Antioxidant Function of the Mitochondrial Protein SP-22 in the
Cardiovascular System*
Masaru
Araki
§,
Hiroki
Nanri
¶,
Kuniaki
Ejima
,
Yoshinobu
Murasato§
,
Toshiyuki
Fujiwara**,
Yasuhide
Nakashima§, and
Masaharu
Ikeda
From the
Department of Health Development,
§ Second Department of Internal Medicine, and
Systems
Physiologya, University of Occupational and Environmental Health,
Kitakyushu 807-8555 and the ** Department of Biochemistry, Fukuoka
University School of Medicine, Fukuoka 814-0180, Japan
 |
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.
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INTRODUCTION |
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 (1, 2). 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 (3-6). 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 (7).
The mitochondrial protein SP-22 was originally isolated from bovine
adrenal cortex as a substrate protein for mitochondrial ATP-dependent protease (8, 9). 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 (10), thiol-specific antioxidant enzyme (11),
23-kDa macrophage stress protein (MSP23) (12), natural killer cell
enhancing factor (NKEF) (13), and MER5 (14) in mammalian cells. MER5 is
92% similar to SP-22 protein and considered to be a mouse homolog of
SP-22 (15). 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 (11, 16). 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.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Materials--
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. (17). 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.
Oxidative Stresses--
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 mM
EDTA, 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 Bergmeyer et al. (17).
Polyclonal Rabbit Antibodies against SP-22--
The peptide
SPTASREYFEKVNQ, corresponding to residues 183-195 of the SP-22 protein
(9), was synthesized and conjugated with hemocyanin. Female Japanese
white rabbits were subcutaneously immunized with the conjugated peptide
(500 µg) emulsified with adjuvant (Titer Max, Sigma). The first
booster injection (250 µg) was given 4 weeks later, and this was
followed by three booster injections (250 µg each) at 2-week
intervals. Sera were obtained 2 weeks after the last booster injection.
Immunocytochemistry--
For immunofluorescence microscopy, KB
cells grown on coverslips in minimal essential medium/10% FCS were
fixed with 4% paraformaldehyde in phosphate-buffered saline and
permeabilized with 0.1% saponine. The permeabilized cells were reacted
with anti SP-22 antibody and then stained with rhodamine-conjugated
second antibody. Electron immunocytochemistry of bovine mitochondria
was carried out as described previously (18). The crude mitochondrial
fraction of KB cells was pelleted, fixed with 8% paraformaldehyde in
0.1 M phosphate buffer, pH 7.4, and embedded in LR white
resin at 50 °C. Thin sections of the mitochondria were incubated
with the antibody against the SP-22, and then incubated with
anti-rabbit IgG-gold. Immunolabeled sections were then stained with
uranyl acetate and lead citrate and examined under a Hitachi HU 12 electron microscope at 100 kV. Control experiments done with preimmune serum gave no immunoreactive signals.
Superoxide Production in BAEC Submitochondrial
Particles--
BAEC submitochondrial particles (SMP) were prepared
essentially as described by Kang et al. (18). Briefly, BAEC
were washed in an isotonic sucrose buffer composed of 10 mM
Tris-HCl, pH 7.4, 1 mM EDTA, 0.25 M sucrose, 15 µg/ml leupeptin, 5 µg/ml
(p-amidinophenyl)methanesulfonyl fluoride hydrochloride, and
50 ng/ml pepstatin, and suspended in the same buffer (1 × 108 cells/ml). The cells were homogenized in a
Potter-Elvehjem homogenizer and centrifuged twice at 600 × g for 10 min to obtain post-nuclear supernatant. The
post-nuclear supernatant was centrifuged at 7000 × g
for 10 min. The pellet (crude mitochondrial fraction) was sonicated and
centrifuged at 320,000 × g for 1 h. The resultant pellet was homogenized in an isotonic sucrose buffer and served as the
SMP fraction. Superoxide production by SMP was determined by the
oxidation of adrenaline to adrenochrome, and was corrected by
subtracting the rate in the presence of 10 µg/ml superoxide dismutase
as described by Takeshige et al. (19).
Immunoblotting Analysis--
SP-22 protein of BAEC was
determined by immunoblotting. Proteins (20 µg) from each sample of
BAEC were separated by 15% SDS-polyacrylamide gel electrophoresis
(PAGE), electrotransferred onto Immobilon P (Millipore), and probed
with anti-SP-22 serum. Immunoreactive proteins were detected using
horseradish peroxidase-conjugated goat anti-rabbit antibody and POD
Immunostain (Wako, Osaka, Japan).
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)--
Semiquantitative RT-RCR was performed to quantitate
SP-22 mRNAs in oxidant-treated BAEC, as described previously (17).
Briefly, total RNA was isolated from confluent BAEC using TRIzol
reagent (Life Technologies, Inc.) based on the method reported by
Chomczynski and Sacchi (20). RT reactions were carried out with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.).
The reverse-transcribed cDNA products were amplified with Taq DNA polymerase in a PJ2000 DNA thermal cycler
(Perkin-Elmer). The conditions for each cycle were 94 °C for 45 s, 55 °C for 1 min, and 72 °C for 2 min (31 cycles). The
following gene-specific primers for SP-22 were used:
5'-tcacgatgtgaactgcgaagttg-3'(bases 366-388, sense) and
5'-ggatggggcttgattgtaggag-3'(bases 710-731, antisense), which
amplified a 366-base pair product. Each primer set yielded a single PCR
product of the predicted size. The identity of the PCR products was
confirmed by direct cycle sequencing. RT-PCR was also performed for the
housekeeping gene
-actin as a control for the amount of RNA used in
the RT reaction. A negative control, in which reverse transcriptase was
omitted, was also performed to exclude the possibility of the
amplification of contaminating genomic DNA. Linear relationships were
observed between the quantity of RNA subjected to the RT reaction and
the amount of amplified PCR product under the PCR conditions used for
SP-22 (31 cycles) and
-actin (24 cycles) (Fig.
1).

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Fig. 1.
Linearity of semiquantitative RT-PCR.
Semiquantitative RT-PCR showed a linear relationship between the amount
of total RNA in the RT reaction and that of the PCR products. The
correlation coefficients for SP-22 and -actin products were 0.992 and 0.994, respectively.
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Northern Blotting Analysis--
SP-22 PCR products (366 base
pairs) were subcloned into the pGEM-T Easy Vector (Promega) by means of
the TA cloning technique. After confirming the direction of the PCR
insert by sequencing, the plasmids with inserts were linearized with
BSP 120I (Fermentas Ltd., Vilnius, Lithuania) and
transcribed in vitro into digoxigenin (DIG)-labeled cRNA
with SP6 RNA polymerase using a DIG RNA labeling kit (Boehringer Mannheim).
Total RNA (5 µg) was dissolved in 12 µl of sample buffer containing
10 mM sodium phosphate, pH 7.0, 50% (w/v) dimethyl
sulfoxide, and 1 M glyoxal. After denaturation (1 h at
50 °C), the samples were electrophoresed in 1% agarose gel with a
10 mM sodium phosphate buffer, and then transferred to a
nylon membrane (Boehringer Mannheim) and immobilized by incubation for
30 min at 121 °C. Hybridization with the DIG-labeled cRNA probe was
carried out overnight at 68 °C in 500 mM sodium
phosphate, 7% SDS, 1 mM EDTA, and 1 mg/ml yeast tRNA (21).
Blots were washed twice at room temperature with 200 mM
sodium phosphate, 5% SDS, and 1 mM EDTA, and for 15 min at
65 °C with 0.2× SSC plus 0.2% SDS before color reaction using a
DIG nucleic acid detection kit (Boehringer Mannheim) according to the
instructions provided by the supplier. The amount of SP-22 mRNA was
normalized by 18 S ribosomal RNA.
Preconditioning of BAEC with a Mild Oxidant--
BAEC were
preincubated with 3.3 µM Fe2+, 330 µM dithiothreitol (DTT) for 24 h, which maximally
increased the expression of SP-22 protein. These treated BAEC were then
exposed to more intense oxidative stress consisting of 3.3 µM Fe2+, 1 mM DTT for 24 h,
and cell viabilities were evaluated by measuring the lactate
dehydrogenase activities of the cell lysate.
Antisense Oligodeoxynucleotide--
A 20-mer antisense
phosphorothioate oligodeoxynucleotide (ODN) was synthesized by Toagosei
Inc. Japan. The first antisense ODN sequence (antisense 1;
5'-catcttcgttatgcagggct-3') is directed against the translation
initiation region of the SP-22 mRNA. The second antisense ODN
(antisense 2; 5'-ccttcaccaagcggagggtc-3') is directed against the
internal region of SP-22 mRNA. We also prepared a sense ODN of
antisense 1 and 2, and several random ODNs for a control experiment.
All of these ODNs were designed to not possess sequences homologous to
other genomic sequences or strong secondary structures (22). For
efficient transfection of BAEC, we used the cationic lipid Tfx-50
reagent (Promega). ODNs that had been diluted in 30 mM
HEPES buffer, pH 7.4 (final concentration of 3.3 µM), and
5 µl of Tfx-50 reagent (final concentration of 5 µM),
which was prepared according to the instructions provided by the
supplier, were mixed and incubated for 15 min at room temperature. The
ODN/Tfx-50 reagent mixture was added to each plate with 1 ml of RPMI
1640 medium without FCS, and the cells were incubated for 1 h at
37 °C in a 5% CO2 atmosphere. At the end of the
incubation period, the cells were gently overlaid with 2 ml of RPMI
1640 medium containing 10% FCS. This process was performed twice more at 24-h intervals.
Experimental Myocardial Infarction and
Immunocytochemistry--
Adult male Wistar rats weighing about
250 g were used for these experiments. The experiments were
performed in accordance with the guidelines specified for institutional
animal care and use of the University of Occupational and Environmental
Health, Japan. The animals were anesthetized and ventilated by a
Harvard small animal respirator. The left descending coronary artery
was ligated via a left-sided thoracotomy, which results in infarction of the free left ventricular wall (23). In a sham procedure, a
superficial suture was placed in the epicardium of the left ventricle,
near the left descending coronary artery. A total of 9 infarcted rats
and 9 sham-operated rats (n = 3 per time point) were
killed either 1 or 2 days after surgery. The heart was frozen in liquid
nitrogen and stored at
80 °C. The hearts from the sham group and
infarct group, at 1 or 2 days after the operation (n = 3), were cut and the sections were fixed with acetone for 5 min.
Endogenous peroxidase was then blocked with 0.3%
H2O2 in methanol. The thin sections were
reacted with the anti-SP-22 polyclonal antibody and anti-HNE monoclonal
antibody, and stained by a Vectastain® Elite
ABC kit (Vector Laboratories, Burlingame, CA). The sections were
counterstained with hematoxylin.
Other Procedures--
Protein concentrations were determined by
the Bio-Rad protein assay kit using bovine serum albumin as a standard.
The intensity of the stained bands in immunoblotting, Northern
blotting, and RT-PCR was quantitated by densitometric analysis using
the public domain computer program NIH Image (Wayne Rasband, NIH,
Research Service Branch, National Institute of Mental Health, Bethesda, MD), and the results are expressed as means ± S.E. A statistical analysis was performed using Student's t-test. Differences
were considered statistically significant at p < 0.05.
 |
RESULTS |
Immunological Detection of SP-22--
We prepared an antibody
against SP-22 protein and used it for immunological analysis. In
immunoblotting, this antibody detected a single band with a molecular
mass corresponding to SP-22 (22 kDa) in total homogenate and the
mitochondrial fraction of BAEC, but not in the cytosol fraction (Fig.
2A). Intracellular
distribution of SP-22 protein was further examined by
immunofluorescence microscopy. When cultured KB cells were stained with
the anti-SP-22 antibody, the signals of immunoreactive SP-22 exhibited
the mitochondrial staining pattern characterized by a reticular
staining appearance (Fig. 2B). Immunostaining signals were
hardly visible when preimmune sera for SP-22 were used. The
localization of the SP-22 protein in the mitochondria was further
confirmed by electron microscopic immunocytochemistry. The
mitochondrial fraction isolated from KB cells was stained with the
anti-SP-22 antibody, followed by gold-labeled second antibody. Proteins
reactive to the antibody were located in the mitochondria (Fig.
2C). These results indicate that SP-22 protein is located in
mitochondria of culture cells, which is consistent with the results of
the biochemical analysis by Watabe et al. (8).

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Fig. 2.
Immunological analysis of subcellular
localization of SP-22. A, proteins in the total
homogenate and subcellular fractions of BAEC were separated on 15%
SDS-PAGE and immunoblotted with the anti-SP-22 antibody
(lanes 2-4). Lane 1, total
BAEC homogenate stained by Coomassie Brilliant Blue; lane
2, total homogenate; lane 3, cytosol;
lane 4, crude mitochondria. Twenty µg of
protein was applied to lanes 1 and 2,
and 60 µg was applied to lanes 3 and
4. Molecular size standards are indicated on the
left (in kDa). B, cultured KB cells were stained
with the anti-SP-22 antibody and examined by immunofluorescence
microscopy as described under "Experimental Procedures."
C, isolated mitochondria from KB cells were reacted with the
anti-SP-22 antibody and then stained with colloidal gold-conjugated
second antibody for immunoelectron microscopy as described under
"Experimental Procedures." The bars in B and
C indicate 10 and 0.2 µm, respectively.
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Induction of the SP-22 Protein by Oxidative Stress--
Exposing
BAEC to Fe2+/DTT, which produces the hydroxyl radical
through the Fenton reaction (24, 25), increased the expression of SP-22
protein in a time-dependent manner, with the maximal elevation occurring after 24 h of exposure. On the other hand, the
expression of MnSOD was induced only slightly in this condition (Fig.
3). SP-22 expression was completely
suppressed with Fe chelators, such as deferoxamine and diethylene
triamineacetic acid (Table I). Table
II summarizes the effects of various
oxidative stresses on the induction of SP-22 protein in BAEC. There was
no apparent difference in the induction profile specific to ROI
species. Peroxides such as hydrogen peroxide,
tert-butylhydroperoxide, and cummene hydroperoxide enhanced
SP-22 protein about 1.5-2.3-fold. Glucose oxidase and
xanthine/xanthine oxidase, which produce hydrogen peroxide and
superoxide (26, 27), enhanced SP-22 protein by about 40% increase.
Sodium arsenite and cadmium chloride, which interact with sulfhydryl
groups (28), also enhanced SP-22 protein by about 50-100% increase.
However, diethylmaleate and buthionine sulfoximine, which are
glutathione depletors that have been shown to induce other
thioredoxindependent peroxide reductase proteins (12),
exhibited a rather poor induction of SP-22 protein. Rotenone, paraquat,
1-methyl-4-phenylpyridinium, antimycin A, and KCN, which are
respiratory chain inhibitors that increase ROI generation in
mitochondria (29), also enhanced SP-22 protein by 1.6-3.2-fold. ROI
generation in SMP treated with antimycin A and rotenone showed 40-100% increase compared with untreated SMP (Table
III). Based on the rate of superoxide
generation by antimycin A-treated SMP (4.1 nmol/min/mg protein), the
antimycin A-treated confluent BAEC (107 cells) produced
superoxide roughly at a rate of 0.13 nmol/min/mg protein (assuming that
the protein content of SMP in 107 cells is about 0.065 mg),
which is about 800-fold lower than the rate of superoxide generation
produced by exogenous xanthine/xanthine oxidase (50 milliunits/ml),
whereas the SP-22 protein induced by antimycin A treatment was about
3-fold higher than that induced by the xanthine/xanthine oxidase
treatment, suggesting that superoxide production in mitochondria
induced SP-22 expression more effectively.

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Fig. 3.
Induction of SP-22 and MnSOD proteins by
Fe2+/DTT in BAEC. BAEC were incubated with 3.3 µM Fe2+, 500 µM DTT for 0, 3, 6, 12, 24, or 48 h. Proteins of BAEC were separated on 15%
SDS-PAGE and immunoblotted with the anti-SP-22 antibody
(upper) and anti-MnSOD antibody (lower). SP-22
and MnSOD in each sample were quantified and are expressed as intensity
of the stained band/mg of protein (arbitrary units). Twenty µg
(upper) and 40 µg (lower) of protein were
applied to each lane, respectively.
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Table I
Induction of SP-22 protein in BAEC exposed to the Fe2+/DTT
system
BAEC were incubated for 24 h with Fe2+/DTT. Proteins of
BAEC total homogenate were separated on 15% SDS-PAGE and immunoblotted
with the anti-SP-22 antibody. Twenty µg of each protein were applied
to each lane. Quantitative data were obtained by a densitometric
analysis of the stained bands. The abundance of SP-22 protein is
expressed as a ratio relative to that of the control BAEC (defined as
1.0). DTT, dithiothreitol; DETAPAC, diethylenetriamineacetic acid.
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Table II
Induction of SP-22 protein in BAEC exposed to various oxidative
stresses
BAEC were incubated with various oxidative stresses. Proteins of BAEC
total homogenate were separated on 15% SDS-PAGE and immunoblotted with
the anti-SP-22 antibody. Twenty µg of each protein were applied to
each lane. Quantitative data were obtained by a densitometric analysis
of the stained bands. The abundance of SP-22 protein is expressed as a
ratio relative to that of the control BAEC (defined as 1.0). The data
reflect the most significant increase in SP-22 protein after
incubation. Incubation times were 24 h except with antimycin A and
KCN. MPP+, 1-methyl-4-phenylpyridinium.
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Table III
Generation of superoxide by SMP treated with respiratory chain
inhibitors
SMP were prepared from BAEC as described under "Experimental
Procedures." Superoxide production by SMP was determined by the
oxidation of adrenaline to adrenochrome and corrected by subtracting
the rate in the presence of 10 µg/ml superoxide dismutase, as
described by Takeshige et al. (19). The control system
contained 0.5 mg/ml SMP and 1 mM adrenaline, 0.25 M sucrose, and 50 mM Hepes/NaOH, pH 7.5, in 1 ml. After preincubation for 5 min, the reaction was started under the
conditions indicated adding of 0.2 mM NADPH, and
adrenochrome formation was determined at 485-575 nm by a
dual-wavelength spectrophotometer. The results represent the means ± S.E. of three separate experiments.
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Respiratory chain inhibitors also deplete ATP by blocking oxidative
energy metabolism in mitochondria (30, 31). To examine the possibility
that the stress of ATP depletion may enhance SP-22 expression, we
compared the protein levels of SP-22 between BAECs grown in medium that
contained either 5 mM glucose or 20 mM
2-deoxyglucose (a glycolytic inhibitor) instead of glucose. Six hours
after the addition of glucose or 2-deoxyglucose, the ATP level of BAEC
incubated with 20 mM 2-deoxyglucose was reduced to
48.1 ± 7.8% of that in the control (with 5 mM
glucose). The expression of SP-22 protein did not differ between two
different media (103 ± 4%, in 20 mM 2-deoxyglucose
medium versus 5 mM glucose). This result
suggests that ATP depletion per se does not influence the
induction of SP-22 expression in BAEC.
Induction of SP-22 mRNA--
The induction of SP-22 protein in
BAEC exposed to oxidative stress was also examined in terms of the
mRNA level. Total RNA was obtained from BAEC exposed to oxidants
for 0, 0.5, 1, 3, 6, and 24 h, and the amount of SP-22 mRNA
was analyzed by Northern blotting and semiquantitative RT-PCR. In
Northern blotting, SP-22 mRNA began to increase after 0.5 h of
exposure to Fe2+/DTT or antimycin A, reached a maximal
level at 3 h, and decreased to the initial level after 24 h
(Fig. 4A). In semiquantitative RT-PCR, almost the same results were obtained (Fig. 4B). To
investigate the mechanism of the apparent induction of SP-22 in
antimycin A-treated BAEC, we examined the effect of actinomycin D on
the increase in SP-22 mRNA expression after antimycin A treatment. As shown in Fig. 4C, actinomycin D suppressed the antimycin
A-mediated increase in SP-22 mRNA expression to the control level.
This result indicates that the observed increase in SP-22 mRNA was
due at least in part to the transcriptional activation of SP-22
mRNA.

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Fig. 4.
Induction of SP-22 and MnSOD mRNAs by
antimycin A and Fe2+/DTT. A and
B, BAEC were incubated with antimycin A (10 µM) for 0, 0.5, 1, 3, 6, and 24 h (lanes
1-6, respectively), or 3.3 µM
Fe2+, 500 µM DTT for 0 and 3 h
(lanes 7 and 8, respectively).
A, 5 µg of total RNA per lane were electrophoresed,
transferred to a nylon membrane, and hybridized as described under
"Experimental Procedures." B, SP-22 mRNA expression
was detected by semiquantitative RT-PCR as described under
"Experimental Procedures." The abundance of SP-22 PCR product
corrected for that of -actin is expressed as a ratio relative to the
level at 0 h (defined as 1.0). Quantitative data from the
densitometric analysis of the stained bands from three independent
experiments are shown as means ± S.E. C, BAEC were
pretreated with actinomycin D (1 µg/ml) or vehicle for 2 h
before the addition of antimycin A (10 µM). SP-22
mRNA expression was detected by semiquantitative RT-PCR as
described above. The values are expressed as a ratio relative to the
level of control (defined as 1.0). Act. D,
actinomycin D; Anti. A, antimycin A.
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Effect of Preconditioning with a Low Dose of Oxidant--
The
pretreatment of BAEC with a low dose of oxidant (3.3 µM
Fe2+, 330 µM DTT), which did not affect cell
viability, increased the level of SP-22 protein about 3.3-fold compared
with that in control cells (Fig.
5A). In contrast to the
untreated control, the cell viability of pretreated BAEC did not
markedly decrease with subsequent treatment with a high dose of oxidant
(Fig. 5B). These results indicate that BAEC in which SP-22
protein had been induced by preconditioning with a mild oxidant became
tolerant to subsequent intense oxidative attack.

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Fig. 5.
Effect of preconditioning with a low dose of
oxidant. A, BAEC were either pretreated with
3.3 µM Fe2+, 330 µM DTT for
24 h or left untreated. Proteins of BAEC total homogenate were
separated on 15% SDS-PAGE and immunoblotted with the anti-SP-22
antibody. SP-22 in each sample was quantified and is expressed as the
intensity of the stained band/mg of protein (arbitrary units). Twenty
µg of protein was applied to each lane. B, before BAEC
were exposed to a high dose of oxidant (3.3 µM
Fe2+, 1 mM DTT) for 24 h, the cells were
pretreated with a low dose of oxidant (3.3 µM
Fe2+, 330 µM DTT) for 24 h. Cell
viabilities are expressed by a ratio relative to the level at 0 h
(defined as 1.0) in terms of the lactate dehydrogenase activities of
BAEC total homogenates. Data are shown as the means ± S.E. of
three separate experiments.
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Antisense ODN for SP-22--
We examined the cell viability of
BAEC with a decreased level of SP-22 due to the treatment with
antisense ODNs against SP-22 mRNA. After BAEC had been exposed to
SP-22 antisense ODNs three times, the protein levels of SP-22 in BAEC
were examined by immunoblotting. The amount of SP-22 in BAEC treated
with antisense 1 and 2 decreased to 43.9% and 72.1%, respectively, of
that in control cells, whereas that of the sense ODN-treated BAEC
remained close to the level in the control (Fig.
6A). As shown in Fig.
6B, oxidative stress (3.3 µM Fe2+,
750 µM DTT) markedly decreased the cell viability of the
antisense 1 ODN-treated BAEC (about 77% decrease), whereas the same
oxidative stress produced only a slight decrease (~20%) in cell
viability for the control and sense ODN-treated cells. Treatment of
BAEC with antisense or sense ODNs by itself did not appreciably affect cell viability (Fig. 6B). These results indicate that BAEC
with a lower level of SP-22 protein were more susceptible to oxidative stress.

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Fig. 6.
Depletion of SP-22 by treatment with
antisense ODN. A, BAEC were treated three times with an
antisense ODN/Tfx-50 reagent mixture for 24 h as described under
"Experimental Procedures." Proteins of BAEC were separated on 15%
SDS-PAGE and immunoblotted with the anti-SP-22 antibody. SP-22 in each
fraction was quantified and is expressed as the intensity of the
stained band/mg of protein (arbitrary units). Thirty-five µg of
protein was applied to each lane. B, after BAEC were treated
with ODNs, they were exposed to oxidative stress (3.3 µM
Fe2+, 750 µM DTT) for 18 h. Cell
viabilities were expressed by a ratio relative to the level at 0 h
(defined as 1.0) in terms of the lactate dehydrogenase activities of
BAEC total homogenates. Data are shown as the means ± S.E. of
three separate experiments.
|
|
Induction of SP-22 in Infarcted Rat Heart--
To examine the
in vivo induction of SP-22 protein, an immunohistochemical
study was carried out using an experimental model of myocardial
infarction in rat heart. Immunohistochemistry with anti-SP-22 and
anti-HNE-protein adducts (32) showed that immunoreactive signals of
both antibodies were markedly increased in the infarcted zone of the
rat heart (Fig. 7, B and
C), indicating that lipid peroxidation occurred in the
infarct zone and SP-22 in rat heart was specifically induced
corresponding to the area exposed to oxidative stress. In sham-operated
animals, immunopositive signals against both antibodies were less
evident, and there were no differences between the infarct and
non-infarct zones (data not shown).

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|
Fig. 7.
Induction of SP-22 proteins in rat ischemic
myocardium. Immunohistology of rat infarcted heart at 24 h
postocclusion of the left ascending artery. Serial sections were
labeled with nonimmune rabbit serum (A), anti-SP-22 antibody
(B), or monoclonal anti-HNE antibody (C) as
described under "Experimental Procedures." The infarcted myocardium
at 24 h postocclusion (left) can be recognized by
staining of the necrotic myocardial fibers (hematoxylin stain; original
magnification, ×25). Immunopositive staining is indicated by a
dark brown color (3,3'-diaminobenzidine).
|
|
 |
DISCUSSION |
In cardiovascular systems, including the heart and vascular
endothelial cells, mitochondria are particularly important for generating ATP. At the same time, the mitochondrial electron transport system consumes a great deal of oxygen and produces ROIs (1, 2). The
generation of ROIs in cardiovascular systems has harmful effects on
tissues and leads to atherosclerosis (33), ischemia reperfusion injury
(34), and hypertension (35). ROIs are removed by various scavenging
systems, such as CuZnSOD or MnSOD (36), glutathione peroxidase (37),
and catalase (38). SOD dismutates superoxide radical and produces
hydrogen peroxide. Although catalase removes hydrogen peroxide in the
cytosolic compartment, there has been no report on mitochondrial
catalase, except for that in rat heart mitochondria (39). SP-22 is a
member of the thioredoxin-dependent peroxide reductase
family (11), and by accepting electrons from the
NADPH-thioredoxin-thioredoxin reductase system, it removes peroxides
in vitro (15). In this study, the induction of SP-22 mRNA and protein were enhanced by various oxidative stresses, including mitochondrial respiratory chain inhibitors, in vascular endothelial cells. It is well known that respiratory chain inhibitors increase ROIs in mitochondria by blocking electron transport (29, 30),
and we confirmed that antimycin A and rotenone actually increased
superoxide production in submitochondrial particles of BAEC. The result
that intramitochondrial ROI generation may be more effective for
enhancing the expression of SP-22 protein than the extracellular
administration of an oxidative stressor can be explained by supposing
that a redox sensor, which exists in or near mitochondria, specifically
responds to intramitochondrial ROIs. Aconitase, an iron-sulfur protein
located in both cytosol and mitochondria, has been reported to serves
as the redox sensor and play a regulatory role in gene expression
(40).
The cytosolic members of the thioredoxin-dependent peroxide
reductase family have been reported to be induced by oxidative stresses. MSP23 is inducibly expressed when mouse peritoneal
macrophages are exposed to diethylmaleate (12). This expression has
also been reported to be enhanced by exposure to oxidized low density lipoprotein, hydrogen peroxide, and heavy metals (12, 41, 42).
Thiol-specific antioxidant from yeast is also induced by high
concentrations of mercaptoethanol, iron and oxygen (43). Yeast
thiol-specific antioxidant exerts a protective effect in vitro on mitochondrial oxidative damage (7). Regarding protein induction by oxidative stresses, SP-22 seems to be induced in a manner
similar to other cytosolic members of the
thioredoxin-dependent peroxide reductase family, except for
its response to sulfhydryl-reactive agents. Diethylmaleate and
buthionine sulfoximine induce the expression of MSP23 in the cytosol
(12), but not of SP-22 protein in the mitochondria. This discrepancy
could be explained by the difference of the subcellular localization of
the two proteins. Extracellularly added sulfhydryl agents has been
reported to exert a different effect on the redox states of glutathione
in the cytosol and mitochondria (44).
It is well known that MnSOD (45) and glutathione peroxidase (37)
function as defensive enzymes against ROIs in mitochondria. Therefore,
we were interested in the relative contribution 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 radical-generating
system, Fe2+/DTT, which only slightly induced MnSOD (Fig.
3), acquired tolerance to a higher concentration of
Fe2+/DTT (Fig. 5). Furthermore, BAEC that had been depleted
of SP-22 by antisense phosphorothioate ODN, were labile to the
Fe2+/DTT system (Fig. 7). These results suggest that SP-22
plays an integral role in antioxidant systems of BAEC. We found that
thioredoxin and thioredoxin reductase, which participate in a cellular
antioxidant defense (46) and are essential for SP-22 activity, are also located in mitochondria of
BAEC.2
Previous reports have mentioned that some cytokines such as tumor
necrosis factor-
and
(TNF-
and
) and interleukin-1
and
regulated the expression of MnSOD (47). Shull et al.
reported xanthine/xanthine oxidase increased MnSOD mRNA in
epithelial cells without induction of catalase, CuZnSOD, and
glutathione peroxidase mRNAs (48). Yamamoto et al.
reported that the promotor region of murine MER5 protein (homolog of
bovine SP-22) had sites for several DNA-binding proteins (AP1, AP2,
SP1) and MER5 protein was enhanced after induction of differentiation
by dimethyl sulfoxide (14). Kang et al. recently reported
overexpression of peroxiredoxin II (thioredoxin-dependent
peroxide reductase) prevented the TNF-
-induced NF
B activation by
removing intracellular hydrogen peroxide (49). These results suggested
that the expression of cellular antioxidant enzymes, such as the
thioredoxin-dependent peroxide reductase and MnSOD, can be
regulated by a cytokine/ROI-mediated signal transduction. We also
observed SP-22 mRNA was induced by
TNF-
.3
In vivo, the expression of SP-22 was enhanced in an
experimental model of rat myocardial infarction. The induction of SP-22 expression peaked at 24 h after ligation, and rapidly decreased at
48 h when necrosis reached a peak. At 24 h after coronary
occlusion, the process of infarction is still incomplete in the rat
heart (23). With the progression of ischemia, ROIs such as superoxide, hydrogen peroxide, and hydroxyl radical are generated through several
pathways, including the leakage of electrons from mitochondria, which
are especially abundant in the heart (29, 50), the metabolism of
arachidonic acid released by activated phospholipases (51), and the
increase in xanthine oxidase activity, which produces superoxide (52).
It has been reported that exogenous SOD and/or catalase reduce the
extent of myocardial infarction (53, 54), suggesting that they may
protect against the progression of myocardial infarction. The present
results obtained in experimental myocardial infarction also suggest
that SP-22 may participate in protecting against myocardial infarction.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Hachisuka (Department
of Rehabilitation Medicine, University of Occupational and
Environmental Health, Kitakyushu, Japan) for technical advice and T. Omae-Sakimura for superb technical assistance.
 |
FOOTNOTES |
*
This work was presented in part at the 71st Scientific
Sessions of the American Heart Association, November 11, 1998, Dallas, TX.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Department of
Health Development, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, 807, Japan. Tel.:
81-93-691-7473; Fax: 81-93-602-6395; E-mail:
nanrih{at}med.uoeh-u.ac.jp.
The abbreviations used are:
ROI, reactive oxygen
intermediate; SP-22, 22-kDa substrate protein; MSP23, 23-kDa macrophage
stress protein; BAEC, bovine aortic endothelial cells; MnSOD, manganese
superoxide dismutase; HNE, 4-hydroxy-2-nonenal; SMP, submitochondrial
particles; RT, reverse transcription; PCR, polymerase chain reaction; DIG, digoxigenin; DTT, dithiothreitol; ODN, oligodeoxynucleotide; CuZnSOD, copper and zinc superoxide dismutase; SOD, superoxide
dismutase; TNF, tumor necrosis factor; PAGE, polyacrylamide gel
electrophoresis; FCS, fetal calf serum.
2
K. Ejima and H. Nanri, submitted for publication.
3
M. Araki and H. Nanri, unpublished results.
 |
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