Antioxidant Function of the Mitochondrial Protein SP-22 in the Cardiovascular System

Abstract

The mitochondrial protein SP-22 has recently been reported to be a member of the thioredoxin-dependent peroxide reductase family, suggesting that it may be one of the antioxidant systems in mitochondria, which are the major site of reactive oxygen intermediate generation. The aim of this study was to examine whether SP-22 is involved in mitochondrial antioxidant mechanisms and whether its expression is induced by oxidative stresses, particularly those in mitochondria. The expression of SP-22 protein was enhanced by about 1.5–4.6-fold when bovine aortic endothelial cells (BAEC) were exposed to various oxidative stresses, including mitochondrial respiratory inhibitors which increased the superoxide generation in BAEC mitochondria. The expression of SP-22 mRNA increased 2.0–3.5-fold with a peak at 3–6 h after exposure to Fe2+/dithiothreitol or a respiratory inhibitor, antimycin A. BAEC with an increased level of SP-22 protein caused by pretreatment with mild oxidative stress became tolerant to subsequent intense oxidative stress. On the other hand, BAEC that had been depleted of SP-22 with an antisense oligodeoxynucleotide against SP-22 mRNA became more labile to oxidative stress than control BAEC. The induction of SP-22 protein by oxidative stress in vivo was demonstrated in an experimental model of myocardial infarction in rat heart. These findings indicate that SP-22 functions as an antioxidant in mitochondria of the cardiovascular system. The mitochondrial protein SP-22 has recently been reported to be a member of the thioredoxin-dependent peroxide reductase family, suggesting that it may be one of the antioxidant systems in mitochondria, which are the major site of reactive oxygen intermediate generation. The aim of this study was to examine whether SP-22 is involved in mitochondrial antioxidant mechanisms and whether its expression is induced by oxidative stresses, particularly those in mitochondria. The expression of SP-22 protein was enhanced by about 1.5–4.6-fold when bovine aortic endothelial cells (BAEC) were exposed to various oxidative stresses, including mitochondrial respiratory inhibitors which increased the superoxide generation in BAEC mitochondria. The expression of SP-22 mRNA increased 2.0–3.5-fold with a peak at 3–6 h after exposure to Fe2+/dithiothreitol or a respiratory inhibitor, antimycin A. BAEC with an increased level of SP-22 protein caused by pretreatment with mild oxidative stress became tolerant to subsequent intense oxidative stress. On the other hand, BAEC that had been depleted of SP-22 with an antisense oligodeoxynucleotide against SP-22 mRNA became more labile to oxidative stress than control BAEC. The induction of SP-22 protein by oxidative stress in vivo was demonstrated in an experimental model of myocardial infarction in rat heart. These findings indicate that SP-22 functions as an antioxidant in mitochondria of the cardiovascular system. reactive oxygen intermediate 22-kDa substrate protein 23-kDa macrophage stress protein bovine aortic endothelial cells manganese superoxide dismutase 4-hydroxy-2-nonenal submitochondrial particles reverse transcription polymerase chain reaction digoxigenin dithiothreitol oligodeoxynucleotide copper and zinc superoxide dismutase superoxide dismutase tumor necrosis factor polyacrylamide gel electrophoresis fetal calf serum. Mitochondria play an important role in aerobic energy metabolism of living cells. The mitochondrial electron transport system consumes approximately 85% of the oxygen utilized by the cell, and about 5% of the oxygen is converted to reactive oxygen intermediates (ROIs)1 (1Forman H.J. Boveris A. Pryor W.A. Free Radicals in Biology. V. Academic Press, New York1982: 65-90Google Scholar, 2Shigenaga M.K. Hagen T.M. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10771-10778Crossref PubMed Scopus (1831) Google Scholar). The generation of ROIs in mitochondria has been reported to impair various cellular functions by attacking reactive moieties of macromolecules such as protein, DNA, and lipid (3Stadtman E.R. Annu. Rev. Biochem. 1993; 62: 797-821Crossref PubMed Scopus (1265) Google Scholar, 4Cahill A. Wang X. Hoek J.B. Biochem. Biophys. Res. Commun. 1997; 235: 286-290Crossref PubMed Scopus (100) Google Scholar, 5Yakes F.M. Van H.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 514-519Crossref PubMed Scopus (1451) Google Scholar, 6Fantone J.C. Ward P.A. Am. J. Pathol. 1982; 107: 395-418PubMed Google Scholar). Mitochondrial permeability transition, which may be an initial event in the process of cell death induced by Ca2+ and inorganic phosphate, has recently been reported to be mediated by ROIs and prevented by antioxidants in vitro (7Kowaltowski A.J. Netto L.E. Vercesi A.E. J. Biol. Chem. 1998; 273: 12766-12769Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The mitochondrial protein SP-22 was originally isolated from bovine adrenal cortex as a substrate protein for mitochondrial ATP-dependent protease (8Watabe S. Kohno H. Kouyama H. Hiroi T. Yago N. Nakazawa T. J. Biochem. (Tokyo). 1994; 115: 648-654Crossref PubMed Scopus (97) Google Scholar, 9Watabe S. Hasegawa H. Takimoto K. Yamamoto Y. Takahashi S.Y. Biochem. Biophys. Res. Commun. 1995; 213: 1010-1016Crossref PubMed Scopus (58) Google Scholar). An analysis of its amino acid sequence revealed that SP-22 is a member of the thioredoxin-dependent peroxide reductase family like the C22 component of alkyl hydroperoxidase in Salmonella typhimurium (10Tartaglia L.A. Storz G. Brodsky M.H. Lai A. Ames B.N. J. Biol. Chem. 1990; 265: 10535-10540Abstract Full Text PDF PubMed Google Scholar), thiol-specific antioxidant enzyme (11Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar), 23-kDa macrophage stress protein (MSP23) (12Ishii T. Yamada M. Sato H. Matsue M. Taketani S. Nakayama K. Sugita Y. Bannai S. J. Biol. Chem. 1993; 268: 18633-18636Abstract Full Text PDF PubMed Google Scholar), natural killer cell enhancing factor (NKEF) (13Shau H. Gupta R.K. Golub S.H. Cell Immunol. 1993; 147: 1-11Crossref PubMed Scopus (134) Google Scholar), and MER5 (14Yamamoto T. Matsui Y. Natori S. Obinata M. Gene (Amst). 1989; 80: 337-343Crossref PubMed Scopus (110) Google Scholar) in mammalian cells. MER5 is 92% similar to SP-22 protein and considered to be a mouse homolog of SP-22 (15Watabe S. Hiroi T. Yamamoto Y. Fujioka Y. Hasegawa H. Yago N. Takahashi S.Y. Eur. J. Biochem. 1997; 249: 52-60Crossref PubMed Scopus (132) Google Scholar). Members of this family have a highly conserved active site sequence among a wide range of species and are believed to act as antioxidant systems together with the NADPH-thioredoxin-thioredoxin reductase system (11Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar, 16Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Among the members of the thioredoxin-dependent peroxide reductase family, SP-22 is the only protein located in mitochondria. To test the hypothesis that SP-22 functions as an antioxidant system in mitochondria, which are the major site of cellular ROI generation, we investigated the oxidant-induced expression of SP-22 using cultured bovine aortic endothelial cells (BAEC) and an in vivo model of experimental myocardial infarction. Furthermore, we also examined the antioxidant function of SP-22 using BAEC with decreased or increased levels of SP-22 protein produced by treatment with antisense oligodeoxynucleotide or mild oxidant preconditioning. The present results indicate that SP-22 plays a crucial role in the antioxidant defense mechanism of mitochondria in the cardiovascular system. BAEC were harvested from bovine thoracic aorta obtained from a local slaughterhouse and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) as described by Kaku et al. (17Kaku Y. Nanri H. Sakimura T. Ejima K. Kuroiwa A. Ikeda M. Biochim. Biophys. Acta. 1997; 1356: 43-52Crossref PubMed Scopus (41) Google Scholar). The identity of endothelial cells was verified by their characteristic morphology and the presence of factor VIII-associated antigen. Endothelial cells at up to passage 10 were used for these experiments. Mouse anti-manganese superoxide dismutase (MnSOD) monoclonal antibody was purchased from Chemicon International (Temecula, CA), anti-4-hydroxy-2-nonenal (HNE) monoclonal antibody was from NOF Co. (Tokyo, Japan), 1-methyl-4-phenylpyridinium was from Aldrich, paraquat from Nacalai Tesque (Kyoto, Japan), antimycin A and superoxide dismutase were from Sigma, and glucose oxidase and xanthine oxidase were from Boehringer Mannheim (Mannheim, Germany). Other chemicals were standard commercial products of analytical grade. Monolayers of confluent BAEC were incubated for the indicated periods with/without oxidative stress agents in Eagle's minimal essential medium (Nissui, Japan) containing 0.5% FCS at 37 °C. After washing the monolayers, cells were disrupted in 150 μl of lysis buffer (50 mm Tris/HCl buffer, pH 7.4, containing 0.2% (w/v) Triton X-100, 1 mmEDTA, 5 μg/ml chymostatin, 10 μg/ml each leupeptin, antipain, and pepstatin, and 20 μm(p-amidinophenyl)methanesulfonyl fluoride hydrochloride) and homogenized with a Teflon homogenizer. After centrifugation at 7000 × g for 10 min at 4 °C, lactate dehydrogenase activity in the supernatant was assayed as described by Bergmeyeret al. (17Kaku Y. Nanri H. Sakimura T. Ejima K. Kuroiwa A. Ikeda M. Biochim. Biophys. Acta. 1997; 1356: 43-52Crossref PubMed Scopus (41) Google Scholar). The peptide SPTASREYFEKVNQ, corresponding to residues 183–195 of the SP-22 protein (9Watabe S. Hasegawa H. Takimoto K. Yamamoto Y. Takahashi S.Y. Biochem. Biophys. Res. Commun. 1995; 213: 1010-1016Crossref PubMed Scopus (58) Google Scholar), was synthesized and conjugated with hemocyanin. Female Japanese were with the conjugated peptide with The was 4 and this was by at were obtained after the cells in minimal essential FCS were with in and with The cells were with SP-22 antibody and with of bovine mitochondria was as described J. A. Y. M. T. M. K. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The mitochondrial of cells was with in buffer, pH 7.4, and in at °C. of the mitochondria were incubated with the antibody against the and incubated with were with and and examined a electron at with serum BAEC submitochondrial particles were as described by et al. J. A. Y. M. T. M. K. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). BAEC were in an buffer of 10 pH 7.4, 1 mm μg/ml leupeptin, 5 μg/ml fluoride and pepstatin, and in the buffer × The cells were homogenized in a and at for 10 min to The supernatant was at 7000 × 10 The mitochondrial was and at × g for 1 The was homogenized in an buffer and as the by was by the of to and was by the in the presence of 10 μg/ml superoxide dismutase as described by et al. K. S. Biochem. J. PubMed Scopus Google Scholar). SP-22 protein of BAEC was by from each of BAEC were by gel electrophoresis and with serum. were using antibody and was to SP-22 in as described (17Kaku Y. Nanri H. Sakimura T. Ejima K. Kuroiwa A. Ikeda M. Biochim. Biophys. Acta. 1997; 1356: 43-52Crossref PubMed Scopus (41) Google Scholar). was isolated from confluent BAEC using the reported by and N. Biochem. PubMed Scopus Google Scholar). were with reverse The products were polymerase in a The for each were for for 1 and for min The for SP-22 were and which a a of the The identity of the products was by was also for the as a control for the of used in the A in which reverse was was also to the of the of were the of to the reaction and the of the used for SP-22 and SP-22 products were the by of the After the of the by the with were and in vitro digoxigenin with polymerase using a was in μl of buffer containing 10 mm phosphate, pH (w/v) and 1 After h at the were in gel with a 10 mm buffer, and to a and by for min at °C. with the was at in mm phosphate, 1 mm and 1 Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). were at with phosphate, 5% and 1 mm and for min at with 0.2% reaction using a acid to the by the The of SP-22 mRNA was by BAEC were with dithiothreitol for which increased the expression of SP-22 These BAEC were exposed to more intense oxidative stress of 1 mm for and cell were by the lactate dehydrogenase of the cell A antisense oligodeoxynucleotide was synthesized by The antisense sequence is against the of the SP-22 The antisense is against the of SP-22 also a of antisense 1 and and for a control of these were to to other or 1994; PubMed Scopus Google Scholar). of we used the lipid that had been in buffer, pH of and 5 μl of of 5 which was to the by the were and incubated for min at The was to each with 1 of RPMI 1640 medium and the cells were incubated for 1 h at 37 in a 5% the of the the cells were with of RPMI 1640 medium containing 10% process was more at about g were used for these experiments. The were in with the for and of the of and The were and by a The was a which results in infarction of the J. PubMed Scopus Google Scholar). a a was in the of the the A of and were 1 or after The was in and at °C. The from the and at 1 or after the were and the were with for 5 was with in The were with the antibody and monoclonal and by a The were with were by the protein using bovine serum as a The of the in and was by analysis using the of and the results are as A analysis was using were considered at an antibody against SP-22 protein and used it for this antibody a with a corresponding to SP-22 in and the mitochondrial of in the of SP-22 protein was examined by cultured cells were with the the of SP-22 the mitochondrial by a were when for SP-22 were The of the SP-22 protein in the mitochondria was by electron The mitochondrial isolated from cells was with the by reactive to the antibody were located in the mitochondria These results indicate that SP-22 protein is located in mitochondria of which is with the results of the analysis by et al. (8Watabe S. Kohno H. Kouyama H. Hiroi T. Yago N. Nakazawa T. J. Biochem. (Tokyo). 1994; 115: 648-654Crossref PubMed Scopus (97) Google Scholar). BAEC to which the the reaction PubMed Scopus Google Scholar, PubMed Scopus Google Scholar), increased the expression of SP-22 protein in a with the after h of On the other hand, the expression of was induced only in this SP-22 expression was with such as and acid the of various oxidative the induction of SP-22 protein in BAEC. was in the induction to ROI such as and enhanced SP-22 protein about oxidase and which peroxide and superoxide Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar), enhanced SP-22 protein by about and which with H. J. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), also enhanced SP-22 protein by about and which are that have been to other peroxide reductase (12Ishii T. Yamada M. Sato H. Matsue M. Taketani S. Nakayama K. Sugita Y. Bannai S. J. Biol. Chem. 1993; 268: 18633-18636Abstract Full Text PDF PubMed Google Scholar), a induction of SP-22 antimycin and which are respiratory chain inhibitors that ROI generation in mitochondria 1982; Google Scholar), also enhanced SP-22 protein by ROI generation in with antimycin A and with the of superoxide generation by antimycin the antimycin confluent BAEC produced superoxide at a of protein that the protein of in cells is about which is about than the of superoxide generation produced by oxidase (50 the SP-22 protein induced by antimycin A treatment was about than that induced by the oxidase suggesting that superoxide in mitochondria induced SP-22 expression more of SP-22 protein in BAEC exposed to the SP-22 protein were incubated for h with of BAEC were and with the of each protein were to each were obtained by a analysis of the The of SP-22 protein is as a to that of the control BAEC as in a of SP-22 protein in BAEC exposed to various oxidative SP-22 protein oxidase (50 oxidase (50 (50 A were incubated with various oxidative of BAEC were and with the of each protein were to each were obtained by a analysis of the The of SP-22 protein is as a to that of the control BAEC as The the in SP-22 protein after were h with antimycin A and in a of superoxide by with respiratory chain A were from BAEC as described by was by the of to and by the in the presence of 10 μg/ml superoxide as described by et al. K. S. Biochem. J. PubMed Scopus Google Scholar). The control system and 1 mm and mm pH in 1 After for 5 the reaction was the indicated of mm and was at by a The results the of experiments. in a BAEC were incubated for h with of BAEC were and with the of each protein were to each were obtained by a analysis of the The of SP-22 protein is as a to that of the control BAEC as BAEC were incubated with various oxidative of BAEC were and with the of each protein were to each were obtained by a analysis of the The of SP-22 protein is as a to that of the control BAEC as The the in SP-22 protein after were h with antimycin A and were from BAEC as described by was by the of to and by the in the presence of 10 μg/ml superoxide as described by et al. K. S. Biochem. J. PubMed Scopus Google Scholar). The control system and 1 mm and mm pH in 1 After for 5 the reaction was the indicated of mm and was at by a The results the of experiments. chain inhibitors also by oxidative energy metabolism in mitochondria G. A. PubMed Scopus Google Scholar, K. T. Y. S. Biochem. Biophys. Res. Commun. 1990; PubMed Scopus Google Scholar). To examine the that the stress of may SP-22 we the protein levels of SP-22 in medium that 5 mm glucose or 20 of after the of glucose or the level of BAEC incubated with 20 mm was to of that in the control 5 The expression of SP-22 protein in 20 mm medium 5 mm that the induction of SP-22 expression in BAEC. The induction of SP-22 protein in BAEC exposed to oxidative stress was also examined in of the mRNA was obtained from BAEC exposed to for and and the of SP-22 mRNA was by and SP-22 mRNA to after h of exposure to or antimycin a level at and decreased to the initial level after h 4 the results were obtained 4 To the mechanism of the induction of SP-22 in antimycin we examined the of the in SP-22 mRNA expression after antimycin A in 4 the antimycin in SP-22 mRNA expression to the control that the in SP-22 mRNA was at in to the of SP-22 The pretreatment of BAEC with a of oxidant which cell increased the level of SP-22 protein about with that in control cells to the the cell of BAEC with subsequent treatment with a of oxidant 5 These results indicate that BAEC in which SP-22 protein had been induced by with a mild oxidant became tolerant to subsequent intense oxidative examined the cell of BAEC with a decreased level of SP-22 to the treatment with antisense against SP-22 After BAEC had been exposed to SP-22 antisense the protein levels of SP-22 in BAEC were examined by The of SP-22 in BAEC with antisense 1 and decreased to and of that in control that of the BAEC to the level in the control in oxidative stress decreased the cell of the antisense 1 BAEC the oxidative stress produced only a in cell for the control and cells. of BAEC with antisense or by cell These results indicate that BAEC with a level of SP-22 protein were more to oxidative stress. To examine vivo induction of SP-22 protein, an study was using an experimental model of myocardial infarction in rat heart. with and K. H.Z. E.R. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar) that of were increased in the of the rat that lipid in the and SP-22 in rat was induced corresponding to the exposed to oxidative stress. against were and were the and cardiovascular including the and endothelial mitochondria are particularly important for the the mitochondrial electron transport system consumes a of oxygen and ROIs (1Forman H.J. Boveris A. Pryor W.A. Free Radicals in Biology. V. Academic Press, New York1982: 65-90Google Scholar, 2Shigenaga M.K. Hagen T.M. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10771-10778Crossref PubMed Scopus (1831) Google Scholar). The generation of ROIs in cardiovascular systems has and to J. W.A. S.H. J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar), J. PubMed Scopus Google Scholar), and U. M. H. 1997; PubMed Scopus Google Scholar). ROIs are by various such as or Annu. Rev. Biochem. 1995; PubMed Scopus Google Scholar), W.A. and Press, New Scholar), and H. Boveris A. Rev. PubMed Scopus Google Scholar). superoxide and peroxide in the has been mitochondrial for that in rat mitochondria J. Biol. Chem. Full Text PDF PubMed Google Scholar). SP-22 is a member of the thioredoxin-dependent peroxide reductase family (11Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar), and by from the NADPH-thioredoxin-thioredoxin reductase it vitro (15Watabe S. Hiroi T. Yamamoto Y. Fujioka Y. Hasegawa H. Yago N. Takahashi S.Y. Eur. J. 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Chem. 1998; 273: 12766-12769Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). protein induction by oxidative stresses, SP-22 to be induced in a similar to other members of the thioredoxin-dependent peroxide reductase family, for its to and the expression of in the (12Ishii T. Yamada M. Sato H. Matsue M. Taketani S. Nakayama K. Sugita Y. Bannai S. J. Biol. Chem. 1993; 268: 18633-18636Abstract Full Text PDF PubMed Google Scholar), of SP-22 protein in the mitochondria. be by the of the of the agents has been reported to a the of in the and mitochondria Res. 115: PubMed Scopus Google Scholar). is that Scholar) and W.A. and Press, New Scholar) function as against ROIs in mitochondria. we were in the of SP-22 to the antioxidant function in mitochondria. BAEC in which the expression of the SP-22 protein was enhanced by exposure to a which only induced to a of Furthermore, BAEC that had been depleted of SP-22 by antisense were labile to the system These results that SP-22 plays an role in antioxidant systems of BAEC. that and which in a cellular antioxidant defense Nanri H. S. K. S. Eur. J. Biochem. PubMed Scopus Google Scholar) and are essential for SP-22 are also located in mitochondria of BAEC. Ejima and H. for have that such as tumor necrosis and and and and the expression of PubMed Scopus Google Scholar). et oxidase increased mRNA in cells induction of and S. M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Yamamoto et that the of MER5 protein of bovine had for and MER5 protein was enhanced after induction of by (14Yamamoto T. Matsui Y. Natori S. Obinata M. Gene (Amst). 1989; 80: 337-343Crossref PubMed Scopus (110) Google Scholar). et al. recently reported of peroxide prevented the by peroxide S.W. H.Z. K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). These results that the expression of cellular antioxidant such as the thioredoxin-dependent peroxide reductase and be by a also SP-22 mRNA was induced by and H. the expression of SP-22 was enhanced in an experimental model of rat myocardial infarction. The induction of SP-22 expression at h after and decreased at h when necrosis a h after the process of infarction is in the rat J. PubMed Scopus Google Scholar). the of ROIs such as and are including the of from mitochondria, which are in the 1982; Google Scholar, Am. J. 1995; Full Text PDF PubMed Scopus Google Scholar), the metabolism of acid by S.W. PubMed Scopus Google Scholar), and the in xanthine oxidase which superoxide J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). has been reported that the of myocardial infarction Res. PubMed Scopus Google Scholar, S.W. Res. PubMed Scopus Google Scholar), suggesting that may against the of myocardial infarction. The present results obtained in experimental myocardial infarction also that SP-22 may in against myocardial infarction. are to of of and Japan) for and T. for