Enhancement of Cyanide-Induced Mitochondrial Dysfunction and Cortical Cell Necrosis by Uncoupling Protein-2
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《毒物学科学杂志》
Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907–1333
Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT
Uncoupling protein 2 (UCP-2) is expressed in the inner mitochondrial membrane and modulates mitochondrial function by partially uncoupling oxidative phosphorylation, and it has been reported to modulate cell death. Cyanide is a potent neurotoxin that inhibits complex IV to alter mitochondrial function to induce neuronal death. In primary rat cortical cells KCN produced an apoptotic death at 200–400 μM. Higher concentrations of potassium cyanide (KCN) (500–600 μM) switched the mode of death from apoptosis to necrosis. In necrotic cells, ATP levels were severely depleted as compared to cortical cells undergoing apoptosis. To determine if UCP-2 expression could alter KCN-induced cell death, cells were transiently transfected with full-length human UCP-2 cDNA (UCP-2+). Overexpression switched the mode of death produced by KCN (400 μM) from apoptosis to necrosis. The change in cell death was mediated by impaired mitochondrial function as reflected by a marked decrease of ATP levels and reduction in mitochondrial membrane potential. RNA interference or transfection with a dominant interfering mutant blocked the necrotic response observed in UCP-2+ cells. Additionally, treatment of UCP-2+ cells with cyclosporin A blocked necrosis, indicating the involvement of mitochondrial permeability pore transition in the necrotic death. These results show that increased expression of UCP-2 alters the response to a potent mitochondrial toxin by switching the mode of cell death from apoptosis to necrosis. It is concluded that UCP-2 levels influence cellular responses to cyanide-induced mitochondrial dysfunction.
Key Words: apoptosis; cyanide; necrosis; mitochondria; uncoupling protein.
INTRODUCTION
Uncoupling protein 2 (UCP-2) is a member of the mitochondrial anion carrier superfamily that is expressed in the inner mitochondrial membrane (Busquets et al., 2001; Rousset et al., 2004). In the mitochondrion, UCP-2 partly dissipates the proton electrochemical gradient by enhancing proton leakage from the inner membrane into the matrix. The mild uncoupling leads to a reduction of mitochondrial membrane potential (m), a decrease of mitochondrial Ca2+ uptake, and a reduction of both cellular ATP production and reactive oxygen species (ROS) generation. Uncoupling proteins are thought to regulate energy metabolism and thermogenesis in peripheral tissue. In brain, UCP-2 is constitutively expressed in hippocampus, amygdala, and limbic cortex, but its physiological function is not known (Sullivan et al., 2003).
UCP-2 is both a death and survival factor, dependent on the initiation stimulus and the death pathway executed (Duval et al., 2002; Rashid et al., 1999). Hypoxic stress can induce expression of UCP-2, which may be an adaptive mechanism to conditions predisposing to oxidative stress (Pecqueur et al., 2002). Activation or overexpression of UCP-2 reduces reactive oxygen species (ROS) generation, thereby protecting against oxidative stress generated in mitochondria (Negre-Salvayre et al., 1997; Teshima et al., 2003). In contrast, decreased expression of UCP-2 by antisense oligonucleotide treatment increases ROS generation and enhances oxidative injury (Duval et al., 2002). UCP-2 is neuroprotective by modulating cellular redox signaling (de Bilbao et al., 2004) or inducing mild mitochondrial uncoupling that prevents release of pro-apoptotic proteins (Mattiasson et al., 2003). Activation of UCP-2 can decrease neuronal apoptosis that is mediated through the caspase cascade (Bechmann et al., 2002). On the other hand, high expression levels of UCP-2 can lead to a rapid fall in m and reduction of both mitochondrial NADH and ATP to selectively produce necrosis (Mills et al., 2002). Expression of a dominant interfering mutant of UCP-2 conferred resistance to necrosis, but not apoptosis.
Cyanide is a rapid-acting mitochondrial toxicant that inhibits cytochrome oxidase, thereby blocking the flow of electrons through complex IV to block oxidative metabolism (Jones et al., 2003) and to enhance ROS generation at complex III (Chen et al., 2003). Depending on cell type and level of oxidative stress, cyanide selectively produces either apoptosis or necrosis (Prabhakaran et al., 2002). Cyanide-induced apoptosis and necrosis share common initiation stimuli, but the death pathways are mediated by divergent intracellular cascades (Prabhakaran et al., 2002; Shou et al., 2003). The apoptotic pathway is caspase-dependent and mediated by mitochrondrial release of apoptogenic proteins (Shou et al., 2004). Cells undergoing necrosis display a high level of oxidative stress and rapid breakdown of mitochondrial function, characterized by onset of mitochondrial membrane permeability transition and ATP depletion (Prabhakaran et al., 2002). Because UCP-2 modulates mitochondrial function and has been linked to regulation of cell death, it was of interest to determine the protein's involvement in cyanide-induced cell death.
MATERIALS AND METHODS
Primary cell culture.
Primary cortical cells (CX) were prepared from embryonic day 16–17 Sprague-Dawley rats. In brief, cerebral cortices were dissected and cells were dissociated in 0.025% trypsin at 37°C for 15 min. Trypsin digestion was stopped by adding trypsin inhibitor and DNase I. Cells were passed through a Pasteur pipette several times and plated at a density of 5 ± 105 cells/cm2 onto culture flasks or 6-well plates precoated with 10 μg/ml poly-L-lysine. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10% horse serum, 22 mM glucose, 2 mM glutamine, and 1 ml penicillin/streptomycin (10,000 U/ml) at 37°C in an atmosphere of 5% CO2 and 95% air. For enriched cultures of post-mitotic cortical neurons, cytosine arabinofuranoside (10 μM) was added at 6 days in vitro (DIV) to suppress proliferation of astrocytes. Medium was changed twice a week until experiments were carried out at 12–14 DIV. For treatment, cells were placed in 6-well plates and/or flasks, and potassium cyanide (KCN) was added in a 10 μl volume so as to yield a 400 μM concentration. Control cells were incubated under the same culture conditions and times as treatment groups.
Evaluation of apoptosis (TUNEL staining).
After transfections, cells were treated with KCN (400 μM for 24 h) and/or pretreated with 1 μM cyclosporin A (CsA) for 30 min before cyanide. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) was performed on paraformaldehyde (4% in phosphate buffered saline) fixed cells using the Apoptag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Briefly, cells were preincubated in equilibration buffer containing 0.1 M potassium cacodylate (pH 7.2), 2 mM CaCl2, and 0.2 mM dithiothreitol for 10 min at room temperature and then incubated in TUNEL reaction mixture at pH 7.2 (containing 200 mM potassium cacodylate, 4 mM MgCl2, 2 mM 2-mercaptoethanol, 30 μM biotin-16-2'-deoxyuridine-5'-triphosphate [dUTP], and 300 U/ml TdT) in a humidified chamber at 37°C for 1 h. After incubation in stop/wash buffer for 10 min, the elongated digoxigenin-labeled DNA fragments were visualized using anti-digoxigenin peroxidase antibody solution, followed by staining with DAB/H2O2 (0.2 mg/ml diaminobenzidine tetrachloride and 0.005% H2O2 in PBS, pH 7.4). Cells were then counterstained with hematoxylin. In the microscopic field, the number of cells undergoing apoptosis was determined by both TUNEL staining and characteristic apoptotic morphology such as chromatin condensation and margination within the nucleus and cell shrinkage. Based on morphology, astrocytic cells were excluded from the counting.
The mode of cell death was confirmed by observation of the cell morphology by electron microscopy. After treatment, cells were fixed by replacing one half of the medium with 3% glutaraldehyde/PBS for 15 min. The fixative–medium mixture was then replaced with fixative solution alone, and cells were processed for transmission electron microscopy and photographed.
Quantitation of necrotic cell death.
Necrotic cell death was quantitated using two DNA fluorescent dyes, SYTO-13 and propidium iodide (PI) by the method of Ankarcrona et al. (1995). Both dyes bind DNA, but only SYTO-13 is membrane permeable. Thus SYTO-13 stains normal cells with a green fluorescence, whereas only cells with disrupted plasma membranes stain red with PI. The percentage of non-astrocytic cells staining positive for PI was determined as an estimate of necrosis. Microscopic examination showed these cells exhibited necrotic morphology characterized by plasma membrane damage and cytoplasmic vacuolization.
UCP-2 transient transfection and RNA interference.
siRNA corresponding to the UCP-2 reporter gene was designed as recommended by Elbashir et al. (2001) and synthesized by Ambion, Inc. (Austin, TX) with 5' phosphate, 3' hydroxyl, and two base overhangs on each strand. The gene-specific sequences were used for UCP-2 interference: sense 5'-GAACGGGACACCUUUAGAGtt-3' and antisense 5'-CUCUAAAGGUGUCCCGUUCtt-3'; annealing for duplex siRNA formation was performed as described by the manufacturer.
The full-length human UCP-2 cDNA (UCP-2+) was subcloned into the expression vector pCDNA3.1 as previously described (Mills et al., 2002). Transient transfections of the UCP-2 plasmid and siRNA were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as described by Krichevsky and Kosik (2002). Briefly, Lipofectamine diluted in Opti-MEM was applied to the plasmids or siRNA and incubated for 45 min. To each microtiter plate well containing cells, 2 μg of plasmid or 0.2 μg of 21-bp siRNA containing Lipofectamine was applied in a final volume of 1 ml. The medium was changed to regular CX cell culture medium 5 h after initiation of transfection, and cells were treated with cyanide 24 h after transfection. The dominant interfering mutant (UCP-2D212N) was a site-directed mutant of UCP-2 created by substitution of position 212 (Asp-Asn) and was subcloned into the expression vector pCDNA3.1 (Mills et al., 2002).
Measurement of mitochondrial membrane potential.
m in cortical cells was determined using the mitochondrial-specific fluorescent probe JC-1 based on the method of Reers et al. (1995). JC-1 exists as a green-fluorescent monomer at low membrane potential (120 mV) and as a red fluorescent dimer (J-aggregate) at membrane potential greater than 180 mV. Following excitation at 485 nm, the ratio of red (595 nm emission) to green (525 nm emission) fluorescence represents the ratio of high to low mitochondrial membrane potential (Reers et al., 1995). After treatment CX cells were incubated with JC-1 (3.0 μM) for 30 min at 37°C in the dark and then washed twice with fresh PBS. Fluorescence was measured with a plate reader at excitation 485 nm, emission 525 nm, and 595 nm.
Western blot analysis.
After various treatments or transient transfection, cells were washed with ice-cold PBS and harvested by scraping and then centrifugation at 500 x g for 5 min. Cell pellets were lysed in a buffer containing 220 mM mannitol; 68 mM sucrose; 20 mM HEPES, pH 7.4; 50 mM KCl; 5 mM EGTA; 1 mM EDTA; 2 mM MgCl2; 1 mM dithiothreitol; 0.1% Triton-X100; and protease inhibitors on ice for 15 min. After centrifugation, supernatants were taken as whole cell protein extraction. The protein content in the extractions was determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA). The samples containing 30 μg protein were boiled in Laemmli buffer for 5 min and subjected to electrophoresis in 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel, followed by transfer to a nitrocellulose membrane. After blocking with Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20, the membrane was exposed to the primary UCP-2 antibody or -actin antibody for 3 h at room temperature on a shaker. The UCP-2 antibody was a rabbit anti-mouse polyclonal antibody (diluted 1:2000) directed toward the c-terminal domain of UCP-2 (Alpha Diagnostic International Inc., San Antonio, TX). Antibody specificity was determined by using a 14 aa UCP-2 blocking peptide according to the manufacturer's protocol. Reactions were detected with a fluorescein-linked anti-mouse IgG (second antibody) conjugated to horseradish peroxidase using enhanced chemiluminescence.
Measurement of caspase-3 activity.
Caspase-3 activity was determined by measuring release of p-nitroanilide (pNA) after cleavage of the caspase tetrapeptide substrate Ac-DEVD-pNA following the protocol provided by the manufacturer (BioVision Inc., Mountain View, CA) and as described by Liao et al. (2003). Briefly, after treatment, cells grown in 6-well culture plates were washed with PBS and lysed with 50 μl of lysis buffer (0.5% Triton X-100; 10 mM Tris-HCl, pH 7.5; 1 mM EDTA) on ice for 10 min. Lysate (50 μl) was added to a reaction mixture containing 100 μl of reaction buffer, 40 μl of H2O, and 5 μl of Ac-DEVD-pNA (final concentration 200 μM) and incubated for 1 h at 37°C. Free pNA was measured at 405 nm in a microtiter plate reader, and caspase activity was calculated after the protein content was normalized.
Measurement of cellular ATP.
Cellular ATP content was determined using a bioluminescence assay according to the manufacturer's instructions (Molecular Probes, Eugene, OR). Cells (2 x 105) were treated with cyanide, and after the indicated times, cells were washed in PBS, lysed in 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and incubated for 10 min on ice. After removal of cell debris by centrifugation (10,000 x g, 15 min, 4°C), the ATP content was measured by the luciferin/luciferase method (Los et al., 2002). The absolute values of ATP content were determined using an ATP standard supplied by the manufacturer.
Statistics.
Data were expressed as mean ± SEM, and statistical significance was assessed by one-way analysis of variance (ANOVA), followed by the Tukey-Kramer multiple range test. Differences were considered significant at p < 0.05.
RESULTS
Cyanide-Induced Cell Death
We have shown that cyanide produces selective cell loss in the CNS in which cortical brain areas undergo apoptotic degeneration and mesencephalic cells undergo necrosis (Mills et al., 1999). The response in primary cultured CX cells parallels cytotoxicity observed in vivo; the concentration-response to cyanide in CX cells is illustrated in Figure 1A and 1B. A concentration-related apoptotic death was observed after 24-h exposure to KCN, with a switch to necrosis at higher concentrations. The apoptosis correlated with a moderate decrease in ATP levels. For concentrations that produced necrosis, cellular ATP declined by more than 35%, which is a severe depletion for neuronal cells (Fig. 1C). It was concluded that the mode of cell death switched from apoptosis to necrosis when cellular energy reserves were depleted below a critical threshold as described by Davey et al. (1998).
Altered UCP-2 Expression and Cell Death
To link UCP-2 expression to the mode of cell death, cells were transfected with UCP-2 cDNA. Under the treatment conditions, UCP-2 expression was increased twofold (Fig. 2A). Electron micrographs of transfected CX cells exposed to 400 μM KCN showed that UCP-2 overexpression produced a necrotic-like morphology characterized by swollen mitochondria, plasma membrane damage, and cytoplasmic vacuolization, as opposed to a morphology characteristic of apoptosis in cells treated with KCN alone, which shows chromatin condensation and margination within the nucleus and cell shrinkage (Fig. 2B). Increased expression of UCP-2 switched the mode of cell death from apoptosis to necrosis at 400 μM cyanide (Fig. 2C). Thus enhanced expression of UCP-2 predisposes to necrotic death.
To confirm the effect of transfection on cell death was due to UCP-2 overexpression, UCP-2 expression was knocked down with RNA interference (McManus and Sharp, 2002). Western analysis of the constructs showed the UCP-2+ plasmid increased expression and RNAi blocked the enhanced expression produced by the plasmid (Fig. 3A). Interestingly, constitutive expression did not change after transfection with siRNA, suggesting that UCP-2 undergoes a slow turnover in wild-type cells. In cells treated with RNAi, the level of cyanide-induced apoptosis was not significantly increased compared to wild-type cells treated with cyanide alone (Fig. 3B). In UCP-2+ cells, RNAi blocked cyanide-induced necrosis, confirming that overexpression of UCP-2 sensitizes the cells to the necrotic effect of cyanide.
Transfection with the dominant interfering mutant UCP-2D212N switched the necrotic response to apoptosis in UCP-2+ cells (Fig. 4). Because previous studies have shown that UCP-2D212N is a functional antagonist to UCP-2 (Mills et al., 2002), it was concluded that activation of UCP-2 plays a pivotal role in cyanide-induced necrosis.
UCP-2 Expression and Caspase Activation
The intrinsic apoptotic pathway is executed by activation of caspase proteases and cellular caspase activity reflects commitment to this mode of death. Exposure to cyanide significantly increased cellular caspase-3 activity, correlating with execution of apoptosis (Fig. 5). On the other hand, transfection with UCP-2 cDNA decreased caspase activation, as expected if the mode of cell death is switched to necrosis. Transfection with UCP-2D212N or with siRNA increased caspase activation, which may explain the blockade of necrosis by these treatments.
UCP-2 Expression and Mitochondrial Function
Mitochondrial dysfunction, characterized by a decline in cellular ATP and loss of m, is central to execution of cell death (Budd Haeberlein, 2004). In necrosis, cells experience a catastrophic loss of mitochondrial function, whereas in apoptosis, a minimal level of mitochondrial function is necessary to execute cell death. In cells transfected with UCP-2, cyanide markedly decreased cellular ATP, correlating with the switch to necrosis (Fig. 6A). ATP levels were not significantly altered by cyanide in cells transfected with the dominant interfering mutant or siRNA, indicating a role for UCP-2 in the response. Changes in m reflect the level of mitochondrial dysfunction and precede final execution of cell death. In UCP-2+ cells, cyanide markedly lowered m, correlating with necrotic death (Fig. 6B).
Opening of the permeability transition pore (PTP) is an important mitochondrial event that mediates cell death. Inhibition of PTP opening with cyclosporin A (CsA) inhibits caspase-independent death (Chang and Johnson, 2002). In UCP2+ cells, pretreatment with CsA blocked the necrotic response to cyanide, indicating that PTP opening is critical to execution of necrosis (Fig. 7). Since CsA also inhibits calcineurin, studies were conducted with bongkrekic acid, a specific inhibitor of PTP opening. Pretreatment with bongkrekic acid produces responses parallel to those of CsA, indicating that the CsA pretreatment blocks PTP opening. It is concluded that the level of UCP-2 expression can influence the level of mitochondrial dysfunction, which in turn leads to either apoptosis or necrosis.
DISCUSSION
In a variety of injury paradigms UCP-2 mediates neuroprotection by uncoupling oxidative metabolism to decrease ROS generation in mitochondria (Mattiasson et al., 2003; Teshima et al., 2003). Similarly, in the present study, decreased levels of UCP-2 increased caspase-mediated apoptotic CX cell death. However, overexpression of UCP-2 potentiated cyanide-induced ATP depletion and loss of m, resulting in increased cortical cell death by necrosis. In the presence of excessive mitochondrial dysfunction, enhanced levels of UCP-2 appear to contribute to execution of the necrotic pathway. These observations have important implications by showing that when overexpressed, UCP-2 can potentiate mitochondrial dysfunction produced by a potent toxin and thus activate the necrotic pathway of cell death.
In neuronal cells, cyanide produces two distinct modes of death, apoptosis and necrosis, depending on the cell type and level of oxidative insult (Prabhakaran et al., 2002). These modes of cell death share common initiation stimuli, but divergent intracellular cascades are activated to produce either apoptosis or necrosis (Prabhakaran et al., 2004; Shou et al., 2003). In both cell death modes, cyanide induces mitochondrial dysfunction by inhibiting cytochrome oxidase (complex IV), leading to reduced oxidative metabolism and enhanced ROS generation at complex III (Davey et al., 1998; Jones et al., 2003). Apoptosis is caspase-dependent and mediated by release of pro-apoptotic proteins from mitochondria. Cyanide-induced apoptosis can be switched to necrosis by blocking caspase activation, which is accompanied by marked reduction in both m and cellular ATP levels (Prabhakaran et al., 2004). Cells undergoing necrosis display an intense level of oxidative stress and experience a rapid breakdown of mitochondrial function, characterized by opening of the PTP and ATP depletion (Prabhakaran et al., 2002).
Present results show that overexpression of UCP-2 switches the mode of death from apoptosis to necrosis by potentiating mitochondrial dysfunction. The enhanced necrosis was due to a catastrophic drop in bioenergy production as reflected by a decrease in m, ATP levels, and PTP. This is a specific interaction between UPC-2 and cyanide since a UCP-2 dominant negative mutant and RNAi blocked the enhanced necrosis, thus eliminating the possibility of nonspecific toxicity associated with the transfection procedure.
Echtay et al. (2002) demonstrated that mitochondrial UCP-2 is activated indirectly by cyanide. In isolated mitochondria, cyanide treatment generated superoxide in the mitochondrial matrix to activate mitochondrial UCP-2, which then increased uncoupling and caused progressive reduction of proton conductance. Apparently this action can also facilitate the catastrophic energy collapse necessary for execution of necrosis as observed in the present study. Other studies have also linked increased UCP-2 expression to cell death. Sriram et al. (2002) showed that two neurotoxicants (methamphetamine and kainic acid) increased UCP-2 expression in mouse brain, and they therefore proposed that abnormal expression of UCP-2 may cause the excessive energy depletion that leads to mitochondrial dysfunction and necrosis. Mattson and Liu (2003) proposed that uncoupling proteins are involved in regulating mitochondrial-mediated cell death. The present results are consistent with these studies.
To prevent apoptosis, UCP-2 may regulate production of superoxide by mitochondria (Horvath et al., 2003; Mattiasson et al., 2003; Sullivan et al., 2003; Teshima et al., 2003). Thus overexpression of human UCP-2 in transgenic mice protected against stroke and brain traumatic injury (Mattiasson et al., 2003). Also, in a seizure model, transgenic mice displayed a lower level of apoptotic hippocampal lesions in CA1 pyramidal cells (Diano et al., 2003). In brains of transgenic mice expressing human UCP-2, decreased oxidative stress and elevated ATP were observed, thus affording neuroprotection. In UCP-2 knockout mice, lack of UCP-2 increased the mitochondrial complex I toxin MPTP-induced nigral dopamine cell death, and overexpression decreased cell loss (Andrews et al., 2005). Furthermore, in cultured cortical cells, UCP-2 inhibited caspase activation and protected against oxygen/glucose deprivation-induced death (Mattiasson et al., 2003). In other studies, the influence of neonatal brain UCP-2 levels on excitotoxic death was determined by altering dietary fat intake (Sullivan et al., 2003). In immature brain with high basal expression of UCP-2, reduced ROS generation and decreased neuronal loss were observed following kainic acid-induced seizures. Also, reduced UCP-2 increased vulnerability to seizure-induced injury, thus supporting a neuroprotectant role for UCP-2. It appears that UCP-2-mediated neuroprotection is related to activation of cellular redox signaling or inhibition of release of apoptogenic factors after mild mitochondrial uncoupling. Recently, de Bilbao et al. (2004) proposed that UCP-2 regulates the response of microglia to ischemia by modulating mitochondrial glutathione levels rather than ROS generation. Regardless of the mechanism of ROS production, the level of ROS generated in mitochondria appears to determine the mode of cortical cell death.
Despite reports that UCP-2 regulates apoptotic neuronal death, a number of studies, including the present work, relate UCP-2 to necrotic cell death. Over-expression of UCP-2 reduces m and decreases mitochondrial NADH and ATP levels to produce necrosis (Mills et al., 2002). Studies in hepatocytes have linked upregulation of UCP-2 to increased vulnerability to necrotic death, possibly related to decreased generation of ATP (Rashid et al., 1999; Uchino et al., 2004). Overexpression of UCP-2 induced marked reductions in m and ATP and produced apoptosis in cultured adipocytes (3T3-L1 cells) (Sun and Zemel, 2004). Expression of a dominant interfering mutant of UCP-2 conferred resistance to necrosis, but not apoptosis, in HeLa cells (Mills et al., 2002). Overexpression of the anti-apoptotic protein Bcl-2 did not influence the level of UCP-2–mediated cell death, suggesting a non-apoptotic mode of death.
Clearly the present results show that overexpression of UCP-2 leads to enhanced cortical cell necrosis, although lowering constitutive levels of UCP-2 reduced necrotic cell death and slightly enhanced apoptosis. In mesencephalic cells, however, reduction of UCP-2 levels markedly decreases cyanide-induced necrosis (Prabhakaran et al., 2003). It is apparent that changes in UCP-2 expression can regulate the response to a neurotoxin in a cell-specific manner. Importantly, the constitutive expression level of UCP-2 may explain the selective vulnerability of the CNS produced by cyanide (Mills et al., 1999). UCP-2 has region-specific expression patterns in the brain (Richard et al., 2001). The present results suggest that higher levels of UCP-2 expression may enhance the potential of mitochondrial inhibitors to initiate necrotic death. It is also interesting to note that, in this study, in the presence of cyanide, the expression level of UCP-2 was greater after transfection. It is possible that cyanide upregulates transcription of UCP-2, thus leading to increased cellular expression. These results raise the possibility that cyanide may upregulate consititutive expression, which in turn may alter the toxic response. Thus, changes in UCP-2 constitutive expression may have important consequences with regard to neuronal sensitivity to mitochondrial active compounds.
It is concluded that UCP-2 can regulate mitochondria-mediated cell death. Elevated levels of UCP-2 enhance nerve cell vulnerability to mitochondrial complex IV inhibition by resulting in a switch from the apoptotic to the necrotic mode of neuronal cell death.
NOTES
2 Present address: University of Texas at Austin, Division of Pharmacology and Toxicology, College of Pharmacy, Austin, TX 7812–1074.
ACKNOWLEDGMENTS
This study was supported in part by National Institutes of Health (NIH) grant ES 04140.
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Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT
Uncoupling protein 2 (UCP-2) is expressed in the inner mitochondrial membrane and modulates mitochondrial function by partially uncoupling oxidative phosphorylation, and it has been reported to modulate cell death. Cyanide is a potent neurotoxin that inhibits complex IV to alter mitochondrial function to induce neuronal death. In primary rat cortical cells KCN produced an apoptotic death at 200–400 μM. Higher concentrations of potassium cyanide (KCN) (500–600 μM) switched the mode of death from apoptosis to necrosis. In necrotic cells, ATP levels were severely depleted as compared to cortical cells undergoing apoptosis. To determine if UCP-2 expression could alter KCN-induced cell death, cells were transiently transfected with full-length human UCP-2 cDNA (UCP-2+). Overexpression switched the mode of death produced by KCN (400 μM) from apoptosis to necrosis. The change in cell death was mediated by impaired mitochondrial function as reflected by a marked decrease of ATP levels and reduction in mitochondrial membrane potential. RNA interference or transfection with a dominant interfering mutant blocked the necrotic response observed in UCP-2+ cells. Additionally, treatment of UCP-2+ cells with cyclosporin A blocked necrosis, indicating the involvement of mitochondrial permeability pore transition in the necrotic death. These results show that increased expression of UCP-2 alters the response to a potent mitochondrial toxin by switching the mode of cell death from apoptosis to necrosis. It is concluded that UCP-2 levels influence cellular responses to cyanide-induced mitochondrial dysfunction.
Key Words: apoptosis; cyanide; necrosis; mitochondria; uncoupling protein.
INTRODUCTION
Uncoupling protein 2 (UCP-2) is a member of the mitochondrial anion carrier superfamily that is expressed in the inner mitochondrial membrane (Busquets et al., 2001; Rousset et al., 2004). In the mitochondrion, UCP-2 partly dissipates the proton electrochemical gradient by enhancing proton leakage from the inner membrane into the matrix. The mild uncoupling leads to a reduction of mitochondrial membrane potential (m), a decrease of mitochondrial Ca2+ uptake, and a reduction of both cellular ATP production and reactive oxygen species (ROS) generation. Uncoupling proteins are thought to regulate energy metabolism and thermogenesis in peripheral tissue. In brain, UCP-2 is constitutively expressed in hippocampus, amygdala, and limbic cortex, but its physiological function is not known (Sullivan et al., 2003).
UCP-2 is both a death and survival factor, dependent on the initiation stimulus and the death pathway executed (Duval et al., 2002; Rashid et al., 1999). Hypoxic stress can induce expression of UCP-2, which may be an adaptive mechanism to conditions predisposing to oxidative stress (Pecqueur et al., 2002). Activation or overexpression of UCP-2 reduces reactive oxygen species (ROS) generation, thereby protecting against oxidative stress generated in mitochondria (Negre-Salvayre et al., 1997; Teshima et al., 2003). In contrast, decreased expression of UCP-2 by antisense oligonucleotide treatment increases ROS generation and enhances oxidative injury (Duval et al., 2002). UCP-2 is neuroprotective by modulating cellular redox signaling (de Bilbao et al., 2004) or inducing mild mitochondrial uncoupling that prevents release of pro-apoptotic proteins (Mattiasson et al., 2003). Activation of UCP-2 can decrease neuronal apoptosis that is mediated through the caspase cascade (Bechmann et al., 2002). On the other hand, high expression levels of UCP-2 can lead to a rapid fall in m and reduction of both mitochondrial NADH and ATP to selectively produce necrosis (Mills et al., 2002). Expression of a dominant interfering mutant of UCP-2 conferred resistance to necrosis, but not apoptosis.
Cyanide is a rapid-acting mitochondrial toxicant that inhibits cytochrome oxidase, thereby blocking the flow of electrons through complex IV to block oxidative metabolism (Jones et al., 2003) and to enhance ROS generation at complex III (Chen et al., 2003). Depending on cell type and level of oxidative stress, cyanide selectively produces either apoptosis or necrosis (Prabhakaran et al., 2002). Cyanide-induced apoptosis and necrosis share common initiation stimuli, but the death pathways are mediated by divergent intracellular cascades (Prabhakaran et al., 2002; Shou et al., 2003). The apoptotic pathway is caspase-dependent and mediated by mitochrondrial release of apoptogenic proteins (Shou et al., 2004). Cells undergoing necrosis display a high level of oxidative stress and rapid breakdown of mitochondrial function, characterized by onset of mitochondrial membrane permeability transition and ATP depletion (Prabhakaran et al., 2002). Because UCP-2 modulates mitochondrial function and has been linked to regulation of cell death, it was of interest to determine the protein's involvement in cyanide-induced cell death.
MATERIALS AND METHODS
Primary cell culture.
Primary cortical cells (CX) were prepared from embryonic day 16–17 Sprague-Dawley rats. In brief, cerebral cortices were dissected and cells were dissociated in 0.025% trypsin at 37°C for 15 min. Trypsin digestion was stopped by adding trypsin inhibitor and DNase I. Cells were passed through a Pasteur pipette several times and plated at a density of 5 ± 105 cells/cm2 onto culture flasks or 6-well plates precoated with 10 μg/ml poly-L-lysine. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10% horse serum, 22 mM glucose, 2 mM glutamine, and 1 ml penicillin/streptomycin (10,000 U/ml) at 37°C in an atmosphere of 5% CO2 and 95% air. For enriched cultures of post-mitotic cortical neurons, cytosine arabinofuranoside (10 μM) was added at 6 days in vitro (DIV) to suppress proliferation of astrocytes. Medium was changed twice a week until experiments were carried out at 12–14 DIV. For treatment, cells were placed in 6-well plates and/or flasks, and potassium cyanide (KCN) was added in a 10 μl volume so as to yield a 400 μM concentration. Control cells were incubated under the same culture conditions and times as treatment groups.
Evaluation of apoptosis (TUNEL staining).
After transfections, cells were treated with KCN (400 μM for 24 h) and/or pretreated with 1 μM cyclosporin A (CsA) for 30 min before cyanide. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) was performed on paraformaldehyde (4% in phosphate buffered saline) fixed cells using the Apoptag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Briefly, cells were preincubated in equilibration buffer containing 0.1 M potassium cacodylate (pH 7.2), 2 mM CaCl2, and 0.2 mM dithiothreitol for 10 min at room temperature and then incubated in TUNEL reaction mixture at pH 7.2 (containing 200 mM potassium cacodylate, 4 mM MgCl2, 2 mM 2-mercaptoethanol, 30 μM biotin-16-2'-deoxyuridine-5'-triphosphate [dUTP], and 300 U/ml TdT) in a humidified chamber at 37°C for 1 h. After incubation in stop/wash buffer for 10 min, the elongated digoxigenin-labeled DNA fragments were visualized using anti-digoxigenin peroxidase antibody solution, followed by staining with DAB/H2O2 (0.2 mg/ml diaminobenzidine tetrachloride and 0.005% H2O2 in PBS, pH 7.4). Cells were then counterstained with hematoxylin. In the microscopic field, the number of cells undergoing apoptosis was determined by both TUNEL staining and characteristic apoptotic morphology such as chromatin condensation and margination within the nucleus and cell shrinkage. Based on morphology, astrocytic cells were excluded from the counting.
The mode of cell death was confirmed by observation of the cell morphology by electron microscopy. After treatment, cells were fixed by replacing one half of the medium with 3% glutaraldehyde/PBS for 15 min. The fixative–medium mixture was then replaced with fixative solution alone, and cells were processed for transmission electron microscopy and photographed.
Quantitation of necrotic cell death.
Necrotic cell death was quantitated using two DNA fluorescent dyes, SYTO-13 and propidium iodide (PI) by the method of Ankarcrona et al. (1995). Both dyes bind DNA, but only SYTO-13 is membrane permeable. Thus SYTO-13 stains normal cells with a green fluorescence, whereas only cells with disrupted plasma membranes stain red with PI. The percentage of non-astrocytic cells staining positive for PI was determined as an estimate of necrosis. Microscopic examination showed these cells exhibited necrotic morphology characterized by plasma membrane damage and cytoplasmic vacuolization.
UCP-2 transient transfection and RNA interference.
siRNA corresponding to the UCP-2 reporter gene was designed as recommended by Elbashir et al. (2001) and synthesized by Ambion, Inc. (Austin, TX) with 5' phosphate, 3' hydroxyl, and two base overhangs on each strand. The gene-specific sequences were used for UCP-2 interference: sense 5'-GAACGGGACACCUUUAGAGtt-3' and antisense 5'-CUCUAAAGGUGUCCCGUUCtt-3'; annealing for duplex siRNA formation was performed as described by the manufacturer.
The full-length human UCP-2 cDNA (UCP-2+) was subcloned into the expression vector pCDNA3.1 as previously described (Mills et al., 2002). Transient transfections of the UCP-2 plasmid and siRNA were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as described by Krichevsky and Kosik (2002). Briefly, Lipofectamine diluted in Opti-MEM was applied to the plasmids or siRNA and incubated for 45 min. To each microtiter plate well containing cells, 2 μg of plasmid or 0.2 μg of 21-bp siRNA containing Lipofectamine was applied in a final volume of 1 ml. The medium was changed to regular CX cell culture medium 5 h after initiation of transfection, and cells were treated with cyanide 24 h after transfection. The dominant interfering mutant (UCP-2D212N) was a site-directed mutant of UCP-2 created by substitution of position 212 (Asp-Asn) and was subcloned into the expression vector pCDNA3.1 (Mills et al., 2002).
Measurement of mitochondrial membrane potential.
m in cortical cells was determined using the mitochondrial-specific fluorescent probe JC-1 based on the method of Reers et al. (1995). JC-1 exists as a green-fluorescent monomer at low membrane potential (120 mV) and as a red fluorescent dimer (J-aggregate) at membrane potential greater than 180 mV. Following excitation at 485 nm, the ratio of red (595 nm emission) to green (525 nm emission) fluorescence represents the ratio of high to low mitochondrial membrane potential (Reers et al., 1995). After treatment CX cells were incubated with JC-1 (3.0 μM) for 30 min at 37°C in the dark and then washed twice with fresh PBS. Fluorescence was measured with a plate reader at excitation 485 nm, emission 525 nm, and 595 nm.
Western blot analysis.
After various treatments or transient transfection, cells were washed with ice-cold PBS and harvested by scraping and then centrifugation at 500 x g for 5 min. Cell pellets were lysed in a buffer containing 220 mM mannitol; 68 mM sucrose; 20 mM HEPES, pH 7.4; 50 mM KCl; 5 mM EGTA; 1 mM EDTA; 2 mM MgCl2; 1 mM dithiothreitol; 0.1% Triton-X100; and protease inhibitors on ice for 15 min. After centrifugation, supernatants were taken as whole cell protein extraction. The protein content in the extractions was determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA). The samples containing 30 μg protein were boiled in Laemmli buffer for 5 min and subjected to electrophoresis in 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel, followed by transfer to a nitrocellulose membrane. After blocking with Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20, the membrane was exposed to the primary UCP-2 antibody or -actin antibody for 3 h at room temperature on a shaker. The UCP-2 antibody was a rabbit anti-mouse polyclonal antibody (diluted 1:2000) directed toward the c-terminal domain of UCP-2 (Alpha Diagnostic International Inc., San Antonio, TX). Antibody specificity was determined by using a 14 aa UCP-2 blocking peptide according to the manufacturer's protocol. Reactions were detected with a fluorescein-linked anti-mouse IgG (second antibody) conjugated to horseradish peroxidase using enhanced chemiluminescence.
Measurement of caspase-3 activity.
Caspase-3 activity was determined by measuring release of p-nitroanilide (pNA) after cleavage of the caspase tetrapeptide substrate Ac-DEVD-pNA following the protocol provided by the manufacturer (BioVision Inc., Mountain View, CA) and as described by Liao et al. (2003). Briefly, after treatment, cells grown in 6-well culture plates were washed with PBS and lysed with 50 μl of lysis buffer (0.5% Triton X-100; 10 mM Tris-HCl, pH 7.5; 1 mM EDTA) on ice for 10 min. Lysate (50 μl) was added to a reaction mixture containing 100 μl of reaction buffer, 40 μl of H2O, and 5 μl of Ac-DEVD-pNA (final concentration 200 μM) and incubated for 1 h at 37°C. Free pNA was measured at 405 nm in a microtiter plate reader, and caspase activity was calculated after the protein content was normalized.
Measurement of cellular ATP.
Cellular ATP content was determined using a bioluminescence assay according to the manufacturer's instructions (Molecular Probes, Eugene, OR). Cells (2 x 105) were treated with cyanide, and after the indicated times, cells were washed in PBS, lysed in 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and incubated for 10 min on ice. After removal of cell debris by centrifugation (10,000 x g, 15 min, 4°C), the ATP content was measured by the luciferin/luciferase method (Los et al., 2002). The absolute values of ATP content were determined using an ATP standard supplied by the manufacturer.
Statistics.
Data were expressed as mean ± SEM, and statistical significance was assessed by one-way analysis of variance (ANOVA), followed by the Tukey-Kramer multiple range test. Differences were considered significant at p < 0.05.
RESULTS
Cyanide-Induced Cell Death
We have shown that cyanide produces selective cell loss in the CNS in which cortical brain areas undergo apoptotic degeneration and mesencephalic cells undergo necrosis (Mills et al., 1999). The response in primary cultured CX cells parallels cytotoxicity observed in vivo; the concentration-response to cyanide in CX cells is illustrated in Figure 1A and 1B. A concentration-related apoptotic death was observed after 24-h exposure to KCN, with a switch to necrosis at higher concentrations. The apoptosis correlated with a moderate decrease in ATP levels. For concentrations that produced necrosis, cellular ATP declined by more than 35%, which is a severe depletion for neuronal cells (Fig. 1C). It was concluded that the mode of cell death switched from apoptosis to necrosis when cellular energy reserves were depleted below a critical threshold as described by Davey et al. (1998).
Altered UCP-2 Expression and Cell Death
To link UCP-2 expression to the mode of cell death, cells were transfected with UCP-2 cDNA. Under the treatment conditions, UCP-2 expression was increased twofold (Fig. 2A). Electron micrographs of transfected CX cells exposed to 400 μM KCN showed that UCP-2 overexpression produced a necrotic-like morphology characterized by swollen mitochondria, plasma membrane damage, and cytoplasmic vacuolization, as opposed to a morphology characteristic of apoptosis in cells treated with KCN alone, which shows chromatin condensation and margination within the nucleus and cell shrinkage (Fig. 2B). Increased expression of UCP-2 switched the mode of cell death from apoptosis to necrosis at 400 μM cyanide (Fig. 2C). Thus enhanced expression of UCP-2 predisposes to necrotic death.
To confirm the effect of transfection on cell death was due to UCP-2 overexpression, UCP-2 expression was knocked down with RNA interference (McManus and Sharp, 2002). Western analysis of the constructs showed the UCP-2+ plasmid increased expression and RNAi blocked the enhanced expression produced by the plasmid (Fig. 3A). Interestingly, constitutive expression did not change after transfection with siRNA, suggesting that UCP-2 undergoes a slow turnover in wild-type cells. In cells treated with RNAi, the level of cyanide-induced apoptosis was not significantly increased compared to wild-type cells treated with cyanide alone (Fig. 3B). In UCP-2+ cells, RNAi blocked cyanide-induced necrosis, confirming that overexpression of UCP-2 sensitizes the cells to the necrotic effect of cyanide.
Transfection with the dominant interfering mutant UCP-2D212N switched the necrotic response to apoptosis in UCP-2+ cells (Fig. 4). Because previous studies have shown that UCP-2D212N is a functional antagonist to UCP-2 (Mills et al., 2002), it was concluded that activation of UCP-2 plays a pivotal role in cyanide-induced necrosis.
UCP-2 Expression and Caspase Activation
The intrinsic apoptotic pathway is executed by activation of caspase proteases and cellular caspase activity reflects commitment to this mode of death. Exposure to cyanide significantly increased cellular caspase-3 activity, correlating with execution of apoptosis (Fig. 5). On the other hand, transfection with UCP-2 cDNA decreased caspase activation, as expected if the mode of cell death is switched to necrosis. Transfection with UCP-2D212N or with siRNA increased caspase activation, which may explain the blockade of necrosis by these treatments.
UCP-2 Expression and Mitochondrial Function
Mitochondrial dysfunction, characterized by a decline in cellular ATP and loss of m, is central to execution of cell death (Budd Haeberlein, 2004). In necrosis, cells experience a catastrophic loss of mitochondrial function, whereas in apoptosis, a minimal level of mitochondrial function is necessary to execute cell death. In cells transfected with UCP-2, cyanide markedly decreased cellular ATP, correlating with the switch to necrosis (Fig. 6A). ATP levels were not significantly altered by cyanide in cells transfected with the dominant interfering mutant or siRNA, indicating a role for UCP-2 in the response. Changes in m reflect the level of mitochondrial dysfunction and precede final execution of cell death. In UCP-2+ cells, cyanide markedly lowered m, correlating with necrotic death (Fig. 6B).
Opening of the permeability transition pore (PTP) is an important mitochondrial event that mediates cell death. Inhibition of PTP opening with cyclosporin A (CsA) inhibits caspase-independent death (Chang and Johnson, 2002). In UCP2+ cells, pretreatment with CsA blocked the necrotic response to cyanide, indicating that PTP opening is critical to execution of necrosis (Fig. 7). Since CsA also inhibits calcineurin, studies were conducted with bongkrekic acid, a specific inhibitor of PTP opening. Pretreatment with bongkrekic acid produces responses parallel to those of CsA, indicating that the CsA pretreatment blocks PTP opening. It is concluded that the level of UCP-2 expression can influence the level of mitochondrial dysfunction, which in turn leads to either apoptosis or necrosis.
DISCUSSION
In a variety of injury paradigms UCP-2 mediates neuroprotection by uncoupling oxidative metabolism to decrease ROS generation in mitochondria (Mattiasson et al., 2003; Teshima et al., 2003). Similarly, in the present study, decreased levels of UCP-2 increased caspase-mediated apoptotic CX cell death. However, overexpression of UCP-2 potentiated cyanide-induced ATP depletion and loss of m, resulting in increased cortical cell death by necrosis. In the presence of excessive mitochondrial dysfunction, enhanced levels of UCP-2 appear to contribute to execution of the necrotic pathway. These observations have important implications by showing that when overexpressed, UCP-2 can potentiate mitochondrial dysfunction produced by a potent toxin and thus activate the necrotic pathway of cell death.
In neuronal cells, cyanide produces two distinct modes of death, apoptosis and necrosis, depending on the cell type and level of oxidative insult (Prabhakaran et al., 2002). These modes of cell death share common initiation stimuli, but divergent intracellular cascades are activated to produce either apoptosis or necrosis (Prabhakaran et al., 2004; Shou et al., 2003). In both cell death modes, cyanide induces mitochondrial dysfunction by inhibiting cytochrome oxidase (complex IV), leading to reduced oxidative metabolism and enhanced ROS generation at complex III (Davey et al., 1998; Jones et al., 2003). Apoptosis is caspase-dependent and mediated by release of pro-apoptotic proteins from mitochondria. Cyanide-induced apoptosis can be switched to necrosis by blocking caspase activation, which is accompanied by marked reduction in both m and cellular ATP levels (Prabhakaran et al., 2004). Cells undergoing necrosis display an intense level of oxidative stress and experience a rapid breakdown of mitochondrial function, characterized by opening of the PTP and ATP depletion (Prabhakaran et al., 2002).
Present results show that overexpression of UCP-2 switches the mode of death from apoptosis to necrosis by potentiating mitochondrial dysfunction. The enhanced necrosis was due to a catastrophic drop in bioenergy production as reflected by a decrease in m, ATP levels, and PTP. This is a specific interaction between UPC-2 and cyanide since a UCP-2 dominant negative mutant and RNAi blocked the enhanced necrosis, thus eliminating the possibility of nonspecific toxicity associated with the transfection procedure.
Echtay et al. (2002) demonstrated that mitochondrial UCP-2 is activated indirectly by cyanide. In isolated mitochondria, cyanide treatment generated superoxide in the mitochondrial matrix to activate mitochondrial UCP-2, which then increased uncoupling and caused progressive reduction of proton conductance. Apparently this action can also facilitate the catastrophic energy collapse necessary for execution of necrosis as observed in the present study. Other studies have also linked increased UCP-2 expression to cell death. Sriram et al. (2002) showed that two neurotoxicants (methamphetamine and kainic acid) increased UCP-2 expression in mouse brain, and they therefore proposed that abnormal expression of UCP-2 may cause the excessive energy depletion that leads to mitochondrial dysfunction and necrosis. Mattson and Liu (2003) proposed that uncoupling proteins are involved in regulating mitochondrial-mediated cell death. The present results are consistent with these studies.
To prevent apoptosis, UCP-2 may regulate production of superoxide by mitochondria (Horvath et al., 2003; Mattiasson et al., 2003; Sullivan et al., 2003; Teshima et al., 2003). Thus overexpression of human UCP-2 in transgenic mice protected against stroke and brain traumatic injury (Mattiasson et al., 2003). Also, in a seizure model, transgenic mice displayed a lower level of apoptotic hippocampal lesions in CA1 pyramidal cells (Diano et al., 2003). In brains of transgenic mice expressing human UCP-2, decreased oxidative stress and elevated ATP were observed, thus affording neuroprotection. In UCP-2 knockout mice, lack of UCP-2 increased the mitochondrial complex I toxin MPTP-induced nigral dopamine cell death, and overexpression decreased cell loss (Andrews et al., 2005). Furthermore, in cultured cortical cells, UCP-2 inhibited caspase activation and protected against oxygen/glucose deprivation-induced death (Mattiasson et al., 2003). In other studies, the influence of neonatal brain UCP-2 levels on excitotoxic death was determined by altering dietary fat intake (Sullivan et al., 2003). In immature brain with high basal expression of UCP-2, reduced ROS generation and decreased neuronal loss were observed following kainic acid-induced seizures. Also, reduced UCP-2 increased vulnerability to seizure-induced injury, thus supporting a neuroprotectant role for UCP-2. It appears that UCP-2-mediated neuroprotection is related to activation of cellular redox signaling or inhibition of release of apoptogenic factors after mild mitochondrial uncoupling. Recently, de Bilbao et al. (2004) proposed that UCP-2 regulates the response of microglia to ischemia by modulating mitochondrial glutathione levels rather than ROS generation. Regardless of the mechanism of ROS production, the level of ROS generated in mitochondria appears to determine the mode of cortical cell death.
Despite reports that UCP-2 regulates apoptotic neuronal death, a number of studies, including the present work, relate UCP-2 to necrotic cell death. Over-expression of UCP-2 reduces m and decreases mitochondrial NADH and ATP levels to produce necrosis (Mills et al., 2002). Studies in hepatocytes have linked upregulation of UCP-2 to increased vulnerability to necrotic death, possibly related to decreased generation of ATP (Rashid et al., 1999; Uchino et al., 2004). Overexpression of UCP-2 induced marked reductions in m and ATP and produced apoptosis in cultured adipocytes (3T3-L1 cells) (Sun and Zemel, 2004). Expression of a dominant interfering mutant of UCP-2 conferred resistance to necrosis, but not apoptosis, in HeLa cells (Mills et al., 2002). Overexpression of the anti-apoptotic protein Bcl-2 did not influence the level of UCP-2–mediated cell death, suggesting a non-apoptotic mode of death.
Clearly the present results show that overexpression of UCP-2 leads to enhanced cortical cell necrosis, although lowering constitutive levels of UCP-2 reduced necrotic cell death and slightly enhanced apoptosis. In mesencephalic cells, however, reduction of UCP-2 levels markedly decreases cyanide-induced necrosis (Prabhakaran et al., 2003). It is apparent that changes in UCP-2 expression can regulate the response to a neurotoxin in a cell-specific manner. Importantly, the constitutive expression level of UCP-2 may explain the selective vulnerability of the CNS produced by cyanide (Mills et al., 1999). UCP-2 has region-specific expression patterns in the brain (Richard et al., 2001). The present results suggest that higher levels of UCP-2 expression may enhance the potential of mitochondrial inhibitors to initiate necrotic death. It is also interesting to note that, in this study, in the presence of cyanide, the expression level of UCP-2 was greater after transfection. It is possible that cyanide upregulates transcription of UCP-2, thus leading to increased cellular expression. These results raise the possibility that cyanide may upregulate consititutive expression, which in turn may alter the toxic response. Thus, changes in UCP-2 constitutive expression may have important consequences with regard to neuronal sensitivity to mitochondrial active compounds.
It is concluded that UCP-2 can regulate mitochondria-mediated cell death. Elevated levels of UCP-2 enhance nerve cell vulnerability to mitochondrial complex IV inhibition by resulting in a switch from the apoptotic to the necrotic mode of neuronal cell death.
NOTES
2 Present address: University of Texas at Austin, Division of Pharmacology and Toxicology, College of Pharmacy, Austin, TX 7812–1074.
ACKNOWLEDGMENTS
This study was supported in part by National Institutes of Health (NIH) grant ES 04140.
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