Increased expression of c-Fos by extracellular
signal-regulated kinase activation under sustained
oxidative stress elicits BimEL upregulation and hepatocyte
apoptosis
Yasuhiro Ishihara
1
, Fumiaki Ito
2
and Norio Shimamoto
1
1 Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences at Kagawa, Tokushima Bunri University, Japan
2 Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan
Introduction
Apoptosis has several morphological features, includ-
ing cell shrinkage, nuclear condensation, and nucleoso-
mal DNA fragmentation. Extensive studies to uncover
the mechanisms underlying the induction of apoptosis
have yielded the generally accepted theory that mito-
chondria play a fundamental role in the process. Apop-
totic stimuli activate the mitochondrial permeability
transition pore and the release of apoptosis-promoting
molecules such as cytochrome c, apoptosis-inducing
factor, and endonuclease G [1]. The pathways
upstream of the mitochondria for apoptotic signal
transduction have recently been identified. Several
Keywords
apoptosis; Bim; c-Fos; extracellular signal-
regulated kinase (ERK); reactive oxygen
species
Correspondence
N. Shimamoto, Laboratory of Pharmacology,

knockdown of c-Fos and c-Jun, respectively. These results indicate that
increases in c-Fos following extracellular signal-regulated kinase activation
are critical for BimEL upregulation and apoptosis.
Abbreviations
AP-1, activator protein-1; ATZ, 3-amino-1,2,4-triazole; ChIP, chromatin immunoprecipitation; ERK, extracellular signal-regulated kinase;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ROS, reactive oxygen species; SE, standard error; siRNA, small interfering RNA.
FEBS Journal 278 (2011) 1873–1881 ª 2011 The Authors Journal compilation ª 2011 FEBS 1873
molecules that are known to be involved in prolifera-
tion and ⁄ or differentiation have been reported to
induce apoptosis [2,3].
Extracellular signal-regulated kinase (ERK) is a
classic mitogen-activated protein kinase that is acti-
vated by growth factors and induces cell cycle pro-
gression via cyclin transcription. However, increasing
evidence shows that ERK is activated by reactive
oxygen species (ROS), and that this is followed by
the induction of apoptosis [4–6]. ERK-dependent
apoptosis induced by ROS has been recognized in
several pathological conditions, such as alcoholic liver
injury [7,8], lung hyperoxia [9], and cisplatin-induced
renal toxicity [10]. However, little is known about the
mechanism responsible for apoptotic signaling elicited
by active ERK, and this process therefore needs to
be investigated.
The mechanism responsible for ERK activation by
ROS is well understood. The phosphorylation of ERK
or its upstream kinases is regulated by phosphatases
such as PTP1B [11], MKP3 [12], and LMW-PTP [13].
The cysteines in the active sites of these phosphatases
are easily inactivated by ROS, resulting in activation

Results
We previously showed that treatment with 3-amino-
1,2,4-triazole (ATZ) and mercaptosuccinic acid
inhibited catalase and glutathione peroxidase, which
are antioxidative enzymes that eliminate hydrogen per-
oxide, and caused sustained increases in ROS levels
and apoptosis in rat primary hepatocytes [22,23]. In
addition, we recently reported that ROS-activated
ERK induces BimEL transactivation, followed by
hepatocyte apoptosis [15]. This study was designed to
examine the mechanism of hepatocyte apoptosis, with
a particular focus on identifying the transcription fac-
tor(s) that activate BimEL transcription downstream
of the ERK pathway.
We cloned a 2.9-kb fragment of the rat bim pro-
moter region from rat primary hepatocytes. The bim
promoter region included an AP-1-binding site, a
FOXO-binding site, and three Myb-binding sites
(Fig. 1A). The bim promoter region was subcloned
into pGL4.24 (pGL4.24-BimProm). pGL4.24-BimProm
mutations were generated at each transcription factor-
binding site (mutated points are indicated in Fig. 1A),
and bim promoter activity in the presence of
ATZ + mercaptosuccinic acid was assessed with a
luciferase reporter assay. The mutations at the binding
sites used in this study reportedly attenuate the activity
of each transcription factor [19,24,25]. When rat pri-
mary hepatocytes were transfected with pGL4.24-Bim-
Prom and treated with ATZ + mercaptosuccinic acid
for 9 h, the luciferase activity increased 3.3 ± 0.3-fold

examined the expression and phosphorylation of c-Fos
and c-Jun. The total amount of nuclear c-Fos
increased over time in the presence of ATZ + merca-
ptosuccinic acid (Fig. 2). Interestingly, phosphorylation
of c-Fos at Ser374 occurred in parallel with increases
in nuclear c-Fos levels (Fig. 2). Pretreatment with
U0126 or vitamin C largely suppressed the accumula-
tion of total and phosphorylated c-Fos in the presence
of ATZ + mercaptosuccinic acid (Fig. 2). In contrast,
there were no changes in the levels of total and phos-
phorylated nuclear c-Jun throughout the 9-h exposure
to ATZ + mercaptosuccinic acid (Fig. 2).
To show that AP-1 proteins directly bind to the con-
sensus AP-1 site in the bim promoter region (from
)2491 to )2497), a chromatin immunoprecipitation
(ChIP) assay was performed. A PCR analysis demon-
strated that c-Fos and c-Jun antibodies apparently pre-
cipitated the bim promoter region from rat primary
hepatocytes treated with ATZ + mercaptosuccinic
acid, whereas untreated hepatocytes and those pretreat-
ed with U0126 or vitamin C showed only slight DNA
binding (Fig. 3). Pretreatment with SP600125, an
inhibitor of c-Jun N-terminal kinase, showed no effect
on the DNA binding of c-Fos and c-Jun induced by
treatment with ATZ + mercaptosuccinic acid, indicat-
ing that JNK is not involved in the binding of AP-1 to
the bim promoter region. Nonspecific IgG also did not
exhibit DNA-binding activity (Fig. 3). These results
indicate that the AP-1 proteins bind specifically to the
AP-1 cis-regulatory region of the bim promoter in

were cultured for 14 h. Cells were treated
with U0126 (40 l
M) or vitamin C (1 mM),
and then incubated for 9 h in the presence
or absence of ATZ (20 m
M) and merca-
ptosuccinic acid (7 m
M). Cell were collected
and lysed, and both firefly and Renilla
luciferase activities were measured. Values
for untreated cells carrying pGL4.24-
BimProm and pRL-RSV were set equal to 1.
The values are the means ± SE of six
separate experiments. Data were analyzed
with Student’s t-test or Dunnett’s test.
**P < 0.01 versus the untreated BimProm
group.
#
P < 0.05 and
##
P < 0.01 versus the
ATZ + mercaptosuccinic acid-treated
BimProm group.
Y. Ishihara et al. Regulation of BimEL expression by c-Fos
FEBS Journal 278 (2011) 1873–1881 ª 2011 The Authors Journal compilation ª 2011 FEBS 1875
condensation and DNA fragmentation were all abro-
gated by knockdown of c-Fos and c-Jun (Fig. 5A–C).
Transfection of scrambled siRNAs showed no effects
on the expression levels of c-Fos, c-Jun, or BimEL,
and did not affect hepatocyte apoptosis (Figs 4 and 5).

but significantly, reduced the luciferase activity in this
study. Therefore, the involvement of FOXO and Myb
in hepatocyte apoptosis should be examined further.
c-Fos is one of the main components of the AP-1
transcription factor complex [30]. Activated ERK
phosphorylates c-Fos at Ser-374, leading to its stabil-
ization [29,31]. Therefore, we examined the expression
and phosphorylation of c-Fos in this study. The total
and phosphorylated c-Fos levels increased over time in
the presence of ATZ + mercaptosuccinic acid, and
this increase was suppressed by pretreatment with
U0126. Therefore, c-Fos is stabilized by phosphoryla-
tion, which is mediated by ERK, allowing c-Fos to
accumulate. In contrast, c-Jun, another major compo-
nent of the AP-1 complex, is reportedly phosphory-
lated at Ser63 and Ser73 by active ERK, and this is
followed by increased c-Jun transcriptional activity
[32,33]. However, the total and phosphorylated c-Jun
levels in nuclei remained unaffected in the presence of
ATZ + mercaptosuccinic acid. Because c-Fos alone
cannot bind to DNA, c-Jun is required for transcrip-
tional activation [27,28]. Thus, BimEL expression is
dependent on both increased levels of c-Fos and basal
levels of c-Jun. This idea is supported by the results of
the ChIP assay, which indicated that both c-Fos and
Fig. 2. Increases in the expression of total and phosphorylated
c-Fos by treatment with ATZ + mercaptosuccinic acid. Primary rat
hepatocytes were treated with U0126 (40 l
M) or vitamin C (1 mM),
and then incubated for 9 h in the presence or absence of ATZ

Fig. 4. Suppression of BimEL expression by knockdown of c-Fos or c-Jun. After transfection of c-Fos or c-Jun siRNA or their scrambled siR-
NAs (Scr siRNA) into hepatocytes, cells were incubated for 14 h, and then further incubated in the presence or absence of ATZ (20 m
M) and
mercaptosuccinic acid (7 m
M) for 9 h. (A) The levels of c-Fos and c-Jun protein were determined by a western blot analysis (Aa), and bands
were then quantified and expressed as the fold change from the density of untreated hepatocytes as determined by densitometry (Ab,c).
The values are the means ± SE of five separate experiments. The data were analyzed with Dunnett’s test. **P < 0.01 versus the
ATZ + mercaptosuccinic acid-treated group. (B) The levels of BimEL mRNA were measured by real-time PCR. BimEL mRNA levels were nor-
malized using GAPDH mRNA. Values for untreated cells were set equal to 1. The values are the means ± SE of five separate experiments.
The data were analyzed with Dunnett’s test. **P < 0.01 versus the ATZ + mercaptosuccinic acid-treated group. (C) The expression of BimEL
proteins was evaluated by a western blot analysis (Ca). The bands were quantified and expressed as the fold change in their density as
compared with untreated hepatocytes (Cb). The values are the means ± SE of five separate experiments. The data were analyzed with
Dunnett’s test. **P < 0.01 versus the ATZ + mercaptosuccinic acid-treated group.
A
B
C
Fig. 5. Suppression of hepatocyte apoptosis by knockdown of c-Fos or c-Jun. After transfection of c-Fos or c-Jun siRNA into hepatocytes,
cells were incubated for 14 h, and then further incubated in the presence or absence of ATZ (20 m
M) and mercaptosuccinic acid (7 mM) for
24 h. Cell viability (A) and chromatin condensation (B) were assayed. The values are the means ± SE of five separate experiments. The data
were analyzed with Dunnett’s test. **P < 0.01 versus the ATZ + mercaptosuccinic acid-treated group. (C) Cellular DNA was extracted and
electrophoresed after a 24-h incubation. The results are representative of four independent experiments.
Y. Ishihara et al. Regulation of BimEL expression by c-Fos
FEBS Journal 278 (2011) 1873–1881 ª 2011 The Authors Journal compilation ª 2011 FEBS 1877
c-Jun localize to the AP-1-binding site in the bim pro-
moter region. Furthermore, knockdown of c-Fos or
c-Jun attenuated BimEL transactivation and apoptosis,
supporting the hypothesis that c-Fos and c-Jun act
coordinately to increase the expression of BimEL.
Increased c-Fos levels are therefore critical for BimEL

accumulation. Therefore, under conditions where ERK
is persistently activated, c-Fos could transcriptionally
activate several genes, together with c-Jun. In this
experimental model, ERK was activated for 9 h after
the addition of ATZ + mercaptosuccinic acid, owing
to inactivation of protein tyrosine phosphatase caused
by sustained increases in intracellular ROS levels [15].
Therefore, we concluded that AP-1-dependent gene
expression occurred under the conditions of sustained
oxidative stress. This idea is supported by data show-
ing that transient oxidative stress for 3 or 6 h did not
induce apoptosis [38].
In conclusion, ERK activation resulting from sus-
tained oxidative stress increased the amounts of total
and phosphorylated nuclear c-Fos. Increased c-Fos
and basal c-Jun localized to the AP-1-binding site in
the bim promoter region and induced transcription of
BimEL mRNA, followed by hepatocyte apoptosis.
Therefore, the increase in c-Fos downstream of ERK
activation is critical for BimEL upregulation and apop-
tosis. The duration of exposure to oxidative stress
affects c-Fos stability and BimEL expression by chang-
ing the duration of the ERK signal. Therefore, the
duration of oxidative stress might be a fundamental
determinant of cellular fate.
Experimental procedures
Materials
ATZ and mercaptosuccinic acid were from Sigma–Aldrich
(St Louis, MO, USA). U0126 was from Promega (Madison,
WI, USA). SP600125 was from Bio Mol (Plymouth Meet-

which contains a BimProm-luc transcriptional fusion.
Mutation of the binding sites for AP-1, Myb and FOXO
in pGL4.24-BimProm was performed by site-directed
mutagenesis with the QuikChange kit (Stratagene, Santa
Regulation of BimEL expression by c-Fos Y. Ishihara et al.
1878 FEBS Journal 278 (2011) 1873–1881 ª 2011 The Authors Journal compilation ª 2011 FEBS
Clara, CA, USA) (primers: AP-1 Se, 5¢-CCGTCAGCGGT
GACTTGGATTCACAGAGAC-3¢; FOXO Se, 5¢-CAAGT
CACTAGGGTACCCACGCCGGGGTGG-3¢; Myb1 Se,
5¢-GACCAAGATGGTCCATC GGTGGGACGA CAG-3¢;
Myb2 Se, 5¢-CTCCCTGGTCTCTCATCTGTCCTTCCCA
CC-3¢; Myb3 Se, 5¢-CCTCCTGAGGCTTCCATCTGGCG
GCCGCGG-3¢). Mutations were confirmed by nucleotide
sequencing.
Transfection and luciferase activity assays
Cells were cotransfected with pGL4.24-BimProm or mutant
pGL4.24-BimProm and with pRL-RSV, using the Nucleo-
fection system (Amaxa, Koln, Germany), as described pre-
viously [40]. Luciferase reporter activity was measured with
the Dual-Glo Luciferase Assay System (Promega). Firefly
luciferase activity was normalized to Renilla luciferase activ-
ity and total protein levels.
Extraction of nuclear proteins and immunoblotting
Nuclear extracts were prepared according to our previous
report, with slight modifications [40]. Briefly, cells were sus-
pended in buffer A (10 mm Hepes, pH 7.8, 10 mm KCl,
2mm MgCl
2
, 0.1 mm EDTA, 0.5 mm dithiothreitol, and
protease inhibitor cocktail) and incubated on ice for

and Rv, 5¢-GGCTAGGTAACAGTTTAGCGAGGA-3¢).
Rat genomic DNA extracted from rat primary hepatocytes
was used as a positive control. PCR products were analyzed
by electrophoresis on 1.5% agarose gels.
Total RNA isolation and real-time PCR
Total RNA extraction from hepatocytes was performed with
an RNeasy Mini Kit (Qiagen). First-strand cDNA was
synthesized from total RNA with a ThermoScript RT-PCR
System (Invitrogen). The level of mRNA for BimEL was
measured by real-time quantitative RT-PCR with a 7500
Real-Time PCR System (Applied Biosystems, Foster City,
CA, USA), according to our previous report [15]. The
sequences of the forward and reverse primers were: Fw,
5¢-CCAGATCCCCACTTTTCATC-3¢; and Rv, 5¢-AAGAG
AAATACCCACTGGAGGA-3¢. The sequence of the Taq-
Man fluorogenic probe was 5¢-TGCTGTCC-3¢ (Universal
ProbeLibrary, Roche Diagnostics, Basel, Switzerland).
BimEL mRNA levels were corrected by glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) mRNA.
Assays for cell death and apoptotic features
Chromatin condensation was assessed with the DNA-bind-
ing fluorochrome Hoechst 33342. Nuclei were visualized
with a BX51WI fluorescence microscope (Olympus, Tokyo,
Japan). To detect DNA fragmentation, an Apoptosis DNA
Ladder Kit (Wako) was used.
RNA interference
The siRNA targeted to rat c-Fos was synthesized by Sigma
Genosys (Ishikari, Japan) (Se: 5¢-CCGAGAUUGCCAAU
CUACUTT-3¢). The siRNAs targeted to rat c-Jun (siTrio,
Cat. No. SRF27A-2035) were purchased from B-Bridge

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