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NGF-induced phosphatidylinositol 3-kinase signaling pathway prevents thapsigargin-triggered ER stress-mediated apoptosis in PC12 cells
Neuroscience Letters 389 (2005) 124–128
NGF-induced phosphatidylinositol 3-kinase signaling pathway prevents thapsigargin-triggered ER stress-mediated apoptosis in PC12 cells Koji Shimoke ∗ , Soichiro Kishi, Takahiro Utsumi, Yuichi Shimamura, Harue Sasaya, Tadao Oikawa, Shinichi Uesato, Toshihiko Ikeuchi Faculty of Engineering and High Technology Research Center (HRC), Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan Received 30 May 2005; received in revised form 28 June 2005; accepted 18 July 2005
Abstract Tunicamycin, an inhibitor of the glycosylation of newly biosynthesized proteins, induces endoplasmic reticulum (ER) stress and subsequent apoptosis, and caspase family proteases are activated during the process of ER stress-mediated apoptosis. In the present study, we showed that thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), also induced ER stress-mediated apoptosis, and nerve growth factor (NGF) prevented the apoptosis in PC12 cells. We also found that LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), reduced the survival of cells treated with NGF for 24 h in the presence of Th. We discovered that the activities of caspase-3, -9 and -12 were increased time-dependently after the treatment with Th, and NGF suppressed the Th-triggered activation of caspase-3, -9 and -12. LY294002 diminished the effect of NGF on the inactivation of all these caspases. These results indicate that the NGF-induced PI3-K signaling pathway prevents Th-triggered ER stress-specific apoptosis via inhibition of caspase-mediated apoptotic signal. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: ER stress; Apoptosis; Caspase; Nerve growth factor; Phosphatidylinositol 3-kinase; PC12 cell
The endoplasmic reticulum (ER) is an important organelle that plays a crucial role in protein biosynthesis. It has been reported that a disruption of the quality control of newly biosynthesized proteins induces ER stress through the accumulation of unfolded protein [4,10–12]. Tunicamycin (Tm), an inhibitor of the glycosylation of newly biosynthesized proteins, induces ER stress [2,8,13]. Thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), also induces ER stress by disrupting the homeostatic balance of the Ca2+ concentration in the ER [3,5,9]. As a result, Th-treated cells die in an apoptotic manner [3,16]. It is of interest to compare the effects of nerve growth factor (NGF) on Tm-induced and Th-induced apoptosis. We first examined whether NGF is capable of preventing Th-induced apoptosis in PC12 cells or not. PC12 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% (v/v) heat∗
Corresponding author. Tel.: +81 6 6368 0853; fax: +81 6 6330 3770. E-mail address: [email protected] (K. Shimoke).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.07.030
inactivated precolostrum newborn calf serum and 5% (v/v) heat-inactivated horse serum. For the measurement of cell viability, PC12 cells were seeded onto collagen-coated 96well plates at 1 × 105 cells/cm2 . The next day, the medium was changed to serum-free DMEM, and 0.5 M Th without or with 100 ng/ml of NGF was added. As shown in Fig. 1A, Th reduced the cell viability, and NGF diminished the effect of Th in the culture for 24 h. For analysis of the mechanism of NGF-mediated cell survival, we administered NGF plus Th with LY294002 (LY) and PD98059 (PD), an inhibitor of phosphatidylinositol 3-kinase (PI3-K) and an inhibitor of upstream kinase (MEK1) of mitogen-activated kinase (MAPK also termed ERK1/2), respectively (Fig. 1A). LY, but not PD, remarkably reduced the NGF-mediated cell survival, suggesting that PI3-K is essential for the NGFmediated prevention of the Th-induced apoptosis. In addition, we found that Th transiently induced the mRNA expression of GRP78 gene, which is one of the target genes of the unfolded protein response (UPR), as an indicator of ER stress response (Fig. 1B). Interestingly, the expression of GRP78
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Fig. 1. Induction of ER stress and subsequent apoptosis in thapsigargin-treated PC12 cells. (A) PC12 cells were plated onto collagen-coated 96-well plates, and 0.5 M thapsigargin (Th) was added to serum-free DMEM (con) without or with 100 ng/mL of NGF (N). 10 M LY294002 (LY) or 50 M PD98059 (PD) was also added in the presence of Th plus N. Cells were cultured for 24 h. Viable cells were measured by alamarblue assay as described previously [13,14]. Values are the mean ± S.E.M. (n = 4) and the statistical analysis was carried out with one-way ANOVA. ** P < 0.01 and # P < 0.05 as compared with control. (B) Cells were plated onto 6 cm diameter dishes and treated with 0.5 M Th for up to 24 h. The specific probes for Northern blotting of GRP78 and 28S rRNA were labeled with [␣-32 P]dCTP as described previously [14]. The specific bands were visualized by autoradiography. The data represents three independent experiments. (C) Cells were plated onto 6 cm diameter dishes and treated with 0.5 M Th for up to 24 h. Then, cells were lysed in lysis buffer comprising 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 g/ml aprotinin and 1% (w/v) SDS. 10 g of protein per lane was used for SDS-PAGE. Next, immunoblotting was carried out using anti-GRP78 antibody (SantaCruz) or anti--actin antibody (Chemicon) as the primary antibody and horseradish peroxidase-conjugated secondary antibody to detect the specific bands. The bands were visualized with a light capture system (Atto) after treatment with Immunostar reagents (WAKO). The data represents four independent experiments.
mRNA by Th had a time course different from the Tminduced time-dependent expression of GRP78 mRNA. We observed a sustained induction of GRP78 protein up to 24 h after the treatment with Th in PC12 cells (Fig. 1C) although the GRP78 mRNA expression was transiently induced by Th. We confirmed that PC12 cells died in an apoptotic manner and NGF clearly prevented the Th-induced apoptosis, judging from the Hoechst 334882 staining (Fig. 2). Furthermore, LY increased the number of condensed nuclei in the presence of NGF (Fig. 2D). These results indicate that NGF attenuates the Th-mediated apoptosis via the PI3-K signaling pathway. It has been reported that caspase family proteases are involved in Tm-mediated apoptosis [13,15]. To clarify the involvement of caspases in Th-mediated apoptosis, we added caspase inhibitors (z-DEVD-fmk, z-VAD-fmk) to Thtreated PC12 cells. As a result, we observed that both caspase inhibitors restored cell viability in the presence of Th, indicating that the caspase cascade is involved in Th-induced apoptosis (Fig. 3). We have reported that caspase-3, -9 and -12 are involved in Tm-induced apoptosis [13]. Then, we analyzed whether NGF attenuated Th-induced apoptosis via suppres-
sion of the activation of caspase-3, -9 and -12, by measuring the caspase activities using fluorogenic substrates (50 M Ac-DEVD-MCA for caspase-3 and 50 M Ac-LEHD-MCA for caspase-9) and by Western blotting with anti-caspase-12 antibody. PC12 cells were treated with Th, Th plus NGF or Th plus NGF in the presence of LY, and lysates were incubated at 37 ◦ C for 1 h with the substrates described above. Fluorescence was detected using a spectrofluorometer with excitation (380 nm) and emission (460 nm) wavelengths for measurement of the caspase-3 and -9 activities. As shown in Fig. 4, Th increased the activities of caspase-3, -9 and -12, and NGF decreased these activities. Moreover, the effect of NGF was diminished by the addition of LY294002. These results clearly indicate that NGF prevents Th-induced apoptosis by suppressing the activation of caspase-3, -9 and -12 via the PI3-K signaling pathway. In the present study, we have shown the NGF-mediated effect on Th-induced apoptosis in PC12 cells. The mitochondrial dysfunction-mediated cell death which is considered as ordinary apoptosis involves the activation of caspase-9 via the formation of a complex between pro-caspase-9, Apaf1, dATP and cytochrome c released from mitochondria [1].
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Fig. 2. Hoechst 334882 staining of thapsigargin-treated PC12 cells. PC12 cells were plated onto 8-well chamber plates, and 0.5 M thapsigargin (Th) was added to serum-free DMEM without or with 100 ng/ml of NGF (N). 10 M LY294002 was added in the presence of Th plus N. Cells were cultured for 24 h. Then, cells were fixed and stained by treatment with 1 g/mL of Hoechst 334882 dye in phosphate-buffered saline (PBS) for 30 min. Results for untreated (A) or Th-treated cells (B) and Th-treated cells in the presence of NGF without (C) or with LY (D) are presented. Apoptotic condensed nuclei were detected and photographs were taken using a fluorescence microscope. Bar denotes 15 m.
Fig. 3. Prevention of thapsigargin-induced apoptosis by caspase inhibitors. PC12 cells were maintained and plated as described in Fig. 1A. 0.5 M thapsigargin (Th) was added to serum-free DMEM (con) in the absence or presence of 100 M z-DEVD-fmk (DEVD) or 50 M z-VAD-fmk (VAD). Viable cells were measured after 24 h by alamarblue assay. Values are the mean ± S.E.M. (n = 4) and the statistical analysis was carried out with oneway ANOVA. ** P < 0.01 and ## P < 0.01 as compared with control.
Although we observed significant activation of caspase-9 in Th-induced apoptosis, we could not clarify the mechanism of the activation. Morishima et al. reported that caspase9 is directly activated by caspase-12 during ER stress [6]. Thus, we consider that caspase-12 is the initiator caspase activating the caspase cascade during ER stress. Furthermore, we previously demonstrated that the activation of caspase-3 and -12 was suppressed by the PI3-K signaling pathway in Tm-treated cerebral cortical neurons and Tmtreated PC12 cells [13,15]. Interestingly, ER stress-mediated apoptosis by Th was also suppressed by the PI3-K signaling pathway in PC12 cells. We consider that there may be a common mechanism underlying Th- and Tm-triggered ER stress-mediated apoptosis although the time courses of GRP78 mRNA expressions during Th- and Tm-induced ER stress are different [13]. We are searching for a specific effector to suppress the caspase-mediated apoptotic pathway downstream of the PI3-K signaling pathway. Given that caspase-12 is the initiator caspase during ER stress, an activator of caspase-12 must exist on the ER membrane. It has also been reported that calpain activates caspase-12 during ER stress [7]. Calpain may be the key to suppression of the activation of caspase-12 through the PI3-K signaling pathway. However, calpain may not be a general mediator of ER stress-induced apoptosis because it is regulated by Ca2+ . Yoneda et al. reported that TNF receptor-associated factor 2 (TRAF2) was important for ER stress-induced
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Acknowledgements This work was supported in part by grants-in-aid for scientific research from MEXT (Ministry of Education, Culture, Sports, Science and Technology of Japan), HAITEKU (2002–2006) from MEXT, a grant from the Kansai University Special Research Fund, 2005 and a research grant from the Smoking Research Foundation, Japan. This research was also financially supported in part by the Sasakawa Scientific Research Grant from Japan Science Society.
References
Fig. 4. Activities of caspases during the thapsigargin-induced apoptosis. (A and B) PC12 cells were maintained as described in Fig. 1A and plated onto 6 cm diameter dishes. The medium was changed to serum-free DMEM without (con) or with 0.5 M thapsigargin (Th), Th + 100 ng/mL NGF (Th + NGF) or Th + NGF + 10 M LY294002 (Th + NGF + LY) for the periods indicated. Then, cells were collected in a lysis buffer comprising 10 mM Hepes–KOH (pH 7.4), 2 mM EDTA and 1 mM PMSF. After centrifugation, an aliquot of the supernatant was mixed with 2× ICE buffer comprising 20 mM Hepes–KOH (pH 7.4), 20% glycerol (v/v), 2 mM PMSF, 4 mM dithiothreitol and 50 M Ac-DEVD-MCA (for caspase-3) or 50 M AcLEHD-MCA (for caspase-9), followed by incubation at 37 ◦ C for 1 h. After addition of 200 l of distilled water, the activity of caspase-3 and -9 was measured by detecting fluorescence using a spectrofluorometer (Dainippon) [13]. Fold increases in the activities of caspase-3 and -9 are shown. Values are the mean ± S.E.M. (n = 3) and the statistical analysis was carried out with one-way ANOVA. * P < 0.05 and ** P < 0.01 as compared with 0 h. (C) Cells untreated (con) or treated with 0.5 M thapsigargin (Th), 0.5 M Th + 100 ng/ml NGF in the absence (Th + N) or presence of 10 M LY294002 (Th + N + LY) for 24 h were lysed in lysis buffer as described in Fig. 1C. Then, immunoblotting was carried out using anti-caspase-12 antibody (Sigma) or anti-actin antibody (SantaCruz) [7,13]. The bands were visualized with a light capture system (Atto) after treatment with Immunostar reagents (WAKO). The apparent molecular masses (kD) measured using molecular weight markers (Amersham) are presented on the right.
apoptosis [17]. Thus, further analyses to identify the activator of caspase-12 and to clarify the exact mechanism underlying the NGF-mediated effect on ER stress-induced apoptosis are necessary.
[1] J.M. Adams, S. Cory, Apoptosomes: engines for caspase activation, Curr. Opin. Cell. Biol. 14 (2002) 715–720. [2] J. Hitomi, T. Katayama, Y. Eguchi, T. Kudo, M. Taniguchi, Y. Koyama, T. Manabe, S. Yamagishi, Y. Bando, K. Imaizumi, Y. Tsujimoto, M. Tohyama, Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death, J. Cell. Biol. 165 (2004) 347–356. [3] J. Hitomi, T. Katayama, M. Taniguchi, A. Honda, K. Imaizumi, M. Tohyama, Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12, Neurosci. Lett. 357 (2004) 127–130. [4] R.J. Kaufman, Orchestrating the unfolded protein response in health and disease, J. Clin. Invest. 110 (2002) 1389–1398. [5] J. Lytton, M. Westlin, M.R. Hanley, Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps, J. Biol. Chem. 266 (1991) 17067–17071. [6] N. Morishima, K. Nakanishi, H. Takenouchi, T. Shibata, Y. Yasuhiko, An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12, J. Biol. Chem. 277 (2002) 34287–34294. [7] T. Nakagawa, J. Yuan, Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis, J. Cell. Biol. 150 (2000) 887–894. [8] T. Nakagawa, H. Zhu, N. Morishima, E. Li, J. Xu, B.A. Yankner, J. Yuan, Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta, Nature 403 (2000) 98–103. [9] H.N. Nguyen, C. Wang, D.C. Perry, Depletion of intracellular calcium stores is toxic to SH-SY5Y neuronal cells, Brain Res. 924 (2002) 159–166. [10] R.V. Rao, S. Castro-Obregon, H. Frankowski, M. Schuler, V. Stoka, G. del Rio, D.E. Bredesen, H.M. Ellerby, Coupling endoplasmic reticulum stress to the cell death program. An Apaf-1-independent intrinsic pathway, J. Biol. Chem. 277 (2002) 21836–21842. [11] R.V. Rao, H.M. Ellerby, D.E. Bredesen, Coupling endoplasmic reticulum stress to the cell death program, Cell Death Differ. 11 (2004) 372–380. [12] R.V. Rao, K.S. Poksay, S. Castro-Obregon, B. Schilling, R.H. Row, G. del Rio, B.W. Gibson, H.M. Ellerby, D.E. Bredesen, Molecular components of a cell death pathway activated by endoplasmic reticulum stress, J. Biol. Chem. 279 (2004) 177–187. [13] K. Shimoke, H. Amano, S. Kishi, H. Uchida, M. Kudo, T. Ikeuchi, Nerve growth factor attenuates endoplasmic reticulum stress-mediated apoptosis via suppression of caspase-12 activity, J. Biochem. 135 (2004) 439–446. [14] K. Shimoke, M. Kudo, T. Ikeuchi, MPTP-induced reactive oxygen species promote cell death through a gradual activation of caspase-3 without expression of GRP78/Bip as a preventive measure against ER stress in PC12 cells, Life Sci. 73 (2003) 581–593. [15] K. Shimoke, T. Utsumi, S. Kishi, M. Nishimura, H. Sasaya, M. Kudo, T. Ikeuchi, Prevention of endoplasmic reticulum stress-
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induced cell death by brain-derived neurotrophic factor in cultured cerebral cortical neurons, Brain Res. 1028 (2004) 105–111. [16] T. Takadera, T. Ohyashiki, Apoptotic cell death and CPP32-like activation induced by thapsigargin and their prevention by nerve growth factor in PC12 cells, Biochim. Biophys. Acta 1401 (1998) 63–71.
[17] T. Yoneda, K. Imaizumi, K. Oono, D. Yui, F. Gomi, T. Katayama, M. Tohyama, Activation of caspase-12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent mechanism in response to the ER stress, J. Biol. Chem. 276 (2001) 13935–13940.
NGF-induced phosphatidylinositol 3-kinase signaling pathway prevents thapsigargin-triggered ER stress-mediated apoptosis in PC12 cells Koji Shimoke ∗ , Soichiro Kishi, Takahiro Utsumi, Yuichi Shimamura, Harue Sasaya, Tadao Oikawa, Shinichi Uesato, Toshihiko Ikeuchi Faculty of Engineering and High Technology Research Center (HRC), Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan Received 30 May 2005; received in revised form 28 June 2005; accepted 18 July 2005
Abstract Tunicamycin, an inhibitor of the glycosylation of newly biosynthesized proteins, induces endoplasmic reticulum (ER) stress and subsequent apoptosis, and caspase family proteases are activated during the process of ER stress-mediated apoptosis. In the present study, we showed that thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), also induced ER stress-mediated apoptosis, and nerve growth factor (NGF) prevented the apoptosis in PC12 cells. We also found that LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), reduced the survival of cells treated with NGF for 24 h in the presence of Th. We discovered that the activities of caspase-3, -9 and -12 were increased time-dependently after the treatment with Th, and NGF suppressed the Th-triggered activation of caspase-3, -9 and -12. LY294002 diminished the effect of NGF on the inactivation of all these caspases. These results indicate that the NGF-induced PI3-K signaling pathway prevents Th-triggered ER stress-specific apoptosis via inhibition of caspase-mediated apoptotic signal. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: ER stress; Apoptosis; Caspase; Nerve growth factor; Phosphatidylinositol 3-kinase; PC12 cell
The endoplasmic reticulum (ER) is an important organelle that plays a crucial role in protein biosynthesis. It has been reported that a disruption of the quality control of newly biosynthesized proteins induces ER stress through the accumulation of unfolded protein [4,10–12]. Tunicamycin (Tm), an inhibitor of the glycosylation of newly biosynthesized proteins, induces ER stress [2,8,13]. Thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), also induces ER stress by disrupting the homeostatic balance of the Ca2+ concentration in the ER [3,5,9]. As a result, Th-treated cells die in an apoptotic manner [3,16]. It is of interest to compare the effects of nerve growth factor (NGF) on Tm-induced and Th-induced apoptosis. We first examined whether NGF is capable of preventing Th-induced apoptosis in PC12 cells or not. PC12 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% (v/v) heat∗
Corresponding author. Tel.: +81 6 6368 0853; fax: +81 6 6330 3770. E-mail address: [email protected] (K. Shimoke).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.07.030
inactivated precolostrum newborn calf serum and 5% (v/v) heat-inactivated horse serum. For the measurement of cell viability, PC12 cells were seeded onto collagen-coated 96well plates at 1 × 105 cells/cm2 . The next day, the medium was changed to serum-free DMEM, and 0.5 M Th without or with 100 ng/ml of NGF was added. As shown in Fig. 1A, Th reduced the cell viability, and NGF diminished the effect of Th in the culture for 24 h. For analysis of the mechanism of NGF-mediated cell survival, we administered NGF plus Th with LY294002 (LY) and PD98059 (PD), an inhibitor of phosphatidylinositol 3-kinase (PI3-K) and an inhibitor of upstream kinase (MEK1) of mitogen-activated kinase (MAPK also termed ERK1/2), respectively (Fig. 1A). LY, but not PD, remarkably reduced the NGF-mediated cell survival, suggesting that PI3-K is essential for the NGFmediated prevention of the Th-induced apoptosis. In addition, we found that Th transiently induced the mRNA expression of GRP78 gene, which is one of the target genes of the unfolded protein response (UPR), as an indicator of ER stress response (Fig. 1B). Interestingly, the expression of GRP78
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Fig. 1. Induction of ER stress and subsequent apoptosis in thapsigargin-treated PC12 cells. (A) PC12 cells were plated onto collagen-coated 96-well plates, and 0.5 M thapsigargin (Th) was added to serum-free DMEM (con) without or with 100 ng/mL of NGF (N). 10 M LY294002 (LY) or 50 M PD98059 (PD) was also added in the presence of Th plus N. Cells were cultured for 24 h. Viable cells were measured by alamarblue assay as described previously [13,14]. Values are the mean ± S.E.M. (n = 4) and the statistical analysis was carried out with one-way ANOVA. ** P < 0.01 and # P < 0.05 as compared with control. (B) Cells were plated onto 6 cm diameter dishes and treated with 0.5 M Th for up to 24 h. The specific probes for Northern blotting of GRP78 and 28S rRNA were labeled with [␣-32 P]dCTP as described previously [14]. The specific bands were visualized by autoradiography. The data represents three independent experiments. (C) Cells were plated onto 6 cm diameter dishes and treated with 0.5 M Th for up to 24 h. Then, cells were lysed in lysis buffer comprising 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 g/ml aprotinin and 1% (w/v) SDS. 10 g of protein per lane was used for SDS-PAGE. Next, immunoblotting was carried out using anti-GRP78 antibody (SantaCruz) or anti--actin antibody (Chemicon) as the primary antibody and horseradish peroxidase-conjugated secondary antibody to detect the specific bands. The bands were visualized with a light capture system (Atto) after treatment with Immunostar reagents (WAKO). The data represents four independent experiments.
mRNA by Th had a time course different from the Tminduced time-dependent expression of GRP78 mRNA. We observed a sustained induction of GRP78 protein up to 24 h after the treatment with Th in PC12 cells (Fig. 1C) although the GRP78 mRNA expression was transiently induced by Th. We confirmed that PC12 cells died in an apoptotic manner and NGF clearly prevented the Th-induced apoptosis, judging from the Hoechst 334882 staining (Fig. 2). Furthermore, LY increased the number of condensed nuclei in the presence of NGF (Fig. 2D). These results indicate that NGF attenuates the Th-mediated apoptosis via the PI3-K signaling pathway. It has been reported that caspase family proteases are involved in Tm-mediated apoptosis [13,15]. To clarify the involvement of caspases in Th-mediated apoptosis, we added caspase inhibitors (z-DEVD-fmk, z-VAD-fmk) to Thtreated PC12 cells. As a result, we observed that both caspase inhibitors restored cell viability in the presence of Th, indicating that the caspase cascade is involved in Th-induced apoptosis (Fig. 3). We have reported that caspase-3, -9 and -12 are involved in Tm-induced apoptosis [13]. Then, we analyzed whether NGF attenuated Th-induced apoptosis via suppres-
sion of the activation of caspase-3, -9 and -12, by measuring the caspase activities using fluorogenic substrates (50 M Ac-DEVD-MCA for caspase-3 and 50 M Ac-LEHD-MCA for caspase-9) and by Western blotting with anti-caspase-12 antibody. PC12 cells were treated with Th, Th plus NGF or Th plus NGF in the presence of LY, and lysates were incubated at 37 ◦ C for 1 h with the substrates described above. Fluorescence was detected using a spectrofluorometer with excitation (380 nm) and emission (460 nm) wavelengths for measurement of the caspase-3 and -9 activities. As shown in Fig. 4, Th increased the activities of caspase-3, -9 and -12, and NGF decreased these activities. Moreover, the effect of NGF was diminished by the addition of LY294002. These results clearly indicate that NGF prevents Th-induced apoptosis by suppressing the activation of caspase-3, -9 and -12 via the PI3-K signaling pathway. In the present study, we have shown the NGF-mediated effect on Th-induced apoptosis in PC12 cells. The mitochondrial dysfunction-mediated cell death which is considered as ordinary apoptosis involves the activation of caspase-9 via the formation of a complex between pro-caspase-9, Apaf1, dATP and cytochrome c released from mitochondria [1].
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Fig. 2. Hoechst 334882 staining of thapsigargin-treated PC12 cells. PC12 cells were plated onto 8-well chamber plates, and 0.5 M thapsigargin (Th) was added to serum-free DMEM without or with 100 ng/ml of NGF (N). 10 M LY294002 was added in the presence of Th plus N. Cells were cultured for 24 h. Then, cells were fixed and stained by treatment with 1 g/mL of Hoechst 334882 dye in phosphate-buffered saline (PBS) for 30 min. Results for untreated (A) or Th-treated cells (B) and Th-treated cells in the presence of NGF without (C) or with LY (D) are presented. Apoptotic condensed nuclei were detected and photographs were taken using a fluorescence microscope. Bar denotes 15 m.
Fig. 3. Prevention of thapsigargin-induced apoptosis by caspase inhibitors. PC12 cells were maintained and plated as described in Fig. 1A. 0.5 M thapsigargin (Th) was added to serum-free DMEM (con) in the absence or presence of 100 M z-DEVD-fmk (DEVD) or 50 M z-VAD-fmk (VAD). Viable cells were measured after 24 h by alamarblue assay. Values are the mean ± S.E.M. (n = 4) and the statistical analysis was carried out with oneway ANOVA. ** P < 0.01 and ## P < 0.01 as compared with control.
Although we observed significant activation of caspase-9 in Th-induced apoptosis, we could not clarify the mechanism of the activation. Morishima et al. reported that caspase9 is directly activated by caspase-12 during ER stress [6]. Thus, we consider that caspase-12 is the initiator caspase activating the caspase cascade during ER stress. Furthermore, we previously demonstrated that the activation of caspase-3 and -12 was suppressed by the PI3-K signaling pathway in Tm-treated cerebral cortical neurons and Tmtreated PC12 cells [13,15]. Interestingly, ER stress-mediated apoptosis by Th was also suppressed by the PI3-K signaling pathway in PC12 cells. We consider that there may be a common mechanism underlying Th- and Tm-triggered ER stress-mediated apoptosis although the time courses of GRP78 mRNA expressions during Th- and Tm-induced ER stress are different [13]. We are searching for a specific effector to suppress the caspase-mediated apoptotic pathway downstream of the PI3-K signaling pathway. Given that caspase-12 is the initiator caspase during ER stress, an activator of caspase-12 must exist on the ER membrane. It has also been reported that calpain activates caspase-12 during ER stress [7]. Calpain may be the key to suppression of the activation of caspase-12 through the PI3-K signaling pathway. However, calpain may not be a general mediator of ER stress-induced apoptosis because it is regulated by Ca2+ . Yoneda et al. reported that TNF receptor-associated factor 2 (TRAF2) was important for ER stress-induced
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Acknowledgements This work was supported in part by grants-in-aid for scientific research from MEXT (Ministry of Education, Culture, Sports, Science and Technology of Japan), HAITEKU (2002–2006) from MEXT, a grant from the Kansai University Special Research Fund, 2005 and a research grant from the Smoking Research Foundation, Japan. This research was also financially supported in part by the Sasakawa Scientific Research Grant from Japan Science Society.
References
Fig. 4. Activities of caspases during the thapsigargin-induced apoptosis. (A and B) PC12 cells were maintained as described in Fig. 1A and plated onto 6 cm diameter dishes. The medium was changed to serum-free DMEM without (con) or with 0.5 M thapsigargin (Th), Th + 100 ng/mL NGF (Th + NGF) or Th + NGF + 10 M LY294002 (Th + NGF + LY) for the periods indicated. Then, cells were collected in a lysis buffer comprising 10 mM Hepes–KOH (pH 7.4), 2 mM EDTA and 1 mM PMSF. After centrifugation, an aliquot of the supernatant was mixed with 2× ICE buffer comprising 20 mM Hepes–KOH (pH 7.4), 20% glycerol (v/v), 2 mM PMSF, 4 mM dithiothreitol and 50 M Ac-DEVD-MCA (for caspase-3) or 50 M AcLEHD-MCA (for caspase-9), followed by incubation at 37 ◦ C for 1 h. After addition of 200 l of distilled water, the activity of caspase-3 and -9 was measured by detecting fluorescence using a spectrofluorometer (Dainippon) [13]. Fold increases in the activities of caspase-3 and -9 are shown. Values are the mean ± S.E.M. (n = 3) and the statistical analysis was carried out with one-way ANOVA. * P < 0.05 and ** P < 0.01 as compared with 0 h. (C) Cells untreated (con) or treated with 0.5 M thapsigargin (Th), 0.5 M Th + 100 ng/ml NGF in the absence (Th + N) or presence of 10 M LY294002 (Th + N + LY) for 24 h were lysed in lysis buffer as described in Fig. 1C. Then, immunoblotting was carried out using anti-caspase-12 antibody (Sigma) or anti-actin antibody (SantaCruz) [7,13]. The bands were visualized with a light capture system (Atto) after treatment with Immunostar reagents (WAKO). The apparent molecular masses (kD) measured using molecular weight markers (Amersham) are presented on the right.
apoptosis [17]. Thus, further analyses to identify the activator of caspase-12 and to clarify the exact mechanism underlying the NGF-mediated effect on ER stress-induced apoptosis are necessary.
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