Activation of signal transducer and activator of transcription 3 protects cardiomyocytes from hypoxia/reoxygenation-induced oxidative stress through the upregulation of manganese superoxide dismutase.
ABSTRACT Mice with cardiac-specific overexpression of signal transducer and activator of transcription 3 (STAT3) are resistant to doxorubicin-induced damage. The STAT3 signal may be involved in the detoxification of reactive oxygen species (ROS).
The effects of leukemia inhibitory factor (LIF) or adenovirus-mediated transfection of constitutively activated STAT3 (caSTAT3) on the intracellular ROS formation induced by hypoxia/reoxygenation (H/R) were examined using rat neonatal cardiomyocytes. Either LIF treatment or caSTAT3 significantly suppressed the increase of H/R-induced ROS evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. To assess whether ROS are really involved in H/R-induced cardiomyocyte injury, the amount of creatine phosphokinase in cultured medium was examined. Both LIF treatment and caSTAT3 significantly decreased H/R-induced creatine phosphokinase release. These results indicate that the gp130/STAT3 signal protects H/R-induced cardiomyocyte injury by scavenging ROS generation. To investigate the mechanism of scavenging ROS, the effects of LIF on the induction of antioxidant enzymes were examined. LIF treatment significantly increased the expression of manganese superoxide dismutase (MnSOD) mRNA, whereas the expression of the catalase and glutathione peroxidase genes were unaffected. This induction of MnSOD mRNA expression was completely blocked by adenovirus-mediated transfection of dominant-negative STAT3. Moreover, caSTAT3 augmented MnSOD mRNA and its enzyme activity. In addition, the antisense oligodeoxyribonucleotide to MnSOD significantly inhibited both LIF and caSTAT3-mediated protective effects.
The activation of STAT3 induces a protective effect on H/R-induced cardiomyocyte damage, mainly by inducting MnSOD. The STAT3-mediated signal is proposed as a therapeutical target of ROS-induced cardiomyocyte injury.
Article: Oxidative stress and heart failure.[show abstract] [hide abstract]
ABSTRACT: Heart failure (HF) is a complex clinical syndrome whose pathogenesis involves an interplay of neurohormonal activation and inflammatory processes at the cellular and molecular levels. Oxidative stress describes an imbalance between antioxidant defense and the production of reactive oxygen species (ROS), which at high levels cause cell damage but at lower levels induce subtle changes in intracellular signaling pathways. Substantial evidence to date suggests the involvement of oxidative stress in the pathophysiology of HF as well as its antecedent conditions such as cardiac hypertrophy and adverse remodeling following myocardial infarction. Oxidative stress may indeed represent one of the common pathways for cell death/apoptosis, mitochondrial dysfunction, cardiac remodeling, and dysfunction. There is increasing evidence implying that ROS play an important role in the development and progression of HF. However, although levels of ROS are elevated in HF, the relative contribution of the different intracellular sources of ROS, the precise mechanisms and their impact on the progression of HF remain unclear. Further delineation of the downstream signaling pathways involved in ROS accumulation is important in order to improve understanding of these processes and also for the development of new therapies. Drugs such as the angiotensin-converting enzyme (ACE) inhibitors and the statins may act in part through such mechanisms. Despite disappointing results in using antioxidants in human studies, it is likely that modulation of endogenous antioxidants in human HF will continue to hold potential for both the treatment and prevention of HF.Coronary Artery Disease 05/2003; 14(2):109-13. · 1.11 Impact Factor
Article: Creatine kinase.[show abstract] [hide abstract]
ABSTRACT: Creatine kinase is present in significant concentrations in skeletal muscle and cardiac muscle and to a lesser extent in gastrointestinal tract and brain tissue. The enzyme has been purified from a variety of tissues and an examination of its kinetic and physical properties reveal that the enzyme consists of two subunits and can exist as three isoenzymes containing essential cysteine residues. These properties are important in understanding its stability, the assay conditions, and the techniques used to identify the different isoenzymes. The relationship between the properties and the determination of the enzyme in biological fluids will be a main thrust of the review. Creatine kinase activity in serum rises rapidly in conditions such as acute myocardial infarction and trauma to skeletal muscle. However, the interpretation of such increases is dependent upon a sound knowledge of the factors which influence both the total and isoenzyme activities. The nature of these factors will be discussed in detail.Critical Reviews in Clinical Laboratory Sciences 07/1982; 16(4):291-335. · 3.78 Impact Factor
Article: Stat3 as an oncogene.[show abstract] [hide abstract]
ABSTRACT: STATs are latent transcription factors that mediate cytokine- and growth factor-directed transcription. In many human cancers and transformed cell lines, Stat3 is persistently activated, and in cell culture, active Stat3 is either required for transformation, enhances transformation, or blocks apoptosis. We report that substitution of two cysteine residues within the C-terminal loop of the SH2 domain of Stat3 produces a molecule that dimerizes spontaneously, binds to DNA, and activates transcription. The Stat3-C molecule in immortalized fibroblasts causes cellular transformation scored by colony formation in soft agar and tumor formation in nude mice. Thus, the activated Stat3 molecule by itself can mediate cellular transformation and the experiments focus attention on the importance of constitutive Stat3 activation in human tumors.Cell 09/1999; 98(3):295-303. · 31.96 Impact Factor
Tadamitsu Kishimoto and Keiko Yamauchi-Takihara
L. Eizirik, Tomoaki Osugi, Masahiro Izumi, Yuichi Oshima, Yoshikazu Nakaoka, Hisao Hirota,
Shinji Negoro, Keita Kunisada, Yasushi Fujio, Masanobu Funamoto, Martine I. Darville, Décio
Upregulation of Manganese Superoxide Dismutase
Cardiomyocytes from Hypoxia/Reoxygenation-Induced Oxidative Stress Through the
Activation of Signal Transducer and Activator of Transcription 3 Protects
Print ISSN: 0009-7322. Online ISSN: 1524-4539
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Activation of Signal Transducer and Activator of
Transcription 3 Protects Cardiomyocytes from
Hypoxia/Reoxygenation-Induced Oxidative Stress Through
the Upregulation of Manganese Superoxide Dismutase
Shinji Negoro, MD, PhD; Keita Kunisada, MD, PhD; Yasushi Fujio, MD, PhD; Masanobu Funamoto, MD, PhD;
Martine I. Darville, MD, PhD; Décio L. Eizirik, MD, PhD; Tomoaki Osugi, MD;
Masahiro Izumi, MD; Yuichi Oshima, MD; Yoshikazu Nakaoka, MD; Hisao Hirota, MD, PhD;
Tadamitsu Kishimoto, MD, PhD; Keiko Yamauchi-Takihara, MD, PhD
Background—Mice with cardiac-specific overexpression of signal transducer and activator of transcription 3 (STAT3) are resistant to
doxorubicin-induced damage. The STAT3 signal may be involved in the detoxification of reactive oxygen species (ROS).
Methods and Results—The effects of leukemia inhibitory factor (LIF) or adenovirus-mediated transfection of constitutively activated
cardiomyocytes. Either LIF treatment or caSTAT3 significantly suppressed the increase of H/R-induced ROS evaluated by
amount of creatine phosphokinase in cultured medium was examined. Both LIF treatment and caSTAT3 significantly decreased
H/R-induced creatine phosphokinase release. These results indicate that the gp130/STAT3 signal protects H/R-induced cardiomyo-
cyte injury by scavenging ROS generation. To investigate the mechanism of scavenging ROS, the effects of LIF on the induction
of antioxidant enzymes were examined. LIF treatment significantly increased the expression of manganese superoxide dismutase
(MnSOD) mRNA, whereas the expression of the catalase and glutathione peroxidase genes were unaffected. This induction of
caSTAT3 augmented MnSOD mRNA and its enzyme activity. In addition, the antisense oligodeoxyribonucleotide to MnSOD
significantly inhibited both LIF and caSTAT3-mediated protective effects.
Conclusions—The activation of STAT3 induces a protective effect on H/R-induced cardiomyocyte damage, mainly by
inducting MnSOD. The STAT3-mediated signal is proposed as a therapeutical target of ROS-induced cardiomyocyte
injury. (Circulation. 2001;104:979-981.)
Key Words: antioxidants ? hypoxia ? signal transduction
tive heart failure, and doxorubicin-induced cardiomyopathy.1–3Re-
cent studies have shown that gp130-mediated signals transduced
both cytoprotective and hypertrophic responses in the heart.4The
signal transducer and activator of transcription-3 (STAT3) is a key
molecule downstream of gp130, which is activated under various
stressful conditions, such as pressure-overload and myocardial
the STAT3 gene are protected against doxorubicin-induced cardio-
myopathy.5Therefore, the activation of STAT3 might induce a
protective effect against oxidative stress–induced cardiomyocyte
damage by scavenging ROS.
n the heart, it has been reported that reactive oxygen species
In the present study, we explored whether the gp130/
STAT3 signal has a protective function against hypoxia/reox-
ygenation (H/R)–induced cardiomyocyte damage.
Cell Culture and H/R Experiments
Primary cultures of neonatal rat cardiomyocytes were prepared from
the ventricles of 1-day-old Sprague-Dawley rats (Kiwa Dobutsu
Wakayama, Japan), as previously described.6Hypoxia was created
by incubating cells in an airtight Plexiglas chamber with ?1% O2
and 5% CO2/95% N2at 37°C for 2 hours using Gas Pak Plus (BBL).
By replacing the medium saturated with 95% air and 5% CO2, the
cells were exposed to normoxic atmosphere (reoxygenation). Anti-
sense oligodeoxyribonucleotides (ODN) corresponding to the initi-
ation sites of manganese superoxide dismutase (MnSOD) translation
Received April 30, 2001; revision received July 9, 2001; accepted July 10, 2001.
From the Department of Molecular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan (S.N., K.K., Y.F., M.F., T.O., M.I., Y.O.,
Y.N., H.H., T.K., K.Y.-T.), and the Gene Expression Unit, Diabetes Research Center, Vrije Universiteit, Brussels, Belgium (M.I.D., D.L.E.).
Correspondence to Keita Kunisada, MD, PhD, Department of Molecular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka,
Suita, Osaka 565-0871, Japan. E-mail: firstname.lastname@example.org
© 2001 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
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(22 mer: CACGCCGCCCGACACAACATTG; 1.0 ?mol/L) or
sense ODN at the same site (22 mer: CAATGTTGTGTCGGGCG-
GCGTG; 1.0 ?mol/L) were added to the cultured medium 18 hours
before hypoxia until 24 hours after reoxygenation.7
Generation of Recombinant Adenovirus and
Protocol of Adenovirus Infection
The recombinant replication-defective adenovirus expressing a
dominant-negative form of STAT3 and constitutively activated
STAT3 (caSTAT3), which were kindly provided by Drs J.F. Brom-
berg and J.E. Darnell, Jr (Laboratory of Molecular Cell Biology, The
Rockefeller University, New York, NY),8were prepared as de-
?-galactosidase (?-gal) was used as a control.
Analysis of Intracellular ROS Generation
The fluorescent probe, 2?,7?-dichlorofluorescin diacetate (DCF-DA),
was used to assess intracellular ROS formation in cultured rat
cardiomyocytes, as previously described.11
Northern Blot Analysis and Western Blot Analysis
Northern and Western blotting were performed as previously de-
scribed.6Specific cDNA probes for the detection of MnSOD, catalase,
and glutathione peroxidase gene transcripts were reported previously.12
Measurement of MnSOD Activity
MnSOD activity was determined by the nitroblue tetrazolium
Measurement of Creatine Phosphokinase
Creatine phosphokinase (CPK) activity in the culture medium was
measured by the MERCK-1-TEST CK kit (Kantou Kagaku Co).13
Statistical analysis was performed by Student’s t test and 1-way
ANOVA followed by Bonferroni collection for multiple group
comparisons. P?0.05 was considered significant.
Activation of Gp130/STAT3 Protects Cardiomyocytes
from H/R Injury by Scavenging ROS
To examine the effects of leukemia inhibitory factor (LIF) or
caSTAT3 on the intracellular ROS formation induced by H/R,
DCF-DA was used as a fluorescent probe. Both LIF treatment
and caSTAT3 significantly suppressed the generation of ROS
induced by H/R (Figure 1A). Furthermore, they both signifi-
cantly decreased H/R-induced CPK release (Figure 1B). These
results indicate that the gp130/STAT3 signal protects H/R-
induced cardiomyocytes injury by scavenging ROS generation.
The Protective Effects of STAT3 Are Involved in
the Upregulation of MnSOD
To explore the mechanism of protection against ROS, the
effects of LIF on the induction of 3 important myocardial
antioxidant enzymes (ie, MnSOD, catalase, and glutathione
peroxidase) were examined. LIF treatment resulted in a
significant increase of MnSOD mRNA level from 1 hour that
continued for up to 24 hours, whereas the expressions of
catalase and glutathione peroxidase mRNAs were unchanged
throughout the time points examined (Figure 2A). Figure 2B
shows the dose-dependent effect of LIF on the induction of
MnSOD mRNA expression. These effects of LIF were
inhibited by dominant-negative form of STAT3 (Figure 2C).
Furthermore, significant enhancement of MnSOD mRNA
expression was detected with caSTAT3, and this was accom-
panied by an increase in its enzyme activity. Thus, STAT3 is
considered essential for LIF-induced MnSOD expression.
To evaluate whether the increase in MnSOD activity is
indeed directly related to the protective effect of STAT3,
antisense ODN against the MnSOD gene were used. Anti-
sense ODN completely cancelled both LIF and caSTAT3-
induced MnSOD activities, and sense ODN did not affect our
experiments (data not shown). As shown in Figure 2D, these
beneficial effects of LIF and caSTAT3 on CPK release were
significantly decreased in the presence of antisense ODN.
Thus, the protective effects of STAT3 against H/R are mainly
related to the induction of MnSOD activity.
protects cardiomyocytes against ROS caused by H/R through the
upregulation of the MnSOD gene and its enzyme activity.
Endotoxin and cytokines such as tumor necrosis factor-?,
several studies have confirmed the cardioprotective role of Mn-
SOD.14–16In view of the transcriptional regulation, 3 interferon-?
activation site motifs (TTCCTCTAA, TTCCTCAA, and TTA-
the region spanning from –2505 to –1104 in the MnSOD promoter
region.17,18This suggests that LIF induce MnSOD mRNA expres-
sion mainly via the STAT3 binding cis-element in cardiac myo-
Figure 1. Activation of gp130/STAT3 protects cardio-
myocytes from H/R injury by scavenging ROS. A, LIF-
treated (preincubated with 103units/mL LIF for 6 hours)
or caSTAT3-transfected cardiomyocytes were sub-
jected to H/R (2 hours of ischemia and 24 hours of
reoxygenation), and incubated with DCF-DA (5 ?mol/L)
for 30 minutes. Two fields were randomly selected in
each well and were examined for each condition. Rep-
resentative cardiomyocytes observed by laser confocal
microscopy are shown. Con indicates control. B, Cardi-
omyocytes were treated with LIF (preincubated with 103
units/mL of LIF for 6 hours) or transfected with ?-gal or
caSTAT3. CPK activity in the culture medium was mea-
sured after H/R. Data are mean?SEM from 3 experi-
ments. *P?0.05 vs ?-gal without H/R; #P?0.05 vs
?-gal with H/R.
August 28, 2001
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cytes. It remains to be identified which motifs of the 3 discussed
To clarify the role of MnSOD in H/R, we used an antisense
strategy to suppress MnSOD induction.7The responses to the
pretreatment with antisense ODN in caSTAT3- or LIF-treated cells
were different. The protective effects observed in caSTAT3 trans-
duction were nearly completely abolished with antisense ODN,
with antisense ODN. These results suggest that induction of Mn-
SOD by caSTAT3 is essential for caSTAT3-induced protection in
H/R. The genes related to cardiac protection, such as the atrial
natriuretic factor and vascular endothelial growth factor genes,
increased in the hearts with cardiac-specific overexpression of
STAT3; however, these genes have little effect on this condition.5
However, LIF stimulation induces other anti-apoptotic molecules,
such as Bcl-xL and Bcl-2, and also activates phosphatidylinositol 3
kinase-Akt and mitogen-activated protein kinase pathways, which
would be involved in a protective effect against ROS.4,19,20There-
fore, LIF presents more protective effects than STAT3 by inducing
other protective factors besides MnSOD.
We propose that the activation of STAT3 could be a thera-
peutic strategy for cardiac protection against ROS-mediated
cytotoxicity in several pathological conditions.
This study was supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Science, and Culture of Japan; grants from the
Ministry of Health and Welfare of Japan; and the Takeda Science Founda-
tion. We thank Jurei Hironaka for her secretarial assistance.
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Figure 2. LIF and caSTAT3 protect from cardio-
myocyte injury after H/R through the induction of
MnSOD. A, Cardiomyocytes were treated with LIF
(103U/mL) and harvested at the indicated time
points. MnSOD, catalase (Cat), and glutathione
peroxidase (GPX) mRNA expressions were exam-
ined by Northern blot analysis. 18S indicates 18S
ribosomal RNA. The results are representative of 3
similar experiments. B, Cardiomyocytes were stim-
ulated with various concentrations of LIF for 3
hours. MnSOD mRNA expression was examined
by Northern blot analysis. The results are represen-
tative of 3 similar experiments. C, Cardiomyocytes
were transfected with adenovirus vectors express-
ing a dominant-negative form of STAT3 (dnSTAT3),
caSTAT3, or ?-gal and stimulated with LIF (103
U/mL) for 3 hours. MnSOD mRNA expression was
examined by Northern blot analysis. After cardio-
myocytes were stimulated with LIF (103U/mL) for 6
hours, MnSOD enzyme activity was determined as
described in Methods. Data are presented as
mean?SEM from 3 samples. *P?0.05 vs ?-gal
without LIF stimulation; #P?0.05 vs ?-gal with LIF
treatment. D, Sense or antisense ODN (1.0 ?mol/L)
were applied to LIF-treated (preincubated with 103U/mL LIF for 6 hours) or ?-gal– or caSTAT3-transfected cardiomyocytes for18 hours
before H/R. CPK activity in the culture medium was measured after H/R. Data are mean?SEM from 3 experiments. *P?0.05 vs LIF
with sense ODN; #P?0.05 vs caSTAT3 with sense ODN.
Negoro et alLIF induces MnSOD via STAT3
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