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, 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. 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 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, Regulation of BimEL expression by c-Fos Y. Ishihara et al. 1874 FEBS Journal 278 (2011) 1873–1881 ª 2011 The Authors Journal compilation ª 2011 FEBS whereas 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 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 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-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 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). These results indicate that c-Fos and c-Hun are crucial for BimEL expression and induction of hepatocyte apoptosis. Discussion 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 mM), and then incubated for 9 h in the presence or absence of ATZ (20 m M) and mercaptosuccinic acid (7 mM). 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 lM), 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. Regulation of BimEL expression by c-Fos Y. Ishihara et al. 1876 FEBS Journal 278 (2011) 1873–1881 ª 2011 The Authors Journal compilation ª 2011 FEBS A B C (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. 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 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 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- 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 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 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 IgG 1 (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 IgG 2a (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 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 1879 from three or more groups were compared by the use of Dun- nett’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. References 1 van Gurp M, Festjens N, van Loo G, Saelens X & Van- denabeele P (2003) Mitochondrial intermembrane pro- teins in cell death. 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