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Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress
Toxicology and Applied Pharmacology 241 (2009) 173–181
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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p
Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress Chih-Hsin Tang a,b,⁎, Yung-Cheng Chiu c,d,e, Chun-Fa Huang f, Ya-Wen Chen g, Po-Chun Chen h a
Department of Pharmacology, China Medical University, Taichung, Taiwan Graduate Institute of Basic Medical Science, China Medical University, Taichung Taiwan Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan d Department of Orthopaedics, Taichung Veterans General Hospital, Taichung, Taiwan e Department of Nursing, Hungkuang University, Taichung County, Taiwan f School of Chinese Medicine, China Medical University, Taichung, Taiwan g Department of Physiology, China Medical University, Taichung, Taiwan h Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan b c
a r t i c l e
i n f o
Article history: Received 9 May 2009 Revised 5 August 2009 Accepted 10 August 2009 Available online 18 August 2009 Keywords: Arsenic Osteoporosis Osteoblast GRP78 ER
a b s t r a c t Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis. © 2009 Elsevier Inc. All rights reserved.
Introduction Bone is a complex tissue composed of several cell types which are continuously undergoing a process of renewal and repair termed “bone remodeling.” The two major cell types responsible for bone remodeling are osteoclasts, which resorb bone, and osteoblasts, which form new bone. Bone remodeling is regulated by several systemic hormones (e.g., parathyroid hormone, 1,25-dihydroxyvitamin D3, sex hormones and calcitonin), and local factors (e.g., nitric oxide, prostaglandins, growth factors and cytokines) (van't Hof and Ralston, 2001). When resorption and formation of bone are not coordinated and bone breakdown overrides bone building, osteoporosis results. Since new bone formation is primarily a function of osteoblasts, agents that act by either increasing apoptosis of the osteoblastic lineage or inducing cell death of osteoblasts can enhance osteoporosis (Khosla et al., 2008; Deal, 2009). Arsenic (As) exists ubiquitously in our environment, and various forms of arsenic circulate in soil, water, air and living organisms. ⁎ Corresponding author. Department of Pharmacology, College of Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung, Taiwan. Fax: +886 4 22053764. E-mail address: [email protected] (C.-H. Tang). 0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2009.08.011
Adverse health effects caused by arsenic compounds have long been recognized, including neurotoxicity, liver injury, peripheral vascular disease (known as blackfoot disease), and increased risk of cancer (Chen et al., 1992, 1995; Lai et al., 2005). Significant exposure to arsenic occurs in a variety of workplaces such as lead smelters, glass works, protect cells from use of arsenic-containing agricultural products such as pesticides and herbicides. Even though the use of arsenicals has been reduced, high occurrence of arsenic in drinking water was found in regions of Canada, Japan, India, and Taiwan. Inhalation and ingestion of arsenic compounds have been primarily associated with increased incidences of various cancers such as skin, lung, bladder, and liver cancer (Bates et al., 1992; Chiou et al., 1995). Endoplasmic reticulum (ER) is a central organelle engaged in lipid synthesis, protein folding and maturation. A variety of toxic insults, including hypoxia, failure of protein synthesis, folding, transport or degradation, and Ca2+ overload, can disturb ER function and result in ER stress (Abcouwer et al., 2002; Soboloff and Berger, 2002; Yung et al., 2007). There is increasing evidence that ER stress plays a crucial role in the regulation of apoptosis. It have been reported that ER stress triggers several specific signaling pathways, including ERassociated protein degradation and unfolded protein response (UPR) (Feldman et al., 2005; Moenner et al., 2007). Glucose-regulated
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proteins (GRP) are the most abundant glycoprotein in ER and play a critical role in regulation of ER. Over-expression antisense and ribozyme approaches in tissues culture systems have directly demonstrated that GRP78 and GRP94 can protect cells against death (Little et al., 1994; Lee, 2001). On the other hand, ER plays a direct role in activating a subset of caspase during activation of apoptosis that occurs during ER stress (Liu et al., 2005). Calpains are a family of Ca2+-dependent intracellular cysteine proteases. Ubiquitously expressed calpain-I (μ-calpain) and calpain-II (m-calpain) proteases have been implicated in development of apoptosis. Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts (Seeman, 2002). Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, while the rate of apoptosis is responsible for the regulation of bone formation (Jilka et al., 1998). However, the effect of As on apoptosis of osteoblasts is mostly unknown. To the best of our knowledge, this study is the first to attempt to determine the apoptosis activity of As in osteoblasts. Our data provide evidence that As reduced cell survival and increased cell apoptosis in osteoblasts. Materials and methods Materials. As2O3 (As) was purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for Bax, Bcl-xL, Bak, GRP78, GRP94, calpain I, calpain II, PARP, caspase 3, caspase 7, caspase 9 and caspase 12 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The GRP78 (-132 to + 7) and GRP94 (-363 to + 34) luciferase plasmids were provided from Dr. Kazutoshi Mori (Kyoto University, Kyoto, Japan) (Yoshida et al., 1998). pSV-β-galactosidase vector and luciferase assay kit were purchased from Promega (Madison, MA). Cell culture. The human osteoblast-like cell line MG-63, hFOB, mouse calvaria osteoblasts MC3T3-E1 and mouse bone marrow stromal cell M2-10B4 were purchased from American Type Culture Collection (ATCC). MG-63 and MC3T3-E1 were cultured in MEM supplemented with 10% FBS and antibiotics (100 U/ml of penicillin G and 100 μg/ml of streptomycin). M2-10B4 cells were maintained in RPMI 1640 with 10% FBS, supplemented with 1 mM sodium pyruvate and antibiotics. The conditionally immortalized human fetal osteoblastic cell line, hFOB, was maintained in a 1:1 mixture of phenol-free DMEM/Ham's F12 medium (GIBCO-BRL) containing 10% FBS supplemented with geneticin (300 μg/ml) and antibiotics at 33.5 °C, the permissive temperature for the expression of the large T antigen. All experiments with hFOB cells were carried out at a permissive temperature of 33.5 °C. MTT assay. Cell viability was determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After treatment with As for 2 days, cultures were washed with PBS. MTT (0.5 mg/ml) was then added to each well and the mixture was incubated for 2 h at 37 °C. Culture medium was then replaced with an equal volume of DMSO to dissolve formazan crystals. After the mixture was shaken at room temperature for 10 min, absorbance of each well was determined at 550 nm using a microplate reader (Bio-Tek, Winooski, VT). Quantification of apoptosis using flow cytometry. Apoptosis was assessed using Annexin V, a protein that binds to phosphatidylserine (PS) residues which are exposed on the cell surface of apoptotic cells, as previously described (Dijkers et al., 2002). Cells were treated with vehicle or As for indicated time intervals. After treatment, cells were washed twice with PBS (pH 7.4), and resuspended in staining buffer containing 1 μg/ml PI and 0.025 μg/ml Annexin V–FITC. Doublelabeling was performed at room temperature for 10 min in the dark
before flow cytometric analysis. Cells were immediately analyzed using FACScan and the Cellquest program (Becton Dickinson). Quantitative assessment of apoptotic cells was also assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick endlabeling (TUNEL) method, which examines DNAstrand breaks during apoptosis by using BD ApoAlert™ DNA Fragmentation Assay Kit. Briefly, cells were incubated with As for the indicated times. The cells were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton-X-100 in 0.1% sodium citrate. After being washed, the cells were incubated with the reaction mixture for 60 min at 37 °C. The stained cells were then analyzed using a flow cytometer. Detection of Ca2+ concentrations. Approximately 2 × 105 cells/well of cells in 12-well plates were incubated with As for 4, 8, 12 and 24 hr to detect changes in Ca2+ levels. Cells were harvested, washed twice, re-suspended in Indo 1/AM (3 μg/ml) at 37 °C for 30 min and analyzed using flow cytometry. Determination of the mitochondrial membrane potential. The mitochondrial membrane potential was assessed using a fluorometric probe, DiOC6 (Molecular Probes), with a positive charge of a mitochondria-specific fluorophore, as previously described (Ye et al., 1999). Briefly, cells were plated in 6-well culture dishes. After reaching confluence, cells were treated with vehicle or As. After incubation, cells were stained with DiOC6 (40 nM) for 15 min at 37 °C. Cells were collected, washed twice in PBS, and analyzed with FACScan flow cytometry. Western blot analysis. The cellular lysates were prepared as described previously (Chiu et al., 2007). Proteins were resolved on SDS-PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes. The blots were blocked with 4% BSA for 1 h at room temperature and then probed with rabbit anti-human antibodies against Bax, Bcl-2, GRP78 or GRP94 (1:1000) for 1 h at room temperature. After three washes, the blots were subsequently incubated with donkey anti-rabbit peroxidase conjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized with enhanced chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY). Caspase activity. The assay is based on the ability of the active enzyme to cleave the chromophore from the enzymesubstrate LEHDpNA (for caspase-9) and Ac-DEVD-pNA (for caspase-3). The cell lysates were prepared and incubated with specific anti-caspase-9 and caspase-3 antibodies. Immunocomplexes were incubated with peptide substrate in assay buffer (100 mM NaCl, 50 mM 4-(2hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES), 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), pH 7.4) for 2 h at 37 °C. The release of p-nitroaniline was monitored at 405 nm. Results are represented as the percentage change in activity compared to untreated control. Calpain activity assays. Suc-Leu-Leu-Val-Tyr-AMC is a calpain protease substrate. Quantitation of 7-amino-4-methylcoumarin (AMC) fluorescence permits the monitoring of enzyme hydrolysis of the peptide-AMC conjugate and can be used to measure enzyme activity. Cells were prepared and treated on 24-well Corning/Costar plates. Prior to addition of inhibitors cells were loaded with 40 M Suc-Leu-Leu-Val-Tyr-AMC (Biomol) and treated with As for the indicated time at 37 °C in a humidified 5% CO2 incubator. Proteolysis of the fluorescent probe was monitored using a fluorescent plate reading system (HTS-7000 Plus Series BioAssay, Perkin Elmer) with filter settings of 360 ± 20 nm for excitation and 460 ± 20 nm for emission.
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siRNA transfection. The siRNAs against human calpain I, calpain II and control siRNA were purchased commercially from Santa Cruz Biotechnology. The ON-TARGET smart pool siRNA of GRP78 and scrambled siRNA were obtained from Dharmacon (Lafayette, CO). Cells were transfected with siRNAs (at a final concentration of 100 nM) using Lipofectamine 2000 (Invitrogen Life Technology) according to the manufacturer's instructions.
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Statistics. The values given are means ± S.D. Statistical analysis between two samples was performed using Student's t test. Statistical comparison of more than two groups was performed using one-way analysis of variance (ANOVA) with Bonferroni's post hoc test. In all cases, P b 0.05 was considered as significant. Results
Reporter assay. The osteoblasts were transfected with reporter plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Twenty-four hours after transfection, the cells were treated with As or vehicle for 24 h. Cell extracts were then prepared, and luciferase and β-galactosidase activities were measured (Chiu et al., 2007). Quantitative real time PCR. Quantitative real time PCR (qPCR) analysis was carried out using Taqman® one-step PCR Master Mix (Applied Biosystems, Foster City CA). Total cDNA (100 ng) was added per 25-μl reaction with sequence-specific primers and Taqman® probes. Sequences for all target gene primers and probes were purchased commercially (β-actin was used as an internal control) (Applied Biosystems, CA). Quantitative RT-PCR assays were carried out in triplicate on StepOnePlus sequence detection system. Cycling conditions were 10 min of polymerase activation at 95 °C followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The threshold was set above the non-template control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected (denoted as CT).
As induced cell apoptosis in osteoblasts Arsenic is a naturally occurring toxic metalloid found in the environment in both inorganic and organic forms. Inorganic As is the predominant form of As in surface and underground water reservoirs. Drinkingwater containing high levels of inorganic As and industrial pollution are major sources of inorganic As exposure for millions of people throughout the world. Therefore, inorganic As (As2O3) was chosen to investigate the effect of arsenic in osteoblastic cells. To investigate the potential cell death of As in osteoblastic cells, we first examined the effect of As on cell survival in human osteoblastic cells (hFOB). Treatment of hFOB cells with As-induced cell death in a concentration-dependent manner using an MTT assay (Fig. 1A). Next we investigated the cell death effect of As in other osteblastic cell lines. Fig. 1B, C shows that As-induced cell death in other osteoblastic cell lines (MC3T3-E1 and MG-63). On the other hand, As also enhanced cell death in bone marrow stromal cell M2-10B4 by MTT assay (Fig. 1D). We next investigated whether As induces cell death through an apoptotic mechanism. Annexin V–PI double-labeling was used for the detection of PS
Fig. 1. As induced cell death of cultured osteoblastic cells. Cells were incubated with various concentrations of As for 48 h, and cell viability was examined by MTT assay (n = 5). Statistical analysis between two samples was performed using Student's t test. Results are expressed as the mean ± S.D.
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externalization, a hallmark of early phase of apoptosis. As compared to vehicle-treated hFOB cells, a high proportion of Annexin V+ labeling was detected in hFOB cells treated with As (Fig. 2A, B). Furthermore, As also increased cell apoptosis in MC3T3-E1, MG-63 and M2-10B4 cells (Fig. 2C–E). Next we investigated the effect of As-induced apoptosis using a TUNEL assay. Compared with vehicletreated cells, those treated with As showed significant cell apoptosis (Fig. 3). These data suggest As increases cell apoptosis in cultured osteoblasts.
As for 24 h induced the loss of the mitochondrial membrane potential in a dose-dependent manner. To further explore whether As-induced cell apoptosis by triggering the mitochondrial apoptotic pathway, we measured the change in expression of Bcl-2 family proteins. Treatment of MC3T3E1 cells with As induced an increase in Bax and Bak protein levels (Fig. 4B). In addition, As decreased the expression of Bcl-2, which led to an increase in the proapoptotic/ antiapoptotic Bcl-2 ratio (Fig. 4B). As caused Ca2+ release and ER stress
As caused mitochondrial dysfunction in cultured osteoblasts To determine whether As induced apoptosis is mediated through mitochondrial dysfunction, we determined the mitochondrial membrane potential with a mitochondria-sensitive dye, DiOC6, using flow cytometry. As shown in Fig. 4A, treatment of cells with
Depletion of luminal ER calcium stores is believed to reflect ER stress, which can promote induction of ER stress (Benali-Furet et al., 2005). We assessed the effect of As on the mobilization of Ca2+. When cells were treated with As, Ca2+ levels significantly increased compared with the vehicle-treated group (Fig. 5A). The results
Fig. 2. As induced apoptosis of cultured osteoblastic cells. Cells were treated with vehicle or As for 24 h, the percentage of apoptotic cells was also analyzed by flow cytometric analysis of Annexin V–PI double staining (n = 4). Results are expressed as the mean ± S.D.
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Fig. 3. As increased TUNEL positive cells of cultured osteoblasts. Cells were treated with vehicle or As for 24 h, the TUNEL positive cells were examined by flow cytometry (n = 4). Results are expressed as the mean ± S.D.
demonstrated that As promoted a Ca2+ flux in a time-dependent manner (Fig. 5A). On the other hand, pretreatment of cells with BAPTA (Ca2+ chelator) reduced As-increased cell apoptosis in osteoblasts (Fig. 5B). Therefore, Ca2+ release and ER stress are involved in As-medicated cell death in osteoblastic cells.
caspase-7 (Fig. 6D). Upstream caspase-9 activities increased significantly, as shown by the observation that treatment with As increased caspase-9 activity in osteoblasts (Fig. 6D, E). It has been
As increased GRP78 expression and calpain activity GRP, a glucose-regulated protein, is a major ER chaperone and plays a critical role in regulating ER homeostasis (Yoshida et al., 1998). We examined the effects of As on the expression of GRP78 and GRP94 in osteoblasts. As markedly increased the level of GRP78 in a time-dependent manner, but GRP94 levels were not affected (Fig. 5C). In addition, As also increased mRNA expression of GRP78 but not GRP94 (Fig. 5D). To directly determine GRP78 activation after As treatment, cells were transiently transfected with GRP78 or GRP94 luciferase plasmids as an indicator of GRP activation. As shown in Fig. 5E, As treatment of cells for 24 h increased GRP78 but not GRP94 luciferase activity. To further investigate whether As induced cell apoptosis through GRP78 activation, GRP78 siRNA was used. Transfection of cells with GRP78 siRNA specifically inhibited GRP78 expression (Fig. 5F, upper panel). On the other hand, GRP78 siRNA reduced As-induced cell apoptosis (Fig. 5F, lower panel). Therefore, GRP78 up-regulation is involved in As-induced cell apoptosis in osteoblasts. We next determined whether the activity of calpain (calcium-dependent thiol proteases) would be induced by As in osteoblasts. As shown in Fig. 6A, As increased calpain I and II expression in a time-dependent manner. Furthermore, As also enhanced calpain activity dose dependently (Fig. 6B). Transfection of cells with calpain I or II siRNA markedly reduced As-mediated cell apoptosis (Fig. 6C). Therefore, our data suggests that GRP78 and calpain activation are involved in As-mediated cell death. One of the hallmarks of the apoptotic process is the activation of cysteine proteases, which represent both initiators and executors of cell death. As increased the activation of caspase-3 in osteoblasts (Fig. 6D, E). On the other hand, As also decreased PARP and increased
Fig. 4. As induced mitochondrial dysfunction in cultured osteoblastic cells. (A) Cells were incubated with As for 24 h, mitochondrial membrane potential was examined by flow cytometry (n = 4). (B) MC3T3-E1 cells were incubated with As (10 μM) for different time intervals, the Bax, Bak and Bcl-2 expressions were examined by Western blot analysis. Results are expressed as the mean ± S.D. ⁎, P b 0.05 compared with control.
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Fig. 5. GRP78 activation is involved in As-mediated cell apoptosis in cultured osteoblastic cells. (A) Cells were incubated with As for different time intervals, Ca2+ flux was examined by flow cytometry (n = 4). (B) Cells were pretreated for 30 min with BATA-AM followed by stimulation with As for 24 h, the percentage of apoptotic cells were then analyzed with flow cytometry analysis of TUNEL-stained cells (n = 5). (C, D) MC3T3-E1 cells were incubated with As (10 μM), the protein and mRNA expression of GRP78 and GRP94 were examined by Western blot and qPCR analysis. (E) MC3T3E-E1 cells transiently transfected with GRP78 or GRP94-luciferase plasmids for 24 h, before incubation with As for 24 h. Luciferase activity was measured, and the results normalized to β-galactosidase activity (n = 5). (F) MC3T3-E1 cells were transfected with GRP78 siRNA, GRP78 expression was examined by Western blot analysis (upper panel). MC3T3-E1 cells were transfected with GRP78 or control siRNA, the percentage of apoptotic cells was also analyzed by flow cytometry analysis of Annexin V–PI double staining (lower panel). Results are expressed as the mean ± S.D. ⁎, P b 0.05 compared with control.
reported that calpains promote caspase-12 activation during ER stress-induced apoptosis (Orrenius et al., 2003). As also triggered the expression of cleaved caspase-12, while the magnitude of increase
was proportional to time (Fig. 6D). In addition, transfection of cells with calpain I or calpain II siRNA reduced As-induced caspase-12 activity. Therefore, these results indicate that caspase-12 may
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Fig. 6. Calpain activation is involved in As-mediated cell apoptosis in cultured osteoblastic cells. (A) MC3T3-E1 cells were incubated with As (10 μM) for different time intervals, calpain I and II expressions were examined by Western blot analysis. (B) Cells were incubated with As for 240 min, calpain activity was measured with the fluorescent calpain substrate (n = 4). (C) Cells were transfected with calpain I, calpain II or control siRNA for 24 h, before incubation with As (10 μM) for 24 h, the percentage of apoptotic cells were then analyzed by flow cytometric analysis of TUNEL-stained cells (n = 5). (D) MC3 T3-E1 cells were incubated with As (10 μM) for different time intervals, the caspase-3, -7, -9, -12 and PARP expressions were examined by Western blot analysis. (E) MC3T3-E1 cells were incubated with As (10 μM) for different time intervals, caspase-3 and caspase-9 activities were examined by caspase ELISA kit (n = 5). Results are expressed as the mean ± S.D. ⁎P b 0.05 compared with control; #P b 0.05 compared with As-treated group.
function as a downstream signaling molecule of calpain in the As signaling pathway (Supplementary data, Fig. S1). Discussion Bone is a living tissue that is continuously being remodeled. This process involves an initial, breaking down of the bone by the action of osteoclasts (Rodan and Martin, 2000). In a second step, differentiated osteoblastic cells are recruited to the resorption lacunae where they secrete a variety of proteins such as type I collagen and noncollagenous proteins to generate a new bone matrix (Rodan and Martin, 2000). Decreased bone formation by osteoblasts may lead to the development of osteoporosis, and the rate of apoptosis is responsible for the regulation of bone formation. Here we found that As increased apoptosis of osteoblastic cells. One of the mechanisms underlying As directed cell apoptosis was through mitochondrial dysfunction and ER stress. To examine whether As induces osteoporosis in vivo, ICR mice received 5 and 10 ppm As in drinking water for 12 weeks. We found that As reduced bone mineral density (vehicle: 0.985 ± 0.002; As 5 ppm: 0.913 ± 0.003; As 10 ppm:
0.881 ± 0.003) and bone mineral content (vehicle: 0.901 ± 0.002; As 5 ppm: 0.852 ± 0.003; As 10 ppm: 0.817 ± 0.003). Therefore, exposure to As may increase resulting osteoporosis. In addition, As induced apoptosis in human cultured chondrocytes (Supplementary data, Fig. S3). As also increased apoptosis of chondrocytes also through GRP78, calpain and ER stress pathways (Supplementary data, Fig. S3). Therefore, a similar pathway may be involved in As induced apoptosis in human chondrocytes. Mitochondrial dysfunction has been implicated as being a key mechanism in apoptosis in various cell death paradigms (Susin et al., 1997). Two major events have been noted in apoptosis involving mitochondrial dysfunction. One event is a change in membrane permeability and subsequent loss of membrane potential (Zamzami et al., 1998). The other is the release of apoptotic proteins including cytochrome c from the intermembrane space of mitochondria into the cytosol (Liu et al., 1996). Here, we also found that As reduced mitochondria membrane potential and increased the release of cytochrome c (Supplementary data, Fig. S2). Bcl-2 family proteins have been found to regulate mitochondria-dependent apoptosis with a balance of anti- and pro-apoptotic members arbitrating life-and-
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death decisions (Adams and Cory, 2001). On the other hand, As treatment results in a significant increase of Bax and Bak expression, and a decrease in Bcl-2, suggesting that changes in the ratio of proapoptotic and anti-apoptotic Bcl-2 family proteins might contribute to apoptosis-promotion activity of As. In agreement with these observations, we noted that mitochondrial dysfunction may be involved in As-induced cell apoptosis of cultured osteoblasts. ER is the primary site for protein synthesis, folding, and trafficking (Kaufman, 1999). Under a variety of stressful conditions, the accumulation of unfolded or misfolded proteins in the ER results in the onset of ER stress (Kaufman, 1999). Elevation of cytosolic-calcium levels or depletion of ER calcium stores represents typical responses of cells to various stimuli. Our study found that As induced a number of ER stress markers, including cytosolic-calcium level elevation and caspase-12 activation. Calcium chelator BAPTA-AM blocked As-induced cell apoptosis in cultured osteoblasts. Together, these findings indicate that As induces apoptotic cell death through ER stress in cultured osteoblastic cells. GRP78, a 78-kDa glucose-regulated protein, is a major ER chaperone and plays a critical role in regulating ER. GRP78 upregulation is believed to increase the capacity to buffer stressful insults initiating from the ER. We demonstrated that As increased GRP78 but not GRP94 expression by Western blot and reporter assay. Furthermore, GRP78 siRNA antagonized the As-mediated potentiation of cell apoptosis, suggesting that GRP78 expression is an obligatory event in As-induced cell death in these cells. Calpains and caspases are two families of cysteine proteases that are involved in regulating pathological cell death (Tan et al., 2006). These proteases share several death-related substrates including caspases themselves, cytoskeletal proteins, Bax, and Bid (Fettucciari et al., 2006). Calpain-mediated proteolysis proceeds in a limited manner but does not require a specific amino acid residue like that of caspases. Although both calpain and caspase have been proposed to play important roles in regulating pathological cell death, the interactions of these two families of proteases under pathological conditions are not clear. In
the present study, we found As increased calpain I and II expression. Treatment of cells with As also increased calpain activity. Knockdown approaches have contributed significantly to our knowledge of calpain's biological properties, particularly with respect to its specific function on cell apoptosis: it is possible that caspase-12 is downstream from calpain in mediating As-induced osteoblastic apoptosis. Osteoblastic apoptosis has been observed following estrogen deprivation in both humans and mice (Kousteni et al., 2002), and the degree of osteoblastic apoptosis is an important determinant of bone formation in postmenopausal osteoporosis (Manolagas, 2000). Apoptosis may be initiated by death receptors, such as Fas or the TNF receptor, which recruit and activate caspases and induce apoptosis upon binding of their cognate ligands (Hock et al., 2001). Previous reports have demonstrated that As increases the risk of neurotoxicity, liver injury, peripheral vascular disease (known as blackfoot disease), and cancer. However, the effect of As on apoptosis of osteoblasts is not well understood. Thus, the results of this study provide evidence for apoptosis caused by As in osteoblatic cells, and more importantly, the molecular basis for its effect. The present study has demonstrated that As causes apoptosis in osteoblasts. As-induced apoptosis in osteoblastic cells through mitochondria dysfunction leads to activation of caspase-9 and involves a caspase-3/-7-mediated mechanism (Fig. 7). As also induced cell death mediated by increasing ER stress, GPR78 activation, and Ca2+ release, which subsequently triggers calpain activity, resulting in apoptosis (Fig. 7). Acknowledgments This work was supported by grants from the National Science Council of Taiwan (96-2320-B-039-028-MY3; 98-2314-B-075A-001MY2) and China Medical University (CMU97-180). We thank Dr. Kazutoshi Mori for providing the GRP78 and GRP94 luciferase plasmids. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.taap.2009.08.011. References
Fig. 7. Schematic diagram of the pathways involved in As-induced cell apoptosis in cultured osteoblasts. Proposed models showing how As affects various biochemical processes and events in osteoblastic cells, resulting in apoptotic cell death, are illustrated in this schematic diagram.
Abcouwer, S.F., Marjon, P.L., Loper, R.K., Vander Jagt, D.L., 2002. Response of VEGF expression to amino acid deprivation and inducers of endoplasmic reticulum stress. Invest Ophthalmol. Vis. Sci. 43, 2791–2798. Adams, J.M., Cory, S., 2001. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem. Sci. 26, 61–66. Bates, M.N., Smith, A.H., Hopenhayn-Rich, C., 1992. Arsenic ingestion and internal cancers: a review. Am. J. Epidemiol. 135, 462–476. Benali-Furet, N.L., Chami, M., Houel, L., De Giorgi, F., Vernejoul, F., Lagorce, D., Buscail, L., Bartenschlager, R., Ichas, F., Rizzuto, R., Paterlini-Brechot, P., 2005. Hepatitis C virus core triggers apoptosis in liver cells by inducing ER stress and ER calcium depletion. Oncogene 24, 4921–4933. Chen, C.J., Chen, C.W., Wu, M.M., Kuo, T.L., 1992. Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br. J. Cancer 66, 888–892. Chen, C.J., Hsueh, Y.M., Lai, M.S., Shyu, M.P., Chen, S.Y., Wu, M.M., Kuo, T.L., Tai, T.Y., 1995. Increased prevalence of hypertension and long-term arsenic exposure. Hypertension 25, 53–60. Chiou, H.Y., Hsueh, Y.M., Liaw, K.F., Horng, S.F., Chiang, M.H., Pu, Y.S., Lin, J.S., Huang, C.H., Chen, C.J., 1995. Incidence of internal cancers and ingested inorganic arsenic: a seven-year follow-up study in Taiwan. Cancer Res. 55, 1296–1300. Chiu, Y.C., Yang, R.S., Hsieh, K.H., Fong, Y.C., Way, T.D., Lee, T.S., Wu, H.C., Fu, W.M., Tang, C.H., 2007. Stromal cell-derived factor-1 induces matrix metalloprotease-13 expression in human chondrocytes. Mol. Pharmacol. 72, 695–703. Deal, C., 2009. Potential new drug targets for osteoporosis. Nat. Clin Pract. Rheumatol. 5, 20–27. Dijkers, P.F., Birkenkamp, K.U., Lam, E.W., Thomas, N.S., Lammers, J.W., Koenderman, L., Coffer, P.J., 2002. FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity. J. Cell Biol. 156, 531–542. Feldman, D.E., Chauhan, V., Koong, A.C., 2005. The unfolded protein response: a novel component of the hypoxic stress response in tumors. Mol. Cancer Res. 3, 597–605. Fettucciari, K., Fetriconi, I., Mannucci, R., Nicoletti, I., Bartoli, A., Coaccioli, S., Marconi, P., 2006. Group B Streptococcus induces macrophage apoptosis by calpain activation. J. Immunol. 176, 7542–7556.
C.-H. Tang et al. / Toxicology and Applied Pharmacology 241 (2009) 173–181 Hock, J.M., Krishnan, V., Onyia, J.E., Bidwell, J.P., Milas, J., Stanislaus, D., 2001. Osteoblast apoptosis and bone turnover. J. Bone Miner. Res. 16, 975–984. Jilka, R.L., Weinstein, R.S., Bellido, T., Parfitt, A.M., Manolagas, S.C., 1998. Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J. Bone Miner Res. 13, 793–802. Kaufman, R.J., 1999. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 13, 1211–1233. Khosla, S., Amin, S., Orwoll, E., 2008. Osteoporosis in men. Endocr. Rev. 29, 441–464. Kousteni, S., Chen, J.R., Bellido, T., Han, L., Ali, A.A., O'Brien, C.A., Plotkin, L., Fu, Q., Mancino, A.T., Wen, Y., Vertino, A.M., Powers, C.C., Stewart, S.A., Ebert, R., Parfitt, A.M., Weinstein, R.S., Jilka, R.L., Manolagas, S.C., 2002. Reversal of bone loss in mice by nongenotropic signaling of sex steroids. Science 298, 843–846. Lai, M.W., Boyer, E.W., Kleinman, M.E., Rodig, N.M., Ewald, M.B., 2005. Acute arsenic poisoning in two siblings. Pediatrics 116, 249–257. Lee, A.S., 2001. The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem. Sci. 26, 504–510. Little, E., Ramakrishnan, M., Roy, B., Gazit, G., Lee, A.S., 1994. The glucose-regulated proteins (GRP78 and GRP94): functions, gene regulation, and applications. Crit. Rev. Eukaryot. Gene Expr. 4, 1–18. Liu, S., Wang, H., Yang, Z., Kon, T., Zhu, J., Cao, Y., Li, F., Kirkpatrick, J., Nicchitta, C.V., Li, C.Y., 2005. Enhancement of cancer radiation therapy by use of adenovirusmediated secretable glucose-regulated protein 94/gp96 expression. Cancer Res. 65, 9126–9131. Liu, X., Kim, C.N., Yang, J., Jemmerson, R., Wang, X., 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147–157. Manolagas, S.C., 2000. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev. 21, 115–137. Moenner, M., Pluquet, O., Bouchecareilh, M., Chevet, E., 2007. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res. 67, 10631–10634.
181
Orrenius, S., Zhivotovsky, B., Nicotera, P., 2003. Regulation of cell death: the calciumapoptosis link. Nat Rev. Mol. Cell Biol. 4, 552–565. Rodan, G.A., Martin, T.J., 2000. Therapeutic approaches to bone diseases. Science 289, 1508–1514. Seeman, E., 2002. Pathogenesis of bone fragility in women and men. Lancet 359, 1841–1850. Soboloff, J., Berger, S.A., 2002. Sustained ER Ca2+ depletion suppresses protein synthesis and induces activation-enhanced cell death in mast cells. J. Biol. Chem. 277, 13812–13820. Susin, S.A., Zamzami, N., Castedo, M., Daugas, E., Wang, H.G., Geley, S., Fassy, F., Reed, J.C., Kroemer, G., 1997. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramideinduced apoptosis. J. Exp. Med. 186, 25–37. Tan, Y., Dourdin, N., Wu, C., De Veyra, T., Elce, J.S., Greer, P.A., 2006. Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stressinduced apoptosis. J. Biol. Chem. 281, 16016–16024. van't Hof, R.J., Ralston, S.H., 2001. Nitric oxide and bone. Immunology 103, 255–261. Ye, J., Wang, S., Leonard, S.S., Sun, Y., Butterworth, L., Antonini, J., Ding, M., Rojanasakul, Y., Vallyathan, V., Castranova, V., Shi, X., 1999. Role of reactive oxygen species and p53 in chromium(VI)-induced apoptosis. J. Biol. Chem. 274, 34974–34980. Yoshida, H., Haze, K., Yanagi, H., Yura, T., Mori, K., 1998. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors. J. Biol. Chem. 273, 33741–33749. Yung, H.W., Korolchuk, S., Tolkovsky, A.M., Charnock-Jones, D.S., Burton, G.J., 2007. Endoplasmic reticulum stress exacerbates ischemia-reperfusion-induced apoptosis through attenuation of Akt protein synthesis in human choriocarcinoma cells. FASEB J. 21, 872–884. Zamzami, N., Brenner, C., Marzo, I., Susin, S.A., Kroemer, G., 1998. Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins. Oncogene 16, 2265–2282.
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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p
Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress Chih-Hsin Tang a,b,⁎, Yung-Cheng Chiu c,d,e, Chun-Fa Huang f, Ya-Wen Chen g, Po-Chun Chen h a
Department of Pharmacology, China Medical University, Taichung, Taiwan Graduate Institute of Basic Medical Science, China Medical University, Taichung Taiwan Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan d Department of Orthopaedics, Taichung Veterans General Hospital, Taichung, Taiwan e Department of Nursing, Hungkuang University, Taichung County, Taiwan f School of Chinese Medicine, China Medical University, Taichung, Taiwan g Department of Physiology, China Medical University, Taichung, Taiwan h Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan b c
a r t i c l e
i n f o
Article history: Received 9 May 2009 Revised 5 August 2009 Accepted 10 August 2009 Available online 18 August 2009 Keywords: Arsenic Osteoporosis Osteoblast GRP78 ER
a b s t r a c t Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis. © 2009 Elsevier Inc. All rights reserved.
Introduction Bone is a complex tissue composed of several cell types which are continuously undergoing a process of renewal and repair termed “bone remodeling.” The two major cell types responsible for bone remodeling are osteoclasts, which resorb bone, and osteoblasts, which form new bone. Bone remodeling is regulated by several systemic hormones (e.g., parathyroid hormone, 1,25-dihydroxyvitamin D3, sex hormones and calcitonin), and local factors (e.g., nitric oxide, prostaglandins, growth factors and cytokines) (van't Hof and Ralston, 2001). When resorption and formation of bone are not coordinated and bone breakdown overrides bone building, osteoporosis results. Since new bone formation is primarily a function of osteoblasts, agents that act by either increasing apoptosis of the osteoblastic lineage or inducing cell death of osteoblasts can enhance osteoporosis (Khosla et al., 2008; Deal, 2009). Arsenic (As) exists ubiquitously in our environment, and various forms of arsenic circulate in soil, water, air and living organisms. ⁎ Corresponding author. Department of Pharmacology, College of Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung, Taiwan. Fax: +886 4 22053764. E-mail address: [email protected] (C.-H. Tang). 0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2009.08.011
Adverse health effects caused by arsenic compounds have long been recognized, including neurotoxicity, liver injury, peripheral vascular disease (known as blackfoot disease), and increased risk of cancer (Chen et al., 1992, 1995; Lai et al., 2005). Significant exposure to arsenic occurs in a variety of workplaces such as lead smelters, glass works, protect cells from use of arsenic-containing agricultural products such as pesticides and herbicides. Even though the use of arsenicals has been reduced, high occurrence of arsenic in drinking water was found in regions of Canada, Japan, India, and Taiwan. Inhalation and ingestion of arsenic compounds have been primarily associated with increased incidences of various cancers such as skin, lung, bladder, and liver cancer (Bates et al., 1992; Chiou et al., 1995). Endoplasmic reticulum (ER) is a central organelle engaged in lipid synthesis, protein folding and maturation. A variety of toxic insults, including hypoxia, failure of protein synthesis, folding, transport or degradation, and Ca2+ overload, can disturb ER function and result in ER stress (Abcouwer et al., 2002; Soboloff and Berger, 2002; Yung et al., 2007). There is increasing evidence that ER stress plays a crucial role in the regulation of apoptosis. It have been reported that ER stress triggers several specific signaling pathways, including ERassociated protein degradation and unfolded protein response (UPR) (Feldman et al., 2005; Moenner et al., 2007). Glucose-regulated
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proteins (GRP) are the most abundant glycoprotein in ER and play a critical role in regulation of ER. Over-expression antisense and ribozyme approaches in tissues culture systems have directly demonstrated that GRP78 and GRP94 can protect cells against death (Little et al., 1994; Lee, 2001). On the other hand, ER plays a direct role in activating a subset of caspase during activation of apoptosis that occurs during ER stress (Liu et al., 2005). Calpains are a family of Ca2+-dependent intracellular cysteine proteases. Ubiquitously expressed calpain-I (μ-calpain) and calpain-II (m-calpain) proteases have been implicated in development of apoptosis. Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts (Seeman, 2002). Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, while the rate of apoptosis is responsible for the regulation of bone formation (Jilka et al., 1998). However, the effect of As on apoptosis of osteoblasts is mostly unknown. To the best of our knowledge, this study is the first to attempt to determine the apoptosis activity of As in osteoblasts. Our data provide evidence that As reduced cell survival and increased cell apoptosis in osteoblasts. Materials and methods Materials. As2O3 (As) was purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for Bax, Bcl-xL, Bak, GRP78, GRP94, calpain I, calpain II, PARP, caspase 3, caspase 7, caspase 9 and caspase 12 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The GRP78 (-132 to + 7) and GRP94 (-363 to + 34) luciferase plasmids were provided from Dr. Kazutoshi Mori (Kyoto University, Kyoto, Japan) (Yoshida et al., 1998). pSV-β-galactosidase vector and luciferase assay kit were purchased from Promega (Madison, MA). Cell culture. The human osteoblast-like cell line MG-63, hFOB, mouse calvaria osteoblasts MC3T3-E1 and mouse bone marrow stromal cell M2-10B4 were purchased from American Type Culture Collection (ATCC). MG-63 and MC3T3-E1 were cultured in MEM supplemented with 10% FBS and antibiotics (100 U/ml of penicillin G and 100 μg/ml of streptomycin). M2-10B4 cells were maintained in RPMI 1640 with 10% FBS, supplemented with 1 mM sodium pyruvate and antibiotics. The conditionally immortalized human fetal osteoblastic cell line, hFOB, was maintained in a 1:1 mixture of phenol-free DMEM/Ham's F12 medium (GIBCO-BRL) containing 10% FBS supplemented with geneticin (300 μg/ml) and antibiotics at 33.5 °C, the permissive temperature for the expression of the large T antigen. All experiments with hFOB cells were carried out at a permissive temperature of 33.5 °C. MTT assay. Cell viability was determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After treatment with As for 2 days, cultures were washed with PBS. MTT (0.5 mg/ml) was then added to each well and the mixture was incubated for 2 h at 37 °C. Culture medium was then replaced with an equal volume of DMSO to dissolve formazan crystals. After the mixture was shaken at room temperature for 10 min, absorbance of each well was determined at 550 nm using a microplate reader (Bio-Tek, Winooski, VT). Quantification of apoptosis using flow cytometry. Apoptosis was assessed using Annexin V, a protein that binds to phosphatidylserine (PS) residues which are exposed on the cell surface of apoptotic cells, as previously described (Dijkers et al., 2002). Cells were treated with vehicle or As for indicated time intervals. After treatment, cells were washed twice with PBS (pH 7.4), and resuspended in staining buffer containing 1 μg/ml PI and 0.025 μg/ml Annexin V–FITC. Doublelabeling was performed at room temperature for 10 min in the dark
before flow cytometric analysis. Cells were immediately analyzed using FACScan and the Cellquest program (Becton Dickinson). Quantitative assessment of apoptotic cells was also assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick endlabeling (TUNEL) method, which examines DNAstrand breaks during apoptosis by using BD ApoAlert™ DNA Fragmentation Assay Kit. Briefly, cells were incubated with As for the indicated times. The cells were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton-X-100 in 0.1% sodium citrate. After being washed, the cells were incubated with the reaction mixture for 60 min at 37 °C. The stained cells were then analyzed using a flow cytometer. Detection of Ca2+ concentrations. Approximately 2 × 105 cells/well of cells in 12-well plates were incubated with As for 4, 8, 12 and 24 hr to detect changes in Ca2+ levels. Cells were harvested, washed twice, re-suspended in Indo 1/AM (3 μg/ml) at 37 °C for 30 min and analyzed using flow cytometry. Determination of the mitochondrial membrane potential. The mitochondrial membrane potential was assessed using a fluorometric probe, DiOC6 (Molecular Probes), with a positive charge of a mitochondria-specific fluorophore, as previously described (Ye et al., 1999). Briefly, cells were plated in 6-well culture dishes. After reaching confluence, cells were treated with vehicle or As. After incubation, cells were stained with DiOC6 (40 nM) for 15 min at 37 °C. Cells were collected, washed twice in PBS, and analyzed with FACScan flow cytometry. Western blot analysis. The cellular lysates were prepared as described previously (Chiu et al., 2007). Proteins were resolved on SDS-PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes. The blots were blocked with 4% BSA for 1 h at room temperature and then probed with rabbit anti-human antibodies against Bax, Bcl-2, GRP78 or GRP94 (1:1000) for 1 h at room temperature. After three washes, the blots were subsequently incubated with donkey anti-rabbit peroxidase conjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized with enhanced chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY). Caspase activity. The assay is based on the ability of the active enzyme to cleave the chromophore from the enzymesubstrate LEHDpNA (for caspase-9) and Ac-DEVD-pNA (for caspase-3). The cell lysates were prepared and incubated with specific anti-caspase-9 and caspase-3 antibodies. Immunocomplexes were incubated with peptide substrate in assay buffer (100 mM NaCl, 50 mM 4-(2hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES), 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), pH 7.4) for 2 h at 37 °C. The release of p-nitroaniline was monitored at 405 nm. Results are represented as the percentage change in activity compared to untreated control. Calpain activity assays. Suc-Leu-Leu-Val-Tyr-AMC is a calpain protease substrate. Quantitation of 7-amino-4-methylcoumarin (AMC) fluorescence permits the monitoring of enzyme hydrolysis of the peptide-AMC conjugate and can be used to measure enzyme activity. Cells were prepared and treated on 24-well Corning/Costar plates. Prior to addition of inhibitors cells were loaded with 40 M Suc-Leu-Leu-Val-Tyr-AMC (Biomol) and treated with As for the indicated time at 37 °C in a humidified 5% CO2 incubator. Proteolysis of the fluorescent probe was monitored using a fluorescent plate reading system (HTS-7000 Plus Series BioAssay, Perkin Elmer) with filter settings of 360 ± 20 nm for excitation and 460 ± 20 nm for emission.
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siRNA transfection. The siRNAs against human calpain I, calpain II and control siRNA were purchased commercially from Santa Cruz Biotechnology. The ON-TARGET smart pool siRNA of GRP78 and scrambled siRNA were obtained from Dharmacon (Lafayette, CO). Cells were transfected with siRNAs (at a final concentration of 100 nM) using Lipofectamine 2000 (Invitrogen Life Technology) according to the manufacturer's instructions.
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Statistics. The values given are means ± S.D. Statistical analysis between two samples was performed using Student's t test. Statistical comparison of more than two groups was performed using one-way analysis of variance (ANOVA) with Bonferroni's post hoc test. In all cases, P b 0.05 was considered as significant. Results
Reporter assay. The osteoblasts were transfected with reporter plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Twenty-four hours after transfection, the cells were treated with As or vehicle for 24 h. Cell extracts were then prepared, and luciferase and β-galactosidase activities were measured (Chiu et al., 2007). Quantitative real time PCR. Quantitative real time PCR (qPCR) analysis was carried out using Taqman® one-step PCR Master Mix (Applied Biosystems, Foster City CA). Total cDNA (100 ng) was added per 25-μl reaction with sequence-specific primers and Taqman® probes. Sequences for all target gene primers and probes were purchased commercially (β-actin was used as an internal control) (Applied Biosystems, CA). Quantitative RT-PCR assays were carried out in triplicate on StepOnePlus sequence detection system. Cycling conditions were 10 min of polymerase activation at 95 °C followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The threshold was set above the non-template control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected (denoted as CT).
As induced cell apoptosis in osteoblasts Arsenic is a naturally occurring toxic metalloid found in the environment in both inorganic and organic forms. Inorganic As is the predominant form of As in surface and underground water reservoirs. Drinkingwater containing high levels of inorganic As and industrial pollution are major sources of inorganic As exposure for millions of people throughout the world. Therefore, inorganic As (As2O3) was chosen to investigate the effect of arsenic in osteoblastic cells. To investigate the potential cell death of As in osteoblastic cells, we first examined the effect of As on cell survival in human osteoblastic cells (hFOB). Treatment of hFOB cells with As-induced cell death in a concentration-dependent manner using an MTT assay (Fig. 1A). Next we investigated the cell death effect of As in other osteblastic cell lines. Fig. 1B, C shows that As-induced cell death in other osteoblastic cell lines (MC3T3-E1 and MG-63). On the other hand, As also enhanced cell death in bone marrow stromal cell M2-10B4 by MTT assay (Fig. 1D). We next investigated whether As induces cell death through an apoptotic mechanism. Annexin V–PI double-labeling was used for the detection of PS
Fig. 1. As induced cell death of cultured osteoblastic cells. Cells were incubated with various concentrations of As for 48 h, and cell viability was examined by MTT assay (n = 5). Statistical analysis between two samples was performed using Student's t test. Results are expressed as the mean ± S.D.
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externalization, a hallmark of early phase of apoptosis. As compared to vehicle-treated hFOB cells, a high proportion of Annexin V+ labeling was detected in hFOB cells treated with As (Fig. 2A, B). Furthermore, As also increased cell apoptosis in MC3T3-E1, MG-63 and M2-10B4 cells (Fig. 2C–E). Next we investigated the effect of As-induced apoptosis using a TUNEL assay. Compared with vehicletreated cells, those treated with As showed significant cell apoptosis (Fig. 3). These data suggest As increases cell apoptosis in cultured osteoblasts.
As for 24 h induced the loss of the mitochondrial membrane potential in a dose-dependent manner. To further explore whether As-induced cell apoptosis by triggering the mitochondrial apoptotic pathway, we measured the change in expression of Bcl-2 family proteins. Treatment of MC3T3E1 cells with As induced an increase in Bax and Bak protein levels (Fig. 4B). In addition, As decreased the expression of Bcl-2, which led to an increase in the proapoptotic/ antiapoptotic Bcl-2 ratio (Fig. 4B). As caused Ca2+ release and ER stress
As caused mitochondrial dysfunction in cultured osteoblasts To determine whether As induced apoptosis is mediated through mitochondrial dysfunction, we determined the mitochondrial membrane potential with a mitochondria-sensitive dye, DiOC6, using flow cytometry. As shown in Fig. 4A, treatment of cells with
Depletion of luminal ER calcium stores is believed to reflect ER stress, which can promote induction of ER stress (Benali-Furet et al., 2005). We assessed the effect of As on the mobilization of Ca2+. When cells were treated with As, Ca2+ levels significantly increased compared with the vehicle-treated group (Fig. 5A). The results
Fig. 2. As induced apoptosis of cultured osteoblastic cells. Cells were treated with vehicle or As for 24 h, the percentage of apoptotic cells was also analyzed by flow cytometric analysis of Annexin V–PI double staining (n = 4). Results are expressed as the mean ± S.D.
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Fig. 3. As increased TUNEL positive cells of cultured osteoblasts. Cells were treated with vehicle or As for 24 h, the TUNEL positive cells were examined by flow cytometry (n = 4). Results are expressed as the mean ± S.D.
demonstrated that As promoted a Ca2+ flux in a time-dependent manner (Fig. 5A). On the other hand, pretreatment of cells with BAPTA (Ca2+ chelator) reduced As-increased cell apoptosis in osteoblasts (Fig. 5B). Therefore, Ca2+ release and ER stress are involved in As-medicated cell death in osteoblastic cells.
caspase-7 (Fig. 6D). Upstream caspase-9 activities increased significantly, as shown by the observation that treatment with As increased caspase-9 activity in osteoblasts (Fig. 6D, E). It has been
As increased GRP78 expression and calpain activity GRP, a glucose-regulated protein, is a major ER chaperone and plays a critical role in regulating ER homeostasis (Yoshida et al., 1998). We examined the effects of As on the expression of GRP78 and GRP94 in osteoblasts. As markedly increased the level of GRP78 in a time-dependent manner, but GRP94 levels were not affected (Fig. 5C). In addition, As also increased mRNA expression of GRP78 but not GRP94 (Fig. 5D). To directly determine GRP78 activation after As treatment, cells were transiently transfected with GRP78 or GRP94 luciferase plasmids as an indicator of GRP activation. As shown in Fig. 5E, As treatment of cells for 24 h increased GRP78 but not GRP94 luciferase activity. To further investigate whether As induced cell apoptosis through GRP78 activation, GRP78 siRNA was used. Transfection of cells with GRP78 siRNA specifically inhibited GRP78 expression (Fig. 5F, upper panel). On the other hand, GRP78 siRNA reduced As-induced cell apoptosis (Fig. 5F, lower panel). Therefore, GRP78 up-regulation is involved in As-induced cell apoptosis in osteoblasts. We next determined whether the activity of calpain (calcium-dependent thiol proteases) would be induced by As in osteoblasts. As shown in Fig. 6A, As increased calpain I and II expression in a time-dependent manner. Furthermore, As also enhanced calpain activity dose dependently (Fig. 6B). Transfection of cells with calpain I or II siRNA markedly reduced As-mediated cell apoptosis (Fig. 6C). Therefore, our data suggests that GRP78 and calpain activation are involved in As-mediated cell death. One of the hallmarks of the apoptotic process is the activation of cysteine proteases, which represent both initiators and executors of cell death. As increased the activation of caspase-3 in osteoblasts (Fig. 6D, E). On the other hand, As also decreased PARP and increased
Fig. 4. As induced mitochondrial dysfunction in cultured osteoblastic cells. (A) Cells were incubated with As for 24 h, mitochondrial membrane potential was examined by flow cytometry (n = 4). (B) MC3T3-E1 cells were incubated with As (10 μM) for different time intervals, the Bax, Bak and Bcl-2 expressions were examined by Western blot analysis. Results are expressed as the mean ± S.D. ⁎, P b 0.05 compared with control.
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Fig. 5. GRP78 activation is involved in As-mediated cell apoptosis in cultured osteoblastic cells. (A) Cells were incubated with As for different time intervals, Ca2+ flux was examined by flow cytometry (n = 4). (B) Cells were pretreated for 30 min with BATA-AM followed by stimulation with As for 24 h, the percentage of apoptotic cells were then analyzed with flow cytometry analysis of TUNEL-stained cells (n = 5). (C, D) MC3T3-E1 cells were incubated with As (10 μM), the protein and mRNA expression of GRP78 and GRP94 were examined by Western blot and qPCR analysis. (E) MC3T3E-E1 cells transiently transfected with GRP78 or GRP94-luciferase plasmids for 24 h, before incubation with As for 24 h. Luciferase activity was measured, and the results normalized to β-galactosidase activity (n = 5). (F) MC3T3-E1 cells were transfected with GRP78 siRNA, GRP78 expression was examined by Western blot analysis (upper panel). MC3T3-E1 cells were transfected with GRP78 or control siRNA, the percentage of apoptotic cells was also analyzed by flow cytometry analysis of Annexin V–PI double staining (lower panel). Results are expressed as the mean ± S.D. ⁎, P b 0.05 compared with control.
reported that calpains promote caspase-12 activation during ER stress-induced apoptosis (Orrenius et al., 2003). As also triggered the expression of cleaved caspase-12, while the magnitude of increase
was proportional to time (Fig. 6D). In addition, transfection of cells with calpain I or calpain II siRNA reduced As-induced caspase-12 activity. Therefore, these results indicate that caspase-12 may
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Fig. 6. Calpain activation is involved in As-mediated cell apoptosis in cultured osteoblastic cells. (A) MC3T3-E1 cells were incubated with As (10 μM) for different time intervals, calpain I and II expressions were examined by Western blot analysis. (B) Cells were incubated with As for 240 min, calpain activity was measured with the fluorescent calpain substrate (n = 4). (C) Cells were transfected with calpain I, calpain II or control siRNA for 24 h, before incubation with As (10 μM) for 24 h, the percentage of apoptotic cells were then analyzed by flow cytometric analysis of TUNEL-stained cells (n = 5). (D) MC3 T3-E1 cells were incubated with As (10 μM) for different time intervals, the caspase-3, -7, -9, -12 and PARP expressions were examined by Western blot analysis. (E) MC3T3-E1 cells were incubated with As (10 μM) for different time intervals, caspase-3 and caspase-9 activities were examined by caspase ELISA kit (n = 5). Results are expressed as the mean ± S.D. ⁎P b 0.05 compared with control; #P b 0.05 compared with As-treated group.
function as a downstream signaling molecule of calpain in the As signaling pathway (Supplementary data, Fig. S1). Discussion Bone is a living tissue that is continuously being remodeled. This process involves an initial, breaking down of the bone by the action of osteoclasts (Rodan and Martin, 2000). In a second step, differentiated osteoblastic cells are recruited to the resorption lacunae where they secrete a variety of proteins such as type I collagen and noncollagenous proteins to generate a new bone matrix (Rodan and Martin, 2000). Decreased bone formation by osteoblasts may lead to the development of osteoporosis, and the rate of apoptosis is responsible for the regulation of bone formation. Here we found that As increased apoptosis of osteoblastic cells. One of the mechanisms underlying As directed cell apoptosis was through mitochondrial dysfunction and ER stress. To examine whether As induces osteoporosis in vivo, ICR mice received 5 and 10 ppm As in drinking water for 12 weeks. We found that As reduced bone mineral density (vehicle: 0.985 ± 0.002; As 5 ppm: 0.913 ± 0.003; As 10 ppm:
0.881 ± 0.003) and bone mineral content (vehicle: 0.901 ± 0.002; As 5 ppm: 0.852 ± 0.003; As 10 ppm: 0.817 ± 0.003). Therefore, exposure to As may increase resulting osteoporosis. In addition, As induced apoptosis in human cultured chondrocytes (Supplementary data, Fig. S3). As also increased apoptosis of chondrocytes also through GRP78, calpain and ER stress pathways (Supplementary data, Fig. S3). Therefore, a similar pathway may be involved in As induced apoptosis in human chondrocytes. Mitochondrial dysfunction has been implicated as being a key mechanism in apoptosis in various cell death paradigms (Susin et al., 1997). Two major events have been noted in apoptosis involving mitochondrial dysfunction. One event is a change in membrane permeability and subsequent loss of membrane potential (Zamzami et al., 1998). The other is the release of apoptotic proteins including cytochrome c from the intermembrane space of mitochondria into the cytosol (Liu et al., 1996). Here, we also found that As reduced mitochondria membrane potential and increased the release of cytochrome c (Supplementary data, Fig. S2). Bcl-2 family proteins have been found to regulate mitochondria-dependent apoptosis with a balance of anti- and pro-apoptotic members arbitrating life-and-
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death decisions (Adams and Cory, 2001). On the other hand, As treatment results in a significant increase of Bax and Bak expression, and a decrease in Bcl-2, suggesting that changes in the ratio of proapoptotic and anti-apoptotic Bcl-2 family proteins might contribute to apoptosis-promotion activity of As. In agreement with these observations, we noted that mitochondrial dysfunction may be involved in As-induced cell apoptosis of cultured osteoblasts. ER is the primary site for protein synthesis, folding, and trafficking (Kaufman, 1999). Under a variety of stressful conditions, the accumulation of unfolded or misfolded proteins in the ER results in the onset of ER stress (Kaufman, 1999). Elevation of cytosolic-calcium levels or depletion of ER calcium stores represents typical responses of cells to various stimuli. Our study found that As induced a number of ER stress markers, including cytosolic-calcium level elevation and caspase-12 activation. Calcium chelator BAPTA-AM blocked As-induced cell apoptosis in cultured osteoblasts. Together, these findings indicate that As induces apoptotic cell death through ER stress in cultured osteoblastic cells. GRP78, a 78-kDa glucose-regulated protein, is a major ER chaperone and plays a critical role in regulating ER. GRP78 upregulation is believed to increase the capacity to buffer stressful insults initiating from the ER. We demonstrated that As increased GRP78 but not GRP94 expression by Western blot and reporter assay. Furthermore, GRP78 siRNA antagonized the As-mediated potentiation of cell apoptosis, suggesting that GRP78 expression is an obligatory event in As-induced cell death in these cells. Calpains and caspases are two families of cysteine proteases that are involved in regulating pathological cell death (Tan et al., 2006). These proteases share several death-related substrates including caspases themselves, cytoskeletal proteins, Bax, and Bid (Fettucciari et al., 2006). Calpain-mediated proteolysis proceeds in a limited manner but does not require a specific amino acid residue like that of caspases. Although both calpain and caspase have been proposed to play important roles in regulating pathological cell death, the interactions of these two families of proteases under pathological conditions are not clear. In
the present study, we found As increased calpain I and II expression. Treatment of cells with As also increased calpain activity. Knockdown approaches have contributed significantly to our knowledge of calpain's biological properties, particularly with respect to its specific function on cell apoptosis: it is possible that caspase-12 is downstream from calpain in mediating As-induced osteoblastic apoptosis. Osteoblastic apoptosis has been observed following estrogen deprivation in both humans and mice (Kousteni et al., 2002), and the degree of osteoblastic apoptosis is an important determinant of bone formation in postmenopausal osteoporosis (Manolagas, 2000). Apoptosis may be initiated by death receptors, such as Fas or the TNF receptor, which recruit and activate caspases and induce apoptosis upon binding of their cognate ligands (Hock et al., 2001). Previous reports have demonstrated that As increases the risk of neurotoxicity, liver injury, peripheral vascular disease (known as blackfoot disease), and cancer. However, the effect of As on apoptosis of osteoblasts is not well understood. Thus, the results of this study provide evidence for apoptosis caused by As in osteoblatic cells, and more importantly, the molecular basis for its effect. The present study has demonstrated that As causes apoptosis in osteoblasts. As-induced apoptosis in osteoblastic cells through mitochondria dysfunction leads to activation of caspase-9 and involves a caspase-3/-7-mediated mechanism (Fig. 7). As also induced cell death mediated by increasing ER stress, GPR78 activation, and Ca2+ release, which subsequently triggers calpain activity, resulting in apoptosis (Fig. 7). Acknowledgments This work was supported by grants from the National Science Council of Taiwan (96-2320-B-039-028-MY3; 98-2314-B-075A-001MY2) and China Medical University (CMU97-180). We thank Dr. Kazutoshi Mori for providing the GRP78 and GRP94 luciferase plasmids. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.taap.2009.08.011. References
Fig. 7. Schematic diagram of the pathways involved in As-induced cell apoptosis in cultured osteoblasts. Proposed models showing how As affects various biochemical processes and events in osteoblastic cells, resulting in apoptotic cell death, are illustrated in this schematic diagram.
Abcouwer, S.F., Marjon, P.L., Loper, R.K., Vander Jagt, D.L., 2002. Response of VEGF expression to amino acid deprivation and inducers of endoplasmic reticulum stress. Invest Ophthalmol. Vis. Sci. 43, 2791–2798. Adams, J.M., Cory, S., 2001. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem. Sci. 26, 61–66. Bates, M.N., Smith, A.H., Hopenhayn-Rich, C., 1992. Arsenic ingestion and internal cancers: a review. Am. J. Epidemiol. 135, 462–476. Benali-Furet, N.L., Chami, M., Houel, L., De Giorgi, F., Vernejoul, F., Lagorce, D., Buscail, L., Bartenschlager, R., Ichas, F., Rizzuto, R., Paterlini-Brechot, P., 2005. Hepatitis C virus core triggers apoptosis in liver cells by inducing ER stress and ER calcium depletion. Oncogene 24, 4921–4933. Chen, C.J., Chen, C.W., Wu, M.M., Kuo, T.L., 1992. Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br. J. Cancer 66, 888–892. Chen, C.J., Hsueh, Y.M., Lai, M.S., Shyu, M.P., Chen, S.Y., Wu, M.M., Kuo, T.L., Tai, T.Y., 1995. Increased prevalence of hypertension and long-term arsenic exposure. Hypertension 25, 53–60. Chiou, H.Y., Hsueh, Y.M., Liaw, K.F., Horng, S.F., Chiang, M.H., Pu, Y.S., Lin, J.S., Huang, C.H., Chen, C.J., 1995. Incidence of internal cancers and ingested inorganic arsenic: a seven-year follow-up study in Taiwan. Cancer Res. 55, 1296–1300. Chiu, Y.C., Yang, R.S., Hsieh, K.H., Fong, Y.C., Way, T.D., Lee, T.S., Wu, H.C., Fu, W.M., Tang, C.H., 2007. Stromal cell-derived factor-1 induces matrix metalloprotease-13 expression in human chondrocytes. Mol. Pharmacol. 72, 695–703. Deal, C., 2009. Potential new drug targets for osteoporosis. Nat. Clin Pract. Rheumatol. 5, 20–27. Dijkers, P.F., Birkenkamp, K.U., Lam, E.W., Thomas, N.S., Lammers, J.W., Koenderman, L., Coffer, P.J., 2002. FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity. J. Cell Biol. 156, 531–542. Feldman, D.E., Chauhan, V., Koong, A.C., 2005. The unfolded protein response: a novel component of the hypoxic stress response in tumors. Mol. Cancer Res. 3, 597–605. Fettucciari, K., Fetriconi, I., Mannucci, R., Nicoletti, I., Bartoli, A., Coaccioli, S., Marconi, P., 2006. Group B Streptococcus induces macrophage apoptosis by calpain activation. J. Immunol. 176, 7542–7556.
C.-H. Tang et al. / Toxicology and Applied Pharmacology 241 (2009) 173–181 Hock, J.M., Krishnan, V., Onyia, J.E., Bidwell, J.P., Milas, J., Stanislaus, D., 2001. Osteoblast apoptosis and bone turnover. J. Bone Miner. Res. 16, 975–984. Jilka, R.L., Weinstein, R.S., Bellido, T., Parfitt, A.M., Manolagas, S.C., 1998. Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J. Bone Miner Res. 13, 793–802. Kaufman, R.J., 1999. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 13, 1211–1233. Khosla, S., Amin, S., Orwoll, E., 2008. Osteoporosis in men. Endocr. Rev. 29, 441–464. Kousteni, S., Chen, J.R., Bellido, T., Han, L., Ali, A.A., O'Brien, C.A., Plotkin, L., Fu, Q., Mancino, A.T., Wen, Y., Vertino, A.M., Powers, C.C., Stewart, S.A., Ebert, R., Parfitt, A.M., Weinstein, R.S., Jilka, R.L., Manolagas, S.C., 2002. Reversal of bone loss in mice by nongenotropic signaling of sex steroids. Science 298, 843–846. Lai, M.W., Boyer, E.W., Kleinman, M.E., Rodig, N.M., Ewald, M.B., 2005. Acute arsenic poisoning in two siblings. Pediatrics 116, 249–257. Lee, A.S., 2001. The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem. Sci. 26, 504–510. Little, E., Ramakrishnan, M., Roy, B., Gazit, G., Lee, A.S., 1994. The glucose-regulated proteins (GRP78 and GRP94): functions, gene regulation, and applications. Crit. Rev. Eukaryot. Gene Expr. 4, 1–18. Liu, S., Wang, H., Yang, Z., Kon, T., Zhu, J., Cao, Y., Li, F., Kirkpatrick, J., Nicchitta, C.V., Li, C.Y., 2005. Enhancement of cancer radiation therapy by use of adenovirusmediated secretable glucose-regulated protein 94/gp96 expression. Cancer Res. 65, 9126–9131. Liu, X., Kim, C.N., Yang, J., Jemmerson, R., Wang, X., 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147–157. Manolagas, S.C., 2000. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev. 21, 115–137. Moenner, M., Pluquet, O., Bouchecareilh, M., Chevet, E., 2007. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res. 67, 10631–10634.
181
Orrenius, S., Zhivotovsky, B., Nicotera, P., 2003. Regulation of cell death: the calciumapoptosis link. Nat Rev. Mol. Cell Biol. 4, 552–565. Rodan, G.A., Martin, T.J., 2000. Therapeutic approaches to bone diseases. Science 289, 1508–1514. Seeman, E., 2002. Pathogenesis of bone fragility in women and men. Lancet 359, 1841–1850. Soboloff, J., Berger, S.A., 2002. Sustained ER Ca2+ depletion suppresses protein synthesis and induces activation-enhanced cell death in mast cells. J. Biol. Chem. 277, 13812–13820. Susin, S.A., Zamzami, N., Castedo, M., Daugas, E., Wang, H.G., Geley, S., Fassy, F., Reed, J.C., Kroemer, G., 1997. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramideinduced apoptosis. J. Exp. Med. 186, 25–37. Tan, Y., Dourdin, N., Wu, C., De Veyra, T., Elce, J.S., Greer, P.A., 2006. Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stressinduced apoptosis. J. Biol. Chem. 281, 16016–16024. van't Hof, R.J., Ralston, S.H., 2001. Nitric oxide and bone. Immunology 103, 255–261. Ye, J., Wang, S., Leonard, S.S., Sun, Y., Butterworth, L., Antonini, J., Ding, M., Rojanasakul, Y., Vallyathan, V., Castranova, V., Shi, X., 1999. Role of reactive oxygen species and p53 in chromium(VI)-induced apoptosis. J. Biol. Chem. 274, 34974–34980. Yoshida, H., Haze, K., Yanagi, H., Yura, T., Mori, K., 1998. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors. J. Biol. Chem. 273, 33741–33749. Yung, H.W., Korolchuk, S., Tolkovsky, A.M., Charnock-Jones, D.S., Burton, G.J., 2007. Endoplasmic reticulum stress exacerbates ischemia-reperfusion-induced apoptosis through attenuation of Akt protein synthesis in human choriocarcinoma cells. FASEB J. 21, 872–884. Zamzami, N., Brenner, C., Marzo, I., Susin, S.A., Kroemer, G., 1998. Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins. Oncogene 16, 2265–2282.