BimEL expression and hepatocyte apoptosis were suppressed by knockdown of c-Fos and c-Jun, respectively.. Results We previously showed that treatment with 3-amino-1,2,4-triazole ATZ and
Trang 1signal-regulated kinase activation under sustained
oxidative stress elicits BimEL upregulation and hepatocyte apoptosis
Yasuhiro Ishihara1, Fumiaki Ito2and Norio Shimamoto1
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,
Faculty of Pharmaceutical Sciences at
Kagawa, Tokushima Bunri University,
1314-1, Shido, Sanuki, Kagawa 769-2193,
Japan
Fax: +81 87 894 0181
Tel: +81 87 894 5111 ext 6513
E-mail: n-shimamoto@kph.bunri-u.ac.jp
(Received 22 December 2010, revised 25
February 2011, accepted 22 March 2011)
doi:10.1111/j.1742-4658.2011.08105.x
We previously reported that the inhibition of catalase and glutathione per-oxidase activities by treatment with 3-amino-1,2,4-triazole (ATZ) and mer-captosuccinic acid evoked sustained increases in the levels of reactive oxygen species and apoptosis in rat primary hepatocytes Apoptosis was accompanied by increased expression of BimEL, following activation of extracellular signal-regulated kinase The aim of this study was to charac-terize the mechanism underlying hepatocyte apoptosis by identifying the transcription factor that induces BimEL expression The bim promoter region was cloned into a promoterless-luc vector, and promoter activity was monitored by a luciferase assay The luciferase activity increased in the presence of ATZ + mercaptosuccinic acid Pretreatment with a MEK inhibitor, U0126, or an antioxidant, vitamin C, suppressed the promoter activity Furthermore, ATZ + mercaptosuccinic acid-induced luciferase activity was attenuated by mutation of the activator protein-1 binding site
in the bim promoter region The amounts of total and phosphorylated c-Fos increased over time in the presence of ATZ + mercaptosuccinic acid, whereas the amounts of total and phosphorylated c-Jun remained unchanged Chromatin immunoprecipitation revealed that both c-Fos and c-Jun localized to the activator protein-1-binding site in the bim promoter region BimEL expression and hepatocyte apoptosis were suppressed by 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.
Trang 2tion 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
of the ERK pathway [14] However, factors that act
on the mitochondria downstream of ERK have been
rarely reported Recently, we showed that
ROS-acti-vated ERK increased the transcriptional expression
of BimEL, a major isoform among the bim gene
products, leading to apoptosis in rat primary
hepato-cytes [15]
Bim is a member of the Bcl-2 family of proteins,
which play a fundamental role in the induction of
mito-chondria-driven apoptosis Under normal conditions,
antiapoptotic Bcl-2 family members such as Bcl-2,
Bcl-xL and Mcl-1 interact with the proapoptotic Bcl-2
family members Bax⁄ Bak, to inhibit the ability of
Bax⁄ Bak to permeabilize the mitochondrial membrane
Bim activates the mitochondrial permeability transition
mediated by Bax⁄ Bak through two different
mecha-nisms [16]: (a) Bim binds to antiapoptotic Bcl-2 family
proteins to liberate Bax⁄ Bak, leading to mitochondrial
permeability transition; and (ii) Bim directly activates
Bax⁄ Bak (induces a conformational change), thus
lead-ing to pore formation
The bim gene is a direct target of transcription
factors such as FOXO3A, Myb and c-Jun [17–21] The
5¢-end of the bim gene contains binding sites for
FOXO, Myb, and activator protein-1 (AP-1) [18]
However, the mechanism underlying the
transcrip-tional activation of BimEL downstream of ERK
acti-vation is not known The aim of this study was to
identify the ERK-responsive transcription factor that
regulates BimEL expression
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
in comparison with untreated cells (Fig 1B) However, pretreatment with U0126, a potent inhibitor of MEK1,
or vitamin C, an antioxidant, largely suppressed ATZ + mercaptosuccinic acid-induced luciferase activ-ity (Fig 1B) In addition, when rat primary hepato-cytes were transfected with a mutated AP-1 (AP-1m) promoter construct, the ATZ + mercaptosuccinic acid-mediated increase in luciferase activity was greatly attenuated (Fig 1B) Transfection with a promoter construct containing myb1m had no effect on the lucif-erase activity, whereas transfection with myb2m, myb3m or FOXOm promoters partially suppressed ATZ + mercaptosuccinic acid-induced luciferase activ-ity (Fig 1B) These results suggest that AP-1 is involved in increasing BimEL expression downstream
of ERK activation in response to treatment with ATZ + mercaptosuccinic acid
The AP-1 transcription factor consists of Fos and Jun proteins [26] Fos and Jun form a dimer, which in turn binds to AP-1 regulatory elements and enhancer regions of numerous mammalian genes Jun forms homodimers and heterodimers with Fos proteins,
Trang 3whereas Fos proteins do not form homodimers, and
require heterodimerization to bind DNA [27,28]
Active ERK phosphorylates one of the major Fos
fam-ily proteins, c-Fos, and stabilizes it [29]; ERK also
phosphorylates c-Jun directly, leading to
transactiva-tion of AP-1 On the basis of these findings, we next
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 showpretreat-ed 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 bindindicat-ing 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 hepatocytes treated with ATZ + mercaptosuccinic acid Next, we examined the effect of c-Fos and c-Jun on BimEL transactivation and apoptosis Transfection with small interfering RNAs (siRNAs) targeted to c-Fos and c-Jun clearly reduced the target protein lev-els (Fig 4A) Elevation of BimEL mRNA expression
by treatment with ATZ + mercaptosuccinic acid was suppressed by transfection with siRNAs against c-Fos and c-Jun (Fig 4B) Increases in BimEL levels caused
by ATZ + mercaptosuccinic acid were also attenuated
by c-Fos or c-Jun knockdown (Fig 4C) AT Z+ mer-captosuccinic acid-induced cell death, chromatin
**
Fig 1 AP-1-dependent Bim transcriptional
activation is induced by treatment with
ATZ + mercaptosuccinic acid (A) A
sche-matic diagram of the rat bim promoter
(BimProm) The positions of the binding
sites for AP-1, Myb and FOXO are shown.
The mutation sequences of each
transcrip-tion factor-binding site are also presented.
(B) After cotransfection with
pGL4.24-BimProm or mutant pGL4.24-pGL4.24-BimProm with
pRL-RSV into rat primary hepatocytes, cells
were cultured for 14 h Cells were treated
with U0126 (40 l M ) or vitamin C (1 m M ),
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.
Trang 4condensation 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)
These results indicate that c-Fos and c-Hun are crucial
for BimEL expression and induction of hepatocyte
apoptosis
The bim promoter activity induced by treatment with ATZ + mercaptosuccinic acid was largely attenuated
by mutating the AP-1-binding site in the bim promoter region Whereas the amounts of total and phosphory-lated c-Fos increased in the presence of ATZ + mer-captosuccinic acid, there was no change in the levels of total and phosphorylated c-Jun throughout the experi-mental period Both c-Fos and c-Jun interacted with the AP-1-binding site in the bim promoter region Knockdown of c-Fos or c-Jun suppressed not only BimEL transactivation, but also hepatocyte apoptosis Pretreatment with U0126 or vitamin C largely abol-ished ATZ + mercaptosuccinic acid-induced luciferase activity, confirming that the ERK pathway elicited by ROS is involved in Bim transcription in this experi-mental system [15,23] In addition, mutation of the AP-1-binding site in the bim promoter region markedly suppressed the luciferase activity induced by ATZ + mercaptosuccinic acid, suggesting that AP-1 is responsible for Bim transcription Biswas et al reported that Bim expression was coregulated by three transcription factors – c-Jun, FOXO, and Myb – when PC12 cells were stimulated by nerve growth factor deprivation, and insisted that the bim promoter acts as
a coincidence detector [18] Interestingly, mutation of the Myb-binding and FOXO-binding sites also slightly, 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 m M ),
and then incubated for 9 h in the presence or absence of ATZ
(20 m M ) and mercaptosuccinic acid (7 m M ) Nuclear proteins were
extracted, and the time courses of c-Fos, c-Fos phosphorylated at
Ser374, c-Jun, c-Jun phosphorylated at Ser63, c-Jun
phosphory-lated at Ser73 and histone H1 were evaluated by immunoblotting.
The results are representative of four independent experiments.
Fig 3 Binding of c-Fos and c-Jun to the AP-1 site of the bim
pro-moter Rat primary hepatocytes were treated with U0126 (40 l M ),
vitamin C (1 m M ), or SP600125 (40 l M ), and then incubated for 9 h
in the presence or absence of ATZ (20 m M ) and mercaptosuccinic
acid (7 m M ) ChIP was used to assess the binding of c-Fos and
c-Jun to the AP-1-binding sites within the rat bim promoter Rat
genomic DNA was used as a positive control, and
immunoprecipita-tion with a nonspecific antibody (IgG) was used as a negative
control The results are representative of three independent
experi-ments.
Trang 5(a)
(a)
(b)
(b) (c)
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.
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 m M ) 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.
Trang 6moter 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
expression and apoptosis in this experimental system
Active ERK is known to phosphorylate BimEL,
resulting in the ubiquitination and degradation of
BimEL [34,35] Therefore, ERK activation was expected
to reduce the level of BimEL, leading to increased cell
survival as long as the proteasome maintains its normal
functions We previously reported that BimEL
degrada-tion was suppressed in this experimental system, because
ROS generated by treatment with ATZ +
merca-ptosuccinic acid inhibited the activities of the
protea-some [15] Namely, BimEL was upregulated by both
increased expression and decreased degradation in this
type of hepatocyte apoptosis c-Fos was also reported to
be degraded by the ubiquitin–proteasome system [36]
In this study, pretreatment with U0126 did not
com-pletely abrogate the c-Fos expression induced by
treat-ment with ATZ + mercaptosuccinic acid Therefore,
proteasome inhibition by ROS might be involved in the
increased expression of c-Fos in this experimental
sys-tem The mechanism(s) underlying the upregulation of
c-Fos should be examined in greater detail
The duration of the ERK signal is reported to be
important for c-Fos stability [37] Transient activation
of ERK could increase c-Fos transcription but could
not lead to c-Fos phosphorylation, because the ERK
signal is inactivated when c-Fos protein is synthesized
Nonphosphorylated c-Fos is rapidly degraded by the
ubiquitin–proteasome system [29,36] In contrast,
sus-tained ERK activation increases c-Fos transcription
and phosphorylation, leading to phosphorylated c-Fos
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
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-ing, PA, USA) Vitamin C was from Wako Pure Industries (Osaka, Japan) All other chemicals were obtained from Sigma–Aldrich or Wako Pure Industries, and were of the highest quality commercially available
Preparation of rat primary hepatocytes All procedures performed on animals were in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry
of Education, Culture, Sports, Science and Technology, Japan, and the Animal Care and Use Committee of Toku-shima Bunri University, Kagawa, Japan
Rat primary hepatocytes were prepared from male Wistar rats (body weight of 150–200 g) (Nippon CLEA, Osaka, Japan) by collagenase perfusion, as described in our previous report [39] Cells were plated onto collagen type I-coated dishes in hepatocyte culture medium (Williams’ medium E containing 10% fetal bovine serum, 300 nm insulin, and
100 nm dexamethasone) After a 2-h attachment period, the medium was exchanged and cells were used for experiments
Cloning and site-directed mutagenesis of the rat bim promoter region
Rat genomic DNA was extracted from rat primary hepato-cytes with the DNeasy Blood & Tissue Kit (Qiagen, Valen-cia, CA, USA) The bim promoter region, including the transcriptional initiation site (2903 bp), was amplified with Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) (primers: Fw, 5¢-GCCAGGCGAGAAATTTAGT GTC-3¢; and Rv, 5¢-CAACAAGCTGTTGACCCAGTG-3¢), and ligated into pGL4.24 to create pGL4.24-BimProm, 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
Trang 7Clara, CA, USA) (primers: AP-1 Se, 5¢-CCGTCAGCGGT
GACTTGGATTCACAGAGAC-3¢; FOXO Se, 5¢-CAAGT
CACTAGGGTACCCACGCCGGGGTGG-3¢; Myb1 Se,
5¢-GACCAAGATGGTCCATCGGTGGGACGACAG-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,
2 mm MgCl2, 0.1 mm EDTA, 0.5 mm dithiothreitol, and
protease inhibitor cocktail) and incubated on ice for
15 min Nonidet-40 at a final concentration of 0.6% was
added to the cell suspension, which was immediately
vor-texed and centrifuged at 18 000 g for 30 sec A white pellet
was washed with buffer A and used as a nuclear fraction
Equal amounts of protein were loaded and separated by
SDS⁄ PAGE with a 10% or 12% (w ⁄ v) polyacrylamide gel
and transferred onto a poly(vinylidene difluoride)
mem-brane The blocked membranes were incubated with
pri-mary antibodies [anti-c-Fos; Rabbit IgG (Cell Signaling
Technology, Danvers, MA, USA); anti-c-Fos pSer374;
Mouse IgG1 (Calbiochem, Darmstadt, Germany);
anti-c-Jun; Rabbit IgG (Cell Signaling Technology); anti-c-Jun
pSer63; Rabbit IgG (Cell Signaling Technology); anti-c-Jun
pSer73; Rabbit IgG (Cell Signaling Technology); anti-Bim;
Rabbit IgG (Cell Signaling Technology); anti-b-actin; Goat
IgG (Santa Cruz, CA, USA); anti-histone H1; Mouse IgG2a
(Santa Cruz)] The membranes were incubated with an
Alexa680-conjugated secondary antibody (Invitrogen) and
visualized
ChIP assay
The cells were fixed in 1% formaldehyde for 10 min at room
temperature, and immunoprecipitation was performed with
antibodies against c-Fos and c-Jun (Santa Cruz), or control
IgG, with the ChIP-IT Express kit (Active Motif, Carlsbad,
CA, USA), according to the manufacturer’s instructions
The immunoprecipitates including DNA were analyzed by
PCR (primers: Fw, 5¢-CCAGACAATCGTCTCGCCCA-3¢; 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 International (Mountain View, CA, USA) Scrambled siRNAs against c-Fos and c-Jun siRNAs were synthesized by Sigma Genosys (scrambled c-Fos siRNA Se, 5¢-GUACGCU ACCACACUUGAUTT-3¢; scrambled c-Jun siRNA1 Se, 5¢-GGGAACAGAGCGGAUAGGATT-3¢; scrambled c-Jun siRNA2 Se, 5¢-GAAAGAUGGCAGAAUAGAATT-3¢; and scrambled c-Jun siRNA3 Se, 5¢-GAAAGCCUUAAGAA UUGUATT-3¢) The transfection of rat primary hepatocytes with siRNA(s) was carried out by electroporation with the Nucleofection system (Amaxa), according to our previous report [40]
Statistical analyses Data for each variable are expressed as the means ± stan-dard error (SE) The data obtained from two groups were compared by the use of Student’s t-test, and data obtained
Trang 8nett’s test P-values < 0.05 were considered to be significant.
Acknowledgements
We thank T Ohshima for helpful discussions, and
T Shinohara for technical contributions
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