopeptides corresponding to residues 179-193 (SFMMT*PY*VVTRYYR), 171-186 (LARHTDDEMT*GY*VATR), and 196-209 (DHTGFLTEY*VATRWC) of JNK, p38, and ERK, respectively. Asterisks represent phosphorylation sites.

Kinase Assays-- Whole cell lysates were prepared in lysis buffer (20 mM Tris (pH 7.5) 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerophosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin). For ERK assay, the lysates were incubated for 4 h at 4 °C with protein A-Sepharose plus 1 µg of anti-phospho-(Tyr-204)-p44/p42 ERK antibody (New England Biolabs), which recognizes activated ERK. The immunoprecipitate was washed as previously outlined and resuspended in 50 µl of kinase buffer containing 200 µM ATP and 2 µg GST-Elk1 fusion protein (New England Biolabs). The reactions were terminated after 30 min by the addition of Laemmli sample buffer (36), and the phosphorylation of GST-Elk1 was examined after SDS-PAGE and immunoblotting with anti-phospho-(Ser-383)-Elk1 antibody (New England BioLabs) by autoradiography. JNK activity was precipitated from whole cell lysates by incubating with 2 µg of GST-c-Jun-(1-89) fusion protein/GSH-Sepharose beads overnight at 4 °C (New England Biolabs). The beads were then washed twice in lysis buffer and twice in kinase buffer (25 mM Tris (pH 7.5), 5 mM beta -glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2) and resuspended in 50 µl of kinase buffer with 100 µM ATP for 30 min at 30 °C. The solid-phase kinase reaction was terminated by the addition of Laemmli sample buffer, and the phosphorylation of GST-c-Jun on Ser-63 was examined after SDS-PAGE and immunoblotting with anti-phospho-(Ser-63) c-Jun antibody (New England BioLabs). For p38 assay, the lysates were incubated with phosphorylated heat and acid-stable protein regulated by insulin (PHAS-I) and [gamma -32P]ATP for 10 min at 30 °C. The kinase reaction was terminated by the addition of Laemmli sample buffer, and the phosphorylation of PHAS-I was examined after SDS-PAGE.

Gel Shift Assay-- Nuclear proteins were prepared as previously reported (35). Binding reactions were 22-µl volumes containing 5 µg of nuclear proteins, 3 µg of poly(dI-dC), and [gamma -32P]ATP-labeled oligonucleotide probe (2 × 104 cpm). After 30 min, the DNA-protein complexes were resolved in a 6% nondenaturing acrylamide gel and electrophoresed at room temperature. The gel was dried and exposed to Kodak film at -80 °C overnight. The oligonucleotide probes used were as follows: AP-1, 5'-CTAGTGATGAGTCAGCCGGATC-3'; AP-2, 5'-GATCGAACTGACCGCCCGCGGCCCGT-3'; AP-3, 5'-CTAGTGGGACTTTCCACAGATC-3'.

In some experiments, 5 µg of nuclear extracts was incubated with 5 µg of highly specific anti-c-Jun, anti-Jun B, anti-Jun D, anti-c-Fos, or anti-Fra-1 antibodies (Santa Cruz Biotechnology) at 4 °C for 1 h and then further incubated with 32P-labeled DNA oligonucleotide for another 30 min. The subsequent supershift complexes were resolved in a 4.5% nondenaturing acrylamide gel and electrophoresed at room temperature as already described.

Immunocytochemistry-- For immunocytochemistry, cells were fixed overnight in PBS containing 42% paraformaldehyde and 0.2% picric acid at 4 OC. Membranes were permeabilized by exposing the fixed cells to PBS containing 0.3% Triton X-100. The cells were then incubated sequentially in PBS solutions containing blocking serum (5% normal swine serum) and primary antibody. The cells were then incubated for 1 h in the presence of fluorescein isothiocyanate-labeled antirabbit igG (Dako), rinsed with PBS containing 0.3% Triton X-100, and covered with anti-fade solution. Primary antibody that recognizes the HNE-protein epitopes was raised by immunizing New Zealand White rabbits with HNE-treated KLH (37). Images of cellular immunofluorescence were aquired using a confocal laser scanning microscope (FLUOROVIEW, Olympus Optical Co., Ltd., Tokyo) with a × 40 objective (488-nm excitation and 518-nm emission).

    RESULTS

HNE Is a Potential Inducer of Oxidative Stress-- Because HNE is a potent electrophile, we first examined the effect of HNE treatment on the change in the cellular redox status. As shown in Fig. 1A, a significant decrease in GSH levels was observed after the first 2 h of the HNE treatment. Interestingly, the amount of GSH began to recover and rose to above the basal level at 24 h, indicating that the cell responded to the GSH depletion. Because GSH is important in metabolism and enzyme regulation as well as detoxification of cytotoxic materials, the GSH depletion may lead to an increase in the susceptibility of the cell to insult by HNE. Indeed, the GSH depletion was associated with the HNE modification of cellular proteins. As shown in Fig. 1B, the protein-bound HNE was mainly detected in the plasma membrane at 30 min (b), and subsequently, the localization gradually shifted to the cytosol (c and d).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   HNE-induced redox alteration and formation of protein-bound HNE in RL34 cells. A, effect of HNE treatment on intracellular GSH levels. The cells were treated with 25 µM HNE for different time intervals as indicated. Intracellular GSH levels were fluorometrically measured as described under "Experimental Procedures." B, immunocytochemical detection of protein-bound HNE. a, untreated control; b, 30 min after HNE treatment; c, 1 h after HNE treatment; d, 4 h after HNE treatment. The cells were fixed in 2% paraformaldehyde and 0.2% picric acid and immunostained with polyclonal antibodies that specifically recognize protein-bound HNE. Images of cellular immunofluorescence were aquired using a confocal laser scanning microscope.

We also found that, in addition to GSH depletion and protein modification, HNE induced intracellular production of pro-oxidants. The production of pro-oxidants was determined using a peroxide-sensitive fluorescent probe, DCF-DA. This chemical is freely permeable to cells. Once inside the cells, it is hydrolyzed to DCF and trapped intracellularly. In the presence of peroxides, especialy H2O2, DCF is oxidized to fluorescent 2',7'-dichlorofluorescein, which can then be readily detected using fluorescence-activated cell sorting. To validate the assay, an experiment with H2O2 was performed. As shown in Fig. 2, treatment of the cells with H2O2 (100 µM) caused an increase in fluorescence intensity as compared with control. Treatment with BSO, a specific inhibitor of GSH biosynthesis, exerted only a slight increase in peroxide production, despite a considerable reduction of intracellular GSH level. In contrast, treatment with HNE (20 and 40 µM) strongly enhanced the peroxide-activated fluorescence intensity of DCF. When these findings are taken together, it is evident that the lipid peroxidation end product HNE is a potential inducer of intracellular oxidative stress.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Changes in intracellular peroxide level after exposure of RL34 cells to exogenous H2O2, BSO, or HNE. RL34 cells were incubated with the dye dichlorofluorescein diacetate (DCF-DA, 50 µM) for 30 min and then treated with 100 µM H2O2, 20 µM HNE, or 40 µM HNE for 30 min. In the case of BSO, cells were treated with 100 µM BSO for 6 h and then incubated with DCF-DA for 30 min. Cells were washed with PBS and resuspended in PBS containing 10 mM EDTA. The fluorescence intensity of more than 10,000 cells was analyzed using a flow cytometer.

HNE Induces c-Jun Expression and AP-1 DNA Binding Activity-- As the earliest events, HNE induced oxidative stress in the cells (Figs. 1 and 2). It was anticipated that this event could be the first signal that might be highly relevant to the onset of the stress response represented by the induction of immediate early genes. This was also suggested by the significant recovery of GSH levels after the depletion. Hence, we examined HNE for the effect on the expression of products of immediate early genes in RL34 cells. As shown in Fig. 3A, HNE dramatically stimulated the expression of c-Jun protein in a time-dependent manner. A maximum 10-fold increase in c-Jun expression in response to HNE was observed at 45 min after initiation of treatment, and this response persisted for at least 8 h. It is noted that HNE had no effect on the level of a wide variety of proteins, except c-Jun. Those failing to respond included other transcription factors responsive to various signals, such as c-Fos, JunB, JunD, Sp1, NF-1, and p53 (data not shown). These data suggest that induction of transcription factors by HNE is highly specific to c-Jun.


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 3.   HNE induces c-Jun expression and AP-1 DNA binding activity. RL34 cells were treated with 25 µM HNE for different time intervals as indicated. A, an equal amount of protein (40 µg) from nuclear extracts and analyzed by Western blotting with anti-c-Jun monoclonal antibody. B, nuclear proteins (5 µg) isolated from untreated control and HNE-treated cells were incubated with 32P-labeled AP-1 oligonucleotides and separated by 4.5% acrylamide gel electrophoresis. C, nuclear proteins (5 µg) isolated from the cells treated with HNE for 1 h were first incubated with antibodies to c-Jun, JunB, JunD, c-Fos, or Fra-1 for 1 h at 4 OC, and 32P-labeled AP-1 oligonucleotides were then added into each reaction and separated by 4.5% acrylamide gel electrophoresis.

Since c-Jun is the major constituent of the transcriptional factor AP-1 and HNE increased the expression of this proto-oncogene, we tested the effect of HNE on AP-1 activation in RL34 cells. AP-1 activity was measured by its ability to bind to the 12-O-tetradecanoylphorbol-13-acetate response element (TRE). Nuclear extracts of HNE-treated cells displayed 3-5-fold greater DNA binding activity than extracts of untreated cells, as determined by gel shift assay (Fig. 3B). Almost maximal increase in AP-1·TRE binding activity in response to HNE treatment was observed by 1 h, and these responses were persistent for at least 2 h. To identify the factors that make up HNE-inducible AP-1 activity, highly specific antibodies directed against individual AP-1 proteins were evaluated for the ability to deplete nuclear extracts of DNA binding activity. The extract used for this analysis was isolated from RL34 cells treated with HNE for 1 h. Antibodies against c-Jun, JunB, and JunD supershifted the HNE-induced AP-1·TRE complex, and anti-Fra-1 antibody significantly reduced the AP-1 binding activity (Fig. 3C), whereas the antibody against c-Fos had little effect. These data suggest that c-Jun, JunB, JunD, and Fra-1 may be important components responsible for AP-1 transactivation by HNE. Moreover, the addition of AP-2 or AP-3 consensus cold oligonucleotide had no effect on HNE-induced AP-1-TRE binding; however, the addition of excess cold AP-1 oligonucleotide probe to the reaction mixture abolished AP-1 binding to 32P-labeled TRE (data not shown). This observation clearly indicates that the AP-1-DNA binding activity observed in the nuclear extracts of HNE-treated cells is specific.

Induction of Tyrosine Phosphorylation by HNE-- To understand the signaling mechanism that triggered this response, we examined the phosphorylation events that may lead to the activation of AP-1 transcription factors. To investigate whether HNE can activate cellular protein kinases, the cells incubated with 25 µM HNE and tyrosine phosphorylation of cellular proteins were determined by Western blot analysis with anti-phosphotyrosine antibody. As shown in Fig. 4, several proteins with apparent molecular mass of 32, 38, 42, 57, and 90 kDa were the dominant proteins phosphorylated after incubation with HNE. The increase in tyrosine phosphorylation was detectable within 15 min during HNE treatment to at least 2 h after the treatment. The early increase in tyrosine phosphorylation led us to hypothesize that the activation of protein-tyrosine kinases is probably one of the upstream signals of the HNE-induced c-Jun expression in the cells. It is also noted that HNE induced an immediate dephosphorylation of a major tyrosine-phosphorylated 60-kDa protein. This may also represent the earliest event that leads to the induction of c-Jun (Fig. 3).


View larger version (93K):
[in this window]
[in a new window]
 
Fig. 4.   HNE-induced tyrosine phosphorylation. RL34 cells were treated with 25 µM HNE for different time intervals as indicated. An equal amount of protein (10 µg) from the cell lysates was analyzed by Western blotting with anti-phosphotyrosine antibodies. M, markers.

A number of transcription factors such as c-Jun and c-Fos have been shown to be phosphorylated in vitro by distinct members of the MAP kinase families triggered by a large variety of extracellular stresses. MAP kinases are important cellular signaling components that convert various extracellular signals into intracellular responses through serial phosphorylation cascades. At the present time, three distinct but parallel MAP kinase cascades have been identified in mammalian cells. It has been established that the activation of MAP kinases culminates in the phosphorylation of downstream cytosolic and nuclear substrates, ultimately leading to changes in gene expression. Based on the findings that HNE is a potential inducer of c-Jun expression, a possible involvement of MAP kinases in the HNE-induced c-Jun expression was examined. To determine the possibility that HNE treatment of the cells results in the phosphorylation of MAP kinases, whole cell lysates were probed with the antibodies specific for phosphorylated JNK, p38, and ERK. As shown in Fig. 5, HNE potently induced phosphorylation of p46 and p54 JNK (A) and p38 (B) and moderately induced phosphorylation of ERK (ERK1 and ERK2) (C). The HNE-induced phosphorylation of JNK and p38 reached a maximum within 30 min and returned to the basal levels at 8 h.


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 5.   Phosphorylation of JNK (A), p38 (B), and ERK (C). RL34 cells were treated with 25 µM HNE for different time intervals as indicated. Phosphorylation of ERK (A), JNK (B), and p38 (C) was monitored by Western blotting of 410 µg of whole cell extracts with highly specific anti-phospho-(Thr-183/Tyr-185) JNK, anti-phospho-(Thr-180/Tyr-182) p38, or anti-phospho-(Tyr-204) ERK MAP kinase antibodies.

HNE Activates JNK and p38 but Not ERK-- To verify that the HNE treatment of the cells resulted in a functional increase in JNK, p38, and ERK activities, we assayed for their activities in the lysates of the cells. As shown in Fig. 6, A and B, the activation of JNK and p38 by HNE began within 30 min and continued for at least 24 h. The activation of ERK by HNE was examined, but no significant change in the substrate phosphorylation was observed (Fig. 5C). The HNE-induced activation of JNK was scarcely affected by actinomycin D, an inhibitor of RNA synthesis, and cycloheximide, an inhibitor of protein sysnthesis (data not shown), indicating that the HNE-mediated JNK activation is not the result of the activation of gene expression.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 6.   Activation of JNK and p38, but not ERK, by HNE. RL34 cells were treated with 25 µM HNE for different time intervals as indicated. Cell lysis and kinase assays were performed as described under "Experimental Procedures." A, JNK assay, showing the phosphorylation of GST-c-Jun. B, p38 assay, showing the phosphorylation of PHAS-I. C, ERK assay, showing the phosphorylation of GST-Elk-1.

The HNE-induced JNK activation was significantly inhibited by a thiol antioxidant NAC (Fig. 7, A and B), whereas BSO that caused a considerable reduction of GSH level only slightly activated JNK (Fig. 7C). This and the observation that GSH depletion by treatment with BSO resulted in a slight increase in intracellular peroxide level (Fig. 2) suggest that the HNE-induced JNK activation may be independent of GSH depletion, whereas the findings that HNE strongly induced intracellular peroxide production (Fig. 2) and H2O2 treatment of the cells significantly activated JNK (Fig. 7D) suggest the involvement of intracellular peroxide production in the JNK activation.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of NAC on HNE-induced JNK activation and H2O2-induced transient activation of JNK. A, effect of NAC treatment on intracellular GSH levels. RL34 cells were incubated with 0-15 mM NAC for 24 h, and intracellular GSH levels were fluorometrically measured as described under "Experimental Procedures." B, effect of NAC pretreatment on the HNE-induced JNK activation. The cells incubated with 0-15 mM NAC for 24 h were treated with 25 µM HNE for 1 h. C, effect of NAC and BSO on JNK activation. The cells were incubated with 10 mM NAC or 1 mM BSO for 6 h. D, effect of H2O2 on JNK activation. The cells were incubated with 40 µM H2O2 for different time intervals as indicated. In B-D, JNK activity in cell lysates was determined by an immunocomplex kinase assay as described under "Experimental Procedures" using GST-c-Jun as the substrate.

Quercetin Is a Specific Inhibitor for the HNE-induced JNK Activation-- Because tyrosine kinases have been shown to be involved in a variety of cellular responses, such as growth factors, oxidative stress, and G protein-coupled signaling cascades (38-40), the effect of tyrosine kinase inhibitors upon HNE-induced JNK activation was tested. As shown in Fig. 8, both herbimycin and genistein that exhibit specific inhibitory activity against src family tyrosine kinases were ineffective; however, the HNE-induced JNK activation was significantly inhibited by quercetin. The results suggest that the src family tyrosine kinases may not be involved in HNE-induced JNK activation and that HNE may activate JNK via activation of protein kinase C, phosphorylase kinase, Na+,K+-ATPase, and/or Ca2+,Mg2+-ATPase, which have been reported to be inhibited by quercetin (41).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of inhibitors on HNE-induced JNK activation. RL34 cells were incubated with inhibitors for 30 min and then treated with 25 µM HNE for 1 h. JNK activity in cell lysates was determined by an immunocomplex kinase assay as described under "Experimental Procedures" using GST-c-Jun as the substrate.

On the other hand, HNE was previously reported to stimulate the Ca2+ influx in rat hepatocytes, leading to the activation of phospholipase C (42, 43). To evaluate the contributions of Ca2+-mediated cell signaling on JNK activation by HNE, cells were treated with the GdCl3, which is known to block the activity of store-operated Ca2+ channels in the hepatocyte plasma membrane, or phospholipase C inhibitor U73122, followed by stimulation with HNE; however, neither of the inhibitors significantly influenced HNE-induced JNK activity (data not shown). The cyclooxygenase inhibitors indomethacin and aspirin were also ineffective (data not shown), suggesting that eicosanoid biosynthesis is not involved in the HNE-induced JNK activation.

    DISCUSSION

In the present study, we have found that HNE is a potential source of intracellular pro-oxidants. As far as we know, this is the first report that clearly demonstrates the intracellular peroxide production by HNE. This finding is consistent with previous studies showing that HNE itself induces lipid peroxidation, as indicated by increased levels of malondialdehyde (44). It is therefore likely that, in addition to GSH depletion and protein modification, the lipid peroxidation end product HNE exerts an increased intracellular peroxide production that further potentiates lipid peroxidation in the cells, while the detailed mechanism for HNE-induced peroxide production is currently unknown.

The major finding of this study is that HNE appears to be a potential inducer of stress signaling pathways. The first experimental evidence that suggested the involvement of HNE-induced signal transduction was the detection of potent and transient expression of transcription factor c-Jun (Fig. 3). It was assumed that activation of protein kinases induced by HNE might cause activation of the signaling pathway, which phosphorylated and activated c-Jun. Being the major component of the AP-1 transcription factor, c-Jun, once activated, can subsequently activate the transcription of several genes. The fact that the c-jun gene itself also contains an AP-1 element in its promoter indicates that phosphorylation of c-Jun allows signals in this pathway to be amplified. First, phosphorylation of c-Jun activates the transcription of c-jun per se, increasing the amount of available c-Jun for the formation of the AP-1 complex. Second, phosphorylation of c-Jun also increases the activity of the newly formed AP-1 factors, allowing the AP-1 transcriptional activity to be further activated. Activation of AP-1 is regulated by complex mechanisms which consist of distinct effects on the preexisting AP-1 complex or on the de novo synthesis of AP-1 subunits.

It has been revealed that ERKs, which are primarily activated in response to growth factor stimulation, are implicated in processes of cellular proliferation (45-48) and differentiation (49, 50), whereas JNK and p38 MAP kinases are characterized by their strong response to cellular stresses such as UV light (51), osmotic stress (25), DNA damaging agents (52), and proinflammatory cytokines (53). Activation of JNK has also been implicated in inducing apoptosis in response to growth factor withdrawal and other environmental stimuli (54, 55) and in stimulating cell proliferation and transformation (56). We found in the present study that a different kind of stress, HNE, also stimulated JNK and p38 (Fig. 6A), which might be directly responsible for the induction of c-Jun expression. In addition to the large body of data demonstrating stimulation of cell signaling, our present findings add HNE to a growing list of chemicals that can trigger stress signaling pathways mediated by JNK and p38 MAP kinases.

It has been shown that the activation of JNK and p38 is dependent on an upstream tyrosine/threonine kinase. One potential upstream kinase responsible for their phosphorylations is MAP kinase kinase 4, which itself must be serine/threonine-phosphorylated for activity (57, 58) and activates both JNK and p38 (58, 59); however, our preliminary experiment demonstrated that HNE does not induce phosphorylation of MAP kinase kinase 4 (data not shown). This implies that HNE acts through a different upstream regulatory element than MAP kinase kinase 4. A similar MAP kinase kinase 4-independent activation of JNK has also been seen in human glomerular mesangial cells treated with diamide or hydrogen peroxide (60). Thus, it remains to be determined whether HNE activates JNK via a known pathway or through an alternative signaling cascade.

Oxidative stress has been reported to be involved in the activation of MAP kinases (60-62). Although the primary target of HNE is still unknown, there is evidence that the intracellular level of GSH, regulating the redox state of the cell (63), may be an important sensor for the initiation of the cellular response to various compounds. In fact, intracellular GSH levels of RL34 cells were readily reduced by treatment with HNE in a time-dependent manner (Fig. 1A). These data led to the assumption that intracellular GSH is a critical parameter for the stress signaling cascade induced by HNE. However, BSO itself did not so effectively enhance intracellular peroxide production (Fig. 2) and only slightly activated JNK (Fig. 7C). These data suggest that the HNE-induced JNK activation may not simply result from GSH depletion alone, whereas the results that HNE strongly induced intracellular peroxide production (Fig. 2) and the HNE-induced JNK activation was significantly blocked by a thiol antioxidant NAC (Figs. 7, A and B) suggest the involvement of pro-oxidants on JNK activation. This was also supported by the result that H2O2 treatment of the cells resulted in a significant activation of JNK (Fig. 7D). Taken together, it is proposed that pro-oxdidants such as peroxides are involved in the HNE-induced activation of stress signaling pathways.

It is noteworthy that the HNE-induced JNK activation was significantly inhibited by quercetin (Fig. 8), which is one of most widely distributed bioflavonoids in the plant kingdom and is a component of most edible fruits and vegetables. It has been demonstrated that quercetin inhibits the growth of malignant cells through various mechanisms: inhibition of glycolysis, macromolecule synthesis and enzymes, freezing cell cycle, and interaction with estrogen type II binding sites (41). In addition, the flavone inhibits the induction of heat shock proteins and thermotolerance without affecting other protein synthesis. The exact mechanisms responsible for the inhibitory effect of quercetin on HNE-induced JNK activation is not yet thoroughly understood, but it is possible that quercetin exerts the activity by inhibiting enzymes involved in the HNE-induced stress signaling pathways. In this regard, it is notable that quercetin is a potential inhibitor of protein kinase C (41). This and the evidence that protein kinase C is activated by H2O2 (64) suggest that HNE-induced peroxide production and the consequential activation of protein kinase C and/or other protein kinases may contribute to the activation of the stress signaling pathways (Scheme 2).


View larger version (10K):
[in this window]
[in a new window]
 
Scheme 2.   A proposed mechanism for the activation of stress signaling pathways by HNE.

It has been suggested that the induction of JNK followed by c-Jun expression may be an important mechanism for controlling the genetic program which cells use to actively and immediately respond to various extracellular stimuli by the induction of proteins that are ultimately responsible for the detoxification of cytotoxic xenobiotics. Previous studies have shown that the expression of many phase II detoxifying enzyme genes is regulated by the AP-1 and AP-1-like elements. In early studies of the stress response system, a wide variety of phase II detoxifying enzymes inducers was found to be electrophiles. The transcriptional activation of response genes by these compounds has been traced to a cis-acting transcriptional enhancer called the antioxidant response element, which has been detected in the promoters of various phase II detoxifying enzymes, including rat and mouse glutathione S-transferase A-1 (65), rat glutathione S-transferase P-1 (66), and NAD(P)H:quinone oxidoreductase (67). Indeed, HNE induced a significant increase in specific binding to the antioxidant response element from the rat glutathione S-transferase P-1 gene, leading to the induction of gene expression (68). Moreover, our preliminary experiments have shown that HNE is a potential inducer of phase II detoxifying enzyme genes including a rate-limiting enzyme of GSH biosynthesis, gamma -glutamylcysteine synthetase.2 Therefore, the HNE-induced gene expression of this enzyme may be responsible for the recovery and subsequent increase in the GSH levels (Fig. 1A). The rise in GSH and glutathione S-transferase contents may allow inactivation of HNE and therefore contribute to the progressive down-regulation of JNK activity poststimulation (Fig. 6A). Our data suggest that the induction of JNK is an important mechanism for controlling the genetic program that cells use to actively and immediately respond to environmental stimuli by the induction of proteins that are responsible for the detoxification of cytotoxic xenobiotics.

Alkylating agents including HNE are versatile mutagens and/or carcinogens. Once formed, they induce diverse aspects of severe cellular stress, including chromosomal aberrations, sister chromatid exchanges, point mutations, and cell killing. These agents react with a variety of nucleophilic sites in DNA and protein, generating various types of adducts. Upon reaction with protein, HNE selectively reacts with the imidazole moiety of histidine residues, the epsilon -amino group of lysine residues, and the sulfhydryl group of cysteine residues (69-71). The primary products are simple Michael addition-type products, which further undergo cyclization between the aldehyde moiety and the C-4 position of HNE to form a hemiacetal structure. Although the primary target molecule resposible for the initiation of HNE-induced signal transduction remains to be identified, it is assumed that the induction of stress-activated cell signaling by HNE may be closely associated with the modification of cellular proteins (Fig. 1B). It has been shown that HNE exogenously added to the cells or endogenously generated in the cells binds to different proteins and impairs their function; examples include the Na+,K+-ATPase (72), neuronal glucose transporter GLUT3 (73), the astrocyte glutamate transporter GLT-1 (44), and the GTP-binding protein Galpha q/11 (74). HNE is relatively stable and can pass among subcellular compartments; thereby, it has the potential to interact with many different cell proteins including Tau (75). Furthermore, the presence of protein-bound HNE in vivo has been assessed in various human tissue samples, including the human aorta with atherosclerotic lesions (76), nigral neurons in Parkinson's disease (77), renal cell carcinomas (78), amyloid deposits in systemic amiloidosis (79), and trophoblast cells of preeclamptic placentas (80).

In summary, this report demonstrates that the potential protein alkylating agent HNE strongly induces peroxide production and activates JNK and p38 MAP kinases in RL34 cells. We have shown that HNE is not only a potential inducer of tyrosine phosphorylation but also a specific activator of JNK and p38 cascades, which may phosphorylate and activate c-Jun. The HNE-mediated activation of c-Jun may eventually lead to the up-regulation of many AP-1-dependent genes. We are currently investigating the further upstream cell signaling induced by HNE. Our future challenge is to define a target molecule that triggers signal transduction pathways leading to JNK activation and to define the biological significance of the activation of these stress signaling pathways mediated by HNE. Studies focusing on these biochemical steps would extend our understanding of the regulation of stress signaling cascades stimulated by various reactive products generated during lipid peroxidation.

    FOOTNOTES

* This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan. Fax: 81-52-789-5741; E-mail: uchidak{at}agr.nagoya-u.ac.jp.

The abbreviations used are: HNE, 4-hydroxy-2-nonenal; MAP, mitogen-activated protein; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; AP, activator protein; ATF-2, activation transcription factor-2; TRE, 12-O-tetradecanoylphorbol-13-acetate response element; NAC, N-acetylcysteine; BSO, L-buthionine-S,R-sulfoximine; GST, glutathione S-transferase; DCF-DA, 2',7'-dichlorofluorescin diacetate; PHAS-I, phosphorylated heat and acid-stable protein regulated by insulin.

2 K. Uchida, M. Shiraishi, Y. Naito, Y. Torii, Y. Nakamura, and T. Osawa, unpublished data.

    REFERENCES
Top
Abstract
Introduction
References

  1. Halliwell, B., and Gutteridge, J. M. C. (1989) Free Radicals in Biology and Medicine, 2nd Ed., Clarendon Press, Oxford
  2. Poli, G., Albano, E., and Dianzani, M. U. (1987) Chem. Phys. Lipids 45, 117-142[Medline] [Order article via Infotrieve]
  3. Esterbauer, H., Schauer, R. J., and Zollner, H. (1991) Free Radicals Biol. Med. 11, 81-128[CrossRef][Medline] [Order article via Infotrieve]
  4. Fazio, V. M., Barrera, G., Martinotti, S., Farace, M. G., Giglioni, B., Frati, L., Manzari, V., and Dianzani, M. U. (1992) Cancer Res. 52, 4866-4871[Abstract]
  5. Parola, M., Pinzani, M., Casini, A., Albano, E., Poli, G., Gentilini, A., Gentilini, P., and Dianzani, M. U. (1993) Biochem. Biophys. Res. Commun. 194, 1044-1050[CrossRef][Medline] [Order article via Infotrieve]
  6. Spycher, S., Tabataba-Vakili, S., O'Donnell, V. B., Palomba, L., and Azzi, A. (1996) Biochem. Biophys. Res. Commun. 226, 512-516[CrossRef][Medline] [Order article via Infotrieve]
  7. Barrera, G., Pizzimenti, S., Serra, A., Ferretti, C., Fazio, V. M., Saglio, G., and Dianzani, M. U. (1996) Biochem. Biophys. Res. Commun. 227, 589-593[CrossRef][Medline] [Order article via Infotrieve]
  8. Leonarduzzi, G., Scavazza, A., Biasi, F., Chiarpotto, E., Camandola, S., Vogl, S., Dargel, R., and Poli, G. (1997) FASEB J. 11, 851-857[Abstract/Free Full Text]
  9. Rao, G., Glasgow, W. C., Eling, T. E., and Runge, M. S. (1996) J. Biol. Chem. 271, 27760-27764[Abstract/Free Full Text]
  10. Angel, P., and Karin, M. (1991) Biochim. Biophys. Acta 1072, 129-157[CrossRef][Medline] [Order article via Infotrieve]
  11. Devary, Y., Gottlieb, R. A., Smeal, T., and Karin, M. (1992) Cell 71, 1081-1091[Medline] [Order article via Infotrieve]
  12. Holbrook, N. J., and Fornace, A. J., Jr. (1991) New Biol. 3, 825-833[Medline] [Order article via Infotrieve]
  13. Morgan, J. I., and Curren, T. (1991) Annu. Rev. Neurosci. 14, 421-451[CrossRef][Medline] [Order article via Infotrieve]
  14. Johnson, R. S., van Lingen, B., Papaioannou, V. E., and Spiegelman, B. M. (1993) Genes Dev. 7, 1309-1317[Abstract]
  15. Schreiber, M., Baumann, B., Cotten, M., Angel, P., and Wagner, E. F. (1995) EMBO J. 14, 5338-5349[Abstract]
  16. Angel, P., Hattori, K., Smeal, T., and Karin, M. (1988) Cell 55, 875-885[Medline] [Order article via Infotrieve]
  17. Karin, M. (1995) J. Biol. Chem. 270, 16483-16486[Free Full Text]
  18. van Dam, H. D., Duyndam, M., Rottier, R., Bosch, A., de Vries-Smits, L., Herrlich, P., Zantema, A., Angel, P., and van der Eb, A. J. (1993) EMBO J. 12, 479-487[Abstract]
  19. van Dam, H., Wilhelm, D., Herr, I., Steffen, A., Herrlich, P., and Angel, P. (1995) EMBO J. 14, 1798-1811[Abstract]
  20. Cahill, M. A., Janknecht, R., and Nordheim, A. (1996) Curr. Biol. 6, 16-19[Medline] [Order article via Infotrieve]
  21. Treisman, R. (1996) Curr. Biol. 8, 205-215
  22. Su, B., and Karin, M. (1996) Curr. Opin. Immunol. 8, 402-411[CrossRef][Medline] [Order article via Infotrieve]
  23. Minden, A., Lin, A., Claret, F.-X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157[Medline] [Order article via Infotrieve]
  24. Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146[Medline] [Order article via Infotrieve]
  25. Galcheva-Gargova, Z., Derijard, B., Wu, J.-H., and Dadis, R. J. (1994) Science 265, 806-808[Medline] [Order article via Infotrieve]
  26. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156-160[CrossRef][Medline] [Order article via Infotrieve]
  27. Freshney, N. W., Rawlinson, L., Guesdon, F., Jones, E., Cowley, S., Hsuan, J., and Saklatvala, J. (1994) Cell 78, 1039-1049[Medline] [Order article via Infotrieve]
  28. Livingstone, C., Patel, G., and Jones, N. (1995) EMBO J. 14, 1785-1797[Abstract]
  29. Gupta, S., Campbell, D., Derijard, B., and Davis, R. J. (1995) Science 267, 389-393[Medline] [Order article via Infotrieve]
  30. Cavigelli, M., Dolfi, F., Claret, F.-X., and Karin, M. (1995) EMBO J. 14, 5957-5964[Abstract]
  31. Whitmarsh, A. J., Shore, P., Sharrocks, A. D., and Davis, R. J. (1995) Science 269, 1760-1763
  32. De Montarby, L., Mosset, P., and Gree, R. (1988) Tetrahedron Lett. 29, 3895[CrossRef]
  33. Yamada, M., Okigaki, T., and Awai, M. (1987) Cell Struct. Funct. 12, 53-62[Medline] [Order article via Infotrieve]
  34. Hissin, P. J., and Hilf, R. (1976) Anal. Biochem. 74, 214-216[Medline] [Order article via Infotrieve]
  35. Schreiber, E., Matthias, P., Müller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Medline] [Order article via Infotrieve]
  36. Laemmli, U. K. (1970) Nature 277, 680-685
  37. Uchida, K., Szweda, L. I., Chae, H. Z., and Stadtman, E. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8742-8746[Abstract/Free Full Text]
  38. Gould, K., and Hunter, T. (1988) Mol. Cell. Biol. 8, 3345-3356[Medline] [Order article via Infotrieve]
  39. Mukhopadhyay, D., Tsiokas, L., Zhou, X., Foster, D., Brugge, J. S., and Suhatme, V. P. (1995) Nature 375, 577-581[CrossRef][Medline] [Order article via Infotrieve]
  40. Wan, Y., Kurosaki, T., and Huang, X. Y. (1996) Nature 380, 541-544[CrossRef][Medline] [Order article via Infotrieve]
  41. Formica, J. V., and Regelson, W. (1995) Food Chem. Toxicol. 33, 1061-1080[CrossRef][Medline] [Order article via Infotrieve]
  42. Carini, R., Bellemo, G., Paradisi, L., Dianzani, M. U., and Albano, E. (1996) Biochem. Biophys. Res. Commun. 218, 772-776[CrossRef][Medline] [Order article via Infotrieve]
  43. Rossi, M. A., Fidale, F., Garramore, A., Esterbauer, H., and Dianzani, M. U. (1990) Biochem. Phamacol. 39, 1715-1719
  44. Keller, J. N., Mark, R. J., Bruce, A. J., Blanc, E. M., Rothstein, J. D., Uchida, K., and Mattson, M. P. (1997) Neuroscience 80, 685-696[CrossRef][Medline] [Order article via Infotrieve]
  45. Boulton, T. G., Nye, S. H., Robbins, D. J., Ip, N. Y., Radziejewska, E., Morgenbesser, S. D., DePinho, R. A., Panayotatos, N., Cobb, M. H., and Yancopoulos, G. D. (1991) Cell 65, 663-675[Medline] [Order article via Infotrieve]
  46. Thomas, G. (1992) Cell 68, 3-6[Medline] [Order article via Infotrieve]
  47. Miltenberger, R. J., Cortner, J., and Farnham, P. J. (1993) J. Biol. Chem. 268, 15674-15680[Abstract/Free Full Text]
  48. Pages, G., Lenormand, P., L'Allemain, G., Chambard, J. C., Meloche, S., and Pouyssegur, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8319-8323[Abstract/Free Full Text]
  49. Han, J., Lee, I.-D., Tobias, P. S., and Ulevitch, R. J. (1993) J. Biol. Chem. 268, 25009-25014[Abstract/Free Full Text]
  50. Alberola-Ila, J., Forbush, K. A., Seger, R., Krebs, E. C., and Perlmutter, R. M. (1995) Nature 373, 620-623[CrossRef][Medline] [Order article via Infotrieve]
  51. Rosette, C., and Karin, M. (1996) Science 274, 1194-1197[Abstract/Free Full Text]
  52. Kharbanda, S., Ren, R., Pandey, P., Shafman, T. D., Feller, S. T., Weichselbaum, R. R., and Kufe, D. W. (1995) Nature 376, 785-788[CrossRef][Medline] [Order article via Infotrieve]
  53. Sluss, H. K., Barrett, T., Derijard, B., and Davis, R. J. (1994) Mol. Cell. Biol. 14, 8376-8384[Abstract]
  54. Chen, Y.-R., Wang, X., Templeton, D., Davis, R. J., and Tan, T.-H. (1997) J. Biol. Chem. 271, 31929-31936[Abstract/Free Full Text]
  55. Xia, Z., Dikens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) Science 270, 1326-1331[Abstract]
  56. Clark, G. J., Westwick, J. K., and Der, C. J. (1997) J. Biol. Chem. 272, 1677-1681[Abstract/Free Full Text]
  57. Yan, M., Dai, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and Templeton, D. J. (1994) Nature 372, 798-800[Medline] [Order article via Infotrieve]
  58. Lin, A., Minden, A., Martinetto, H., Claret, F. X., Lange-Carter, C., Mercurio, F., Fohnson, G. L., and Karin, M. (1995) Science 268, 286-290[Medline] [Order article via Infotrieve]
  59. Derijard, B., Raingeaud, J., Barrett, T., Wu, I. H., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) Science 267, 682-685[Medline] [Order article via Infotrieve]
  60. Wilmer, W. A., Tan, L. C., Dickerson, J. A., Danne, M., and Rovin, B. H. (1997) J. Biol. Chem. 272, 10877-10881[Abstract/Free Full Text]
  61. Guyton, K. Z., Liu, Y., Gorospe, M., Xu, Q., and Holbrook, N. J. (1996) J. Biol. Chem. 271, 4138-4142[Abstract/Free Full Text]
  62. Tourinier, C., Thomas, G., Pierre, J., Jacquemin, C., Pierre, M., and Saunier, B. (1997) Eur. J. Biochem. 244, 587-595[Abstract]
  63. Meister, A. (1983) Science 220, 472-477[Medline] [Order article via Infotrieve]
  64. Konishi, H., Tanaka, M., Takemura, Y., Matsuzaki, H., Ono, Y., Kikkawa, U., and Nishizuka, Y. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11233-11237[Abstract/Free Full Text]
  65. Rushmore, T. H., King, R. G., Paulson, K. E., and Pickett, C. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3826-3830[Abstract]
  66. Okuda, A., Imagawa, M., Maeda, Y., Sakai, M., and Muramatsu, M. (1989) J. Biol. Chem. 264, 16919-16926[Abstract/Free Full Text]
  67. Li, Y., and Jaiswal, A. K. (1992) J. Biol. Chem. 267, 15097-15104[Abstract/Free Full Text]
  68. Fukuda, A., Nakamura, Y., Ohigashi, H., Osawa, T., and Uchida, K. (1997) Biochem. Biophys. Res. Commun. 236, 505-509[CrossRef][Medline] [Order article via Infotrieve]
  69. Uchida, K., and Stadtman, E. R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4544-4548[Abstract]
  70. Uchida, K., and Stadtman, E. R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5611-5615[Abstract]
  71. Uchida, K., and Stadtman, E. R. (1993) J. Biol. Chem. 268, 6388-6393[Abstract/Free Full Text]
  72. Mark, R. J., Lovell, M. A., Markesbery, W. R., Uchida, K., and Mattson, M. P. (1997) J. Neurochem. 68, 255-264[Medline] [Order article via Infotrieve]
  73. Mark, R. J., Pang, Z., Geddes, J. W., Uchida, K., and Mattson, M. P. (1997) J. Neurosci. 17, 1046-1054[Abstract/Free Full Text]
  74. Blanc, E. M., Kelly, J. F., Mark, R. J., and Mattson, M. P. (1997) J. Neurochem. 69, 570-580[Medline] [Order article via Infotrieve]
  75. Mattson, M. P., Fu, W., Waeg, G., and Uchida, K. (1997) NeuroReport 8, 2275-2281[Medline] [Order article via Infotrieve]
  76. Uchida, K., Itakura, K., Kawakishi, S., Hiai, H., Toyokuni, S., and Stadtman, E. R. (1995) Arch. Biochem. Biophys. 324, 241-248[CrossRef][Medline] [Order article via Infotrieve]
  77. Yoritaka, A., Hattori, N., Uchida, K., Tanaka, M., Stadtman, E. R., and Mizuno, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2696-2701[Abstract/Free Full Text]
  78. Okamoto, K., Toyokuni, S., Uchida, K., Ogawa, O., Takenawa, J., Kakehi, Y., Kinoshita, H., Hattori-Nakakuki, Y., Hiai, H., and Yoshida, O. (1994) Int. J. Cancer 58, 825-829[Medline] [Order article via Infotrieve]
  79. Ando, Y., Nyhlin, N., Suhr, O., Holmgren, G., Uchida, K., El Sahly, M., Yamashita, T., Terasaki, H., Nakamura, M., Uchino, M., and Ando, M. (1997) Biochem. Biophys. Res. Commun. 232, 497-502[CrossRef][Medline] [Order article via Infotrieve]
  80. Morikawa, S., Kurauchi, O., Tanaka, M., Yoneda, M., Uchida, K., Itakura, A., Furugori, K., Kuno, N., Mizutani, S., and Tomoda, Y. (1997) Biochem. Mol. Biol. Int. 41, 767-775[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.