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Keywords:

  • c-Jun N-terminal kinase;
  • extracellular signal-regulated kinase;
  • mitogen-activated protein kinases;
  • reactive oxygen species;
  • Zn2+

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia- and seizure-induced neuronal death. However, the intracellular signaling pathways involved in Zn2+-induced cell death are largely unknown. In the present study, we investigated the roles of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+-induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 µm), a Zn2+ ionophore, and Zn2+ (10 µm) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+-induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol-3 kinase had little effect on Zn2+-induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+-induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.

Abbreviations used
DCF

2′,7′-dichlorodihydrofluorescein diacetate

ERK

extracellular signal-regulated kinase

GSH

glutathione

HBSS

Hank's balanced salt solution

JNK

c-Jun N-terminal kinase

JNKK

JNK kinase

MAPKs

mitogen-activated protein kinases

MnTBAP

manganese (III) tetrakis (4-benzoic acid) porphyrin

NAC

N-acetyl-cysteine

NGF

using nerve growth factor

PDTC

pyrrolidine dithiocarbamate

PKC

protein kinase C (PKC)

ROS

reactive oxygen species

TPEN

N,N,N′,N′-tetrakis-2-pyridylmethyl ethylenediamine.

Neurological insults, such as ischemia and seizures, can trigger selective neuronal cell death in various brain areas (Meldrum 1993; Choi 1996). It is well established that post-synaptic Ca2+ accumulation, mediated by the activation of glutamate receptors, plays a crucial role in ischemia- and seizure-induced neuronal cell death (Hartley et al. 1993; Choi 1995). However, it has also been shown that the release of high concentrations of Zn2+ from excitatory nerve terminals followed by an influx across the post-synaptic neuronal membrane contributes to neuronal cell death (Choi and Koh 1998; Weiss and Sensi 2000).

The mechanisms responsible for cell death following intracellular Zn2+ accumulation have not been fully elucidated. The inhibition of energy production is suggested to be an important contributory factor to Zn2+-induced neuronal death (Choi and Koh 1998; Sheline et al. 2000). Recently, however, some studies have shown that generation of reactive oxygen species (ROS) is enhanced by intracellular Zn2+ overload (Kim et al. 1999b; Sensi et al. 1999), although the involvement of oxidative stress in Zn2+-induced cell death is controversial (Sheline et al. 2000). Oxidative stress is associated with several neurodegenerative diseases (Murphy et al. 1989; Foley and Riederer 2000) and has been found to trigger cell death via various signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways (Lander 1997).

The MAPK family, which includes c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), is comprised of serine/threonine kinases that have fundamental roles in both the maintenance of cell survival and the induction of cell death. c-Jun N-terminal kinase and p38 MAPK mediate cellular responses to stress and have often been demonstrated to be involved in cell death in many cell types, including PC12 cells (Xia et al. 1995; Verheij et al. 1996; Basu and Kolesnick 1998). In contrast, ERK is mainly activated by growth factors and has been shown to be associated with cell proliferation and differentiation (Xia et al. 1995; Derkinderen et al. 1999). However, this is not always the case and growing evidence suggests that activation of ERK also contributes to neuronal death (Murray et al. 1998; Runden et al. 1998). Interestingly, both JNK and ERK have been reported to participate in cell death induced by ROS. c-Jun N-terminal kinase was shown to be involved in the ROS-mediated cell death induced by daunorubicin, β-lapachone and auto-oxidized dopamine in U937, HL-60 and PC12 cells, respectively (Kang et al. 1998; Mansat-de Mas et al. 1999; Shiah et al. 1999), while ERK contributed to glutamate-induced oxidative toxicity in neurons (Stanciu et al. 2000).

Given the widespread involvement of MAPKs in cell death pathways, in the present study we investigated the roles of these kinases in Zn2+-induced cell death using nerve growth factor (NGF)-differentiated PC12 cells. In addition, we examined the role of ROS and their interaction with MAPKs in Zn2+-induced cell death. Our results indicate that JNK and ERK are activated by the intracellular accumulation of Zn2+, but that only ERK, and not JNK, contributes to Zn2+-induced cell death. Furthermore, they indicate that the Zn2+-induced activation of ERK is mediated by the enhanced accumulation of ROS via the Ras/Raf/MEK/ERK signaling cascade.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

The PC12 cells were obtained from ATCC (Manassas, VA, USA). Anti-phospho ERK, anti-ERK, anti-phospho JNK, anti-JNK, anti-phospho p38 MAPK and anti-p38 MAPK polyclonal antibodies were purchased from New England Biolabs (Beverly, MA, USA). PD98059, wortmannin, LY294002, GF109203X and manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) were purchased from Calbiochem (San Diego, CA, USA). Magfura-2AM, 2′,7′-dichlorodihydrofluorescein diacetate (DCF) and N,N,N′,N′-tetrakis -2-pyridylmethyl ethylenediamine (TPEN) were purchased from Molecular Probes (Eugene, OR, USA). Other reagents including ZnCl2 and 1-hydroxypyridine-2-thione (pyrithione) were obtained from Sigma (St Louis, MO, USA), unless indicated otherwise.

Cell culture

To obtain neuronally differentiated PC12 cultures, cells were grown on collagen (10 µg/mL; Upstate Biotechnology, Lake Pacid, NY, USA) coated plates for 7 days in Dulbecco's modified Eagle's medium (Life Technologies Inc., Grand Island, NY, USA). The cultures were supplemented with 2% heat-inactivated horse serum and 1% fetal bovine serum (Life Technologies, Inc.) containing 50 ng/mL of 2.5S mNGF (Alomone Laboratory, Jerusalem, Israel). The medium, including NGF, was replaced every 2 days. Cultures were maintained at 37°C in a humidified, 5% CO2 incubator. All experiments were performed in the presence of NGF to exclude the possibility of NGF-deprived cell death signaling pathways.

Cell transfection

The PC12 cells were transiently transfected with Ras dominant negative mutant pMT3-RasN17 (kindly provided by C. J. Marshall, Institute of Cancer Research, London, UK) or SEK1 dominant negative mutant pCMVSEKAL (kindly provided by D. Templeton, Case Western University, Cleveland, OH, USA) using LipofectAMINE 2000 transfection reagent (Life Technologies, Inc.). LipofectAMINE 2000-mediated transfections were performed according to the manufacturer's instructions. In each experiment, the transfection efficiency was determined by the cotransfection of green fluorescent protein expression construct pEGFP-N1 (GFP; CLONTECH, Palo Alto, CA, USA). Following a two-day expression period in the presence of 50 ng/mL NGF, more than 50% of transfected cells were verified by fluorescence microscopy. For analysis of nuclear morphology, cells were stained with 1 µg/mL Hoechst 33258 and examined under UV illumination using fluorescence microscope.

Western blot analysis

Following the indicated treatments, cells were washed with ice-cold phosphate-buffered saline. Cells were then lysed in buffer containing 10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1 mm EDTA, 1 mm EGTA, 1 mm PMSF, 1 µg leupeptin and 1 µg aprotinin per mL. The protein concentration of lysate was normalized with BCA reagent (Pierce, Rockford, IL, USA). Equal amounts of proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred to nitrocellulose membranes. Blots were blocked with 5% non-fat dried milk in TBST (20 mm Tris-HCl, 150 mm NaCl, and 0.1% Tween 20, pH 7.5) for 1 h and then incubated overnight with anti-phospho ERK, anti-phospho JNK or anti-phospho p38 MAPK antibody diluted 1 : 1000 in blocking solution at 4°C. After being washed with TBST and incubated with 1: 2000 diluted horseradish peroxidase-conjugated anti-rabbit IgG antibody for 1 h at room temperature (22°C), the blots were immuno-detected with an ECL detection system (Amersham-Pharmacia Biotech, Braunschweig, Germany). To verify equivalent sample loading, membranes were stripped and re-probed with anti-ERK, anti-JNK and anti-p38 MAPK antibodies, respectively. Relative levels of each MAPK activated by intracellular Zn2+ were determined by the densitometric scanning of enhanced chemiluminescence-exposed film.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay

Approximately 5 × 103 cells were plated onto collagen-coated 48-well plates and allowed to differentiate to neuronal cells over 7 days. After each indicated treatment, 20 µL stock 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, 5 mg/mL in sterile phosphate-buffered saline) was added and incubated for 1 h at 37°C. Finally, 200 µL of solubilizing solution (50% dimethylformamide and 20% sodium dodecyl sulfate, pH 4.8) was added. After incubating overnight, the absorption value at 570 nm was determined. Viability was determined as percentage survival =[(experimental–blank)/(control–blank)], where experimental, control and blank were the readings for the treated cells, untreated cells and the MTT added to the medium, respectively.

Measurement of intracellular Zn2+

Intracellular Zn2+ concentration ([Zn2+]i) in differentiated PC12 cells was measured using a modification of a method described previously (Cheng and Reynolds 1998). Briefly, cells were loaded with magfura-2 at 37°C in a 5% CO2 incubator by including 3 µm magfura-2AM for 20 min in a HCO3-buffered solution containing (in mm): 110 NaCl, 4.5 KCl, 1 NaH2PO4, 1 MgSO4, 1.5 CaCl2, 5 HEPES-Na, 5 HEPES free acid, 25 NaHCO3 and 10 d-glucose (pH 7.4). Cells were then rinsed twice and incubated in the HCO3-buffered solution for at least 20 min before use.

The [Zn2+]i was measured on the stage of an inverted microscope (Nikon, Tokyo, Japan) by spectrofluorometry (Photon Technology International, Brunswick, NJ, USA), while cells were superfused at a constant perfusion rate of 2 mL/min with the HCO3-buffered solution equilibrated with 95% O2, 5% CO2 to maintain a pH of 7.4. All experiments were performed at 37°C. The excitation wavelength was alternated between 340 and 380 nm and the emission fluorescence was recorded at 510 nm. The values of [Zn2+]i were calculated using a modification of the equation described by Grynkiewicz (Grynkiewicz et al. 1985):

[Zn2 + ]i  =  Kd . Sf2/Sb2[(R − Rmin)/(Rmax − R)]

in which Kd of magfura-2 for Zn2+ was assumed to be 20 nm (Simons 1993), R is the observed 340/380 fluorescence ratio, Rmin the 340/380 fluorescence ratio value determined in magfura-2 loaded differentiated PC12 cells exposed to 100 µm of TPEN, and Rmax the 340/380 fluorescence ratio value (obtained by adding 20 µm pyrithione, a Zn2+ ionophore, in the presence of 1 mm Zn2+). Sf2 is the fluorescence intensity at 380 nm at Rmin and Sb2 the fluorescence intensity at 380 nm at Rmax.

Visualization of intracellular reactive oxygen species

Reactive oxygen species (ROS) levels were measured using a fluorescence probe, DCF (Greenlund et al. 1995). In brief, cells were incubated for 5 min in the presence of 5 µg/mL of DCF and washed in Hank's balanced salt solution (HBSS). The DCF fluorescence was measured using a confocal microscope (Leica, Buffalo, NY, USA) with an excitation wavelength at 488 nm and an emission at 525 nm. To avoid photo-oxidation of DCF, the fluorescence images were collected using a single rapid scan, identical settings were used for all samples. All experiments were repeated five times.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Intracellular accumulation of Zn2+ induces cell death in differentiated PC12 cells

In order to increase [Zn2+]i, we treated differentiated PC12 cells with 5 µm pyrithione in the presence of 10 µm Zn2+. This allowed us to achieve rapid and relatively constant elevated levels of [Zn2+]i without depolarizing the cells. As shown in Fig. 1(a), 10 µm Zn2+ or 5 µm pyrithione alone did not affect the fluorescence ratio in magfura-2-loaded PC12 cells. However, the fluorescence ratio began to increase immediately after the addition of 5 µm pyrithione in the presence of 10 µm Zn2+, and reached a plateau within 20 min. Application of 10 µm TPEN, a membrane permeable Zn2+ specific chelator, promptly decreased the fluorescence ratio to the basal level. This result indicated that, although magfura-2 could detect Ca2+ and Mg2+, the increased fluorescence ratio caused by combined application of 10 µm Zn2+ and 5 µm pyrithione (Zn2+ pyrithione) resulted solely from the influx of Zn2+. The elevated [Zn2+]i achieved by applying Zn2+ pyrithione was calculated to be 220 ± 10 nm (mean ± SEM, n = 4). However, we do not rule out the possibility that the value may be slightly underestimated, because the high-affinity Zn2+ probe magfura-2 (Kd = 20 nm) was used.

image

Figure 1.  Combined application of Zn2+ and pyrithione induces intracellular accumulation of Zn2+ and cell death in differentiated PC12 cells. (a) Changes in [Zn2+]i were measured in magfura-2 loaded differentiated PC12 cells using ratiometric fluorescence recording techniques. Cells were exposed to 5 µm pyrithione, 10 µm Zn2+ or 5 µm pyrithione in the presence of 10 µm Zn2+. The TPEN (10 µm) was added at the indicated time to chelate Zn2+ when cells were exposed to Zn2+ followed by pyrithione. The result is representative of four independent experiments. (b) Differentiated PC12 cells were incubated with 5 µm pyrithione, 10 µm Zn2+ or 5 µm pyrithione in the presence of 10 µm Zn2+ for 12 h and cell viability was assessed by MTT assay as described in Methods and materials. Results are presented as mean ± SD (n = 4 cultures per condition). ▪, Zn2+●, pyrithione, ▴, Zn2++ pyrithione.

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To investigate the effect of intracellular Zn2+ on cell death, cell viability was measured using the MTT assay. As shown in Fig. 1(b), treatment with Zn2+ pyrithione caused a time-dependent decrease in the cell viability of differentiated PC12 cells (n = 4). After 12 h, approximately 90% of the cells were dead. Exposure of cells to 10 µm Zn2+ alone did not induce cell death and 5 µm pyrithione caused a small decrease in cell viability at 12 h.

Intracellular accumulation of Zn2+ activates JNK and ERK, but not p38 MAPK, in differentiated PC12 cells

To assess the activation of MAPKs in differentiated PC12 cells, antibodies were used that recognize the activated forms of MAPKs. We first examined whether the intracellular accumulation of Zn2+ induced an activation of JNK (i.e. p46 JNK-1 and p54 JNK-2). As shown in Fig. 2(a), this proved to be the case. A maximum 28.9-fold increase in JNK activation was observed 3 h after Zn2+ pyrithione treatment, and decreased thereafter. This transient increase in JNK activation was observed in each independent experiment, although total JNK levels were unaltered (n = 5). We then examined whether intracellular Zn2+ activates ERK. As shown in Fig. 2(b), ERK (i.e. p42 ERK-1 and p44 ERK-2) was also activated by intracellular Zn2+ accumulation. However, the activation of ERK continued to increase upon Zn2+ pyrithione treatment and a 75.3-fold increase in ERK activation was observed after 4 h. Total ERK levels were unaffected by intracellular Zn2+ accumulation (n = 5). In contrast to JNK and ERK, p38 MAPK was not activated by the application of Zn2+ pyrithione (n = 4; Fig. 2c).

image

Figure 2.  Intracellular accumulation of Zn2+ activates JNK and ERK, but not p38 MAPK, in PC12 cells. Differentiated PC12 cells were incubated with Zn2+ (10 µm) and pyrithione (5 µm) for the indicated times. Cells were then analyzed by western blot using an anti-phospho JNK (a, n = 5), anti-phospho ERK (b, n = 5) or anti-phospho p38 MAPK (c, n = 4) antibody (upper panel). To verify equivalent sample loading, membranes were stripped and re-probed with anti-JNK (a), anti-ERK (b) and anti-p38 MAPK (c) antibodies, respectively (lower panel). In (c), cells were exposed to 10 µg/mL anisomycin (An) for 4 h as a positive control.

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Inhibition of JNK activation does not prevent Zn2+-induced PC12 cell death

To investigate the role of JNK in Zn2+-induced PC12 cell death, cells were transiently transfected with dominant negative JNK kinase (JNKK)/SAPK kinase (SEK1) mutant (SEKAL). The Zn2+-induced JNK activation was found to be inhibited in SEKAL transfected PC12 cells compared with the level of JNK activation in the empty vector-transfected cells (n = 3; Fig. 3a). We then tested whether the inhibition of JNK activation modulated the cell death induced by intracellular Zn2+ accumulation. Assessment of cell viability using the MTT assay revealed that there was no significant difference in the cell viabilities of SEKAL-transfected and empty vector-transfected cells (n = 4; Fig. 3b). This result was confirmed by a further experiment in which the nuclear morphology of SEKAL-transfected cells was compared with that of GFP-positive control cells. As shown in Fig. 3(c), the chromatin of GFP-positive control cells was condensed and fragmented by Zn2+, which was not affected by the transfection of SEKAL, i.e. transfection of SEKAL did not exert any protective effect on Zn2+-induced cell death. These results indicate that JNK is not an active participant in the Zn2+-induced PC12 cell death program.

image

Figure 3.  Inhibition of JNK does not prevent Zn2+-induced PC12 cell death. (a) Where indicated, PC12 cells were transiently transfected with 5 µg of empty vector (vector) or SEK1 dominant negative mutant vector (SEKAL). After a two-day expression period, PC12 cells were incubated with (+) or without (–) Zn2+ (10 µm) and pyrithione (5 µm) for 3 h. Cells were then analyzed by western blot with an anti-phospho JNK antibody (upper panel) and re-probed with an anti-JNK antibody (lower panel). Similar results were observed in three independent experiments. (b) PC12 cells were transfected using the method described in (a), and incubated with (+) or without (–) Zn2+ (10 µm) and pyrithione (5 µm) for 8 h. Viable cells were assessed by the MTT method. Results are expressed as mean ± SD of four independent experiments. *Indicates the difference from the empty vector-transfected control level (p < 0.05); N.S. indicates that there is no significant difference from the Zn2+ pyrithione-treated level in empty vector-transfected cells (p > 0.05), as determined by anova and Bonferroni's test. (c) PC12 cells were transfected using the method described in (a) and incubated with Zn2+ (10 µm) and pyrithione (5 µm) for 3 h. Nuclear morphology was determined by Hoechst 33258 staining. The GFP-positive cells were observed by fluorescence microscopy and the same field was visualized by phase-contrast microscopy. An arrowhead indicates a GFP-positive cell.

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Inhibition of ERK activation attenuates Zn2+-induced PC12 cell death

The activation of ERK requires phosphorylation of both threonine and tyrosine residues by the dual specificity MAP kinase/ERK kinase (MEK1/2; Anderson et al. 1990). Extra cellular signal-regulated kinase is the only known substrate for MEK1/2 (Seger and Krebs 1995). A synthetic inhibitor of MEK1/2, PD98059, binds to the dephosphorylated form of MEK1/2, preventing its phosphorylation and activation, and the subsequent activation of ERK (Dudley et al. 1995). In vitro and in vivo studies have shown that PD98059 is highly specific, with no known effects on at least 18 other kinases including JNK and p38 MAPK (Alessi et al. 1995; Pang et al. 1995). Here we examined the effect of PD98059 on Zn2+-induced ERK activation. Treatment of differentiated PC12 cells with PD98059 exerted concentration-dependent inhibition of Zn2+-induced ERK activation in the concentration range 1–50 µm (n = 3; Fig. 4a). To determine whether ERK activation contributed to Zn2+-induced cell death, cells were pre-treated with 50 µm PD98059 for 10 min and cell viability was measured using MTT assay, 8 h after Zn2+ pyrithione treatment. As shown in Fig. 4(b), Zn2+-induced cell death was significantly inhibited by 50 µm PD98059 (n = 3, p < 0.05). This result suggests that strong prolonged activation of ERK caused by the intracellular accumulation of Zn2+ contributes to the induction of cell death.

image

Figure 4.  Inhibition of ERK prevents Zn2+-induced PC12 cell death. (a) PC12 cells were incubated with or without Zn2+ (10 µm) and pyrithione (5 µm) for 3 h. Zn2+ pyrithione-applied cells were pre-treated with PD98059, an MEK1/2 inhibitor, for 10 min at the indicated concentrations. Cells were then analyzed by western blot with an anti-phospho ERK antibody (upper panel) and re-probed with an anti-ERK antibody (lower panel). Similar results were observed in three independent experiments. (b) PC12 cells were incubated with (+) or without (–) Zn2+ (10 µm) and pyrithione (5 µm) for 8 h in the presence (+) or absence (–) of 50 µm PD98059. PD98059 was applied 10 min prior to Zn2+ pyrithione treatment when present. Cell viability was measured using the MTT method. Results are presented as mean ± SD of three independent experiments. *Indicates difference from the untreated level (p < 0.05); #indicates difference from the Zn2+ pyrithione-treated but PD98059-untreated level (p < 0.05), as determined by anova and Bonferroni's test.

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ERK activation induced by Zn2+ is Ras-mediated

We next sought to determine which signaling pathways were required for the activation of ERK induced by intracellular Zn2+ accumulation. As the most extensively characterized regulator of the ERK signaling pathway is the small GTP-binding protein, Ras (Pawson 1995), we first determined whether active Ras was necessary for Zn2+ to activate ERK, using cells transfected with Ras dominant negative (RasN17) mutant. We observed that Zn2+-induced ERK activation was strongly reduced in RasN17-transfected PC12 cells compared with the level of ERK activation in the empty vector-transfected cells (n = 4; Fig. 5a). Furthermore, over-expression of RasN17 significantly prevented Zn2+-induced cell death (n = 4, p < 0.05; Fig. 5b) and nuclear condensation and fragmentation (Fig. 5c). These results indicate that Zn2+-induced ERK activation and PC12 cell death are mediated by the activation of Ras.

image

Figure 5.  Zn2+-induced ERK activation and cell death are mediated by Ras. (a) PC12 cells were transiently transfected with empty vector (vector) or Ras dominant negative mutant (RasN17) vector. After a two-day expression period, PC12 cells were incubated with (+) or without (–) Zn2+ (10 µm) and pyrithione (5 µm) for 3 h. Cells were then analyzed by western blot with an anti-phospho ERK anti-body (upper panel) and re-probed with an anti-ERK antibody (lower panel). Similar results were observed in four independent experiments. (b) Transfected PC12 cells were incubated with (+) or without (–) Zn2+ (10 µm) and pyrithione (5 µm) for 8 h. Cell viability was determined by MTT assay. Results are presented as mean ± SD of four independent experiments. *Indicates difference from the empty vector-transfected level (p < 0.05); #indicates difference from the Zn2+ pyrithione-treated level in empty vector-transfected cells (p < 0.05), as determined by anova and Bonferroni's test. (c) PC12 cells were transfected using the method described in (a), and incubated with Zn2+ (10 µm) and pyrithione (5 µm) for 3 h. Nuclear morphology was determined by Hoechst 33258 staining. The GFP-positive cells were observed by fluorescence microscopy and the same field was visualized by phase-contrast microscopy. An arrowhead indicates a GFP-positive cell.

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The protein kinase C (PKC)- and phosphatidylinositol (PI) 3-kinase-dependent pathways are not involved in Zn2+-induced ERK activation

As Ras-independent pathways, like the protein kinase C (PKC)- and PI3-kinase-dependent pathways, have also been reported to activate ERK (Kolch et al. 1993; Duckworth and Cantley 1997; Grammer and Blenis 1997; Marais et al. 1998; Formisano et al. 2000), we examined the involvement of these kinases in Zn2+-induced ERK activation. As shown in Fig. 6(a and b), the PKC inhibitor GF109203X (1–5 µm), and the PI3-kinase inhibitors wortmannin (100 nm) and LY294002 (50 µm) failed to inhibit Zn2+-induced ERK activation (n = 5 and 4, respectively). Wortmannin, GF109203X and LY294002 were previously shown to inhibit the activation of PKC and PI3-kinase in PC12 cells at similar concentrations (Hundle et al. 1995; Haring et al. 1998; Neri et al. 1999; Venkateswarlu et al. 1999). Treatment with each inhibitor alone failed to induce ERK activation (data not shown). These results suggest that neither PKC nor PI3-kinase modulates the activation of ERK induced by intracellular Zn2+ accumulation.

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Figure 6.  Zn2+-induced ERK activation is independent of PKC and PI-3 kinase. PC12 cells were incubated with or without Zn2+ (10 µm) and pyrithione (5 µm) for 3 h. Cells were pre-treated with a PKC specific inhibitor GF109203X at the indicated concentrations (a, n = 5) or with PI-3 kinase inhibitors, 100 nm wortmannin or 50 µm LY294002 (b, n = 4) for 10 min. They were then analyzed by western blot with an anti-phospho ERK antibody (upper panels) and re-probed with an anti-ERK antibody (lower panels).

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ROS are responsible for ERK activation in Zn2+-induced cell death

To determine whether ROS contribute to Zn2+-induced cell death in PC12 cells, we first examined the effect of various antioxidants, such as 1 mmN-acetyl-cysteine (NAC), 1 mm glutathione (GSH), and 10 µm MnTBAP, on Zn2+-induced cell death. Pretreatment with antioxidants for 10 min greatly attenuated Zn2+-induced cell death as shown in Fig. 7(a) (n = 4, p < 0.05). As we had found that cell death caused by intracellular Zn2+ accumulation was mediated by ERK activation, we next tested whether the antioxidants also prevented ERK activation caused by Zn2+ accumulation. As shown in Fig. 7(b), all antioxidants examined in this study decreased the activation level of ERK (n = 4). These results suggest that ROS may mediate ERK activation and cell death caused by Zn2+ accumulation. Therefore, to confirm that Zn2+ accumulation enhanced ROS generation in differentiated PC12 cells, ROS levels were determined by DCF fluorescence after incubation with Zn2+ pyrithione for 1 h. As shown in Fig. 8(a and b), elevated levels of ROS were observed in cells treated with Zn2+ pyrithione compared with untreated cells (n = 4). Pre-treatment of cells with NAC (1 mm) for 10 min almost completely prevented the accumulation of ROS (n = 4; Fig. 8c). On the other hand, the accumulation of ROS was not significantly affected by the 10 min pretreatment with PD98059 (n = 4; Fig. 8d), suggesting that ERK activation does not contribute to the generation of ROS.

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Figure 7.  Zn2+-induced cell death and ERK activation are blocked by antioxidants. (a) PC12 cells were incubated with (+) or without (–) Zn2+ (10 µm) and pyrithione (5 µm) for 8 h. Cells were pre-treated with (+) or without (–) antioxidants, 1 mmN-acetyl-cystein (NAC), 1 mm GSH and 10 µm MnTBAP, for 10 min. The viability of cells was assessed by the MTT assay. Results are mean ± SD of four independent experiments. (b) PC12 cells were incubated with (+) or without (–) Zn2+ (10 µm) and pyrithione (5 µm) for 3 h. Cells were pre-treated with (+) or without (–) the same antioxidants as used in (a) for 10 min, and then analyzed by western blot with an anti-phospho ERK antibody (upper panel) and re-probed with an anti-ERK antibody (lower panel). Similar results were observed in four independent experiments. *Indicates difference from the control level (p < 0.05); # indicates difference from the Zn2+ pyrithione-treated level (p < 0.05), as determined by anova and Bonferroni's test.

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image

Figure 8.  Intracellular Zn2+ enhances ROS generation. PC12 cells were incubated with (b, c and d) or without (a) Zn2+ (10 µm) and pyrithione (5 µm) for 1 h. Cells were pre-treated with 1 mm NAC (c) or 50 µm PD98059 (d) for 10 min, and then loaded with a ROS fluorescence probe DCF for 10 min. After washing with HBSS, ROS were detected by confocal microscopy. Similar results were observed in four independent experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In the present study, we found that the application of Zn2+ pyrithione led to time-dependent cell death over a 12-h period in differentiated PC12 cells. Exposure of cells to Zn2+ (10 µm) in the absence of pyrithione did not increase [Zn2+]i and had no apparent toxic effects, implying that Zn2+ entry is necessary for cell death. These results are in agreement with previous reports that the neurotoxic effects of Zn2+ occur after Zn2+ entry via various routes, such as, voltage-sensitive Ca2+ channels, NMDA channels and Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid/kainate channels (Weiss et al. 1993; Yin and Weiss 1995; Choi and Koh 1998). Using the Zn2+-sensitive fluorescence dye, magfura-2, we confirmed that the application of Zn2+ pyrithione caused a rapid accumulation of Zn2+, from 17 nm to 220 nm, whereas both 10 µm Zn2+ and 5 µm pyrithione alone had almost no effect on [Zn2+]i. The Zn2+ pyrithione-induced increase in the magfura-2 fluorescence ratio was completely reversed by a Zn2+ specific chelator, TPEN, confirming that the Zn2+ pyrithione-induced increase in fluorescence ratio specifically represented an increase in [Zn2+]i. Application of pyrithione (5 µm) alone induced a small increase in cell death. This increase was probably the result of Zn2+ entry from the culture media; this source of Zn2+ has been shown to be responsible for pyrrolidine dithiocarbamate (PDTC)-induced cell death in bovine cerebral endothelial cells (Kim et al. 1999a). Therefore, these findings suggest that the observed Zn2+ pyrithione-induced cell death was caused entirely by the intracellular accumulation of Zn2+.

We found that Zn2+-induced cell death was accompanied by the activation of both JNK and ERK in differentiated PC12 cells. However, inhibition of JNK activation by the expression of dominant negative SEK1 (SEKAL) did not reduce Zn2+-induced cell death, indicating that the activation of JNK plays little role in Zn2+-induced cell death in our system. On the other hand, our results showed that the inhibition of ERK by PD98059, an MEK1/2 inhibitor, significantly attenuated Zn2+-induced cell death. Our findings are contrary to those previously reported by Xia and colleagues (Xia et al. 1995), which demonstrated that the activation of JNK and p38 MAPK and the concurrent inhibition of ERK were critical for the induction of apoptosis induced by NGF withdrawal in PC12 cells. Furthermore, they reported that the activation of the ERK pathway prevented apoptosis and promoted the survival of differentiated PC12 cells. Taken together, our study and that of Xia and colleagues imply that, even in the same cell types, ERK may have a dual role in the regulation of cell survival and death. The way in which ERK mediates these opposing cellular processes is unknown. One possibility is that strong and persistent activation of ERK leads to cell death (Stanciu et al. 2000), whereas a short-lived activation of ERK is associated with proliferation (Fukunaga and Miyamoto 1998). In support of this, our data showed that Zn2+-induced ERK activation continued to increase for 4 h and reached a level approximately some 75-fold higher than that observed before Zn2+ pyrithione treatment. Although the sustained activation of ERK has also been reported to mediate NGF-induced differentiation of PC12 cells (Marshall 1995), we found a marked difference in the kinetics of ERK activation between NGF and Zn2+ treatment. Stimulation of PC12 cells with NGF induced a biphasic increase in ERK activity, i.e. an 81-fold increase in ERK activity at 5 min followed by a decrease to a sustained elevated level that was approximately 10-fold higher than the value obtained before NGF stimulation (data not shown). This is in contrast to the activation pattern of ERK caused by Zn2+. In addition to this, the intensity of ERK activation by Zn2+ was much greater than that induced by NGF. Considering that PC12 cells had been exposed to NGF for 7 days before Zn2+ pyrithione was treated, the magnitude of ERK activation induced by Zn2+ was 75 times greater than that needed for differentiation of PC12 cells. Thus, the magnitude and duration of ERK activation might be an important factor that determines whether cells undergo survival or death. Prolonged strong activation of ERK may switch on a downstream signal leading to cell death, whereas transient or biphasic activation of ERK appears to be required for the proliferation and differentiation of PC12 cells. Our results indicate that the intracellular accumulation of Zn2+ resulted in a sustained and strong activation of ERK and that the over-stimulation of ERK might have been responsible for the cell death.

It has been reported that sustained ERK activation is associated with translocation of ERK to the nucleus, whereas transient activation does not lead to nuclear translocation (Marshall 1995). Therefore, it is possible that Zn2+-induced sustained and strong activation of ERK leads to translocation and accumulation of active ERK in the nucleus, which might play a critical role in Zn2+-induced cell death. This possibility is supported by the report that focal ischemia and reperfusion caused nuclear accumulation of pERK and neuronal cell damage, which was attenuated by PD98059 (Alessandrini et al. 1999). Although the downstream target of ERK has not been identified, it was reported that induction of an immediate early gene, egr-1, through ERK activation might be involved in the Zn2+-induced cell death of cortical neurons (Park and Koh 1999).

In contrast to ERK and JNK, p38 MAPK was not activated by Zn2+. Such differential activation of MAPKs was also reported previously, i.e. PDTC induced an activation of ERK and JNK, but not p38 MAPK in PC12 cells (Chung et al. 2000). As PDTC was shown to mediate the influx of Zn2+ and Cu2+ and the divalent metal chelators, such as EDTA and bathocuproline disulfonic acid, modulated the activities of ERK and JNK (Kim et al. 1999a; Chung et al. 2000), these metal ions were thought to be responsible for the differential activation of MAPKs caused by PDTC. However, the role of each MAPK on Zn2+- and Cu2+-induced cell death has not been elucidated yet. Now, our data indicate that although JNK is also activated by Zn2+, activation of ERK is mainly responsible for Zn2+-induced cell death and that when ERK plays a critical role in cell death, JNK and p38 MAPK contribute little to the signaling pathway leading to cell death.

The predominant signaling pathway for ERK activation has been proposed to involve the Ras/Raf/MEK/ERK cascade (Greene and Kaplan 1995). The small GTP-binding protein, Ras, is activated by guanine nucleotide exchange factors recruited to the membrane by various adaptor proteins in response to receptor tyrosine kinase stimulation (Pawson 1995). Transmission of signals from Ras is achieved by sequential phosphorylation and activation of kinases consisting of Raf (MEKK), MEK and ERK. However, it has been reported that Ras-independent pathways also lead to ERK activation. For example, PKC has been identified as an activator of MEK and ERK via the stimulation of Raf-1 kinase (Kolch et al. 1993; Marais et al. 1998; Formisano et al. 2000). Furthermore, PI 3-kinase has been reported to regulate the ERK pathway (Duckworth and Cantley 1997; Grammer and Blenis 1997). In the present study, we have demonstrated that the expression of dominant negative Ras mutant (RasN17) strongly reduced ERK activation and cell death caused by Zn2+ accumulation. However, the inhibitors of PKC (GF109203X) and PI3-kinase (LY294002 and wortmannin) did not inhibit ERK activation, which suggested that PKC and PI3-kinase did not participate in modulating ERK activation. Thus, our data suggest that the activation of the Ras/Raf/MEK/ERK cascade is associated with Zn2+-induced cell death in differentiated PC12 cells.

Having established the involvement of the Ras/Raf/MEK/ERK signaling cascade in Zn2+-induced cell death mechanisms, we sought to determine whether Zn2+-induced activation of ERK was mediated by ROS. As ROS have been suggested to be involved in Zn2+-induced cell death in cortical neurons (Kim et al. 1999b; Sensi et al. 1999), and shown to activate Ras in PC12 cells and fibroblasts (Lander et al. 1995; Abe and Berk 1999), it was tempting to speculate that the Zn2+-induced activation of the Ras/Raf/MEK/ERK signaling cascade might be mediated by ROS generation. Indeed, in our system, ROS appeared to be a key trigger for ERK activation in the Zn2+-induced cell death of differentiated PC12 cells, because the accumulation of Zn2+ in the cells resulted in the generation of ROS. Moreover, pre-treatment with antioxidants, such as NAC, GSH and MnTBAP, almost completely inhibited both ERK activation and the cell death induced by Zn2+. In addition, by showing that the inhibition of ERK activation with PD98059 did not prevent ROS generation, we confirmed that ERK is a downstream target of ROS.

It remains unknown how Zn2+ enhances the accumulation of ROS, which is produced during respiration under physiological conditions. However, the excessive production of ROS is harmful and, thus, biochemical antioxidants and enzymes, such as glutathione reductase and peroxidase, provide antioxidant defense mechanisms to maintain ROS homeostasis. Zn2+ was shown to trigger prolonged mitochondrial superoxide production in cortical neurons (Sensi et al. 1999) and to inhibit glutathione reductase and peroxidase in hepatocytes (Mize and Langdon 1962; Splittgerber and Tappel 1979). Therefore, the increased production of ROS and the dysfunction of antioxidant defense mechanisms might contribute to the enhanced accumulation of ROS induced by Zn2+.

We therefore conclude that prolonged accumulation of Zn2+ generates ROS that induce cell death, at least in part, by activating the Ras/Raf/MEK/ERK signaling cascade. Although JNK is also activated by intracellular Zn2+ accumulation, it appears to play little role in the mechanisms leading to cell death. Given that Zn2+ is a key mediator of cell death caused by ischemia and seizure (Choi and Koh 1998), our results suggest that ROS-induced ERK activation caused by Zn2+ accumulation may play a critical role in ischemia- and seizure-induced cell death.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by the Research Fund from Yonsei University College of Dentistry for 1997. SAC and JYS are graduate students supported by BK21 Project for Medical Science, Yonsei University. We thank Dr C. J. Marshall for providing Ras dominant negative mutant expression vector (pMT3-RasN17) and Dr D. Templeton for providing SEK1 dominant negative mutant expression vector (pCMVSEKAL).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References