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• W2093009462 abstract "Nitrosative stress with subsequent inflammatory cell death has been associated with many neurodegenerative disorders. Expression of inducible nitric-oxide synthase and production of nitric oxide (NO) have been frequently elevated in many inflammatory disorders. NO can rapidly react with superoxide anion, producing more reactive peroxynitrite. In the present study, exposure of rat pheochromocytoma (PC12) cells to the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1) induced apoptosis, which accompanied depletion of intracellular glutathione (GSH), c-Jun N-terminal kinase activation, mitochondrial membrane depolarization, the cleavage of poly(ADP-ribose)polymerase, and DNA fragmentation. During SIN-1-induced apoptotic cell death, expression of inducible cyclooxygenase (COX-2), and peroxisome proliferator-activated receptor-γ (PPARγ) was elevated. SIN-1 treatment resulted in elevated production of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous PPARγ activator. Preincubation with 15d-PGJ2 rendered PC12 cells resistant to nitrosative stress induced by SIN-1. 15d-PGJ2 fortified an intracellular GSH pool through up-regulation of glutamylcysteine ligase, thereby preventing cells from SIN-1-induced GSH depletion. The above findings suggest that 15d-PGJ2 may act as a survival mediator capable of augmenting cellular thiol antioxidant capacity through up-regulation of the intracellular GSH synthesis in response to the nitrosative insult. Nitrosative stress with subsequent inflammatory cell death has been associated with many neurodegenerative disorders. Expression of inducible nitric-oxide synthase and production of nitric oxide (NO) have been frequently elevated in many inflammatory disorders. NO can rapidly react with superoxide anion, producing more reactive peroxynitrite. In the present study, exposure of rat pheochromocytoma (PC12) cells to the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1) induced apoptosis, which accompanied depletion of intracellular glutathione (GSH), c-Jun N-terminal kinase activation, mitochondrial membrane depolarization, the cleavage of poly(ADP-ribose)polymerase, and DNA fragmentation. During SIN-1-induced apoptotic cell death, expression of inducible cyclooxygenase (COX-2), and peroxisome proliferator-activated receptor-γ (PPARγ) was elevated. SIN-1 treatment resulted in elevated production of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous PPARγ activator. Preincubation with 15d-PGJ2 rendered PC12 cells resistant to nitrosative stress induced by SIN-1. 15d-PGJ2 fortified an intracellular GSH pool through up-regulation of glutamylcysteine ligase, thereby preventing cells from SIN-1-induced GSH depletion. The above findings suggest that 15d-PGJ2 may act as a survival mediator capable of augmenting cellular thiol antioxidant capacity through up-regulation of the intracellular GSH synthesis in response to the nitrosative insult. Inflammation is a central feature of neurodegenerative disorders, such as Alzheimer's disease, stroke, and neurovascular disease (1Breitner J.C. Neurobiol. Aging. 1996; 17: 789-794Crossref PubMed Scopus (172) Google Scholar, 2McGeer E.G. McGeer P.L. Exp. Gerontol. 1998; 33: 371-378Crossref PubMed Scopus (297) Google Scholar). In Alzheimer tissue, pro-inflammatory enzymes, such as cyclooxygenase-2 (COX-2) 1The abbreviations used are: COX-2, cyclooxygenase 2; NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; DMEM, Dulbecco's modified Eagle's medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DHR 123, dihydrorhodamine 123; TMRE, tetramethylrhodamine ethyl ester; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; JNK, Jun N-terminal kinase; BSO, buthionine-[S,R]-sulfoximine; PPARγ, peroxisome proliferator-activated receptor-γ; 15d-PGJ2, 15-deoxy-Δ12,14-PGJ2; SIN-1, 3-morpholinosydnonimine hydrochloride; RNS, reactive nitrogen species; PARP, poly-(ADP-ribose)polymerase; GCLC, glutamylcysteine ligase catalytic; GCLM, glutamylcysteine ligase modulatory.1The abbreviations used are: COX-2, cyclooxygenase 2; NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; DMEM, Dulbecco's modified Eagle's medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DHR 123, dihydrorhodamine 123; TMRE, tetramethylrhodamine ethyl ester; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; JNK, Jun N-terminal kinase; BSO, buthionine-[S,R]-sulfoximine; PPARγ, peroxisome proliferator-activated receptor-γ; 15d-PGJ2, 15-deoxy-Δ12,14-PGJ2; SIN-1, 3-morpholinosydnonimine hydrochloride; RNS, reactive nitrogen species; PARP, poly-(ADP-ribose)polymerase; GCLC, glutamylcysteine ligase catalytic; GCLM, glutamylcysteine ligase modulatory. and inducible nitric-oxide synthase (iNOS), are notably up-regulated (3Colangelo V. Schurr J. Ball M.J. Pelaez R.P. Bazan N.G. Lukiw W.J. J. Neurosci. Res. 2002; 70: 462-473Crossref PubMed Scopus (447) Google Scholar, 4Luth H.J. Munch G. Arendt T. Brain Res. 2002; 953: 135-143Crossref PubMed Scopus (203) Google Scholar). Excessive nitric oxide (NO) production is considered as one of the main causes of neuronal cell death (5Heales S.J. Bolanos J.P. Stewart V.C. Brookes P.S. Land J.M. Clark J.B. Biochim. Biophys. Acta. 1999; 1410: 215-228Crossref PubMed Scopus (426) Google Scholar). Many studies suggest that the cytotoxic effect of NO is mediated via peroxynitrite formed by the reaction between NO and superoxide anion (O2−) (6Brune B. Gotz C. Messmer U.K. Sandau K. Hirvonen M.R. Lapetina E.G. J. Biol. Chem. 1997; 272: 7253-7258Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 7Szabo C. Day B.J. Salzman A.L. FEBS Lett. 1996; 381: 82-86Crossref PubMed Scopus (213) Google Scholar). The level of 3-nitrotyrosine, a relatively specific marker of nitrosative damage caused by peroxynitrite, has been reported to increase in Alzheimer's disease as well as other neurodegenerative disorders (8Beal M.F. Free Radic. Biol. Med. 2002; 32: 797-803Crossref PubMed Scopus (686) Google Scholar).Up-regulation of COX has also been frequently observed in patients with neurological symptoms (9Kitamura Y. Shimohama S. Koike H. Kakimura J. Matsuoka Y. Nomura Y. Gebicke-Haerter P.J. Taniguchi T. Biochem. Biophys. Res. Commun. 1999; 254: 582-586Crossref PubMed Scopus (209) Google Scholar). COX is a rate-limiting enzyme responsible for the synthesis of a series of prostaglandins (PGs) and thromboxanes that have multiple physiological functions. Two isoforms of COX have been described: COX-1 and COX-2. COX-1 is ubiquitously expressed in most tissues and produces prostanoids that are involved in maintaining homeostatic functions. COX-2 is induced under proinflammatory conditions and can mediate deleterious effects in the neurodegenerative disorders (9Kitamura Y. Shimohama S. Koike H. Kakimura J. Matsuoka Y. Nomura Y. Gebicke-Haerter P.J. Taniguchi T. Biochem. Biophys. Res. Commun. 1999; 254: 582-586Crossref PubMed Scopus (209) Google Scholar, 10Nogawa S. Zhang F. Ross M.E. Iadecola C. J. Neurosci. 1997; 17: 2746-2755Crossref PubMed Google Scholar). However, the role of COX-2 in neuronal cell death is conflicting. As prolonged intake of non-steroidal anti-inflammatory drugs (NSAIDs) with COX-2 inhibitory effects has been reported to reduce the risk of Alzheimer's disease and delay its onset (2McGeer E.G. McGeer P.L. Exp. Gerontol. 1998; 33: 371-378Crossref PubMed Scopus (297) Google Scholar, 11Stewart W.F. Kawas C. Corrada M. Metter E.J. Neurology. 1997; 48: 626-632Crossref PubMed Scopus (1042) Google Scholar), a number of studies have focused on the neuroprotective effects of NSAIDs. Recently, it has been recognized that the neuroprotective effects of NSAIDs are mediated via peroxisome proliferator-activated receptor-γ (PPARγ) activation, independently of COX-2 inhibition (12Asanuma M. Miyazaki I. Tsuji T. Ogawa N. Nihon Shinkei Seishin Yakurigaku Zasshi. 2003; 23: 111-119PubMed Google Scholar).Cyclopentenone PGs have various biological effects, such as growth inhibition, induction of apoptosis, differentiation, antioxidant, and anti-inflammation etc. depending on its concentrations and the cell type (13Straus D.S. Glass C.K. Med. Res. Rev. 2001; 21: 185-210Crossref PubMed Scopus (544) Google Scholar, 14Levonen A.L. Dickinson D.A. Moellering D.R. Mulcahy R.T. Forman H.J. Darley-Usmar V.M. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1846-1851Crossref PubMed Scopus (145) Google Scholar). Among them, PGJ2 is synthesized by non-enzymatic dehydration within the cyclopentane ring of PGD2, one of the major products of COX-2. PGJ2 is metabolized further to yield Δ12-PGJ2 and 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2). Members of the J2 series PGs are characterized by the presence of a reactive α,β-unsaturated carbonyl functional group in the cyclopentenone ring that can bind covalently by means of the Michael addition reaction with nucleophiles such as the free sulfhydryls of GSH and cysteine residues in cellular proteins (15Fukushima M. Eicosanoids. 1990; 3: 189-199PubMed Google Scholar, 16Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1200) Google Scholar). Interestingly, 15d-PGJ2 has been reported to exert dual effects on cell survival and apoptosis (14Levonen A.L. Dickinson D.A. Moellering D.R. Mulcahy R.T. Forman H.J. Darley-Usmar V.M. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1846-1851Crossref PubMed Scopus (145) Google Scholar, 17Emi M. Maeyama K. Biochem. Pharmacol. 2004; 67: 1259-1267Crossref PubMed Scopus (27) Google Scholar, 18Na H.K. Surh Y.J. Biochem. Pharmacol. 2003; 66: 1381-1391Crossref PubMed Scopus (112) Google Scholar). Thus, 15d-PGJ2 at relatively high concentrations causes cell death, whereas at submicromolar concentrations this cyclopentenone PG elicits cytoprotective effects. However, molecular mechanisms underlying cytoprotective effects of 15d-PGJ2 are poorly understood. In this study we hypothesize that 15d-PGJ2 rescues PC12 cells from the nitrosative stress induced by peroxynitrite through up-regulation of intracellular GSH biosynthesis.EXPERIMENTAL PROCEDURESMaterials—15d-PGJ2 and GW9662 were obtained from Cayman Chemical Co. (Ann Arbor, MI). 3-Morpholinosydnonimine hydrochloride (SIN-1) and Mn (II) tetrakis (4-benzoic acid) prophyrin (MnTBAP) were purchased from BIOMOL (Plymouth Meeting, PA). Dihydrorhodamine 123 (DHR 123) was supplied from Fluka and Riedel-de Haën (Buchs, Switzerland). Tetramethylrhodamine ethyl ester (TMRE) was obtained from Molecular Probes, Inc. (Eugene, OR). The in situ cell death detection kit was supplied by Roche Applied Science. GSH and buthionine-[S,R]-sulfoximine (BSO) were purchased from Sigma. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, horse serum, F-12, and N-2 supplement were obtained from Invitrogen Life Technologies, Inc.Cell Culture and Viability Measurement—PC12 cells were maintained routinely in DMEM supplemented with heat-inactivated 10% horse serum and 5% fetal bovine serum at 37 °C in a humidified atmosphere of 10% CO2, and 90% air. All cells were cultured in poly-d-lysinecoated culture dishes. Cells were cultured for 1 day before treatment, and switched to serum-free N-2-defined medium. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (19Jang J.H. Surh Y.J. Free Radic Biol. Med. 2003; 34: 1100-1110Crossref PubMed Scopus (362) Google Scholar).Measurement of Peroxynitrite—Generation of peroxynitrite from SIN-1 was determined spectrophotometrically using DHR 123. The peroxynitrite-dependent oxidation of DHR 123 to rhodamine 123 was measured according to the previously reported method (20Crow J.P. Nitric Oxide. 1997; 1: 145-157Crossref PubMed Scopus (551) Google Scholar). At pH 7.4 and 37 °C, peroxynitrite production from SIN-1 decomposition reaches a steady state after ∼3 min and remains essentially linear for up to 60 min. One hour after SIN-1 addition, the medium was collected and mixed with phosphate-buffered saline containing 20 μm DHR 123. After 10 min of incubation at 22 °C, the fluorescence intensity of DHR 123 was measured using a microplate reader (TECAN GmbH, Salzburg, Austria) at an excitation wavelength of 500 nm and an emission at 536 nm (ϵ500 nm = 78,800 m-1 cm-1). Peroxynitrite concentrations were calculated from the DHR 123 fluorescence intensity (21Takakura K. Beckman J.S. MacMillan-Crow L.A. Crow J.P. Arch. Biochem. Biophys. 1999; 369: 197-207Crossref PubMed Scopus (187) Google Scholar). Synthetic peroxynitrite oxidizes DHR 123 to rhodamine 123 with 44% efficiency (20Crow J.P. Nitric Oxide. 1997; 1: 145-157Crossref PubMed Scopus (551) Google Scholar). Thus, formation of rhodamine 123 from DHR 123 after reaction with SIN-1 was corrected to total peroxynitrite production by dividing the value by 0.44.Western Blot Analysis—Western blot analysis was performed with anti-poly-(ADP-ribose)polymerase (PARP), anti-phospho-c-Jun N-terminal kinase (JNK), anti-JNK, and anti-PPARγ polyclonal antibodies (1:500 dilution, all products of Santa Cruz Biotechnology) as described previously (19Jang J.H. Surh Y.J. Free Radic Biol. Med. 2003; 34: 1100-1110Crossref PubMed Scopus (362) Google Scholar). An anti-COX-2 polyclonal antibody (1:1000, Transduction Laboratories, Lexington, KY) was used for detection of COX-2.PGE2 Enzyme Immunoassay—PC12 cells were plated at a density of 2 × 105 cells/4 ml in 60-mm plates and incubated for the desired times after 3 mm SIN-1 treatment. PGE2 released into incubation medium was measured by using an enzyme immunoassay kit (Amersham Biosciences). 50 μl of culture medium was used for an assay. PGE2 concentration was determined spectrophotometrically according to the instructions provided from the supplier.15d-PGJ2 Enzyme Immunoassay—PC12 cells were plated at a density of 5 × 106 cells/7 ml in 100-mm plates, and the 15d-PGJ2 production was determined by using an enzyme immunoassay kit according to the instructions provided from the supplier (Assay Designs, Inc., Ann Arbor, MI). In brief, the culture media and cell lysates were transferred to a goat anti-rabbit IgG microtiter plate and incubated with anti-15d-PGJ2 polyclonal antibody and alkaline phosphatase conjugated with 15d-PGJ2 at room temperature for 2 h. The plate was rinsed three times with washing solution and further incubated with 200 μl of p-nitrophenyl phosphate substrate at 37 °C for 3 h. The reaction was terminated by addition of 50 μl of stop solution. The changes in absorbance at 405 nm were measured using a spectrophotometric microplate reader.Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End-labeling (TUNEL)—The commercially available in situ Cell Death Detection kit was used to assess the internucleosomal DNA fragmentation. PC12 cells (5 × 105 cells/3 ml in chamber slide) were fixed for 30 min in 10% neutral buffered formalin solution at room temperature. Endogenous peroxidase was inactivated by incubation with 0.3% hydrogen peroxide in methanol for 30 min at room temperature and further incubated in a permeabilizing solution (0.1% sodium citrate and 0.1% Triton X-100) for 2 min at 4 °C. The cells were incubated with the TUNEL reaction mixture for 60 min at 37 °C followed by labeling with peroxidase-conjugated anti-goat antibody (Fab fragment) for additional 30 min. After staining with diaminobenzidine for 10 min, cells were rinsed with phosphate-buffered saline and mounted with 50% glycerol.Measurement of Mitochondrial Transmembrane Potential (ΔΨm)— The fluorescent probe TMRE was used to measure the mitochondrial transmembrane potential (ΔΨm). This dye binds in a manner dependent on the mitochondrial electric potential. PC12 cells were plated at a density of 4 × 105 cells/2.4 ml in chamber slides. The cells were pretreated with 2 μm 15d-PGJ2 for 24 h, and SIN-1 was then added to the medium at the final concentration of 3 mm. After 5 h of incubation with SIN-1, PC12 cells were washed with DMEM and incubated with TMRE (150 nm) in DMEM for additional 30 min at 37 °C, then washed twice with DMEM, and examined under a confocal microscope (Leica Microsystems Heidelberg GmbH, Heidelberg, Germany). TMRE rapidly equilibrates between cellular compartments due to potential differences. A decrease in TMRE fluorescence is indicative of a reduction in ΔΨm. The fluorescence was monitored as red fluorescence (excitation: 555 nm, emission: >590 nm) at hyperpolarized membrane potentials. For the quantification of the fluorescence intensity, four fields containing about 400 cells were analyzed, and the fluorescence intensity of the red was determined as the indicator of ΔΨm.GSH Measurement—The GSH content was measured using a commercial kit according to the manufacturer's protocol (GSH-400 method, OXIS International, Portland, OR) using 4-chloro-1-methyl-7-trifluoromethyl-quinolinum methylsulfate. Cells were harvested and homogenized in metaphosphoric working solution. After centrifugation, 50 μl of R1 solution (solution of chromogenic reagent in HCl) was added to the 700 μl of supernatant, followed by gentle vortex mixing. Following the addition of 50 μl of R2 solution (30% NaOH), the mixtures were incubated at 25 ± 3 °C for 10 min. After centrifugation, the absorbance of the clear supernatant was read at 400 nm. The protein concentration was determined using the BCA protein assay kit.Reverse Transcription-Polymerase Chain Reaction (RT-PCR)—Total RNA was isolated from PC12 cells using TRIzol® (Invitrogen Life Technologies, Inc.) following the manufacturer's instructions. RT-PCR was performed according to standard techniques. PCR conditions for glutamylcysteine ligase catalytic (GCLC), glutamylcysteine ligase modulatory (GCLM), and the housekeeping, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were as follows: for GCLC and GAPDH, 35 cycles of 94 °C for 30 s; 55 °C for 30 s; and 72 °C for 45 s and for GCLM, 35 cycles of 95 °C for 1 min; 60 °C for 1 min; 68 °C for 2 min; and 68 °C for 7 min (22Tian L. Shi M.M. Forman H.J. Arch. Biochem. Biophys. 1997; 342: 126-133Crossref PubMed Scopus (130) Google Scholar). The sequence of each primer used is as follows (forward and reverse, respectively): GCLC subunit, 5′-GCC AAG GTC ATC CAT GAC AAC-3′ and 5′-AGT GTA GCC CAG GAT GCC CTT-3′; GCLM subunit, 5′-AGA CCG GGA ACC TGC TCA AC-3′ and 5′-CAT CAC CCT GAT GCC TAA GC-3′; GAPDH, 5′-AGT GTA GCC CAG GAT GCC CTT-3′ and 5′-GCC AAG GTC ATC CAT GAC AAC-3′. Amplification products were resolved by 1.0% agarose gel electrophoresis, stained with ethidium bromide, and photographed under ultraviolet light. All primers were purchased from Bionics (Seoul, Korea).GCL Reporter Gene Assay—The luciferase reporter construct pGL3 containing the rat GCLC promoter was prepared as reported previously (23Yang H. Wang J. Huang Z.Z. Ou X. Lu S.C. Biochem. J. 2001; 357: 447-455Crossref PubMed Scopus (48) Google Scholar). Transient transfection was carried out by using DOTAP Liposomal Transfection Reagent (Roche Applied Science). Transfection was performed at 1–3×105 cells per well in 6-well plates. Twelve hours later, the transfection medium was removed and replaced with N-2 medium containing Me2SO or 2 μm 15d-PGJ2 and incubated for additional 24 h. Cell extracts were assayed for luciferase and β-galactosidase activities. Luciferase activity was normalized to β-galactosidase activity. The result was expressed as means ± S.D. of three independent experiments.Measurement of GCL Activity—GCL activity was determined as described by Seelig and Meister (24Seelig G.F. Meister A. J. Biol. Chem. 1984; 259: 3534-3538Abstract Full Text PDF PubMed Google Scholar) with slight modifications. The assay measures the rate of oxidation of NADH (assumed to be equal to the rate of formation of ADP) from the change in absorbance at 340 nm in reaction mixtures containing the following: 0.1 m Tris-HCl (pH 8.0), 150 mm KCl, 5 mm Na2ATP, 2 mm phosphoenolpyruvate, 10 mm l-glutamate, 10 mm l-α-aminobutyrate, 20 mm MgCl2,2mm Na2EDTA, 0.2 mm NADH, 17 μg/ml of pyruvate kinase, and 17 μg/ml of lactate dehydrogenase. Cells were harvested and lysed in 0.1 m Tris-HCl (pH 8.0) containing 0.1% Triton X-100, and the supernatant was used for the assay. The protein concentration was determined using the BCA protein assay kit. The enzyme activity was expressed in international units per mg of protein and calculated by using the molar extinction coefficient, 6.22 for NADH.RESULTSSIN-1 Caused Apoptotic Death in PC12 Cells—To assess the nitrosative stress-induced cytotoxicity, PC12 cells were treated for 24 h with varying concentrations of the peroxynitrite donor SIN-1. Peroxynitrite released from SIN-1 was quantified by measuring the fluorescence intensity derived from DHR 123 oxidized by peroxynitrite (Fig. 1A). In parallel with increased peroxynitrite release, SIN-1 treatment decreased the cell viability as determined by the MTT reduction assay (Fig. 1A). Cells treated with 3 mm SIN-1 exhibited gradual cleavage of PARP, one of the common biochemical features of apoptosis (Fig. 1B). The SIN-1-induced cytotoxicity was abolished by cotreatment with the membrane permeable peroxynitrite scavenger MnTBAP (Fig. 1C). Activation of the JNK-signaling cascades has been frequently implicated in neuronal cell death induced by a wide variety of toxicants (25Kang C.D. Jang J.H. Kim K.W. Lee H.J. Jeong C.S. Kim C.M. Kim S.H. Chung B.S. Neurosci. Lett. 1998; 256: 37-40Crossref PubMed Scopus (61) Google Scholar). JNK activation via phosphorylation during the SIN-1-induced PC12 cell death coincided with the PARP cleavage (Fig. 1D). Both events were blocked by the peroxynitrite scavenger MnTBAP, in a concentration-dependent manner (Fig. 1D).SIN-1 Treatment Induced Activation of COX-2 and PPARγ— To investigate the probable involvement of inflammatory pathways in nitrosative PC12 cell death induced by SIN-1, COX-2 expression was measured by Western blot analysis. Fig. 2A illustrates a time-related up-regulation of COX-2 in cells exposed to peroxynitrite. The release of PGE2, one of the major COX-2 products, was also substantially elevated by SIN-1 treatment (Fig. 2A). To clarify the role of COX-2 during the nitrosative cell death, two selective COX-2 inhibitors, SC-58635, and NS-398, were coincubated with 2 mm SIN-1 for 24 h. As shown in Fig. 2B, both of these COX-2 selective inhibitors aggravated SIN-1-induced cytotoxicity.Fig. 2COX-2 and PPARγ expression induced by cytotoxic concentrations of SIN-1 in PC12 cells.A, SIN-1-induced COX-2 expression and PGE2 production in PC12 cells. Cells were exposed to 3 mm SIN-1 for indicated time periods. COX-2 expression was examined by Western blot analysis, and PGE2 production was measured 24 h later by using the PGE2 enzyme immunoassay kit. B, aggravation of SIN-1-induced cytotoxicity by COX-2 inhibitors. PC12 cells were treated with SC-58635 (15 and 30 μm) or NS-398 (30 μm) in the presence or absence of 2 mm SIN-1 for 24 h. The cell viability was measured by the MTT reduction assay. C, SIN-1-induced PPARγ expression. PC12 cells were exposed to 3 mm SIN-1 for indicated time periods. PPARγ induction was examined by Western blot analysis. D, enhancement of SIN-1-induced cytotoxicity by the PPARγ antagonist GW9662. PC12 cells were treated with GW9662 (1, 5, or 10 μm) together with 2 mm SIN-1 for 24 h. The results are presented as means ± S.D. (n = 3). *, significantly different from the group treated with SIN-1 alone (p < 0.01).View Large Image Figure ViewerDownload (PPT)PPARγ is implicated in anti-inflammatory and proapoptotic properties of several NSAIDs. PPARγ expression was increased in cells treated with a proapoptotic concentration of SIN-1 (Fig. 2C). To determine the role of PPARγ during the nitrosative PC12 cell death, a PPARγ antagonist GW9662 was added to the media together with SIN-1. As illustrated in Fig. 2D, GW9662 aggravated SIN-1-induced cytotoxicity. Since pharmacologic inhibition of both COX-2 and PPARγ further enhanced the SIN-1-induced cell death, it is likely that the product of COX-2 capable of acting as a PPARγ ligand exerts cytoprotective effects. Based on the fact that 15d-PGJ2 produced in the arachidonic cascade mediated by COX-2 is an endogenous activator of PPARγ, the possible involvement of this cyclopentenone PG in the cellular defense mechanism against SIN-1-induced cell death was explored.15d-PGJ2 was hence proposed as a probable common denominator in the COX-2- and PPARγ-mediated processes that confer cell survival or adaptation in response to the nitrosative insult. The levels of 15d-PGJ2 was elevated in both culture media and cell lysates after SIN-1 stimulation (Fig. 3, A and B). To clarify the role of elevated 15d-PGJ2 during the SIN-1-induced nitrosative cell death, two selective COX-2 inhibitors (SC-58635 and NS-398) were coincubated with SIN-1. As illustrated in Fig. 3, A and B, both of SC-58635 and NS-398 significantly reduced SIN-1-induced 15d-PGJ2 production, at the concentration that aggravated nitrosative cell death.Fig. 3Protective effect of 15d-PGJ2 against SIN-1-induced apoptosis in PC12 cells.A and B, SIN-1-induced 15d-PGJ2 production and the effect of COX-2 inhibitors. PC12 cells were treated with SC-58635 (30 μm) or NS-398 (30 μm) in the absence or presence of SIN-1 (2 mm) for 24 h. 15d-PGJ2 production in the culture media (A) and cell lysates (B) was measured by using the 15d-PGJ2 enzyme immunoassay kit. C, 15d-PGJ2-mediated protection of PC12 cells from SIN-1-induced cytotoxicity. After 24 h of preincubation with 15d-PGJ2 (0.5 or 2 μm), PC12 cells were exposed to 3 mm SIN-1 for additional 24 h, and the cell viability was determined by the MTT reduction assay. The results are presented as means ± S.D. (n = 3). *, significantly different from the SIN-1 alone group (p < 0.01). D, attenuation of SIN-1-induced internucleosomal DNA fragmentation by 15d-PGJ2 in PC12 cells. a, untreated control cells. b, cells treated with 3 mm SIN-1 for 10 h. c, cells treated with 3 mm SIN-1 after 24 h of preincubation with 2 μm 15d-PGJ2. E, amelioration of SIN-1-induced perturbation of mitochondrial transmembrane permeability (ΔΨm) by 15d-PGJ2. ΔΨm was assessed by changes in the TMRE fluorescence as described under “Experimental Procedures.” Images of the cellular fluorescence were acquired by using a confocal laser-scanning microscope. a, untreated control cells. b, cells treated with 3 mm SIN-1 for 5 h. c, cells treated with 3 mm SIN-1 after 24 h preincubation with 2 μm 15d-PGJ2. ΔΨm was quantified by measuring average values of the red fluorescence intensity of four fields, each containing about 400 cells.View Large Image Figure ViewerDownload (PPT)Though relatively high concentrations (>10 μm) of 15d-PGJ2 alone elicited a proapoptotic response in PC12 cells (data not shown), 24 h treatment with nontoxic concentrations (0.5 or 2 μm) of this cyclopentenone PG prior to SIN-1 exposure markedly restored the viability (Fig. 3C). Co-treatment of 15d-PGJ2 failed to protect PC12 cells against the SIN-1-induced cytotoxicity (data not shown), suggesting that 15d-PGJ2 itself is not a direct peroxynitrite scavenger.The protective effect of 15d-PGJ2 against SIN-1-induced apoptosis was further confirmed by analyzing several hallmarks of apoptosis. As illustrated in Fig. 3D, a nontoxic concentration (i.e. 2 μm) of 15d-PGJ2 ameliorated SIN-1-induced internucleosomal DNA fragmentation as revealed by TUNEL staining. Mitochondrial depolarization occurs in the cells undergoing apoptosis. TMRE is detected as red fluorescence in the intact mitochondrial membrane. In contrast, in cells treated with a proapoptotic concentration (i.e. 3mm) of SIN-1 alone for 5 h, the red staining disappeared, indicating the perturbation of the mitochondrial transmembrane potential (Fig. 3E). 15d-PGJ2 pretreatment restored the mitochondrial membrane potential (ΔΨm). Changes in ΔΨm values were quantified by measuring the average values of the red fluorescence intensity of four fields, each containing about 400 cells (Fig. 3E). It has been known that peroxynitrite may directly induce mitochondrial dysfunction through nitration of mitochondrial proteins, such as complex I, II, V respiratory system (26Boyd C.S. Cadenas E. Biol. Chem. 2002; 383: 411-423Crossref PubMed Scopus (172) Google Scholar, 27Brown G.C. Borutaite V. Biochem. Soc. Symp. 1999; 66: 17-25Crossref PubMed Scopus (171) Google Scholar, 28Leist M. Single B. Naumann H. Fava E. Simon B. Kuhnle S. Nicotera P. Exp. Cell Res. 1999; 249: 396-403Crossref PubMed Scopus (247) Google Scholar). Preservation of ΔΨm by 15d-PGJ2 implies that this cyclopentenone PG may increase the intracellular antioxidant capacity, which can efficiently quench peroxynitrite.15d-PGJ2 Rescued PC12 Cells from Nitrosative Stress through Augmentation of Cellular GSH—To elucidate the plausible mechanism by which 15d-PGJ2 protected against apoptosis induced by SIN-1, the levels of peroxynitrite were initially compared in the cells with and without 15d-PGJ2 pretreatment (Fig. 4A). As illustrated in Fig. 4A, the accumulation of peroxynitrite in SIN-1-treated cells was significantly reduced by 2 μm 15d-PGJ2. The free thiol group of GSH, the most abundant cellular antioxidant, is a target for peroxynitrite-mediated oxidation. In support of this notion, intracellular GSH was substantially depleted at 6 h after SIN-1 treatment (Fig. 4B). Preincubation of PC12 cells with 2 μm 15d-PGJ2 for 24 h restored the GSH to the control level (SIN-1 alone, 64.8 ± 5.55% of the control GSH level versus SIN-1 plus 15d-PGJ2, 102.1 ± 6.51% of control, p < 0.05). The GSH depletion coincided with the cytotoxici" @default.
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