Overexpression of Mitochondrial Methionine Sulfoxide Reductase B2 Protects Leukemia Cells from Oxidative Stress-induced Cell Death and Protein Damage
2008; Elsevier BV; Volume: 283; Issue: 24 Linguagem: Inglês
10.1074/jbc.m708580200
ISSN1083-351X
AutoresFilipe Cabreiro, Cédric R. Picot, Martine Perichon, Julien Castel, Bertrand Friguet, Isabelle Petropoulos,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoAccording to the mitochondrial theory of aging, mitochondrial dysfunction increases intracellular reactive oxidative species production, leading to the oxidation of macromolecules and ultimately to cell death. In this study, we investigated the role of the mitochondrial methionine sulfoxide reductase B2 in the protection against oxidative stress. We report, for the first time, that overexpression of methionine sulfoxide reductase B2 in mitochondria of acute T-lymphoblastic leukemia MOLT-4 cell line, in which methionine sulfoxide reductase A is missing, markedly protects against hydrogen peroxide-induced oxidative stress by scavenging reactive oxygen species. The addition of hydrogen peroxide provoked a time-gradual increase of intracellular reactive oxygen species, leading to a loss in mitochondrial membrane potential and to protein carbonyl accumulation, whereas in methionine sulfoxide reductase B2-overexpressing cells, intracellular reactive oxygen species and protein oxidation remained low with the mitochondrial membrane potential highly maintained. Moreover, in these cells, delayed apoptosis was shown by a decrease in the cleavage of the apoptotic marker poly(ADP-ribose) polymerase-1 and by the lower percentage of Annexin-V-positive cells in the late and early apoptotic stages. We also provide evidence for the protective mechanism of methionine sulfoxide reductase B2 against protein oxidative damages. Our results emphasize that upon oxidative stress, the overexpression of methionine sulfoxide reductase B2 leads to the preservation of mitochondrial integrity by decreasing the intracellular reactive oxygen species build-up through its scavenging role, hence contributing to cell survival and protein maintenance. According to the mitochondrial theory of aging, mitochondrial dysfunction increases intracellular reactive oxidative species production, leading to the oxidation of macromolecules and ultimately to cell death. In this study, we investigated the role of the mitochondrial methionine sulfoxide reductase B2 in the protection against oxidative stress. We report, for the first time, that overexpression of methionine sulfoxide reductase B2 in mitochondria of acute T-lymphoblastic leukemia MOLT-4 cell line, in which methionine sulfoxide reductase A is missing, markedly protects against hydrogen peroxide-induced oxidative stress by scavenging reactive oxygen species. The addition of hydrogen peroxide provoked a time-gradual increase of intracellular reactive oxygen species, leading to a loss in mitochondrial membrane potential and to protein carbonyl accumulation, whereas in methionine sulfoxide reductase B2-overexpressing cells, intracellular reactive oxygen species and protein oxidation remained low with the mitochondrial membrane potential highly maintained. Moreover, in these cells, delayed apoptosis was shown by a decrease in the cleavage of the apoptotic marker poly(ADP-ribose) polymerase-1 and by the lower percentage of Annexin-V-positive cells in the late and early apoptotic stages. We also provide evidence for the protective mechanism of methionine sulfoxide reductase B2 against protein oxidative damages. Our results emphasize that upon oxidative stress, the overexpression of methionine sulfoxide reductase B2 leads to the preservation of mitochondrial integrity by decreasing the intracellular reactive oxygen species build-up through its scavenging role, hence contributing to cell survival and protein maintenance. Oxidized protein repair plays a major role in protein maintenance during aging or in certain conditions of oxidative stress (1Berlett B.S. Stadtman E.R. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2763) Google Scholar, 2Stadtman E.R. Free Radic. Res. 2006; 40: 1250-1258Crossref PubMed Scopus (660) Google Scholar). An important repair system is the Msr (methionine sulfoxide reductase) system, composed of MsrA and MsrB, which can catalyze the reversion of the methionine S-sulfoxide and the methionine R-sulfoxide, respectively, to the reduced form of methionine within proteins (3Grimaud R. Ezraty B. Mitchell J.K. Lafitte D. Briand C. Derrick P.J. Barras F. J. Biol. Chem. 2001; 276: 48915-48920Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 4Jung S. Hansel A. Kasperczyk H. Hoshi T. Heinemann S.H. FEBS Lett. 2002; 527: 91-94Crossref PubMed Scopus (65) Google Scholar, 5Kuschel L. Hansel A. Schonherr R. Weissbach H. Brot N. Hoshi T. Heinemann S.H. FEBS Lett. 1999; 456: 17-21Crossref PubMed Scopus (93) Google Scholar, 6Lescure A. Gautheret D. Carbon P. Krol A. J. Biol. Chem. 1999; 274: 38147-38154Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 7Weissbach H. Etienne F. Hoshi T. Heinemann S.H. Lowther W.T. Matthews B. St. John G. Nathan C. Brot N. Arch. Biochem. Biophys. 2002; 397: 172-178Crossref PubMed Scopus (257) Google Scholar). Methionine can be easily oxidized into methionine sulfoxide (8Davies M.J. Biochim. Biophys. Acta. 2005; 1703: 93-109Crossref PubMed Scopus (1074) Google Scholar), and its reduction by Msr could represent an efficient antioxidant system, since in proteins, the surface-exposed methionine residues can act as scavengers of a variety of oxidants (9Levine R.L. Berlett B.S. Moskovitz J. Mosoni L. Stadtman E.R. Mech. Ageing Dev. 1999; 107: 323-332Crossref PubMed Scopus (291) Google Scholar, 10Levine R.L. Mosoni L. Berlett B.S. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15036-15040Crossref PubMed Scopus (870) Google Scholar). An age-related decrease in Msr level and activity was previously shown in rat organs (11Petropoulos I. Mary J. Perichon M. Friguet B. Biochem. J. 2001; 355: 819-825Crossref PubMed Scopus (112) Google Scholar) and in human fibroblasts submitted to replicative senescence (12Picot C.R. Perichon M. Cintrat J.C. Friguet B. Petropoulos I. FEBS Lett. 2004; 558: 74-78Crossref PubMed Scopus (65) Google Scholar). As a consequence, this decline in Msr activity may contribute to the age-associated accumulation of oxidized proteins (13Petropoulos I. Friguet B. Free Radic. Res. 2006; 40: 1269-1276Crossref PubMed Scopus (64) Google Scholar).Although the role of MsrA in cellular protection against oxidative stress is now well documented in bacteria, plants, yeasts, flies, and mammals (14Cabreiro F. Picot C.R. Friguet B. Petropoulos I. Ann. N. Y. Acad. Sci. 2006; 1067: 37-44Crossref PubMed Scopus (90) Google Scholar, 15Moskovitz J. Biochim. Biophys. Acta. 2005; 1703: 213-219Crossref PubMed Scopus (250) Google Scholar), little is known about the role of MsrB in this process as well as its importance in aging and longevity. In mammals, overexpression of MsrA in human T-lymphocytes (16Moskovitz J. Berlett B.S. Poston J.M. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9585-9589Crossref PubMed Scopus (296) Google Scholar), PC-12 (17Yermolaieva O. Xu R. Schinstock C. Brot N. Weissbach H. Heinemann S.H. Hoshi T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1159-1164Crossref PubMed Scopus (136) Google Scholar), lens (18Kantorow M. Hawse J.R. Cowell T.L. Benhamed S. Pizarro G.O. Reddy V.N. Hejtmancik J.F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9654-9659Crossref PubMed Scopus (153) Google Scholar), and WI-38 SV40 fibroblast cells (19Picot C.R. Petropoulos I. Perichon M. Moreau M. Nizard C. Friguet B. Free Radic. Biol. Med. 2005; 39: 1332-1341Crossref PubMed Scopus (63) Google Scholar) protects them against oxidative stress. By contrast, msra null mice exhibit increased sensitivity to oxidative stress and a shortened life span (20Moskovitz J. Bar-Noy S. Williams W.M. Requena J. Berlett B.S. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12920-12925Crossref PubMed Scopus (544) Google Scholar). There are three different mammalian MsrBs, localized in distinct cellular compartments, MsrB1 (a selenoprotein also called SelX in humans) in the cytosol and the nucleus, MsrB2 (previously named hCBS-1 in humans) in the mitochondria, and MsrB3 in the mitochondria and the endoplasmic reticulum (21Kim H.Y. Gladyshev V.N. Mol. Biol. Cell. 2004; 15: 1055-1064Crossref PubMed Scopus (250) Google Scholar). The short interfering RNA-mediated gene silencing of each of the three MsrBs in human lens cells resulted in increased oxidative stress-induced cell death (22Marchetti M.A. Pizarro G.O. Sagher D. Deamicis C. Brot N. Hejtmancik J.F. Weissbach H. Kantorow M. Invest. Ophthalmol. Vis. Sci. 2005; 46: 2107-2112Crossref PubMed Scopus (70) Google Scholar). Since the MsrB2 protein is localized in the mitochondria, it would be a key protein for the maintenance of mitochondrial functions due to its antioxidant properties through protein-exposed methionine residue scavenging of ROS. 5The abbreviations and trivial names used are: ROS, reactive oxygen species; XTT, sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)benzene sulfonic acid hydrate; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; PI, propidium iodide; DHE, dihydroethidium; CT-L, chymotrypsin-like; LLVY-AMC, succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin; MG132, N-benzyloxycarbonyl-Leu-Leu-leucinal; siRNA, small interfering RNA; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; CT-L, chymotrypsin-like. 5The abbreviations and trivial names used are: ROS, reactive oxygen species; XTT, sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)benzene sulfonic acid hydrate; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; PI, propidium iodide; DHE, dihydroethidium; CT-L, chymotrypsin-like; LLVY-AMC, succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin; MG132, N-benzyloxycarbonyl-Leu-Leu-leucinal; siRNA, small interfering RNA; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; CT-L, chymotrypsin-like. The mitochondrion is, according to the mitochondrial theory of aging, responsible for the generation of cellular ROS (23Harman D. J. Gerontol. 1956; 11: 298-300Crossref PubMed Scopus (6396) Google Scholar, 24Harman D. J. Am. Geriatr. Soc. 1972; 20: 145-147Crossref PubMed Scopus (1500) Google Scholar). The steady-state level generation of ROS, resulting from the electron leaking from the respiratory chain, is properly counterbalanced by the presence of critical scavenging enzymes, providing minimal damage to the mitochondria. However, according to the "vicious cycle" concept, any perturbation to the oxidative phosphorylation process would increase the production of ROS and breakdown of membrane potential, leading to further protein, lipid, and nucleic acid mitochondrial damage (24Harman D. J. Am. Geriatr. Soc. 1972; 20: 145-147Crossref PubMed Scopus (1500) Google Scholar). Thus, it was of interest to study the role of MsrB2 in cellular protection against oxidative stress. To address this question, we have overexpressed and silenced MsrB2 in human T-lymphocytic leukemia MOLT-4 cells, a cell line that does not express MsrA (5Kuschel L. Hansel A. Schonherr R. Weissbach H. Brot N. Hoshi T. Heinemann S.H. FEBS Lett. 1999; 456: 17-21Crossref PubMed Scopus (93) Google Scholar). In this study, we have investigated the role of MsrB2 in resistance to oxidative stress-induced cell death and to oxidant-mediated mitochondrial and protein damage. Although no increased susceptibility to cell death could be evidenced in silencing MsrB2 cells, we have shown that the overexpression of MsrB2 in MOLT-4 cells leads to a protection against H2O2-mediated cell death and mitochondrial damage. Moreover, we have demonstrated that, when overexpressed, MsrB2 can maintain a low level of intracellular ROS, can prevent oxidized protein accumulation and can partly protect the proteasome against oxidative stress-induced inactivation.EXPERIMENTAL PROCEDURESTransfection of MOLT-4 Cell Line by Human MsrB2 (hCbs-1) cDNA—The human MOLT-4 cell line (ATCC; CRL 1582) (LGC Promochem, Molsheim, France) was stably transfected with the pLXSN retroviral expression vector (BD Biosciences) based on the Moloney murine leukemia virus and Moloney murine sarcoma virus to generate a replication-deficient recombinant retrovirus containing the human MsrB2 cDNA. The oligonucleotide primers flanking the open reading frame (5′-CGGAATTCATGGCGCGGCTCCTCTGGTT-3′) and (5′-GCAGATCTTCAGTGTTTCCTTGGTTTGAACTTCAAAGC-3′) were used in a PCR, and the amplified DNA fragment was cloned into the EcoRI-BamHI site of the pLXSN vector. The recombinant pLXSN vector was transfected using the liposome transfection reagent FuGene (Roche Applied Science) into RetroPack PT67 packaging cells cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum (Invitrogen), 100 units/ml penicillin, and 0.1 mg/ml streptomycin. To select stable transfectants, cells were grown in complete medium supplemented with 200 μg/ml Geneticin G418 antibiotics (Invitrogen). The medium from the positive transfected cells was collected, filtered through a 0.45-μm filter, and diluted 2-fold with fresh medium. The high titer retroviral enriched medium was subsequently used to transduce the MOLT-4 cell line, cultivated in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) fetal calf serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, 100 units/ml penicillin, 100 μg/ml streptomycin at 37 °C, 5% CO2, and 95% humidity. Positive and stably transfected MOLT-4 cells were selected in RPMI 1640 complete medium with 200 μg/ml Geneticin G418 and tested for functional protein expression levels by immunoblot and Msr activity analysis.MsrB2 siRNA-targeted Gene Silencing—One micromolar siGENOME ON-TARGETplus MsrB2-directed synthetic siRNA (5′-GGUCAGAGGUUUUGCAUCAUU-3′ and 5′-PUGAUGCAAACCUCUGACCUU-3′) and nonsilencing control siRNA (5′-UCAUCUAGGUCACGUGUUUUU-3′ and 5′-PAAACACGUGACCUAGAUGAUU-3′) obtained from Dharmacon RNA Technologies (Lafayette, CO) were nucleofected into 2 × 106 MOLT-4 cells using the AMAXA basic nucleofection protocol for suspension cell lines (AMAXA AG, Köln, Germany).Cell Culture and Oxidative Stress—Stably transfected MOLT-4 cell lines with either pLXSN/MsrB2 or pLXSN empty vector were propagated in 75-cm3 plastic flasks (Greiner, VWR, Fontenay-sous-Bois, France) at 37 °C, 5% CO2 and 95% humidity. siRNA-targeted cell lines were cultivated in the same conditions in 6-well plates for 48 h after nucleofection, for an effective suppression of MsrB2. For oxidative stress conditions, cells in exponential growth were collected by centrifugation for 5 min at 200 × g and seeded at a density of 1.3 × 106 cells/ml in fresh medium supplemented with 2% fetal calf serum in 6- or 24-well plates. Control and MsrB2-overexpressing cells were submitted to various concentrations of H2O2 (0, 75, 150, and 225 μm) for 4 h for PARP-1 (poly(ADP-ribose) polymerase-1) protein expression levels, 12 h for MsrB2 protein expression levels and 24 h for the rest of the experiments. For siRNA experiments, cells were submitted to the following concentrations of H2O2: 0, 75, and 150 μm. MG132 (Sigma) was used at a 90 nm final concentration for cell treatment for 24 h. Experiments using fluorescent probes were performed with RPMI 1640 medium without phenol red.XTT Viability Assay—Cytotoxicity was determined by a colorimetric assay based on the cleavage of the yellow tetrazolium salt XTT (Roche Applied Sciences) according to the manufacturer's protocol. Briefly, cells were harvested from the exponential phase maintenance cultures and dispersed in 96-well plates at a 1.4 × 105 cells/well density. Formation of an orange formazan dye by metabolic active cells was measured using a microplate reader at 450 nm (Fluostar Galaxy, bMG, Stuttgart, Germany). The relative number of viable cells as compared with the nontreated cells was expressed as the percentage of proliferative cells.Analysis of MsrB Transcripts in MOLT-4 Cells—The relative levels of MsrB1 and MsrB2 were estimated in the siRNA control and siRNA MsrB2 cell lines by semiquantitative real time PCR. 2 μg of total RNA was reverse transcribed using SuperScript™ III (Invitrogen) and the cDNA was amplified by real time PCR using primers for MsrB1 (forward, 5′-GCGAGGTTTTCCAGAATCAC-3′; reverse, 5′-GGACACCTTCAAGGCTTCAG-3′) and for MsrB2 (forward, 5′-GCGACAGTCCACTCTTCAGTT-3′; reverse, 5′-CCCAGGTCCATCAGGAAACA-3′). The MsrB2 transcript was amplified for 29 and 32 PCR cycles with an annealing temperature of 65 °C for siRNA control and siRNA MsrB2 cells, respectively. MsrB1 transcripts were amplified for 30 PCR cycles with annealing temperatures of 60 °C. The reactions were performed using LightCycler® FastStart DNA MasterPlus SYBR Green I (Roche Applied Sciences) with an efficiency over 96%. The PCR values were normalized to those produced with primers for the S26 gene.ROS Measurements—After removal of 24-h H2O2 treatment medium, cells were incubated with 10 μm dihydroethidium (DHE) (Molecular Probes) for 45 min at 37 °C, 5% CO2. For ROS kinetics measurements, control cells were loaded with DHE after harvesting at different H2O2 treatment times. Analysis of 1.5 × 104 individual cells was performed on a FACStarPlus flow cytometer with a 488-nm excitation and a 610-nm emission filter for DHE.Evaluation of Apoptosis and Necrosis by Flow Cytometry— Cells undergoing apoptosis or necrosis in control and MsrB2-overexpressing cells after 24 h of H2O2 treatment were determined by FACS using an Annexin-V-FITC apoptosis detection kit (Calbiochem) according to the manufacturer's protocol. After this time period, cells were harvested, and ∼5 × 105 cells were stained with Annexin-V-FITC during a 15-min incubation period at room temperature. Cells were centrifuged at 1000 × g for 5 min at room temperature. Cells were washed once and then stained with propidium iodide (PI). Analysis of 1.5 × 104 individual cells was performed on a FACStarPlus flow cytometer (BD Biosciences) with a 488-nm excitation and a 530-nm filter for Annexin-V-FITC and a 610-nm filter for PI.ΔΨm Measurements—Mitochondrial membrane potential was assayed with the mitoProbe™ JC-1 assay kit for flow cytometry (Molecular Probes). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) shows a potential-dependent accumulation in mitochondria, indicated by a fluorescence shift from green to red. The measurements were performed according to the manufacturer's protocol. Briefly, after treatment, cells were gently resuspended in 1 ml of prewarmed phosphate-buffered saline containing 2 μm final concentration JC-1 probe and incubated at 37 °C, 5% CO2 for 20 min. As a positive control, nontreated control cells were incubated for 5 min, previous to the addition of JC-1, with 50 μm uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Analysis of 1.5 × 104 individual cells was performed on a FACStarPlus flow cytometer with a 488-nm excitation and 530- and 610-nm emission filters for green and red fluorescence, respectively. Fluorescence imaging was performed in parallel to the FACS analysis. One aliquot of each sample was analyzed on a Nikon Eclipse TE2000-U microscopy (Nikon S.A.S, Champigny-sur-Marne, France) with excitation/emission wavelengths of 480 ± 15/535 ± 20 nm for green fluorescence and 535 ± 25/610 ± 30 nm for red fluorescence.Subcellular Fractioning, Immunoblot, and Oxyblot Analysis— Subcellular fractioning was achieved using the mitochondria isolation kit for mammalian cells (Perbio Sciences, Brebières, France) and performed according to the manufacturer's protocol. Alternatively, cellular homogenates were obtained using the CelLytic™ M mammalian cell lysis/extraction reagent (Sigma) at 4 °C. The lysed cells were centrifuged for 15 min at 15,000 × g to pellet the cellular debris, and the protein concentration of the supernatant was determined by the Bradford method (Bio-Rad). Total proteins were separated on SDS-PAGE and then electrotransferred onto a Hybond nitrocellulose membrane (GE Healthcare Europe GmbH). Western blotting experiments were performed with anti-actin monoclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:500 dilution, anti-MsrB2 at a 1:2500 dilution, anti-PARP-1 (Santa Cruz Biotechnology) at a 1:1000 dilution, anti-aconitase at a 1:5000 dilution, anti-20 S proteasome at a 1:2000 dilution, and anti-β5 proteasome subunit (Biomol International, L.P., Exeter, UK) at a 1:1000 dilution. Detection of carbonyl groups was performed with the OxyBlot oxidized protein detection kit (Chemicon International, Ltd., Hampshire, UK) according to the manufacturer's protocol. 2.5 μg of total extract proteins were incubated for 15 min at room temperature with 2,4-dinitrophenylhydrazine to form the carbonyl derivative dinitrophenylhydrazone before SDS-PAGE separation. After transfer onto nitrocellulose, modified proteins were revealed by antidinitrophenol antibodies. Blots were developed with chemiluminescence using the SuperSignal West Pico chemiluminescent substrate (Perbio Sciences). Films were scanned, and the amount of signal was quantified by densitometric analysis using ImageMaster 1D software (GE Healthcare Europe).Determination of Msr and Proteasome Activities—Total Msr enzymatic activity was measured in cellular homogenates using N-acetyl-[3H]methionine R,S-sulfoxide substrate (GE Healthcare Europe), as previously described (25Brot N. Werth J. Koster D. Weissbach H. Anal. Biochem. 1982; 122: 291-294Crossref PubMed Scopus (72) Google Scholar). Peptidase activities of the proteasome were assayed using fluorogenic peptide, succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (LLVY-AMC) (Sigma) for the chymotrypsin-like (CT-L) activity, as described previously (26Bulteau A.L. Moreau M. Nizard C. Friguet B. Free Radic. Biol. Med. 2002; 32: 1157-1170Crossref PubMed Scopus (74) Google Scholar) with minor modifications. The mixture, containing 20 μg of crude protein cellular extracts in proteasome buffer (25 mm Tris-HCl, pH 7.5), was incubated at 37 °C with the substrate LLVY-AMC at 25 μm in a 200-μl final volume. Enzymatic kinetics were conducted in a temperature-controlled microplate fluorimetric reader (Fluostar Galaxy, bMG, Stuttgart, Germany). Excitation/emission wavelengths were 350/440 nm for aminomethylcoumarin. Proteasome activities were determined as the difference between total activity and the remaining activity of the crude extract in the presence of 20 μm proteasome inhibitor MG132.Statistical Analysis—All results are expressed as the means ± S.E. Comparisons between control, MsrB2-overexpressing, and silencing cells under the different H2O2 concentration treatments were analyzed with Student's t test and were assumed to be statistically significant if p was ≤0.05.RESULTSOverexpression of MsrB2 in Mitochondria Protects MOLT-4 Cells against H2O2-mediated Death—To assess the role of the mitochondrial Msr in protection against oxidative stress, MsrB2 was stably transfected in MOLT-4 cells by using the retroviral method described under "Experimental Procedures." Interestingly, the MOLT-4 cell line does not contain MsrA RNA and protein at detectable levels. Moreover, real time quantitative PCR experiments did not show the presence of MsrB3 in MOLT-4 cells (data not shown), indicating that the Msr activity detected in the mitochondrial and cytosolic fractions was most likely due to MsrB2 and MsrB1, respectively. The level of Msr activity was found to be similar in cytosol and mitochondrial fractions of MOLT-4 cells (Fig. 1A). When overexpressed, the MsrB2 protein was essentially targeted to the mitochondria, as shown by Western blot (Fig. 1A), indicating that the human MsrB2 cDNA contains a mitochondrial signal peptide, MARLLWLLRGLTLGTAPRRAVRG, as previously evidenced for the mouse MsrB2 (21Kim H.Y. Gladyshev V.N. Mol. Biol. Cell. 2004; 15: 1055-1064Crossref PubMed Scopus (250) Google Scholar). Furthermore, the Msr activity assessed in transfected cells exhibited a 4-fold increase in mitochondria, whereas no increase was detected in the cytosol (Fig. 1A). To study whether oxidative stress can modulate Msr activity in MsrB2-overexpressing MOLT-4 cells as well as in control cells transfected with the empty vector, cells were exposed for 24 h to increasing concentrations of H2O2 (Fig. 1B). In the presence of a low level of oxidant (75 μm H2O2), Msr activity was slightly activated in both cell lines but more significantly in MsrB2-overexpressing cells. At 150 and 225 μm H2O2 treatment, Msr activity was decreased but was maintained at a very high level in MsrB2-overexpressing cells compared with the control cells (Fig. 1B, top), whereas the MsrB2 protein level followed the same trend (Fig. 1B, bottom). To analyze the role of MsrB2 in cellular protection against cell death induced by H2O2, cell survival was assayed by the XTT test in MsrB2-overexpressing and control MOLT-4 cells. As shown in Fig. 1C, MsrB2-overexpressing cells were more resistant to H2O2 treatment for all of the tested concentrations. Maximum protection conferred by overexpressing MsrB2 was on the order of 30% and observed when both cell types were treated with 150 μm H2O2.MsrB2 Silencing Does Not Affect Sensitivity of MOLT-4 Cells to H2O2-mediated Cell Death—We have shown that the overexpression of mitochondrial MsrB2 protects MOLT-4 cells against H2O2-induced oxidative stress. To further evaluate the role of MsrB2 in this protection, an MsrB2-deficient cell line was established by RNA silencing (small interfering RNA) technology. siRNA-mediated transient gene silencing of MsrB2 results in a significant decrease in the protein expression levels of MsrB2 when compared with the nonsilencing siRNA control cell line 48 h after transfection (Fig. 2A). Interestingly, although the levels of MsrB2 protein were heavily reduced in the siRNA MsrB2 cells compared with the control ones, the levels of Msr activity remained almost unchanged (Fig. 2B). Therefore, in order to check for a possible Msr activity compensatory mechanism by any other Msr enzyme, we measured the transcription levels of MsrB1, the only known Msr present in this cell line besides MsrB2. No differences were observed for MsrB1 at the transcription level when comparing both siRNA cell lines (Fig. 2C). Moreover, MsrB2 depletion did not result in a significant change in the cellular protective response of siRNA MsrB2 cells compared with siRNA control cells in both basal and H2O2-induced oxidative stress conditions, as evaluated by the XTT test (Fig. 2D).FIGURE 2MsrB2 silencing and induction of cell death by H2O2 treatment.A, MOLT-4 cells were transfected with 1 μm of siRNA negative control (siRNA control) or MsrB2 targeting siRNA (siRNA MsrB2). Cell lysates collected 48 h after transfection were blotted with anti-MsrB2. Immunodetection of actin was used as loading control. Immunoblots are representative of n = 3 independent experiments. B, MsrB activity levels in both siRNA control and siRNA MsrB2 cells were checked 48 h after transfection using N-acetyl-[3H]methionine R,S-sulfoxide as substrate (n = 3). C, RNA transcription level measurement of msrb2 and msrb1 genes by real time PCR in siRNA control and siRNA MsrB2 cells after 48 h of transfection. The real time PCR values were normalized to those produced with primers for the S26 gene (n = 3). D, measurement of cell survival following an oxidative stress mediated by a 24-h H2O2 treatment. Cell survival was assayed in control, siRNA control, and siRNA MsrB2 cells using the XTT viability assay (n = 3). The data in bar graphs represent means ± S.E. Asterisks indicate the level of statistical significance: ***, p < 0.001 according to Student's t test. au, arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MsrB2 Overexpression Decreases Intracellular ROS Production— In order to determine whether H2O2 treatment induces ROS production by the mitochondria, DHE was used to detect the generation of superoxide ions over treatment time. As measured by FACS at different time points, H2O2 induced a massive increase in ROS within the first hours of treatment in the 225 μm H2O2-treated control cells and reached a plateau at 9 h (Fig. 3A). To evaluate whether MsrB2, as has been previously demonstrated for MsrA (17Yermolaieva O. Xu R. Schinstock C. Brot N. Weissbach H. Heinemann S.H. Hoshi T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1159-1164Crossref PubMed Scopus (136) Google Scholar, 19Picot C.R. Petropoulos I. Perichon M. Moreau M. Nizard C. Friguet B. Free Radic. Biol. Med. 2005; 39: 1332-1341Crossref PubMed Scopus (63) Google Scholar, 27Marchetti M.A. Lee W. Cowell T.L. Wells T.M. Weissbach H. Kantorow M. Exp. Eye Res. 2006; 83: 1281-1286Crossref PubMed Scopus (63) Google Scholar, 28Sreekumar P.G. Kannan R. Yaung J. Spee C.K. Ryan S.J. Hinton D.R. Biochem. Biophys. Res. Commun. 2005; 334: 245-253Crossref PubMed Scopus (60) Google Scholar), can affect the intracellular ROS production, we measured the level of intracellular ROS after 24 h of treatment in control and MsrB2-overexpressing and -silencing cells treated with different H2O2 concentrations. Interestingly, the basal level of superoxide is slightly but significantly lower in MsrB2-overexpressing cells, and this level remains smaller when assayed 24 h after treatment of the cells with increasing concentrations of H2O2 (Fig. 3B). In contrast, no differences in ROS modulation were observed between the siRNA MsrB2 cell line and the siRNA control cell line (Fig. 3C). The use of DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) for the determination of ROS other than superoxide indicated that ROS were already reduced in
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