Calcium-independent Phospholipase A2 Localizes in and Protects Mitochondria during Apoptotic Induction by Staurosporine
2006; Elsevier BV; Volume: 281; Issue: 31 Linguagem: Inglês
10.1074/jbc.m604330200
ISSN1083-351X
AutoresKonstantin Seleznev, Chunying Zhao, Xu Hannah Zhang, Keying Song, Zhongmin Alex,
Tópico(s)ATP Synthase and ATPases Research
ResumoMitochondria-mediated production of reactive oxygen species (ROS) plays a key role in apoptosis. Mitochondrial phospholipid cardiolipin molecules are likely the main target of ROS because they are particularly rich in polyunsaturated fatty acids. They are also located in the inner mitochondrial membrane near the ROS-producing sites. Under physiological conditions mitochondria can repair peroxidative damage in part through a remodeling mechanism via the deacylation-reacylation cycle mediated by phospholipase A2 (PLA2) and acyl-coenzyme A-dependent monolysocardiolipin acyltransferase. Here we investigate whether group VIA Ca2+-independent PLA2 (iPLA2) plays a role in the protection of mitochondrial function from damage caused by mitochondrially generated ROS during apoptotic induction by staurosporine (STS). We show that iPLA2-expressing cells were relatively resistant to STS-induced apoptosis. iPLA2 localized to mitochondria even before apoptotic induction, and most iPLA2-associated mitochondria were intact in apoptotic resistant cells. Expression of iPLA2 in INS-1 cells prevented the loss of mitochondrial membrane potential, attenuated the release of cytochrome c, Smac/DIABLO, and apoptosis inducing factor from mitochondria, and reduced mitochondrial reactive oxygen species production. Inhibition of caspase 8 has little effect on STS-induced apoptosis in INS-1 cells. Finally, we found that STS down-regulated endogenous iPLA2 transcription in both INS-1 and iPLA2-expressing INS-1 cells without affecting the expression of group IV Ca2+-dependent PLA2. Together, our data indicate that iPLA2 is important for the protection of mitochondrial function from oxidative damage during apoptotic induction. Down-regulation of endogenous iPLA2 by STS may result in the loss of mitochondrial membrane repair functions and lead to mitochondrial failure and apoptosis. Mitochondria-mediated production of reactive oxygen species (ROS) plays a key role in apoptosis. Mitochondrial phospholipid cardiolipin molecules are likely the main target of ROS because they are particularly rich in polyunsaturated fatty acids. They are also located in the inner mitochondrial membrane near the ROS-producing sites. Under physiological conditions mitochondria can repair peroxidative damage in part through a remodeling mechanism via the deacylation-reacylation cycle mediated by phospholipase A2 (PLA2) and acyl-coenzyme A-dependent monolysocardiolipin acyltransferase. Here we investigate whether group VIA Ca2+-independent PLA2 (iPLA2) plays a role in the protection of mitochondrial function from damage caused by mitochondrially generated ROS during apoptotic induction by staurosporine (STS). We show that iPLA2-expressing cells were relatively resistant to STS-induced apoptosis. iPLA2 localized to mitochondria even before apoptotic induction, and most iPLA2-associated mitochondria were intact in apoptotic resistant cells. Expression of iPLA2 in INS-1 cells prevented the loss of mitochondrial membrane potential, attenuated the release of cytochrome c, Smac/DIABLO, and apoptosis inducing factor from mitochondria, and reduced mitochondrial reactive oxygen species production. Inhibition of caspase 8 has little effect on STS-induced apoptosis in INS-1 cells. Finally, we found that STS down-regulated endogenous iPLA2 transcription in both INS-1 and iPLA2-expressing INS-1 cells without affecting the expression of group IV Ca2+-dependent PLA2. Together, our data indicate that iPLA2 is important for the protection of mitochondrial function from oxidative damage during apoptotic induction. Down-regulation of endogenous iPLA2 by STS may result in the loss of mitochondrial membrane repair functions and lead to mitochondrial failure and apoptosis. Mitochondria play a central role in the control of apoptosis (1Orrenius S. Toxicol. Lett. 2004; 149: 19-23Crossref PubMed Scopus (337) Google Scholar, 2Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2751) Google Scholar). Apoptotic induction triggers the permeabilization of the outer mitochondrial membrane and the release of cytochrome c, which interacts with and causes the oligomerization of monomeric apoptotic protease activating factor-1 (Apaf-1) and the subsequent recruitment of caspase-9 to form the apoptosome (3Acehan D. Jiang X. Morgan D.G. Heuser J.E. Wang X. Akey C.W. Mol. Cell. 2002; 9: 423-432Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar). Activated caspase-9 then cleaves and activates downstream caspases to amplify the death process (4Budihardjo I. Oliver H. Lutter M. 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Mitochondria are also considered to be the most important cellular source of reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; PLA2, phospholipase A2; iPLA2, group VI Ca2+-independent PLA2; cPLA2, cytosolic Ca2+-dependent PLA2; STS, staurosporine; FACS, fluorescence activated cell sorting; PS, phosphatidylserine; TBARS, thiobarbituric acid reactive substances; HE, hydroethidium; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine; BEL, bromoenol lactone; CHO, Chinese hamster ovary; PI, propidium iodide; PBS, phosphate-buffered saline; Z-, benzyloxycarbonyl; FMK, fluoromethyl ketone; AIF, apoptosis inducing factor; RT, reverse transcription; GFP, green fluorescent protein.2The abbreviations used are: ROS, reactive oxygen species; PLA2, phospholipase A2; iPLA2, group VI Ca2+-independent PLA2; cPLA2, cytosolic Ca2+-dependent PLA2; STS, staurosporine; FACS, fluorescence activated cell sorting; PS, phosphatidylserine; TBARS, thiobarbituric acid reactive substances; HE, hydroethidium; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine; BEL, bromoenol lactone; CHO, Chinese hamster ovary; PI, propidium iodide; PBS, phosphate-buffered saline; Z-, benzyloxycarbonyl; FMK, fluoromethyl ketone; AIF, apoptosis inducing factor; RT, reverse transcription; GFP, green fluorescent protein. (5Cadenas E. Mol. Aspects Med. 2004; 25: 17-26Crossref PubMed Scopus (340) Google Scholar). Some ROS, such as superoxide (O2.¯ ) or hydroxyl radicals (·OH), are extremely unstable, whereas others, such as hydrogen peroxide (H2O2), are freely diffusible and relatively long-lived. If not adequately neutralized, ROS can damage cells by promoting DNA fragmentation, sulfhydryl-mediated protein cross-linking, and peroxidation of membrane phospholipids. Peroxidation of membrane phospholipids is a major mechanism of ROS attack. Phospholipids, the major building blocks of membranes, are essential for cell life. They are rich in polyunsaturated fatty acids and vulnerable to attack by ROS (6Girotti A.W. J. Lipid Res. 1998; 39: 1529-1542Abstract Full Text Full Text PDF PubMed Google Scholar, 7Nigam S. Schewe T. Biochim. Biophys. Acta. 2000; 1488: 167-181Crossref PubMed Scopus (173) Google Scholar). It is well known that peroxidation of membrane phospholipids alters membrane fluidity, ion permeability, surface charge, passive electric properties, membranous enzyme activity, and cell signaling (6Girotti A.W. J. Lipid Res. 1998; 39: 1529-1542Abstract Full Text Full Text PDF PubMed Google Scholar, 8Sevanian A. Davies K. Oxidative Damage and Repair. Pergamon Press, New York1988: 543-549Google Scholar). The inner membrane of mitochondria contains a high proportion of the "double" phospholipid cardiolipin, which is particularly rich in unsaturated fatty acids and, thus, particularly susceptible to ROS attack (9Kagan V.E. Borisenko G.G. Tyurina Y.Y. Tyurin V.A. Jiang J. Potapovich A.I. Kini V. Amoscato A.A. Fujii Y. Free Radic. Biol. Med. 2004; 37: 1963-1985Crossref PubMed Scopus (286) Google Scholar). Indeed, cardiolipin peroxidation in mitochondria has been suggested to play a role in the initiation of the apoptotic signal (10Nakagawa Y. Ann. N. Y. Acad. 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It is possible that the iPLA2-mediated remodeling and repair of membrane phospholipids plays a role in protecting mitochondrial membranes from peroxidative damage. Staurosporine (STS), a potent protein kinase C inhibitor with a broad spectrum of activity (23Ruegg U.T. Burgess G.M. Trends Pharmacol. Sci. 1989; 10: 218-220Abstract Full Text PDF PubMed Scopus (833) Google Scholar), is known to induce apoptosis through a mitochondria-mediated pathway (24Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4362) Google Scholar) and to cause oxidative stress through mitochondrially generated ROS in a variety cells (25Cai J. Jones D.P. J. Biol. Chem. 1998; 273: 11401-11404Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar, 26Kruman I. Guo Q. Mattson M.P. J. Neurosci. Res. 1998; 51: 293-308Crossref PubMed Scopus (342) Google Scholar). This stress, which can be blocked by Bcl-2 (25Cai J. Jones D.P. J. Biol. Chem. 1998; 273: 11401-11404Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar) and antioxidants (27Ahlemeyer B. Bauerbach E. Plath M. Steuber M. Heers C. Tegtmeier F. Krieglstein J. Free Radic. Biol. Med. 2001; 30: 1067-1077Crossref PubMed Scopus (120) Google Scholar, 28Pong K. Doctrow S.R. Huffman K. Adinolfi C.A. Baudry M. Exp. Neurol. 2001; 171: 84-97Crossref PubMed Scopus (82) Google Scholar, 29Gil J. Almeida S. Oliveira C.R. Rego A.C. Free Radic. Biol. Med. 2003; 35: 1500-1514Crossref PubMed Scopus (91) Google Scholar), causes peroxidation of membrane phospholipids, including mitochondrial cardiolipin (12Kagan V.E. Tyurin V.A. Jiang J. Tyurina Y.Y. Ritov V.B. Amoscato A.A. Osipov A.N. Belikova N.A. Kapralov A.A. Kini V. Vlasova I.I. Zhao Q. Zou M. Di P. Svistunenko D.A. Kurnikov I.V. Borisenko G.G. Nat. Chem. Biol. 2005; 1: 223-232Crossref PubMed Scopus (951) Google Scholar, 26Kruman I. Guo Q. Mattson M.P. J. Neurosci. Res. 1998; 51: 293-308Crossref PubMed Scopus (342) Google Scholar, 28Pong K. Doctrow S.R. Huffman K. Adinolfi C.A. Baudry M. Exp. Neurol. 2001; 171: 84-97Crossref PubMed Scopus (82) Google Scholar, 30Matsura T. Serinkan B.F. Jiang J. Kagan V.E. FEBS Lett. 2002; 524: 25-30Crossref PubMed Scopus (53) Google Scholar). Therefore, STS-induced apoptosis is a useful model for the study of the effects of mitochondrially generated ROS on membrane phospholipid peroxidation. In the present study we use this model to investigate the role of iPLA2-mediated phospholipid repair of oxidatively damaged mitochondrial membranes during apoptosis in rat insulinoma INS-1. Cells and Transfection—Rat insulinoma INS-1 cells or CHO cells were cultured as described previously (31Ma Z. Bohrer A. Wohltmann M. Ramanadham S. Hsu F.F. Turk J. Lipids. 2001; 36: 689-700Crossref PubMed Scopus (43) Google Scholar, 32Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 33Song K. Zhang X. Zhao C. Ang N.T. Ma Z.A. Mol. Endocrinol. 2005; 19: 504-515Crossref PubMed Scopus (49) Google Scholar, 34Zhang X.H. Zhao C. Seleznev K. Song K. Manfredi J.J. Ma Z.A. J. Cell Sci. 2006; 119: 1005-1015Crossref PubMed Scopus (47) Google Scholar). Stable expression of iPLA2 in an INS-1 cell line (iPLA2-INS) was previously established using the retroviral vector pMSCVneo (Clontech, Palo Alto, CA) containing iPLA2 cDNA downstream of a 5′ long terminal repeat from the PCMV virus as described (31Ma Z. Bohrer A. Wohltmann M. Ramanadham S. Hsu F.F. Turk J. Lipids. 2001; 36: 689-700Crossref PubMed Scopus (43) Google Scholar, 35Ma Z. Ramanadham S. Wohltmann M. Bohrer A. Hsu F.F. Turk J. J. Biol. Chem. 2001; 276: 13198-13208Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 36Ma Z. Zhang S. Turk J. Ramanadham S. Am. J. Physiol. Endocrinol. Metab. 2002; 282: 820-833Crossref PubMed Scopus (39) Google Scholar). For expression of iPLA2, we used the expression vector pcDNA3-iPLA2 to transfect the cells with a FuGENE 6 transfection reagent (Roche Applied Science) as described (34Zhang X.H. Zhao C. Seleznev K. Song K. Manfredi J.J. Ma Z.A. J. Cell Sci. 2006; 119: 1005-1015Crossref PubMed Scopus (47) Google Scholar). For expression of iPLA2-GFP fusion protein, the expression vector pEGFP-N2-iPLA2 was constructed by subcloning fulllength rat iPLA2 cDNA in-frame upstream of the GFP cassette in the pEGFP-N2 vector (Clontech). Transfected cells stably expressing iPLA2-GFP were selected with G418 (active concentration, 400 μg/ml). Detection of Apoptosis—Apoptosis was induced by treating the cells with STS (Sigma), actinomycin D, etoposide, camptothecin (CHEMICON International, Inc.), or H2O2 (Promega). Multiple methods were used to detect apoptosis. For detection of phosphatidylserine (PS) externalization by flow cytometry, an annexin V-FLUOS staining kit (Roche Applied Science) was used to stain cells with fluorescent isothiocyanate-conjugated annexin V and propidium iodide (PI) according to the manufacturer's protocol. Briefly, ∼106 cells were harvested, washed with PBS by centrifugation at 200 × g for 5 min, and resuspended in 100 μl of annexin V-FLUOS labeling solution containing annexin V and PI. Cells were incubated for 10-15 min at 15-25 °C and immediately analyzed by flow cytometry on a BD Biosciences FACSCalibur (34Zhang X.H. Zhao C. Seleznev K. Song K. Manfredi J.J. Ma Z.A. J. Cell Sci. 2006; 119: 1005-1015Crossref PubMed Scopus (47) Google Scholar). PS externalization is a unique marker for apoptosis. Combined staining with annexin V and PI can be used to determine the early and late apoptosis. Cells were considered early apoptotic when they were annexin V-positive and PI-negative and late-apoptotic when they were both annexin V- and PI-positive. For detection of DNA fragmentation, fragmentized DNA was purified using the apoptotic DNA ladder kit (Roche Applied Science) and analyzed by electrophoresis in 1% agarose gel with ethidium bromide staining. Measurement of Caspase Activity—For determination of caspase 3 activity, cells were pretreated with or without 20 μm Z-DEVD-FMK (BD Biosciences), a caspase 3 specific inhibitor, or 20 μm Z-IETD-FMK (BD Biosciences), a caspase 8 specific inhibitor, and followed by apoptotic induction. Then the cells were counted with hemacytometer. 106 cells were resuspended in cell lysis buffer (Promega, Madison, WI) and analyzed with the CaspACE Assay System Colorimetric (Promega) according to the manufacturer's protocol. Western Blot Analysis—The cytosol and mitochondrial fractions of cells were obtained with mitochondria/cytosol fractionation kit following the manufacturer's protocol (BioVIsion, Mountain View, CA). The protein concentration of the supernatant was determined by Bradford assay using bovine serum albumin as the standard. Aliquots (containing 60 μg for cytoplasmic fraction or 15 μg for mitochondrial fraction) were separated by SDS-PAGE, transferred to nitrocellulose, blotted with corresponding antibodies, and detected by ECL WESTERN blotting detection system (Amersham Biosciences). Anti-iPLA2, anti-cPLA2, anti-cytochrome c, anti-Smac/DIABLO, anti-AIF, anti-actin antibodies (Santa Cruz), and anti-histone H4 (Upstate, Waltham, MA) antibodies were used. Confocal Microscopy—Confocal fluorescence microscopy was performed using a Zeiss LSM 510 META confocal laser scanning microscope (Carl Zeiss MicroImaging, Inc. Thornwood, NY). INS-1 cells were transfected with pEGFP-N2-iPLA2 construct and grown on cover slides. After induction of apoptosis, the cells were incubated in 50 nm Mito-Tracker Red CMXRos (Molecular Probes) for 15 min, washed with PBS, fixed in 3.8% paraformaldehyde (Fisher), and stained with 300 nm 4′,6-diamidino-2-phenylindole (Molecular Probes). The samples were embedded in Vectashield Mounting Medium H-1000 (Vector Laboratories, Burlingame, CA) and analyzed on a Zeiss LSM 510 META confocal laser-scanning microscope. Measurement of Mitochondrial Membrane Potential—Mitochondrial membrane potentials were determined by the JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine) mitochondrial membrane potential detection kit according to the manufacturer's protocol (Cell Technology, Mountain View, CA). Briefly, after induction of apoptosis, cells were washed with PBS and labeled with the JC-1 reagent for 15 min. After washing, the mitochondrial membrane potential was measured on a BD Biosciences FACSCalibur flow cytometer. Mitochondria containing red JC-1 aggregates in healthy cells were detectable in the FL2 channel, and green JC-1 monomers in apoptotic cells were detectable in the fluorescein isothiocyanate channel. The loss of mitochondrial membrane potential was calculated as the percentage of green cells versus total cells. Measurement of Mitochondrial Generation of ROS—Intramitochondrial generation of superoxide (O2.) was determined by the superoxide-induced conversion of the superoxide-sensitive dye, dihydroethidium (HE) to the highly fluorescent ethidium (37Bucana C. Saiki I. Nayar R. J. Histochem. Cytochem. 1986; 34: 1109-1115Crossref PubMed Scopus (97) Google Scholar, 38Rothe G. Valet G. J. Leukocyte Biol. 1990; 47: 440-448Crossref PubMed Scopus (778) Google Scholar). Briefly, after the treatment of STS, cells were stained with 2 μm HE (Molecular Probes) at 37 °C for 15 min. The cells were collected by trypsinization, washed once with PBS, and analyzed on a BD Biosciences FACSCalibur. Data were visualized using Windows Multiple Document Interface (WinMDI) (Joseph Trotter, Salk Institute for Biological Studies, La Jolla, CA) or FlowJo (Tree Star, Inc., Ashland, OR) flow-cytometry software. Values were expressed as a percentage of total cell counts. Measurement of Lipid Peroxidation—The Oxltek TBARS assay kit (ZeptoMetrix) was used to assess lipid peroxidation according to the manufacturer's protocol. Briefly, samples of cell suspension were separated into aliquots, and 2.5 ml of trichloroacetic acid/TBARS reagent was added. Each sample was incubated at 95 °C for 60 min. Supernatants were analyzed by a spectrophotometer at 532 nm, and values were expressed as a percentage of values in controls. iPLA2 Activity Assay—iPLA2 activity was determined using a modified kit originally designed for cytosolic Ca2+-dependent PLA2 (cPLA2) (cPLA2 assay kit, Cayman Chemicals) as described (33Song K. Zhang X. Zhao C. Ang N.T. Ma Z.A. Mol. Endocrinol. 2005; 19: 504-515Crossref PubMed Scopus (49) Google Scholar, 39Smani T. Zakharov S.I. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (234) Google Scholar). Briefly, after specific treatments, cultured cells were collected and homogenized in buffer (50 mm Hepes, pH 7.4, and 1 mm EDTA) followed by centrifugation at 14,000 × g for 20 min at 4 °C. The supernatant was removed, and the protein concentration was determined. iPLA2 activity was assayed by incubating the samples with arachidonoyl thio-PC for 1 h at 25°C in Ca2+-free buffer (4 mm EGTA, 160 mm Hepes, pH 7.4, 300 mm NaCl, 8 mm Triton X-100, 60% glycerol, and 2 mg/ml bovine serum albumin). The reaction was stopped by the addition of 5,5′-dithiobis(nitrobenzoic acid) for 5 min, and absorbance was determined at 414 nm using a μQuant microplate reader (BIO-TEK Instruments, Inc., Winooski, VT). The specific activity of iPLA2 was calculated and expressed in nmol/min/mg of total proteins. The background basal lipase activity, which was determined by inhibiting all specific iPLA2 activity in control samples with bromoenol lactone (BEL), was subtracted from all readings as described (33Song K. Zhang X. Zhao C. Ang N.T. Ma Z.A. Mol. Endocrinol. 2005; 19: 504-515Crossref PubMed Scopus (49) Google Scholar, 39Smani T. Zakharov S.I. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (234) Google Scholar). Isolation of RNA and RT-PCR Analysis—Total RNA was isolated from INS-1 cells using the RNeasy Mini Kit (Qiagen, Valencia, CA). RT-PCRs were performed using the Qiagen OneStep RT-PCR Kit (Qiagen), and the RT-PCR products were analyzed by electrophoresis in a 1% agarose gel with ethidium bromide staining. Primers for amplification of a fragment (505 bp) of rat iPLA2 were sense (5′-ATGCAGTTCTTTGGACGC-3′) and antisense (5′-CCAGAATCTCACTGTCAC-3′) and for amplification of rat glyceraldehyde-3-phosphate dehydrogenase were sense (5′-TGTCAGCAATGCATCCTG-3′) and antisense (5′-AACACGGAAGGCCATGCC-3′). Statistical Analysis—Data were expressed as the mean ± S.D. The statistical significance of differences was analyzed using Student's t test, where p < 0.05 was considered significant. iPLA2 Protects Cells from Mitochondrial Stress-induced Apoptosis—STS has previously been shown to induce apoptosis (24Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4362) Google Scholar), mitochondrial superoxide (O2.) generation (25Cai J. Jones D.P. J. Biol. Chem. 1998; 273: 11401-11404Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar), and membrane phospholipid peroxidation (26Kruman I. Guo Q. Mattson M.P. J. Neurosci. Res. 1998; 51: 293-308Crossref PubMed Scopus (342) Google Scholar, 28Pong K. Doctrow S.R. Huffman K. Adinolfi C.A. Baudry M. Exp. Neurol. 2001; 171: 84-97Crossref PubMed Scopus (82) Google Scholar, 30Matsura T. Serinkan B.F. Jiang J. Kagan V.E. FEBS Lett. 2002; 524: 25-30Crossref PubMed Scopus (53) Google Scholar). To verify these findings in rat insulinoma INS-1 cells, we treated INS-1 cells with 1 μm STS and analyzed for apoptosis by flow cytometry after staining with annexin V-FLUOS (Fig. 1A). We also used HE staining to assess mitochondrial superoxide generation (Fig. 1B) and the TBARS assay to measure lipid peroxidation levels (Fig. 1C) in these cells. As in other cells, STS induced both early (annexin V-positive and PI-negative cells) and late apoptosis (annexin V- and PI-positive cells) in INS-1 cells as well as mitochondrial ROS production and membrane lipid peroxidation. To investigate the role of iPLA2 in apoptosis induced by mitochondrially generated ROS, we compared the effect of STS on INS-1 cells with its effect on INS-1 cells stably expressing iPLA2 (iPLA2-INS) (31Ma Z. Bohrer A. Wohltmann M. Ramanadham S. Hsu F.F. Turk J. Lipids. 2001; 36: 689-700Crossref PubMed Scopus (43) Google Scholar, 35Ma Z. Ramanadham S. Wohltmann M. Bohrer A. Hsu F.F. Turk J. J. Biol. Chem. 2001; 276: 13198-13208Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). We first rigorously analyzed the time course of STS-induced apoptosis in each cell line using annexin V labeling followed by flow cytometric analysis. As shown in Fig. 2, iPLA2-INS cells were relatively resistant to STS-induced apoptosis compared with parental INS-1 cells. We found that the peak of early apoptosis (annexin V-positive and PI-negative) occurred between 8 to 12 h in both cell lines, and many of these cells progressed into late apoptosis (annexin V- and PI-positive) (Fig. 2A). However, in contrast to the INS-1 cells, which were nearly all dead 36 h after STS treatment, about 40% of iPLA2-INS cells remained alive (Fig. 2B), and many cells were still in early apoptosis after the same amount of time (Fig. 2A). These results clearly show that although STS induces a considerable amount of cell death in iPLA2-INS cells, their apoptotic progression is significantly delayed in comparison to INS-1 cells. To evaluate whether the degree of apoptotic resistance correlates with the level of iPLA2 expression, we first analyzed the effect of iPLA2 on apoptosis in a population of cells. INS-1 cells were transfected with iPLA2-GFP, and apoptosis was induced with STS. After PI staining, cells were sorted via FACS using two gates, one for PI and one for GFP. After sorting, cells were divided into groups according to their levels of GFP expression (inset in Fig. 2C), and the extent of cell death within each group (PI-positive cells) was determined. As shown in Fig. 2C, groups with no or low levels of GFP expression exhibited the highest proportions of dead cells. As iPLA2-GFP fusion protein expression increased, the proportion of PI-positive cells in each group fell. These results suggest that resistance to STS-induced apoptosis is positively correlated with higher iPLA2 levels. To confirm this finding, we selected and expanded individual colonies expressing variable levels of iPLA2. Once again, as shown in Fig. 2D, as iPLA2 expression increased, the proportion of dead cells decreased. To determine the extent of resistance of iPLA2-INS cells to apoptotic induction by STS, we treated both cell lines with increasing concentrations of STS for 8 h. As shown in Fig. 2E, when STS concentration was increased to 5 μm, we saw no significant difference in cell death levels between INS-1 cells and those expressing iPLA2, indicating that the stronger apoptotic induction overcame the protective function of iPLA2. To confirm that iPLA2-INS cells were relatively resistant to STS-induced apoptosis, we also looked for the appearance of DNA ladders. DNA fragmentation was apparent in INS-1 within4hofSTS treatment and intensified with time. However, DNA fragmentation was significantly attenuated in iPLA2-INS cells (Fig. 3A). We also compared STS-induced caspase-3 activation between the two cell lines (Fig. 3B). A difference in the degree of caspase 3 activation could be detected within2hofSTS treatment and became significant after 4 h. Although the activity increased prominently in a time-dependent manner in INS-1 cells, there was no significant increase in iPLA2-INS cells after 4 h, indicating that elevated levels of iPLA2 prevent the activation of caspase 3 in STS-induced apoptosis. It has been reported that STS causes the release of histones into the cytoplasm during apoptosis (40Wu D. Ingram A. Lahti J.H. Mazza B. Grenet J. Kapoor A. Liu L. Kidd V.J. Tang D. J. Biol. Chem. 2002; 277: 12001-12008Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). As shown in Fig. 3C, this release of histones from nucleosomes to cytoplasm was very prominent in INS-1 cells; they were evident on the Coomassie Blue-stained SDS-PAGE and reacted strongly with anti-histone H4 antibodies on the Western blot (Fig. 3C). Furthermore, the timing of histone release correlated well with the progression of apoptosis in the INS-1 cells. In contrast, many fewer histones were released in the iPLA2-INS cells in response to STS treatment. Taken together, our results unambiguously demonstrate that iPLA2 protects β-cells from apoptosis induced by mitochondrial stress. To determine whether iPLA2 can also protect other types of cells from apoptosis, we transfected CHO cells with a pDNA3-iPLA2 construct. As a control, a separate group of CHO cells were mock-transfected. Both groups of cells were then treated with 1 μm STS. Similar to our results in INS-cells, DNA fragmentation in iPLA2-expressing CHO cells was also dramatically less tha
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