Mitogen-activated protein kinase/extracellular ... - Wiley Online Library

Journal of Neurochemistry, 2004, 88, 647–656

doi:10.1046/j.1471-4159.2003.02191.x

Mitogen-activated protein kinase/extracellular signal-regulated kinase attenuates 3-hydroxykynurenine-induced neuronal cell death Hyun Jung Lee,* Jae-Hyung Bach,* Hee-Sun Chae,* Sang Hyung Lee, à Wan Seok Joo, Se Hoon Choi, Kyung Yong Kim,* Won Bok Lee* and Sung Su Kim*, *Department of Anatomy, College of Medicine, BioGrand Inc., Chung-Ang University, àDepartment of Neurosurgery, College of Medicine, Seoul National University, Seoul, South Korea

Abstract 3-Hydroxykynurenine (3-HK), an endogenous tryptophan metabolite, is known to have toxic effects in brain. However, the molecular mechanism of the toxicity has not been well identified. In this study, we investigated the involvement of MAPK/extracellular signal-regulated kinase (ERK) in the 3-HK-induced neuronal cell damage. Our results showed that 3-HK induced apoptotic neuronal cell death and ERK phosphorylation occurred during cell death. Inhibition of ERK activation using PD98059 considerably increased cell death. Furthermore, cell death was preceded by mitochondrial

malfunction including collapse of mitochondrial membrane potential (DYm) and cytochrome c release from mitochondria to the cytosol. Interestingly, inhibition of ERK dramatically increased mitochondrial malfunction, and enhanced caspase activation, resulting in enhanced neuronal cell death. Thus, our results show that ERK plays a protective role by maintaining mitochondrial function and regulating caspase activity under conditions of cellular stress. Keywords: apoptosis, extracellular signal-regulated kinase, 3-hydroxykynurenine, mitochondrial malfunction. J. Neurochem. (2004) 88, 647–656.

The kynurenine pathway, the major metabolic pathway of the amino acid tryptophan, produces 3-hydroxykynurenine (3-HK) as a metabolic intermediate (Okuda et al. 1996). An increase of 3-HK was detected in the cortical and striatal regions of patients with Huntington’s disease (Pearson and Reynolds 1992). Another study has revealed that the level of 3-HK in the brain considerably increased in other pathological disorders such as hepatic encephalopathy (Pearson and Reynolds 1991), Parkinson’s disease (Ogawa et al. 1992) and acquired immunodeficiency syndrome dementia (Sardar et al. 1995). Thus, it has been suggested that 3-HK is one of the causal mediators of neurotoxicity. All of the above diseases are known to be tightly linked to neuronal cell death, which subsequently causes dysfunction in neuronal circuitry. A large body of evidence suggests that oxidative stress may be a pivotal factor leading to neuronal cell death in these disorders (Coyle and Puttfarcken 1993). 3-Hydroxykynurenine is one of the possible metabolites that increase reactive oxygen species in the brain (Nakagami et al. 1996; Okuda et al. 1996). However, the molecular mechanisms of cell death triggered by endogenous 3-HK are not well elucidated.

MAPKs are a family of related serine/threonine protein kinases that transduce several signals responsible for cell proliferation or cellular stress (Davis 1993; Cobb and Goldsmith 1995; Karin and Hunter 1995). In general, MAPK is activated by phosphorylation of tyrosine and threonine residues by MAPKK (Boulton et al. 1991), which is activated through phosphorylation by MAPKKK (Yamaguchi et al. 1995), sequentially. Roughly, the MAPK family can be Received April 24, 2003; revised manuscript received July 18, 2003; accepted September 26 2003. Address correspondence and reprint requests to Sung Su Kim, Department of Anatomy, College of Medicine, Chung-Ang University, 221 Huksuk-dong, Dongjak-ku, Seoul, 156-756, Korea. E-mail: [email protected] Abbreviations used: Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-7amino-4-methylcoumarin; Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Aspaldehyde; Ac-LEHD-AMC, N-acetyl-Asp-Leu-Val-His-7-amino-4methylcoumarin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate; ECL, enhanced chemiluminescent reagent; ERK, extracellular signal-regulated kinase; 3-HK, 3-hydroxykynurenine; ROI, reactive oxygen intermediate; TMRE, tetramethylrhodamine ethyl ester; Z-LEHD-fmk, Leu-Glu-His-Asp-fluoromethylketone; Z-VAD-PNA, ValAla-Asp-p-nitro-aniline.

2004 International Society for Neurochemistry, J. Neurochem. (2004) 88, 647–656

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classified into three major kinase groups, the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase or stress-activated protein kinase and the p38 MAPK (Davis 1993). Each MAPK member has different substrates that mediate diverse biological effects (Davis 1993). Activation of c-Jun N-terminal kinase/stress-activated protein kinase and p38 MAPK is often linked to neuronal degeneration. For example, the p38 inhibitor, SB203580, attenuates cell death induced by stimulation of N-methyl-D-aspartate receptor in cultured rat cerebellar granule neurons (Kawasaki et al. 1997). CEP1347/KT7515, a specific inhibitor of c-Jun N-terminal kinase, protected Ab-induced neuronal apoptosis in neuronal PC12 cells and sympathetic neurons (Troy et al. 2001). Persistent activation of ERK also contributed to glutamateinduced oxidative toxicity in a mouse neuronal cell line, HT22, and primary cortical neuron cultures (Stanciu et al. 2000). However, other studies have suggested that ERK activation is associated with enhanced cell survival in hypoxia, staurosporine-induced cell death and ischemia/reperfusion (Deng et al. 2000; Yue et al. 2000; Kunlin et al. 2002). In the present study, we investigated if and how ERK may be involved in 3-HK-induced neuronal cell death using human neuroblastoma SK-N-SH cells. We also studied whether a functional loss of the mitochondria would be involved in the cell death. Our results suggest that ERK plays a protective role by protecting mitochondrial function, damaged by 3-HK.

Experimental procedures Cell culture SK-N-SH human neuroblastoma cells were maintained at 37C in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL, CA, USA) in a humidified 95% air, 5% CO2 incubator. The medium was changed to RPMI 1640 containing 1% fetal bovine serum for 2 h before 3-HK (250 lM) treatment. Pharmacological treatments 3-Hydroxykynurenine was obtained from Sigma (St Louis, MO, USA) and dissolved in dimethylsulfoxide. A dose of 250 lM 3-HK was used, a dose that induces cell death by 50% within 36 h. PD98059 (2¢-amino-3¢-methoxyflavone), a specific inhibitor of MEK1/2, was purchased from TOCRIS (Bristol, UK) and was used at the indicated concentration 2 h before 3-HK treatment. Cell viability assay (AlamarBlue test) SK-N-SH cells were plated on 96-well plates (Nunc, Slangerup, Denmark) at a density of 15 000 cells/well in 100 lL of RPMI 1640/ 10% fetal bovine serum and incubated for 24 h. The medium was replaced with 1% fetal bovine serum/RPMI 1640 before the 2 h 3-HK treatment. At the end of the treatment, 10 lL of AlamarBlue (Serotec, Oxford, UK) were added aseptically. The cells were incubated for 3 h and the absorbance was measured at a wavelength of 570 nm with an ELISA Reader (Molecular Devices, Sunnyvale, CA, USA). The background absorbance was measured at 600 nm and subtracted. Cell

viability was defined as [(test sample count) ) (blank count)/ (untreated control count) ) (blank count)] · 100 (Shimoke and Chiba 2001). Hoechst 33258 staining SK-N-SH cells were fixed with 4% paraformaldehyde for 20 min and then stained with 8 lg/mL of Hoechst dye 33258 (Sigma) for 5 min. They were washed twice with phosphate-buffered saline and then observed using an IX70 microscope (Olympus, Tokyo, Japan) equipped with attachments for fluorescence microscopy. Dead cells and apoptotic bodies were characterized by condensed or fragmented nuclei. Immunoblotting After treatment with 3-HK and/or pharmacological inhibitors, the cells were washed twice with ice-cold phosphate-buffered saline and then collected using a cell scraper followed by a brief centrifugation. Collected cells were divided into two groups, one for whole cell lysates to detect ERK and another for fractionation of cytosol to detect cytochrome c release from mitochondria. Detection of extracellular signal-regulated kinase phosphorylation Cells were dissolved in RIPA buffer (1% TX-100, 20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM Na3VO4, 40 mM NaF, 5 mM EGTA, 0.2% sodium dodecyl sulfate, 0.5% sodium deoxycholate and 0.2 mM phenylmethylsulfonyl fluoride) and then dispersed by sonication. The cell homogenate was centrifuged for 10 min at 1000 g to discard unbroken or coarse cell debris and the resulting supernatant fluid (RIPA lysate) was used for immunoblotting. Protein concentrations of RIPA lysates were determined by a modified Bradford method (Bradford 1976) using bovine serum albumin as a standard. Sample buffer was added to the aliquots (50 lg of protein) of the lysates, boiled for 3 min and then resolved by sodium dodecyl sulfate– polyacrylamide gel electrophoresis under reducing conditions (Laemmli 1970). The resolved proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Little Chalfont, UK) and probed with anti-phospho-p44/42 or anti-p44/42 (Cell Signaling Technology, Beverly, MA, USA). The blots were followed by detection with an ECL detection system (Amersham Pharmacia Biotech). The relative levels of p44/42 phosphorylation were determined using densitometric Bio1D software (Vilber Lourmat, Torcy, France) normalizing to the p44/42 band on a duplicate blot. Detection of cytochrome c release from cytosol Collected cells were resuspended in 100 lL of fractionation buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and 5 lg/mL of each aprotinin and leupeptin) containing 250 mM sucrose. After a 5-min incubation on ice, cells were homogenized with 25 strokes in a dounce homogenizer (Wheaton, NJ, USA). Nuclei and unbroken cell debris were removed by centrifugation at 1000 g for 10 min at 4C and the supernatant fluids were centrifuged again at 14 000 g for 20 min at 4C. The resulting supernatant fluid was used as the soluble cytosolic fraction (Wang et al. 2000). Immunoblotting and evaluation procedures were performed as above except that primary goat anti-cytochrome c antibody and secondary horseradish peroxidase-conjugated antibody against goat (Santa Cruz Biotechnology, Santa Cruz, CA,, USA) were used.


Protective role of ERK in 3-HK-induced neuronal cell death 649

Analysis of mitochondrial membrane potential The changes in mitochondrial membrane potential (DYm) were estimated using tetramethylrhodamine ethyl ester (TMRE; Molecular Probes, Eugene, OR, USA) which is a cationic potentiometric dye that accumulates preferentially into energized mitochondria driven by the membrane potential. To estimate DYm, cells were incubated with 100 nM TMRE for 15 min at 37C and the TMRE fluorescein intensity was then measured with excitation at 549 nm and emission at 574 nm using a fluorometer (TECAN, GENios, Maennedort, Switzerland). The intensity of DYm is expressed as arbitrary units of relative value.

fluorometer (TECAN, GENios). Enzymatic activity is expressed as arbitrary units of relative value.

Caspase substrate cleavage assay For assaying caspase activity in SK-N-SH cells, monolayers of cultured cells (2 · 106 cells) were harvested from a 60-mm dish and lysed with 1 mL of cell lysis buffer. A part (50 lg) of the lysed sample was incubated with 100 lL of HEPES buffer (100 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mM dithiothreitol) containing 0.5 mM each of Ac-DEVD-AMC, Ac-LEHD-AMC and 0.25 mM zVAD-PNA (Pharmingen, San Diego, USA) for 30 min to 1 h. Pan-caspase activity was measured at 405 nm with an ELISA Reader (Molecular Devices). Caspase 3 and 9 activities were measured with excitation at 380 nm and emission at 460 nm using a

Results

Statistical analysis Unless specifically indicated, all data are expressed as mean ± SEM values. To determine the significance of differences between the means of two groups, an unpaired two-tailed Student’s t-test was applied. To determine the significance of differences among the means of several groups, one-way ANOVA followed by Scheffe’s post-hoc tests were applied. p < 0.05 was considered statistically significant.

3-Hydroxykynurenine-induced activation of extracellular signal-regulated kinase in SK-N-SH cells To investigate whether the ERK pathway is involved in 3-HK-induced neuronal cell death, we assessed the levels of active ERK by detecting the phosphorylation of ERK by immunoblotting with anti-phospho-ERK1/2 antibody. As shown in Fig. 1, treating SK-N-SH with 250 lM 3-HK induced activation of ERK1/2. Phosphorylation of ERK1/2 peaked at 30 min after 3-HK treatment and decreased (a)

(b)

Fig. 1 Immunoblot analysis of phospho-activation of extracellular signal-regulated kinase (ERK) 1/2 by 3-hydroxykynurenine (3-HK). (a) SK-N-SH cells were treated with 250 lM of 3-HK for the indicated times and the presence of dually phosphorylated ERK1/2 in whole cell lysate was determined by immunoblotting with anti-phospho-ERK1/2 monoclonal antibody. The same blot was stripped and reprobed with an antibody recognizing non-phosphorylated ERK1/2 (total ERK) to ensure equal protein loading amounts. Blots shown are representative of at least three independent experiments. (b) Quantification of phosphorylated ERK; total levels of ERK1/2 were used as an internal standard in quantification of phosphorylated ERK1/2. The levels of phosphorylated ERK1/2 are expressed as arbitrary units of relative value. *p < 0.05 versus 0 h. ANOVA followed by Scheffe’s post-hoc tests.

Fig. 2 Effects of PD98059 on 3-hydroxykynurenine (3-HK)-induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in SK-N-SH. (a) PD98059 (10 lM) was used for pre-treatment for 2 h and 250 lM of 3-HK was incubated with the cells for the indicated times. Active ERKs in equivalent amounts of total protein lysate (50 lg) were visualized by immunoblot analysis using anti-phospho-ERK1/2 monoclonal antibody. (b) Relative active ERK levels, shown in the graph below the blot, were determined from densitometric analysis of ECL-exposed film. Relative active ERK levels in control cells were given a value of 1. Analogous inhibition of active ERKs by PD98059 was observed in three independent experiments. *p < 0.05 versus 0 h. ANOVA followed by Scheffe’s post-hoc tests.



gradually. Total ERK1/2 levels were not affected by 3-HK treatment (Fig. 1). PD98059 increased 3-hydroxykynurenine-induced neurotoxicity To identify why ERK was activated, we investigated the effects of ERK inhibition on 3-HK-induced cell death. Cotreatment with PD98059, a specific ERK pathway inhibitor, significantly reduced 3-HK-induced ERK activation (Fig. 2). The cell viability, according to AlarmaBlue assay at 24 h, was reduced to 63% by 3-HK alone whereas cotreatment with PD98059 and 3-HK decreased the cell viability further to 30% at the same time point (Fig. 3). We consistently observed morphological changes, such as cell shrinkage and membrane blebbing indicative of 3-HKinduced apoptotic features (Fig. 3d), by phase-contrast microscopy (Fig. 3). Cotreatment with PD98059 exacerbated the morphological changes more severely (Fig. 3e). Similarly, nuclear condensation and fragmentation were detected

Fig. 3 The effects of PD98059 on 3-hydroxykynurenine (3-HK)induced SK-N-SH cell death. PD98059 (10 lM) was used for pretreatment for 2 h and 250 lM of 3-HK for treatment for 24 h. (a) The cell viability was detected using the AlarmaBlue assay (Materials and methods). The relative cell viability of treated cells was decreased compared with the untreated control. Data are the mean value obtained from three independent experiments and the bars represent the SD. Morphological changes associated with 3-HK toxicity and the effect of pre-incubation with PD98059. SK-N-SH cells were treated with 3-HK for 24 h in the presence and absence of PD98059 and viewed under a

36 h after treatment with 3-HK (Fig. 3h) and those changes became more prominent by cotreatment with 3-HK and PD98059 (Fig. 3i). Extracellular signal-regulated kinase 1/2 attenuated increase of mitochondrial cytochrome c release into cytosol and collapse of mitochondrial membrane potential (Dwm) in 3-hydroxykynurenine-induced apoptosis The integrity of mitochondrial function is important to maintain cell viability (Whitlock et al. 2000). So far, it has not been well defined if mitochondrial damage is related significantly in 3-HK-induced neuronal cell death. Mitochondrial damage was detected as a loss of mitochondrial membrane potential (Dwm) and release of cytochrome c. We used TMRE staining to detect Dwm changes. Tetramethylrhodamine ethyl ester, a cationic lipophilic dye, accumulates only in the negatively charged functional mitochondrial matrix (Ehrenberg et al. 1998). As 3-HK-induced neuronal

phase-contrast microscope. (b) Control; (c) PD98059; (d) 3-HK and (e) PD98059 + 3-HK. Scale bar, 20 lm. Nuclear morphological changes of 3-HK- and/or PD98059-treated SK-N-SH cells were detected at the same incubation periods by Hoechst 33258 staining. (f) Control; (g) PD98059; (h) 3-HK and (i) PD98059 + 3-HK. These images were visualized by fluorescence microscopy (Materials and methods). Scale bar, 10 lm. The figures are representative of four independent experiments. *p < 0.05 versus vehicle alone; **p < 0.05 versus 250 lM of 3-HK alone. ANOVA followed by Scheffe’s post-hoc tests.



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Fig. 4 The effects on mitochondrial membrane potential (Dwm) of pretreatment with PD98059 (10 lM) in 3-hydroxykynurenine (3-HK; 250 lM)-treated SK-N-SH cells. (a) The cell viability of SK-N-SH cells was detected using AlarmaBlue assay. (b) Quantification of Dwm with a fluorometer (TECAN GENios). The intensity of tetramethylrhodamine ethyl ester (TMRE) fluorescence was detected with excitation at 549 nm and emission at 574 nm. Fluorescent levels are expressed as arbitrary units of relative value. SK-N-SH cells plated on glass

coverslips were maintained for 6 h at 37C in either (c) culture medium, (d) medium containing 10 lM PD98059, (e) medium containing 250 lM 3-HK or (f) medium containing 250 lM 3-HK + 10 lM PD98059. Cells were then loaded with 100 nM TMRE to measure Dwm (Materials and methods). DYm, as TMRE fluorescence, was evaluated in SK-N-SH by fluorescence microscopy. The data are the mean ± SEM of four separate experiments. Scale bar, 10 lm. *p < 0.05 versus 0 h. ANOVA followed by Scheffe’s post-hoc tests.

cell apoptosis occurs in a time- and dose-dependent manner (Fig. 4a), Dwm also decreased gradually (Fig. 4b). Incubating cells with 3-HK for 6 h reduced the fluorescent intensity of TMRE significantly compared with the control (Figs 4c and e). Whereas PD98059 alone did not damage the Dwm (Fig. 4d), cotreatment with 3-HK and PD98059 increased the Dwm reduction significantly (Fig. 4f). As ERK activation peaks within 30 min of 3-HK treatment, while the effects of 3-HK on cell death are visible between 12 and 24 h and the effects on mitochondrial activity are visible at 3 h, all after ERK levels have returned to baseline, questions as to whether ERK activation is directly linked to protection of mitochondria and whether PD98059 action is through specific inhibition of the ERK pathway have been raised. Therefore, we examined whether severe mitochondrial damage induced by cotreatment with 3-HK and PD98059 was a consequence of ERK inhibition. Pre-incubation of PD98059 perfectly inhibited 3-HKinduced activation of ERK (Fig. 5a, I and II). However, removal of PD98059 after cotreatment with 3-HK and PD98059 for 2 h resulted in moderate phosphorylation of ERK (Fig. 5a, III). In addition, Dwm was also largely recovered by ERK reactivation (Fig. 5b, III) compared with cotreatment with 3-HK and PD98059 (Fig. 5b, II). To exclude the possibility of an effect of PD98059 other than on ERK, PD98059 was used 2 h after 3-HK treatment. As a result, post-treatment with PD98059 2 h after 3-HK treat-

ment could not affect Dwm compared with 3-HK alone treatment groups (Fig. 5b, II and IV). This result suggests that early ERK activation is essential for the self-protective mechanism and attenuation of mitochondrial damage. The other sign of mitochondrial damage is release of cytochrome c from the mitochondria to the cytosol (Green and Reed 1998). However, it is reported that release of cytochrome c is not always linked to mitochondrial damage at the breakdown of Dwm (Colombaioni et al. 2002; Gibson et al. 2002). Thus, to investigate the involvement of cytochrome c in 3-HK-induced neuronal cell death, we estimated the cytochrome c release into cytosol using fractionation and immunoblotting techniques. Treatment of SK-N-SH cells with 3-HK alone for 6 h increased cytochrome c levels 2.1-fold whereas cotreatment with PD98059 increased the levels 1.6 times further compared with 3-HK alone (Fig. 6a). The caspase 9 activity was measured under identical conditions (Fig. 6b). Activation of caspase 9 is caused by cytochrome c released from the mitochondria to the cytoplasm. Extracellular signal-regulated kinase acted as an upstream regulator of caspase in 3-hydroxykynurenineinduced neuronal cell death In a previous study, we reported that 3-HK-induced neuronal apoptosis occurred via increasing caspase activity (Lee et al. 2001). The caspase activity was increased in a time- and dose-dependent manner in 3-HK-induced cell death. Thus,



Fig. 5 Regulation of extracellular signal-regulated kinase (ERK) against mitochondrial membrane potential (Dwm) collapse in 3-hydroxykynurenine (3-HK)-induced apoptosis. (a) Phosphorylation of ERK by either the presence or absence of PD98059. After the treatment with PD98059 for the indicated time, PD98059 was removed and the level of active ERK was determined using western blotting. The cells were harvested at 2 h (I and II) or 3 h (III). (b) The effect of reactivated ERK on Dwm. The intensity of tetramethylrhodamine ethyl ester (TMRE) fluorescence was detected with a fluorometer after a 4-h (I and II) or 6-h (III and IV) 3-HK treatment. The experimental illustration below shows incubation times. A dotted line indicates incubation with 3-HK and solid line indicates the presence of PD98059. Experimental data from one representative experiment are displayed. *p < 0.05 versus 250 lM of 3-HK alone. Unpaired two-tailed Student’s t-tests.

we confirmed the role of active ERK against pan-caspase and caspase 3 activity. As shown in Fig. 7(a), pan-caspase activity increased 4.9-fold by incubating with 3-HK for 6 h, while ERK inhibition significantly increased 7.8-fold compared with the control groups. In addition, 3-HK elevated caspase 3 activity 5.2 times compared with the control groups, whereas pre-incubation of PD98059 enhanced caspase 3 activity 8.8-fold. These results suggest that ERK activation is involved in the negative regulation of apoptosis through inhibiting activation of caspases, such as caspase 3 in 3-HK-induced cell death.

Fig. 6 Release of cytochrome c from mitochondria to cytosol by PD98059 and/or 3-hydroxykynurenine (3-HK) treatment of SK-N-SH cells. (a) Treatment of cells with either 3-HK (250 lM) or PD98059 (10 lM) followed by 3-HK (250 lM) 2 h later increased the level of cytochrome c as indicated. Cell lysates (20 lg) from cultured SK-N-SH cells were loaded on 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting using antibodies against cytochrome c. The data represent three independent experiments. (b) The caspase 9 activities were detected using the fluorogenic substrate Ac-LEHD-AMC. Cellular extracts (20 lg) were incubated with 0.5 mM Ac-LEHD-AMC in 100 lL of total volume at 37C for 30 min. Excitation at 380 nm and emission at 460 nm were measured with a fluorometer (see Materials and methods). The data are shown as means ± SEM for four independent experiments. *p < 0.05 versus vehicle alone; **p < 0.05 versus 250 lM of 3-HK alone. ANOVA followed by Scheffe’s post-hoc tests.

Discussion

There have been several studies that suggest that 3-HK induces apoptotic cell death in neuronal cells. However, the molecular mechanisms of apoptosis induced by 3-HK are not well understood. Moreover, studies on mitochondrial malfunction in 3-HK-induced cell death have not been reported. In the present study, we investigated whether the functional loss of the mitochondria is involved in 3-HK-induced cell death. Our results showed that Dwm was disrupted and cytochrome c was released from the mitochondria to the cytoplasm in 3-HK-induced neuronal apoptosis. These results suggest that the 3-HK-induced neuronal apoptosis goes through mitochondrial dysfunction.



Fig. 7 The effects of PD98059 and caspase inhibitors on 3-hydroxykynurenine (3-HK)-induced caspase activation. The pan-caspase activity and caspase 3 activity induced by 3-HK (250 lM) were measured using the chromogenic substrate, zVAD-PNA, and fluorogenic substrate, Ac-DEVD-AMC (see Materials and methods). The effects of pre-/cotreatment with either PD98059 (10 lM) or zVAD-fmk (10 lM)/Ac-DEVD-CHO (10 lM) are presented. For pan-caspase activity, 20 lg of cellular extracts were incubated with 0.25 mM zVADPNA in 100 lL of total volume at 37C for 1 h. (a) Absorbance at 405 nm was measured with an ELISA Reader. (b) For caspase 3 activity, the same concentration of cellular extracts was incubated with 0.5 mM Ac-DEVD-AMC in 100 lL of total volume at 37C for 30 min. Excitation at 380 nm and emission at 460 nm were measured with a fluorometer. The data are the mean ± SEM of three separate experiments. *p < 0.05 versus vehicle alone; **p < 0.05 versus 250 lM of 3-HK alone. ANOVA followed by Scheffe’s post-hoc tests.

In addition, it is not known whether ERK is involved in 3-HK-induced cell death. It has been suggested that ERK plays a protective role in various systems (Chang and Karin 2001). For example, several studies showed that ERK exerts a protective effect against either neuronal hypoxia or ischemia (Han and Holtzman 2000; Sugino et al. 2000). In other studies, it was reported that ERK acted as a mediator of cell death. Persistent activation of ERK contributes to glutamate-induced oxidative toxicity in a mouse neuronal cell line, HT22, and primary cultures (Stanciu et al. 2000). In addition, PD98059, a specific inhibitor of ERK, enhanced the

survival of an oligodendrocyte cell line, CG4, in hydrogen peroxide-induced cell death (Bhat and Zhang 1999). Therefore, it is essential to study the potential role of ERK in order to understand the cellular mechanism of 3-HK-induced neuronal cell death. Our results showed that a specific inhibitor of the ERK pathway, PD98059, significantly exacerbated the 3-HKinduced cell death, which means that active ERK attenuates 3-HK-induced neuronal apoptosis. We also obtained similar results using another ERK pathway inhibitor, U126 (data not shown). Thus, exacerbation of 3-HK-induced cell death with ERK inhibition is not due to unknown minor actions of ERK inhibitors on mitochondria. Our TMRE results with PD98059 alone also support this idea. Previous reports have implicated ERK in exerting a protective role through activation of transcription factors including cAMP response element modulation protein, Elk1, c-Myc, heat shock protein 25 as well as others (Franklin and McCubrey 2000; Sweatt 2001; Geum et al. 2002). However, it is not clear that the mitochondrial functional changes are associated with ERK activity. Our results showed that inhibition of active ERKs significantly increased the breakdown of Dwm and release of cytochrome c from the mitochondria, which suggests that ERK activation attenuates neuronal cell death by suppressing mitochondrial malfunction induced by 3-HK. Dwm is essential for various mitochondrial functions, including production of ATP by oxidative phosphorylation (Waterhouse et al. 2001). If Dwm is not kept intact, ATP production would be halted, which could possibly lead to cell death. It is reported that ATP depletion leads to cell death as a result of Dwm breakdown. As a result, cytochrome c would be released from the mitochondria to the cytoplasm and the released cytochrome c possibly turns on the apoptotic cascades. It has been reported that released cytochrome c binds to Aparf-1 and that this complex activates caspase 9. The active caspase 9 then cleaves procaspase 3 to caspase 3, which eventually leads to cell death (Li et al. 1997; Srinivasula et al. 1998; Zou et al. 1999; Cain et al. 2000). The mitochondria are closely tied to the Bcl-2 family. In the mitochondrial outer membrane, pro-apoptotic Bcl-2 family proteins, such as Bax and Bak, form large pores through which apoptotic factors are released (Green and Reed 1998; Desagher and Martinou 2000; Korsmeyer et al. 2000). Bax inserts itself into the outer membrane and the assembly of bax oligomers makes the channel composed of a large protein complex (Antonsson et al. 2000; Saito et al. 2000). Translocation of truncated BH3-interacting death agonist (tBid) from the cytosol to the mitochondria induces pore formation to allow the release of cytochrome c (Wei et al. 2000). Thus, Bid and Bax increase the release of cytochrome c through the outer mitochondrial membrane (von Ahsen et al. 2000). On the other hand, Bcl-XL forms a complex with cytochrome c and thus the cytochrome c



Fig. 8 Proposed signal transduction scheme in 3-hydroxykynurenine (3-HK)-induced neuronal cell death and involvement of extracellular signal-related protein kinase (ERK). A temporal or mild 3-HK insult induces ERK activation by protecting the mitochondria, which is critical for cell death commitment. If 3-HK insult is prolonged or the insults are strong, 3-HK-induced ERKmediated mitochondrial protection cannot stand up to the insults further and the characteristic signs of the activation of the apoptotic pathway, such as collapse of mitochondrial potential, cytochrome c release into cytosol and activation of the caspase cascade, occur.

binding activity with the mitochondrial inner membrane is increased (Skulachev 1998). If anti-apoptotic proteins (Bcl-2 and Bcl-XL) failed to block the release of cytochrome c from the mitochondria to the cytosol, then cells will undergo apoptosis in response to apoptogenic treatments such as ionizing radiation (Kharbanda et al. 1997). Thus, mitochondrial malfunction is related to an interaction between antiapoptotic and pro-apoptotic Bcl-2 family members. In this study, it may be suggested that ERK attenuates mitochondrial dysfunction by regulating the Bcl-2 family, such as Bcl2, Bcl-XL, Bax and Bid. Previous results show that ERK could phosphoregulate a pro-apoptotic Bcl-2 family member, Bad (Kunlin et al. 2002). Phosphorylation of Bad on Ser 112 dissociates Bcl-2/Bad heterodimers and unmasks the anti-apoptotic effect of Bcl-2 (Bonni et al. 1999; Scheid et al. 1999). Activation of ERK with high glucose also suppresses apoptosis through up-regulation of Bcl-XL in human coronary artery smooth muscle cells (Sakuma et al. 2002). However, the mechanism by which ERK plays a protective role against mitochondria remains to be further elucidated. It is plausible that activated caspase 9, induced by the release of cytochrome c from the mitochondria, evoked the caspase 3 activation in the present study. Many previous reports have suggested that caspase 9 cleaved procaspase 3 to active caspase 3 and led to cell death. However, another possibility that cannot be excluded is that caspases other than caspase 9 and 3 are involved in the 3-HK-induced cell death because various caspase activities, measured by using a pancaspase substrate (zVAD-PNA) together with the caspase 9 (z-LEHD-fmk) and caspase 3 (Ac-DEVD-CHO) inhibitors, were increased by 3-HK (data not shown). Furthermore,

those activities were also increased by PD98059, which implies that other caspase activities may be associated with ERK. In summary, 3-HK causes neuronal cell death by inducing the dysfunction of mitochondria. In addition, in this mechanism, activation of ERK by 3-HK blocks mitochondrial malfunction and activation of several caspases, therefore, attenuates cell death (as schematically depicted in Fig. 8). An increased level of 3-HK was detected in several neurodegenerative brains from patients with Alzheimer’s disease, Parkinson’s disease and Huntington’s disease (Ogawa et al. 1992; Pearson and Reynolds 1992; Sardar et al. 1995). Consequently, in order to develop new strategies for treating diseases associated with kynurenine metabolites and neuronal cell death, it may be very valuable to elucidate the detailed functional role of ERK in 3-HK-induced neuronal cell death. Acknowledgements This research was supported by grants from the Korea Health 21 R & D Project funded by the Ministry of Health and Welfare of the Korean government (01-PJ8-PG1-01 CN2-0003) and Rural Development Administration (02-N-I-02).

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