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Involvement of mitochondrial- and Fas-mediated dual mechanism in CoCl2-induced apoptosis of rat PC12 cells
Neuroscience Letters 371 (2004) 85–90
Involvement of mitochondrial- and Fas-mediated dual mechanism in CoCl2-induced apoptosis of rat PC12 cells Ji-Yeon Jung, Won-Jae Kim∗ College of Dentistry, Dental Science Research Institute, Chonnam National University, Gwang Ju 500-757, South Korea Received 6 April 2004; received in revised form 19 June 2004; accepted 27 June 2004
Abstract Hypoxic/ischemic condition induces neuronal apoptotic events, which consequently lead to neuronal cell death. However, its specific mechanistic pathways remain obscure. Cobalt chloride (CoCl2 ) could mimic the hypoxic condition including the production of reactive oxygen species (ROS). In this report, we investigated the signal pathway of CoCl2 -induced apoptosis in PC12 cells. The main mechanism for these apoptosis appeared to be mitochondria-mediated pathway accompanied with loss of the mitochondrial transmembrane potential (m) followed by cytochrome c release from the mitochondria into the cytosol, resulting in the activation of caspase-9 and caspase-3. Also, upregulation of pro-apoptotic protein Bax, and downregulation of anti-apoptotic protein Bcl-2 by presence of CoCl2 appeared significantly and it might result in activating mitochondria-mediated apoptosis. We showed that expression of Fas and Fas ligand was upregulated and caspase-8 was significantly activated in CoCl2 -induced apoptotic cells. In addition, ZB4, an antagonistic Fas-antibody, inhibited the activation of caspase-8 by CoCl2 , indicating that Fas receptor was involved in this pathway. These results demonstrate that CoCl2 induce apoptosis in PC12 cells via different dual apoptosis pathway through death receptor as well as mitochondria. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Rat PC12 cells; CoCl2 ; Hypoxia; Apoptosis; Caspase; Cytochrome c; Bcl-2 family; Mitochondria; Fas
Apoptosis is a gene-regulated mechanism of cell death and it plays a pivotal role in physiological and pathological processes [2]. Hypoxic/ischemic condition has long been recognized as an important mediator or modulator of apoptosis because this condition is accompanied with the production of reactive oxygen species (ROS) which can attack nucleic acids, proteins and membrane phospholipids [6,23, 27]. Hypoxia/ischemia-induced apoptosis is a major concern in various clinical entities such as ischemic disease, organ transplantation and other disease. However, the exact apoptotic mechanisms in hypoxic/ischemic condition and its treatments have not been settled yet. In general, apoptosis is driven from the activation of a family of cysteine protease called caspases, which cleave a critical set of cellular proteins to initiate apoptotic cell death [9,19]. These family are expressed as proenzymes and activated by upstream stimuli. Among mammalian caspases ∗
Corresponding author. Tel.: +82 62 530 4881; fax: +82 62 530 4885. E-mail address: [email protected] (W.-J. Kim).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.06.069
of at least 14 known members, those involved with apoptosis can be further subdivided into the initiator caspases (-2, -8, -9, and -10) and the effector caspases (-3, -6, and -7) [20]. Two main pathways of activating caspases are death receptor-mediated mechanism and mitochondria-mediated mechanism. Both pathways share the activation of caspase-3 as an executioner caspase, which activates caspase-activated DNase, causing apoptotic DNA fragmentation. The mitochondrial pathway is stimulated by hypoxic/ischemic condition, cytotoxic reagents, radiation, and growth factor deprivation [28,29]. These stimuli induce generation of ROS, leading to loss of mitochondrial transmembrane potential (m), and release of cytochrome c from mitochondria into cytosol. The subsequent interaction of cytochrome c with Apaf-1 protein results in the recruitment of procaspase-9. Activation of caspase-9 results in the activation of caspase-3 and subsequently contributes to apoptotic cell death [10,11,17]. Death receptor pathway is stimulated by cell surface death receptor such as Fas and tumor necrosis factor (TNF) receptor [5]. The receptors activated by ligands lead to activation of caspase-8,
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with subsequent activation of caspase-3. There have been no investigations regarding the mechanism of the death receptormediated apoptosis in hypoxic/ischemic condition. Besides the caspase, members of the Bcl-2 family are also critical for the regulation of apoptosis [21]. Bcl-2 family control the release of mitochondrial cytochrome c by modulating the permeability of the outer mitochondrial membrane. Bcl-2 family members are functionally divided into antiapoptotic molecules (Bcl-2, Bcl-XL , Bcl-W, Mcl-1, A1) and pro-apoptotic molecules (Bax, Bcl-Xs, Bak, Bid, Bad, Bim, Bik) [1,8,21]. However, the roles of Bcl-2 family and caspases in hypoxic/ischemic-induced neuronal apoptosis have not been well elucidated. Cobalt chloride (CoCl2 ) can mimic hypoxic/ischemic conditions, including generation of reactive oxygen species and transcriptional change of some genes such as hypoxia inducible factor (HIF-1␣), and p53, p21, and pCNA in promoting the cell death [7,14]. PC12 is a cell line derived from rat pheochromocytoma widely used for investigating neuronal apoptosis [22]. Therefore, CoCl2 -induced apoptosis may serve as a simple and convenient in vitro model to elucidate molecular mechanism in hypoxia-linked neuronal cell death and to search its treatment methods. In the present study, we investigated whether CoCl2 -induced apoptosis is accompanied by Bcl-2 family and associated with mitochondria- and death receptor-mediated pathway in neuronal hypoxic/ischemic condition. PC12 rat adrenal pheochromocytoma cells were maintained in RPMI 1640 medium supplemented with 10% horse serum (Gibco, USA) and 5% fetal bovine serum (Gibco, USA) under 5% CO2 at 37 ◦ C. Cell viability was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra zolium bromide) assay (Sigma, USA). ROS production was monitered by fluoresence spectrophotometer (Hitachi F4500, Japan) using DCF-DA (2 ,7 -dichlorofluorescin diacetate). Cells were 1 h pretreated with 5 mM NAC (N-acetylcystein) before CoCl2 treatment. Morphological changes of apoptotic cells were investigated by Diff-Quik (Kuk Jae, Japan) under microscope (Olympus, USA). Also, Induction of apoptosis was detected by DNA fragmentation assay (Takara, Japan). The m was determined by FACScan (Becton Dikinson, CA) using 5,5 ,6,6 -tetrachloro-1,1 ,3,3 tetrathyl-benzimidazol carbocyanine iodide (JC-1, Molecular Probes). Caspase activities were assayed using the Colorimetric Caspases-3, -9 Assay Kit (Calbiochem, CA) and Fluorometric caspase-8 Assay Kit (Santa Cruz, USA) according to the manufacturer’s instructions. Western blot were performed with antibody against cytochrome c (Pharmingen, CA), Bax, Bid, and Bcl-2 (Santa Cruz, USA). Antagonistic effect using ZB4, Fas-antibody (Upstate Biotechnology, KY), or IgG (Sigma) was performed. Expression of HIF-1␣, Fas and Fas-L was analyzed by RT-PCR. Statistical analysis was performed by Student’s t-test and presented as mean ± standard deviations (S.D.). To verify that exposure of PC12 cells to CoCl2 induces a hypoxia-mimicking response, expression of hypoxia-
Fig. 1. CoCl2 increased expression of HIF-1␣ and ROS generation in PC12 cells. (A) Cells were incubated in the absence, or in the presence of 150 M CoCl2 for 12 h. Cells were harvested and subjected to RT-PCR analysis for HIF-1␣. (B) DCF-loaded cells were incubated with 150 M CoCl2 alone or preincubated with 5 mM NAC 1 h prior to CoCl2 treatment. The intracellular levels of ROS were detected by measuring the DCF fluorescence. Data are mean ± S.D. from five independent experiments. * P < 0.05 vs. the corresponding control value, # P < 0.01 vs. the corresponding 150 M CoCl2 -treated cells.
inducible factor-1␣ (HIF-1␣), a marker of hypoxia, was examined and significantly elevated in CoCl2 -treated cells (Fig. 1A). Hypoxic signal increases the generation of ROS, which mediates cell injury. In this study, PC12 cells treated with 150 M CoCl2 showed an increment in intracellular ROS compared with control cells within 0.5 h. However, treatment with 5 mM NAC, free radical scavenger, significantly attenuated an increment of ROS level induced by CoCl2 in PC12 cells (Fig. 1B). After PC12 cells were exposed to CoCl2 , cell viability determined by MTT assay was markedly decreased in a dose and time-dependent manner (Fig. 2A and B). The cell viability was less than about 50% when the cells were treated with 150 M CoCl2 for 8 h. Apoptosis was assessed in terms of both morphological changes and DNA ladder formation. Morphological evaluation in 150 M CoCl2 -treated cells using Diff-Quik stain method revealed an increment in the number of cells with chromatin condensation and nuclear fragmentation (Fig. 2C). As shown in Fig. 2D, DNA fragmentation was confirmed by electrophoresis of genomic DNA extracted from PC12 cells treated with 150 M CoCl2 for 12 h. These results indicate that CoCl2 led to cell death as apoptosis in PC12 cells. Generally, expression ratio of Bax to Bcl-2 has proven to be significant for apoptosis determination, since a high ratio denotes a lower apoptotic threshold, while a low ratio indicates a higher apoptotic threshold. We investigated whether CoCl2 induce apoptosis by modulating the expression of Bcl2 family. As shown in Fig. 3A, treatment with 150 M CoCl2 caused an increment of Bax and a decrement of Bcl-2 in
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Fig. 2. CoCl2 decreased cell viability and increased the number of cells with apoptotic morphology and fragmented DNA in PC12 cells. (A) Cells were incubated with different concentration of CoCl2 for 8 h and with (B) 150 M CoCl2 for various time periods. Cell viability was measured by MTT assay. Data are mean ± S.D. from three independent experiments. * P < 0.05 and ** P < 0.01 vs. the corresponding control value. (C) Cells were treated with 150 M CoCl2 for 8 h and stained using the Diff-Quik method. Arrows indicate cells with chromatin condensation and nuclear fragmentation. (D) Cells were incubated in the absence (lane 1), or in the presence of 150 M CoCl2 for 12 h (lane 2). Fragmentations of genomic DNA were detected on 1.8% agarose electrophoresis.
Fig. 3. Expression of Bcl-2 family was modulated in CoCl2 -treated PC12 cells. CoCl2 upregulated the expression of Bax, while it downregulated that of Bcl-2. (A) After incubating with 150 M CoCl2 for 4, 8, 24 h, Bax and Bcl-2 in protein level was analyzed by Western blot analysis. (B) The ratio of Bax and Bcl-2 determined by densitometer was then calculated and graphed. Data are representative the mean ± S.D. for three independent experiments. * P < 0.05 vs. the corresponding control value.
Fig. 4. CoCl2 -induced loss of m and release of cytochrome c from mitochondria into cytosol. (A) The m in 150 M CoCl2 -treated cells for 8 or 24 h was measured using JC-1 (10 g/ml) and by flow cytometry. (B) Subcellular fractions (fr.) of cells treated with 150 M CoCl2 for indicated times were prepared by previous described method. Western blot analysis was performed with anti-cytochrome c antibody.
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protein level. Correspondingly a densitometric analysis of the bands revealed the increment of Bax/Bcl-2 ratio that favors apoptosis (Fig. 3B). As shown in Fig. 4A, exposure to 150 M CoCl2 caused the change of m, representing the increment of cells with depolarized mitochondrial membranes. However, pretreatment of 5 mM NAC for 1 h inhibited loss of m by CoCl2 . Cytochrome c was known to be released from mitochondria into cytosol response to death signal and active caspase cascade, we therefore, examined the release of cytochrome c from mitochondria into cytosol by Western blot analysis in cytosolic and mitochondrial fraction prepared by previous described report [3]. As shown in Fig. 4B, CoCl2 -treated cells released the cytochrome c from mitochondria into cytosol in a time-dependent manner. The complex cascade of caspase activation is specific to apoptosis. Among the caspases, caspases-3, -8, and -9 have been shown to play pivotal roles in apoptosis. In the present study, caspases-8 and -9 activities were examined in PC12 cells treated with 150 M CoCl2 for 8 or 24 h and compared to that in control cultures. As shown in Fig. 5, the caspases-8 and -9 activities were increased in CoCl2 -treated PC12 cells.
To confirm the involvement of caspase-8 and caspase-9 in CoCl2 -induced apoptosis of PC12 cells, we investigated the effect of caspase inhibitors on activity of caspase-3. PC12 cells were treated with 150 M CoCl2 for 8 h, in combination 1 h with 50 M Z-IETD-FMK, or 50 M LEHD-CHO prior to CoCl2 treatment. Significant elevation of caspase3 activity was also observed in PC12 cells with 150 M CoCl2 . However, caspase inhibitors inhibited the activation of caspase-3. To make sure the presence of active caspase3 in CoCl2 -treated cells, the cleavage of poly (ADP-ribose) polymerase (PARP) was investigated by Western blot analysis. PARP fragments (85 kDa) were detected in CoCl2 -treated cells. These results suggest that CoCl2 -induced apoptosis is associated with the activation of caspases-8, -9, and -3. To investigate whether death receptor-mediated apoptosis pathway are involved in CoCl2 -treated PC12 cells, we observed modulation of the mRNA levels of Fas and FasL by RT-PCR analysis. Upregulation of Fas and Fas-L levels was showed in 150 M CoCl2 -treated cells for 8 or 24 h (Fig. 6A), suggesting that the apoptotic pathway related to Fas/Fas ligand also seems to be involved in CoCl2 -induced apoptosis. Ligation of Fas by Fas ligand leads to cleavage
Fig. 5. CoCl2 enhanced caspases-3, -8, and -9 activity. Cultured cells were treated with 150 M CoCl2 for 8 or 24 h, cells were harvested and caspase-8 (A) and caspase-9 (B) activity were each measured according to manufacturer’s instructor. Data are representative the mean ± S.D. for three independent experiments. * P < 0.05 and ** P < 0.01 vs. the corresponding control value. (C) Caspase-3 activity was measured in cells treated with or without 150 M CoCl for 8 h 2 and in cells pretreated for 1 h with 50 M Z-IETD-FMK, a caspase-8 inhibitor, and 50 M LEDH-CHO, a caspase-9 inhibitor, prior to CoCl2 treatment. Data are representative the mean ± S.D. for three independent experiments. ** P < 0.01 vs. the corresponding control value. # P< 0.01 vs. the corresponding the CoCl2 -treated cells. (D) Cultured cells were treated with or without 150 M CoCl2 for 24 h and cleavage of PARP was measured using Western blot analysis. The lower band (85 kDa) indicates cleavage fragment of PARP associated with apoptosis.
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Fig. 6. (A) Fas and Fas-L expression were examined using RT-PCR after treating with 150 M CoCl2 for 8 h or 24 h. Fas and Fas-L were upregulated by CoCl2 . (B) Bid expression was examined using Western blot after treating with 150 M CoCl2 for different time. * P < 0.05 and ** P < 0.01 vs. the corresponding control value.
of procaspase-8, with the consequent generation of active caspase-8. Caspase-8 then activates downstream effector caspase through cleavage of Bid, commuting the cell to apoptosis [4]. We examined the expression of Bid pro-form during treatment with 150 M CoCl2 at different times using Western blot analysis. As shown in Fig. 6B, loss of Bid pro-form was showed in CoCl2 -treated PC12 cells, suggesting that Bid was activated in CoCl2 -induced apoptosis. To confirm the involvement of the Fas-mediated pathway in CoCl2 -induced apoptosis, we examined the effects of ZB4, an antagonistic Fas-antibody, on caspase-8 activity in CoCl2 -treated cells. As shown in Fig. 7, pretreatment of ZB4 ameliorated the increment of caspase-8 activity by CoCl2 and also recovered the decrement of cell viability by CoCl2 , suggesting that Fas receptor–ligand interaction was involved in CoCl2 -induced apoptosis pathway. We demonstrated that CoCl2 suppressed the cellular growth of PC12 cells via induction of apoptosis. Previous studies have shown that caspase-3 is activated in response to various hypoxia and expressed in PC12 cells, indicating that caspase-3 may play a pivotal role in hypoxia-induced neuronal apoptosis [24,25]. The present study showed that caspase-3 activity was upregulated and PARP was cleaved in CoCl2 -treated cells, which is consistent with result of the previous report. However, the upstream mechanism which activates caspase-3 in CoCl2 -induced apoptosis has not been still examined, even if some pathways are suggested in various hypoxic model. Caspase-3 is a key executioner caspase involved in neuronal apoptosis and caspase-3 activity is controlled by upstream regulatory, such as caspase-8 or caspase-9, which modulates the mitochondria- and death receptor-dependent pathway, respectively [15]. We detected the loss of m, release of cytochrome c from mitochondria into cytosol, and enhanced caspase-9 activity in CoCl2 -treated cells. Taken together, these results suggest that CoCl2 -induced apoptosis in PC12 cells is associated with the loss of m and the activation of caspases,
Fig. 7. (A) Cells were pretreated for 30 min with 500 ng/ml ZB4, an antagonistic Fas-antibody or 500 ng/ml IgG, and followed by 150 M CoCl2 treatment for indicated time and then caspase-8 activity was measured. Data are representative the mean ± S.D. for three independent experiments. * P < 0.05 vs. the corresponding control value. (B) Cells were treated with same condition mentioned above, and cell viability was measured by MTT assay. Data are representative the mean ± S.D. for three independent experiments. * P < 0.05 vs. the corresponding control value.
probably via mitochondria-mediated apoptosis pathway. We further demonstrated that Bax was upragulated by CoCl2 . Bax translocation to the mitochondria has been influenced to reduce m, release cytochrome c from mitochondria into cytosol, and activate caspases [18,26,27]. However, roles of Bcl-2 family in CoCl2 -induced apoptosis of PC12 cells may be a debate which Bcl-2 family regulates the production of ROS and cytochrome c release from mitochondria into cytosol in hypoxic/ischemic condition, or ROS could conversely regulates the expression of Bcl-XL mRNA [12]. Further researches for the roles of the Bcl-2 family will be followed in CoCl2 -induced apoptosis. The pathway of death receptor-mediated apoptosis is known to related to caspase-8 activation. On activation of death receptors by their ligation, the death receptors recruit the adaptor molecule FADD, followed by activation of caspase-8. We demonstrated that Fas and Fas ligand levels were upregulated in CoCl2 -treated PC12 cells. Besides, ZB4, an antagonistic Fas antibody, ameliorated the increment of caspase-8 activities by CoCl2 . These results indicate that Fasmediated signaling plays an important role in CoCl2 -induced apoptosis in PC12 cells. The link between the extrinsic apoptotic pathway initiated by death receptors and the intrinsic pathway mediated
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by mitochondria is provided by caspase-8, which activates pro-apoptotic Bid protein [16]. Subsequently, truncated Bid is inserted into the mitochondrial outermembrane and releases cytochrome c, resulting in activation of caspase-9 [13]. We demonstrated that loss of Bid pro-form was detected in CoCl2 -treated PC12 cells, suggesting that Bid was activated in CoCl2 -induced apoptosis. From these results, it was suggested that death receptor-mediated pathway is involved in CoCl2 -induced apoptosis. Prospectively, the studies for interaction between death receptor pathway and mitochondriamediated pathway will be need in CoCl2 -induced apoptosis. In summary, the present studies suggest that CoCl2 induced apoptosis in PC12 cells was mediated by dual pathways through mitochondria and death receptor with a regulatory role of Bcl-2 family and caspases (-3, -8, -9).
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Acknowledgment This study was financially supported by research fund of Chonnam National University in 2003.
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Involvement of mitochondrial- and Fas-mediated dual mechanism in CoCl2-induced apoptosis of rat PC12 cells Ji-Yeon Jung, Won-Jae Kim∗ College of Dentistry, Dental Science Research Institute, Chonnam National University, Gwang Ju 500-757, South Korea Received 6 April 2004; received in revised form 19 June 2004; accepted 27 June 2004
Abstract Hypoxic/ischemic condition induces neuronal apoptotic events, which consequently lead to neuronal cell death. However, its specific mechanistic pathways remain obscure. Cobalt chloride (CoCl2 ) could mimic the hypoxic condition including the production of reactive oxygen species (ROS). In this report, we investigated the signal pathway of CoCl2 -induced apoptosis in PC12 cells. The main mechanism for these apoptosis appeared to be mitochondria-mediated pathway accompanied with loss of the mitochondrial transmembrane potential (m) followed by cytochrome c release from the mitochondria into the cytosol, resulting in the activation of caspase-9 and caspase-3. Also, upregulation of pro-apoptotic protein Bax, and downregulation of anti-apoptotic protein Bcl-2 by presence of CoCl2 appeared significantly and it might result in activating mitochondria-mediated apoptosis. We showed that expression of Fas and Fas ligand was upregulated and caspase-8 was significantly activated in CoCl2 -induced apoptotic cells. In addition, ZB4, an antagonistic Fas-antibody, inhibited the activation of caspase-8 by CoCl2 , indicating that Fas receptor was involved in this pathway. These results demonstrate that CoCl2 induce apoptosis in PC12 cells via different dual apoptosis pathway through death receptor as well as mitochondria. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Rat PC12 cells; CoCl2 ; Hypoxia; Apoptosis; Caspase; Cytochrome c; Bcl-2 family; Mitochondria; Fas
Apoptosis is a gene-regulated mechanism of cell death and it plays a pivotal role in physiological and pathological processes [2]. Hypoxic/ischemic condition has long been recognized as an important mediator or modulator of apoptosis because this condition is accompanied with the production of reactive oxygen species (ROS) which can attack nucleic acids, proteins and membrane phospholipids [6,23, 27]. Hypoxia/ischemia-induced apoptosis is a major concern in various clinical entities such as ischemic disease, organ transplantation and other disease. However, the exact apoptotic mechanisms in hypoxic/ischemic condition and its treatments have not been settled yet. In general, apoptosis is driven from the activation of a family of cysteine protease called caspases, which cleave a critical set of cellular proteins to initiate apoptotic cell death [9,19]. These family are expressed as proenzymes and activated by upstream stimuli. Among mammalian caspases ∗
Corresponding author. Tel.: +82 62 530 4881; fax: +82 62 530 4885. E-mail address: [email protected] (W.-J. Kim).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.06.069
of at least 14 known members, those involved with apoptosis can be further subdivided into the initiator caspases (-2, -8, -9, and -10) and the effector caspases (-3, -6, and -7) [20]. Two main pathways of activating caspases are death receptor-mediated mechanism and mitochondria-mediated mechanism. Both pathways share the activation of caspase-3 as an executioner caspase, which activates caspase-activated DNase, causing apoptotic DNA fragmentation. The mitochondrial pathway is stimulated by hypoxic/ischemic condition, cytotoxic reagents, radiation, and growth factor deprivation [28,29]. These stimuli induce generation of ROS, leading to loss of mitochondrial transmembrane potential (m), and release of cytochrome c from mitochondria into cytosol. The subsequent interaction of cytochrome c with Apaf-1 protein results in the recruitment of procaspase-9. Activation of caspase-9 results in the activation of caspase-3 and subsequently contributes to apoptotic cell death [10,11,17]. Death receptor pathway is stimulated by cell surface death receptor such as Fas and tumor necrosis factor (TNF) receptor [5]. The receptors activated by ligands lead to activation of caspase-8,
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with subsequent activation of caspase-3. There have been no investigations regarding the mechanism of the death receptormediated apoptosis in hypoxic/ischemic condition. Besides the caspase, members of the Bcl-2 family are also critical for the regulation of apoptosis [21]. Bcl-2 family control the release of mitochondrial cytochrome c by modulating the permeability of the outer mitochondrial membrane. Bcl-2 family members are functionally divided into antiapoptotic molecules (Bcl-2, Bcl-XL , Bcl-W, Mcl-1, A1) and pro-apoptotic molecules (Bax, Bcl-Xs, Bak, Bid, Bad, Bim, Bik) [1,8,21]. However, the roles of Bcl-2 family and caspases in hypoxic/ischemic-induced neuronal apoptosis have not been well elucidated. Cobalt chloride (CoCl2 ) can mimic hypoxic/ischemic conditions, including generation of reactive oxygen species and transcriptional change of some genes such as hypoxia inducible factor (HIF-1␣), and p53, p21, and pCNA in promoting the cell death [7,14]. PC12 is a cell line derived from rat pheochromocytoma widely used for investigating neuronal apoptosis [22]. Therefore, CoCl2 -induced apoptosis may serve as a simple and convenient in vitro model to elucidate molecular mechanism in hypoxia-linked neuronal cell death and to search its treatment methods. In the present study, we investigated whether CoCl2 -induced apoptosis is accompanied by Bcl-2 family and associated with mitochondria- and death receptor-mediated pathway in neuronal hypoxic/ischemic condition. PC12 rat adrenal pheochromocytoma cells were maintained in RPMI 1640 medium supplemented with 10% horse serum (Gibco, USA) and 5% fetal bovine serum (Gibco, USA) under 5% CO2 at 37 ◦ C. Cell viability was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra zolium bromide) assay (Sigma, USA). ROS production was monitered by fluoresence spectrophotometer (Hitachi F4500, Japan) using DCF-DA (2 ,7 -dichlorofluorescin diacetate). Cells were 1 h pretreated with 5 mM NAC (N-acetylcystein) before CoCl2 treatment. Morphological changes of apoptotic cells were investigated by Diff-Quik (Kuk Jae, Japan) under microscope (Olympus, USA). Also, Induction of apoptosis was detected by DNA fragmentation assay (Takara, Japan). The m was determined by FACScan (Becton Dikinson, CA) using 5,5 ,6,6 -tetrachloro-1,1 ,3,3 tetrathyl-benzimidazol carbocyanine iodide (JC-1, Molecular Probes). Caspase activities were assayed using the Colorimetric Caspases-3, -9 Assay Kit (Calbiochem, CA) and Fluorometric caspase-8 Assay Kit (Santa Cruz, USA) according to the manufacturer’s instructions. Western blot were performed with antibody against cytochrome c (Pharmingen, CA), Bax, Bid, and Bcl-2 (Santa Cruz, USA). Antagonistic effect using ZB4, Fas-antibody (Upstate Biotechnology, KY), or IgG (Sigma) was performed. Expression of HIF-1␣, Fas and Fas-L was analyzed by RT-PCR. Statistical analysis was performed by Student’s t-test and presented as mean ± standard deviations (S.D.). To verify that exposure of PC12 cells to CoCl2 induces a hypoxia-mimicking response, expression of hypoxia-
Fig. 1. CoCl2 increased expression of HIF-1␣ and ROS generation in PC12 cells. (A) Cells were incubated in the absence, or in the presence of 150 M CoCl2 for 12 h. Cells were harvested and subjected to RT-PCR analysis for HIF-1␣. (B) DCF-loaded cells were incubated with 150 M CoCl2 alone or preincubated with 5 mM NAC 1 h prior to CoCl2 treatment. The intracellular levels of ROS were detected by measuring the DCF fluorescence. Data are mean ± S.D. from five independent experiments. * P < 0.05 vs. the corresponding control value, # P < 0.01 vs. the corresponding 150 M CoCl2 -treated cells.
inducible factor-1␣ (HIF-1␣), a marker of hypoxia, was examined and significantly elevated in CoCl2 -treated cells (Fig. 1A). Hypoxic signal increases the generation of ROS, which mediates cell injury. In this study, PC12 cells treated with 150 M CoCl2 showed an increment in intracellular ROS compared with control cells within 0.5 h. However, treatment with 5 mM NAC, free radical scavenger, significantly attenuated an increment of ROS level induced by CoCl2 in PC12 cells (Fig. 1B). After PC12 cells were exposed to CoCl2 , cell viability determined by MTT assay was markedly decreased in a dose and time-dependent manner (Fig. 2A and B). The cell viability was less than about 50% when the cells were treated with 150 M CoCl2 for 8 h. Apoptosis was assessed in terms of both morphological changes and DNA ladder formation. Morphological evaluation in 150 M CoCl2 -treated cells using Diff-Quik stain method revealed an increment in the number of cells with chromatin condensation and nuclear fragmentation (Fig. 2C). As shown in Fig. 2D, DNA fragmentation was confirmed by electrophoresis of genomic DNA extracted from PC12 cells treated with 150 M CoCl2 for 12 h. These results indicate that CoCl2 led to cell death as apoptosis in PC12 cells. Generally, expression ratio of Bax to Bcl-2 has proven to be significant for apoptosis determination, since a high ratio denotes a lower apoptotic threshold, while a low ratio indicates a higher apoptotic threshold. We investigated whether CoCl2 induce apoptosis by modulating the expression of Bcl2 family. As shown in Fig. 3A, treatment with 150 M CoCl2 caused an increment of Bax and a decrement of Bcl-2 in
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Fig. 2. CoCl2 decreased cell viability and increased the number of cells with apoptotic morphology and fragmented DNA in PC12 cells. (A) Cells were incubated with different concentration of CoCl2 for 8 h and with (B) 150 M CoCl2 for various time periods. Cell viability was measured by MTT assay. Data are mean ± S.D. from three independent experiments. * P < 0.05 and ** P < 0.01 vs. the corresponding control value. (C) Cells were treated with 150 M CoCl2 for 8 h and stained using the Diff-Quik method. Arrows indicate cells with chromatin condensation and nuclear fragmentation. (D) Cells were incubated in the absence (lane 1), or in the presence of 150 M CoCl2 for 12 h (lane 2). Fragmentations of genomic DNA were detected on 1.8% agarose electrophoresis.
Fig. 3. Expression of Bcl-2 family was modulated in CoCl2 -treated PC12 cells. CoCl2 upregulated the expression of Bax, while it downregulated that of Bcl-2. (A) After incubating with 150 M CoCl2 for 4, 8, 24 h, Bax and Bcl-2 in protein level was analyzed by Western blot analysis. (B) The ratio of Bax and Bcl-2 determined by densitometer was then calculated and graphed. Data are representative the mean ± S.D. for three independent experiments. * P < 0.05 vs. the corresponding control value.
Fig. 4. CoCl2 -induced loss of m and release of cytochrome c from mitochondria into cytosol. (A) The m in 150 M CoCl2 -treated cells for 8 or 24 h was measured using JC-1 (10 g/ml) and by flow cytometry. (B) Subcellular fractions (fr.) of cells treated with 150 M CoCl2 for indicated times were prepared by previous described method. Western blot analysis was performed with anti-cytochrome c antibody.
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protein level. Correspondingly a densitometric analysis of the bands revealed the increment of Bax/Bcl-2 ratio that favors apoptosis (Fig. 3B). As shown in Fig. 4A, exposure to 150 M CoCl2 caused the change of m, representing the increment of cells with depolarized mitochondrial membranes. However, pretreatment of 5 mM NAC for 1 h inhibited loss of m by CoCl2 . Cytochrome c was known to be released from mitochondria into cytosol response to death signal and active caspase cascade, we therefore, examined the release of cytochrome c from mitochondria into cytosol by Western blot analysis in cytosolic and mitochondrial fraction prepared by previous described report [3]. As shown in Fig. 4B, CoCl2 -treated cells released the cytochrome c from mitochondria into cytosol in a time-dependent manner. The complex cascade of caspase activation is specific to apoptosis. Among the caspases, caspases-3, -8, and -9 have been shown to play pivotal roles in apoptosis. In the present study, caspases-8 and -9 activities were examined in PC12 cells treated with 150 M CoCl2 for 8 or 24 h and compared to that in control cultures. As shown in Fig. 5, the caspases-8 and -9 activities were increased in CoCl2 -treated PC12 cells.
To confirm the involvement of caspase-8 and caspase-9 in CoCl2 -induced apoptosis of PC12 cells, we investigated the effect of caspase inhibitors on activity of caspase-3. PC12 cells were treated with 150 M CoCl2 for 8 h, in combination 1 h with 50 M Z-IETD-FMK, or 50 M LEHD-CHO prior to CoCl2 treatment. Significant elevation of caspase3 activity was also observed in PC12 cells with 150 M CoCl2 . However, caspase inhibitors inhibited the activation of caspase-3. To make sure the presence of active caspase3 in CoCl2 -treated cells, the cleavage of poly (ADP-ribose) polymerase (PARP) was investigated by Western blot analysis. PARP fragments (85 kDa) were detected in CoCl2 -treated cells. These results suggest that CoCl2 -induced apoptosis is associated with the activation of caspases-8, -9, and -3. To investigate whether death receptor-mediated apoptosis pathway are involved in CoCl2 -treated PC12 cells, we observed modulation of the mRNA levels of Fas and FasL by RT-PCR analysis. Upregulation of Fas and Fas-L levels was showed in 150 M CoCl2 -treated cells for 8 or 24 h (Fig. 6A), suggesting that the apoptotic pathway related to Fas/Fas ligand also seems to be involved in CoCl2 -induced apoptosis. Ligation of Fas by Fas ligand leads to cleavage
Fig. 5. CoCl2 enhanced caspases-3, -8, and -9 activity. Cultured cells were treated with 150 M CoCl2 for 8 or 24 h, cells were harvested and caspase-8 (A) and caspase-9 (B) activity were each measured according to manufacturer’s instructor. Data are representative the mean ± S.D. for three independent experiments. * P < 0.05 and ** P < 0.01 vs. the corresponding control value. (C) Caspase-3 activity was measured in cells treated with or without 150 M CoCl for 8 h 2 and in cells pretreated for 1 h with 50 M Z-IETD-FMK, a caspase-8 inhibitor, and 50 M LEDH-CHO, a caspase-9 inhibitor, prior to CoCl2 treatment. Data are representative the mean ± S.D. for three independent experiments. ** P < 0.01 vs. the corresponding control value. # P< 0.01 vs. the corresponding the CoCl2 -treated cells. (D) Cultured cells were treated with or without 150 M CoCl2 for 24 h and cleavage of PARP was measured using Western blot analysis. The lower band (85 kDa) indicates cleavage fragment of PARP associated with apoptosis.
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Fig. 6. (A) Fas and Fas-L expression were examined using RT-PCR after treating with 150 M CoCl2 for 8 h or 24 h. Fas and Fas-L were upregulated by CoCl2 . (B) Bid expression was examined using Western blot after treating with 150 M CoCl2 for different time. * P < 0.05 and ** P < 0.01 vs. the corresponding control value.
of procaspase-8, with the consequent generation of active caspase-8. Caspase-8 then activates downstream effector caspase through cleavage of Bid, commuting the cell to apoptosis [4]. We examined the expression of Bid pro-form during treatment with 150 M CoCl2 at different times using Western blot analysis. As shown in Fig. 6B, loss of Bid pro-form was showed in CoCl2 -treated PC12 cells, suggesting that Bid was activated in CoCl2 -induced apoptosis. To confirm the involvement of the Fas-mediated pathway in CoCl2 -induced apoptosis, we examined the effects of ZB4, an antagonistic Fas-antibody, on caspase-8 activity in CoCl2 -treated cells. As shown in Fig. 7, pretreatment of ZB4 ameliorated the increment of caspase-8 activity by CoCl2 and also recovered the decrement of cell viability by CoCl2 , suggesting that Fas receptor–ligand interaction was involved in CoCl2 -induced apoptosis pathway. We demonstrated that CoCl2 suppressed the cellular growth of PC12 cells via induction of apoptosis. Previous studies have shown that caspase-3 is activated in response to various hypoxia and expressed in PC12 cells, indicating that caspase-3 may play a pivotal role in hypoxia-induced neuronal apoptosis [24,25]. The present study showed that caspase-3 activity was upregulated and PARP was cleaved in CoCl2 -treated cells, which is consistent with result of the previous report. However, the upstream mechanism which activates caspase-3 in CoCl2 -induced apoptosis has not been still examined, even if some pathways are suggested in various hypoxic model. Caspase-3 is a key executioner caspase involved in neuronal apoptosis and caspase-3 activity is controlled by upstream regulatory, such as caspase-8 or caspase-9, which modulates the mitochondria- and death receptor-dependent pathway, respectively [15]. We detected the loss of m, release of cytochrome c from mitochondria into cytosol, and enhanced caspase-9 activity in CoCl2 -treated cells. Taken together, these results suggest that CoCl2 -induced apoptosis in PC12 cells is associated with the loss of m and the activation of caspases,
Fig. 7. (A) Cells were pretreated for 30 min with 500 ng/ml ZB4, an antagonistic Fas-antibody or 500 ng/ml IgG, and followed by 150 M CoCl2 treatment for indicated time and then caspase-8 activity was measured. Data are representative the mean ± S.D. for three independent experiments. * P < 0.05 vs. the corresponding control value. (B) Cells were treated with same condition mentioned above, and cell viability was measured by MTT assay. Data are representative the mean ± S.D. for three independent experiments. * P < 0.05 vs. the corresponding control value.
probably via mitochondria-mediated apoptosis pathway. We further demonstrated that Bax was upragulated by CoCl2 . Bax translocation to the mitochondria has been influenced to reduce m, release cytochrome c from mitochondria into cytosol, and activate caspases [18,26,27]. However, roles of Bcl-2 family in CoCl2 -induced apoptosis of PC12 cells may be a debate which Bcl-2 family regulates the production of ROS and cytochrome c release from mitochondria into cytosol in hypoxic/ischemic condition, or ROS could conversely regulates the expression of Bcl-XL mRNA [12]. Further researches for the roles of the Bcl-2 family will be followed in CoCl2 -induced apoptosis. The pathway of death receptor-mediated apoptosis is known to related to caspase-8 activation. On activation of death receptors by their ligation, the death receptors recruit the adaptor molecule FADD, followed by activation of caspase-8. We demonstrated that Fas and Fas ligand levels were upregulated in CoCl2 -treated PC12 cells. Besides, ZB4, an antagonistic Fas antibody, ameliorated the increment of caspase-8 activities by CoCl2 . These results indicate that Fasmediated signaling plays an important role in CoCl2 -induced apoptosis in PC12 cells. The link between the extrinsic apoptotic pathway initiated by death receptors and the intrinsic pathway mediated
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by mitochondria is provided by caspase-8, which activates pro-apoptotic Bid protein [16]. Subsequently, truncated Bid is inserted into the mitochondrial outermembrane and releases cytochrome c, resulting in activation of caspase-9 [13]. We demonstrated that loss of Bid pro-form was detected in CoCl2 -treated PC12 cells, suggesting that Bid was activated in CoCl2 -induced apoptosis. From these results, it was suggested that death receptor-mediated pathway is involved in CoCl2 -induced apoptosis. Prospectively, the studies for interaction between death receptor pathway and mitochondriamediated pathway will be need in CoCl2 -induced apoptosis. In summary, the present studies suggest that CoCl2 induced apoptosis in PC12 cells was mediated by dual pathways through mitochondria and death receptor with a regulatory role of Bcl-2 family and caspases (-3, -8, -9).
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Acknowledgment This study was financially supported by research fund of Chonnam National University in 2003.
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