Mitochondrial Targeted Cyclophilin D Protects Cells from Cell Death by Peptidyl Prolyl Isomerization
2002; Elsevier BV; Volume: 277; Issue: 34 Linguagem: Inglês
10.1074/jbc.m112035200
ISSN1083-351X
AutoresDa‐Ting Lin, James D. Lechleiter,
Tópico(s)Peptidase Inhibition and Analysis
ResumoCyclophilin D (CyPD) is thought to sensitize opening of the mitochondrial permeability transition pore (mPTP) based on the findings that cyclosporin A (CsA), a pseudo-CyPD substrate, hyperpolarizes the mitochondrial membrane potential (ΔΨ) and inhibits apoptosis. We provide evidence that contrasts with this model. Using live cell imaging and two photon microscopy, we report that overexpression of CyPD desensitizes HEK293 and rat glioma C6 cells to apoptotic stimuli. By site-directed mutagenesis of CyPD that compromises peptidyl-prolyl cis-trans isomerase (PPIase) activity, we demonstrate that the mechanism involved in this protective effect requires PPIase activity. Furthermore, we show that, under resting conditions, ΔΨ is hyperpolarized in CyPD wild type-overexpressing cells but not in cells overexpressing mutant forms of CyPD that lack PPIase activity. Finally, in glutathioneS-transferase (GST) pull-down assays, we demonstrate that CyPD binding to the adenine nucleotide translocator (ANT), which is considered to be the core component of the mPTP, is not affected by the loss of PPIase activity. Collectively, our data suggest that CyPD should be viewed as a cell survival-signaling molecule and indicate a protective role of CyPD against apoptosis that is mediated by one or more targets other than the ANT. Cyclophilin D (CyPD) is thought to sensitize opening of the mitochondrial permeability transition pore (mPTP) based on the findings that cyclosporin A (CsA), a pseudo-CyPD substrate, hyperpolarizes the mitochondrial membrane potential (ΔΨ) and inhibits apoptosis. We provide evidence that contrasts with this model. Using live cell imaging and two photon microscopy, we report that overexpression of CyPD desensitizes HEK293 and rat glioma C6 cells to apoptotic stimuli. By site-directed mutagenesis of CyPD that compromises peptidyl-prolyl cis-trans isomerase (PPIase) activity, we demonstrate that the mechanism involved in this protective effect requires PPIase activity. Furthermore, we show that, under resting conditions, ΔΨ is hyperpolarized in CyPD wild type-overexpressing cells but not in cells overexpressing mutant forms of CyPD that lack PPIase activity. Finally, in glutathioneS-transferase (GST) pull-down assays, we demonstrate that CyPD binding to the adenine nucleotide translocator (ANT), which is considered to be the core component of the mPTP, is not affected by the loss of PPIase activity. Collectively, our data suggest that CyPD should be viewed as a cell survival-signaling molecule and indicate a protective role of CyPD against apoptosis that is mediated by one or more targets other than the ANT. mitochondrial permeability transition pore adenine nucleotide translocator cyclosporin A cyclophilin D differential interference contrast enhanced yellow fluorescent protein glutathioneS-transferase peptidyl-prolyl cis-transisomerase tert-butyl hydroperoxide tetramethyl rhodamine ethyl ester voltage-dependent anion channel mitochondrial membrane potential diethylpyrocarbonate Focht Chamber System 2 propidium iodide analysis of variance In multicellular organisms, programmed cell death (apoptosis) is an essential process of normal development, tissue maintenance, and aging (1Meier P. Finch A. Evan G. Nature. 2000; 407: 796-801Crossref PubMed Scopus (818) Google Scholar, 2Yuan J. Yankner B.A. Nature. 2000; 407: 802-809Crossref PubMed Scopus (1608) Google Scholar). Abnormal regulation of apoptosis results in multiple human diseases, including cancer, AIDS (3Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6205) Google Scholar), and many neurological disorders (2Yuan J. Yankner B.A. Nature. 2000; 407: 802-809Crossref PubMed Scopus (1608) Google Scholar, 4Honig L.S. Rosenberg R.N. Am. J. Med. 2000; 108: 317-330Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Mitochondria provide a key regulatory role in cell death by releasing apoptogenic proteins into the cytosol that initiate the caspase cascade (5Korsmeyer S.J. Harvey Lect. 1999; 95: 21-41PubMed Google Scholar, 6Waterhouse N.J. Green D.R. J. Clin. Immunol. 1999; 19: 378-387Crossref PubMed Scopus (58) Google Scholar, 7Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6296) Google Scholar, 8Boya P. Roques B. Kroemer G. EMBO J. 2001; 20: 4325-4331Crossref PubMed Scopus (110) Google Scholar). Release of these factors is thought to occur via a tightly regulated increase in the permeability of the inner mitochondrial membrane, due to the opening of the mitochondrial permeability transition pore (mPTP)1 (9Lemasters J.J. Qian T. Bradham C.A. Brenner D.A. Cascio W.E. Trost L.C. Nishimura Y. Nieminen A.L. Herman B. J. Bioenerg. Biomembr. 1999; 31: 305-319Crossref PubMed Scopus (347) Google Scholar, 10Halestrap A.P. Biochem. Soc. Symp. 1999; 66: 181-203Crossref PubMed Scopus (166) Google Scholar, 11Bernardi P. Petronilli V., Di Lisa F. Forte M. Trends Biochem. Sci. 2001; 26: 112-117Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 12Zamzami N. Kroemer G. Nat. Rev. Mol. Cell. Biol. 2001; 2: 67-71Crossref PubMed Scopus (898) Google Scholar). The primary components of the mPTP include the voltage-dependent anion channel (VDAC) in the outer membrane, ANT in the inner membrane, and CyPD within the mitochondrial matrix (10Halestrap A.P. Biochem. Soc. Symp. 1999; 66: 181-203Crossref PubMed Scopus (166) Google Scholar, 13Crompton M. J. Physiol. 2000; 529: 11-21Crossref PubMed Scopus (277) Google Scholar). Bcl-2 family members can regulate the release of apoptotic factors from mitochondria through direct interaction with VDAC and regulation of ANT activity during apoptosis (14Brenner C. Cadiou H. Vieira H.L. Zamzami N. Marzo I. Xie Z. Leber B. Andrews D. Duclohier H. Reed J.C. Kroemer G. Oncogene. 2000; 19: 329-336Crossref PubMed Scopus (309) Google Scholar, 15Shimizu S. Matsuoka Y. Shinohara Y. Yoneda Y. Tsujimoto Y. J. Cell Biol. 2001; 152: 237-250Crossref PubMed Scopus (332) Google Scholar, 16Vander Heiden M.G., Li, X.X. Gottleib E. Hill R.B. Thompson C.B. Colombini M. J. Biol. Chem. 2001; 276: 19414-19419Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Cyclophilins are a group of PPIases with highly conserved protein sequences (17Harding M.W. Pharmacotherapy. 1991; 11: 142S-148SPubMed Google Scholar). These proteins are thought to be important for protein folding (18Gothel S.F. Marahiel M.A. Cell. Mol. Life Sci. 1999; 55: 423-436Crossref PubMed Scopus (527) Google Scholar). CyPD is a mitochondrial-targeted PPIase (19Woodfield K.Y. Price N.T. Halestrap A.P. Biochim. Biophys. Acta. 1997; 1351: 27-30Crossref PubMed Scopus (66) Google Scholar). Although its specific physiological function remains largely unknown, CyPD is considered critical for the opening of the mPTP (20Nicolli A. Basso E. Petronilli V. Wenger R.M. Bernardi P. J. Biol. Chem. 1996; 271: 2185-2192Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 21Tanveer A. Virji S. Andreeva L. Totty N.F. Hsuan J.J. Ward J.M. Crompton M. Eur. J. Biochem. 1996; 238: 166-172Crossref PubMed Scopus (146) Google Scholar). This view is based on the observations that CsA, a potent inhibitor of mitochondrial-mediated apoptosis (9Lemasters J.J. Qian T. Bradham C.A. Brenner D.A. Cascio W.E. Trost L.C. Nishimura Y. Nieminen A.L. Herman B. J. Bioenerg. Biomembr. 1999; 31: 305-319Crossref PubMed Scopus (347) Google Scholar), blocks the mPTP at concentrations similar to those needed to inhibit the enzymatic activity of CyPD. This suggested that the PPIase activity was a necessary step in the opening of the mPTP (22Halestrap A.P. Davidson A.M. Biochem. J. 1990; 268: 153-160Crossref PubMed Scopus (672) Google Scholar, 23McGuinness O. Yafei N. Costi A. Crompton M. Eur. J. Biochem. 1990; 194: 671-679Crossref PubMed Scopus (93) Google Scholar). Cyclophilins bind CsA via a conserved hydrophobic pocket, which is critical for PPIase activity (24Kallen J. Spitzfaden C. Zurini M.G. Wider G. Widmer H. Wuthrich K. Walkinshaw M.D. Nature. 1991; 353: 276-279Crossref PubMed Scopus (242) Google Scholar, 25Ke H. Mayrose D. Cao W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3324-3328Crossref PubMed Scopus (84) Google Scholar, 26Theriault Y. Logan T.M. Meadows R., Yu, L. Olejniczak E.T. Holzman T.F. Simmer R.L. Fesik S.W. Nature. 1993; 361: 88-91Crossref PubMed Scopus (170) Google Scholar). Thus, early models of CsA inhibition of the permeability transition suggested that CsA acted as a pseudo-substrate of CyPD that prevented it from interacting with the mPTP. However, the addition of CsA was shown not to disrupt the binding of CyPD and ANT (27Crompton M. Virji S. Ward J.M. Eur. J. Biochem. 1998; 258: 729-735Crossref PubMed Scopus (407) Google Scholar). In addition, Scorranoet al. (28Scorrano L. Nicolli A. Basso E. Petronilli V. Bernardi P. Mol. Cell. Biochem. 1997; 174: 181-184Crossref PubMed Scopus (58) Google Scholar) reported that CsA inhibited mPTP opening, whereas diethylpyrocarbonate (DPC) induced mPTP opening. Because both CsA and DPC inhibit PPIase activity, the authors argued that CyPD must regulate the mPTP independently of this enzymatic activity. In this manuscript, we examine the role of CyPD in the regulation of cell death. Overexpression of CyPD was used to ascertain its physiological function in response to oxidative stress and staurosporin-induced cell death. Two-photon imaging was employed to reduce photo-bleaching of fluorophores and photo-toxicity in live cells, which was important for the prolonged imaging periods in our experiments. The collapse of ΔΨ and the loss of membrane integrity were monitored to assess the initiation of cell death (29Lemasters J.J. Nieminen A.L. Qian T. Trost L.C. Elmore S.P. Nishimura Y. Crowe R.A. Cascio W.E. Bradham C.A. Brenner D.A. Herman B. Biochim. Biophys. Acta. 1998; 1366: 177-196Crossref PubMed Scopus (1230) Google Scholar, 30Huser J. Blatter L.A. Biochem. J. 1999; 343: 311-317Crossref PubMed Scopus (233) Google Scholar). In contrast to the current view of CyPD acting as a key accessory protein that sensitizes the mPTP to opening, we demonstrate that overexpression of CyPD delays both the onset of ΔΨ collapse under oxidative stress and the loss of membrane integrity induced by staurosporin. The protective effect of CyPD was dependent on its PPIase activity as demonstrated by site-directed mutagenesis. Furthermore, under resting conditions, the ΔΨ was significantly higher in cells overexpressing CyPD. Taken together, these data suggest that CyPD is an important factor with functional consequences in the regulation of programmed cell death. The coding fragment of rat CyPD cDNA (gift of Dr. Halestrap, University of Bristol, UK) was amplified using a forward primer, 5′-cgggatccatgctagctctgcgctgcgg-3′, containing a BamHI site and a reverse primer, 5′-agtctctagaggctgtgacttagctcaactg-3′, containing an XbaI site. The PCR product was subcloned into theXenopus expression vector pGEM-HNb between theBamHI and XbaI sites (31John L.M. Mosquera-Caro M. Camacho P. Lechleiter J.D. J. Physiol. 2001; 535: 3-16Crossref PubMed Scopus (25) Google Scholar). The CyPD coding region was subsequently subcloned into pcDNA3.1/zeo(+) (Invitrogen, Carlsbad, CA) between the BamHI and XbaI sites. All restriction enzymes are from Life Technologies (Rockville, MD). CyPD R96G and H167Q mutants were generated by QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA) using plasmid pGEM-HNb-CyPD as template. The forward primer for the R96G mutant was 5′-ccttccacggggtcatcccagccttcatgtgcc-3′ and the reverse complement primer was 5′-ggcacatgaaggctgggatgaccccgtggaagg-3′. The forward primer for the H167Q was 5′-ggctggatggcaagcaagttgtgtttggccatg-3′ and the reverse complement primer was 5′-catggccaaacacaacttgcttgccatccagcc-3′. Both mutants were subsequently subcloned into pcDNA3.1/zeo(+) between the BamHI and XbaI sites. To generate the GST-CyPD fusion vector, the CyPD fragment was subcloned into pGEX-4T-2 (Amersham Biosciences, Piscataway, NJ) betweenBamHI and NotI sites from pGEM-HNb-CyPD vector. The CyPD-enhanced yellow fluorescent protein (EYFP) fusion fragment was engineered by a two-stage fusion PCR strategy. First, full-length coding regions of CyPD and EYFP were amplified in two separate reactions. The 3′ primer for CyPD had 21 bp of the 5′ of EYFP, and the 5′ primer for EYFP contained 20 bp of the 3′ of CyPD. These complementary sequences were annealed in the second PCR stage to generate the CyPD plus EYFP fusion fragment. The CyPD stop codon was removed from the PCR primers. A BamHI site was attached to the forward primer of CyPD, and an XbaI site was added to the reverse primer of EYFP. The fusion PCR fragment was then subcloned into pcDNA3.1/zeo(+) between BamHI and XbaI sites. The CyPD and EYFP bicistronic expression vector was generated by digesting pGEM-HNb-CyPD vector with SmaI and NotI. The CyPD fragment was subsequently subcloned into pIRES-EYFP (CLONTECH, Palo Alto, CA) betweenEcoRV and NotI sites. The rat ANT1 cDNA (gift of Dr. Y. Shinohara, University of Tokushima, Japan) was subcloned into the pGEM-HNb vector by PCR with a forward primer, 5′-actgcccgggatgggggatcaggctttgagc-3′, and a reverse primer, 5′-cggaattcttacacatattttttgatctcatcatac-3′. An SmaI site was attached to the 5′-end of the forward primer, and anEcoRI site was attached to the 5′-end of the reverse primer. The PCR fragment was subcloned into pGEM-HNb betweenSmaI and EcoRI sites. All vectors were automatically sequenced by the University of Texas Health Science Center at San Antonio sequencing core facility to demonstrate correct engineering. The oligonucleotides used were purchased from Operon Technologies (Alameda, CA). Cells were maintained at 37 °C in Dulbecco's modified Eagle's medium/F-12 (Invitrogen, Rockville, MD) supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 200 μg/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2/95% air. Cells were subcultured 1:10 by incubating in 0.05% Trypsin, 0.53 mm EDTA (Invitrogen, Rockville, MD) for 5 min when they were 70% confluent. DNA was transfected into cells by LipofectAMINE reagent (Invitrogen, Rockville, MD) according to the instructions of the manufacturer. After 72 h of transfection, HEK293 cells were trypsinized and transferred from a six-well dish to a 100-mm dish with medium containing 200 μg/ml Zeocin (Invitrogen, Carlsbad, CA). Surviving cells were grown for 2–3 weeks, and symmetrical colonies were marked under a microscope. A grease boundary was drawn around marked colonies. A solution of 5 μl of 0.05% Trypsin and 0.53 mm EDTA was briefly applied to the colony, and the cell suspension was transferred to 96-well plates. 24 colonies were picked for each construct to examine their protein expression level. Cells transiently expressing CyPD-EYFP were transferred to a 35-mm dish with a coverslip bottom 24 h after transfection and allowed to re-attach for another 24 h. Five minutes prior to imaging, the growth medium was replaced with recording buffer (120 mm NaCl, 4.5 mm KCl, 1 mm CaCl2, 2 mm MgCl2, 10 mm HEPES, pH 7.4) containing 50 nm tetramethyl rhodamine ethyl ester (TMRE, Molecular Probes, Eugene, OR) at room temperature. Fluorescent images were acquired using an Olympus Fluoview 300 confocal microscope with a 40× oil objective (numerical aperture (N.A.) = 1.35) at zoom 5. CyPD-EYFP was excited at 488 nm and fluorescence was detected between 510 and 550 nm. TMRE was excited at 568 nm, and fluorescence was detected beyond a 585-nm long pass barrier filter. A Focht Chamber System 2 (FCS2, Bioptechs Inc., Butler, PA) was used for time-lapse two-photon imaging. Cells were transferred to the FCS2 coverslips 24 h prior to each experiment and maintained in the cell culture incubator. The chamber system was subsequently assembled and mounted on an Olympus Fluoview 300 confocal microscope customized for two-photon excitation and external photo-multiplier detection. Chamber temperature was maintained at 37 °C during imaging, and cells were constantly perfused with phenol red-free Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 25 mm HEPES, pH 7.4, 2 mm l-glutamine, 200 μg/ml penicillin, 100 μg/ml streptomycin, 50 nmTMRE, and 100 μmtert-butyl hydroperoxide (t-BuOOH) at 15 ml/h. TMRE was excited at 820 nm using a Ti-sapphire Coherent Mira 900 laser pumped with a 5-watt Verdi laser (Coherent Inc., Santa Clara, CA). Laser intensity was attenuated with a neutral-density filter wheel such that no significant photo-bleaching of TMRE was observed during a 24-h recording period. Signal was collected through an Olympus 60× oil objective (N.A. = 1.4) at zoom 1. For CyPD and EYFP bicistronic expression, the FCS2 chamber was mounted on a Zeiss 510 confocal microscope adapted for two-photon imaging. EYFP was excited at 488 nm, and emission was collected through a 505- to 550-nm band pass barrier filter. PI was excited at 543 nm and emission was collected through a 560-nm long pass filter. TMRE was excited at 820 nm, and emission was collected through a 565- to 615-nm barrier filter. Confocal and two-photon imaging was performed in two tracks alternating in the line-by-line mode. Differential interference contrast (DIC) images were collected simultaneously with either the PI or TMRE track. Signal was collected through a Zeiss 63× oil objective (N.A. = 1.4) at zoom 1. Once an EYFP-positive cell was identified, 488-nm excitation was turned off, and only the TMRE/PI and DIC signals were acquired in the single-track setting. Image analysis was performed using the public domain program ImageJ (National Institutes of Health, available at rsb.info.nih.gov/ij/) and the software ANALYZE (Mayo Foundation, Rochester, MN). PPIase activity was estimated according to Kofron et al. (32Kofron J.L. Kuzmic P. Kishore V. Colon-Bonilla E. Rich D.H. Biochemistry. 1991; 30: 6127-6134Crossref PubMed Scopus (486) Google Scholar). Briefly, purified GST-CyPD or GST (10 nm final concentration) was pre-equilibrated with buffer (50 mm HEPES, 100 mm NaCl, pH 8.0). Immediately before assaying activity, chymotrypsin (60 mg/ml in 1 mm HCl, final concentration 6 mg/ml, Calbiochem, La Jolla, CA) was added. The PPIase substrate I (Calbiochem) was dissolved in the solvent trifluoroethanol with LiCl (470 mm) to a 3 mm stock concentration. The PPIase substrate I was added to the reaction to give a final concentration of 75 nm. Absorption at 380 nm was measured in a BioSpec-1601 Spectrophotometer (Shimadzu, Columbia, MD). Reactions with no PPIase were used as background controls. Western blot procedures were essentially performed as previously described (33John L.M. Lechleiter J.D. Camacho P. J. Cell Biol. 1998; 142: 963-973Crossref PubMed Scopus (181) Google Scholar). Briefly, cells were harvested by trypsinization and washed three times with phosphate-buffered saline. Subsequently, cells were solubilized in 20 mm Tris-HCl, pH 7.6, 140 mm NaCl, 1 mm EDTA, 0.5% Triton X-100, 1 μm pepstatin A, 20 μm leupeptin, 0.2 mm4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride at 4 °C for 30 min. The cell lysate was centrifuged at 900 × gfor 5 min to separate the nuclei. The concentration of total protein was determined by the BCA assay kit (Pierce, Rockford, IL). For SDS-PAGE, 10 μg of total protein was loaded per lane. CyPD was detected with polyclonal rabbit anti-CyPD antibody (Pocono Rabbit Farm & Laboratory, Inc., Canadensis, PA). Horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was used and visualized by chemiluminescence (PerkinElmer Life Sciences, Boston, MA). Goat polyclonal anti-actin antibody sc-1615 and donkey anti-goat IgG sc-2304 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used to show the equal loading of proteins. BL21(DE3) bacteria transformed with pGEX-4T-2-CyPD construct were grown until a 0.6 OD at 600 nm was reached. Isopropylthio-β-d-galactoside (Invitrogen, Rockville, MD) was added to a final concentration of 1 mm to induce GST-CyPD expression for 4 h at 37 °C. Bacteria were harvested and lysed by sonication in 1× phosphate-buffered saline containing 1 mmphenylmethylsulfonyl fluoride, 100 mm EDTA. Bacterial lysate was centrifuged at 22,000 × g, and the supernatant was collected. Binding of GST or GST fusion protein to glutathione-Sepharose 4B (Amersham Biosciences) was performed at 4 °C for 1 h followed by three washes with equilibration buffer (0.5 m Tris-HCl, pH 8.0, 4 mm EDTA, 0.1% 2-mercaptoethanol, 5% glycerol, 1 mm phenylmethylsulfonyl fluoride). Elution of bound protein was performed in equilibration buffer with 15 mm glutathione. Preparation of synthetic mRNAs and in vitro translations were performed as previously described (34Camacho P. Lechleiter J.D. Cell. 1995; 82: 765-771Abstract Full Text PDF PubMed Scopus (200) Google Scholar,35Roderick H.L. Lechleiter J.D. Camacho P. J. Cell Biol. 2000; 149: 1235-1248Crossref PubMed Scopus (201) Google Scholar). Briefly, mRNAs were diluted to 0.1 μg/μl and translated for 45 min in a rabbit reticulocyte lysate translation system (Promega, Madison, WI) supplemented withl-[35S]methionine (PerkinElmer Life Sciences, Boston, MA). Binding of in vitro translated ANT1 to GST-CyPD fusion proteins was performed in phosphate-buffered saline, pH 7.2, at room temperature for 1 h, followed by three washes in equilibration buffer containing 0.5 m NaCl and 1% Triton X-100. Proteins bound to glutathione-Sepharose were resolved by SDS-PAGE followed by autoradiography. CyPD is thought to be an accessory protein that controls the opening of the mPTP in response to apoptosis-inducing factors (36Halestrap A.P. Doran E. Gillespie J.P. O'Toole A. Biochem. Soc. Trans. 2000; 28: 170-177Crossref PubMed Scopus (289) Google Scholar). Because CsA is known to bind CyPD and, thereby, reduce the probability of mPTP opening, we hypothesized that overexpression of CyPD would increase sensitivity of cells to oxidative stress. Our approach to test this hypothesis was 2-fold. First, we tested whether cells overexpressing CyPD exhibited the correct targeting of this protein to the mitochondria. For this purpose, we created a fusion protein consisting of CyPD tagged with EYFP at its COOH-terminal and then transiently transfected this construct into HEK293 cells. A cell expressing CyPD-EYFP is presented in Fig.1A. When cells expressing this fusion protein were also labeled with the mitochondrial marker TMRE (Molecular Probes, Eugene, OR), we found that CyPD-EYFP was correctly targeted to the mitochondria (Fig. 1A). The second step in our experimental design was to create stable cell lines overexpressing wild type CyPD (non-tagged). A Western blot analysis of CyPD expression in one of these cells lines is presented in Fig. 1B. The physiological effects of CyPD overexpression were tested in this stable cell line and compared with a control cell line, expressing vector alone. Oxidative stress was induced by exposure to t-BuOOH and measured by monitoring the ΔΨ (37Nieminen A.L. Byrne A.M. Herman B. Lemasters J.J. Am. J. Physiol. 1997; 272: C1286-C1294Crossref PubMed Google Scholar). Briefly, cells were seeded on glass coverslips and allowed to adhere for 24 h. Plated cells were initially labeled with TMRE (50 nm) in a culture incubator for 5 min. The glass coverslip was then sealed in a closed perfusion chamber, and the cells were imaged on a two-photon confocal microscope for 12 h. Two-photon imaging was used to simultaneously excite the membrane potential indicator TMRE and to produce a DIC image. An overlay of both the DIC image and the TMRE-labeled mitochondria is presented in Fig. 1C. Throughout the course of the experiment, a complete z section (six images in 1-μm steps) of the cells was taken every 5 min. The cells were then monitored for ΔΨ collapse, which was measured by the loss of TMRE fluorescence. Simultaneous imaging with DIC optics insured that a loss of fluorescence was not due to a change in the focal plane or to the loss of the cell. Spatial-temporal stacks were created with only a single image from each z section (Fig. 1D). The chosen image from each z section was selected on the basis of brightest TMRE fluorescence, which generally coincided with the middle image. These data reveal that cells overexpressing CyPD maintain their mitochondrial membrane potentials in the presence of t-BuOOH for significantly longer periods of time compared with cells expressing vector alone (p < 0.0005, t test). A histogram of the average time of ΔΨ collapse is presented in Fig. 1E. The average time until the ΔΨ collapsed for CyPD-overexpressing cells was 9.9 ± 0.3 h (n = 69 cells, pooled from four separate experiments). The average time until ΔΨ collapsed was 6.2 ± 0.1 h in control cells expressing vector alone (n = 85 cells, pooled from five separate experiments). We conclude from these data that overexpression of CyPD significantly prolongs the time until ΔΨ collapses. Thus, rather than sensitizing cells to oxidative stress, CyPD appears to significantly protect the cells. CsA is a well-established inhibitor of mPTP opening (38Bernardi P. Biochim. Biophys. Acta. 1996; 1275: 5-9Crossref PubMed Scopus (378) Google Scholar). Because the action of CsA on mPTP is thought to be mediated by CsA binding to CyPD, we examined how CsA exposure affected the collapse of ΔΨ in response to oxidative stress. HEK293 cells were plated on glass coverslips, labeled with TMRE, sealed in the perfusion chamber, and imaged as described above. In addition, cells were pre-treated with CsA (10 μm) for 30 min and then continuously perfused with Dulbecco's modified Eagle's medium containing CsA (10 μm), TMRE (50 nm), and t-BuOOH (100 μm). Under these experimental conditions, we found that the average time for ΔΨ to collapse in CsA treated cells was 20.5 ± 0.1 h (n = 52 cells, pooled from two experiments) (Fig. 2). In comparison, the average time to ΔΨ collapse in control cells (vehicle only) exposed to t-BuOOH was 6.2 ± 0.1 (n = 54, pooled from two experiments). Because it is known that CsA also has non-mitochondrial targets that can affect cell viability (39Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3634) Google Scholar), we also tested how the immunosuppressant FK506 affected the collapse of ΔΨ in response to oxidative stress. FK506 is an immunosuppressant that, like CsA, prevents activation of calcineurin. However, FK506 has no effect on mitochondria (40Kay J.E. Biochem. J. 1996; 314: 361-385Crossref PubMed Scopus (149) Google Scholar). Pre-treatment of cells with FK506 (1 μm, 30 min) had no protective effect on the collapse of ΔΨ in cells exposed to t-BuOOH (Fig. 2). The average time to ΔΨ collapse was 6.0 ± 0.1 h (n = 58 cells, pooled from two experiments). We conclude from these data that CsA inhibits the collapse of ΔΨ when cells are exposed to oxidative stress. However, the effects of CsA are unlikely to be mediated by calcineurin inhibition, because treatment of cells with FK506 did not affect the collapse of ΔΨ. To determine whether the protective effect of CyPD on ΔΨ collapse was dependent on the cell-type, we re-examined the effects of CyPD overexpression in another cell line. Rat C6 glioma cells were transiently transfected with a bicistronic vector for independent expression of CyPD and EYFP (see “Materials and Methods”). Cells were allowed to express the proteins for 48 h, and positively transfected cells were identified by EYFP fluorescence (Fig. 3A, left panels). Plated cells were then labeled with TMRE, treated with t-BuOOH, and imaged as described above. Consistent with our findings in the HEK293 cells, we found that C6 glioma cells overexpressing CyPD maintained their ΔΨ for significantly longer periods than non-transfected cells when exposed to t-BuOOH (Fig. 3A, upper panels). C6 glioma cells transfected with the bicistronic vector containing EYFP alone exhibited no significant difference from control, non-transfected cells (Fig. 3A, lower panels). The average time until the ΔΨ collapsed in the presence of t-BuOOH was 2.2 ± 0.1 h in control, non-transfected cells (n = 10 experiments); 3.5 ± 0.2 h in CyPD and EYFP expressing cells (n = 5 experiments) and 2.2 ± 0.1 h for EYFP alone expressing cells (n = 5 experiments) (Fig. 3B). We conclude from these data that, under oxidative stress, overexpression of CyPD prolongs the time until ΔΨ collapses in both HEK293 and rat C6 glioma cells, suggesting that it might be a generalized mechanism operating in many cell types. To determine whether CyPD protects cells against other apoptotic stimuli, the effects of CyPD overexpression were examined in staurosporin-treated cells. To induce cell death, rat C6 glioma cells were treated with 1 μm staurosporin for 6 h. Apoptosis was confirmed by staining the cells with Annexin V-FITC (Molecular Probes), which detects phosphatidylserine in the outer leaflet of the plasma membrane. Phosphatidylserine flips to the outer leaflet of the plasma membrane preceding the loss of membrane integrity and is an early indication of apoptosis (41van Engeland M. Nieland L.J. Ramaekers F.C. Schutte B. Reutelingsperger C.P. Cytometry. 1998; 31: 1-9Crossref PubMed Scopus (1553) Google Scholar). Co-staining with propidium iodide (PI, Molecular Probes) identified those cells with compromised plasma membrane integrity and that had progressed through apoptosis (Fig. 4A). These data demonstrate that staurosporin induces apoptosis in these cells. To test whether CyPD confers protection under these conditions, rat C6 glioma cells were transiently transfected with the bicistronic vector for independent expression of CyPD and EYFP. Plated cells were treated with staurosporin in the presence of PI and imaged as described above following 48 h of expression. Positively transf
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