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Cystatin C induces apoptosis and tyrosine hydroxylase gene expression through JNK-dependent pathway in neuronal cells
Neuroscience Letters 496 (2011) 100–105
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Cystatin C induces apoptosis and tyrosine hydroxylase gene expression through JNK-dependent pathway in neuronal cells XueYun Liang a,b , Atsushi Nagai a,∗ , Masaharu Terashima c , Abdullah Md. Sheikh a , Yuri Shiota a , Shingo Mitaki d , Seung U. Kim e,f , Shuhei Yamaguchi d a
Department of Laboratory Medicine, Shimane University Faculty of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan Department of Laboratory Medicine, Affiliated Hospital of Ningxia Medical University, Yinchuan, PR China c Department of Registered Dietitian and Nutritional Science, Tokaigakuen University Faculty of Human Wellness, Nagoya, Japan d Department of Internal Medicine III, Shimane University Faculty of Medicine, Izumo, Japan e Department of Neurology, UBC Hospital, University of British Columbia, Vancouver, Canada f Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 20 December 2010 Received in revised form 28 March 2011 Accepted 29 March 2011 Keywords: Cystatin C Neuron Apoptosis Differentiation JNK
a b s t r a c t Cystatin C (CysC), an endogenous cysteine protease inhibitor, has been implicated in the apoptosis and differentiation processes of neuronal cells. In this study, we have investigated the pathway involved in the process. A human neuronal hybridoma cell line (A1 cell) was treated with CysC in both undifferentiated and retinoic acid (RA)-induced differentiated conditions, which decreased overall process length in both conditions. Also, CysC increased apoptotic cell number time-dependently, as revealed by TUNEL assay. Western blot analysis demonstrated that in differentiated A1 cells, CysC treatment decreased Bcl-2 and increased active caspase-9 protein level time-dependently. Immunocytochemistry results revealed that, CysC treatment significantly increased active form of Bax expressing cell number, which co-localized with mitochondria. Mitogen activated protein (MAP) kinase inhibition experiments showed that Bax mRNA induction and Bcl-2 mRNA inhibition by CysC treatment were c-Jun N-terminal kinase (JNK)-dependent. After RA-induced differentiation, choline acetyltransferase (ChAT) and neurofilament (NF) mRNA levels were increased in A1 cells. CysC treatment inhibited NF mRNA level in both undifferentiated and RA-differentiated, and increased TH mRNA in differentiated A1 neurons. Analysis of signal transduction pathway demonstrated that TH gene induction was also JNK-dependent. Thus, our results demonstrated the significance of JNK-dependent pathways on CysC-induced apoptosis and TH gene expression in neuronal cells, which might be an important target in the management of CysC dependent neurodegenerative processes. © 2011 Elsevier Ireland Ltd. All rights reserved.
Cystatin C (CysC) is an endogenous cysteine protease inhibitor, which is ubiquitously expressed in the nucleated cells and secreted in the body fluids. By inhibiting cysteine proteases such as cathepsins, it plays important roles in the regulation of diverse biological functions including inflammation, tumor invasion and neuronal cell differentiation. In the central nervous system (CNS), where CysC concentration is 5.5 times higher in cerebrospinal fluid (CSF) compared to serum [1], it might play an important role in CNS pathophysiology by balancing the protease activities. Indeed,
Abbreviations: CysC, cystatin C; RA, retinoic acid; ChAT, choline acetyltransferase; NF, neurofilament; TH, tyrosin hydroxylase; JNK, c-Jun N-terminal kinase; CNS, central nervous system; CSF, cerebrospinal fluid; ER, endoplasmic reticulum; DMEM, Dulbecco’s modified eagle’s medium; TUNEL, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling; RT, room temperature; MAPK, mitogenactivated protein kinase. ∗ Corresponding author. Tel.: +81 853 20 2409; fax: +81 853 20 2409. E-mail address: [email protected] (A. Nagai). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.091
its concentration is reported to be decreased in neuroinflammatory disease conditions [14], leptomeningeal metastasis [13] and cerebral amyloid angiopathy [19]. Conversely, enhanced CysC expression is observed in cell-stress conditions such as facial nerve axotomy [10], hypophysectomy [6], transient forebrain ischemia [17] and 6-OHDA-induced niagrostraiatal neuronal degeneration in vivo [22]. It has been shown that CysC possesses functions other than lysosomal cysteine protease inhibition. Such as, it could influence the fibrillation process of Alzheimer’s amyloid peptide [11], or deposited intracellularly in endoplasmic reticulum (ER) [9]. Moreover, there are reports that have shown that CysC provides neuroprotection in serum deprived neuroblastoma cells [21], and increases nestin-positive neuronal progenitor cells and neurosphere [7]. On the other hand, we and others have found that CysC treatment increases the apoptosis of neuronal cells [2,11]. Interestingly, using CysC knockout mice, a report showed that CysC modulates both neurodegeneration and neurogenesis in a status
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Fig. 1. Effects of CysC on the morphological changes and the apoptosis of A1 cells. The effects of CysC on the morphological changes are shown in (A); where photomicrographs of undifferentiated (a–c) and differentiated (d–f) A1 cells in normal culture condition (a and d), and cultured in DMEM medium containing 0.5% FCS alone (b and e) or 50 nM CysC (c and f) for 48 h are shown. (B) Shows the confocal microscope images of TUNEL+ undifferentiated (a and b) and differentiated (c and d) A1 cells cultured in DMEM medium containing 0.5% FCS alone (a and c) or 50 nM CysC (b and d) for 48 h. Arrows indicate TUNEL positive cells. The quantification data of TUNEL+ cells, counted in a microscopic field at 200× magnification, is presented in (C). TUNEL+ cells were counted in 5 random microscopic fields of a slide, averaged and expressed as % of total cells in a field. The data presented in (C) are average ± SEM of 3 independent experiments. *p < 0.05 vs medium-treated A1 cells for 48 h. Scale bar = 20 m.
epilepticus model [18]. These results are implicating the diverse function of CysC, involving several signaling pathways in different cellular conditions. In this study, our aim was to investigate the signaling mechanisms of CysC-induce neuronal apoptosis and differentiation-specific gene expression in both undifferentiated and differentiated neuronal cells. A1 neuronal hybrid cell line, generated by fusion of human cerebral neurons and human neuroblastoma cells [12], was cultured in DMEM supplemented with 10% fetal calf serum (Invitrogen, Camarillo, Canada). For differentiation, A1 neuron was treated with 1 M retinoic acid (RA; Sigma, St. Louis, MO, USA), as described previously [12]. To analyze the apoptotic condition, we performed TUNEL assay using a kit (In Situ Cell Death Detection kit, POD, Roche, Mannheim, Germany), according to the manufacturer’s instruction. After TUNEL reaction, cells were washed with PBS, mounted and examined under a laser-scanning confocal microscope (Olympus FV300). Total RNA was isolated from A1 neurons using RNA Iso Plus (TaKaRa, Shigaken, Japan), according to the manufacturer’s instructions. Then, to determine the mRNA level of target genes, real time PCR was performed with gene specific primers and SyBr green PCR master mix (power SyBr green, ABI systems, Foster, USA), using an ABI Prism 7000 Sequence Detector system (Applied Biosystems). GAPDH mRNA level was used as an internal control, and the target gene mRNA level in a sample was quantified by relative quantification method. The primer sequences used for PCR were, Bax: 5 -TGGAGCTGCAGAGGATGATTG(forward) and 5 -AGCTGCCACTCGGAAAAAGAC-3 3 (reverse); Bcl-2: 5 -GCCCCCGTTGCTTTTCC-3 (forward) (reverse); tyrosine and 5 -CCGGTTATCGTACCCTGTTCTC-3 hydroxylase: 5 -TGTCCACGCTGTACTGGTTCAC-3 (forward) and 5 -CGGCACCATAGGCCTTCA-3 (reverse); low molecular weight-neurofilament (NFL): 5 -GATCTGCCTACGGCGGTTTA-3 (forward) and 5 -TGGTGTAGTAGGACGGGAAGGA-3 (reverse); choline acetyltransferase (ChAT): 5 -CGCTGGTGGCTAGAACA3 (forward) and 5 -TGATTGCAGCAGGCTACGAT-3 (reverse);
GAPDH: 5 -GCACCGTCAAGGCTGAGAA-3 (forward) and 5 TCTCGCTCCTGGAAGATGGT-3 (reverse). Total cellular protein was isolated as described previously [12]. Forty microgram of total protein was separated by SDS PAGE, using 10% polyacrylamide gel. The separated protein was transferred to a PVDF membrane, immunoblotted with primary antibody against Bax (rabbit, 1:1000, Abcam, Tokoyo, Japan), Bcl2 (mouse, 1:1000, Upstate, NY, USA), caspase-8 (mouse, 1:1000, MBL, Nagoya, Japan), caspase-9 (mouse, 1:1000, MBL, Nagoya, Japan) and -actin (mouse, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), followed by HRP-conjugated species-specific IgG, and immunoreactive proteins were visualized using an enhanced chemiluminescence kit (Amersham, Little Chalfont, Buckinghamshire, UK). Densitometric analysis of expressed protein was done using NIH image software. For immunocytochemistry, cultured cells on the cover slip were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Cells were blocked with 5% normal goat serum for 30 min and then incubated with monoclonal mouse anti-Bax (6A7, 1:50, Santa Cruz) antibody overnight at 4 ◦ C. The immunoreactive protein was detected with FITC-conjugated goat anti-mouse IgG (1: 100, Santa Cruz), and the fluorescence signals were visualized using a laser-scanning confocal microscope. Mitochondria were stained with MitoTracker Red (Lonza, Walkersville, USA) according to the manufacturer’s protocol. Data are expressed as means ± SEM (standard error mean). The statistical significance of the numerical data between groups was determined by one-way ANOVA or Student’s t test. The significance level was defined as p values less than 0.05. First, to analyze the effects of CysC on neuronal morphology and cellular apoptosis, both undifferentiated and RA-differentiated A1 neurons were treated with CysC (R&D Systems, Minneapolis, USA) up to 48 h. Undifferentiated A1 cells possess mostly a few short neuritis that were found extended after differentiation (Fig. 1A). CysC treatment for 48 h decreased the process length in both undifferentiated and differentiated conditions. TUNEL assay
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Fig. 2. CysC induces apoptosis through activating intrinsic pathway of apoptosis in A1 cells. (A) Differentiated A1 cells were treated with CysC (50 nM) for indicated time, and Bax, Bcl-2, caspase-8 and caspase-9 proteins were analyzed by Western blot. -Actin protein of corresponding samples was used as a loading control. Normalized desitometric data of Bax protein of 3 independent experiments were averaged and shown with SEM in (B). (C) The localization of active form of Bax was analyzed by immunocytochemistry using an active Bax specific antibody (6A7). Mitochondria were identified using Mito Tracker Red. Representative photomicrograph of A1 cells treated with 0.5% FCS/DMEM alone (a–f) or with 50 nM CysC (g–l) are shown, of which (d–f) and (j–l) are higher magnification photomicrograph of (a–c) and (g–i), respectively. Localization of active Bax are shown in (a, d, g and j), mitochondria in (b, e, h and k), and co-localization of active Bax with mitochondria in (c, f, i and l). The quantification data of active Bax positive cells, counted in a microscopic field at 200× magnification, are presented in (D). Active Bax+ cells were counted in 5 random microscopic fields of a slide, averaged and expressed as % of total cells in a field. The data presented in (D) are average ± SEM of 3 independent experiments. *p < 0.05 vs medium-treated A1 cells for 24 h. Scale bar = 20 m.
results showed that, CysC treatment increased TUNEL-positive cells time-dependently up to 48 h (Fig. 1B and C). The number of apoptotic cells between undifferentiated and differentiated conditions at each time point was similar (Fig. 1C). Our previous study demonstrated that CysC treatment increases proapoptotic Bax mRNA and decreases antiapoptotic Bcl-2 mRNA in RA-differentiated A1 cells [14], suggesting that intrinsic pathway of apoptosis might be involved in CysC-induced A1 apoptosis. To further examine that observation, we investigated the apoptotic proteins profile by Western blot. CysC treatment decreased Bcl-2 protein level at 4 h, and from 8 h treatment, it was almost undetectable (Fig. 2A). CysC treatment slightly increased Bax protein level, nevertheless such increment was not statistically significant (Fig. 2A and B). While pro and intermediate form of caspase-8 was
detectable, we could not detect active caspase-8. However, both pro and active form of caspase-9 was detectable, and CysC treatment increased the protein level of active caspase-9 (Fig. 2A). As CysC did not change Bax protein expression significantly, next we investigated its effects on Bax protein activation. Immunocytochemical analysis of active Bax protein showed that CysC treatment for 24 h significantly increased active Bax-positive cell number (Fig. 2D). The active Bax was mainly co-localized with mitochondria (Fig. 2C). Furthermore, we investigated the mechanisms involved in Bax mRNA induction and Bcl-2 mRNA inhibition by CysC in differentiated A1 neurons, mainly focusing on mitogen activated protein kinase (MAPK) pathways. JNK inhibitor (SP600125) inhibited CysCinduced Bax mRNA level (Fig. 3A). Bcl-2 mRNA inhibition by CysC
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Fig. 3. CysC modulates apoptosis-related genes expression in A1 cells in a JNK-dependent manner. A1 cells were treated with CysC (50 nM) in the presence or absence of a JNK-specific inhibitor (SP 600125, 1 M) for 8 h, total RNA was isolated, and Bax (A) and Bcl-2 (B) mRNA was analyzed by real time PCR, calculated as fold induction relative to medium stimulated condition. The data are presented here as average ± SEM of 3 independent experiments. GAPDH mRNA of corresponding samples was used as loading control. *p < 0.05 vs CysC treated A1 cells.
was partially rescued by JNK inhibition (Fig. 3B). Those data suggests the CysC-regulated apoptotic gene expressions are mediated through JNK-dependent pathway. CysC is mainly known as endogenous cysteine protease inhibitor. However, some functions of CysC have been reported to be independent of its cysteine protease inhibitory function [4]. Hence, we investigated whether another chemical cysteine protease inhibitor, E-64 (Sigma, St. Louis, MO, USA) has the ability to induce A1 cell apoptosis. Our findings demonstrated that E-64 also induced apoptosis of both differentiated and undifferentiated A1 neurons with similar efficacy as CysC (Fig. 3). Combined CysC and E-64 treatment for 48 h significantly increased the apoptotic cell number than CysC- or E-64-alone-treated condition, although combined treatment did not show any synergistic effect (Fig. 4). Next, we investigated whether CysC affects neuronal differentiation marker mRNA expression in A1 cells. Undifferentiated A1 cells changed the morphology (Fig. 1A), and ChAT and NF mRNA expres-
Fig. 4. Effects of cystein protease inhibition on A1 cell apoptosis. Undifferentiated (A) and differentiated (B) A1 cells were treated with 0.5% FCS/DMEM alone or 0.5% FCS/DMEM containing E-64 (10 M), or cystatin C (40 nM), or E-64 and CysC for 48 h. The cellular apoptosis was analyzed by TUNEL assay. The TUNEL+ cells were counted in 5 random microscopic field at 200× magnification, averaged and expressed as % of total cells in a field, and presented as average ± SEM of 3 independent experiments. *p < 0.05 vs medium stimulated condition, # p < 0.05 vs CysC- or E-64-alone treated condition.
sion was increased in the differentiation process (Fig. 5A). We treated both undifferentiated and differentiated A1 cells with CysC. Interestingly, CysC treatment increased TH mRNA with decreased NF mRNA levels in differentiated A1 neurons in time and dose dependent manner (Fig. 5C). Although such changes of TH mRNA expression did not observed in undifferentiated A1 cells, here also CysC similarly inhibited NF mRNA expression (Fig. 5B). Since it was previously shown that a member of cystatin family protein influence neuronal differentiation through modulation of MAPK activity [5], we investigated whether CysC-induced TH mRNA by was regulated through this pathway. As shown in Fig. 5D, JNK inhibitor showed the inhibitory effects on CysC-induced TH mRNA expression. p38 inhibitor also showed inhibitory effects on TH mRNA expression; however, that inhibitory effect did not reached a significant level. These results indicated that intracellular CysC signaling to induce TH gene is mediated through the JNK signaling pathway. The principal findings of our study are: (1) CysC induces apoptosis of neuronal cells through activating intrinsic pathway of apoptosis. (2) JNK-dependent pathways play important roles in CysC-modulated neuronal cellular functions. A recent report pointed out the importance of JNK-mediated cytochrome c release and activation of intrinsic pathway of apoptosis in the process of nerve growth factor-deprived neuronal apoptosis [8]. In the present study, CysC also affected the intrinsic pathway to regulate the expression of Bcl-2 in a JNK-dependent manner. We did not identified how JNK is activated by CysC in neuronal cells. Possible mechanism might be the oxidative stress, which has been implicated in JNK dependent, intrinsic pathway mediated apoptosis of cardiomyocytes in mice [16]. Indeed, a report has demonstrated the CysC-induced apoptosis of neuronal cells are mediated through increasing oxidative stress [15]. Our previous report showed that the CysC-induced neuronal cell apoptosis was cathepsin inhibition-dependent. In this report, a chemical inhibitor of cysteine protease, E64 has similar effects as CysC to increase the apoptotic cells number. However, combined CysC and E-64 treatment increased only 3–4% of apoptotic cells than CysC- or E-64-alone treatment. These findings suggest that, although cystein protease inhibition might play a role in CysCinduced neuronal apoptosis, it seems likely that there might be other factors involved in this process. Further studies should be carried out to elucidate cysteine protease inhibition-independent function of CysC [4,20]. In the present experiments, we have found that CysC has a function to induce TH gene expression through JNK-dependent pathway in differentiated A1 cells. It has been shown that nicotine-induced TH gene expression in PC12 cells is ATF-2 transcription factordependent and phosphorylation of ATF-2 is also JNK-dependent [3], indicating that ATF-2 phosphorylation by JNK might play an important role in the process.
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In conclusion, our study demonstrates the significance of JNKdependent pathways on CysC-induced apoptosis and TH gene expression in neuronal cells, which might be an important target for the management of CysC-dependent neurodegenerative processes.
Acknowledgment This work was supported by Grants from Society for Catecholamines and Neurologic Diseases (AN).
References
Fig. 5. Effects of CysC on neuronal differentiation markers expression in A1 cells. The mRNA level of neuronal-cell-type markers in undifferentiated and differentiated A1 cells in normal culture condition are shown in (A), calculated relatively as mRNA copy number of target gene per 106 GAPDH mRNA. To analyze the effects of CysC on neuronal-cell-type markers mRNA expression, undifferentiated (B) and differentiated (C) A1 cells were treated with indicated concentrations of CysC for indicated times, and neuronal-cell-type markers mRNA was analyzed by real time PCR, calculated relatively using 24 h medium-only stimulated sample of 1 experiment as calibrator, and presented as fold induction relative to calibrator sample. The data are presented here as average ± SEM of 3 independent experiments. (D) The regulation of CysC-induced TH mRNA expression was analyzed by treating differentiated A1 cells with CysC (50 nM) in the presence or absence of p38 (SB 203580, 10 M), JNK (SP 600125, 1 M) or MEK (U0126, 10 M) inhibitor, or above mentioned inhibitors alone, and TH mRNA expression was analyzed real time PCR. The TH mRNA level was calculated as fold induction relative to medium-only stimulated condition. The data are presented here as average ± SEM of 3 independent experiments. GAPDH mRNA of corresponding samples was used as loading control. † p < 0.05 vs undifferentiated A1 cells, *p < 0.05 vs corresponding medium-only unstimulated condition, # p < 0.05 vs CysC stimulated condition.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Cystatin C induces apoptosis and tyrosine hydroxylase gene expression through JNK-dependent pathway in neuronal cells XueYun Liang a,b , Atsushi Nagai a,∗ , Masaharu Terashima c , Abdullah Md. Sheikh a , Yuri Shiota a , Shingo Mitaki d , Seung U. Kim e,f , Shuhei Yamaguchi d a
Department of Laboratory Medicine, Shimane University Faculty of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan Department of Laboratory Medicine, Affiliated Hospital of Ningxia Medical University, Yinchuan, PR China c Department of Registered Dietitian and Nutritional Science, Tokaigakuen University Faculty of Human Wellness, Nagoya, Japan d Department of Internal Medicine III, Shimane University Faculty of Medicine, Izumo, Japan e Department of Neurology, UBC Hospital, University of British Columbia, Vancouver, Canada f Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 20 December 2010 Received in revised form 28 March 2011 Accepted 29 March 2011 Keywords: Cystatin C Neuron Apoptosis Differentiation JNK
a b s t r a c t Cystatin C (CysC), an endogenous cysteine protease inhibitor, has been implicated in the apoptosis and differentiation processes of neuronal cells. In this study, we have investigated the pathway involved in the process. A human neuronal hybridoma cell line (A1 cell) was treated with CysC in both undifferentiated and retinoic acid (RA)-induced differentiated conditions, which decreased overall process length in both conditions. Also, CysC increased apoptotic cell number time-dependently, as revealed by TUNEL assay. Western blot analysis demonstrated that in differentiated A1 cells, CysC treatment decreased Bcl-2 and increased active caspase-9 protein level time-dependently. Immunocytochemistry results revealed that, CysC treatment significantly increased active form of Bax expressing cell number, which co-localized with mitochondria. Mitogen activated protein (MAP) kinase inhibition experiments showed that Bax mRNA induction and Bcl-2 mRNA inhibition by CysC treatment were c-Jun N-terminal kinase (JNK)-dependent. After RA-induced differentiation, choline acetyltransferase (ChAT) and neurofilament (NF) mRNA levels were increased in A1 cells. CysC treatment inhibited NF mRNA level in both undifferentiated and RA-differentiated, and increased TH mRNA in differentiated A1 neurons. Analysis of signal transduction pathway demonstrated that TH gene induction was also JNK-dependent. Thus, our results demonstrated the significance of JNK-dependent pathways on CysC-induced apoptosis and TH gene expression in neuronal cells, which might be an important target in the management of CysC dependent neurodegenerative processes. © 2011 Elsevier Ireland Ltd. All rights reserved.
Cystatin C (CysC) is an endogenous cysteine protease inhibitor, which is ubiquitously expressed in the nucleated cells and secreted in the body fluids. By inhibiting cysteine proteases such as cathepsins, it plays important roles in the regulation of diverse biological functions including inflammation, tumor invasion and neuronal cell differentiation. In the central nervous system (CNS), where CysC concentration is 5.5 times higher in cerebrospinal fluid (CSF) compared to serum [1], it might play an important role in CNS pathophysiology by balancing the protease activities. Indeed,
Abbreviations: CysC, cystatin C; RA, retinoic acid; ChAT, choline acetyltransferase; NF, neurofilament; TH, tyrosin hydroxylase; JNK, c-Jun N-terminal kinase; CNS, central nervous system; CSF, cerebrospinal fluid; ER, endoplasmic reticulum; DMEM, Dulbecco’s modified eagle’s medium; TUNEL, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling; RT, room temperature; MAPK, mitogenactivated protein kinase. ∗ Corresponding author. Tel.: +81 853 20 2409; fax: +81 853 20 2409. E-mail address: [email protected] (A. Nagai). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.091
its concentration is reported to be decreased in neuroinflammatory disease conditions [14], leptomeningeal metastasis [13] and cerebral amyloid angiopathy [19]. Conversely, enhanced CysC expression is observed in cell-stress conditions such as facial nerve axotomy [10], hypophysectomy [6], transient forebrain ischemia [17] and 6-OHDA-induced niagrostraiatal neuronal degeneration in vivo [22]. It has been shown that CysC possesses functions other than lysosomal cysteine protease inhibition. Such as, it could influence the fibrillation process of Alzheimer’s amyloid peptide [11], or deposited intracellularly in endoplasmic reticulum (ER) [9]. Moreover, there are reports that have shown that CysC provides neuroprotection in serum deprived neuroblastoma cells [21], and increases nestin-positive neuronal progenitor cells and neurosphere [7]. On the other hand, we and others have found that CysC treatment increases the apoptosis of neuronal cells [2,11]. Interestingly, using CysC knockout mice, a report showed that CysC modulates both neurodegeneration and neurogenesis in a status
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Fig. 1. Effects of CysC on the morphological changes and the apoptosis of A1 cells. The effects of CysC on the morphological changes are shown in (A); where photomicrographs of undifferentiated (a–c) and differentiated (d–f) A1 cells in normal culture condition (a and d), and cultured in DMEM medium containing 0.5% FCS alone (b and e) or 50 nM CysC (c and f) for 48 h are shown. (B) Shows the confocal microscope images of TUNEL+ undifferentiated (a and b) and differentiated (c and d) A1 cells cultured in DMEM medium containing 0.5% FCS alone (a and c) or 50 nM CysC (b and d) for 48 h. Arrows indicate TUNEL positive cells. The quantification data of TUNEL+ cells, counted in a microscopic field at 200× magnification, is presented in (C). TUNEL+ cells were counted in 5 random microscopic fields of a slide, averaged and expressed as % of total cells in a field. The data presented in (C) are average ± SEM of 3 independent experiments. *p < 0.05 vs medium-treated A1 cells for 48 h. Scale bar = 20 m.
epilepticus model [18]. These results are implicating the diverse function of CysC, involving several signaling pathways in different cellular conditions. In this study, our aim was to investigate the signaling mechanisms of CysC-induce neuronal apoptosis and differentiation-specific gene expression in both undifferentiated and differentiated neuronal cells. A1 neuronal hybrid cell line, generated by fusion of human cerebral neurons and human neuroblastoma cells [12], was cultured in DMEM supplemented with 10% fetal calf serum (Invitrogen, Camarillo, Canada). For differentiation, A1 neuron was treated with 1 M retinoic acid (RA; Sigma, St. Louis, MO, USA), as described previously [12]. To analyze the apoptotic condition, we performed TUNEL assay using a kit (In Situ Cell Death Detection kit, POD, Roche, Mannheim, Germany), according to the manufacturer’s instruction. After TUNEL reaction, cells were washed with PBS, mounted and examined under a laser-scanning confocal microscope (Olympus FV300). Total RNA was isolated from A1 neurons using RNA Iso Plus (TaKaRa, Shigaken, Japan), according to the manufacturer’s instructions. Then, to determine the mRNA level of target genes, real time PCR was performed with gene specific primers and SyBr green PCR master mix (power SyBr green, ABI systems, Foster, USA), using an ABI Prism 7000 Sequence Detector system (Applied Biosystems). GAPDH mRNA level was used as an internal control, and the target gene mRNA level in a sample was quantified by relative quantification method. The primer sequences used for PCR were, Bax: 5 -TGGAGCTGCAGAGGATGATTG(forward) and 5 -AGCTGCCACTCGGAAAAAGAC-3 3 (reverse); Bcl-2: 5 -GCCCCCGTTGCTTTTCC-3 (forward) (reverse); tyrosine and 5 -CCGGTTATCGTACCCTGTTCTC-3 hydroxylase: 5 -TGTCCACGCTGTACTGGTTCAC-3 (forward) and 5 -CGGCACCATAGGCCTTCA-3 (reverse); low molecular weight-neurofilament (NFL): 5 -GATCTGCCTACGGCGGTTTA-3 (forward) and 5 -TGGTGTAGTAGGACGGGAAGGA-3 (reverse); choline acetyltransferase (ChAT): 5 -CGCTGGTGGCTAGAACA3 (forward) and 5 -TGATTGCAGCAGGCTACGAT-3 (reverse);
GAPDH: 5 -GCACCGTCAAGGCTGAGAA-3 (forward) and 5 TCTCGCTCCTGGAAGATGGT-3 (reverse). Total cellular protein was isolated as described previously [12]. Forty microgram of total protein was separated by SDS PAGE, using 10% polyacrylamide gel. The separated protein was transferred to a PVDF membrane, immunoblotted with primary antibody against Bax (rabbit, 1:1000, Abcam, Tokoyo, Japan), Bcl2 (mouse, 1:1000, Upstate, NY, USA), caspase-8 (mouse, 1:1000, MBL, Nagoya, Japan), caspase-9 (mouse, 1:1000, MBL, Nagoya, Japan) and -actin (mouse, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), followed by HRP-conjugated species-specific IgG, and immunoreactive proteins were visualized using an enhanced chemiluminescence kit (Amersham, Little Chalfont, Buckinghamshire, UK). Densitometric analysis of expressed protein was done using NIH image software. For immunocytochemistry, cultured cells on the cover slip were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Cells were blocked with 5% normal goat serum for 30 min and then incubated with monoclonal mouse anti-Bax (6A7, 1:50, Santa Cruz) antibody overnight at 4 ◦ C. The immunoreactive protein was detected with FITC-conjugated goat anti-mouse IgG (1: 100, Santa Cruz), and the fluorescence signals were visualized using a laser-scanning confocal microscope. Mitochondria were stained with MitoTracker Red (Lonza, Walkersville, USA) according to the manufacturer’s protocol. Data are expressed as means ± SEM (standard error mean). The statistical significance of the numerical data between groups was determined by one-way ANOVA or Student’s t test. The significance level was defined as p values less than 0.05. First, to analyze the effects of CysC on neuronal morphology and cellular apoptosis, both undifferentiated and RA-differentiated A1 neurons were treated with CysC (R&D Systems, Minneapolis, USA) up to 48 h. Undifferentiated A1 cells possess mostly a few short neuritis that were found extended after differentiation (Fig. 1A). CysC treatment for 48 h decreased the process length in both undifferentiated and differentiated conditions. TUNEL assay
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Fig. 2. CysC induces apoptosis through activating intrinsic pathway of apoptosis in A1 cells. (A) Differentiated A1 cells were treated with CysC (50 nM) for indicated time, and Bax, Bcl-2, caspase-8 and caspase-9 proteins were analyzed by Western blot. -Actin protein of corresponding samples was used as a loading control. Normalized desitometric data of Bax protein of 3 independent experiments were averaged and shown with SEM in (B). (C) The localization of active form of Bax was analyzed by immunocytochemistry using an active Bax specific antibody (6A7). Mitochondria were identified using Mito Tracker Red. Representative photomicrograph of A1 cells treated with 0.5% FCS/DMEM alone (a–f) or with 50 nM CysC (g–l) are shown, of which (d–f) and (j–l) are higher magnification photomicrograph of (a–c) and (g–i), respectively. Localization of active Bax are shown in (a, d, g and j), mitochondria in (b, e, h and k), and co-localization of active Bax with mitochondria in (c, f, i and l). The quantification data of active Bax positive cells, counted in a microscopic field at 200× magnification, are presented in (D). Active Bax+ cells were counted in 5 random microscopic fields of a slide, averaged and expressed as % of total cells in a field. The data presented in (D) are average ± SEM of 3 independent experiments. *p < 0.05 vs medium-treated A1 cells for 24 h. Scale bar = 20 m.
results showed that, CysC treatment increased TUNEL-positive cells time-dependently up to 48 h (Fig. 1B and C). The number of apoptotic cells between undifferentiated and differentiated conditions at each time point was similar (Fig. 1C). Our previous study demonstrated that CysC treatment increases proapoptotic Bax mRNA and decreases antiapoptotic Bcl-2 mRNA in RA-differentiated A1 cells [14], suggesting that intrinsic pathway of apoptosis might be involved in CysC-induced A1 apoptosis. To further examine that observation, we investigated the apoptotic proteins profile by Western blot. CysC treatment decreased Bcl-2 protein level at 4 h, and from 8 h treatment, it was almost undetectable (Fig. 2A). CysC treatment slightly increased Bax protein level, nevertheless such increment was not statistically significant (Fig. 2A and B). While pro and intermediate form of caspase-8 was
detectable, we could not detect active caspase-8. However, both pro and active form of caspase-9 was detectable, and CysC treatment increased the protein level of active caspase-9 (Fig. 2A). As CysC did not change Bax protein expression significantly, next we investigated its effects on Bax protein activation. Immunocytochemical analysis of active Bax protein showed that CysC treatment for 24 h significantly increased active Bax-positive cell number (Fig. 2D). The active Bax was mainly co-localized with mitochondria (Fig. 2C). Furthermore, we investigated the mechanisms involved in Bax mRNA induction and Bcl-2 mRNA inhibition by CysC in differentiated A1 neurons, mainly focusing on mitogen activated protein kinase (MAPK) pathways. JNK inhibitor (SP600125) inhibited CysCinduced Bax mRNA level (Fig. 3A). Bcl-2 mRNA inhibition by CysC
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Fig. 3. CysC modulates apoptosis-related genes expression in A1 cells in a JNK-dependent manner. A1 cells were treated with CysC (50 nM) in the presence or absence of a JNK-specific inhibitor (SP 600125, 1 M) for 8 h, total RNA was isolated, and Bax (A) and Bcl-2 (B) mRNA was analyzed by real time PCR, calculated as fold induction relative to medium stimulated condition. The data are presented here as average ± SEM of 3 independent experiments. GAPDH mRNA of corresponding samples was used as loading control. *p < 0.05 vs CysC treated A1 cells.
was partially rescued by JNK inhibition (Fig. 3B). Those data suggests the CysC-regulated apoptotic gene expressions are mediated through JNK-dependent pathway. CysC is mainly known as endogenous cysteine protease inhibitor. However, some functions of CysC have been reported to be independent of its cysteine protease inhibitory function [4]. Hence, we investigated whether another chemical cysteine protease inhibitor, E-64 (Sigma, St. Louis, MO, USA) has the ability to induce A1 cell apoptosis. Our findings demonstrated that E-64 also induced apoptosis of both differentiated and undifferentiated A1 neurons with similar efficacy as CysC (Fig. 3). Combined CysC and E-64 treatment for 48 h significantly increased the apoptotic cell number than CysC- or E-64-alone-treated condition, although combined treatment did not show any synergistic effect (Fig. 4). Next, we investigated whether CysC affects neuronal differentiation marker mRNA expression in A1 cells. Undifferentiated A1 cells changed the morphology (Fig. 1A), and ChAT and NF mRNA expres-
Fig. 4. Effects of cystein protease inhibition on A1 cell apoptosis. Undifferentiated (A) and differentiated (B) A1 cells were treated with 0.5% FCS/DMEM alone or 0.5% FCS/DMEM containing E-64 (10 M), or cystatin C (40 nM), or E-64 and CysC for 48 h. The cellular apoptosis was analyzed by TUNEL assay. The TUNEL+ cells were counted in 5 random microscopic field at 200× magnification, averaged and expressed as % of total cells in a field, and presented as average ± SEM of 3 independent experiments. *p < 0.05 vs medium stimulated condition, # p < 0.05 vs CysC- or E-64-alone treated condition.
sion was increased in the differentiation process (Fig. 5A). We treated both undifferentiated and differentiated A1 cells with CysC. Interestingly, CysC treatment increased TH mRNA with decreased NF mRNA levels in differentiated A1 neurons in time and dose dependent manner (Fig. 5C). Although such changes of TH mRNA expression did not observed in undifferentiated A1 cells, here also CysC similarly inhibited NF mRNA expression (Fig. 5B). Since it was previously shown that a member of cystatin family protein influence neuronal differentiation through modulation of MAPK activity [5], we investigated whether CysC-induced TH mRNA by was regulated through this pathway. As shown in Fig. 5D, JNK inhibitor showed the inhibitory effects on CysC-induced TH mRNA expression. p38 inhibitor also showed inhibitory effects on TH mRNA expression; however, that inhibitory effect did not reached a significant level. These results indicated that intracellular CysC signaling to induce TH gene is mediated through the JNK signaling pathway. The principal findings of our study are: (1) CysC induces apoptosis of neuronal cells through activating intrinsic pathway of apoptosis. (2) JNK-dependent pathways play important roles in CysC-modulated neuronal cellular functions. A recent report pointed out the importance of JNK-mediated cytochrome c release and activation of intrinsic pathway of apoptosis in the process of nerve growth factor-deprived neuronal apoptosis [8]. In the present study, CysC also affected the intrinsic pathway to regulate the expression of Bcl-2 in a JNK-dependent manner. We did not identified how JNK is activated by CysC in neuronal cells. Possible mechanism might be the oxidative stress, which has been implicated in JNK dependent, intrinsic pathway mediated apoptosis of cardiomyocytes in mice [16]. Indeed, a report has demonstrated the CysC-induced apoptosis of neuronal cells are mediated through increasing oxidative stress [15]. Our previous report showed that the CysC-induced neuronal cell apoptosis was cathepsin inhibition-dependent. In this report, a chemical inhibitor of cysteine protease, E64 has similar effects as CysC to increase the apoptotic cells number. However, combined CysC and E-64 treatment increased only 3–4% of apoptotic cells than CysC- or E-64-alone treatment. These findings suggest that, although cystein protease inhibition might play a role in CysCinduced neuronal apoptosis, it seems likely that there might be other factors involved in this process. Further studies should be carried out to elucidate cysteine protease inhibition-independent function of CysC [4,20]. In the present experiments, we have found that CysC has a function to induce TH gene expression through JNK-dependent pathway in differentiated A1 cells. It has been shown that nicotine-induced TH gene expression in PC12 cells is ATF-2 transcription factordependent and phosphorylation of ATF-2 is also JNK-dependent [3], indicating that ATF-2 phosphorylation by JNK might play an important role in the process.
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In conclusion, our study demonstrates the significance of JNKdependent pathways on CysC-induced apoptosis and TH gene expression in neuronal cells, which might be an important target for the management of CysC-dependent neurodegenerative processes.
Acknowledgment This work was supported by Grants from Society for Catecholamines and Neurologic Diseases (AN).
References
Fig. 5. Effects of CysC on neuronal differentiation markers expression in A1 cells. The mRNA level of neuronal-cell-type markers in undifferentiated and differentiated A1 cells in normal culture condition are shown in (A), calculated relatively as mRNA copy number of target gene per 106 GAPDH mRNA. To analyze the effects of CysC on neuronal-cell-type markers mRNA expression, undifferentiated (B) and differentiated (C) A1 cells were treated with indicated concentrations of CysC for indicated times, and neuronal-cell-type markers mRNA was analyzed by real time PCR, calculated relatively using 24 h medium-only stimulated sample of 1 experiment as calibrator, and presented as fold induction relative to calibrator sample. The data are presented here as average ± SEM of 3 independent experiments. (D) The regulation of CysC-induced TH mRNA expression was analyzed by treating differentiated A1 cells with CysC (50 nM) in the presence or absence of p38 (SB 203580, 10 M), JNK (SP 600125, 1 M) or MEK (U0126, 10 M) inhibitor, or above mentioned inhibitors alone, and TH mRNA expression was analyzed real time PCR. The TH mRNA level was calculated as fold induction relative to medium-only stimulated condition. The data are presented here as average ± SEM of 3 independent experiments. GAPDH mRNA of corresponding samples was used as loading control. † p < 0.05 vs undifferentiated A1 cells, *p < 0.05 vs corresponding medium-only unstimulated condition, # p < 0.05 vs CysC stimulated condition.
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