Enhancement of oxidative stress-induced apoptosis by Hsp105a in mouse embryonal F9 cells Nobuyuki Yamagishi, Youhei Saito, Keiichi Ishihara and Takumi Hatayama Department of Biochemistry, Kyoto Pharmaceutical University, Japan Hsp105a is one of the major mammalian heat shock proteins that belongs to the HSP105/110 family, and is expressed at especially high levels in the brain as compared with other tissues in mammals. Previously, we showed that Hsp105a prevents stress-induced apoptosis in neuronal PC12 cells, and is a novel anti-apoptotic neuroprotective factor in the mammalian brain. On the other hand, we have also demonstrated that Hsp105a is expressed transiently at high levels during mouse embryogenesis and is found not only in various tissues but also in apoptotic cells. In the present study, to elucidate the role of Hsp105a during mouse embryogenesis, we established mouse embryonal F9 cell lines that constitutively over-express Hsp105a.Over- expression of Hsp105a enhanced hydrogen peroxide- induced apoptosis by enhancing the activation of caspase-3, poly(ADP-ribose)polymerasecleavage,cytochrome crelease and activation of p38 mitogen-activated protein kinase (p38). Furthermore, oxidative stress-induced apoptosis was suppressed by SB202190, a potent inhibitor of p38, in F9 cells. These findings indicated that the activation of p38 is an essential step for apoptosis in F9 cells and that Hsp105a enhances activation of p38, release of cytochrome c and caspase activation. Hsp105a may play important roles in organogenesis, during which marked apoptosis occurs, by enhancing apoptosis during mouse embryogenesis. Keywords: apoptosis; F9 cells; Hsp105; p38 MAPK; oxida- tive stress. Cell death is classified into two major morphologically and biochemically distinct modes, necrosis and apoptosis [1]. Necrosis is characterized by swelling of organelles and cells, followed by lysis of the plasma membrane and random DNA degradation. In contrast, apoptosis is a process that is characterized by cell shrinkage, plasma membrane blebbing, nuclear condensation and endonucleolytic cleavage of DNA into fragments of oligonucleosomal length, and is a funda- mental and indispensable process during normal embryonic development, tissue homeostasis and regulation of the immune system [2–4]. In addition, environmental stresses such as heat shock, radiation, chemical agents and oxidative stress can also induce apoptosis. Heat shock proteins (Hsps) are a set of highly conserved proteins that are induced in response to physiological and environmental stress, and are classified into several families on the basis of their apparent molecular weights, such as HSP105/110, HSP90, HSP70, HSP60, HSP40 and HSP27 [5,6]. Several studies have shown that Hsp70, Hsp90 and Hsp27 protect against cell death through apoptosis by a variety of stressors, such as heat shock, oxidative stress and chemotherapeutic agents [7–9]. In addition, recent studies have demonstrated that Hsp70, Hsp90, Hsp60 and Hsp27 can modulate the functions of several major components of apoptotic processes, including the caspase cascade and the c-Jun N-terminal kinase (JNK) signalling pathway [10–19]. We have previously characterized two heat shock pro- teins, Hsp105a and Hsp105b, which belong to the HSP105/ 110 family and are expressed in various mammals including human, mouse and rat [20–22]. Hsp105a is a constitutively expressed 105-kDa stress protein and is induced by a variety of stressors, whereas Hsp105b is an alternatively spliced form of Hsp105a that is specifically induced by heat shock at 42 °C. These proteins exist as complexes associated with Hsp70/Hsc70 [23,24], and negatively regulate Hsp70/Hsc70 chaperone activity [25]. In addition, our recent study demon- strated that Hsp105a protects neuronal cells against the apoptosis induced by various stresses [26]. On the other hand, we have shown previously that the level of Hsp105a increases transiently in most tissues of mouse embryos from gestational day 9–11, and that Hsp105a is localized not only in various tissues, but also in apoptotic cells and apoptotic bodies at the interdigital regions of limbs, suggesting that Hsp105a may play important roles in apoptosis dur- ing mouse embryogenesis [27]. In the present study, to examine the role of Hsp105a during mouse embryogenesis, we established mouse embry- onal F9 cells that constitutively express Hsp105a,and showed that Hsp105a enhanced the oxidative stress-induced apoptosis at or upstream of p38 mitogen-activated protein kinase (p38) activation. EXPERIMENTAL PROCEDURES Cell culture Mouse teratocarcinoma F9 cells were obtained from the Japanese Cancer Research Resources Bank and maintained Correspondence to T. Hatayama, Department of Biochemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan. Fax: + 81 75 595 4758, Tel.: + 81 75 595 4653, E-mail: hatayama@mb.kyoto-phu.ac.jp Abbreviations: HSP, heat shock protein; JNK, c-Jun N-terminal kinase; PARP, poly (ADP-ribose) polymerase; Ac-DEVD-pNA, N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide. (Received 19 March 2002, revised 19 June 2002, accepted 11 July 2002) Eur. J. Biochem. 269, 4143–4151 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03109.x in Dulbecco’s modified Eagle’s minimal essential medium (Nissui Pharmaceutical) supplemented with 10% foetal bovine serum (Life Technologies) in a humidified atmo- sphere of 5% CO 2 in air at 37 °C. To induce cell differen- tiation, cells grown on collagen-coated culture dishes were incubated in the presence of 100 n M retinoic acid or 1 m M dibutyryl-cAMP/100 n M retinoic acid at 37 °Cfor6days. Cell morphology was examined using a difference interfer- ence contrast microscope. Construction of mouse Hsp105a expression plasmid and isolation of Hsp105a-over-expressing cells Plasmid pcDNA105a wasusedtoexpressmouseHsp105a in F9 cells. To construct this plasmid, the mouse Hsp105a cDNA derived from pB105-1 plasmid [21] was subcloned into EcoRV–XbaI sites of the mammalian expression vector pcDNA3 (Invitrogen). F9 cells were transfected with pcDNA105a or pcDNA3 empty vector by lipofection using Superfect reagent (Qiagen) according to the manufacturer’s instructions. Forty-eight hours after transfection, the cells were maintained in complete medium containing 400 lgÆmL )1 geneticin (Life Technologies) for 3 weeks to select geneticin-resistant cells. The surviving cell clones were isolated, grown in complete medium containing 200 lgÆmL )1 geneticin, and the expres- sion levels of Hsp105a were analysed by Western blotting using anti-mouse Hsp105 Ig [28]. Oxidative stress treatment Cells were grown exponentially on culture dishes at 37 °C for 24 h and then treated with 0.25–2 m M hydrogen peroxide in NaCl/P i containing 0.9 m M CaCl 2 and 0.5 m M MgCl 2 at 37 °C for 30–60 min, washed with NaCl/P i and further incubated in fresh medium at 37 °Cfor24h. Cell viability assay After stress treatment, cells were incubated in medium containing 50 lgÆmL )1 neutral red at 37 °C for 3 h, then fixed with 1% formaldehyde containing 1% CaCl 2 for 1 min. The dye incorporated into viable cells was extracted with 50% ethanol containing 1% acetic acid, and absorb- ance at 540 nm was measured. DNA fragmentation analysis DNA fragmentation was analysed essentially as described by Ishizawa et al. [29]. Cells were lysed at 37 °Cfor30min in 200 lLlysisbuffer(10m M Tris/HCl pH 8.0, 150 m M NaCl, 10 m M EDTA, 0.1% SDS, 0.5 mgÆmL )1 ribonuc- lease A, 0.5 mgÆmL )1 proteinase K), and the cell lysates were mixed with 300 lLNaIsolution(6 M NaI, 10 m M Tris/HCl pH 8.0, 13 m M EDTA, 0.5% sodium N-lauroyl- sarcosine, 30 lgÆmL )1 glycogen) and incubated at 60 °Cfor 15 min. An equal volume of isopropanol was added to the mixtures, which were shaken vigorously and kept for 15 min at room temperature. After centrifugation at 15 000 g for 15 min, the precipitate was washed successively with 50% and 100% isopropanol, dried in air, and resolved in Tris/HCl/EDTA buffer (10 m M Tris/HCl pH 8.0, 1 m M EDTA). Aliquots of 5 lg of DNA were electrophoresed on 2% agarose gels and stained with 1 lgÆmL )1 ethidium bromide. Morphological examination of apoptotic cells Cells were plated onto coverslips at a density of 1 · 10 5 cellsÆcm )2 and grown at 37 °C for 24 h. After treatment, cells were washed with NaCl/P i , fixed with 3.7% formalde- hyde for 30 min at room temperature, and stained with 10 l M Hoechst 33342 for 10 min in the dark. After washing with NaCl/P i , the stained cells were observed using a fluorescence microscope (Zeiss). Cells were scored as apoptotic if they displayed nuclear fragmentation and/or chromatin condensation. Assay for poly (ADP-ribose) polymerase (PARP) cleavage Cells (1 · 10 6 cells) were lysed with 200 lLlysisbuffer (50 m M Tris/HCl pH 8.0, 150 m M NaCl, 1% NP-40, 0.1% SDS, 5 lgÆmL )1 aprotinin, 5 lgÆmL )1 leupeptin, 2 lgÆmL )1 pepstatin A, 1 m M phenylmethanesulfonyl fluoride) on ice for 1 h. The lysate was then sonicated for 10 s and centrifuged at 20 000 g for 15 min at 4 °C. The supernatant was recoverd as the cell extract. Aliquots (20 lgprotein)of cell extracts in SDS sample buffer containing urea (62.5 m M Tris/HCl pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 5% 2- mercaptoethanol, 0.00125% Bromophenol blue) were sub- jected to 7.5% SDS/PAGE, then transferred onto nitrocel- lulose membranes by electrotransfer. The membranes were blocked with 10% skim milk in NaCl/Tris (20 m M Tris/HCl pH 7.6, 137 m M NaCl) containing 0.1% Tween 20 (NaCl/ Tris/Tween), and incubated with anti-PARP Ig (Santa Cruz). Then, the membranes were incubated with horse- radish peroxidase-conjugated anti-rabbit IgG, and the antibody–antigen complexes were detected using the ECL- Western blot detection system (Amersham Pharmacia Biotech). Measurement of caspase-3 activity Caspase-3 activity was measured using the colorimetric CaspACE assay system according to the manufacturer’s instructions (Promega). Briefly, cells (1 · 10 6 cells) were suspended in 50 lL cell lysis buffer on ice for 10 min, and lysed by freezing and thawing. After centrifugation at 20 000 g for 20 min at 4 °C, the supernatants were recov- ered as cell extracts. Cell extracts (50 lgprotein)were incubated in caspase assay buffer (100 m M Hepes pH 7.5, 10 m M sucrose, 0.1% Chaps, 10 m M dithiothreitol) con- taining 200 l M N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA) at 25 °C for 2 h, and then absorbance at 405 nm was measured using a microplate reader. Release of cytochrome c from mitochondria Release of cytochrome c was analysed essentially as des- cribed by Yano et al. [30]. Briefly, cell suspensions were mixed with an equal volume of 100 lgÆmL )1 digitonin in NaCl/P i , and incubated at 25 °C for 5 min. After centrif- ugation at 15 000 g for 2 min, supernatants were recovered as cytosolic fractions. Pellets were dissolved in NaCl/P i containing 0.5% Triton X-100, and centrifuged, then supernatants were recovered as mitochondrial fractions. 4144 N. Yamagishi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Both fractions were subjected to 15% SDS/PAGE, and analysed by Western blotting using anti-cytochrome c Ig (Santa Cruz). Activation of JNK and p38 Phosphoryled JNK and p38 were detected by Western blotting using PhosphoPlus JNK (Thr183/Tyr185) and PhosphoPlus p38 (Thr180/Tyr182) antibody kits (Cell Signaling Technology, Inc.), respectively. Cell extracts (20 lg protein) were separated by SDS/PAGE (10% polyacrylamide), and analysed by Western blotting using phosphorylation state-specific anti-JNK Ig or anti-p38 Ig. Then, the membranes were incubated at 50 °Cfor30minin stripping buffer (62.5 m M Tris/HCl pH 6.7, 2% SDS, 100 m M 2-mercaptoethanol), and total JNK or p38 on the same membranes were detected using anti-JNK Ig or anti- p38 Ig, respectively. Kinase activity of p38 was assayed by its ability to phosphorylate MAPKAPK-2. Cell extracts (20 lgprotein) were separated by SDS/PAGE (10% polyacrylamide), and analysed by Western blotting using phosphorylation state- specific anti-(MAPKAPK-2) Ig (Cell Signaling Technology, Inc.). RESULTS Characterization of stable transfectants over-expressing Hsp105a To determine the role of Hsp105a in embryonal cells, we established mouse embryonal F9 cell clones that express Hsp105a at high levels. In this study, we used two Hsp105a over-expressing F9 cell lines, S3 and S23, in which the expression levels of Hsp105a were about two- and threefold higher than that in parental F9 cells or controls transfected with empty pcDNA3 vector (V1), respectively (Fig. 1A). The growth rates of S3 and S23 cells were not significantly different from those of F9 and V1 cells (Fig. 1B). F9 cells can be differentiated toward primitive endoderm-like and parietal endoderm- like cells by exposure to retinoic acid and dibutyryl- cAMP/retinoic acid, respectively [31,32]. Upon exposure to retinoic acid, S3 and S23 cells showed an enlarged and flattened morphology as seen in F9 or V1 cells, which is characteristic of primitive endoderm-like cells (Fig. 1C, e–h). In addition, the Hsp105a over-expressing cells were also induced to differentiate toward parietal endo- derm-like cells by treatment with retinoic acid and dibutyryl-cAMP; they also showed the typical changes in morphology (Fig. 1C, i–l). Thus, over-expression of Hsp105a did not affect cell growth and differentiation toward primitive endoderm-like and parietal endoderm- like phenotypes in F9 cells. Enhancement of oxidative stress-induced apoptosis by over-expression of Hsp105a in F9 cells As Hsp105a is expressed transiently at high levels during mouse embryogenesis and is localized in apop- totic cells [27], we next examined the effects of over- expression of Hsp105a on stress-induced cell death in embryonal F9 cells. When F9, V1, S3 and S23 cells weretreatedwith0.25–1m M hydrogen peroxide for 1 h, Fig. 1. Over-expression of Hsp105a in F9 cells and its effects on growth and differentiation. (A) Cell extracts (20 lg protein) from parental F9 cells (F9) and cell clones stably transfected with either pcDNA3 vector (V1) or pcDNA105a (S3 and S23) were separated by SDS/PAGE, and the levels of Hsp105a were determined by Western blotting using anti-mouse Hsp105 Ig. (B) F9. V1, S3 and S23 cells were plated into 35 mm culture dishes at adensityof1· 10 4 cells per dish, and cultured for 6 days at 37 °C. At the indicated times, cell numbers were counted. (C) Cells (1 · 10 5 cells per dish) were cultured in the presence of 100 n M retinoic acid (+ RA) or 1 m M dibutyryl-cAMP/100 n M retinoic acid (+ RA/cAMP) at 37 °Cfor 6 days. Cell morphology was observed using a difference interference contrast microscope. Scale bars ¼ 10 lm. Ó FEBS 2002 Hsp105 enhances oxidative stress-induced apoptosis (Eur. J. Biochem. 269) 4145 S3 and S23 cells were more sensitive to the oxidative stress than F9 or V1 cells (Fig. 2A). Therefore, we further analysed the hydrogen peroxide-induced cell death of F9 cells. Cell death is classified into two morphologically and biochemical distinct modes, apoptosis and necrosis [1]. To characterize the hydrogen peroxide-induced cell death in F9 cells, we examined whether DNA fragmen- tation, characteristic of apoptosis, occurred in these cells. As shown in Fig. 2B, nucleosomal-length DNA frag- mentation was observed in the hydrogen peroxide- treated F9 cells, and the amounts of fragmented DNA caused by low doses of hydrogen peroxide were increased in the Hsp105a over-expressing cells as compared with those in S3 and S23 cells. In addition, apoptotic morphology such as nuclear condensation and chromatin fragmentation was also prominently observed by Hoechst 33342 staining in these cells (Fig. 3A), and the rate of apoptotic cells was approximately threefold higher in the Hsp105a over-expressing cells than in F9 or V1 cells (Fig. 3B). However, these morphological changes were suppressed by treatment with a cell- permeable caspase inhibitor zVAD-fmk. Thus, Hsp105a was demonstrated to enhance oxidative stress-induced apoptosis in F9 cells. Over-expression of Hsp105a enhances caspase-3 activity and PARP cleavage after treatment with hydrogen peroxide A common event in the apoptotic pathway is the activation of caspases. These enzymes participate in a cascade that is triggered in response to pro-apoptotic signals and results in cleavage of a set of proteins, resulting in disassembly of the cells. Caspase-3 is a major effector caspase, and induces cleavage of several substrate proteins and is responsible for several apoptotic processes. We next assayed caspase-3 activity in extracts from cells treated with oxidative stress. As shown in Fig. 4A, although caspase-3 activity increased slightly in F9 and V1 cells treated with hydrogen peroxide, its activity was increased markedly in S3 and S23 cells by this treatment. In addition, the activation of caspase-3 was suppressed by the treatment with a cell-permeable caspase inhibitor zVAD-fmk. PARP, a DNA repair-related enzyme, is an important substrate of caspase-3, and is cleaved from a 116-kDa protein to an 85-kDa fragment and inactivated during apoptosis [33,34]. Western blotting analysis clearly revealed cleavage of PARP in F9 cells treated with hydrogen peroxide (Fig. 4B). Furthermore, in accordance with the Fig. 2. Effects of Hsp105a over-expression on the sensitivity of F9 cells to hydrogen peroxide. F9, V1, S3 and S23 cells were exposed to 0.1–1 m M hydrogen peroxide for 1 h, and further incubated for 24 h at 37 °C. (A) Cell viability was then determined by neutral red assay. Experiments were repeated at least three times and essentially the same results were obtained each time. (B) DNA was extracted from the cells treated with hydrogen peroxide, and aliquots (5 lg each) of DNA were electrophoresed on a 2% agarose gel and visualized by staining with ethidium bromide. 4146 N. Yamagishi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 enhancement of caspase-3 activity, the cleavage of PARP by hydrogen peroxide was more intense in S3 and S23 cells than in F9 or V1 cells. Thus, over-expression of Hsp105a was shown to markedly enhance the activation of procaspase-3 during apoptosis induced by hydrogen peroxide. Over-expression of Hsp105a enhances release of cytochrome c from mitochondria after treatment with hydrogen peroxide In mammalian cells, one of the main pathways that activates caspase-3 is via mitochondria. When mitochondria receive appropriate signals from a variety of stresses or are damaged irreversibly, pro-apoptotic molecules such as cytochrome c are released from mitochondria into the cytosol [35–37]. In the cytosol, cytochrome c forms a complex with Apaf-1 and procaspase-9, and activates caspase-9, which in turn converts procaspase-3 into its active form, resulting in apoptosis [38–40]. We next analysed whether cytochrome c is released from mitochondria in F9 cells by oxidative stress. When F9 cells were fractionated into mitochondrial and cytosolic fractions and cytochrome c was examined by Western blotting, a large amount of cytochrome c was found in mitochondria with only a small amount in the cytosolic fraction of control cells under these experimental conditions (Fig. 5A). However, although the amounts of cytochrome c released by hydrogen peroxide increased slightly in F9 or V1 cells, the release was increased to a greater extent in S3 and S23 cells than in control and V1 cells (Fig. 5B). Thus, over-expression of Hsp105a seemed to enhance the release of cytochrome c from mitochondria in F9 cells by hydrogen peroxide. Fig. 3. Enhancement of oxidative stress-induced apoptosis by over-expression of Hsp105a. (A) F9, V1, S3 and S23 cells were grown on coverslips, exposedto1m M hydrogen peroxide for 1 h, and further incubated at 37 °C for 24 h. Ten l M z-VAD-fmk was added to the medium 1 h before treatment. Cells were washed with NaCl/P i , fixed with 3.7% formaldehyde, and stained with 10 l M Hoechst 33342. Nuclear morphology of cells was observed using a fluorescence microscope. (B) Rates of apoptotic cells were obtained from at least 200 cells in each experiment. Data are shown as the means ± SD of three independent experiments. The significance of differences was assessed by unpaired Student’t t-test. Fig. 4. Effects of over-expression of Hsp105a on caspase-3 activity. F9, V1, S3 and S23 cells were exposed to 1 m M hydrogen peroxide for 1 h, and further incubated for 24 h at 37 °C. (A) Caspase-3 activity in cell extracts was measured with caspase-3 substrate, Ac-DEVD-pNA. Data represent the means ± SD of three independent experiments. (B) Aliquots (20 lg protein) of cell extracts were separated by SDS/ PAGE, and PARP (116 kDa) and the cleaved fragment (85 kDa) were detected by Western blotting using anti-PARP Ig. Ó FEBS 2002 Hsp105 enhances oxidative stress-induced apoptosis (Eur. J. Biochem. 269) 4147 Over-expression of Hsp105a enhances activation of p38 after treatment with hydrogen peroxide JNK and p38 pathways are activated by cellular stresses and inflammatory cytokines, resulting in growth arrest and apoptosis, and have been implicated as key regulators of stress-induced apoptosis in many cell types [41,42]. Fur- thermore, mitochondria are influenced by proapoptotic signals through the JNK pathway [43]. To determine the effects of Hsp105a on JNK and p38 signalling pathways, we examined the activation of JNK and p38 in F9 cells by oxidative stress. As shown in Fig. 6A, JNK was not activated in control F9 or V1 cells by treatment with hydrogen peroxide. In contrast, hydrogen peroxide-treat- ment induced marked activation of JNK within 30 min in S23 cells, but not in S3 cells. Therefore, the enhancement of the oxidative stress-induced JNK-activation seemed not to be solely due to the over-expression of Hsp105a in F9 cells. On the other hand, p38 was activated at low levels within 1–2 h in F9 and V1 cells after treatment with hydrogen peroxide, and the activation of p38 by hydrogen peroxide was enhanced in both S3 and S23 cells compared with F9 and V1 cells (Fig. 6B). Thus, over-expression of Hsp105a seemed to enhance the oxidative stress-induced activation of p38 but not JNK in F9 cells. As Hsp105a enhances the activation of p38 induced by oxidative stress, we further examined whether its activation is responsible for the induction of apoptosis in F9 cells using SB202190, a potent inhibitor of p38. As shown in Fig. 7A, although hydrogen peroxide-treatment induced apoptosis in F9 cells as described above, the apoptosis was significantly suppressed by SB202190. Under these conditions, although phosporylation of MAPKAPK-2, a substrate of p38, was enhanced by the hydrogen peroxide treatment, it was suppressed to basal level by treatment with 10 l M SB202190 (Fig. 7B). These findings indicate that the activation of p38 is an essential step for induction of apoptosis by hydrogen peroxide, and Hsp105a is suggested to enhance the oxida- Fig. 6. Effects of Hsp105a on activation of JNK and p38 by oxidative stress. F9, V1, S3 andS23cellswereexposedto1m M hydrogen peroxide for 30 min and further incubated for 0.5, 1 or 2 h at 37 °C. Aliquots (20 lgprotein) of cell extracts were separated by SDS/PAGE. Activated and total JNK or p38 were detected by Western blotting, as described in Experi- mental procedures. (A) JNK, (B) p38. Upper and lower panels represent activated and total JNK or p38, respectively. Fig. 5. Effects of Hsp105a over-expression on release of cytochrome c from mitochondria. F9, V1, S3 and S23 cells were exposed to 1 m M hydrogen peroxide for 45 min, and further incubated for 24 h at 37 °C. Cells were then fractionated into cytosolic and mitochondrial fractions, as described in Experimental procedures. (A) Both fractions were subjected to 15% SDS/PAGE, and analysed by Western blotting using anti-cytochrome c Ig.C,cytosolicfraction;M,mitochondrial fraction. (B) The densities the cytochrome c bands were quantified by densitometry, and the rates release into the cytosol are shown. Data represent the means ± SD of three independent experiments. Filled bars, untreated cells; open bars, hydrogen peroxide-treated cells. 4148 N. Yamagishi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 tive stress-induced apoptosis at or upstream of p38 activa- tion in F9 cells. DISCUSSION Hsp105a is expressed in most tissues, but its levels are especially high in the brain of adult mammals such as rats, mice and humans [21–23]. We have shown that Hsp105a plays an important role in protection of neuronal cells against stress-induced apoptosis [26]. In accordance with these findings, ischemia/reperfusion in the rat forebrain induces the expression of HSP105/110 family proteins, Hsp105a, APG-1 (testis-specific homologue of Hsp105) and APG-2 [44,45], and Hsp110 (hamster homologue of Hsp105a) confers heat resistance on rat fibroblasts and human epithelial carcinoma cells [46]. On the other hand, we have also shown that the levels of Hsp105a increase transiently in embryonic tissues during mouse embryogen- esis, and this protein is localized not only in various tissues, but also in apoptotic cells and apoptotic bodies at the interdigital regions of limbs [27]. In the present study, to explore the function of Hsp105a in embryogenesis, we established mouse embryonal F9 cell lines that constitutively express Hsp105a. Although growth rate and differentiation of F9 cells were not affected by the over-expression of Hsp105a, the sensitivity of cells to oxidative stress was enhanced by the over-expression of Hsp105a,andthese findings were in clear contrast with those in neuronal PC12 cells. However, as sensitivity of F9 cells to stresses such as heat shock, etoposide, actinomycin D and serum depriva- tion was also enhanced by over-expression of Hsp105a (unpublished data), the enhancement of cell death by Hsp105a seemed to be a general phenomenon in embryonal F9 cells. The present findings together with previous observations in neuronal PC12 cells suggested that Hsp105a has a pro-apoptotic effect in embryonal cells and an anti- apoptotic effect in neuronal cells. Thus, these observations provide the first evidence that Hsp105a can function as an enhancer or suppressor of apoptosis depending on the cell type in mammals. Apoptosis is an active process resulting in characteristic morphological changes such as cell shrinkage, condensation of chromatin and membrane blebbing [2–4]. The common pathway of apoptosis involves a family of proteases known as the caspases, which are activated in a proteolytic cascade to cleave specific substrates. The release of cytochrome c from mitochondria triggers the formation of apoptosome complex with Apaf-1 and pro-caspase-9, and activates caspase-9, which then in turn activates downstream effector caspases such as caspase-3 [38–40]. Active caspase-3 cleaves several substrates such as PARP [33,34], and activates death effector molecules or triggers the structural changes char- acteristic of apoptotic cells. Here, we showed that over- expression of Hsp105a enhances PARP cleavage, caspase-3 activation and release of cytochrome c from mitochondria in F9 cells exposed to hydrogen peroxide, and our results Fig. 7. Suppression of oxidative stress-induced apoptosis in F9 cells by a potent p38 inhibitor. F9, V1, S3 and S23 cells were grown on coverslips, treated with or without 10 l M SB202190 for 1 h before and during the 1 h-treatment with 1 m M hydrogen peroxide, and further incubated at 37 °C for 24 h (A) or 2 h (B). (A) Cells were then washed with NaCl/P i , fixed with 3.7% formaldehyde, and stainedwith10l M Hoechst 33342. Nuclear morphology of cells was observed using a fluorescence microscope. Rates of apoptotic cells were obtained from at least 300 cells in each experiment. Data are shown as the means ± SD of at least three independent experi- ments. The significance of differences was assessed by unpaired Student’t t-test. (B) Cell extracts (20 lg proteins) of S23 cells were separated by SDS/PAGE, and phosphory- lated MAPKAPK-2 was detected by Western blotting using anti-phospho-MAPKAPK-2 Ig. Ó FEBS 2002 Hsp105 enhances oxidative stress-induced apoptosis (Eur. J. Biochem. 269) 4149 suggested that Hsp105a enhances the apoptosis at or upstream of cytochrome c release from mitochondria. Furthermore, the transmission of signals from external stresses is accompanied by the activation of a family of stress-activated protein kinases, JNK and p38. Activation of these signalling pathways leads to apoptosis [41,42], and mitochondria is influenced by proapoptotic signal trans- duction through the JNK pathway [43]. As the activation of p38 is an essential step for apoptosis induced by hydrogen peroxide in F9 cells as shown in Fig. 7, Hsp105a was suggested to enhance the oxidative stress-induced apoptosis directly or indirectly at or upstream of activation of p38. In contrast, although Hsp105a prevents the apoptosis induced by several stresses including hydrogen peroxide in neuronal PC12 cells, p38 is not activated by these stresses in neuronal cells [26]. The p38 signalling pathway may be a possible target at which Hsp105a enhances the apoptosis induced by hydrogen peroxide in embryonal cells. Several Hsps have been shown to modulate the pathway of apoptosis positively or negatively. Hsp60 with or without Hsp10 directly stimulates apoptosis by promoting the proteolytic maturation of caspase-3 [17,18]. In contrast, Hsp70, Hsp90 and Hsp27 exert negative influences on apoptotic signalling. In particular, Hsp70 has been shown to protect against apoptosis by a variety of stressors through suppression of JNK activation [10–13] and apoptosome formation [14,15]. Interestingly, a recent study demonstra- ted that the chaperone activity of Hsp70 is required for protection against heat-induced apoptosis [47]. In contrast, we cannot detect the chaperone activity of Hsp105a, but the protein exists as complexes associated with Hsp70/Hsc70 [23,24] and suppresses the Hsc70 chaperone activity [25]. 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Enhancement of oxidative stress-induced apoptosis by over-expression of Hsp105a in F9 cells As Hsp105a is expressed transiently. Enhancement of oxidative stress-induced apoptosis by Hsp105a in mouse embryonal F9 cells Nobuyuki Yamagishi, Youhei Saito,