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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.
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.
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- Materials and methods
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.