Thioredoxin Peroxidase Is a Novel Inhibitor of Apoptosis with a Mechanism Distinct from That of Bcl-2

Thioredoxin peroxidase (TPx) is a member of a newly discovered family of proteins that are conserved from yeast to mammals and to which natural killer enhancing factor belongs. These proteins are antioxidants that function as peroxidases only when coupled to a sulfhydryl reducing system. The physiological function of TPx in cells is not yet known. Here we demonstrate that when the human TPx II, a member of this family, is stably overexpressed in Molt-4 leukemia cells, it protects from apoptosis induced by serum deprivation, ceramide, or etoposide. TPx II, like Bcl-2, is able to inhibit release of cytochrome c from mitochondria to cytosol, and it inhibits lipid peroxidation in cells. TPx II, unlike Bcl-2, could prevent hydrogen peroxide accumulation in cells, suggesting that it functions upstream of Bcl-2 in the protection from apoptosis and may be implicated as an endogenous regulator of apoptosis. Thioredoxin peroxidase (TPx) is a member of a newly discovered family of proteins that are conserved from yeast to mammals and to which natural killer enhancing factor belongs. These proteins are antioxidants that function as peroxidases only when coupled to a sulfhydryl reducing system. The physiological function of TPx in cells is not yet known. Here we demonstrate that when the human TPx II, a member of this family, is stably overexpressed in Molt-4 leukemia cells, it protects from apoptosis induced by serum deprivation, ceramide, or etoposide. TPx II, like Bcl-2, is able to inhibit release of cytochrome c from mitochondria to cytosol, and it inhibits lipid peroxidation in cells. TPx II, unlike Bcl-2, could prevent hydrogen peroxide accumulation in cells, suggesting that it functions upstream of Bcl-2 in the protection from apoptosis and may be implicated as an endogenous regulator of apoptosis. Thioredoxin peroxidase is a member of a family of proteins initially discovered from yeast (1Kim K. Kim I.H. Lee K.Y. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1988; 263: 4704-4711Abstract Full Text PDF PubMed Google Scholar) and rat (2Kim I.H. Kim K. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6018-6122Crossref PubMed Scopus (131) Google Scholar) to be important in protecting glutamate synthetase oxidation by a metal ion catalyzed reaction, and this protection required the presence of a thiol (1Kim K. Kim I.H. Lee K.Y. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1988; 263: 4704-4711Abstract Full Text PDF PubMed Google Scholar). Later it was discovered that this protein indeed acts as a peroxidase but requires thioredoxin or a thiol-containing intermediate to carry on its peroxidase function (3Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar). The gene encoding TPx 1The abbreviations used are: TPx, thioredoxin peroxidase; NKEF, natural killer enhancing factor; PARP, poly(ADP-ribose)polymerase; FBS, fetal bovine serum; PBS, phosphate-buffered saline. II has been demonstrated to be identical to the gene encoding natural killer enhancing factor B (NKEF-B) (4Pahl P. Berger R. Hart I. Chae H.Z. Rhee S.G. Patterson D. Genomics. 1995; 26: 602-606Crossref PubMed Scopus (24) Google Scholar) and highly homologous to the gene encoding natural killer enhancing factor A (NKEF-A) (5Shau H. Butterfield L.H. Chiu R. Kim A. Immumogenetics. 1994; 40: 129-134Crossref PubMed Scopus (119) Google Scholar). NKEF is a cytosolic factor found in human red blood cells and thought to be a major antioxidant protecting red blood cells from oxidative injury (6Shau H. Gupta R.K. Golub S.H. Cell. Immunol. 1993; 147: 1-11Crossref PubMed Scopus (134) Google Scholar, 7Shau H. Kim A. Biochem. Biophys. Res. Commun. 1994; 199: 83-88Crossref PubMed Scopus (63) Google Scholar). The yeast Saccharomyces cerevisiae thioredoxin peroxidase has been demonstrated to have hydrogen peroxidase function, to act by dimerization as a thio-specific antioxidant, and to increase 2–3-fold by Western blot determination when S. cerevisiae cells undergo oxidative stress, suggesting that it is of physiological importance (8Chae H.Z. Kim I.H. Kim K. Rhee S.G. J. Biol. Chem. 1993; 268: 16815-16821Abstract Full Text PDF PubMed Google Scholar). The function of this family of proteins in cells, however, remains unknown. Apoptosis is known to be induced by oxidative damage either from oxygen free radicals or hydrogen peroxide directly or from their generation in cells by injurious agents (9Sarafian T.A. Bredesen D.E. Free Radical Res. 1994; 21: 1-8Crossref PubMed Scopus (186) Google Scholar, 10Slater A.F. Stefan C. Nobel C. Van Den Dobbelsteen D.J. Orrenius S. Toxicol. Lett. 1995; 82–83: 149-153Crossref PubMed Scopus (284) Google Scholar, 11Satch T. Sakai N. Enokido Y. Uchiyama Y. Hatanaka H. J. Biochem. (Tokyo). 1996; 120: 540-546Crossref PubMed Scopus (145) Google Scholar, 12Dobmeyer T.S. Findhammer S. Dobmeyer J.M. Klein S.A. Raffel B. Hoelzer D. Helm E.B. Kabelitz D. Rossol R. Free Radical Biol. Med. 1997; 22: 775-785Crossref PubMed Scopus (105) Google Scholar). Bcl-2 has been demonstrated to protect cells from apoptosis, and it is thought that Bcl-2 can protect cells from oxidative stress (13Hockenbery D. Nunez G. Milliman C. Schreiber R.D. Korsmeyer S.J. Nature. 1990; 348: 334-336Crossref PubMed Scopus (3544) Google Scholar, 14Korsmeyer S.J. Yin X.-M. Oltvai Z.N. Veis-Novack D.J. Linette G.P. Biochim. Biophys. Acta. 1995; 1271: 63-66Crossref PubMed Scopus (256) Google Scholar). Catalase and superoxide dismutase have also been extensively demonstrated to protect cells from different inducers of apoptosis, again suggesting a role for oxidation products in regulating cell death (15Sandstrom P.A. Buttke T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4708-4712Crossref PubMed Scopus (188) Google Scholar, 16Rabizadeh S. Gralla E.B. Borchelt E.R. Gwinn R. Valentine J.S. Sisodia S. Wong P. Lee M. Hahn H. Bredesen D.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3024-3028Crossref PubMed Scopus (328) Google Scholar). These considerations prompted us to investigate whether thioredoxin peroxidase could also function as an inhibitor of apoptosis. Molt-4 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37 °C in an environment containing 5% CO2 and 95% air. Monoclonal anti-cytochrome cantibody was purchased from Pharmagen. Polyclonal antibody against TPx II was prepared as described (3Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar). 2,7-Dichloro-fluorescin diacetate was from Molecular Probes (Eugene, OR). Etoposide was obtained from Sigma. Molt-4 cells (3 × 107/ml) were transfected by electroporation with PCRTM 3.1-Uni vector (Invitrogen) with and without full-length TPx II DNA (10 μg) (17Chae H,Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (706) Google Scholar). A heterogeneous population of positive clones was selected with G418 (Life Technologies, Inc.) and maintained in RPMI 1640 containing 10% FBS, 25 mm HEPES, pH 7.4, and 0.5 mg/ml of G418. To determine the expression of TPx II, whole cell extracts (10 μg/lane) were separated on a 12% SDS-polyacrylamide gel, Western blotted with the polyclonal anti-TPx II antibody, and visualized with the ECL reagents (Amersham) as described (3Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar). For serum deprivation experiments, cells were washed twice with phosphate-buffered saline (PBS) and once with serum free RPMI 1640 containing 25 mm HEPES, pH 7.5. Washed cells were seeded at a density of 5 × 105/ml in serum free RPMI 1640 containing 25 mm HEPES, pH 7.5. To study the effects of ceramide, etoposide, or H2O2, cells were washed once and seeded (at a density of 5 × 105/ml) with RPMI 1640 containing 2% FBS and 25 mm HEPES, pH 7.5. Control cells were treated with appropriate amounts of vehicle (ethanol for ceramide and Me2SO for etoposide) diluted in the same culture medium as that for the drugs. At desired timed intervals, aliquots of cells were removed for various assays. Cell viability was assessed by trypan blue exclusion. Cells (5 × 106) were pelleted by centrifugation, rinsed once with ice-cold PBS, resuspended in 100 μl of PBS containing 1 mm EDTA, 1 mmEGTA, and 1 mm phenylmethylsulfonyl fluoride, diluted with 5 × SDS sample buffer, and boiled for 10 min. Intact and cleaved PARP were detected by Western blot as described (18Smyth M.J. Perry D.K. Zhang J. Poirier G.G. Hannun Y.A. Obeid L.M. Biochem. J. 1996; 316: 25-28Crossref PubMed Scopus (198) Google Scholar) using anti-PARP anti-serum from Enzyme System Products (Dublin, CA). Cells were washed twice with ice-cold PBS and resuspended in 100 μl of 20 mm HEPES, pH 7.5, containing 10 mm KCl, 1.5 mm MgCl2, 5 mm EDTA, 5 mm dithiothreitol, 2 mm phenylmethylsulfonyl fluoride, and 250 mmsucrose. Cytosol was prepared, and the levels of cytosolic cytochromec were determined by Western blot exactly as described (19Yang J. Liu X.S. Bhalla K. Kim C.N. Ibrado A.M. Cai J.Y. Peng T.I. Jones D.P. Wang X.D. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar) using a monoclonal anti-cytochrome c antibody from PharMingen (San Diego, CA). Cells (5 × 106) were washed twice with PBS and lysed by three cycles of repeated freezing and thaw in 300 μl of 200 mm Tris-HCl, pH 7.4. The amounts of representative lipid peroxide, malonaldehyde, were determined spectrophotometrically with a kit from Calbiochem exactly as described by the manufacturer and normalized to total protein, determined by the Bio-Rad dye binding assay using bovine serum albumin as standard. Cellular H2O2 was determined following a protocol described by Hockenbery et al. (20Hockenbery D.M. Oltvai Z.N. Yin X.M. Milliman C.L. Korsmeyer S.J. Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3297) Google Scholar). Briefly, cells (5 × 106) were loaded with vehicle Me2SO (0.05%) or 50 μm 2,7-dichloro-fluorescin diacetate for 1 h at 37 °C. Afterward, cells were treated with H2O2 (80 μm). At the indicated time, cells were washed twice and resuspended in 5 ml of PBS buffer. Cells were analyzed using a Becton Dickson fluorophotometer with excitation and emission wavelength settings of 495 and 525 nm, respectively. We stably transfected Molt-4 leukemia cells with vector control or cDNA encoding the mammalian thioredoxin peroxidase protein TPx II. After selection for stable transfectants, we demonstrated the overexpression of TPx II protein using Western blot analysis (Fig.1). We next evaluated the cells for their sensitivity to inducers of apoptosis. The cells overexpressing TPx II were resistant to a variety of inducers of apoptosis. Fig.2 A demonstrates that vector-transfected cells were ∼25 and ∼40% dead by 24 and 48 h following serum deprivation, respectively, whereas cells overexpressing TPx II were significantly resistant to death in response to serum deprivation. Similarly, vector-transfected cells were easily induced to undergo cell death in response to other well known inducers of apoptosis such as ceramide treatment (Fig. 2 B), whereas TPx II again protected cells from ceramide-induced cell death. We also tested the ability of TPx II to protect from apoptosis in response to the pharmacologic agent, etoposide, a chemotherapeutic agent that has been demonstrated to induce cell death by apoptosis (21Kaufmann S. Cancer Res. 1989; 49: 5870-5878PubMed Google Scholar). TPx II, but not vector control, protected cells from undergoing cell death in response to etoposide treatment (Fig. 2 C). Bcl-2 has been well documented to protect cells from apoptosis (13Hockenbery D. Nunez G. Milliman C. Schreiber R.D. Korsmeyer S.J. Nature. 1990; 348: 334-336Crossref PubMed Scopus (3544) Google Scholar). As Fig.2 D demonstrates, Bcl-2 was as effective as TPx II at protecting cells from ceramide-induced apoptosis. To confirm that cell death in response to these inducers was occurring by apoptosis, we evaluated the ability of these inducers to cleave the death substrate PARP (22Kaufmann S.H. Desnoyers S. Ottaviano Y. Davidson N.E. Poirier G.G. Cancer Res. 1993; 53: 3976-3985PubMed Google Scholar). TPx II, but not control vector, inhibited PARP cleavage in response to up to 72 h of serum deprivation as well as in response to ceramide treatment (Fig. 2 E). These studies demonstrate that TPx II inhibits apoptosis, and it appears to be as effective as Bcl-2.Figure 2Effect of TPx on apoptosis and PARP cleavage. TPx II protects cells from apoptosis in response to serum deprivation (A), C6-ceramide (20 μm) (B), or etoposide (40 μm) (C); Bcl-2 protects cells from apoptosis induced by ceramide (20 μm) (D). All treatments were for the indicated times. E, TPx II inhibited PARP cleavage.Top panel, lane 1, TPx II cells serum deprived for 24 h; lane 2, vector cells serum deprived for 24 h; lane 3, TPx II cells serum deprived for 48 h; lane 4, vector cells serum deprived for 48 h;lane 5, TPx II cells serum deprived for 72 h;lane 6, vector cells serum deprived for 72 h.Bottom panel, lane 1, vector cells treated with ethanol (0.1%); lane 2, vector cells treated with C6-ceramide (20 μm); lane 3, TPx II cells treated with ethanol (0.1%); lane 4, TPx II cells treated with C6-ceramide (20 μm). Results inA–D are the means ± S.D. of duplicate determinations from three separate experiments. PARP data shown in E are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cytochrome c has recently been implicated in the apoptotic process, and it has been identified as a component required in a cell free system to induce activation of the protease cascade (23Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar). Cytochrome c appears to be released from the mitochondria into the cytosol in response to several apoptotic stimuli. Bcl-2 has recently been demonstrated to block cytochrome c release and consequently rescues cells from activation of the caspases and cell death (19Yang J. Liu X.S. Bhalla K. Kim C.N. Ibrado A.M. Cai J.Y. Peng T.I. Jones D.P. Wang X.D. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar, 24Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar). Up to this point of evaluation, TPx II appeared to act similar to Bcl-2 in protecting cells from apoptosis; however, it was unclear if TPx II functioned upstream, downstream, or at the same site as Bcl-2 in the apoptotic pathway. Therefore, in an attempt to order thioredoxin peroxidase in the apoptotic pathway with respect to Bcl-2, we next evaluated its ability to modulate cytochromec translocation. Cytochrome c release into the cytosol was evaluated in cells that overexpress Bcl-2 as well as cells that overexpress TPx II. We first evaluated if the cell-permeable ceramide analog C6-ceramide is able to induce cytochromec release into the cytosol, because ceramide has been demonstrated to function upstream of Bcl-2 in the apoptotic pathway (25Zhang J. Alter N. Reed J.C. Borner C. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5325-5328Crossref PubMed Scopus (294) Google Scholar). The addition of C6-ceramide (20 μm) resulted in release of cytochrome c from mitochondria to cytosol seen as early as 2 h. Importantly, Bcl-2, protected cells from cytochrome c release in response to ceramide (Fig.3 A). Interestingly, TPx II also protected cells as effectively as Bcl-2 from cytochromec release (Fig. 3 B). We also assayed cytochromec release in response to the chemotherapeutic agent etoposide, and the results show that TPx II (Fig. 3 D) was as effective as Bcl-2 (Fig. 3 C) in protecting cells from cytochrome c release. Because release of cytochromec is considered an early event in the commitment to apoptosis, this shows that TPx II functions similar to Bcl-2 in this phase. To further understand the mechanism by which TPx II inhibits apoptosis and to compare this with Bcl-2, we next evaluated the effects of TPx II on lipid peroxidation, given the biochemical function of TPx II as a peroxidase. Lipid peroxidation has been implicated as an intermediate in the apoptotic pathway that is thought to precede DNA fragmentation and morphological changes of apoptosis (20Hockenbery D.M. Oltvai Z.N. Yin X.M. Milliman C.L. Korsmeyer S.J. Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3297) Google Scholar, 26Sandstrom P.M. Pardi D. Tebbey P.W. Dudek R.W. Terrian D.M. Folks T.M. Buttke T.M. FEBS Lett. 1995; 365: 66-70Crossref PubMed Scopus (109) Google Scholar), but it is not completely clear if it occurs prior to or after shows that TPx II was able to inhibit lipid peroxidation in response to serum deprivation (Fig. ceramide (Fig. as well as etoposide (Fig. C) that TPx II indeed can function as a peroxidase to prevent lipid peroxidation and that lipid peroxidation is an intermediate in cell death. lipid peroxidation was also inhibited by Bcl-2 as demonstrated in D as well as by Hockenbery et al. (20Hockenbery D.M. Oltvai Z.N. Yin X.M. Milliman C.L. Korsmeyer S.J. Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3297) Google Scholar). These results that lipid peroxidation is of the site of of Importantly, these results indicated to us that TPx II may act as a it inhibited lipid peroxidation at by release of cytochrome c and was from Bcl-2 has been demonstrated to hydrogen peroxide accumulation apoptosis and to prevent the effects of hydrogen peroxide on cell death S.J. Yin X.-M. Oltvai Z.N. Veis-Novack D.J. Linette G.P. Biochim. Biophys. Acta. 1995; 1271: 63-66Crossref PubMed Scopus (256) Google Scholar), suggesting that Bcl-2 functions of an oxidative evaluated the effects of TPx II on the accumulation of hydrogen peroxide and the ability of TPx II to protect from hydrogen apoptosis. A shows that TPx II protected from hydrogen apoptosis as well as Bcl-2 (Fig. 5 B). Interestingly, hydrogen peroxide was at cytochrome c release (Fig. 5 that hydrogen peroxide generation in cells may be upstream of of apoptosis. TPx II was also able to protect from cytochrome c release in response to hydrogen peroxide C). we evaluated the ability of TPx II to block accumulation of hydrogen peroxide in cells. TPx II inhibited accumulation of endogenous hydrogen peroxide in response to hydrogen peroxide treatment (Fig. 5 D) the other Bcl-2 not inhibit this accumulation the ability of TPx II and Bcl-2 to inhibit hydrogen peroxide induced cell death. These results that TPx II may function directly as a peroxidase to inhibit accumulation of in apoptosis, whereas Bcl-2 functions further by the ability of these to induce cell death. this study we demonstrate several and and of the apoptotic pathway. we demonstrate that the protein thioredoxin peroxidase is a inhibitor of apoptosis. we demonstrate that TPx II inhibits cell death by a mechanism from Bcl-2 and upstream of the site of of We also demonstrate that hydrogen peroxide generation in cells is upstream of cytochrome c release. the results show that TPx II but not Bcl-2 is able to prevent hydrogen peroxide accumulation in cells. us to order the peroxidase upstream of Bcl-2 and cytochrome c release in the apoptotic pathway as in I. These studies have several and important these data demonstrate that a protein with a function is in protecting cells from apoptosis in response to oxygen protection is at a point from that of this protein is to thioredoxin and thioredoxin in (3Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar). has recently been demonstrated that the factor was described as a factor that of by by the human 1, and is thought to act like a is the human of thioredoxin G. Biochim. Biophys. Acta. 1994; PubMed Scopus Google Scholar). factor has recently been demonstrated to inhibit factor and apoptosis M. H. H. S. A. S. A. K. A. J. Immunol. 147: Google Scholar). has also been implicated in to chemotherapeutic agents of cell J. M. K. M. T. M. 1997; PubMed Google Scholar). These coupled with data on the protection of apoptosis by thioredoxin II in a in the of apoptosis. I. 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