Oxidative Stress Is Associated With XIAP and Smac/DIABLO Signaling Pathways in Mouse Brains After Transient Focal Cerebral Ischemia
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Oxidative Stress Is Associated With XIAP and
Smac/DIABLO Signaling Pathways in Mouse Brains After
Transient Focal Cerebral Ischemia
Atsushi Saito, MD; Takeshi Hayashi, MD, PhD; Shuzo Okuno, MD;
Tatsuro Nishi, MD, PhD; Pak H. Chan, PhD
Background and Purpose—The interaction of X chromosome-linked inhibitor-of-apoptosis protein (XIAP) with second
mitochondria-derived activator of caspase (Smac)/direct inhibitor-of-apoptosis protein-binding protein with low pI
(DIABLO) contributes to regulation of apoptosis after a variety of cell death stimuli, and in our reported in vivo transient
focal cerebral ischemia (tFCI) model. We have also reported that overexpression of copper/zinc superoxide dismutase
(SOD1) reduces apoptotic cell death after tFCI. Our present study was designed to clarify the relationship between the
XIAP signaling pathway and oxidative stress in the regulation of apoptosis after tFCI.
Methods—We used a tFCI model of SOD1 transgenic mice and wild-type littermates to examine the expression of XIAP
and Smac/DIABLO by Western blotting and the interaction of XIAP with Smac/DIABLO (XIAP/Smac) or caspase-9
(XIAP/caspase-9) by coimmunoprecipitation. The direct oxidation of carbonyl groups, an indication of oxidative injury
to total and individual proteins caused by tFCI, was examined using a 2,4-dinitrophenylhydrazone reaction assay.
Results—Direct oxidative injury to cytosolic and mitochondrial proteins was reduced by SOD1 after tFCI. The individual
oxidized carbonyls in XIAP, mitochondrial Smac/DIABLO, and caspase-9 were also reduced by SOD1. Expression of
XIAP and XIAP/caspase-9 was promoted, whereas translocation of Smac/DIABLO and XIAP/Smac was reduced, by
SOD1 after tFCI.
Conclusions—These results suggest that overexpression of SOD1 may affect the XIAP pathway after tFCI by reducing the
direct oxidative reaction to XIAP regulators after reperfusion injury. (Stroke. 2004;35:1443-1448.)
Key Words: cerebral ischemia 䡲 superoxide dismutase 䡲 apoptosis
T he apoptotic signaling pathway is regulated by a variety
of factors and is based on the balance between cell death
and survival factors.1,2 The central players in apoptosis are the
BIR domain of XIAP in particular inhibits cleaved
caspase-9.15,16 We have shown that the interaction of XIAP
with Smac/DIABLO and the caspases plays a critical role
caspases, and one important route to caspase activation in the regulation of apoptotic neuronal cell death after in
involves the translocation of cytochrome c from mitochondria vivo cerebral ischemia.11
to the cytosol.3,4 An apoptosome forms when released cyto- Reactive oxygen species have been implicated in the
chrome c interacts with Apaf-1 and caspase-9, both of which mechanism of reperfusion injury after cerebral ische-
play essential roles in the mitochondrial apoptotic pathway.5 mia.17,18 The electron flow in isolated brain mitochondria
Second mitochondria-derived activator of caspases (Smac)/ produces superoxide anions, which are scavenged by
direct inhibitor-of-apoptosis protein (IAP)-binding protein superoxide dismutase (SOD).19 We have shown that
with low pI (DIABLO) is released from mitochondria into the copper/zinc-SOD (SOD1), a cytosolic isoenzyme, is highly
cytosol concurrently with cytochrome c and eliminates the protective against ischemia and reperfusion injury after
inhibitory effects of IAP.6 –11 ischemia.20,21 Our studies have demonstrated that SOD1
The IAP family includes cIAP1/2, X chromosome- affected the early release of cytochrome c and Smac/
linked IAP (XIAP), NIAP, and survivin.12 These proteins DIABLO from mitochondria in ischemia models in
bind to and inhibit both initiator caspases such as vivo.10,22 However, whether SOD1 affects IAP reaction
caspase-9 and effector caspases.13 All IAPs contain bacu- remains unknown. The present study was designed to
lovirus IAP repeat (BIR) domains.14 Among the IAPs, clarify the role of SOD1 in the XIAP pathway after
XIAP is the most potent inhibitor of caspases.12 The third transient focal cerebral ischemia (tFCI).
Received December 16, 2003; final revision received February 27, 2004; accepted March 10, 2004.
From the Department of Neurosurgery, Department of Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University
School of Medicine, Stanford, Calif.
Correspondence to Dr Pak H. Chan, Neurosurgical Laboratories, Stanford University, 1201 Welch Rd, MSLS, #P314, Stanford, CA 94305-5487.
E-mail phchan@stanford.edu
© 2004 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org DOI: 10.1161/01.STR.0000128416.28778.7a
1443
Downloaded from http://stroke.ahajournals.org/ by guest on May 21, 20151444 Stroke June 2004
Figure 1. Western blotting demonstrated
that XIAP immunoreactivity (molecular
weight [MW]⫽57 kDa) was stronger in the
SOD1 Tg mice than in the wild-type mice 8
hours after reperfusion (A) (*P⬍0.05). Trans-
located cytosolic Smac/DIABLO (MW⫽25
kDa) significantly decreased in the SOD1 Tg
mice compared with the wild-type mice 24
hours after tFCI (B) (*P⬍0.05). Coimmuno-
precipitation revealed that XIAP/Smac
(MW⫽25 kDa) was significantly reduced in
the SOD1 Tg mice compared with the wild-
type mice 8 hours after reperfusion (C)
(*P⬍0.05). XIAP/caspase-9 (MW⫽15 kDa)
significantly increased in the SOD1 Tg mice
compared with the wild-type mice 8 hours
after reperfusion (D) (*P⬍0.05). There was
no significant difference in the nonischemic
controls (cont) between the wild-type and
SOD1 Tg mice. Beta-actin was used as an
internal control. OD indicates optical den-
sity; wt, wild-type.
Materials and Methods were immunoblotted with anti-Smac/DIABLO antibody (Chemicon)
and anticaspase-9 antibody (Santa Cruz Biotechnology) as described
SOD1 Transgenic Mice in the Western blot method.
All animals were treated in accordance with Stanford University
guidelines and an animal care protocol approved by Stanford’s Detection of Oxidized Carbonyls in Protein
Administrative Panel on Laboratory Animal Care. Heterozygous A commercial detection assay was used with 2,4-dinitrophenylhydrazone
SOD1 transgenic (Tg) mice of the SOD1 TGHS/SF-218-3 strain with
(DNP) to examine the direct oxidative injury to proteins (Chemicon).
a CD-1 background and carrying human SOD1 genes with a 3-fold
The manufacturer’s methodological protocols were followed. DNP
increase in SOD1 were derived from the founder stock described
specifically reacted with the oxidized carbonyl residues on the
previously.17 There were no differences in the phenotypes or in the
protein samples.26 The DNP binding sites of the oxidized proteins
regional cerebral blood flow before or after FCI between the SOD1
were specifically detected using an anti-DNP antibody by Western
Tg mice and their wild-type littermates.23
blot or coimmunoprecipitation.26,27 We analyzed the density of each
lane by the total expression of DNP.
Focal Cerebral Ischemia
Adult male mice (3 months old, 35 to 40 g) were subjected to tFCI
by intraluminal middle cerebral artery blockade with a 6-0 nylon
Quantification and Statistical Analysis
suture as described previously.18,20 The mice were anesthetized with The data are expressed as mean⫾SD. We performed 1-way analysis
1.5% isoflurane in 30% oxygen and 70% nitrous oxide using a face of variance followed by Fisher protected least-significant difference
mask. After 60 minutes of middle cerebral artery occlusion, blood test for multiple-group analysis (SigmaStat software; Jandel Corpo-
flow was restored by withdrawal of the nylon suture. ration). Comparisons between 2 groups were achieved using Stu-
dent’s t test. Significance was accepted with P⬍0.05.
Western Blot Analysis
Protein extraction was performed to obtain the mitochondrial and Results
cytosolic fractions as described previously.16 Equal amounts of the
samples were loaded per lane. The primary antibodies were 1:1000 Effect of SOD1 Overexpression on the
dilution of antibody against Smac/DIABLO (Chemicon Interna- XIAP Pathway
tional), 1:1000 dilution of antibody against XIAP (BD Transduction In our previous study, XIAP transiently increased 8 hours
Laboratories), or 1:10 000 dilution of anti–-actin monoclonal anti- after tFCI and Smac/DIABLO translocated from mitochon-
body (Sigma Chemical Co). Western blots were performed with dria into the cytosol 24 hours after tFCI.11 The direct binding
horseradish peroxidase-conjugated immunoglobulin G (Cell Signal-
ing Technology) with the use of enhanced chemiluminescence of XIAP to both Smac/DIABLO (XIAP/Smac) and caspase-9
detection reagents (Amersham International). (XIAP/caspase-9) significantly increased 8 hours after tFCI.11
In the present study, we have now demonstrated that XIAP
Coimmunoprecipitation immunoreactivity was stronger in the SOD1 Tg mice than in
Protein extraction and immunoprecipitation were performed as the wild-type mice 8 hours after reperfusion (Figure 1A; n⫽4,
described previously.24,25 Two hundred micrograms of the protein
sample were incubated with protein G-Sepharose (Amersham Bio-
42.5%⫾30.8%; *P⬍0.05). Translocated Smac/DIABLO sig-
sciences) and with an anti-XIAP antibody (BD Transduction Labo- nificantly decreased in the SOD1 Tg mice compared with the
ratories) for 3 hours at 4°C. The pellets were used as the samples and wild-type mice 24 hours after tFCI (Figure 1B; n⫽4,
Downloaded from http://stroke.ahajournals.org/ by guest on May 21, 2015Saito et al Oxidative Stress Affects XIAP/Smac After Stroke 1445
50.5%⫾34.3%; *P⬍0.05). XIAP/Smac was significantly re-
duced in the SOD1 Tg mice compared with the wild-type
mice 8 hours after reperfusion (Figure 1C; n⫽4,
68.6%⫾28.8%; *P⬍0.05). XIAP/caspase-9 significantly in-
creased in the SOD1 Tg mice compared with the wild-type
mice 8 hours after reperfusion (Figure 1D; n⫽4,
70.5%⫾41.3%; *P⬍0.05).
Direct Oxidative Injury to Cytosolic and
Mitochondrial Proteins After tFCI
We used Western blot analysis of DNP to examine oxidative
injury to cytosolic and mitochondrial proteins by detecting
the DNP binding sites. Total oxidative injury to cytosolic
carbonyl proteins was reduced in the SOD1 Tg mice com-
pared with the wild-type mice 1, 4, and 24 hours after tFCI
(Figure 2A; 1 hour: n⫽4, 28.1%⫾13.7%, *P⬍0.05; 4 hours:
n⫽4, 28.9%⫾9.6%, *P⬍0.05; 24 hours: n⫽4, 40.2%⫾9.3%,
*P⬍0.05). Total oxidative injury to mitochondrial proteins
was reduced in the SOD1 Tg mice compared with the
wild-type mice 1, 4, and 24 hours after tFCI (Figure 2B; 1
hour: n⫽4, 31.2%⫾8.2%, *P⬍0.05; 4 hours: n⫽4,
32.9%⫾12.1%, *P⬍0.05; 24 hours: n⫽4, 59.0%⫾6.0%,
*P⬍0.05).
Direct Oxidative Injury to the Individual
Regulators of the XIAP/Smac Pathway
We examined oxidative injury to the individual proteins,
indicated by oxidation of the carbonyl groups, with the use of
samples precipitated by the DNP antibody after DNP binding.
XIAP expression in the proteins bound to DNP (XIAP/DNP)
remarkably increased 1 and 2 hours after tFCI (Figure 3A and
B; n⫽4, *P⬍0.05). Smac/DIABLO in the DNP-bound pro-
teins (Smac/DNP) was not detected in the cytosolic proteins
after tFCI (data not shown); however, in the mitochondrial
fraction, Smac/DNP was detected and increased 1 and 2 hours
after tFCI (Figure 3A and B; n⫽4, *P⬍0.05). Caspase-9 in
the DNP-bound proteins (caspase-9/DNP) significantly in-
creased 8 hours after tFCI (Figure 3A and B; n⫽4, *P⬍0.05).
SOD1 Overexpression Reduced Oxidative Injury
to the Individual Binding Proteins of the Figure 2. Western blot analysis revealed that oxidative injury to
XIAP Pathway the total cytosolic proteins was reduced in the SOD1 Tg mice
compared with the wild-type mice 1, 4, and 24 hours after tFCI
Cytosolic XIAP/DNP (n⫽4, 63.7%⫾15.4%, *P⬍0.05) and (A) (*P⬍0.05). The total oxidative injury to mitochondrial proteins
mitochondrial Smac/DNP (n⫽4, 73.8%⫾15.5%, *P⬍0.05) was reduced in the SOD1 Tg mice compared with the wild-type
were both significantly reduced in the SOD1 Tg mice mice 1, 4, and 24 hours after tFCI (B) (*P⬍0.05). Fifty micro-
compared with the wild-type mice 1 hour after tFCI (Figure grams of protein for the cytosolic samples and 20 g of protein
for the mitochondrial samples were used. The densitometry of
4A and B). Caspase-9/DNP was also reduced in the SOD1 Tg each smear band was calculated and quantitatively examined.
mice compared with the wild-type mice 8 hours after tFCI Cont indicates control.
(Figure 4C; n⫽4, 75.5%⫾32.5%, *P⬍0.05).
Discussion strated that the interaction among XIAP, Smac/DIABLO, and
IAPs are potent regulators of cell death and cell survival after caspases plays a critical role in regulation of apoptotic
a variety of apoptotic stimuli.9,12 XIAP interacts with other neuronal cell death after tFCI.11 However, how oxidative
protein regulators via dimerization, and BIR domains play a stress affects the XIAP pathway after reperfusion injury
critical role in protein binding.6,12 In the cytosol, Smac/ remains unknown. In the present study, we demonstrated the
DIABLO binds BIR domains of XIAP to eliminate any following points for the first time: (1) SOD1 overexpression
inhibitory effect and other BIR domains of XIAP bind the increased XIAP and reduced translocated Smac/DIABLO
initiator or the effector of caspase to inhibit them in many after tFCI; and (2) SOD1 overexpression reduced binding of
apoptotic models.8,9,28 In our previous study, we demon- XIAP to Smac/DIABLO and increased the interaction of
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Figure 3. Coimmunoprecipitation demonstrated that XIAP/DNP
remarkably increased 1 and 2 hours after tFCI (A, top row;
*P⬍0.05). Mitochondrial Smac/DNP remarkably increased 1 and
2 hours after tFCI (A, middle row; *P⬍0.05). Caspase-9/DNP
remarkably increased 8 hours after tFCI (A, bottom row;
*P⬍0.05). We confirmed equal amounts of the precipitated pro-
teins among all samples. The optical densities (OD) are demon-
strated and show the significant difference (B) (*P⬍0.05). P.C.
indicates positive control; N.C., negative control; sham, sham-
operated control sample. Figure 4. Coimmunoprecipitation revealed that XIAP/DNP was
significantly reduced in the SOD1 Tg mice compared with the
wild-type mice 1 hour after tFCI (A) (*P⬍0.05). Mitochondrial
Smac/DNP was significantly reduced in the SOD1 Tg mice com-
XIAP with caspase-9 after tFCI. We used methods for pared with the wild-type mice 2 hours after tFCI (B) (*P⬍0.05).
detecting DNP-binding sites on proteins to examine the Caspase-9/DNP was significantly reduced in the SOD1 Tg mice
compared with the wild-type mice 8 hours after tFCI (C)
targets of oxidative injury in the XIAP pathway and to find (*P⬍0.05). OD indicates optical density.
where SOD1 could protect against oxidative injury to the
XIAP pathway. With the DNP assays, we demonstrated the
following: (1) direct oxidative injuries to the cytosolic and was detected 1 hour after reperfusion in our model.18 The
mitochondrial proteins increased after tFCI, but were reduced total cytosolic and mitochondrial oxidized carbonyl proteins
by SOD1 overexpression; and (2) formation of oxidized
were shown as smear bands, the expression of which began to
carbonyls in XIAP, mitochondrial Smac/DIABLO, and
increase concurrently with superoxide production, which
caspase-9 was detected during the early period of reperfusion
matches results of our previous report.25 Each band changed
injury and these oxidized carbonyl proteins were all signifi-
in a variety of ways, some being time-dependent and others
cantly reduced by SOD1 overexpression after tFCI. The
precise physiological mechanisms of superoxide interaction not. These results suggest that total oxidation of proteins may
with the carbonyl residues of those individual proteins remain accumulate in a time-dependent manner; however, oxidative
unclear. Our results, however, suggest that overexpression of stress may not injure each protein uniformly and the injuries
SOD1 might inhibit oxidative injury to neuronal cells at the may progress at various time points. Apoptotic cell death
level of intracellular proteins after in vivo tFCI. induced by oxidative injury is known to proceed to chain
Our previous studies demonstrated that SOD1 overexpres- reactions. Our results suggest that not only an upstream
sion has protective effects against ischemic damage.18,20 –22 In substrate but also individual downstream substrates might be
this study, we examined the role of SOD1 in the XIAP directly injured by oxidative stress and that each individual
pathway after tFCI by focusing on the formation of oxidized protein injured by oxidative stress might be rescued by SOD.
carbonyls in the individual proteins. Superoxide production Protein oxidation was quantitatively analyzed by measuring
Downloaded from http://stroke.ahajournals.org/ by guest on May 21, 2015Saito et al Oxidative Stress Affects XIAP/Smac After Stroke 1447
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Downloaded from http://stroke.ahajournals.org/ by guest on May 21, 2015Oxidative Stress Is Associated With XIAP and Smac/DIABLO Signaling Pathways in
Mouse Brains After Transient Focal Cerebral Ischemia
Atsushi Saito, Takeshi Hayashi, Shuzo Okuno, Tatsuro Nishi and Pak H. Chan
Stroke. 2004;35:1443-1448; originally published online April 29, 2004;
doi: 10.1161/01.STR.0000128416.28778.7a
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