ToxSci Advance Access originally published online on June 16, 2006
Toxicological Sciences 2006 93(1):136-145; doi:10.1093/toxsci/kfl039
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PPAR
-Mediated Upregulation of Uncoupling Protein-2 Switches Cyanide-Induced Apoptosis to Necrosis in Primary Cortical Cells
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-1333
1 To whom correspondence should be addressed. Fax: (765) 494-1414. E-mail: geisom{at}purdue.edu.
Received March 30, 2006; accepted June 5, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peroxisome proliferator–activated receptor alpha (PPAR
) is a member of the nuclear factor PPAR family that regulates a variety of cellular functions, including lipid metabolism, cellular oxidative stress defense, and inflammatory responses. Based on the report that Wy14,643, a PPAR agonist, can upregulate uncoupling protein-2 (UCP-2), this study was conducted in primary cortical cells to determine if PPAR activation enhances cyanide-induced neurotoxicity through changes in the level of UCP-2. PCR and Western blot analysis showed that Wy14,643 upregulated UCP-2 transcriptionally over a 12-h period. This response was mediated by PPAR since it was blocked by MK886, a selective PPAR antagonist. The effect of UCP-2 upregulation on the cytotoxic response to cyanide was quantitated by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (apoptosis) and propidium iodide staining (necrosis). Wy14,643 switched the mode of cyanide-induced cell death from apoptosis to necrosis. Cell death was preceded by marked mitochondrial dysfunction, as reflected by depletion of ATP and reduction of the mitochondrial membrane potential (
m). Knock down of UCP-2 expression by RNA interference blocked the Wy14,643-mediated enhancement of cyanide-induced mitochondrial dysfunction and the switch of the cell death mode, thus confirming that the response was mediated by upregulation of UCP-2. This study shows that PPAR activation can upregulate UCP-2 expression, which in turn enhances cyanide-induced necrotic cell death through an increase of mitochondrial dysfunction.
Key Words: PPAR; cyanide; cell death; apoptosis; necrosis; UCP-2; mitochondrial function.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Uncoupling protein-2 (UCP-2) is a member of the family of proton transporters located in the mitochondrial inner membrane and regulates superoxide and ATP synthesis (Jacobsson et al., 1985
). UCP-2 can also be activated by superoxide and can dissipate the proton gradient between the mitochondrial intermembrane space and matrix to uncouple electron transport from ATP synthesis (Argiles et al., 2002; Echtay et al., 2002; Ledesma et al., 2002). Recent studies have shown that UCP-2 expression is upregulated transcriptionally in several models of brain damage and neurodegenerative diseases (MacManus et al., 2004; Sullivan et al., 2003). Mild uncoupling by UCP-2 attenuates mitochondrial ROS production to provide protection against oxidative stress (Diano et al., 2003; Paradis et al., 2003). On the other hand, overexpression and activation may sensitize the cell to apoptotic or necrotic cell death through depletion of mitochondrial GSH and/or a decrease in efficiency of cellular ATP synthesis (de Bilbao et al., 2004; Li et al., 2005; Prabhakaran et al., 2005). The effect of UCP-2 on cell survival is dependent on the level of expression and activation, which in turn modulates the mitochondrial membrane potential and bioenergetics.
Cyanide is a potent neurotoxin that inhibits complex IV (cytochrome oxidase) leading to altered mitochondrial function and subsequent activation of intrinsic cell death cascades (Li et al., 2005). Cyanide-induced cell death is enhanced by increasing UCP-2 expression. In primary rat cortical cells, cyanide produces apoptotic death and forced overexpression of UCP-2 switches the death mode to necrosis (Li et al., 2005). On the other hand, in primary cultured mesencephalic cells, enhanced expression of UCP-2 reduced mitochondrial ATP generation and membrane potential (m) leading to increased necrotic death (Prabhakaran et al., 2005). Thus, the level of UCP-2 expression can influence cellular responses to mitochondrial toxins through modulation of mitochondrial function.
UCP-2 expression can be regulated by a variety of transcription factors, including the peroxisome proliferators–activated receptors (PPARs). The PPARs are ligand-activated transcription factors belonging to the nuclear receptor superfamily and regulate the expression of target genes involved in lipid and energy metabolism, including UCP-2 (Murray et al., 2005). PPAR, an isotype of PPARs, is found predominantly in the liver, heart, and kidney. PPAR has also been characterized in the brain where it is expressed in neurons and glia (Cullingford et al., 1998). In the CNS, PPAR regulates neural cell differentiation, excitatory amino acid neurotransmission, and oxidative stress defense (Cimini et al., 2005). PPAR activation has also been associated with inflammation and neurodegeneration. Smith et al. (2001) showed that ligand-mediated activation of PPAR enhanced cerebellar granule cell death. Interestingly, PPAR activation by Wy14,643 (4-chloro-6-(2,3-xylidino)2-pyrimidinylthioacetic acid), a selective and high-affinity PPAR ligand, upregulated UCP-2 expression in liver and pancreatic ß cells (Nakatani et al., 2002; Ravnskjaer et al., 2005). However, in the CNS, a link between PPAR activation and enhanced UCP-2 expression has not been shown. In this study, we demonstrate in primary cortical cells that Wy14,643-induced upregulation of UCP-2 expression is mediated by activation of PPAR. The upregulation of UCP-2 enhances cyanide-induced neurotoxicity by increasing mitochondrial dysfunction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture.
Primary cortical cells were prepared from embryonic day 17 Sprague-Dawley rats as previously described (Li et al., 2005
). In brief, cerebral cortices were dissected and cells 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 x 105 cells/cm2 onto 75-cm2 culture flasks or plates precoated with 10 µg/ml poly-L-lysine. Cells were grown in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10% horse serum, 2mM glutamine, and 1 ml penicillin/streptomycin (5000 U/ml) at 37°C in an atmosphere of 5% CO2 and 95% air. To limit growth of glial cells, cytosine arabinofuranoside (10µM) was added at day 6 in vitro (DIV) for 24 h and the medium was changed. Medium was also changed twice a week until experiments were carried out at 10–12 DIV. At the time of experimentation, the percentage of astrocytes in the culture was approximately 30% as determined by immunostaining of the astrocyte-specific marker glial fibrillary acidic protein and 70% neurons as determined by immunostaining of the neuron-specific marker microtubule associated protein-2.
siRNA preparation and transient transfection.
siRNA corresponding to the UCP-2 reporter gene and a negative control siRNA (Silencer Negative Control #1), which does not target any genes of rat, mouse, or humans, was 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. Lipofectamine was used to transfect siRNA into cortical cells (Krichevsky and Kosik, 2002).
Evaluation of apoptosis (TUNEL staining).
Terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick end labeling (TUNEL) was performed on paraformaldehyde (4% in phosphate-buffered saline [PBS]) fixed cells using the Apoptag in situ apoptosis detection kit (Oncor, Gaithersburg, MD) as previously described (Li et al., 2005). Briefly, cells were preincubated in equilibration buffer containing 0.1M potassium cacodylate (pH 7.2), 2mM CaCl2, and 0.2mM dithiothreitol for 10 min at room temperature and then incubated in TUNEL reaction mixture (containing 200mM potassium cacodylate [pH 7.2], 4mM MgCl2, 2mM 2-mercaptoethanol, 30µM biotin-16-dUTP, and 300 U/ml TdT) in a humidified chamber at 37°C for 1 h. After incubating in stop/wash buffer for 10 min, the elongated digoxigenin-labeled DNA fragments were visualized using antidigoxigenin 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. Selectivity of the assay is based on the presence of 3-OH DNA fragment ends in apoptotic cells. In four or more random microscopic fields, the number of cells undergoing apoptosis was determined by TUNEL staining. Based on morphology, astrocytic cells were excluded from the counting.
Quantitation of necrotic cell death.
Apoptosis and necrosis were distinguished by using combined staining of chromatin dye, Hoechst 33342 and propidium iodide (PI) (Saito et al., 2001). Both dyes bind DNA but only Hoechst 33342 is membrane permeable. Hoechst 33342 (excitation: 360 nm, emission: 490 nm) freely enters living cells and therefore stains the nuclei of viable cells as well as those that died by apoptosis or necrosis. Apoptotic cells can be distinguished from viable ones on the basis of nuclear condensation and fragmentation; PI (excitation: 536 nm, emission: 620 nm) enters only cells with damaged cell membranes and viable cells are PI negative. In brief, at the end of the treatment, Hoechst 33342 was added to the culture medium at 1 µg/ml for 10 min, and cells were incubated with PI at 1 µg/ml for 10 min. The percentage of cells stained positive for PI was determined as an estimate of necrosis.
Western blot analysis.
UCP-2 expression was assessed with Western blot analysis. After various treatments, cells were washed with ice-cold PBS and harvested by centrifugation at 500 x g for 5 min. Cell pellets were lysed in a buffer containing 220mM mannitol, 68mM sucrose, 20mM HEPES, pH 7.4, 50mM KCl, 5mM EGTA, 1mM EDTA, 2mM MgCl2, 1mM dithiothreitol, 0.1% Triton X-100, 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 60 µg protein were boiled in Laemmli buffer for 5 min and subjected to electrophoresis in 12% SDS-polyacrylamide gel, followed by transfer to a nitrocellulose membrane. After blocking with 5% nonfat dry milk, the membrane was exposed to the primary antibody for 3 h at room temperature on a shaker. The UCP-2 antibody was a rabbit anti-mouse polyclonal antibody (1:2000) (Alpha Diagnostic International, Inc., San Antonio, TX). The PPAR antibody was a rabbit anti-human polyclonal antibody (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA). Reactions were detected with a fluorescein-linked anti-mouse IgG (second antibody) conjugated to horseradish peroxidase using enhanced chemiluminescence. Densitometric analysis of Western blots was done with Scion Image software and the data shown in the figures are the relative density of each band compared with the respective control band. For each study, Western blot analysis was conducted two to three times and representative blots are shown.
Reverse transcriptase–PCR analysis.
Total RNA was isolated from cortical cells using RNeasy Mini Kit (Qiagen, Valencia, CA), reverse transcribed into cDNA, and used as template for the PCR. RNA samples (1 µg) with oligo (dt) were denatured at 75°C for 5 min followed by addition of 1 x reverse transcriptase (RT) buffer, 4 µl dNTP mix, 1 µl RNase inhibitor, and 1 µl RT for a total of 20 µl (Retroscript Kit, Ambion). After gently mixing, samples were incubated at 42°C for 1 h and the reaction was stopped by heating to 92°C for 10 min. PCR amplimer pairs for analysis of UCP-2 cDNA were 5'-CGA CAG TGC TCT GGT ATC TCC-3' (sense) and 5'-ACA TCA ACG GGG GAG GCA ATG-3' (antisense). ß-Actin cDNA amplimer pairs were 5'-GTG GGC CGC TCT AGG CAC CAA-3' (sense) and 5'-CTC TTT GAT GTC ACG CAC GAT-3' (antisense). ß-Actin mRNA was assessed to control for the amount and integrity of RNA in each sample. Each PCR was performed on 1 µl cDNA sample using Taq DNA polymerase (Promega, Madison, WI) in a total volume of 25 µl in a Perkin-Elmer-Cetus 2400 DNA thermal cycler (Norwalk, CT). Each cycle consisted of a denaturation step (94°C for 30 s), an annealing step (45 s), and a primer extension step (72°C for 1 min). Annealing temperature and cycle number for UCP-2 were 54°C and 30 cycles and for ß-actin were 53°C and 23 cycles. PCR products were separated by electrophoresis on 1.5% agarose gel and detected by ethidium bromide staining using a UV transilluminator.
Measurement of mitochondrial membrane potential.
m was determined using JC-1, a mitochondrial-specific fluorescent probe. JC-1 exists as a green-fluorescent monomer at low membrane potential less than 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 (325 nm emission) fluorescence represents the relative mitochondrial membrane potential (Reers et al., 1995).
Measurement of cellular ATP.
ATP content was determined using a bioluminescence assay according to the manufacturer's instructions (Sigma Chemical, St Louis, MO). Pretreated cells (1 x 106) were incubated with cyanide for 24 h, then washed in PBS twice and lysed in buffer (0.5% Triton X-100, 10mM Tris-HCl, pH 7.5, 1mM EDTA), followed by incubation on ice for 10 min. 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).
Statistics.
Statistical evaluations were performed using one-way ANOVA with the Tukey-Krammer multiple range tests. Differences were considered as significant at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Influence of Wy14,643 on Cyanide-Induced Cell Death
In primary rat cortical cells, KCN produces predominately apoptotic death at 200–400µM and higher concentrations of KCN (500–600µM) switch the mode of death from apoptosis to necrosis (Li et al., 2005
). Cyanide induced predominately an apoptotic death in primary cortical cells (Fig. 1A), consistent with our previous report (Li et al., 2005). Wy14,643 (100µM) produced a low-level cytotoxicity which was slightly elevated above the level of cell death observed in control cultures. When cells were exposed to cyanide (400µM) following pretreatment for 5 h with Wy14,643, the mode of cell death produced by cyanide switched from apoptosis to necrosis (Fig. 1A). Note that the total time of exposure to Wy14,643 in the cytotoxicity studies was 29 h. Exposure to higher concentrations of KCN (> 600µM) in the presence of Wy14,643 enhanced the level of necrotic death (data not shown). To confirm the cell death mode, multiple histological staining methods were used (TUNEL and dye-staining techniques). Hoechst 33342 and PI double staining showed that the number of PI-positive cells (necrotic) produced by cyanide was significantly increased after Wy14,643 pretreatment (Fig. 1B). However, in cells treated with Wy14,643 and cyanide, total cell death (apoptotic + necrotic) was not increased above that produced by cyanide alone.
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Effect of Wy14,643 on UCP-2 Expression
PPAR
agonists have been shown to upregulate uncoupling protein expression in a variety of tissues (Nakatani et al., 2002; Young et al., 2001). Since our previous study showed that overexpression of UCP-2 switched cyanide-induced cortical cell death from apoptosis to necrosis (Li et al., 2005), the effect of Wy14,643 on UCP-2 expression was determined. Western blot analysis showed that Wy14,643 produced a concentration-dependent upregulation of UCP-2 (Fig. 2A). The increased expression was initiated within 1 h of exposure to Wy14,643 and continued to increase over the 12-h observation period (Fig. 2A). RT-PCR analysis showed that Wy14,643 upregulated UCP-2 at the mRNA level in a concentration- and time-dependent manner (Fig. 2B). Since the RT-PCR methodology is semiquantitative, the analysis provides a relative measure of the UCP-2 mRNA upregulation.
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Selective PPAR
Antagonism Blocks UCP-2 UpregulationIn order to determine if upregulation of UCP-2 expression is mediated by an enhanced cellular level of PPAR
, the effect of varying concentrations of Wy14,643 on PPAR expression was determined by Western blot analysis. Over a 12-h period Wy14,643 did not change the level of PPAR expression (data not shown). On the other hand, preincubation with MK886 (5µM), a specific antagonist of PPAR, blocked Wy14,643-induced UCP-2 expression at both the mRNA and protein levels (Figs. 3A and 3B). It was concluded that Wy14,643-induced upregulation of UCP-2 was mediated by ligand activation of constitutive PPAR.
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Wy14,643-Mediated Mitochondrial Dysfunction
To link the effect of Wy14,643 to cyanide cytotoxicity, cellular ATP levels and the relative
m were determined as indicators of mitochondrial dysfunction. Cyanide alone (400µM) produced a slight decrease of ATP levels and m, whereas Wy14,643 pretreatment enhanced the mitochondrial dysfunction produced by cyanide (Figs. 4A and 4B). In the case of ATP levels and m, the effect of KCN and Wy14,643 appears to be additive. It should be noted that Wy14,643 alone did reduce both the ATP levels and m. Pretreatment with the PPAR antagonist MK886 attenuated the Wy14,643 enhancement of cyanide-induced mitochondrial dysfunction and cell death (Fig. 5), paralleling the suppression of Wy14,643-induced upregulation of UCP-2 (Fig. 3). These observations further confirmed that PPAR-mediated UCP-2 expression played a role in the enhancement of KCN neurotoxicity.
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Our previous studies in cortical cells showed that cyanide induced mitochondrial permeability transition (MPT) pore opening in cells overexpressing UCP-2 (Li et al., 2005
). To determine if the Wy14,643-mediated mitochondrial dysfunction was associated with MPT pore opening, cyclosporin A (CsA) was used to block MPT. In cells pretreated with Wy14,643, CsA blocked the reduction of ATP and m produced by cyanide (Figs. 6A and 6B). Importantly, CsA reversed the cyanide-induced necrotic cell death back to apoptosis in Wy14,643-treated cells (Fig. 6C). In this study, CsA significantly reduced the level of KCN-induced necrosis and increased the level of apoptosis in cells pretreated with Wy14,643 as compared to cells not treated with CsA. It is concluded that the mode of cell death is determined by the level of mitochondrial dysfunction, in which opening of the MPT pore and loss of the membrane potential leads rapidly to a critical loss of bioenergetic homeostasis and then necrosis predominates over apoptosis.
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Knock down of UCP-2 Expression Prevents Wy14,643-Mediated Necrosis
To further confirm that Wy14,643-mediated necrosis was due to upregulation of UCP-2, gene-specific RNA sequences (siRNA) were used for UCP-2 RNA interference (RNAi) to knock down expression. We have previously shown that this specific RNAi can be used to knock down UCP-2 expression (Li et al., 2005
). Western blot analysis of UCP-2 expression showed that RNAi effectively knocked down UCP-2 in control cells and blocked the enhanced expression produced by Wy14,643 (Fig. 7A). Transfection with negative control RNAi containing a 21-base mRNA sequence starting with the AA dinucleotide did not significantly alter induction of UCP-2 expression by Wy14,643 (data not shown). RNAi prevented the increase of cyanide-induced mitochondrial dysfunction (reduced ATP levels and m) in cells treated with Wy14,643 (Figs. 7B and 7C). Moreover, in cells treated with Wy14,643, RNAi reversed the cyanide-induced necrosis back to apoptosis (Fig. 7D), thus showing that the necrosis is mediated via upregulation of UCP-2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PPAR
agonist Wy14,643 switched the mode of cyanide-induced cell death from apoptosis to necrosis by stimulating transcription of the UCP-2 gene to increase expression levels. Subsequently, the upregulation of UCP-2 enhanced the cyanide-induced mitochondrial dysfunction leading to execution of necrosis. Knock down of UCP-2 expression by RNAi attenuated Wy14,643-enhanced reduction of cellular ATP and m, confirming that increased expression of UCP-2 was responsible for the response. The effect was mediated by PPAR since the selective receptor antagonist MK886 reversed the upregulation of UCP-2 by Wy14,643.
In this study, upregulation of UCP-2 expression was linked with switching of cyanide-induced cell death from apoptosis to necrosis. In many cell models, the mode of death produced by an initiation stimulus may be switched by inhibiting control points or key execution steps in the death pathway, such as caspase activation, cytochrome c release from mitochondria, or ROS generation (Kalai et al., 2002; Nicotera, 2003). Inhibition of caspases or transient inhibition of their expression can switch the mode of death from apoptosis to necrosis (Denecker et al., 2001), and changes in the generation level of ROS and NO can either inhibit apoptosis or switch to necrosis (Melino et al., 2000). We have previously shown that cyanide-induced apoptosis in primary cultured cortical cells can be switched to necrosis by inhibiting caspase function (Prabhakaran et al., 2004). Apoptosis requires energy since it is a highly regulated process involving a number of ATP-dependent steps such as caspase activation, enzymatic hydrolysis, chromatin condensation, and apoptotic body formation (Skulachev, 2006). Marked depletion of cellular ATP by mitochondrial inhibitors such as cyanide can cause a switching of the cell death mechanism from apoptosis to necrotic cell death (Li et al., 2005). It is apparent that upregulation of UCP-2 by PPAR activation enhances the cytotoxic response to a mitochondrial toxin such as cyanide by producing a catastrophic decline of mitochondrial function.
PPAR, expressed in cerebral cells including neurons and astrocytes, can be activated by both natural and synthetic ligands including Wy14,643 (Cimini et al., 2005; Cullingford et al., 1998). Ligand-mediated activation of PPAR stimulates the formation of a heterodimeric transcription factor complex with retinoid X receptor alpha, another member of the nuclear receptor superfamily (Gearing et al., 1993). The heterodimer then binds to specific peroxisome proliferator–responsive elements of target genes to regulate transcription. PPAR ligands can stimulate the expression of a variety of genes involved in lipid homeostasis, glucose metabolism, and inflammation (Abumrad, 2004; Kersten et al., 2000; Patsouris et al., 2004; Ravnskjaer et al., 2005). Previous studies have shown that UCP-2 is a target gene of PPAR and UCP-2 expression can be upregulated by Wy14,643 (Nakatani et al., 2002; Ravnskjaer et al., 2005). Treatment of cultured mouse or rat hepatocytes with 50–100µM Wy14,643 produced appropriately a fivefold increase in UCP-2 mRNA (Nakatani et al., 2002). In the present study, 100µM Wy14,643 was used since it did not produce toxicity in the cortical cells and previous studies have shown that this concentration did not exhibit inherent toxicity in cultured neurons (Smith et al., 2001, 2004). Present results demonstrate that the PPAR agonist Wy14,643 induced a concentration-dependent upregulation of UCP-2 in primary cortical cells at both the mRNA and protein levels. The UCP-2 upregulation was dependent on PPAR activation since inhibition by the PPAR-specific antagonist MK886 blocked UCP-2 upregulation. Previous studies have shown that continuous exposure to Wy14,643 can induce expression of PPAR (Santo et al., 2005); however, in this study the response was due to activation of constitutively expressed PPAR since the level of the receptor did not change over the treatment period (data not shown).
Other reports have shown that activation of PPAR by Wy14,643 can induce apoptosis in glial cell lines (Strakova et al., 2005) and enhance cell death in cultured cerebellar granule cells (Smith et al., 2001). In the present study, Wy14,643 switched the mode of cyanide-induced cell death from apoptosis to necrosis. This is consistent with our previous studies in which overexpression of UCP-2 by transient transfection potentiated cyanide-induced ATP depletion and lowered m, resulting in increased necrotic cell death (Li et al., 2005). Moreover, RNAi knock down of UCP-2 upregulation blocked the effects of Wy14,643 on cyanide-induced mitochondrial dysfunction and cell death, thus confirming the role of PPAR. It should be noted that neither MK886 nor siRNA could totally restore the cyanide-induced decrease in ATP levels and m in cells pretreated with Wy14,643. It is possible that Wy14,643 directly inhibits mitochondrial respiration independent of PPAR activation and UCP-2 upregulation. It has recently been reported that PPAR ligands can produce dysfunction of the mitochondrial respiratory chain independent of PPAR binding (Scatena et al., 2004).
UCP-2 is expressed in a variety of tissues, including brain (Andrews et al., 2005; Kim-Han and Dugan, 2005; Richard et al., 2001). Constitutive expression or low-level upregulation of UCP-2 affords neuroprotection by decreasing mitochondrial ROS generation via uncoupling oxidative phosphorylation (Conti et al., 2005; Horvath et al., 2003). However, overexpression of UCP-2 can enhance mitochondrial dysfunction by depleting GSH and decreasing cellular ATP synthesis (de Bilbao et al., 2004; Li et al., 2005; Prabhakaran et al., 2005). In a number of pathological conditions, including transient ischemia (Horvath et al., 2002; MacManus et al., 2004) and chemical-induced seizures (Diano et al., 2003), UCP-2 mRNA is upregulated in the brain regions affected by the injury, suggesting that overexpression may be involved in the degenerative responses. Upregulation of UCP-2 in brain may lead to a dual effect on neuronal cells depending on the level of UCP-2 expression and the basal ATP levels and mitochondrial function. There appears to be a balance between beneficial and detrimental effects of UCP-2 overexpression (Horvath et al., 2003; Mills et al., 2002). Low-level upregulation and activation of UCP-2 decreases ROS production and may decrease ATP generation (Miwa and Brand, 2003). Under pathological conditions, such as ischemia and chemically induced seizures (Daino et al., 2003), overexpression of UCP-2 can enhance mitochondrial dysfunction, leading to marked depletion of cellular ATP. Similarly, Wy14,643-induced UCP-2 upregulation decreased intracellular ATP and mitochondrial membrane potential and enhanced cyanide-induced necrosis.
Cyanide is a rapidly acting neurotoxin that induces mitochondrial dysfunction by inhibiting cytochrome oxidase (complex IV) to reduce oxidative metabolism and enhance ROS generation at complexes I and III (Davey et al., 1998; Jones et al., 2003). In neuronal cells, cyanide produces two distinct modes of death, apoptosis and necrosis, depending on cell type and the level of oxidative insult (Mills et al., 1999; Prabhakaran et al., 2002). These modes of cell death appear to share common initiation stimuli, but divergent intracellular cascades are activated to produce either apoptosis or necrosis. In cyanide-induced neuronal death, apoptosis is caspase dependent and mediated by release of proapoptotic proteins from mitochondria (Shou et al., 2003). Cells undergoing necrosis display a more intense level of oxidative stress and experience a rapid breakdown of mitochondrial function characterized by the onset of mitochondrial membrane permeability transition and ATP depletion (Prabhakaran et al., 2002). We have previously shown that forced overexpression of UCP-2 by transient transfection with UCP-2 cDNA alters the response to cyanide by switching the mode of cell death from apoptosis to necrosis. In this study, Wy14,643-induced upregulation of UCP-2 expression potentiated cyanide-induced ATP depletion and m reduction, resulting in increased cortical cell death by necrosis.
Results of the present study may have important pathological significance. It is apparent that changes in UCP-2 expression can regulate the response to a neurotoxin in a cell-specific manner (Li et al., 2005; Prabhakaran et al., 2005). Changes in constitutive expression of UCP-2 in select brain areas may explain their selective vulnerability to injury by mitochondrial active compounds. This study showed that upregulation of UCP-2 expression by PPAR activation was associated with enhanced mitochondrial dysfunction and a switch in the mode of cell death. Thus, it is tempting to speculate that through this mechanism a variety of environmental and chemical agents that can activate PPAR could influence the level of neuronal sensitivity to mitochondrial toxins. Several studies have shown that UCP-2 upregulation may alter cellular responsiveness to excess energy depletion. Sriram et al. (2002) showed that the neurotoxicants methamphetamine and kainic acid increased UCP-2 expression in mouse brain which was linked with marked mitochondrial dysfunction and necrosis. Mills et al. (2002) reported that overexpression of UCP-2 leads to a rapid fall in m and a reduction of both mitochondrial NADH and ATP levels to selectively produce necrosis. Expression of a UCP-2–dominant interfering mutant conferred resistance to necrosis, but not apoptosis.
It is concluded that Wy14,643-mediated upregulation of UCP-2 expression in cultured cortical cells is through activation of PPAR. MK886, a specific antagonist of PPAR, and UCP-2 knockdown by RNAi blocked the effect of Wy14,643. Cyanide-induced mitochrondrial dysfunction was enhanced by upregulation of UCP-2 and resulted in a switch of cell death mode from apoptosis to necrosis. The increased mitochondrial dysfunction was reflected by reduced cellular ATP levels and reduced m.
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ACKNOWLEDGMENTS |
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This work was support by NIH grant ES04140.
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