ToxSci Advance Access originally published online on December 1, 2005
Toxicological Sciences 2006 90(1):120-132; doi:10.1093/toxsci/kfj055
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Resveratrol Protects Against 4-Hydroxynonenal-Induced Apoptosis by Blocking JNK and c-JUN/AP-1 Signaling
* Biological Sciences and Bioengineering Program, Sabanci University, 34956 Orhanli, Tuzla Istanbul, Turkey; and Department of Clinical and Biological Sciences,University of Turin at St. Luigi Gonzaga Hospital 10043-Orbassano, Turin, Italy
1 To whom correspondence should be addressed at Sabanci University Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, 34956 Orhanli, Tuzla Istanbul-Turkey. Fax: + 90 216 483 9550. E-mail: huveyda{at}sabanciuniv.edu.
Received July 1, 2005; accepted November 12, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study we have studied the effect of resveratrol in signal transduction mechanisms leading to apoptosis in 3T3 fibroblasts when exposed to 4-hydroxynonenal (HNE). In order to gain insight into the mechanisms of apoptotic response by HNE, we followed MAP kinase and caspase activation pathways; HNE induced early activation of JNK and p38 proteins but downregulated the basal activity of ERK
. We were also able to demonstrate HNE-induced release of cytochrome c from mitochondria, caspase-9, and caspase-3 activation. Resveratrol effectively prevented HNE-induced JNK and caspase activation, and hence apoptosis. Activation of AP-1 along with increased c-Jun and phospho-c-Jun levels could be inhibited by pretreatment of cells with resveratrol. Moreover, Nrf2 downregulation by HNE could also be blocked by resveratrol. Overexpression of dominant negative c-Jun and JNK1 in 3T3 fibroblasts prevented HNE-induced apoptosis, which indicates a role for JNK-c-Jun/AP-1 pathway. In light of the JNK-dependent induction of c-Jun/AP-1 activation and the protective role of resveratrol, these data may show a critical potential role for JNK in the cellular response against toxic products of lipid peroxidation. In this respect, resveratrol acting through MAP kinase pathways and specifically on JNK could have a role other than acting as an antioxidant-quenching reactive oxygen intermediate. Key Words: 4-hydroxynonenal; lipid peroxidation; apoptosis; AP-1; MAP kinases; resveratrol.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is increasing evidence that aldehydes generated endogenously during the process of lipid peroxidation are casually involved in most pathophysiological effects associated with oxidative stress in cells and tissues. 4-Hydroxyonenal (HNE) is a highly reactive and stable end product of lipid oxidative degradation of arachidonic acid, linoleic acid, or their hydroperoxides (Esterbauer et al., 1991
; Schneider et al., 2001). HNE induces apoptosis in a variety of cells from different origins, induces various enzymes including protein kinase c (PKC), phospholipase C and A, adenylate cyclase, and stress-activated protein kinases involved in signal transduction cascades (Parola et al., 1999; Poli et al., 2004; Poli and Schaur, 2000). HNE concentration increases during oxidative stress, and HNE in varying amounts is present in all cell lines and tissues. In addition, HNE modulates the expression of various genes including c-myc and globin genes, procollagen type I, c-myb, and the transforming growth factor B1 gene (Barrera et al., 2000, 2004; Parola et al., 1996; Zanetti et al., 2003). HNE is a potent alkylating agent that reacts with nucleophilic sites in DNA and protein, generating various types of adducts and leading to the assumption that the adducts cause a specific cellular response. This may include the transcriptional induction of proto-oncogenes, including c-Jun, which is a member of multiprotein family that has been implicated in a number of signal transduction pathways associated with cellular growth, differentiation, and neuronal excitation. Induction of c-Jun in response to genotoxic agents and cellular stress is mediated by two AP-1 like sites in its promoter, and this the transcriptional activity is regulated by upstream protein kinases related to the MAP kinase superfamily. To date at least three different MAP kinases are known (Schaeffer and Weber, 1999). 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. Mitogens and growth factors lead to the activation of protein kinase cascades, resulting in activation of extracellular signal-regulated kinases 1 and 2 (ERK ) (Robinson and Cobb, 1997; Schaeffer and Weber, 1999). On the other hand many forms of cellular stress preferentially trigger two related signaling pathways centered on MAP kinases: JNK and p38 (Davis, 2000). There is strong evidence that a sustained activation of JNK precedes apoptosis (Davis, 2000; Zhang et al., 2004). It has been shown that the prolongation of TNF-
-induced JNK and c-Jun activation can lead to apoptosis, and cells can be protected from apoptosis by blocking JNK-c-Jun activation pathway (Liu et al., 2002).
Studies have shown that HNE has a specific role for JNK and caspase-3 activation in several cell lines of diverse origin (Ji et al., 2001; Soh et al., 2000; Uchida et al., 1999). HNE-induced apoptosis in human myeloid HL-60 is preceded by a sustained activation of JNK and an increase in AP-1 binding (Cheng et al., 2001). Various treatments that induce cellular stress such as nerve growth factor (NGF)-withdrawal or ultraviolet (UV) radiation mediate apoptosis through induction of HNE formation (Yang et al., 2003; Bruckner et al., 2003). Together, these studies strongly suggest that the products of lipid peroxidation, HNE inclusive, are involved in stress-induced apoptosis.
Resveratrol was reported to exhibit protective effects against H2O2-induced and ß-amyloid–induced cell death in rat pheochromocytoma cells by increasing the resistance of these cells to oxidative stress and cell death. This is accomplished by attenuating intracellular ROS accumulation and restoring the levels of some marker proteins of apoptosis such as Bax, Bcl-XL, JNK, and PARP (Jang and Surh, 2001, 2003). Resveratrol has also been shown to protect against H2O2-induced PC12 cell death through activation of Nrf2 (NF-E2-related factor 2) and increased HO-1 expression (Chen et al., 2005).
In this study we found that resveratrol strongly prevents c-Jun expression induced by HNE. To fully understand the signaling mechanism that exerted this response, we investigated the phosphorylation events that lead to the activation of AP-1. The study reported here demonstrates that HNE is a potential inducer of signaling pathways that involve JNK activation, and that resveratrol is able to reverse this situation; thus preventing AP-1 activation and apoptosis. The identification of the proto-oncoprotein AP-1 as a nuclear target of HNE-mediated signaling and resveratrol acting along the same pathway may provide new clues for the regulatory role of this molecule.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents and antibodies.
Caspase-3 inhibitor (Z-DEVD-FMK), caspase-9 inhibitor (Z-LEHD-FMK), and general caspase inhibitor (Z-VAD-FMK) were obtained from BD Biosciences Pharmingen (San Diego, CA, USA). JNK inhibitor (SP600125), p38 inhibitor (SB203580), and MEK1/ERK
inhibitor (PD98059) were from Calbiochem (San Diego, CA, USA). JNK, phospho-JNK (Thr 183/Tyr 185), p38, phospho-p38, ERK , phospho-ERK , and cytochrome c antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Caspase-9, caspase-3, c-Jun, phospho-c-Jun, c-Fos, and ß-actin antibodies were from Cell Signaling Technology Inc. (Beverly, MA, USA). Cox IV (cytochrome c oxidase subunit IV) antibody was purchased from Abcam (Cambridge, UK). The Milk Diluent Concentrate Kit was obtained from KPL (Gaithersburg, MD, USA). 4-Hydroxynonenal (HNE) was obtained from Calbiochem (La Jolla, CA) and dissolved in ethanol. Dulbecco's Modified Eagles' Medium (DMEM), Phosphatase Inhibitor Cocktail 1, Phosphatase Inhibitor Cocktail 2, digitonin, fetal bovine serum, and other chemicals were purchased from Sigma (Darmstadt, Germany) otherwise indicated.
Cell culture and treatments.
Swiss 3T3 fibroblasts were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 IU/ml penicillin and streptomycin. Cultures were maintained at 37°C in a humidified 5% CO2 atmosphere. Cells were seeded in 6-well culture plates (1 x 106 cells/well), 60-mm culture flasks (1 x 107 cells/well) or 96-well plates (1 x 104 cells/well) and treated as indicated in the experimental protocols. Ethanol (
0.05%, v/v) was added to all control wells in each experiment.
Transfection and plasmids.
Plasmid containing dominant form of c-Jun, pcDNAFLAG-D169 (c-JunD169; DN-c-Jun) was kindly provided by Dr. J. Ham (University College London, UK) and was described before (Ham et al., 1995). The DN-JNK1 [pcDNA3-Flag-JNK1-APF] construct was kindly provided by Dr. R. Davis (Howard Hughes Medical Institute, Cambridge, MA, USA) and has the tyrosine 185 and threonine 183 amino acids, which require phosphorylation for activity replaced with alanine and phenylalanine, respectively (Gupta et al., 1995). The empty pcDNA1 and pcDNA3 vectors were used as mock transfections and were purchased from Invitrogen (Gronmgen, the Netherlands). For transient transfections, Swiss 3T3 fibroblasts were plated in 60-mm dishes and transfected with plasmids (2–4 µg) using Lipofectamine Plus reagent (Gibco Europe, Breda, the Netherlands) according to the manufacturer's recommendations. The transfection medium was removed and replaced with fresh culture medium after 4 h, and the cells were incubated for another 16 h prior to treatments. The transfection efficiency was monitored via detection of FLAG-tag by immunofluorescence and immunoblot analysis.
Nuclear and cytoplasmic protein extraction.
Nuclear and cytoplasmic proteins were isolated as described before, with minor modifications (Kutuk and Basaga, 2003). Briefly, cells were treated as indicated and washed with ice-cold phosphate buffered saline (PBS), then scraped and harvested by centrifugation. They were resuspended in 1 ml of cold PBS and transferred to 1.5-ml microfuge tubes. After centrifugation at 300 x g for 30 s, cells were lysed by incubation for 10 min in 200 µl of cold hypotonic buffer (10 mM Hepes/KOH [pH 7.9], 10 mM KCl, 2 mM MgCl2, 0.1 mM ethylene diamine tetraacetic acid [EDTA], 1 mM dithiothreitol [DTT], 0.5 mM phenylmethanesulfonyl fluoride (PMSF), protease inhibitors, and Nonidet P-40 [0.2%]). After centrifugation at 13,000 x g for 30 s, supernatants containing cytoplasmic proteins were removed and stored at –70°C. The nuclear pellet was washed, and nuclear protein isolation was carried out by incubation for 20 min on ice in a cold saline buffer (20 mM Hepes/KOH [pH 7.9], 1.5 mM MgCl2, 0,2 mM EDTA, 650 mM NaCl, glycerol [25%, v/v], 1 mM DTT, 0.5 mM PMSF, and protease inhibitors). After centrifugation at 13,000 x g for 20 min at 4°C, supernatants containing nuclear proteins were removed and stored at –70°C. Protein concentrations were determined by Bradford reagent (Bio-Rad, Munich, Germany).
Gel shift assay.
The gel shift method was performed as described elsewhere (Kutuk and Basaga, 2003). Briefly, the oligonucleotide probes (AP-1 and ARE) were labeled with 
-32P-dATP (3000 Ci/mmol) using T4 polynucleotide kinase, and then labeled oligonucleotide was purified on a Sephadex G-25 column. Next, 5 µg of nuclear proteins were incubated for 20 min at room temperature with 0.2 ng of 32P-labeled oligonucleotide probe in gel shift binding buffer (1.25 µg of BSA and 1.25 µg of poly [dI-dC]. poly [dI-dC] in 20 mM Hepes/KOH, 75 mM NaCl, 1 mM EDTA, 5% v/v glycerol, 0.5 mM MgCl2, 1 mM DTT [pH 7.9], final volume 10 µl). DNA–protein complexes then resolved on a non-denaturating 6% polyacrylamide gel run for 3 h at 180 V. The gel was then dried and autoradiographed on Kodak x-ray film.
For competition experiments unlabeled consensus or mutated probe was added in excess (50x) in the binding buffer and for supershift experiment incubation with anti-c-Jun and anti-c-Fos antibodies was performed before addition of 32P-labelled oligonucleotide probe into the binding buffer.
Cell death and apoptosis assays.
Cell death was determined using an MTT assay kit (Roche, Mannheim, Germany) according to the manufacturer's protocol. Briefly, Swiss 3T3 cells in 96-well plates were treated as indicated, and 10 µl of MTT labeling reagent was added to each well, after which the plates were incubated for 4 h. The cells were then incubated in 100 µl of the solubilization solution for 12 h, and the absorbance was measured with a microtiter plate reader (Bio-Rad, CA, USA) at a test wavelength of 550 nm and a reference wavelength of 650 nm. Percent viability was calculated as (OD of drug-treated sample/control OD) x 100.
For evaluation of apoptosis, a Cell Death Detection ELISAPLUS kit (Roche, Mannheim, Germany) was used according to the instructions of the manufacturer. This kit detects cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) after induced cell death. Briefly, cells were washed with PBS and incubated with 200 µl of lysis buffer for 30 min and centrifuged for 10 min at 300 x g, and 20 µl of the supernatant (cytoplasmic fraction) was assayed in the ELISA. The reaction was developed with a peroxidase system, and development of color was measured with a microtiter plate reader (Bio-Rad, Hercules, CA, USA) at a test wavelength of 405 nm and a reference wavelength of 490 nm. Results were determined as fold increases in absorbance over untreated control cells (enrichment factor).
Immunoblot analysis.
Treated and control 3T3 fibroblasts were harvested, washed with ice-cold PBS, and lysed on ice in a solution containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, Nonidet P-40 0.5%, (v/v), 1 mM EDTA, 0.5 mM PMSF, 1 mM DTT protease inhibitor cocktail (Complete from Roche, Mannheim, Germany), and phosphatase inhibitors (Phosphatase inhibitor cocktail 1 and 2, Sigma, Darmstadt, Germany). After cell lysis, cell debris was removed by centrifugation 15 min at 13,000 x g and protein concentrations were determined with the Bradford protein assay. Proteins (40 µg) were separated on a 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto Polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% dried milk in PBS-Tween 20 and incubated with appropriate primary and horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Pharmacia Biotech, Freiburg, Germany) in antibody buffer containing 10% (v/v) milk diluent/blocking concentrate. After required washes with PBS-Tween 20, proteins were finally analyzed using an enhanced chemiluminescence detection system (ECL-Plus, Amersham Pharmacia Biotech, Freiburg, Germany) and exposed to Hyperfilm-ECL (Amersham Pharmacia Biotech, Freiburg, Germany).
Detection of cytochrome c release.
Release of cytochrome c from mitochondria was detected as described previously (Pique et al., 2000). 3T3 fibroblasts cells were seeded in 6-well plates (1 x 106 cells/well) and after indicated treatments, cells were harvested, washed once with PBS, and lysed for 30 s in 100 µl ice-cold lysis buffer (250 mM sucrose, 1 mM EDTA, 0.05% digitonin, 25 mM Tris, pH 6.8, 1 mM DTT, 0.1 mM PMSF, and protease inhibitor cocktail (CompleteMini, Roche, Germany)]. Cell lysates were centrifuged at 13,000 x g at 4°C for 5 min, and supernatants (mitochondria-free cytosolic extracts) and pellets (mitochondrial fraction) were separately obtained. Cytosolic and mitochondrial fractions were separated on a 15% SDS-PAGE gel and then analyzed by Western blot using anti-cytochrome c antibody and HRP-conjugated secondary antibody. Proteins were finally developed using an ECL-Plus enhanced chemiluminescence detection system and exposed to Hyperfilm-ECL.
Caspase activation assays.
The enzymatic activity of caspase-3 and caspase-9 was determined by using a caspase activation assay kit (Sigma, Darmstadt, Germany). 3T3 fibroblasts were treated as indicated, washed twice with ice-cold PBS, and then resuspended in lysis buffer (250 mM HEPES, pH 7.4; 25 mM CHAPS; 25 mM DTT). After 15 min of incubation on ice, samples were centrifuged for 10 min at 10,000 x g at 4°C; supernatants were collected, and protein concentrations were determined by Bradford protein assay, after which 10 µg of protein were assayed in 200 µl of reaction solution containing Ac-DEVD-AMC for caspase-3-like DEVDase activity and Ac-LEHD-AMC for caspase-9 activity. The released fluorescent AMC was monitored at an excitation of 360 nm and emission of 460 nm using a Spectramax Gemini XS multiplate spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA). Results were calculated from a standard curve of AMC, and specific caspase activities were derived as mean relative fluorescence units (RFU)/mg protein. The specificity of fluorometric caspase assays was always checked by inhibitor studies and internal positive controls. Data shown are mean ± SEM of three independent experiments performed in triplicate.
Statistical analysis.
The results are expressed as mean ± SEM, and the mean values were compared using Students t-tail test. Values of p < 0.05 and p < 0.01 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HNE Induces Increased c-Jun Expression/Phosphorylation and AP-1 Binding, but Decreased c-Fos Expression
To investigate the effect of HNE on AP-1 proteins in 3T3 fibroblasts, we evaluated the levels of c-Jun, phospho-c-Jun, and c-Fos proteins. 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. As shown in Figure 1A, HNE (20 µM) dramatically stimulated the expression of c-Jun protein in a time-dependent manner with a maximal response by 1 h; in addition, increased phospho-c-Jun levels were detected at 1 h following HNE treatment. The increased expression of c-Jun/phospho-c-Jun was sustained until 4 h after treatment. In contrast, time-course experiments up to 4 h have shown that downregulation of c-Fos by HNE (20 µM) was evident at 30 min and remained downregulated until 4 h. Furthermore, the effect of HNE on AP-1 c-Jun, phospho-c-Jun, and c-Fos was concentration-dependent, in which HNE at 20 µM concentration induced increased c-Jun and phospho-Jun, and decreased c-Fos levels efficiently after 1 h of treatment (Fig. 1A).
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Our data indicate that induction of AP-1 proteins by HNE is highly specific to c-Jun. Because c-Jun is the major constituent of the transcription factor AP-1, and because HNE increased the expression of this proto-oncogene, we tested the effect of HNE on AP-1 activation.
AP-1 activity was measured by its ability to bind to the palindromic TPA response element (TRE). The nuclear extracts of HNE-treated cells displayed greater binding activity than extracts of untreated cells, as determined by gel shift assay. The maximum increase in AP-1 binding activity in response to HNE (20 µM) treatment was observed by 1 h, and these responses persisted for at least 2 h (Fig. 1B). HNE also induced a dose-dependent activity in AP-1 binding, and an increased AP-1 binding could be observed with as little as 5 µM HNE treatment for 1 h. A clearly significant degree of binding was detected when cells were treated with 20 µM HNE for 1 h as compared to control untreated cells (Fig. 1B).
These results suggest that HNE induces activation of AP-1 transcription factor complexes along with increased expression and phosphorylation of c-Jun and decreased expression of c-Fos.
HNE Activates JNK and p38 MAP Kinases, but Inhibits Basal ERK Activation
A number of transcription factors such as c-Jun/AP-1 and c-Fos/AP-1 have been shown to be phosphorylated by distinct members of MAP kinase proteins triggered by a large variety of extracellular stresses. Based on the finding that HNE is a potential inducer of c-Jun expression and phosphorylation, a possible involvement of MAP kinases in HNE-induced c-Jun expression/phosphorylation was examined.
To determine the possibility that HNE treatment of the cells results in the activation/phosphorylation of MAP kinases, whole cell lysates were probed with the antibodies specific for JNK, phospho-JNK, p38, phospho-p38, ERK , phospho-ERK by means of immunoblot analysis. As shown in Figure 2A, HNE (20 µM) induced an abrupt increase in phosphorylation of JNK, which peaked at 15 min after stimulation without any significant change in total JNK protein levels. The activity of JNK began to diminish at 1 h of HNE treatment and became undetectable at 4 h. Interestingly, ERK 1 phosphorylation became undetectable after 30 min of HNE treatment, but we were able to observe phosphorylated ERK 2 until 2 h after HNE stimulation. HNE induced a complete inhibition of ERK phosphorylation after 2 h of treatment without any significant change in total ERK protein levels. Immunoblot analysis of phospho-p38 proteins revealed an early and transiently increased level of phospho-p38, which could be exclusively detected at 15–30 min after HNE treatment. HNE did not influence the total p38 protein level, as shown in Figure 2A. In addition, loading of proteins for all immunoblots were checked by reprobing of membranes by b-actin antibody; a representative b-actin blot is shown in lower panel of Figure 2A. These findings demonstrate that HNE alternates the activity of all three MAP kinases in 3T3 fibroblasts with different time kinetics and characteristics.
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Effect of HNE on Caspase Activation and Cytochrome c Release from Mitochondria
We have previously shown that HNE-induced apoptosis in 3T3 fibroblasts (Kutuk et al., 2004
). To identify the possible role of caspases in this apoptotic process, we investigated the activation of caspase-9 and caspase-3 in response to HNE treatment by using immunoblot analysis for active caspase fragments and fluorometric caspase assays. HNE (20 µM) induced the appearance of an active caspase-9 fragment (35 kDa) at 1 h, which was followed by a more evident and strong band in immunoblot analysis at 4–8 h after HNE treatment (Fig. 2B). The parallel analysis of caspase-9 activation through its ability to cleave its specific substrate (Ac-LEHD-AMC) and formation of the fluorogenic AMC compound displayed a quite different caspase-9 activation pattern compared to immunoblot analysis (Fig. 2B). Although we could detect a pronounced active caspase-9 band at 4 h of HNE treatment, a complete activation of caspase-9 activation could only be detected at 8 h of HNE treatment in fluorometric caspase assays. In the mitochondrial apoptosis pathway, caspase-3 operates downstream of cytoplasmic cytochrome c translocation and caspase-9 activation. As shown in Figure 2B, HNE (20 µM) induced the appearance of cleaved active caspase-3 fragments within 4 h, and complete active bands were detected at 8 h post-treatment. The fluorometric caspase assays of caspase-3 activation through its ability to cleave its specific substrate (Ac-DEVD-AMC) and formation of the fluorogenic AMC compound presented a time-course similar to immunoblot analysis for caspase-3 activation (Fig. 2B). The release of cytochrome c from mitochondria in response to apoptosis inducers (such as chemotherapeutics, UV radiation, growth factor withdrawal) and its binding to Apaf-1 leads to formation of apoptosome complex and caspase-9 activation. As shown in Figure 2C, HNE treatment resulted in an early and complete release of cytochrome c from mitochondria and its appearance in cytoplasm 1 h after HNE treatment, which is compatible with caspase-9 and caspase-3 activation patterns. Taken together, these results present evidence for intrinsic mitochondrial apoptotic machinery involved in HNE-induced apoptosis.
JNK and Caspases are Functionally Involved in HNE-Induced Apoptosis; Protective Effect of Resveratrol
Activation of MAP kinases either upstream or downstream of caspase has been postulated in many in vitro and in vivo experimental apoptosis models (Davis, 2000; Robinson and Cobb, 1997; Schaeffer and Weber, 1999). Regarding HNE-induced modulation of MAP kinases and caspases, we investigated the functional involvement of MAP kinases in HNE-induced apoptosis. Others and we have previously reported that HNE-induced cell death is predominantly apoptotic rather than necrotic (Choudhary et al., 2002; Kutuk et al., 2004; Liu et al., 2000; Zhang et al., 2001). Therefore, we utilized MTT assay for determination of cell viability in response to specific MAP kinase or caspase inhibitors and HNE treatment. Treatment of cells with HNE (20 µM) reduced cell viability to 37.39 ± 1.68% (Fig. 3A, lane 1), and pretreatment of cells with 10 µM SP600125 (JNK inhibitor) for 1 h protected 3T3 fibroblasts against HNE-induced apoptosis (Fig. 3A, lane 1, **p < 0.01, compared to HNE-treated cells), but 10 µM SB203580 (p38 inhibitor) or 10 µM PD98059 (ERK inhibitor) did not exhibit any significant protective effect (Fig. 3A, lanes 3 and 4, respectively). Pretreatment of cells with 20 µM pancaspase inhibitor Z-VAD-FMK (Fig. 3A, lane 5); 20 µM caspase-9 inhibitor, Z-LEHD-FMK (Fig. 3A, lane 6); or 20 µM caspase-3 inhibitor, Z-DEVD-FMK (Fig. 3A, lane 7) for 30 min significantly prevented HNE-induced apoptosis (**p < 0.01 for Z-VAD-FMK and Z-LEHD-FMK; *p < 0.05 for Z-DEVD-FMK, compared to HNE-treated cells). The protective effect of resveratrol (20 µM), a polyphenol with anti-carcinogenic and anti-inflammatory properties, is also comparable to protective effects of SP600125 and caspase inhibitors (Fig. 3A, lane 8, **p < 0.01, compared to HNE-treated cells).
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To determine the interdependency of MAP kinases and caspases, as well as to identify the critical steps involved in resveratrol-mediated protection against HNE-induced apoptosis, we followed HNE-induced activation of caspases after pretreatment with specific MAP kinase inhibitors and resveratrol. As demonstrated in Figure 3B, pretreatment of cells with 10 µM SP600125 or 20 µM resveratrol significantly inhibited both caspase-3 and caspase-9 activation with similar efficiencies. In contrast, 10 µM SB203580 or 10 µM PD98059 did not confer any effect on caspase activation induced by HNE consistent with MTT results. Pretreatment of cells with caspase inhibitors did not show any effect on modulation of MAP kinases with HNE treatment (data not shown). We next explored the involvement of cytochrome c release in inhibitory effect of JNK and resveratrol on caspase activation. Figure 3C shows that pretreatment of cells with 10 µM SP600125 or 20 µM resveratrol led to blockage of cytochrome c release from mitochondria and thereby disrupted formation of the apoptosome complex and caspase-9 activation. Our results suggest a JNK-dependent caspase-activation module in HNE-induced apoptosis in which JNK acts upstream of mitochondrial apoptotic machinery and regulates cytochrome c release in response to HNE treatment. Resveratrol also acts upstream of mitochondrial apoptotic machinery and exerts its protective effect through prevention of cytochrome c release and consequent caspase activation.
c-Jun/AP-1 Transcriptional Activity Is Involved in HNE-Induced Apoptosis
The protective effect of a pharmacological inhibitor of JNK (SP600125) against HNE- induced apoptosis has put forth functional involvement of c-Jun/AP-1 activation for consideration in this process. To examine whether c-Jun/AP-1 activation has a similar role in HNE-induced apoptosis, we transfected 3T3 cells with the expression vector for DN-c-Jun mutant. DN-c-Jun mutant lacking the N-terminal c-Jun transactivation domain (amino acids 1–168) can efficiently dimerize, but it cannot activate transcription and will prevent any member of the Jun and Fos family from activating transcription of AP-1–dependent target genes and may do so by occupying AP-1 binding sites in the place of functional Jun/Jun or Jun/Fos dimers. We also transfected 3T3 fibroblasts with the expression vector for DN-JNK1 to verify the protective effect of the pharmacological JNK inhibitor. The transfected cells were treated with HNE (20 µM) for 24 h and assayed for apoptosis using the Cell Death Detection ELISAPLUS kit. As shown in Figure 4, lane 3, overexpression of DN c-Jun significantly attenuated apoptosis when compared to HNE-treated untransfected cells (Fig. 4, lane 1), but the protective effect of DN JNK1 overexpression against HNE-induced apoptosis was more pronounced (Fig. 4, lane 5). The expression of mock vectors did not exhibit any effect on HNE-induced apoptosis (Fig. 4, lanes 2 and 4). These results demonstrate that HNE-induced apoptosis in 3T3 fibroblasts involve c-Jun/AP-1 activation downstream of JNK, but JNK also has downstream targets other than c-Jun/AP-1.
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Resveratrol Inhibits HNE-Induced JNK and p38 Activation, c-Jun Expression, and Phosphorylation
To address the mechanisms of protection by resveratrol against HNE-induced apoptosis, 3T3 cells were pretreated with or without resveratrol (1, 10, or 20 µM) for 4 h, followed by treatment with HNE (20 µM). Total protein lysates were analyzed for phosphorylation and expression of MAP kinases and c-Jun and expression of c-Fos by means of immunoblot analysis. Resveratrol attenuated HNE-induced JNK activation/phosphorylation in a dose-dependent manner, and it completely blocked HNE-induced p38 activation/phosphorylation at 20 µM concentration without any effect on total JNK and p38 levels (Fig. 5). Resveratrol also restored basal ERK
activation/phosphorylation downregulated by HNE without any effect on total ERK protein level (Fig. 5). Immunoblot analysis also revealed that resveratrol pretreatment efficiently inhibited HNE-induced increase in c-Jun expression, even at the 10 µM concentration, but HNE-induced phospho-c-Jun levels could only be attenuated when cells were pretreated with 20 µM resveratrol (Fig. 5).
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Resveratrol pretreatment also restored c-Fos protein level, which was downregulated by HNE treatment. Although a moderate effect of resveratrol was observed even at 1 µM concentration, a complete restoration of c-Fos protein levels similar to untreated cells could be achieved when cells were pretreated with 20 µM resveratrol (Fig. 5). These results suggest that resveratrol could protect against HNE-induced apoptosis through alteration of cellular signaling modules formed by MAP kinases and their downstream targets.
Modulation of HNE-Induced AP-1 Activation by SP600125 and Resveratrol
To examine whether AP-1 activation is a target for resveratrol, we evaluated the effect of resveratrol on HNE-induced AP-1 activation. 3T3 cells were pretreated with resveratrol (1, 5, 10, 15, or 20 µM) for 4h, followed by HNE (20 µM) for 1 h, and nuclear proteins were analyzed by means of AP-1 gel shift assay. Resveratrol dose-dependently inhibited HNE-induced AP-1 activation with a maximum efficiency at 20 µM concentration, and resveratrol (20 µM) alone did not trigger any significant effect on AP-1 activation (Fig. 6A). Because MAP kinases reside upstream of AP-1 activation, we next investigated the inhibitory potential of specific MAP kinase inhibitors on AP-1 activation compared to resveratrol. Pretreatment of cells with SP600125 (10 µM) prior to treatment with HNE (20 µM) attenuated AP-1 activation at a comparable efficiency to resveratrol (20 µM) (Fig. 6B, lanes 4 and 3, respectively). In contrast, SB203580 (10 µM) and PD98059 (10 µM) did not exhibit any prominent effect on HNE-induced AP-1 activation (Fig. 6B, lanes 5 and 6, respectively). The specificity of AP-1 band was confirmed by competition experiments with an excess of unlabeled AP-1 oligonucleotide probe (Fig. 6B, lane 7), and by the ability of highly specific antibodies directed against individual AP-1 proteins to deplete nuclear extracts of DNA-binding activities, which confirmed that AP-1 complexes are mainly formed by c-Jun/c-Jun dimers (Fig. 6B, lanes 8, 9, and 10).
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Resveratrol Increases ARE Binding Activity
To determine whether resveratrol could protect against HNE-induced apoptosis through activation of Nrf2, 3T3 cells were pretreated with resveratrol (20 µM) for 4 h with or without HNE (20 µM) for 30 min, 1 h, and 2 h. Nuclear proteins were analyzed by means of gel shift assay using ARE consensus oligonucleotide. As illustrated in Figure 7, HNE induced a moderate decrease in ARE binding activity of Nrf2, with the most prominent effect at 1 h treatment, which could be blocked by resveratrol pretreatment. Furthermore, resveratrol (20 µM) treatment for 4 h alone induced an increase in ARE-binding activity. The specificity of the ARE band was confirmed by competition experiments with an excess of unlabeled consensus and mutated oligonucleotide probe.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HNE is a lipid peroxidation end product of arachidonic and linoleic acids. Because of its relatively high lipophilicity, HNE tends to concentrate within cell membranes where it reaches steady-state amounts over 10–5 M (Esterbauer et al., 1991
). Further, HNE may readily pass across the cellular membranes to modify critical molecules such as proteins and DNA (Grune and Davies, 2003; Kowalczyk et al., 2004). Increased levels of HNE adducts have been detected in various human pathologies, such as neurodegenerative disorders, atherosclerosis, and chronic obstructive pulmonary disease (Esterbauer et al., 1991). At the cellular level, HNE has been shown to reduce intracellular glutathione levels and modulate intracellular signaling pathways (Leonarduzzi et al., 2004; Zhang et al., 2005). HNE was shown to induce either apoptosis or proliferation in a concentration-dependent manner (Choudhary et al., 2002; Liu et al., 2000; Ruef et al., 1998; Zhang et al., 2001). In spite of intense research conducted, the exact mechanisms involved in HNE-induced apoptosis remain to be elucidated.
We have previously shown that HNE treatment induces apoptosis in 3T3 cells in a dose- and time-dependent manner, along with production of reactive oxygen intermediates (Kutuk et al., 2004). HNE at 20 µM concentration efficiently induced apoptosis in 3T3 cells through the release of cytochrome c from mitochondria and caspase-9 and caspase-3 activation.
AP-1 is a major transcription factor complex composed mainly of homodimers of c-Jun and heterodimers of c-Jun/c-Fos. AP-1 transcriptional activity may be related to either apoptosis or proliferation in different cellular systems (Shaulian and Karin, 2002). It was assumed that induction of protein kinases by HNE might cause activation of signaling pathways that phosphorylated and activated c-Jun (Uchida et al., 1999; Kikuta et al., 2004). Another major member of the AP-1 transcription factor protein complexes, c-Fos, has been proposed to be necessary for cell proliferation and cell cycle progression in response to growth factor stimulation (Gao et al., 2004; Tanos et al., 2005). In HL60 leukemia cells, HNE-induced apoptosis has been shown to take place after JNK activation, c-Jun phosphorylation, and AP-1 activation, which could be attenuated by transfection of HL60 cells with glutathione S-transferase isozymes (Cheng et al., 2001). The experimental evidence presented here indicates the involvement of HNE in AP-1 activation, as shown by the phosphorylation and potent expression of c-Jun. Interestingly, in our experimental conditions, HNE induced a moderate decrease in c-Fos protein levels, but the involvement of this downregulation in the apoptotic process remains to be elucidated. Amino-terminal phosphorylation of c-Jun at serines 63 and 73 has been reported to regulate both proliferation and stress-induced apoptosis (Behrens et al., 1999). Furthermore, overexpression of a dominant-negative c-Jun (DN-c-Jun) mutant with a cleaved N-terminal fragment has been shown to attenuate neuronal apoptosis induced by survival factor withdrawal (Ham et al., 1995). Additionally, overexpression of wild type c-Jun has been shown to trigger an apoptotic response in 3T3 fibroblasts and human vascular endothelial cells (Bossy-Wetzel et al., 1997; Wang et al., 1999). Our results indicate a pro-apoptotic role for c-Jun/AP-1 activation by HNE, as transfection of 3T3 fibroblasts with a DN-c-Jun mutant significantly attenuated HNE-induced apoptosis in 3T3 fibroblasts.
Because we had demonstrated the requirement of c-Jun in HNE-induced apoptosis, we investigated the activation of upstream MAP kinases, which are proline-directed protein kinases involved in the regulation of critical cellular processes such as proliferation, cell cycle progression, gene expression, and apoptosis. It has been suggested that ERK , which are primarily activated in response to growth factor stimulation, are involved in process of cellular proliferation and differentiation, whereas JNK and p38 MAP kinases are characterized by their strong response to cellular stresses such as UV light, serum deprivation, DNA-damaging agents, and pro-inflammatory cytokines (Robinson and Cobb, 1997; Schaeffer and Weber, 1999). Previous reports have shown differential regulation or activation of MAP kinase pathways by HNE. HNE-induced activation of JNK has been initially presented on hepatic stellate cells, and direct modification of p46 and p54 isoforms of JNK has been demonstrated (Parola et al., 1998). In addition, in hepatic stellate cells no alteration of the ERK pathway or c-Fos protein level has been observed. In vascular endothelial cells, stimulation of cells with HNE resulted in activation of ERK , p38, and JNK MAP kinases (Usatyuk and Natarajan, 2004). In addition, HNE has been shown to induce significant phosphorylation of JNK and p38, but not ERK in rat hepatic epithelial cells (Uchida et al., 1999). Interestingly, it has been demonstrated that HNE induced a strong activation of JNK in PC12 cells during HNE-induced apoptosis, while ERK and p38 MAP kinases remained unaffected (Soh et al., 2000). JNK3 has also been reported to mediate a caspase-dependent HNE-induced apoptotic pathway in sympathetic neurons (Bruckner and Estus, 2002). Furthermore, treatment of IMR-90 human lung fibroblasts with 25 µM HNE markedly activated ERK and p38, without any effect on JNK MAP kinases (Tsukagoshi et al., 2002). The present findings suggest that HNE leads to activation of JNK and p38 MAP kinases, but with different time kinetics and amplitudes. In contrast, phospho-ERK downregulation was induced after HNE treatment, and no phospho-ERK could be detected at 4 h following HNE treatment. Pretreatment with JNK inhibitor (SP600125) effectively prevented the apoptosis induced by HNE through blocking the release of cytochrome c and caspase-9 and caspase-3 activation, which indicates that JNK acts upstream of mitochondria in the apoptotic signaling machinery. Furthermore, pretreatment of cells with JNK inhibitor prior to HNE stimulation attenuated AP-1 DNA binding activity, but we were not able to observe any significant effect with p38 or ERK inhibitors. Additionally, overexpression of a dominant-negative JNK1 mutant efficiently inhibited HNE-induced apoptosis, which confirmed the involvement of JNK in HNE-mediated apoptosis in 3T3 fibroblasts. The protective effect of DN-JNK1 overexpression was more pronounced than DN-c-Jun overexpression, which underlines JNK targets other than c-Jun in the HNE-induced apoptotic pathway, such as Bcl-2 protein members. Interestingly, a recent report has proposed the involvement of the Bcl-2 family member Bim in c-Jun-mediated apoptosis and cytochrome c release in neuronal cells (Whitfield et al., 2001), but the c-Jun target genes that are involved in HNE-induced apoptosis have yet to be identified. In addition to a 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 at least partially by c-Jun/AP-1.
Resveratrol is a phytoalexin, which is one of most widely distributed flavones in nature, and highly concentrated in grape skin and seed, mulberries, and peanuts. Resveratrol has been shown to exert anti-inflammatory and anti-proliferative effects in various cellular systems (Alarcon de la Lastra and Villegas, 2005). The cardioprotective effect of resveratrol as an important constituent of red wine has been proposed to be responsible for the "French paradox," characterized by a low incidence of cardiovascular diseases along with a diet rich in fat (Bradamante et al., 2004). Apoptosis and inflammation have been shown to be involved in the pathogenesis of atherosclerosis, and this anti-apoptotic effect of resveratrol may be involved in its protective function against atherosclerosis (Delmas et al., 2005). Recent reports have mainly demonstrated that resveratrol acts as a cancer chemopreventive molecule, mainly acting through triggering of apoptosis. Resveratrol was shown to induce pro-apoptotic p53 pathway through activation of ERK and p38 MAP kinases in a mouse epidermal cell line (She et al., 2001). In contrast, resveratrol has been shown to suppress TNF-induced NF-
B and AP-1 activation and apoptosis (Manna et al., 2000). Ischemia-reperfusion- and ß-amyloid-mediated cellular injuries were shown to be prevented by resveratrol pretreatment (Dernek et al., 2004; Jang and Surh, 2003). As shown in a recent report, resveratrol inhibited PMA-induced AP-1 and MAP kinase activation (Yu et al., 2001). We have previously shown that resveratrol protects against HNE-induced ROI formation and cell death in 3T3 fibroblasts (Kutuk et al., 2004).
To investigate the mechanisms of this protection by resveratrol, we evaluated its effects on HNE-induced c-Jun/AP-1 activation. Resveratrol prevents increased c-Jun expression and phosphorylation, as well as AP-1 DNA binding activity at a level of efficiency comparable with the JNK inhibitor. It is noteworthy that HNE-induced p38 and JNK activation could also be inhibited efficiently by resveratrol. Resveratrol has also restored the decreased c-Fos and phospho-ERK levels. The exact mechanism responsible for the inhibitory effect of resveratrol on HNE-induced JNK activation remains to be elucidated, but it is possible that it exerts its activity either by inhibiting upstream kinases of JNK such as PKC or by activating JNK-specific protein phosphatases.
Caspase activation and cytochrome c release from mitochondria have also been attenuated by resveratrol pretreatment. Considering the involvement of the c-Jun/AP-1 and JNK pathways in HNE-induced apoptosis, resveratrol blocks the apoptotic machinery through inhibition of these pathways upstream of mitochondria. In a recent report, resveratrol has also been demonstrated to protect against oxidative stress via activation of Nrf2 and upregulation of heme oxygenase-1 (HO-1) (Chen et al., 2005). In the present study, Nrf2 ARE-binding activity was found to be upregulated by resveratrol, which may be responsible, at least in part, for its protective effect against HNE-induced apoptosis.
In conclusion, we report here on the activation of an apoptotic signaling pathways by HNE, which involves the activation of the JNK-c-Jun/AP-1 signaling cascade, mitochondrial cytochrome c release, and caspase-9/3 activation. Resveratrol prevents JNK, c-Jun/AP-1, caspase activation, and apoptosis induced by HNE. Thus, our studies provided new insight into the molecular mechanisms of HNE-induced apoptosis and the cytoprotective properties of resveratrol. The present results argue strongly that c-Jun-mediated genes as well as JNK target proteins other than the c-Jun/AP-1 pathways, which are h involved in HNE-induced apoptosis, still remain to be identified. Moreover, identification of these mechanisms, along with utilization small-molecule inhibitors such as resveratrol, may enable the development of specific and rationale therapies against human diseases caused by deregulation of apoptosis.
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ACKNOWLEDGMENTS |
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This work was supported by Sabanci University Research Funds. The authors thank Dr. Jonathan Ham and Dr. Roger Davis for the generous gift of plasmids, and Ozgur Gul for his excellent technical assistance.
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