ARTICLE

Journal of Cellular Biochemistry 117:1078–1091 (2016)

TRAIL-Induced Caspase Activation Is a Prerequisite for Activation of the Endoplasmic Reticulum Stress-Induced Signal Transduction Pathways Dae-Hee Lee,1,2 Ki Sa Sung,3,4 Zong Sheng Guo,1 William Taehyung Kwon,1 David L. Bartlett,1 Sang Cheul Oh,2 Yong Tae Kwon,4 and Yong J. Lee1,5* 1

Department of Surgery, School of Medicine, University of Pittsburgh, Pittsburgh 15213, Pennsylvania Division of Oncology/Hematology, Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea 3 Center for Pharmacogenetics and Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh 15261, Pennsylvania 4 Protein Metabolism Medical Research Center and Department of Biomedical Science, College of Medicine, Seoul National University, Seoul 110-799, Korea 5 Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh 15213, Pennsylvania 2

ABSTRACT It is well known that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis can be initially triggered by surface death receptors (the extrinsic pathway) and subsequently amplified through mitochondrial dysfunction (the intrinsic pathway). However, little is known about signaling pathways activated by the TRAIL-induced endoplasmic reticulum (ER) stress response. In this study, we report that TRAIL-induced apoptosis is associated with the endoplasmic reticulum (ER) stress response. Human colorectal carcinoma HCT116 cells were treated with TRAIL and the ER stress-induced signal transduction pathway was investigated. During TRAIL treatment, expression of ER stress marker genes, in particular the BiP (binding immunoglobulin protein) gene, was increased and activation of the PERK (PKR-like ER kinase)eIF2a (eukaryotic initiation factor 2a)-ATF4 (activating transcription factor 4)-CHOP (CCAAT-enhancer-binding protein homologous protein) apoptotic signal transduction pathway occurred. Experimental data from use of a siRNA (small interfering RNA) technique, caspase inhibitor,

Abbreviations: Apaf1, apoptosis signal-regulating kinase; APC, allophycocyanin; ASK1, apoptosis signal-regulating kinase 1; ATCC, American Tissue Type Culture Collection; ATF4, activating transcription factor 4; ATF6, activating transcription factor-6; BAP31, B cell receptor-associated protein 31; Bax, Bcl-2–associated X protein; Bcl-xL, B-cell lymphoma-extra large; BH3, Bcl-2 homology domain 3; BiP, binding immunoglobin protein; CDIP1, cell deathinducing p53 target 1; CHOP, CCAAT-enhancer-binding protein homologous protein; CPC, colorectal peritoneal carcinomatosis; CRS, cytoreductive surgery; DAPI, 40 ,6-diamidino-2-phenylindole; DcR, decoy receptor; DD, death domain; DISC, death-inducing signaling complex; DMEM, Dulbecco0 s modified eagle medium; DR4, death receptor 4, DR5: death receptor 5; eIF2a, eukaryotic initiation factor 2a; ER, endoplasmic reticulum; FACS, fluorescenceactivated cell sorting; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GRP78, 78 kDa glucose regulated protein; HA, hemagglutinin; HIPEC, hyperthermic intraperitoneal chemotherapy; IRE1, inositol-requiring enyzme-1; JNK, c-Jun NH2-terminal kinase; KD, knock-down; KI, knock-in; KO, knock-out; MEF, mouse embryo fibroblast; MOI, multiplicity of infection; NBS1, Nijmegen breakage syndrome; PAGE, polyacrylamide gel electrophoresis; PARP, poly (ADP-ribose) polymerase; PBS, phosphate-buffered saline solution; PERK, PKR-like ER kinase; PI, propidium iodide; PID, protein disulfide isomerase; PUMA, p53 upregulated modulator of apoptosis; RPMI, Roswell Park Memorial Institute medium; S2P, Site-2 protease; SDS, sodium dodecyl sulfate; shRNA, small hairpin RNA; SIP, Site-1 protease; siRNA, small interfering RNA; tBid, truncated Bid; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; UPR, unfolded protein response; WT, wild-type. Conflict of interest disclosure: The authors declare no competing financial interests. Grant sponsor: National Cancer Institute; Grant numbers: CA140554, P30CA047904, NRF-2013R1A2A2A01014170. *Correspondence to: Dr. Yong J. Lee, Department of Surgery, University of Pittsburgh, Hillman Cancer Center, 5117 Centre Ave. Room 1.46C, Pittsburgh, PA 15213. E-mail: [email protected] Manuscript Received: 2 April 2015; Manuscript Accepted: 20 July 2015 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 24 July 2015 DOI 10.1002/jcb.25289  © 2015 Wiley Periodicals, Inc.

1078

and caspase-3-deficient cell line revealed that TRAIL-induced caspase activation is a prerequisite for the TRAIL-induced ER stress response. TRAIL-induced ER stress was triggered by caspase-8-mediated cleavage of BAP31 (B cell receptor-associated protein 31). The involvement of the proapoptotic PERK-CHOP pathway in TRAIL-induced apoptosis was verified by using a PERK knockout (PERK/) mouse embryo fibroblast (MEF) cell line and a CHOP/ MEF cell line. These results suggest that TRAIL-induced the activation of ER stress response plays a role in TRAIL-induced apoptotic death. J. Cell. Biochem. 117: 1078–1091, 2016. © 2015 Wiley Periodicals, Inc.

KEY WORDS:

TRAIL-INDUCED CASPASE-8 ACTIVATION; TRAIL-INDUCED ER STRESS; PERK-eIF2a-ATF4-CHOP SIGNALS; CLEAVAGE OF BAP31

C

olorectal cancer is one of the most common cancers in both men and women in the United States. The American Cancer Society estimates 132,700 colorectal cancer cases in the United States in 2015 [American Cancer Society, 2015]. Colorectal peritoneal carcinomatosis (CPC), which affects approximately 10% of colorectal cancer patients, is one manifestation of metastatic colorectal cancer. CPC is regarded as a lethal condition, with a poor prognosis and a median survival time of approximately six to nine months [Confuorto et al., 2007]. In the past, CPC was considered a terminal disease stage, but a new therapeutic approach based on a combination of cytoreductive surgery and hyperthermic intraperitoneal chemoperfusion has been introduced over the past two decades. However, two-thirds of patients still develop recurrence. Thus, more effective novel regimens are needed to improve survival from this disease. Our previous studies suggest that use of the biologic agent TRAIL (tumor necrosis factor-related apoptosisinducing ligand) is a very attractive anti-cancer treatment strategy [Song et al., 2012; Kim et al., 2014]. TRAIL (Apo2L) is a type II integral membrane protein belonging to the tumor necrosis factor (TNF) family. TRAIL is a 281-amino acid protein, most closely related to a Fas/APO-1 ligand. As in Fas ligand (FasL) and TNF, the C-terminal extracellular region of TRAIL (amino acids 114-281) exhibits a homotrimeric subunit structure [Pitti et al., 1996]. However, several studies have revealed that unlike FasL and TNF, TRAIL induces apoptosis in a wide variety of tumor cells, but does not cause toxicity to most normal cells [Ashkenazi and Dixit, 1999; Walczak et al., 1999]. Current knowledge about TRAILinduced apoptotic signaling pathways includes the following findings: TRAIL-induced apoptosis can be initiated via the extrinsic (death receptor) pathway followed by the intrinsic (mitochondrial) pathway with divergent death signals activated as a consequence of the stimulators [Ashkenazi, 2002]. TRAIL initiates the extrinsic pathway by binding to death receptors (DRs) such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5) and inducing the apoptotic signal. Both DR4 and DR5 contain a cytoplasmic death domain, which is required for TRAIL receptor-induced apoptosis. However, in normal cells, TRAIL also binds to highly expressed decoy receptors (DcR1 and DcR2), which results in inhibition of TRAIL signaling [Pan et al., 1997; Sheridan et al., 1997]. Activation of death domains (DDs) leads to the formation of the death-inducing signaling complex (DISC) [Ganten et al., 2004]. Caspase-8 recruitment to and its activation at the DISC leads to further activation of signaling molecules downstream which results in the activation of the executioner caspase-3, -6, and -7, which culminates in apoptotic death [Li et al., 1997]. Activated caspase-8 also cleaves the proapoptotic molecule Bid and truncated Bid (tBid) translocates to the mitochondria and then tBid induces Bcl-2-associated X protein (Bax) and Bak oligomerization [Wei et al., 2000; Grinberg et al., 2002]. Insertion of oligomerized Bax and Bak

JOURNAL OF CELLULAR BIOCHEMISTRY

into the outer mitochondrial membrane, permeabilization, and depolarization of the mitochondria promote cytochrome c release [Eskes et al., 2000; Logue et al., 2013a]. Released cytochrome c facilitates the formation of the Apaf1 (apoptosis signal-regulating kinase)/caspase-9 apoptosome which activates caspase-9 and subsequently caspase-3 [Baliga and Kumar, 2003]. In understanding the effects of TRAIL treatment, although much is currently known about the signaling pathways involving external signals through cell surface receptors and subsequent internal signals through mitochondrial dysfunction, little is known about the involvement of endoplasmic reticulum (ER) stress-induced signal transduction pathways in apoptosis during TRAIL treatment. Previous studies have revealed that upon ER stress, BiP (also referred to as GRP78, 78 kDa glucose regulated protein) senses ER stress and dissociates from three ER-resident transmembrane proteins: IRE1 (inositol-requiring enyzme-1), PERK (protein kinase RNA-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor-6) [Lee, 2005; Li and Lee, 2006]. After the disassociation from BiP, these three proteins are activated and subsequently activate downstream signal transduction pathways [Leem and Koh, 2012]. IRE1 can recruit TRAF2 (TNF receptorassociated factor 2) and ASK1 (apoptosis signal-regulating kinase 1), which activates the MEK-JNK1-Bcl-2/Bcl-xL-Bax/Bak signal transduction pathway [Nishitoh et al., 2002]. PERK activates the eIF2aATF4 pathway which leads to upregulation of CHOP and subsequent upregulation of PUMA (p53 upregulated modulator of apoptosis) and Noxa (Latin for damage), which is involved in the ER stress-mediated apoptosis pathway [Li et al., 2006]. ATF6 (p90ATF6) is cleaved by Site-1 protease (S1P) and Site-2 protease (S2P) and releases its cytoplasmic domain, and is then converted to a 50-kDa protein (p50ATF6) [Ye et al., 2000]. p50ATF6, a nuclear protein, enters the nucleus and acts as a transcription factor in response to ER stress [Haze et al., 1999]. In this study, we observed that TRAIL induces caspase-mediated ER stress which facilitates apoptotic death through the activation of the PERK-eIF2a-ATF4-CHOP signal transduction pathways. We clearly demonstrate that TRAIL-induced caspase activation is a prerequisite for the activation of the ER stress-induced signal transduction pathways. We believe that modulation of ER stressinduced signal transduction pathways may affect the clinical efficacy of TRAIL in cancer patients.

MATERIALS AND METHODS CELL CULTURE DJ-1/ and corresponding wild-type (WT) mouse embryo fibroblast (MEF) cell lines were provided by Dr. Mark R. Cookson (NIH,

TRAIL-INDUCED ER STRESS

1079

Washington DC, USA). PERK/ and corresponding wild-type MEF cell lines were provided by Dr. J. Alan Diehl (University of Pennsylvania, PA, USA). CHOP/ and corresponding wild-type MEF cell lines were provided by Dr. Randal J. Kaufman (Sanford Burnham Medical Research Institute, CA, USA). MEF cells were maintained in Dulbecco0 s modified Eagle0 s medium (DMEM) with the addition of 55 mM b-mercaptoethanol (Invitrogen, Carlsbad, CA). Cells were seeded and cultured to 60–80% confluency. Human colon cancer HCT116, SW480, and HT-29 cells were purchased from American Tissue Type Culture Collection (ATCC) (Manassas, VA). Human colorectal carcinoma CX-1 cells were obtained from Dr. John M. Jessup (NIH). HCT116 cells were cultured in McCoy0 s 5A. SW480 and HT-29 cells were cultured in DMEM, and CX-1 cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI) medium (Invitrogen). All cell lines were cultured with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1 mM L-glutamine, and 26 mM sodium bicarbonate. The dishes containing cells were kept in a 37°C humidified incubator with 5% CO2. REAGENTS AND ANTIBODIES For production of human TRAIL for human cancer cells, a human TRAIL cDNA fragment (amino acids 114–281) obtained by RT-PCR was cloned into a pET-23d (Novagen, Madison, WI) plasmid, and His-tagged TRAIL protein was purified using the Qiagen express protein purification system (Qiagen, Valencia, CA). Soluble recombinant murine TRAIL and recombinant human TNF-a were purchased from R&D Systems (Minneapolis, MN). Vaccinia virus expressing murine TRAIL (vvmTRAIL) and its parental thymidine kinase-deleted virus VJS6 for MEF cells was described previously [Ziauddin et al., 2010]. Anti-BAP31, anti-DJ-1, and anti-CHOP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho PERK, anti-PERK, anti-phospho IRE1a, anti-IRE1a, anti-phospho eIF2a, anti-eIF2a, anti-ATF4, antiCHOP, anti-DJ-1, anti-PDI, anti-Bax, anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-phospho JNK, anti-JNK, anti-cytochrome c, and anti-PARP-1 were purchased from Cell Signaling (Beverly, MA). Anti-actin antibody was purchased from MP Biomedicals (Solon, OH). For the secondary antibodies, antimouse-IgG-HRP and anti-rabbit-IgG-HRP were purchased from Santa Cruz Biotechnology. MEASUREMENT OF CYTOCHROME C RELEASE HCT116 cells growing in 100 mm dishes were treated with TRAIL. Using the Mitochondrial Fractionation Kit (Active Motif, Carlsbad, CA), mitochondria and cytosol fractions were prepared from treated cells using instructions and reagents included in the kit. SMALL INTERFERING RNA (siRNA) Caspase-8 siRNA (Cat. No. SC-29930), BAP31 siRNA (Cat. No. SC-37283), and negative control siRNA (Cat. No. SC-37007) were obtained from Santa Cruz Biotechnology. Cells were transfected with siRNA oligonucleotides using LipofectAMINE RNAi Max reagents (Invitrogen) according to the manufacturer0 s instructions. After transfection, cells were treated with TRAIL for further analysis.

1080

TRAIL-INDUCED ER STRESS

qRT-PCR ANALYSIS Total RNA was isolated from untreated or TRAIL-treated cells using the RNAeasy Kit (Qiagen) according to the manufacturer0 s protocol. Total RNA (2 mg) was used to generate complementary DNA using SuperScript III reverse transcriptase (Invitrogen). All PCR reactions were performed in triplicate, and PCR products were subjected to a melting curve analysis. The abundance of specific mRNAs was determined by comparison with a standard curve constructed by serial dilution of the sample and normalized to GAPDH. Primers used for PCR reactions are listed in Table I. SURVIVAL ASSAY MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium) studies were carried out using the Promega CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). Cells were grown in tissue culture-coated 96-well plates and treated as described in Results. Cells were then treated with MTS/phenazine methosulfate solution for 2 h at 37°C. Absorbance at 490 nm was determined using an enzyme-linked immunosorbent assay plate reader. APOPTOSIS ASSAY The translocation of phosphatidylserine, one of the markers of apoptosis, from the inner to the outer leaflet of the plasma membrane was detected by binding of fluorescein isothiocyanate (FITC)conjugated annexin V. Briefly, colorectal cancer cell lines untreated or treated with TRAIL were resuspended for 4 h in the binding buffer provided in the annexin V-FITC Detection Kit II (BD Biosciences Pharmingen, San Diego, CA). Cells were mixed with 5 mL annexin VFITC reagent and incubated for 30 min at room temperature in the dark. The staining was terminated and cells were immediately analyzed using flow cytometry. CONFOCAL MICROSCOPE Cells were stained with 200 nM MitoTracker (Life Technologies). Cells were washed three times with 0.5% bovine serum albumin (BSA) in phosphate buffered saline (PBS), followed by fixation in 4% paraformaldehyde for 15 min. CHOP was stained with anti-CHOP antibody. Nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI) (Cell Signaling). Slides were mounted and visualized in 0.4-mm sections using an Olympus Fluoview 1000 confocal microscope and the companion software FV10-ASW2.1 under a 63X oil immersion objective. WESTERN BLOTTING Western blotting was carried out as previously described [Lee et al., 2015]. Cells were lysed with Laemmli lysis buffer and boiled for 7 min. Protein content was measured with BCA Protein Assay

TABLE I. Primer Sequences for Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT – PCR) Human Bip Human PDI Human GAPDH

Forward: 50 -GCTATTGCTTATGGCCTGGA-30 Reverse: 50 -CGCTGGTCAAAGTCTTCTCC-30 Forward: 50 -GTTCTTCCGCAATGGGAACC-30 Reverse: 50 -CCTGCAGGTCCTGGAAGAAG-30 Forward: 50 -AATCCCATCACCATCTTCCA-30 Reverse: 50 -TGGACTCCACGACGTACTCA-30

JOURNAL OF CELLULAR BIOCHEMISTRY

Reagent (Thermo Scientific, Hudson, NH), separated by SDS–PAGE and electrophoretically transferred to nitrocellulose membrane. Immunoreactive proteins were visualized by the chemiluminescence protocol (ECL, Amersham, Arlington Heights, IL). ImageJ software (NIH) was used to quantify area intergration of optical density of western blot bands. STATISTICAL ANALYSIS Statistical analysis was carried out using GraphPad Prism6 software (GraphPad Software, Inc., San Diego, CA). The results were expressed as the mean of arbitrary values  SD. All results were evaluated using an unpaired Student0 s t test in which a P-value of less than 0.05 was considered significant.

RESULTS TRAIL-INDUCED APOPTOSIS It is well known that TRAIL selectively induces apoptosis in a variety of cancer cells, but not in most normal cells. To assess the role of the ER in TRAIL-induced apoptosis, we first examined the effect of TRAIL on a variety of cancer cells. Data from annexin V/PI (propidium iodide) assay show TRAIL-induced apoptosis in human colorectal carcinoma HCT116 cells (Fig. 1a; early apoptotic death cells in lower right plot quadrants and late apoptotic death cells in upper right plot quadrants). Apoptosis occurred in a TRAIL

dose-dependent manner (Fig. 1b). However, differences in TRAIL sensitivity were observed in a variety of colon cancer cells (Fig. 1b). TRAIL-INDUCED ER STRESS RESPONSE Next, we examined a possible involvement of ER stress in TRAILinduced apoptosis by measuring the expression of BiP and PDI (protein disulfide isomerase) genes, markers of ER stress, during TRAIL treatment. Figure 2a and b show an increase in the expression of these genes, in particular BiP, in a time-dependent manner in human colon cancer HCT116 cells. Similar results were observed in CX-1 cells (Fig. 2b). A TRAIL-induced ER stress response was also confirmed by examining ER stress-related proteins in HCT116 cells (Fig. 2c–e). TRAIL increased the levels of p-PERK, p-IRE1a, p-eIF2a (phosphorylated eukaryotic initiation factor 2a), ATF4 (activating transcription factor 4), and CHOP in a dose- and time-dependent manner (Fig. 2c and d). Images obtained by confocal microscopy confirmed an increase in the intracellular level of CHOP during treatment with TRAIL (Fig. 2e). As shown in Figure 2f, similar results were obtained by treatment with other caspase-8 activator such as tumor necrosis factor-a (TNF-a). Several researchers have reported that the ER stress response is initiated by activation of three molecules, PERK, IRE1a, and ATF6 [Liu and Kaufman, 2003]. Activated PERK phosphorylates eIF2a which promotes translation initiation of ATF4. ATF4 leads to transcription of the ATF4 downstream target CHOP [Woo et al., 2009; Saito et al., 2011].

Fig. 1. TRAIL-induced apoptosis. (a) HCT116 cells were treated with PBS (sham control) or various concentrations (2.5–10 ng/mL) of TRAIL for 4 h. The cells were stained with annexin V and propidium iodide (PI), followed by FACS (fluorescence-activated cell sorting) analysis. (b) Four different human colorectal cancer cell lines were untreated or treated with TRAIL for 4 h at the indicated concentration. Apoptosis was determined by FACS analysis and plotted. Error bars represent the SD (standard deviation) from three separate experiments.  or   , statistically significant difference compared with the control at P < 0.05 or P < 0.01, respectively.

JOURNAL OF CELLULAR BIOCHEMISTRY

TRAIL-INDUCED ER STRESS

1081

Fig. 2. TRAIL-induced ER stress response. (a and b) Cells were treated with TRAIL (5, 10, or 100 ng/ml) for various times (1–4 h). (a) Expressions of BiP and PDI genes were analyzed by qRT-PCR in HCT116 cells. The levels of BiP and PDI mRNAs were normalized to the level of GAPDH. Error bars represent the SD from triplicate samples ( P < 0.05;  P < 0.01). (b) HCT116 and CX-1 cell lysates were analyzed by immunoblotting using indicated antibodies. Actin was used to confirm the equal amount of proteins loaded in each lane. Relative area integration of optical density of western blot bands was plotted. (c and d) HCT116 cells were treated with various concentrations (2.5–10 ng/ml) of TRAIL for 4 h (c) or various times (1–4 h) with 10 ng/ml TRAIL (d). Cell lysates were analyzed with immunoblotting using indicated antibodies. Actin was used to confirm the equal amount of proteins loaded in each lane. Relative area integration of optical density of western blot bands was plotted. (e) HCT116 cells were treated with TRAIL (5 ng/ml) for 4 h followed by immunocytochemical staining analysis for CHOP expression. Cells were stained with anti-CHOP (green) and nuclei were stained with DRAQ5 (blue). Cells were examined under a confocal microscope. (f) HCT116 cells were treated with 100 ng/mL of TNF-a for 4 h. Cell lysates were analyzed with immunoblotting using indicated antibodies. Actin was used to confirm that similar amounts of protein were loaded in each lane. Relative area integration of optical density of western blot bands was plotted.

1082

TRAIL-INDUCED ER STRESS

JOURNAL OF CELLULAR BIOCHEMISTRY

Fig. 2. Continued.

Our data show that TRAIL significantly activated (phosphorylated) PERK and IRE1a. We also observed that activation of the ER stressassociated PERK-eIF2a-ATF4-CHOP apoptotic signal transduction pathway occurred during TRAIL treatment (Fig. 2c and d, Li et al., 2006; Logue et al., 2013b). These data clearly demonstrate that TRAIL induces ER stress in cancer cells.

JOURNAL OF CELLULAR BIOCHEMISTRY

TRAIL-INDUCED CASPASE ACTIVATION IS A PREREQUISITE FOR TRAIL-INDUCED ER STRESS It is well known that TRAIL induces apoptosis via cleavage (activation) of caspases, Bax activation and cytochrome c release (Fig. 3a and b, Petak et al., 2003; Kim and Lee, 2005). We examined whether TRAIL-induced caspase activation is a prerequisite for

TRAIL-INDUCED ER STRESS

1083

Fig. 3. TRAIL-induced caspase activation is a prerequisite for TRAIL-induced ER stress. (a and b) HCT116 cells were treated with various concentrations (2.5–10 ng/mL) of TRAIL for 4 h or various times (1–4 h) with 10 ng/mL TRAIL and cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, Bax activation, and cytochrome c release were detected by immunoblotting. Actin was used to confirm equal amounts of proteins loaded in each lane. Relative area integration of optical density of western blot bands was plotted. (c and d) HCT116 cells were transfected with control siRNA (siCon) or caspase-8 siRNA (siCas-8) for 48 h and then treated with 5 ng/ml TRAIL for 4 h. (c) The cells were collected, lysed, and subjected to immunoblot analysis with indicated antibodies. Relative area integration of optical density of western blot bands was plotted. (d) Cell viability was determined using the MTS assay. Error bars represent the mean  SD from three separate experiments ( P < 0.05). (e) HCT116 cells were pretreated with caspase-8 inhibitor (z-IETD-fmk) for 30 min and treated with TRAIL (5 ng/ml) for 4 h. Cell lysates were analyzed by immunoblotting using indicated antibodies. Relative area integration of optical density of western blot bands was plotted. (f) MCF-7 cells were stably transfected with control vector (pBABE) or pcDNA-caspase-3 (pCas-3) (upper panel) and then treated with TRAIL (50 ng/ml) for 24 h. The activation of caspase-8, CHOP, and the cleavage of PARP were detected using immunoblot analysis with the indicated antibodies (lower panel). Actin was shown as an internal standard. Relative area integration of optical density of western blot bands was plotted.

1084

TRAIL-INDUCED ER STRESS

JOURNAL OF CELLULAR BIOCHEMISTRY

Fig. 3. Continued.

JOURNAL OF CELLULAR BIOCHEMISTRY

TRAIL-INDUCED ER STRESS

1085

Fig. 4. TRAIL induces cleavage of BAP31. (a) HCT116 cells were treated with dimethyl sulfoxide (DMSO) (sham control) or various concentrations (2.5–10 ng/mL) of TRAIL for 4 h. The activation of BAP31 was detected using immunoblot analysis with anti-BAP31 antibody. Relative area integration of optical density of western blot bands was plotted. (b and c) HCT116 cells were transfected with control siRNA (siCon) or BAP31 siRNA (siBAP31) for 48 h and then treated with 5 or 50 ng/mL TRAIL for 4 h. The cells were collected, lysed, and subjected to immunoblot analysis with indicated antibodies. Relative area integration of optical density of western blot bands was plotted. (d) Cell viability was determined using the MTS assay. Error bars represent the mean  SD from three separate experiments ( P < 0.05).

1086

TRAIL-INDUCED ER STRESS

JOURNAL OF CELLULAR BIOCHEMISTRY

TRAIL-induced ER stress by using a siRNA technique, a caspase inhibitor, and a caspase-3 deficient cell line. As shown in Fig. 3c and d, knockdown of caspase-8 protected cells from TRAIL-induced PARP-1 (poly [ADP-ribose] polymerase 1, the hallmark feature of

JOURNAL OF CELLULAR BIOCHEMISTRY

apoptosis) cleavage, JNK (c-Jun N-terminal protein kinase) activation (phosphorylation of JNK), phosphorylation of PERK and eIF2a, elevation of ATF4 and CHOP levels, and TRAIL cytotoxicity. Similar results were observed in caspase-8 inhibitor

TRAIL-INDUCED ER STRESS

1087

Fig. 6. Schematic diagram of the working model of ER stress involvement in TRAIL-induced apoptosis.

pretreated cells (Fig. 3e). We also observed an involvement of ER stress in TRAIL-induced apoptosis in caspase-3-deficient MCF-7 cells (Fig. 3f, upper panel). MCF-7 cells were resistant to TRAIL and restoration of caspase-3 led to an increase in apoptotic death (PARP1 cleavage), activation of caspase-8, phosphorylation of PERK and p-eIF2a, and elevation of ATF and CHOP levels during TRAIL treatment (Fig. 3f, lower panel). TRAIL-INDUCED CLEAVAGE OF BAP31 We further investigated TRAIL-induced ER stress with B cell receptor-associated protein 31 (BAP31), which is known as the primary initiator of the ER stress response [Liu et al., 2006]. BAP31 is cleaved by activated caspase-8 and moves between the peripheral ER and a juxtanuclear compartment in response to ER stress [Ng et al., 1997; Breckenridge et al., 2003; Wakana et al., 2008]. As shown in Figure 4a, TRAIL (2.5–10 ng/ml) induced cleavage of BAP31 in a

dose-dependent manner. Knockdown of BAP31 protected cells from PARP-1 cleavage without changing the activation of caspase-8; knockdown of BAP31 also protected cells from TRAIL-induced cytotoxicity (Fig. 4b and d). Similar results were obtained with higher concentration (50 ng/ml) of TRAIL treatment (Fig. 4C). These data clearly demonstrate that TRAIL-induced caspase activation is a prerequisite for TRAIL-induced ER stress and that ER stress-mediated signals promote apoptosis. THE PROAPOPTOTIC PERK-CHOP PATHWAY IS INVOLVED IN TRAIL-INDUCED APOPTOSIS As shown in Figure 2c and d, TRAIL activates the PERK-eIF2a-ATF4CHOP signal transduction pathways; this pathway is known to upregulate the expression of the proaptototic proteins PUMA and Noxa [Saito et al., 2011]. We further examined the role of ER stressmediated signals in TRAIL-induced apoptosis. For this study, we

Fig. 5. The proapoptotic PERK-CHOP pathway is involved in TRAIL-induced apoptosis. (a) PERK WT (wild-type) and PERK KO (knockout) MEF cells were infected with control vaccinia virus vJS6 (0.5–2 multiplicity of infection (MOI)) or vaccinia virus expressing murine TRAIL vvmTRAIL (0.5–2 MOI) for 24 h. Cell viability was determined using the MTS assay. Error bars represent the mean  SD from three separate experiments ( P < 0.05). (b and c) WT and PERK KO MEF cells were infected with vJS6 (1 MOI) and vvmTRAIL (1 MOI) for 24 h. (b) Cell lysates were subjected to immunoblot analysis with anti-PERK or anti-PARP-1 antibody. Actin was used to confirm the equal amount of proteins loaded in each lane. Relative area integration of optical density of western blot bands was plotted. (c) Cell viability was determined using the MTS assay. Error bars represent the mean  SD from three separate experiments. (d and e) WT and CHOP KO MEF cells were infected with vJS6 (1 MOI) and vvmTRAIL (1 MOI) for 24 h. (d) The cells were collected, lysed, and subjected to immunoblot analysis with indicated antibodies. Relative area integration of optical density of western blot bands was plotted. (e) Cell viability was determined using the MTS assay. Error bars represent the mean  SD from three separate experiments ( P < 0.05). (f and g) HCT116 cells were transfected with control siRNA (siCon) or CHOP siRNA (siCHOP) for 48 h and then treated with 5 ng/mL TRAIL for 4 h. (f) The cells were collected, lysed, and subjected to immunoblot analysis with indicated antibodies. Relative area integration of optical density of western blot bands was plotted. (g) Cell viability was determined using the MTS assay. Error bars represent the mean  SD from three separate experiments ( P < 0.05). (h and i) WT and PERK KO or CHOP KO MEF cells were treated with recombinant murine TRAIL (200 ng/mL) for 24 h. Cell lysates were subjected to immunoblot analysis with anti-PERK, anti-CHOP or anti-PARP-1 antibody. Actin was shown as an internal standard. Relative area integration of optical density of western blot bands was plotted.

1088

TRAIL-INDUCED ER STRESS

JOURNAL OF CELLULAR BIOCHEMISTRY

employed a PERK knockout (PERK/) mouse embryo fibroblast (MEF) cell line and a CHOP/ MEF cell line. We also employed murine TRAIL gene-armed vaccinia virus [Ziauddin et al., 2010]. Figure 5a–c show that PERK/ MEF cells were resistant to murine TRAIL. Figures 5d and e show that CHOP/ MEF cells were resistant to murine TRAIL. Similar results were obtained with murine TRAIL treatment (Fig. 5h and i). Similar results were also observed in human colon cancer HCT116 cells. Knockdown of CHOP protected cells from TRAIL-induced apoptotic death (Fig. 5f and g).

DISCUSSION In the study of TRAIL-induced cell death, although much is known about the signaling pathways that involve external signals through cell surface receptors and subsequent internal signals through mitochondrial dysfunction, little is known about the signaling pathways activated by ER stress during TRAIL treatment. Our data clearly illustrate that TRAIL-induced ER stress activates the PERKeIF2a-ATF4-CHOP signal transduction pathway, which is involved, at least partially, in TRAIL-induced apoptosis. TRAIL-induced caspase activation is a prerequisite for the ER stress response (Fig. 6). TRAIL exerts its effect through the activation of caspase-8 which is located at distinct cellular loci and plays a key role in mediating death receptor-induced apoptosis [Yin et al., 1999]. In this study, we investigated a consequence of TRAIL-induced activation of caspase-8 (Figs. 3 and 4). BAP31, an integral membrane protein of the ER, is known to be a preferred substrate for caspase-8. Cleaved BAP31 binds to cell death-inducing p53 target 1 (CDIP1) at the ER membrane and the BAP31-CDIP1 complex induces mitochondrial fission through ER signals and enhances cytochrome c release from mitochondria [Nguyen et al., 2000; Breckenridge et al., 2003; Namba et al., 2013]. The simultaneous cleavage of BAP31 through the ER pathway and Bid through the intrinsic pathway by caspase-8 may lead to a dual attack on mitochondria, with cleaved BAP31 causing mitochondrial fission and tBid inducing cristae remodeling which promotes Bax/Bak oligomerization [Scorrano et al., 2002; Namba et al., 2013]. The ER is the main secretory protein-folding locus in the cell [Urade, 2007]. Stressful environmental conditions in the ER lead to ER stress. This stress triggers the unfolded protein response (UPR) in the cell and can be characterized by changes in the expression of ER-stress-related genes (Fig. 2a, Liu et al., 2007). Also, accumulation of unfolded proteins in the ER may generate reactive oxygen species (ROS) and make proteins more susceptible to oxidative modifications because of exposure of interior portions of unfolded proteins [Ozgur et al., 2014]. Dysfunction of the mitochondria, like that of ER, elicites ROS production. Mitochondrial membrane potential is critical for maintaining the physiological function of the respiratory chain to generate ATP [Joshi and Bakowska, 2011]. However, during TRAIL treatment, depolarization of mitochondrial membrane potential occurs and elevates ROS generation [Chaudhari et al., 2007]. ROS can be sensed through thioredoxin (TRX) and glutaredoxin (GRX). These sensor molecules may be converted to the oxidized intramolecular disulfide form of TRX-(S-S) and GRX(S-S) during treatment with TRAIL. The oxidized form of TRX and

JOURNAL OF CELLULAR BIOCHEMISTRY

GRX may dissociate from ASK1 to activate the MEK-JNK1 signal transduction pathway [Song et al., 2002; Song and Lee, 2003,2005]. Our data and literature suggest that TRAIL induces the ER stress response and activates the ROS-ASK-JNK-CHOP pathway (Fig. 3b, Trivedi et al., 2014). It is well known that TRAIL can activate several caspases that can mediate death receptors and mitochondria. In this study, we observed that the ER stress response also plays an important role in TRAIL-induced apoptosis. Our data clearly demonstrate that the TRAIL-induced ER stress response is involved in apoptosis through the PERK-eIF2a-ATF4-CHOP apoptotic signal transduction pathways (Fig. 2c and d). However, several other possibilities exist. Previous studies have demonstrated communication and coordination between the ER and mitochondria during pathological apoptosis [Pinton et al., 2008]. The mitochondrial-associated ER membrane (MAM) interconnects between two organelles and serves as a signaling juncture to facilitate signal transfer and coordinate apoptosis. It is well known that Ca2þ is the most prominent signaling factor that is maintained at all times within the lumen of the ER [Pinton et al., 2008; Grimm, 2012]. It is possible that during TRAIL treatment, Ca2þ is released from the ER, transferred to the mitochondria through MAM, and causes apoptosis [Pinton et al., 2008; Grimm, 2012]. MAM mediates the transfer of TRAIL-induced apoptosis signals to the mitochondria. Another possibility is that MAM is known to form a quasisynaptic structure containing highly active sphingolipid-specific glycosyltransferases and exchange lipids and lipid-derived molecules such as ceramide for apoptosis induction [Ardail et al., 2003; Bionda et al., 2004; Fujimoto and Hayashi, 2011]. During TRAIL treatment, the ER stress response may generate ceramide and induce apoptosis [Nam et al., 2002; White-Gilbertson et al., 2009; Skender et al., 2014]. At the present time, we can only speculate on the involvement of MAM in ER stress response-induced apoptosis during TRAIL treatment. Obviously, this possibility needs to be further examined to understand the role of ER stress in TRAIL-induced apoptosis.

REFERENCES American Cancer Society. Cancer Facts & Figures. Atlanta, American Cancer Society, 2015:1–52. Ardail D, Popa I, Bodennec J, Louisot P, Schmitt D, Portoukalian J. 2003. The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases. Biochem J 371:1013–1019. Ashkenazi A. 2002. Targeting death and decoy receptors of the tumournecrosis factor superfamily. Nat Rev Cancer 2:420–430. Ashkenazi A, Dixit VM. 1999. Apoptosis control by death and decoy receptors. Curr Opin Cell Biol 11:255–260. Baliga B, Kumar S. 2003. Apaf-1/cytochrome c apoptosome: An essential initiator of caspase activation or just a sideshow? Cell Death Differ 10:16–18. Bionda C, Portoukalian J, Schmitt D, Rodriguez-Lafrasse C, Ardail D. 2004. Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria-associated membrane) and/or mitochondria? Biochem J 382:527–533. Breckenridge DG, Stojanovic M, Marcellus RC, Shore GC. 2003. Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol. J Cell Biol 160:1115–1127.

TRAIL-INDUCED ER STRESS

1089

Chaudhari AA, Seol JW, Kim SJ, Lee YJ, Kang HS, Kim IS, Kim NS, Park SY. 2007. Reactive oxygen species regulate Bax translocation and mitochondrial transmembrane potential, a possible mechanism for enhanced TRAILinduced apoptosis by CCCP. Oncol Rep 18:71–76.

Logue S, Cleary P, Saveljeva S, Samali A. 2013a. New directions in ER stressinduced cell death. Apoptosis 18:537–546.

Confuorto G, Giuliano ME, Grimaldi A, Viviano C. 2007. Peritoneal carcinomatosis from colorectal cancer: HIPEC? Surg Oncol 16(Suppl1):S149–S152.

Nam SY, Amoscato AA, Lee YJ. 2002. Low glucose-enhanced TRAIL cytotoxicity is mediated through the ceramide-Akt-FLIP pathway. Oncogene 21:337–346.

Eskes R, Desagher S, Antonsson B, Martinou JC. 2000. Bid induces the oligomerization and insertion of bax into the outer mitochondrial membrane. Mol Cell Biol 20:929–935. Fujimoto M, Hayashi T. 2011. New insights into the role of mitochondriaassociated endoplasmic reticulum membrane. Int Rev Cell Mol Biol 292:73–117. Ganten TM, Haas TL, Sykora J, Stahl H, Sprick MR, Fas SC, Krueger A, Weigand MA, Grosse-Wilde A, Stremmel W, Krammer PH, Walczak H. 2004. Enhanced caspase-8 recruitment to and activation at the DISC is critical for sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by chemotherapeutic drugs. Cell Death Differ 11:S86–S96. Grimm S. 2012. The ER-mitochondria interface: The social network of cell death. Biochim Biophys Acta 1823:327–334. Grinberg M, Sarig R, Zaltsman Y, Frumkin D, Grammatikakis N, Reuveny E, Gross A. 2002. TBID Homooligomerizes in the mitochondrial membrane to induce apoptosis. J Biol Chem 277:12237–12245. Haze K, Yoshida H, Yanagi H, Yura T, Mori K. 1999. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell 10:3787–3799. Joshi DC, Bakowska JC. 2011. Determination of mitochondrial membrane potential and reactive oxygen species in live rat cortical neurons. J Vis Exp 51:2704. Kim KM, Lee YJ. 2005. Amiloride augments TRAIL-induced apoptotic death by inhibiting phosphorylation of kinases and phosphatases associated with the P13K-Akt pathway. Oncogene 24:355–366. Kim SY, Lee DH, Song X, Bartlett DL, Kwon YT, Lee YJ. 2014. Role of Bcl-xL/ Beclin-1 in synergistic apoptotic effects of secretory TRAIL-armed adenovirus in combination with mitomycin C and hyperthermia on colon cancer cells. Apoptosis 19:1603–1615. Lee AS. 2005. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35:373–381. Lee DH, Sung KS, Bartlett DL, Kwon YT, Lee YJ. 2015. HSP90 inhibitor NVPAUY922 enhances TRAIL-induced apoptosis by suppressing the JAK2STAT3-Mcl-1 signal transduction pathway in colorectal cancer cells. Cell Signal 27:293–305. Leem J, Koh EH. 2012. Interaction between mitochondria and the endoplasmic reticulum: Implications for the pathogenesis of Type 2 diabetes mellitus. Exp Diabetes Res 2012:242984. Li J, Lee AS. 2006. Stress induction of GRP78/BiP and its role in cancer. Curr Mol Med 6:45–54. Li J, Lee B, Lee AS. 2006. Endoplasmic reticulum stress-induced apoptosis: Multiple pathways and activation of p53-up-regulated modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem 281:7260–7270. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/Caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479–489. Liu CY, Kaufman RJ. 2003. The unfolded protein response. J Cell Sci 116:1861–1862.

Logue SE, Gorman AM, Cleary P, Keogh N, Samali A. 2013b. Current concept in ER stress-induced apoptosis. J Carcinogene Mutagene S6:2157–2158.

Namba T, Tian F, Chu K, Hwang SY, Yoon KW, Byun S, Hiraki M, Mandinova A, Lee SW. 2013. CDIP1-BAP31 complex transduces apoptotic signals from endoplasmic reticulum to mitochondria under endoplasmic reticulum stress. Cell Rep 5:331–339. Ng FW, Nguyen M, Kwan T, Branton PE, Nicholson DW, Cromlish JA, Shore GC. 1997. P28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum. J Cell Biol 139:327–338. Nguyen M, Breckenridge DG, Ducret A, Shore GC. 2000. Caspaseresistant BAP31 inhibits fas-mediated apoptotic membrane fragmentation and release of cytochrome c from mitochondria. Mol Cell Biol 20: 6731–6740. Nishitoh H, Matsuzawa A, Tobiume K, Saegusa K, Takeda K, Inoue K, Hori S, Kakizuka A, Ichijo H. 2002. ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16:1345–1355. Ozgur R, Turkan I, Uzilday B, Sekmen AH. 2014. Endoplasmic reticulum stress triggers ROS signalling, changes the redox state, and regulates the antioxidant defence of Arabidopsis thaliana. J Exp Bot 65:1377–1390. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. 1997. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 277:815–818. Petak I, Vernes R, Szucs KS, Anozie M, Izeradjene K, Douglas L, Tillman DM, Phillips DC, Houghton JA. 2003. A caspase-8-independent component in TRAIL/Apo-2L-induced cell death in human rhabdomyosarcoma cells. Cell Death Diff 10:729–739. Pinton P, Giorgi C, Siviero R, Zecchini E, Rizzuto R. 2008. Calcium and apoptosis: ER-mitochondria Ca2þ transfer in the control of apoptosis. Oncogene 27:6407–6418. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A. 1996. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271:12687–12690. Saito A, Ochiai K, Kondo S, Tsumagari K, Murakami T, Cavener DR, Imaizumi K. 2011. Endoplasmic reticulum stress response mediated by the PERK-eIF2 (alpha)-ATF4 pathway is involved in osteoblast differentiation induced by BMP2. J Biol Chem 286:4809–4818. Scorrano L, Ashiya M, Buttle K, Weiler S, Oakes SA, Mannella CA, Korsmeyer SJ. 2002. A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Dev Cell 2:55–67. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ashkenazi A. 1997. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 277:818–821. Skender B, Hofmanova J, Slavık J, Jelınkova I, Machala M, Moyer MP, Kozubık A, Hyrslova Vaculova A. 2014. DHA-mediated enhancement of TRAIL-induced apoptosis in colon cancer cells is associated with engagement of mitochondria and specific alterations in sphingolipid metabolism. Biochim Biophys Acta 1841:1308–1317.

Liu JX, Srivastava R, Che P, Howell SH. 2007. Salt stress responses in Arabidopsis utilize a signal transduction pathway related to endoplasmic reticulum stress signaling. Plant J 51:897–909.

Song JJ, Rhee JG, Suntharalingam M, Walsh SA, Spitz DR, Lee YJ. 2002. Role of glutaredoxin in metabolic oxidative stress. Glutaredoxin as a sensor of oxidative stress mediated by H2 O2. J Biol Chem 277: 46566–46575.

Liu N, Scofield VL, Qiang W, Yan M, Kuang X, Wong PK. 2006. Interaction between endoplasmic reticulum stress and caspase 8 activation in retrovirus MoMuLV-ts1-infected astrocytes. Virol 348:398–405.

Song JJ, Lee YJ. 2003. Differential role of glutaredoxin and thioredoxin in metabolic oxidative stress-induced activation of apoptosis signal-regulating kinase 1. Biochem J 373:845–853.

1090

TRAIL-INDUCED ER STRESS

JOURNAL OF CELLULAR BIOCHEMISTRY

Song JJ, Lee YJ. 2005. Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: A negative feedback loop. J Cell Biol 170:61–72.

Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M, Thompson CB, Korsmeyer SJ. 2000. TBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 14:2060–2071.

Song X, Kim HC, Kim SY, Basse P, Park BH, Lee BC, Lee YJ. 2012. Hyperthermia-enhanced TRAIL- and mapatumumab-induced apoptotic death is mediated through mitochondria in human colon cancer cells. J Cell Biochem 113:1547–1558.

White-Gilbertson S, Mullen T, Senkal C, Lu P, Ogretmen B, Obeid L, VoelkelJohnson C. 2009. Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase-3 in colon cancer cells. Oncogene 28:1132–1141.

Trivedi R, Maurya R, Mishra DP. 2014. Medicarpin, a legume phytoalexin sensitizes myeloid leukemia cells to TRAIL-induced apoptosis through the induction of DR5 and activation of the ROS-JNK-CHOP pathway. Cell Death Dis 5:e1465.

Woo CW, Cui D, Arellano J, Dorweiler B, Harding H, Fitzgerald KA, Ron D, Tabas I, . 2009. Adaptive suppression of the ATF4-CHOP branch of the unfolded protein response by toll-like receptor signalling. Nat Cell Biol 11:1473–1480.

Urade R. 2007. Cellular response to unfolded proteins in the endoplasmic reticulum of plants. FEBS J 274:1152–1171. Wakana Y, Takai S, Nakajima K, Tani K, Yamamoto A, Watson P, Stephens DJ, Hauri HP, Tagaya M. 2008. Bap31 is an itinerant protein that moves between the peripheral endoplasmic reticulum (ER) and a juxtanuclear compartment related to ER-associated Degradation. Mol Biol Cell 19:1825–1836. Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH. 1999. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 5:157–163.

JOURNAL OF CELLULAR BIOCHEMISTRY

Ye J, Rawson RB, Komuro R, Chen X, Dave UP, Prywes R, Brown MS, Goldstein JL. 2000. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell 6:1355–1364. Yin XM, Wang K, Gross A, Zhao Y, Zinkel S, Klocke B, Roth KA, Korsmeyer SJ. 1999. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400:886–891. Ziauddin MF, Guo ZS, O0 Malley ME, Austin F, Popovic PJ, Kavanagh MA, Li J, Sathaiah M, Thirunavukarasu P, Fang B, Lee YJ, Bartlett DL. 2010. TRAIL gene-armed oncolytic poxvirus and oxaliplatin can work synergistically against colorectal cancer. Gene Ther 17:550–559.

TRAIL-INDUCED ER STRESS

1091