Essential role of p38 MAPK in caspase-independent, iPLA2-dependent cell
death under hypoxia/low glucose conditions
Mamoru Aotoa,*, Koei Shinzawab,c, Yoji Suzukia, Nobutaka Ohkuboa, Noriaki Mitsudaa,
aDepartment of Physiology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan
bLaboratory of Molecular Genetics, Department of Medical Genetics, Osaka University Medical School, Osaka 565-0871, Japan
cSolution Oriented Research for Science and Technology (SORST) of the Japan Science and Technology Agency (JST), Osaka, Japan
a r t i c l ei n f o
Received 16 March 2009
Revised 16 April 2009
Accepted 16 April 2009
Available online 24 April 2009
Edited by Vladimir Skulachev
Caspase-independent cell death
p38 mitogen-activated protein kinase
Calcium-independent phospholipase A2
Reactive oxygen species
a b s t r a c t
The mechanisms of cell death induced by hypoxia or ischemia are not yet fully understood. We have
previously demonstrated that cell death induced by hypoxia occurs independently of caspases, and
is mediated by phospholipase A2(PLA2).
Here, we show that p38 mitogen-activated protein kinase is activated under hypoxia. A selective
inhibitor of p38 or decrease in the p38alpha protein level prevents hypoxia-induced cell death.
The p38 inhibitor abolishes PLA2activation by hypoxia, indicating that p38 acts upstream of PLA2.
The antioxidant N-acetyl-cysteine inhibits activation of p38 and cell death induced by hypoxia, indi-
cating that reactive oxygen species (ROS) are responsible for p38 activation. These results demon-
strate that the ROS/p38/PLA2signaling axis has a crucial role in caspase-independent cell death
induced by hypoxia.
Crown Copyright ? ? 2009 Published by Elsevier B.V. on behalf of Federation of European Biochemical
society. All rights reserved.
Programmed cell death is a biological process that eliminates
unwanted cells and it plays a crucial role in various events, includ-
ing morphogenesis during normal development and maintenance
of homeostasis. Apoptosis is one form of programmed cell death
for which the molecular mechanisms have been well investigated.
Apoptosis depends on the activation of caspases, a unique family of
cysteine proteases that cleave specific substrates and thereby
mediate many of the characteristic biochemical and morphological
changes of apoptotic cells, such as chromatin condensation, nucle-
ar DNA fragmentation, and formation of apoptotic bodies .
Recent studies have revealed other forms of regulated cell death
[2,3] that are caspase-independent and show necrosis-like fea-
tures, although the molecular mechanisms involved have not been
Cell death due to hypoxia is a major concern in various clinical
settings, such as in patients with ischemic diseases and during or-
gan transplantation. However, the mechanisms of hypoxic cell
death have not been fully elucidated. One of the reasons for this
is that apoptosis and necrosis occur simultaneously in many cell
lines under hypoxic conditions, with the percentage of apoptotic
and necrotic cells varying among different cell lines . Recently,
we found that exposure to hypoxia/low glucose stress causes al-
most all cells to undergo caspase-independent death , which is
characterized by loss of plasma membrane integrity and a shrun-
ken round nucleus. These characteristic morphological changes
and the occurrence of cell death can be suppressed by inhibitors
of phospholipase A2(PLA2), an enzyme that we initially identified
as inducing nuclear shrinkage.
PLA2is a family of esterases that hydrolyze the sn-2 ester bond
in phospholipids to release free fatty acids and lysophospholipids.
A number of mammalian PLA2isotypes have been identified, and
these are divided into three major subfamilies known as (1) secre-
tory PLA2(sPLA2), (2) cytosolic Ca2+-dependent PLA2(cPLA2), and
0014-5793/$36.00 Crown Copyright ? 2009 Published by Elsevier B.V. on behalf of Federation of European Biochemical society. All rights reserved.
Abbreviations: AA, arachidonic acid; ATF-2, activating transcription factor-2;
BEL, bromoenol lactone; cPLA2, calcium-dependent PLA2; iPLA2, calcium-indepen-
dent PLA2; MAPK, mitogen-activated protein kinase; LDH, lactate dehydrogenase;
ro-3-phosphoethanolamine; PLA2, phospholipase A2; ROS, reactive oxygen species;
siRNA, small interfering RNA; TPA, 12-O-tetradecanoylphorbol 13-acetate.
* Corresponding authors. Address: Laboratory of Molecular Genetics, Department
of Medical Genetics, Osaka University Medical School, Osaka 565-0871, Japan (Y.
Tsujimoto). Fax: +81 89 960 5246 (M. Aoto), +81 6 6879 3369 (Y. Tsujimoto).
E-mail addresses: email@example.com (M. Aoto), firstname.lastname@example.org.
ac.jp (Y. Tsujimoto).
FEBS Letters 583 (2009) 1611–1618
journal homepage: www.FEBSLetters.org
(3) Ca2+-independent PLA2 (iPLA2). By studies employing small
interfering RNA (siRNA), we have shown that iPLA2b plays the ma-
jor role in hypoxia/low glucose-induced cell death, at least in the
cell lines tested so far . Although there have been several reports
suggesting a crucial role of PLA2s in ischemic cell death [6–8], the
mechanisms regulating the activity of these enzymes under hyp-
oxic/ischemic conditions are not well known.
The p38 mitogen-activated protein kinase (MAPK) family has
four members that are known as p38a, b, c, and d. These p38 MAP-
Ks are important stress kinases with a role in inflammation, cell
growth and differentiation, cell cycle regulation, and apoptotic cell
death . The role of p38 MAPKs in apoptotic cell death varies with
the type of cell and stimulus studied . An anti-apoptotic effect
of p38 MAPKs has been described in cardiomyocytes , fibro-
blasts with DNA damage , endothelial cells exposed to anox-
ia-reoxygenation , differentiating neurons , and activated
macrophages . On the other hand, there is also good evidence
that p38 MAPKs mediate apoptosis in certain types of cells, includ-
ing neurons  and cardiomyocytes . Moreover, studies of
cardiomyocytes and embryonic fibroblasts derived from p38a ?/
? mice have suggested that p38a sensitizes cells to apoptosis via
both up-regulation of proapoptotic proteins and down-regulation
of survival pathways . However, the role of p38 MAPKs in the
process of caspase-independent, non-apoptotic cell death is largely
The purpose of this study was to investigate the molecular
mechanism of PLA2 activation during caspase-independent cell
death induced by hypoxia/low glucose stress. We demonstrated
that PLA2was activated via p38 MAPK, which in turn was activated
by reactive oxygen species produced under hypoxia/low glucose
2. Materials and methods
Bromoenol lactone (BEL, a selective inhibitor of iPLA2) was pur-
chased from Cayman Chemical (Ann Arbor, MI) and a p38 MAPK
inhibitor (SB203580) was obtained from Calbiochem (Darmstadt,
Germany). BEL and SB203580 were dissolved in DMSO. An anti-
p38a antibody (#9218), anti-p38b antibody (#2339), anti-phos-
pho-p38 (T180/Y182) antibody (#9211), anti-ATF-2 antibody
(#9226), anti-phospho-ATF-2 (T71) antibody (#9221), anti-p44/
42 MAPK (T202/Y204) antibody (#4695), anti-phospho-p44/42
MAPK antibody (#9101), anti-JNK (T183/Y185) antibody (#9252),
and anti-phospho-JNK (#9251) were purchased from Cell Signaling
Technology (Danvers, MA). An anti-ASK1 (C2) antibody was ob-
tained from AnaSpec Inc. (San Jose, CA). An anti-GAPDH antibody
(MAB374) and an anti-iPLA2b antibody (160507) were obtained
from Millipore (Billerica, MA) and Cayman Chemical, respectively.
2.2. Cell culture
A mouse neuroblastoma cell line (Neuro2a) was purchased from
the RIKEN Cell Bank (Tsukuba, Japan) and routinely incubated at
37 ?C in a humidified atmosphere of 5% CO2in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% FBS, 100 lg/ml penicillin,
100 lg/ml streptomycin, and 4.5 g/l glucose.
2.3. Hypoxic treatment
Cells (3.5 ? 105) were seeded in 3.5 cm dishes containing
DMEM as described above. After 16 h, the medium was changed
to DMEM containing 2.2 g/l glucose and 10% dialyzed FBS, and
the cells were exposed to normoxic or hypoxic conditions at
37 ?C. Hypoxia was achieved by using a BD BBL GasPakTMPouch
Anaerobic System (Becton Dickinson, Franklin Lakes, NJ), which
catalytically reduces oxygen to undetectable levels (less than
0.1%) as assessed with an oxygen electrode. After trypsinization,
cells were harvested and resuspended in PBS. After staining with
10 lM Hoechst 33342 and 10 lg/ml propidium iodide, nuclear
morphology was assessed under a fluorescence microscope (Key-
ence, BZ-8000). The extracts of cells exposed to hypoxia were col-
lected inside a glove box equilibrated with 100% N2to avoid any
possibility of re-oxygenation. The glove box was equipped with
an oxygen sensor that continuously monitored the oxygen tension.
2.4. Measurement of cell death
Cell death was measured using a commercial kit (Kyokuto
Chemical Co., Osaka, Japan) according to the manufacturer’s proto-
col. This kit is based on the measurement of lactate dehydrogenase
(LDH) that is released into the medium from damaged cells. Cell
death is presented as the amount of LDH measured in the medium
divided by the total LDH released after treatment of control cells
with 1% Triton X-100.
2.5. Western blot analysis
Cells were lysed in lysis buffer (10 mM HEPES, pH 7.5, 150 mM
NaCl, 5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 50 mM NaF, and
20 mM b-glycerophosphate) and then centrifuged (14 000?g, 4 ?C,
20 min), after which the supernatant was collected as the cell ly-
sate. Lysates were subjected to SDS–polyacrylamide gel electro-
phoresis on 15% polyacrylamide gel for 1 h at 30 mA. Proteins
were transferred from the gels to PVDF membranes (Millipore,
Billerica, MA) by a semi-dry blotting method for 2 h at 90 mA. Then
membranes were blocked by incubation for 1 h at room tempera-
ture in 5% skim milk dissolved in 0.1% Tween-20/PBS (PBS-T),
and were subsequently incubated overnight at 4 ?C with the pri-
mary antibody. Next, the membranes were washed with PBS-T (4
times for 5 min each) and incubated with a secondary antibody
coupled to horseradish-peroxidase for 1 h at room temperature.
After further washing in PBS-T, immunoreactive bands were visu-
alized by using the ECL plus Western blotting Detection System
(GE Healthcare, Chalfont St Giles, England).
2.6. Gene silencing
siRNAs for mouse iPLA2b and control siRNA (SXXC-0600) were
purchased from B-Bridge International Inc. (Mountain View, CA)
and siRNAs for mouse p38a (Mm_MAPK14_2) and mouse ASK1
(Mm_3k5_1, Mm_3k5_2) were obtained from QIAGEN (Tokyo,
CATCTA-30(#2725). The sequences of mouse p38a siRNA, mouse
ASK1 siRNAs and the control siRNA were 50-TCAGATAATAC-
3k5_2), and 50-ATCCGCGCGATAGTACGTA-30, respectively. Trans-
fection of Neuro2a cells was performed with a ‘Cell Line Nucleofec-
tor kit V’ (Amaxa Biosystems, Gaithersburg, MD) according to the
general protocol for adherent cell lines described in the manufac-
turer’s instructions. Cells (1 ? 106) were transfected with 10 lg
of siRNA as follows. In brief, suspended cells were transferred to
an Amaxa cuvette and nuclear transfection was done using the
H-22 program. Immediately after transfection, the cells were trans-
ferred with the recommended plastic pipettes into the culture
dishes. Forty-eight hours after transfection with siRNA, cells were
used for these experiments.
M. Aoto et al./FEBS Letters 583 (2009) 1611–1618
2.7. Arachidonic acid release assay
Arachidonic acid release was assessed as described previously
. Briefly, cells were labeled for 16 h with 0.1 lCi/ml [3H] ara-
chidonic acid (AA) in serum-free DMEM containing 0.2% (wt/vol)
fatty acid-free bovine serum albumin (BSA/DMEM). After washing
three times with BSA/DMEM, the cells were exposed to hypoxia.
The radioactivity of [3H] AA released into the medium was mea-
sured using a scintillation counter. Release of AA is presented as
the radioactivity of [3H] AA measured in the medium divided by
the total radioactivity of [3H] AA released after treatment of control
cells with 1% Triton X-100.
2.8. Fluorometric assay of PLA2activity
A mixture of 1,2-dipalmitoylphosphatidylserine (DPPS), choles-
terol, phosphatidylglycerol, and PED6 (107:31:20:1; a total of
287 nmol) was dried and resuspended in 1 ml of PBS, after which
liposomes were prepared by sonication on ice for 5 min. Then the
liposomes were added to cultures at a final medium concentration
of 50 ll/ml, and incubation was done for 10 min at 37 ?C. Cells
were subsequently washed with PBS, stained with 10 lM Hoechst
33342, and placed on a glass slide for observation under a fluores-
cence microscope. The fluorescence intensity was measured with
Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA).
2.9. Measurement of reactive oxygen species (ROS)
Intracellular ROS was measured using of the oxidant-sensitive
fluorescent probe 5-(and-6)-chloromethyl-20,70-dichlorodihydro-
fluorescein diacetate acetyl ester (CM-H2DCFDA, Invitrogen, Carls-
bad, CA). Cells were exposed to hypoxia in the presence of 10 lM
CM-H2DCFDA. Subsequently, cells were washed with PBS and lysed
in 1% Triton X-100. After centrifugation, the fluorescence of the
supernatant was measured in a Cyto Fluor?multi-well plate reader
series 4000 (Perseptive Biosystems, Foster City, CA) with excitation
of 488 nm and emission of 530 nm.
3.1. Hypoxia/low glucose induces caspase-independent, iPLA2-
dependent death of mouse Neuro2a cells
In our previous study of PC12 cells , we found that caspase-
independent death was induced by hypoxia in the presence of a
low glucose concentration (such as 2.2 g/l). To determine whether
such caspase-independent, iPLA2-dependent death also occurred in
other neuronal cells under the same conditions, we studied the
mouse neuroblastoma cell line Neuro2a. Cell death was assessed
by detecting lactate dehydrogenase (LDH) that was released into
the culture medium and from changes of nuclear morphology.
When Neuro2a cells were exposed to hypoxia in the presence of
a low glucose concentration (2.2 g/l), a significant fraction of cells
died (Fig. 1A), and a pan-caspase inhibitor (zVAD-fmk) did not in-
hibit this cell death (Fig. 1A). When stained with propidium iodide
(PI), the nuclei of cells exposed to hypoxia incorporated the dye
(Fig. 1B), demonstrating loss of membrane integrity that is a char-
acteristic of necrotic death. On the other hand, in the presence of
an iPLA2 inhibitor, bromoenol lactone (BEL), hypoxia/low glu-
cose-induced cell death was strongly inhibited (Fig. 1A). As shown
in Fig. 1B, the number of cells with nuclei stained by PI was also
markedly decreased in the presence of BEL. It was noteworthy that
BEL also inhibited hypoxia/low glucose-induced nuclear shrinkage.
These findings indicated that hypoxia/low glucose-induced death
of Neuro2a cells was mediated by a caspase-independent and
iPLA2-dependent mechanism like that acting in PC12 cells.
To confirm the responsibility of iPLA2b for hypoxia/low glucose-
induced cell death of Neuro2a cells, we reduced the iPLA2b level
using siRNA. Neuro2a cells were treated with siRNAs for iPLA2b,
and the expression of iPLA2b protein was assessed by Western
blotting. After transfection with siRNAs of iPLA2b, the level of
iPLA2b protein decreased significantly (Fig. 1C). As shown in
Fig. 1D, reduction of the iPLA2b level by siRNAs suppressed the hy-
poxia/low glucose-induced cell death. These results suggested that
iPLA2b plays a major role in hypoxia/low glucose-induced, caspase-
independent cell death of Neuro2a cells.
3.2. p38 MAPK is involved in hypoxia/low glucose-induced death
We next investigated the influence of hypoxia/low glucose
stress on p38 MAPK activity. It is known that p38 MAPK is acti-
vated by upstream kinases through phosphorylation of threonine
and tyrosine residues residing within the activation loop of the cat-
alytic domain (Thr180/Tyr182 in human p38 MAPK) . As
shown in Fig. 2A, exposure to hypoxia/low glucose conditions
resulted in transient phosphorylation of p38 MAPK at the sites cor-
responding to Thr180/Tyr182 of human p38 MAPK (p-p38). Phos-
phorylation of ATF-2 (p-ATF-2), one of the targets of p38 MAPK,
was also detected in a similar manner. On the other hand, we could
not detect the phosphorylation of p44/42 MAPK (ERK) and JNK
during hypoxia/low glucose treatment (Fig. 2A). These results indi-
cated that p38 MAPK was activated under hypoxia/low glucose
We also examined whether p38 MAPK was required for hypox-
ia/low glucose-induced death by using a p38 MAPK inhibitor or
RNAi. Treatment with a p38 MAPK inhibitor (SB203580, 10 lM)
inhibited cell death due to hypoxia/low glucose stress (Fig. 2B).
SB203580 is a specific inhibitor of p38a and p38b, rather than
p38c and p38d, and Neuro2a cells predominantly expressed
p38a (Fig. 2C). Reduction of the p38a level by siRNA also inhibited
hypoxia/low glucose-induced cell death and the phosphorylation
of p38 MAPK and ATF-2 (Fig. 2D–F). These results demonstrated
that p38 MAPK, particularly p38a, mediated cell death induced
by hypoxia/low glucose stress in Neuro2a cells.
3.3. A p38 MAPK inhibitor prevents PLA2activation induced by
We have previously reported that PLA2activity is elevated by
hypoxia/low glucose stress and is essential for cell death to occur
. Therefore, we examined the relationship between PLA2
activation and p38 MAPK activation by testing whether PLA2
activation was blocked by a p38 MAPK inhibitor. PLA2 activity
was monitored with a fluorescent PLA2reporter, N-((6-(2,4-dini-
nolamine (PED6). The fluorescent BODIPYTMmoiety at the sn-2 po-
sition of PED6 is quenched by the dinitrophenyl group in the head
group, but emits fluorescence when released by PLA2catalysis .
As shown in Fig. 3A and B, strong BODIPYTMfluorescence was de-
tected in cells exposed to hypoxia/low glucose, but only weak fluo-
rescence was found in control cells and p38 MAPK inhibitor-
treated cells. To further confirm the inhibition of hypoxia/low glu-
cose-induced PLA2activation by the p38 inhibitor, arachidonic acid
(AA) release into the culture medium was measured. AA release in-
duced by hypoxia/low glucose was inhibited by the p38 inhibitor
as well as BEL (Fig. 3C).
To investigate whether activation of iPLA2has a positive feed-
back effect on p38 MAPK activation under hypoxia/low glucose
conditions, we examined the effect of an iPLA2inhibitor on the
phosphorylation of p38 MAPK and its downstream target ATF-2.
As shown in Fig. 3D, the iPLA2 inhibitor had no effect on the
M. Aoto et al./FEBS Letters 583 (2009) 1611–1618
phosphorylation of p38 MAPK and ATF-2. Taken together with the
results described above, these findings suggested that p38 MAPK
acted upstream of iPLA2.
3.4. Increase of ROS under hypoxia/low glucose stress causes p38
It has been shown that hypoxia induces the generation of ROS
[22–24] and that ROS activate p38 MAPK . We investigated
whether hypoxia stimulates ROS production in Neuro2a cells
by measuring ROS production through the use of oxidant-sensi-
tive fluorescent probe CM-H2DCFDA. As shown in Fig. 4A, a sig-
nificant increase of fluorescence intensity was observed in
lysates from cells treated with hypoxia, but not with normoxia.
Hypoxia/low glucose-induced ROS production was not inhibited
by zVAD-fmk and p38 inhibitor (Fig. 4A). However, when cells
treated with hypoxia in the presence of NAC (the antioxidant
N-acetyl-cysteine), an increase of fluorescence intensity was
not observed (Fig. 4A). Moreover, NAC (25 mM) significantly
reduced hypoxia/low glucose-induced cell death, as well as
phosphorylation of p38 MAPK and ATF-2 (Fig. 4B and C). These
results indicate that hypoxia/low glucose-induced death is med-
iated by ROS.
It is known that ROS activate MAPKKK ASK1  and ASK1 acti-
vates p38 MAPK through the activation of MKK3 or MKK6. We
investigated whether ASK1 contributes to hypoxia/low glucose-
Fig. 1. Induction and characterization of caspase-independent cell death by exposure to hypoxia/low glucose conditions. Neuro2a cells were incubated in the absence or
presence of a pan-caspase inhibitor (zVAD-fmk, 50 lM) or an iPLA2inhibitor (BEL, 40 lM), and then were exposed to normoxia or hypoxia at 37 ?C. (A) The percentage of dead
cells was evaluated by measuring the release of lactate dehydrogenase into the culture medium as described in Section 2. (B) At 24 h after exposure to hypoxia/low glucose
conditions, Neuro2a cells were stained with Hoechst 33342 and PI before being viewed under a fluorescence microscope. Scale bar: 50 lm. (C) siRNAs for iPLA2b (#874,
#2725) or the control siRNA were transfected into Neuro2a cells as described in Section 2. Expression of iPLA2b protein was assessed by Western blotting. Lysates of
untreated, iPLA2b siRNA-treated, or control siRNA-treated Neuro2a cells were subjected to 15% SDS–PAGE followed by Western blotting with antibodies for iPLA2b or GAPDH.
(D) As described above, Neuro2a cells were transfected with siRNAs for iPLA2b (#874, #2725) or the control siRNA, and then were exposed to hypoxia/low glucose conditions.
Cell death was measured as described in Section 2. The data shown in A and D represent the means ± S.D. of four independent experiments.
M. Aoto et al./FEBS Letters 583 (2009) 1611–1618
induced cell death. Silencing of ASK1 inhibited hypoxia/low
glucose-induced cell death (Fig. 4D and E). It also suppressed the
phosphorylation of p38 and ATF-2 and the release of arachidonic
acid into the culture medium (Fig. 4F and G), indicative of suppres-
sion of PLA2activation.
These results suggested that the increase of ROS under hypoxia/
low glucose conditions led to cell death through activation of
We previously developed an experimental model using PC12
cells, in which almost all of the cells exposed to hypoxia/low glu-
cose stress died and showed morphologic changes like shrunken
round nuclei and plasma membrane disruption that were clearly
different from apoptosis. In this model, a pan-caspase-inhibitor
(z-VAD-fmk) causes little inhibition of cell death, while an iPLA2
Fig. 2. Requirement of p38 MAPK for hypoxia/low glucose-induced cell death. (A) Neuro2a cells were incubated under hypoxic conditions for the indicated time. Neuro2a
cells were also treated with 1 lM 12-O-tetradecanoylphorbol 13-acetate for 10 min or incubated at 42 ?C for 60 min as the control for p44/42 MAPK activation or JNK
activation, respectively. Cell lysates were subjected to 15% SDS–PAGE followed by Western blot analysis. The membrane was incubated with antibodies for phospho-p38 (p-
p38), p38a, phospho-ATF-2 (p-ATF-2), ATF-2, phospho-p44/42 MAPK, p44/42 MAPK, phospho-JNK, JNK and GAPDH (loading control). (B) Neuro2a cells were exposed to
hypoxia/low glucose conditions. A p38 MAPK inhibitor (10 lM) was added to the culture medium before hypoxic stress. Cell death was monitored by LDH release. (C) Lysates
of Neuro2a cells and HeLa cells were subjected to Western blot analysis with anti-p38a antibody, anti-p38b antibody, and anti-GAPDH antibody. (D) siRNA for p38a or the
control siRNA was transfected into Neuro2a cells as described in Section 2. Expression of p38a protein was assessed by Western blotting. Lysates of untreated, p38a siRNA-
treated, or control siRNA-treated Neuro2a cells were subjected to 15% SDS–PAGE followed by Western blotting with antibodies for p38a or GAPDH. (E) As described above,
Neuro2a cells were transfected with siRNA for p38a or the control siRNA, and then were exposed to hypoxia/low glucose conditions. Cell death was measured as described in
Section 2. (F) Neuro2a cells, transfected the control siRNA or siRNA for p38a, were incubated under hypoxic conditions for the indicated time. Cell lysates were subjected to
15% SDS–PAGE followed by Western blot analysis. The data shown in B and E represent the means ± S.D. of four independent experiments.
M. Aoto et al./FEBS Letters 583 (2009) 1611–1618
inhibitor (BEL) plus z-VAD-fmk efficiently inhibit it, suggesting
that the cells undergo caspase-independent and iPLA2-dependent
cell death . Exposure to hypoxia in the presence of BEL alone
leads to a significant level of apoptosis. In order to determine
whether other cell lines were also affected in the same way as
PC12 cells, we applied the same experimental conditions to Neu-
ro2a mouse neuroblastoma cells. When exposed to hypoxia/low
glucose, these cells also died in a caspase-independent and PLA2-
dependent manner like PC12 cells. Unlike PC12 cells, however,
BEL inhibited the death of Neuro2a cells without the addition of
z-VAD-fmk. Using siRNAs for iPLA2b, we showed that hypoxia/
low glucose-induced cell death was primarily due to activation of
When we investigated the molecular mechanism underlying
dependent death of Neuro2a cells in more detail, we found evi-
dence for a role of p38 MAPK in this form of cell death. Mito-
gen-activated protein kinases (MAPKs), including p38 MAPK,
Fig. 3. Inhibition of hypoxia/low glucose-induced activation of PLA2by a p38 MAPK inhibitor. (A) Neuro2a cells were exposed to hypoxia for 24 h in the absence or presence
of a p38 MAPK inhibitor (10 lM), and cells were also incubated under normoxic condition. Liposomes containing PED6 (a fluorescent PLA2reporter) were prepared as
described in Section 2, and added to the culture medium with incubation for 10 min. After nuclear staining, fluorescence due to PED6 (a–c) and Hoechst 33342 (d–f) was
visualized under a fluorescence microscope. Scale bar: 50 lm. (B) The fluorescence intensity was measured as described in Section 2. The data represent the means ± S.D. of
five independent experiments.*P < 0.001. (C) Neuro2a cells were incubated in the absence or presence of a p38 MAPK inhibitor (10 lM) or BEL (40 lM), and then were
exposed to normoxia or hypoxia at 37 ?C. AA released into the culture medium by hypoxia was measured as described in Section 2. The data represent the means ± S.D. of four
independent experiments. (D) Neuro2a cells were exposed to hypoxia in the absence or presence of BEL (40 lM) for the indicated time. Cell lysates were subjected to 15%
SDS–PAGE, followed by Western blot analysis.
M. Aoto et al./FEBS Letters 583 (2009) 1611–1618
p44/42 MAPK, and JNK, play important roles in survival and cell
death . Phosphorylation of p38 MAPK and its substrate,
ATF-2, was transiently elevated by hypoxia/low glucose stress,
but not p44/42 MAPK and JNK. A specific inhibitor of p38 MAPK
or siRNA prevented cell death. The p38 MAPK inhibitor also pre-
vented the activation of PLA2, indicating that p38 MAPK acts up-
stream of iPLA2b. We also demonstrated that ROS produced
under hypoxia/low glucose conditions induced the activation of
p38 MAPK, while an antioxidant (NAC) abolished the activation
of p38 MAPK and inhibited hypoxia/low glucose-induced cell
death. Our results suggest that the ROS-p38 MAPK-PLA2signaling
axis has an important role in hypoxia/low glucose-induced, cas-
pase-independent cell death.
In our system, glucose in culture media was perhaps completely
consumed during hypoxia treatment. It was reported that glucose
deprivation induces oxidative stress . It has also been shown
that production of ROS is induced during hypoxia via complex III
of the mitochondrial electron transport chain [23,24,28]. Such
ROS reportedly activate MAPKKK ASK1  and then initiate p38
MAPK activation mediated by the upstream MAPK kinases MKK3
and MKK6 [29,30]. Our data agree with the above-mentioned find-
ings. Under normal conditions, ASK1 is inhibited by binding to thi-
Fig. 4. Activation of p38 MAPK by ROS production under hypoxia/low glucose conditions. (A) Neuro2a cells were exposed to normoxia or hypoxia in the presence of the
oxidant-sensitive fluorescent probe (CM-H2DCFDA, 10 lM). Then the fluorescence in each cell lysate was measured as described in Section 2. (B) Neuro2a cells were exposed
to hypoxia in the absence or presence of an antioxidant (NAC, 25 mM). Cell death was monitored by LDH release. (C) Neuro2a cells were incubated under hypoxic conditions
in the absence or presence of 25 mM NAC for the indicated time. Cell lysates were subjected to 15% SDS–PAGE, followed by Western blot analysis. (D) siRNAs for ASK1 (#1,
#2) or the control siRNA were transfected into Neuro2a cells as described in Section 2. Lysates of untreated, ASK1 siRNA-treated, or control siRNA-treated Neuro2a cells were
subjected to 15% SDS–PAGE followed by Western blotting with antibodies for ASK1 or GAPDH. (E) Neuro2a cells were transfected with siRNAs for ASK1 (#1, #2) or the control
siRNA, and then were exposed to hypoxia/low glucose conditions. Cell death was measured as described in Section 2. (F) Neuro2a cells, which were transfected with the
control siRNA or siRNA for ASK1 (#2), were incubated under hypoxic conditions for the indicated time. Cell lysates were subjected to 15% SDS–PAGE followed by Western blot
analysis. (G) Neuro2a cells were transfected with the control siRNA or siRNA for ASK1 (#2), and then were exposed to hypoxia. AA released into the culture medium was
measured as described in Section 2. The data shown in A, B, and E represent the means ± S.D. of four independent experiments.
M. Aoto et al./FEBS Letters 583 (2009) 1611–1618
oredoxin (Trx). However, ASK1 only binds to the reduced form of
Trx and not to the oxidized form, suggesting that ASK1 activity de-
pends on the redox status of Trx. ROS cause dissociation of Trx
from ASK1 and thereby activate ASK1. Thus, the Trx–ASK1 system
serves as a molecular switch that converts the redox signal evoked
by oxidative stress to activation via kinase cascades. This Trx-ASK1
system may be active in our hypoxia model.
An important question to be answered is how p38 MAPK upreg-
ulates iPLA2b. It might increase the expression level of iPLA2b pro-
tein or might phosphorylate and activate this enzyme directly or
indirectly. We did not find any change in iPAL2b levels during hy-
poxia/low glucose conditions [5 and unpublished data]. Although
no previous studies have been found on the phosphorylation-
dependent regulation of iPLA2b activity, it has been reported that
cPLA2a activity is enhanced by phosphorylation of Ser727, which
prevents association with some cPLA2a-binding proteins . In
this study, we could not determine whether there was direct phos-
phorylation of iPLA2b by p38 MAPK. It was previously shown that
p38 MAPK played a role in thrombin-induced activation of iPLA2in
vascular smooth muscle cells , although the mechanism was
In summary, the present study demonstrated for the first time
that p38 MAPK plays an important role in caspase-independent,
iPLA2b-dependent death of Neuro2a cells under hypoxia/low glu-
cose conditions, with the changes being mediated by ROS
This work was supported in part by a grant for Creative Scien-
tific Research from Japan Society for the Promotion of Science, a
grant for the 21st Century COE Program, a grant for Scientific Re-
search from the Ministry of Education, Science, Sports and Culture,
a grant for the Global COE program, and a grant for Research on
Dementia and Bone Fracture from the Ministry of Health, Labor
and Welfare, Japan.
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