activated protein kinase 1
TRANSCRIPT
1
Hydrogen Peroxide Stimulates c -Src-Mediated Big Mitogen-
Activated Protein Kinase 1 (BMK1) and the MEF2C Signaling
Pathway in PC12 Cells
POTENTIAL ROLE IN CELL SURVIVAL FOLLOWING OXIDATIVE INSULTS
Yuki Suzaki*, Masanori Yoshizumi*‡, Shoji Kagami†, A. Hajime Koyama¶, Yutaka
Taketani§, Hitoshi Houchi, Koichiro Tsuchiya, Eiji Takeda§, and Toshiaki Tamaki
Department of Pharmacology, Pediatrics†, Virology¶ and Clinical Nutrition§, The University
of Tokushima School of Medicine, Tokushima 770-8503, Japan
*The first two authors contributed equally to this work
Running title: Anti-apoptotic role of BMK1 in PC12 cells
‡Address correspondence to: Masanori Yoshizumi, M.D., Ph.D.
Department of Pharmacology
The University of Tokushima School of Medicine
3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
Phone: 81-886-33-7061
Fax: 81-886-33-7062
E-mail: [email protected]
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on January 8, 2002 as Manuscript M111790200 by guest on A
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Abstract
Reactive oxygen species (ROS), generated by reduction-oxidation (redox) reactions,
have been recognized as one of the major mediators of ischemia and reperfusion injury in the
brain. ROS-induced cerebral events are attributable, in part, to the change in intracellular
signaling molecules including mitogen-activated protein (MAP) kinases. Big MAP kinase 1
(BMK1), also known as ERK5, is a newly identified member of MAP kinase family and has
been reported to be sensitive to oxidative stress. In the present study, we examined the effect
of H2O2 on BMK1 activity in PC12 cells and investigated the pathophysiological implication of
BMK1. Findings showed that BMK1 was rapidly and significantly activated by H2O2 in a
concentration-dependent manner in PC12 cells. BMK1 activation by H2O2 was inhibited by
both PD98059 and U0126, which were reported to inhibit MEK5 as well as MEK1/2. c-Src
was suggested to be involved in BMK1 activation from the experiments with herbimycin A and
PP2, specific inhibitors of Src family kinases. Transfection of kinase inactive Src also
inhibited H2O2–induced BMK1 activation. In addition, H2O2 treatment of cells induced an
enhancement of DNA binding activity of MEF2C, a downstream transcription factor of BMK1
in PC12 cells. Finally, pretreatment of cells with PD98059 and U0126 resulted in an increase
in cell death including apoptosis by H2O2 in ERK1/2 down-regulated cells as well as in intact
PC12 cells. These findings suggest that c-Src mediated BMK1 activation by H2O2 may
counteract ischemic cellular damage probably through the activation of MEF2C transcription
factor.
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Reactive oxygen species (ROS) have been proposed to be involved in the
pathogenesis of cerebral ischemia and reperfusion injury (1, 2). ROS including hydrogen
peroxide (H2O2), superoxide radical (O2 . -), hydroxyl radical (. OH), and peroxynitrate
(ONOO -) have been shown to increase upon reperfusion of the brain following ischemia (3).
In the ischemia and reperfused brain, many alterations such as the change in neurotransmitter
release, change in gene expression, and neuronal cell death have been observed (4, 5). These
alterations in the brain during ischemia-reperfusion were suggested to be attributable to the
change in the intracellular signaling mechanisms. Among many intracellular signaling
molecules, ROS-induced cellular events have been implicated, in part, to the activation of
mitogen-activated protein (MAP) kinases (6, 7).
Four subfamilies of MAP kinases that are sensitive to ROS have been identified:
extracellular-signal regulated kinase (ERK1/2), c-Jun NH2-terminal kinase (JNK), p38 kinase,
and big MAP kinase 1 (BMK1 or ERK5) (8, 9). Each subfamily may be regulated via
different signal transduction pathways and modulate specific cell functions (10). ERK1/2 is
activated by an upstream kinase (MAPK kinase 1, or MEK1) via dual phosphorylation of the
TEY motif, whereas JNK and p38 kinases are activated by MKK4/7 and MKK3/6 via
phosphorylation of the TPY and TGY motifs, respectively. BMK1 is the newest MAP
kinase family member with a large C-terminal and a unique loop-12 sequence that shares the
TEY activation motif with ERK1/2 but is activated by MAPK kinase 5 (MEK5) (11, 12).
The role of BMK1 in neuronal cells as well as in PC12 cells was recently investigated and it
was reported that BMK1 was activated by serum, epidermal growth factor (EGF), nerve
growth factor (NGF) (13-15). BMK1 has been reported to contribute to EGF-induced cell
proliferation and cell cycle progression (14) as well as Ras-dependent cellular transformation
(16).
PC12 cells are useful for studying the intracellular signaling mechanisms, and are
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regarded as a model for catecholamine-containing neurons. Recently, several studies
reported the roles of MAP kinase activation in both the protection and the injury of
postischemic neuronal cells including PC12 cells (17-19). It was reported that activation of
three classical MAP kinase family members, ERK1/2, JNK, and p38 kinase was observed
after ischemia and reperfusion of neurons (6, 7). These studies suggested that the activation
of ERK1/2 made neuronal cells protective against ischemia and reperfusion, whereas the
activation of JNK and p38 kinase led to apoptosis (17, 18). However, changes in BMK1
activity and its role in oxidative stress-induced neuronal cell damage have not yet been reported.
In the ischemia and reperfused heart, activation of BMK1 was reported (20). In addition,
ROS-mediated BMK1 activation was observed in cultured fibroblasts (21). Therefore, we
hypothesized that BMK1 may be activated by oxidative stress in neuronal cells and play some
role in ROS-mediated cellular injury.
In the present study, we examined the effect of H2O2, as an oxidative stress, on the
change in BMK1 activity in PC12 cells. Furthermore, we investigated the signaling
pathways that are involved in BMK1 activation and their physiological implications in ROS-
mediated cellular injury.
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MATERIALS AND METHODS
Che mi cals - All chemicals were purchased from Sigma (St. Louis, MO) except
where indicated. Herbimycin A and PP2 were purchased from Calbiochem (San Diego, CA).
U0126 was from Promega (Madison, WI). H2O2 and PD98059 were from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). Anti-ERK5 and anti-ERK1/2 antibodies were
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Src antibody was from Upstate
Biotechnology (Lake Placid, NY). Anti-Src phosphospecific antibody (Tyr418) was from
BIOSOURCE (Camarillo, CA). All other chemicals were commercial products of reagent
grade.
Ce ll cult ure and t ransfe ct i on - Rat pheochromocytoma (PC12) cells were
grown in RPMI 1640 medium supplemented with 10% donor horse serum, 5% new born calf
serum and antibiotics (100 units/ml penicillin, 100 µg/ml streptomycin) in flasks precoated
with collagen. The culture was replaced after 2 months of passage by thawing a fresh aliquot
of frozen cells. The cultures were maintained in a humidified atmosphere containing 5% CO2
at 37°C. Cells at 70-80% confluence in 100-mm, 60-mm and 35-mm dishes were growth
arrested by incubation in serum-free RPMI 1640 medium for 24 h prior to use. All
experiments were performed with growth-arrested cells to minimize basal BMK1 activity.
For transfection of wild type (WT) or kinase inactive (KI) Src, commercially available pUSE
mammalian expression vectors encoding pp60c-Src (WT) or catalytically inactive Src (K297R)
were used (Upstate Biotechnolgy). For transient expression experiments, cells in serum-free
Opti-MEM medium (Gibco BRL, Rockville, MD) were transfected 1 day after replating by
SuperFector (B-Bridge International, Inc., San Jose, CA) as described previously (22).
After 24 h of incubation, cells were harvested for experiments.
Immunopre ci pi t at i on - Subconfluent PC12 cells in 100-mm culture dishes were
made quiescent by placing them in serum-free medium. PC12 cells in the serum-free medium
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were treated with or without H2O2 at each indicated time or dose-point. After treatment, the
cells were washed once with PBS, then lysed with 0.5 ml of lysis buffer (20 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium
pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin and 1 mM
PMSF) and flash frozen using liquid nitrogen. After allowing the cells to thaw, cells were
scraped off the dish and centrifuged at 14,000 x g for 30 min at 4°C, and the protein
concentration was determined using the Bradford protein assay kit (Bio-Rad, Hercules, CA).
For immunoprecipitation, cell lysates were incubated with anti-ERK5 antibody (Santa Cruz) or
anti-Src antibody (Upstate Biotechnology) overnight and then incubated with protein A agarose
beads or protein G agarose beads, respectively (Gibco BRL) for 2 h on a roller system at
4°C.
BMK1 ki nase assay - BMK1 kinase activity was measured by
autophosphorylation as described previously (21). For the kinase assay of BMK1,
subconfluent cells on a 100-mm dish in the serum free medium were treated with or without
H2O2. Then the cells were washed once with ice-cold PBS, scraped in a lysis buffer
consisting of 20 mM Tris-HCl (pH 7.5), 5 mM EGTA, 25 mM ß-glycerophosphate, 1% Triton
X-100, 2 mM DTT, and 1% aprotinin (200 µl of buffer/60-mm dish or 400 µl/100-mm dish)
and extracted by pipetting 20 times, followed by centrifugation at 14,000 x g for 20 min.
Protein concentrations were assayed by the Bradford methods as described above. Equal
amounts of protein extract were used for immunoprecipitation as described above. The
immune complex on the beads were washed two times with lysis buffer, two times with LiCl
wash buffer (500 mM LiCl, 100 mM Tris-HCl, pH 7.6, 0.1% Triton X-100, and 1 mM DTT),
and two times in washing buffer (20 mM HEPES, pH 7.2, 2 mM EGTA, 10 mM MgCl2, 1
mM DTT, and 0.1% Triton X-100). To detect the kinase activity, the immune complex was
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washed once with a reaction buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM EGTA, 2 mM
DTT, and 1 mM PMSF and incubated for 30 min at 30°C in buffer containing 20 mM Tris-HCl
(pH 7.5), 10 mM MgCl2 and 100 µM ATP (2 µCi of [γ-32P]ATP). After SDS-polyacrylamide
gel electrophoresis, the gel was dried and subjected to autoradiography and analyzed with a
bioimaging analyzer (BAS-1500; Fuji film, Tokyo, Japan).
Src ki nase assay - Src kinase activity was quantified using the Universal
Tyrosine Kinase Assay Kit (TaKaRa, Tokyo, Japan) as described previously (23). PC12
cells were immediately solubilized with an equal volume of lysis buffer for 30 min, and the
resulting lysates were centrifuged at 14,000 x g for 20 min at 4°C. Cell lysates were
immunoprecipitated with anti-Src antibody (Upstate Biotechnology) as described above.
Following immunoprecipitation, Src immune complexes were washed twice with lysis buffer
and then Src kinase activities of each sample was assayed using the kit according to the
manufacturer' s instructions.
We st e rn blot analy s i s - Using goat polyclonal ERK5 antibody (Santa Cruz),
ERK5/BMK1 proteins in PC12 cells were measured with Western blot analysis as described
previously (24). For evaluation of activated Src, phosphospecific-Src antibody at Tyr418
(BIOSOURCE) was used. ERK1/2 proteins in antisense oligonucleotides transfected cells
were determined with anti-ERK1/2 antibody (Santa Cruz). For Western blot analysis, the
PC12 cells protein extract (30 µg) was boiled for 5 min in Laemmli sample buffer and then
subjected to SDS-PAGE, and the protein extracts were transferred to a nitrocellulose
membrane (HybondTM-ECL, Amersham Pharmacia Biotech, Buckinghamshire, England).
Complete protein transfer to the membrane was verified by staining the gel with Coommasie
blue. The membrane was blocked for 1 h at room temperature with 5% BSA in PBS-T. The
blots were incubated overnight at 4°C with ERK5 antibody or phospho-Src antibody, followed
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by incubation for 1 h with secondary antibody (horseradish peroxidase-conjugated).
Immunoreactive bands were visualized using enhanced chemiluminescence with ECL reagent
(Amersham) and exposure to Hyperfilm-ECL. The intensity of the bands was measured
using a Macintosh i-Mac computer with an optical UMAX Astra 2200 scanner, and the public
domain NIH Image program.
Gel mobi li ty shift a s s a y - Gel mobility shift assay was measured as described
previously (25). For the assay, nuclear protein extracts were prepared from PC12 cells in
100-mm dishes after stimulation with H2O2 at the indicated time points. The cells were
washed with ice-cold PBS and were scraped off the plate into 1 ml PBS, then the cells were
centrifuged at 5,000 x g for 5 min and supernatants were removed. The samples were
homogenized in 0.4 ml of 10 mM Hepes (pH 7.9) containing 10 mM KCl, 0.1 mM EDTA, 0.1
mM EGTA, 0.5 mM PMSF, 1 mM DTT, 10 µg/µl chymostatin, 1 µg/ml aprotinin, 1 µg/ml
pepstatin and 1 µg/ml leupeptin; incubated on ice for 15 min; and then added to a 1/100 volume
of 10% Nonidet P-40 centrifuged at 5,000 x g for 30 sec at 4°C. The resulting precipitations
were homogenized in 50 µl of 20 mM Hepes (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM
EGTA, 1 mM PMSF, 1 mM DTT, 10 µg/ml chymostatin, 1 µg/ml aprotinin, 1 µg/ml pepstatin
and 1 µg/ml leupeptin; stired at 4°C for 15 min. The resulting supernatants were assayed for
protein concentrations and stored at –80°C before use. The procedure for the assay was
described previously (25). In brief, the gel mobility shift assay of PC12 cells nuclear
MEF2C binding activity was performed with an oligonucleotide probe containing the MEF2C
binding sequence (5’-GATGCGTCTAAAAATAACCCTGTCG-3’) (Santa Cruz
Biotechnology, Inc.). The probe was end-labeled with [γ-3 2P]-ATP (Amersham) using T4
polynucleotide kinase, and purified by chromatography on a Bio-Spin column (Roche
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Molecular Biochemicals, Mannheim, Germany). For the DNA-protein binding reaction, the
samples of PC12 cell nuclear extracts (10 µg protein) were incubated with 10 fmol of a 3 2P-
labeled oligonucleotide containing the consensus MEF2C binding site at room temperature for
30 min, in 20 µl of binding buffer consisting of 20 mM Hepes (pH 7.9), 0.2 mM EDTA, 0.2
mM EGTA, 80 mM NaCl, 0.3 mM MgCl2, 1 mM DTT, 0.2 mM PMSF, 6% glycerol and 2 µg
of polydeoxyinosinic deoxycytidylic acid (poly[dl-dc] )(Roche Molecular Biochemicals).
The DNA-protein complexes were separated from the free DNA probe using electrophoresis
on 7% nondenaturing poryacrylamide gels in 6.7 mM Tris-HCl (pH 7.5), 3.3 mM sodium
acetate, 0.1 mM EDTA, and 2.5% glycerol. Gels were run at 160 V at 4°C for 3 h, dried,
then subjected to autoradiography and analyzed with a bioimaging analyzer (BAS-1500). To
demonstrate the specificity of DNA-protein binding, binding reactions were performed as
described above, in the presence of a 100-fold molar excess of a non-labeled MEF2C
consensus oligonucleotide competitor followed by electrophoresis. In addition, supershift
assays were performed with rabbit polyclonal anti-MEF2C antibody (Santa Cruz
Biotechnology). Anti-MEF2C antibody was added to samples after the initial binding
reaction between PC12 cells nuclear protein extracts and 3 2P-labeled consensus MEF2C
oligonucleotide, the reaction was allowed to proceed at room temperature for 1 h, and then the
samples were subjected to electrophoresis, as described above.
Cell viability determination with MTT assay - Measurement of cellular
MTT reduction was carried out as described previously (26). PC12 cells were cultured at
70-80% confluency. Thereafter, cells were growth arrested by serum-free RPMI 1640
medium. Following the indicated incubation time with H2O2, MTT was added to a final
concentration of 0.5 mg/ml, and after a further 1 h incubation PC12 cells were lysed with
isopropanol that contained 0.04N HCl. The MTT reduction was read at 550 nm using a
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spectrophotometer.
Nucle ar morphology -Hoe chs t 33248 s t ai ni ng - PC12 cells were stained
with Hoechst 33248 dye (27). PC12 cells were cultured on a coverglass in a 35-mm dish and
then the cell-attached coverglass was removed, washed in PBS, transferred into MeOH/Acetic
acid (3:1) and submerged for 2 min, then cells were dried at room temperature. Then, the
cells were fixed on a coverglass at room temperature and stained with Hoechst 33248 working
solution (0.05 µg/ml) for 10 min. Next, the coverglass was washed with distilled water and
mounted in buffered glycerol. Fluorescence was visualized using a fluorescent microscope
(OLYMPUS, Tokyo, Japan).
Agarose gel electrophoresis for DNA fragmentation - DNA
fragmentation was detected by the methods of Wyllie et al. (28) with minor modifications.
Cells were pelleted at 400 x g and washed twice with ice-cold Tris-buffered saline (137 mM
NaCl, 2.7 mM KCl, 25 mM Tris, pH 7.0). The pellets were resuspended in 50 µl of Tris-
EDTA (1 mM EDTA, 10 mM Tris, pH 8.0) and lysed with 0.5 ml of an extraction buffer (0.1
M EDTA, 0.5% SDS, 10 mM Tris, pH 8.0) containing 0.5 mg/ml proteinase K. After
overnight incubation at 50°C, DNA was extracted from the samples and ethanol preincubated
for agarose gel electrophoresis. The DNA separated in the 2% agarose gel was visualized by
UV fluorescence.
Antisense oligonucleotide treatment of ce l ls - Antisense oligonucleotide
treatment of cells was carried out as described previously (29). Cells (70% confluent in 35-
mm dishes) were carefully washed with RPMI 1640 medium. ERK1/2 oligonucleotides
(antisense and control) (BIOMOL, Plymouth Meeting, PA) and DOTAP solution (15 µg)
(Roche) diluted with HEPES-buffered saline (20 mM HEPES, 150 mM NaCl) were mixed and
incubated at room temperature for 15 min. The final concentrations of oligonucleotides are as
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indicated. Transfected cells were incubated for 8 h at 37°C in the presence of 5% CO2.
After this time the medium containing DOTAP was removed and the incubation continued for
24 h using fresh serum-free medium containing appropriate oligonucleotide concentrations.
After 24 h, cells were replaced with fresh serum-free medium, stimulated by H2O2 and were
assayed for cell viability.
Sta t i s t i c s - Values are presented as means ± S.D. for 3-5 separate experiments.
One-way analysis of variance was used to determine significance among groups, after which
the modified ttest with the Bonferroni correction were used for comparison between individual
groups. A value at p<0.05 was considered to be significant.
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RESULTS
H2 O2 -induced BMK1 a c t i v a ti o n in PC12 cel ls – We first examined
whether BMK1 was activated by H2O2 in PC12 cells. Growth arrested PC12 cells were
treated with various time and concentrations of H2O2. Endogenous BMK1 activity was
measured by in vitro kinase assay based on BMK1 autophosphorylation as described in
"Materials and Methods". The H2O2 treatment of cells resulted in a maximal activation of
BMK1 at 5 min (Fig. 1A). In the examined concentration range of H2O2 (0.01-1 mM), the
BMK1 activity increased in a dose-dependent manner and maximal activation occurred at 300
µM of H2O2 (Fig.1B).
U0126 and PD98059 both inhibited H2 O2 -induced BMK1 activation -
Recently, the MEK1/2 inhibitors, U0126 and PD98059 were reported shown to inhibit MEK5,
which is known as an upstream regulator of BMK1 in many cells (15, 30). Therefore, we
investigated whether H2O2-induced BMK1 activation was also inhibited by U0126 and
PD98059 in PC12 cells. Pretreatment of the cells with U0126 resulted in an inhibition of
BMK1 activation by H2O2 in a concentration-dependent manner (Fig. 2A). Another MEK1/2
inhibitor, PD98059 also inhibited the H2O2-induced BMK1 activation in a concentration-
dependent manner (Fig. 2B).
c -Src tyrosin kinase is involved in the H2 O2 -induced BMK1
act iva t ion - Many tyrosine kinases including receptor and nonreceptor type tyrosine kinases
have been reported to activate ERKs in many cell types (31, 32). It was reported that H2O2
stimulates ERK1/2 activation via the Src tyrosine kinase dependent pathway (33). It has also
been reported that c-Src is involved in H2O2-induced BMK1 activation in cultured fibroblasts
(21). To elucidate whether H2O2 activates BMK1 through the Src dependent pathway in
PC12 cells, we first examined the effect H2O2 on c-Src tyrosine kinase activity in PC12 cells.
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Application of 300 µM H2O2 caused rapid and significant activation of c-Src (Fig. 3B) as well
as its phosphorylation (Fig. 3A) at Tyrosine 418, an autophosphorylation that leads to full
catalytic activity of the kinase. A tyrosine kinase inhibitor, herbimycin A (HA)(0.3 µM) and
Src kinase inhibitor, PP2 (30 µM) both inhibited H2O2-induced Src kinase activation and its
phosphorylation (Fig. 3A and 3B). In addition, HA and PP2 both inhibited H2O2-induced
BMK1 activation in a dose-dependent manner (Fig. 4). Ttransfection of kinase inactive Src
(KI Src) also inhibited H2O2-induced BMK1 activation in PC12 cells (Fig. 5). These
findings strongly suggest that c-Src is involved in H2O2-induced BMK1 activation in PC12
cells.
H2 O2 -i nduce d BMK1 act i v at i on re gulat e s DNA bi ndi ng act i v i t y of
MEF2C, a dow nst re am t ranscri pt i on fact or of BMK1 i n P C12 ce lls - It was
reported that BMK1 activation leads to transcription factor MEF2C DNA binding in several cell
types (13, 30). Therefore, we examined whether H2O2-induced BMK1 activation regulates
MEF2C DNA binding activity in PC12 cells evaluated with gel mobility shift assay. The
nuclear protein extracts from PC12 cells treated with H2O2 at different time points were
assayed. As shown Fig. 6A, MEF2C DNA binding activity increased time-dependently,
reached a peak at 8 h and was sustained for up to 24 h incubation of PC12 cells with 300 µM
H2O2. This band was found to have specific binding for MEF2C, because the addition of
unlabeled MEF2C consensus oligonucleotide competitor resulted in a decrease in the formation
of MEF2C complexes. Furthermore, the addition of anti-MEF2C antibody induced
supershifted band complexes (Fig. 6B). The H2O2-induced increase in MEF2C DNA
binding activity was inhibited by U0126 and PD98059 similar to the inhibition of BMK1
activation as shown in Fig. 2 (Fig. 6C). These findings suggested that H2O2-induced BMK1
activation affected MEF2C DNA binding in PC12 cells. However, the treatment with
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inhibitors did not show a complete inhibition of the increase in MEF2C DNA binding caused
by H2O2.
H2 O2 -induced BMK1/MEF2C activation influences the viability o f
PC12 cel ls - The transcription factor MEF2C was reported to relate to cell survival (30).
Therefore, we investigated the pathophysiological implications of BMK1/MEF2C activation in
PC12 cells after H2O2 treatment. As shown in Fig. 7, MTT assay revealed that the
stimulation with H2O2 significantly decreased PC12 cell viability by 24 h treatment. In
addition, pretreatment of cells with U0126 and PD98059 significantly augmented H2O2-
induced cell death (Fig. 7). Both U0126 and PD98059 alone at 30 µM did not affect the cell
viability up to 24 h (data not shown). In addition, we also examined with Hoechst 33248
staining and DNA fragmentation whether these cell death are due to apoptosis evoked by H2O2.
As shown in Fig. 8, H2O2-induced cell deaths were almost found to be apoptosis as determined
by both apoptotic nuclei staining and DNA fragmentation analysis. Moreover, pretreatment
of cells with U0126 and PD98059 both increased PC12 cell apoptosis by H2O2 (Fig. 8).
Effects of U0126 and PD98059 on H2 O2 -induced PC12 cell death i n
which ERK1/2 was down-regulated - Since it has been reported that neuronal cell
survival is augmented by the activation of ERK1/2 (17), which is also inhibited by PD98059
and U0126, involvement of ERK1/2 as well as BMK1 against cell death cannot be ruled out.
Therefore, we examined the effects of U0126 and PD98059 on antisense oligonucleotides
transfected PC12 cells in which ERK1/2 expression was diminutive (Fig. 9A). Transfection
of ERK1/2 antisense oligonucleotides caused an increase in H2O2-induced cell death compared
with that of intact PC12 cells; however, the ficilitatory effect of ERK1/2 antisense on H2O2-
induced cell death was less potent than the angmentation by U0126 and PD98059 of intact
PC12 cell death by H2O2 (Fig. 9B). Moreover, pretreatment with U0126 and PD98059
augmented cell death by H2O2 even in the ERK1/2 down-regulated PC12 cells as well as in
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intact cells.
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DISCUSSION
The major findings of the present study were that H2O2 stimulated BMK1 via a
pathway that was dependent on c-Src in PC12 cells. PD98059 and U0126, which have been
shown to inhibit MEK5 as well as MEK1/2, inhibited H2O2-induced BMK1 activation in a
concentration-dependent manner. Herbimycin A and PP2, specific inhibitors of Src family
kinases, both inhibited H2O2-mediated c-Src and BMK1 activation. Transfection of kinase
inactive Src also inhibited H2O2-induced BMK1 activation. It was suggested that c-Src is
involved in BMK1 activation in PC12 cells. Gel mobility shift analysis revealed that DNA
binding activity of MEF2C, a transcription factor which exists downstream of BMK1, was
increased by H2O2 treatment. Finally, pretreatment of cells with PD98059 and U0126
resulted in an increase in cell death including apoptosis by H2O2 in ERK1/2 down-regulated
cells as well as in intact PC12 cells. From the findings of these studies, it was suggested that
BMK1 may work as an anti-apoptotic signaling molecule in PC12 cells. Based on the present
study, we propose a scheme (Fig. 10) for ROS-mediated signal transduction leading to
activation of Src, BMK1 and MEF2C.
Recently, the role of BMK1 in neuronal cells as well as PC12 cells has been
investigated in several studies. BMK1 was reported to be activated by neurotrophins in
cortical neurons and in PC12 cells (30). It was also reported that EGF and NGF stimulated
BMK1 activation via Ras-dependent pathways in PC12 cells (15). However, many of these
studies were performed with artificially transfected cells. In the present study, we found, for
the first time, that endogenous BMK1 was rapidly and significantly activated by oxidative
stress in intact PC12 cells, which was inhibited by U0126 and PD98059 in a concentration-
dependent manner (Figs. 1 and 2). We utilized H2O2 as an oxidative stress to the cells
because the examined concentrations of H2O2 were in the range of those reached under
pathophysiological conditions, such as during transient cerebral ischemia (34). Therefore,
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the findings of the present study may be applied to cerebral ischemia and reperfusion in the
clinical setting. It was reported that neuronal ischemia and reperfusion both in in vivo and in
vitro induce activation of classical MAP kinase family members, i.e. ERK1/2, JNK and p38
kinase (6, 7). From the findings of these studies, it was suggested that ERK1/2 activation
works as a cell survival factor against oxidative stress, whereas activation of JNK and p38
kinase contributes to cell death machinery. Rapid activation of BMK1 by oxidative stress
may also play some pathophysiological role during cerebral ischemia and reperfusion. The
present findings that H2O2 stimulated rapid and significant activation of BMK1 are consistent
with those of Abe e t al. who reported that 200 µM H2O2 activated BMK1 at a 5 min peak in
vascular smooth muscle cells (35). In addition, H2O2-induced rapid activation of BMK1 in
cultured fibroblasts was also reported (21). Therefore, activation of BMK1 by oxidative
stress may be common in many cell types and may be involved in the cell fate determination
after oxidative insults.
The intracellular signaling mechanisms that lead to BMK1 activation have been
investigated. Using the yeast two-hybrid system, MEK5 was identified by Zhou e t al. (12)
as the molecule responsible for regulating BMK1 activity. Kato e t al. reported that MEK5
specifically activates BMK1 but not other mammalian MAP kinases in vivo (13). Recently,
interest has been focused on the upstream regulators of MEK5 and several molecules have
been identified to activate MEK5 in many cells. Ras and raf-1 mediated BMK1 activation via
MEK5 has been reported (16) as well as MEKK3 directly activating MEK5 in 293T cells (36,
37). It was also reported that Cot oncoprotein regulates MEK5 (37). In addition, c-Src
tyrosine kinase has also been shown to mediate BMK1 activation in response to oxidative
stress in fibroblasts (21). Since previous studies have shown that c-Src is involved in
signaling events stimulated by oxidative stress (38), we examined whether c-Src mediates
H2O2-induced BMK1 in the present study. As shown in Fig. 3, c-Src was rapidly and
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significantly activated by H2O2 in PC12 cells, which was inhibited by herbimycin A and PP2,
specific inhibitors of Src family tyrosine kinases. Furthermore, H2O2-induced BMK1
activation was also inhibited by herbimycin A and PP2 at similar concentrations as those of the
inhibition of Src kinase activity (Fig. 4). Experiments on KI Src transfection also strongly
suggest that c-Src is involved in H2O2-induced BMK1 activation in PC12 cells (Fig. 5).
Since we previously reported that c-Src meidates H2O2-induced JNK activation in vascular
smooth muscle cells (24), c-Src may have a diverse role in activating MAP kinase family
members in response to oxidative stress. However, since we did not determine whether c-
Src directly regulates MEK5 activity in this study, further studies are required to define the
precise nature of Src kinases.
The downstream effector of BMK1 has also been investigated. Kato et al. reported
that BMK1 activation by EGF leads to the activation of MEF2C transcription factor (13, 14).
It was also reported that BMK1 activation is required for neurotrophin stimulation of MEF2C
in both cortical neurons and PC12 cells, distinct from ERK1/2-induced activation of Elk1 and
CREB transcription factors (30). The MEF2 proteins constitute a family of transcription
factors: MEF2A, MEF2B, MEF2C, and MEF2D. They cooperate with members of the
MyoD family in muscle differentiation (39, 40). In addition to muscle, MEF2A and MEF2C
are expressed in developing and adult brain including cortex and cerebellum (41-44). It was
also reported that the cerebral cortex contains a high level of MEF2C protein (45). Therefore,
we examined the effect of H2O2 on MEF2C DNA binding activity in PC12 cells using gel
mobility shift assay. As shown in Fig. 6, H2O2 stimulated time-dependent activation of
MEF2C DNA binding, peaked at 8 h and was sustained, which was then inhibited by U0126
and PD98059. Although it was reported that p38 kinase also activates MEF2C (46), it is
unlikely because SB203058, a specific inhibitor of p38 kinase, failed to inhibit MEF2C
activation in the present experimental conditions (data not shown). In addition, only slight
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activation of p38 kinase by 300 µM H2O2 stimulation compared to BMK1 activation was
observed in PC12 cells (unpublished data). Since it was reported that ERK1/2 does not
activate MEF2C (13), it appears reasonable to suggest that MEF2C activation by H2O2 in PC12
cells depends on BMK1 activation. However, since the inhibitions by U0126 and PD98059
of BMK1 activation and MEF2C DNA binding were incomplete, other possible mechanism(s)
for BMK1 activation and for the increase of MEF2C DNA binding activity with a mechanism
via MEK5 cannot be excluded.
The pathophysiological role of MEF2C in the nervous system has not been
extensively studied. Although MEF2 mediates T cell receptor-induced apoptosis in T cells
(47), a recent study suggested that MEF2 mediates activity-dependent survival of cortical and
cerebellar neurons (30). Thus, we investigated the involvement of BMK1 activation, which
leads to MEF2C activation, in PC12 cell death machinery induced by H2O2. We utilized
U0126 and PD98059 as BMK1 inhibitors, and we found that H2O2-induced cell death was
augmented by treatment with these inhibitors (Fig. 7). These findings are consistent with
those shown in Fig. 8 in which H2O2-induced PC12 cell apoptosis was increased by both
U0126 and PD98059, and suggest that BMK1 activation works against cell death machinery.
However, since neuronal cell survival is also augmented by the activation of ERK1/2 (17),
which is also inhibited by U0126 and PD98059, involvement of ERK1/2 against H2O2-
induced PC12 cell death cannot be ruled out. Therefore, we next utilized ERK1/2 down-
regulated PC12 cells with ERK1/2 antisense oligonucletides and found that U0126 and
PD98059 still increased cell death by H2O2 even though ERK1/2 protein expression was
diminutive (Fig. 9). These findings suggest that BMK1/MEF2C pathway works against cell
death machinery induced by H2O2 although BMK1 activation could not overcome the oxidative
insults. Since it was suggested that cell fate, such as apoptosis, proliferation, and
differentiation, after oxidative insults would be determined by the balance of survival signals
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and death signals (5), an H2O2-induced gradual decrease in cell viability may suggest that the
death signals are stronger than the survival signals which are up-regulated by BMK1 activation.
Future studies should be conducted to use gene-transfer of the constitutive-active form of
MEK5 or BMK1 to prevent or overcome cell death pathways by oxidative insults.
In summary, the present findings demonstrate that BMK1 is activated by H2O2 in
PC12 cells. c-Src activation was suggested to be involved in oxidative stress-induced BMK1
activation. The downstream transcriptional target of BMK1 is suggested to be MEF2C,
which may be stimulated by oxidative insults. Although the physiological role of BMK1
remains to be elucidated, it may work in complement or in coordination when or where
ERK1/2 activity is low (48, 49).
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Footnotes: This study was supported, in part, by the Japan Heart Foundation/IBM Japan
Research Grant (to M.Y.).
The abbreviations used were: ROS, reactive oxygen species; MAP, mitogen-activated
protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MEK,
MAP kinase/ERK kinase; MEF2C, myocyte enhancer factor 2C; PBS, phosphate-buffered
saline; SDS, sodium dodecylsulfate; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl
fluoride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NGF, nerve
growth factor; EGF, epidermal growth factor; MEKK, MAP kinase/ERK kinase kinase.
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FIGURE LEGENDS
Fig. 1. H2 O2 -induced BMK1 activation in PC12 cel ls . (A) Time course of
H2O2-induced BMK1 activation in PC12 cells. Cells were treated with 300 µM H2O2 for the
indicated periods of time. (B) Dose-response of H2O2-induced BMK1 activation in PC12
cells. Cells were exposed to the indicated concentrations of H2O2 for 5 min. Cells were
harvested, lysed, and used for subsequent analysis. Lysates were prepared and
immunoprecipitated with anti-ERK5 antibody. BMK1 activation was determined by the in
vitro kinase assay as described in ”Materials and Methods” using [γ-3 2P]ATP and measured
after SDS-PAGE by autoradiography. No difference in the amount of BMK1 was observed
in samples by Western blot analysis with anti-ERK5 antibody (panels A and B, middle).
Values were normalized by arbitrarily setting the densitometry of control cells (time = 0 or
without H2O2) to 1.0 (values are the mean ± S.D., n = 3, panels A and B, lower).
Fig. 2. U0126 and PD98059 both inhibit BMK1 activation by H2 O2 in PC12
cells. Cells were preincubated with various concentrations of U0126 (A) and PD98059 (B)
for 30 min. After stimulation with 300 µM H2O2 for 5 min, cells were harvested, lysed, and
used for subsequent analysis. BMK1 activity was assayed as described in ”Materials and
Methods”. No difference in the amount of BMK1 was observed in samples by Western blot
analysis with anti-ERK5 antibody (panels A and B, middle). Values were normalized by
arbitrarily setting the densitometry of control cells (without H2O2) to 1.0 (values are the mean
± S.D., n = 3, panels A and B, lower). The asterisks represent significant differences
compared with the value of H2O2 stimulation (*p<0.05, **p<0.01).
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Fig. 3. c -Src tyrosine kinase was phosphorylated and activated by H2 O2 in
PC12 cells and its inhibition by herbimycin A (HA) and PP2 . Cells were treated
with 300 µM H2O2 for indicated periods of time. Cells were harvested, lysed, and used for
subsequent analysis. (A) The phosphorylation of c-Src at Tyr418 was determined by Western
blot analysis with phospho-Src antibody as described in “Materials and Methods”. (B) The
activity of c-Src was measured with an immune-complex kinase assay with anti-Src antibody
as described in “Materials and Methods”. Values are expressed as the % of controls which
was defined from the basal Src kinase activity without H2O2 stimulation (values are the mean
± S.D., n = 3). Cells were pretreated with 0.3 µM of herbimycin A (HA) for 16 h and 30 µM
of PP2 for 15 min. The asterisks represent significant differences compared with the value of
H2O2 stimulation (*p<0.05).
Fig. 4. H2 O2 -induced BMK1 activation was inhibited by herbimycin A (HA)
and PP2 in PC12 cells. Cells were pretreated with various concentrations of herbimycin
A (HA) for 16 h (A) and PP2 for 15 min (B). After stimulation with 300 µM H2O2 for 5 min,
cells were harvested, lysed, and used for subsequent analysis. BMK1 activity was assayed
as described in ”Materials and Methods”. No differences in the amount of BMK1 were
observed in samples by Western blot analysis with anti-ERK5 antibody (panels A and B,
middle). Values were normalized by arbitrarily setting the densitometry of control cells
(without H2O2) to 1.0 (values are the mean ± S.D., n = 3, panels A and B, lower). The
asterisks represent significant differences compared with the value of H2O2 stimulation
(*p<0.05, **p<0.01).
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Fig. 5. Efect of transfection of kinase inactive Src (KI Src) on H2 O2 -induced
BMK1 activation in PC12 cells. Cells were transfected with pUSE vector alone, wild
type Src (WT Src), or kinase inactive Src (KI Src) and 24 h later washed with media and
maintained in serum-free medium for 24 h. Cells were then treated with 300 µM H2O2 for 5
min, then cells were harvested in lysis buffer, and BMK1 activity was assayed as described
in ”Materials and Methods”. No differences in the amount of BMK1 were observed in
samples by Western blot analysis with anti-ERK5 antibody (middle). Values were
normalized by arbitrarily setting the densitometry of control cells (without H2O2) to 1.0 (values
are the mean ± S.D., n = 3, lower). The asterisks represent significant differences compared
with the value of H2O2 stimulation in cells transfected with vector alone (*p<0.05).
Fig. 6. Time course for the activation of MEF2C DNA binding from PC12
cell nuclear extracts stimulated with 300 µµµµM H2 O2 and its inhibition by U0126
and PD98059. (A) The bracket in the panel indicates PC12 cell nuclear extracts MEF2C
DNA binding complexes induced by H2O2 (300 µM) stimulation at the indicated time points.
(B) Cells were stimulated with H2O2 (300 µM) for 8 h. The competition assay for MEF2C
was carried out in the presence of a 100-fold molar excess of unlabeled MEF2C
oligonucleotide (competitor). Supershift analysis was performed with specific anti-MEF2C
antibody (SS). (C) Effects of U0126 and PD98059 on H2O2-induced increases in MEF2C
DNA binding activity of PC12 cell nuclear extracts. For the experiments of U0126 and
PD98059 against H2O2-induced increases in MEF2C DNA binding activity, these inhibitors
were added to the incubation medium 30 min prior to H2O2 (300 µM) stimulation for 8 h.
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The asterisks represent significant differences compared with the value of H2O2 stimulation
(*p<0.05).
Fig. 7. H2 O2 -induced PC12 cell death and the effects of U0126 and PD98059.
Cell viability was evaluated by MTT reduction as described in “Materials and Methods”.
Cells were treated with or without 300 µM H2O2 for 24 h, then assayed. U0126 (30 µM) and
PD98059 (30 µM) were added to the incubation medium 30 min prior to H2O2 stimulation.
Both U0126 and PD98059 alone at 30 µM did not affect the cell viability up to 24 h (data not
shown). Values are the means ± S.D. of three experiments performed in triplicate. The
asterisks represent significant differences compared with the value of control (*p<0.05) and
Η2Ο2 stimulation (§p<0.05).
Fig. 8. U0126 and PD98059 promote H2 O2 -induced PC12 cell apoptosis
assessed by apoptotic nuclei staining with Hoechst 33248 dye and agarose gel
electrophoretic analysis of DNA fragmentation. (A) Cells were treated with (b) or
without (a) 300 µM H2O2 for 24 h. U0126 (30 µM) (c) and PD98059 (30 µM) (d) were
added to the incubation medium 30 min prior to H2O2 stimulation. (B) DNA was extracted
from the cells as described in ”Materials and Methods” then the DNA was separated in agarose
gel electrophoresis. Representative photograph from three separate experiments is shown. M,
marker.
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Fig. 9. ERK1/2 antisense oligonucleotides selectively reduce ERK1/2
expression and the effects of U0126 and PD98059 on H2 O2 -induced apoptosis
in ERK1/2 down-regulated cel ls . (A) Lysates from control PC12 cells or from treated
with control oligonucleotides, or ERK1/2 antisense oligonucleotides, for 8 h, were placed in
serum-free RPMI 1640 medium overnight. Representative Western blots using anti-ERK1/2
antibody are shown. (B) Effect of H2O2 on the viability of ERK1/2 down-regulated PC12
cells as well as intact cells with or without control oligonucleotides were evaluated by MTT
reduction. ERK1/2 down-regulated cells and intact cells were treated with or without 300
µM H2O2 for 24 h, then assayed. U0126 (30 µM) and PD98059 (30 µM) were added to the
incubation medium 30 min prior to H2O2 stimulation. Values are the means ± S.D. of three
experiments preformed in triplicate. The asterisks represent significant differences compared
with the value of H2O2 stimulated intact PC12 cells (*p<0.05).
Fig. 10. Diagram of H2 O2 -mediated s ignal tarnsduction pathways . ROS
activate multiple MAP kinases in a variety of cells. In the present study, we found that c-Src
was required for H2O2–mediated BMK1 activation in PC12 cells. MEF2C would be a target
molecule of BMK1 that may have a role in cell survival following oxidative insults.
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0
1
2
3
4
0 2 5 10 30 60 (min)
0 2 5 10 30 60 (min)
H2O2 (300 µM)
IB:BMK1
BMK1activity
tpBMK1
0
1
2
3
4
0 0.01 0.03 0.1 0.3 1 (mM)
H2O2 (5 min)
0 0.01 0.03 0.1 0.3 1 (mM)
t
BMK1
(A) (B)
Fig.1
BM
K1
ac
tiv
ity
(fo
ldin
cre
as
e)
BM
K1
ac
tiv
i ty
(fo
ldin
cre
as
e)
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(A) (B)
(µ )
control H2O2 (300 µM)
BMK1activity
IB:BMK1
U0126 : 0 100 0 1 3 10 30 100 (µM)
control H2O2 (300 µM)
(µ01 3 1U0126 : 0 100 0 1 3 10 30 100 M PD98059 : 0 100 0 1 3 0 0 0 M)
control H2O2 (300 µM)
PD98059 : 0 100 0 1 3 10 30 100 (µM)
t
BMK1
t
pBMK1
control H2O2 (300 µM)
0
1
2
3
4
0
1
2
3
4
Fig.2
BM
K1
ac
tiv
ity
(fo
ldin
cre
as
e)
BM
K1
ac
tiv
i ty
(fo
ldin
cre
as
e)
********
**
****
**
*
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(A)
0 2 5 10 30 60 (min)
activatedSrc
c-Src
control H2O2 H A PP2(300 µM)
IB: Src
IB: pY418-Src
(0.3 µM) (30 µM)
H2O2
H2O2 (300 µM)
activatedSrc
c-Src
IB: Src
IB: pY418-Src
Fig.3A
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(B)
0
100
200
300
400
500
H2O2 (300 µM)
(300 µM)
0
100
200
300
400
500
(0.3 µM) (30 µM)
H2O2
control H2O2 H A PP2
0 2 5 10 30 60 (min)
Fig.3B
Src
kin
as
ea
cti
vit
y(%
of
co
ntr
ol)
Src
kin
as
ea
cti
vit
y(%
of
co
ntr
ol)
**
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(A) (B)
t
pBMK1
t
BMK1
HA : 0 1 0 0.01 0.03 0.1 0.3 1 (µM)
control H2O2 (300 µM)
PP2 : 0 100 0 1 3 10 30 100 (µM)
PP2 : 0 100 0 1 3 10 30 100 (µM)
control H2O2 (300 µM)
BMK1activity
IB:BMK1
HA : 0 1 0 0.01 0.03 0.1 0.3 1 (µM)
control H2O2 (300 µM)
control H2O2 (300 µM)
0
1
2
3
4
0
1
2
3
4
Fig.4
BM
K1
ac
tiv
ity
(fo
ldin
cre
as
e)
BM
K1
ac
tiv
i ty
(fo
ldin
cre
as
e)
******
***
*
**
*
**
**
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Fig.5
H O (300 M)2 2 µ - + + +
vector WT Src KI Src
BM
K1
ac
tiv
ity
(fo
ldin
cre
as
e)
BMK1activity
IB:BMK1
pBMK1
BMK1
tt
*
- -
H O (300 M)2 2 µ - + + +vector WT Src KI Src
- -
0
1
2
3
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MEF2C
0 1 2 4 6 8 12 24 ( h )
H2O2 (300 µM)
(A) (B)
t
SS
control H2O2 U 10 26 PD98059
(C)
(300 µM)
0
1
2
3
4
5
MEF2C
H2O2
*
H2O2 (300 µM)
U0126 (30 µM)
PD98059 (30 µM)
+-
++-
+
+
Fig.6
co
ntr
ol
X1
00
co
mp
eti
tor
an
ti-M
EF
2C
HO
(30
0M
)2
2µ
ME
F2
Cb
ind
ing
ac
tiv
ity
(fo
ldin
cre
as
e)
--- -
-
*
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Fig.7
H2O2 + PD98059 (30 µM)H2O2 + U0126 (30 µM)
H2O2 (300 µM)control
0
25
50
75
100
0 2 8 12 24 (h)
*§
§
§ §
§
§
ce
llv
iab
ilit
y(%
of
co
ntr
ol)
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a
b
c
d
a: control
b: H2O2 (300 µM)
c: H2O2 + U0126 (30 µ M)
d: H2O2 + PD98059 (30 µM)
M control H2O2 U0126 PD98059(300 µM) (30 µM) (30 µM)
H2O2
(A)
(B)
Fig. 8
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(A)
(B)
control oligo antisense oligo
0 1 5 10 1 5 10 (µM)
0
25
50
75
100antisense oligo
control oligo
without oligo
H2O2 (300 µM)
U0126 (30 µM)
PD98059 (30 µM)
+ ++
+
+
Fig. 9
IB:ERK1/2
ERK1/2
ERK1/2
*
tc
ell
via
bil
ity
(%o
fc
on
tro
l)
---
-- --
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c-Src
MEK5
BMK1/ERK5
MEF2C
cell survival
H2O2
MEK1/2 MKK4/7 MKK3/6
ERK1/2 JNK/SAPK p38
cell membrane
Fig.10
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Taketani, Hitoshi Houchi, Koichiro Tsuchiya, Eiji Takeda and Toshiaki TamakiYuki Suzaki, Masanori Yoshizumi, Shoji Kagami, A. Hajime Koyama, Yutaka
survival following oxidative insults1 (BMK1) and the MEF2C signaling pathway in PC12 cells - potential role in cell
Hydrogen peroxide stimulates c-Src-mediated big mitogen-activated protein kinase
published online January 8, 2002J. Biol. Chem.
10.1074/jbc.M111790200Access the most updated version of this article at doi:
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