Distinct Endoplasmic Reticulum Signaling Pathways Regulate

Chem. Res. Toxicol. 1999, 12, 943-951

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Distinct Endoplasmic Reticulum Signaling Pathways Regulate Apoptotic and Necrotic Cell Death following Iodoacetamide Treatment Bob van de Water,† Yuping Wang,‡ Senait Asmellash,‡ Hong Liu,‡ Yi Zhan,‡ Ellen Miller,‡ and James L. Stevens*,§ Adirondack Biomedical Research Institute, 10 Old Barn Road, Lake Placid, New York 12946, Division of Toxicology, Leiden Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands, and Department of Pathology, College of Medicine, University of Vermont, Burlington, Vermont 05405 Received March 31, 1999

Environmental stress induces the synthesis of glucose-regulated proteins (Grps) in the endoplasmic reticulum (ER) and heat shock proteins (Hsps) in the cytoplasm. Iodoacetamide (IDAM), a prototypical alkyating agent, induces both Grp and Hsp synthesis in renal epithelial cells and causes necrosis which is prevented by prior activation of the ER stress response (preER stress) [Liu, H., et al. (1997) J. Biol. Chem. 272, 21751-21759]. In this study, we examined the biochemical pathways leading to IDAM-induced apoptosis and investigated the role of the ER stress response in apoptotic cell death. The antioxidant N,N′-diphenyl-p-phenylenediamine (DPPD) prevented necrosis after IDAM treatment, but the cells went on to die with hallmarks of apoptosis, i.e., cell detachment, caspase-3 activation, cleavage of poly(ADP-ribose)polymerase (PARP), and DNA-ladder formation, all of which were blocked by the general caspase inhibitor zVAD. As with IDAM-induced necrosis, dithiothreitol protected against apoptosis, but cell permeable calcium chelators did not, suggesting that distinct biochemical pathways mediate these two forms of cell death. Pre-ER stress, but not heat shock, prevented IDAM-induced apoptosis. pkASgrp78 cells are deficient in Grp78 induction due to expression of a grp78 antisense RNA and are more sensitive to necrosis. However, these cells were resistant to IDAMinduced apoptosis and had increased basal levels of Grp94 and a KDEL-containing protein of about 50 kDa. Thus, the expression of grp78 antisense perturbs ER functions and activates expression of other ER stress genes accounting for the resistance to apoptosis. Taken together, the data describe functionally distinct signaling pathways through which the ER regulates apoptosis and necrosis caused by chemical toxicants.

Introduction Toxicant exposure and environmental stress activate expression of stress response genes, including members of the heat shock (Hsp)1 and glucose-regulated protein (Grp) families (1-3). Grp78/BiP and Grp94, the prototypical members of the Grp family, are resident proteins of the endoplasmic reticulum (ER) (4, 5), where they function as chaperones to mediate protein folding and maturation (4, 6, 7). Agents that perturb ER protein folding, e.g., thapsigargin, tunicamycin, and calcium * To whom correspondence should be addressed: Department of Pathology, College of Medicine, University of Vermont, A249 Soule Medical Alumni Building, Burlington, VT 05405. Phone: (802) 6569495. Fax: (802) 656-8892. E-mail: [email protected]. † Leiden University. ‡ Adirondack Biomedical Research Institute. § University of Vermont. 1 Abbreviations: IDAM, iodoacetamide; ER, endoplasmic reticulum; Grp, glucose-regulated protein; Hsp, heat shock protein; pkASgrp78, LLC-PK1 cells expressing antisense RNA to grp78; pkNEO, LLC-PK1 cells selected for the expression of a G418 resistance marker; LDH, lactate dehydrogenase; DMEM, Dulbecco’s Modified Eagle’s Medium; DPPD, N,N′-diphenyl-p-phenylenediamine; DTT, dithiothreitol; DTTox, oxidized DTT; Thaps, thapsigargin; Tun, tunicamycin; zVAD, carbobenzoxy-Val-Ala-Asp-fluromethyl ketone; EGTA/AM, acetoxy methyl ester of EGTA; DFOM, desferroxamine; CHX, cycloheximide; LDH, lactate dehydrogenase.

ionophores, cause unfolded proteins to accumulate, thereby activating transcription of ER stress protein genes (5, 6). Increasing Grp levels render cells tolerant to various insults, including calcium overload, oxidative stress, attack by cytotoxic T-cells, antineoplastic etoposides and doxorubicin, nephrotoxic cysteine conjugates, alkylating agents, and organic oxidants (8-19). Conversely, preventing synthesis of Grp78 or Grp94 sensitizes cells to calcium overload and chemical toxicants (9, 10, 18, 19). Thus, enhanced expression of grp genes helps maintain ER function during stress and protects cells from injury. Apoptosis is a controlled form of cell death that occurs under physiological conditions and as a pathological response to injury in all organs (20). The kidney is a particularly important target for a variety of stressful insults, including toxic drugs, chemicals, and environmental pollutants, all of which can cause renal damage and cell death by necrosis and/or apoptosis (21). The pig kidney epithelial cell line, LLC-PK1, is a useful model for investigating cell death induced by nephrotoxicants and cytokines as well as the general cellular response to stress (17-19, 22-28). IDAM is a prototypical soft electrophile that reacts with cellular thiols, as do many chemical toxicants and toxicant metabolites. In LLC-PK1 cells, IDAM exposure causes rapid depletion of gluta-

10.1021/tx990054q CCC: $18.00 © 1999 American Chemical Society Published on Web 08/25/1999

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thione (GSH), protein thiol oxidation, an uncontrolled rise in calcium levels, oxidative stress, and lipid peroxidation (18, 23). Antioxidants block lipid peroxidation, preventing plasma membrane rupture and IDAM-induced cell death (23). Similar pathways of cell killing have been described in renal epithelial cells and other cell types; the subject has been reviewed in refs 21, 29, and 30. IDAM activates the expression of stress response genes, including hsp70 and grp78 in LLC-PK1 cells (18, 24). However, an increasing level of expression of heat shock proteins with prior heat shock does not protect against IDAM necrosis. By contrast, inducing ER stress proteins protects cells from necrosis caused by IDAM or tert-butyl hydroperoxide and prevents the uncontrolled rise in the level of intracellular free calcium that follows toxicant exposure (18, 19). Elevated cytosolic free calcium plays a critical role in cell death in a variety of cells (29, 31, 32), including renal epithelial cells (33). The ER is the major intracellular calcium storage site (34), and a high level of lumenal calcium is essential for normal ER function (35-37). Grp78, Grp94, and calreticulin are calcium binding proteins and help maintain ER calcium levels (18, 19, 38-41). Both ER stress and calreticulin overexpression prevent IDAM-induced necrosis, strongly suggesting that the ability of the ER and ER stress proteins to control cellular calcium levels is important in resting cells and in injured cells destined to undergo necrosis. ER stress can induce apoptosis directly in fibroblasts in vitro and in the kidney epithelium in vivo (42, 43). A number of studies have shown that the levels of ER stress proteins and Bcl-2 can regulate apoptotic cell death in response to ER calcium depletion (44-47). However, the role of ER stress proteins in apoptosis induced by reactive chemical species has not been considered. Moreover, it is not clear if ER stress proteins can regulate apoptosis and necrosis via similar signaling pathways. If the cell death pathway could be switched from necrosis to apoptosis with a single agent, it would provide a unique opportunity to investigate the role of ER stress in both pathways. In this regard, we have shown that antioxidants block necrosis after LLC-PK1 cells are treated with the nephrotoxicant S-(1,2-dichlorovinyl)-Lcysteine (DCVC), but the cells go on to die by apoptosis (48, 49). Since IDAM-induced necrosis is prevented by ER stress (18), we examined whether antioxidants also facilitate apoptosis after IDAM treatment, and if so whether it could be prevented by pre-ER stress. Herein, we show that antioxidants “switch” the cell death pathway after IDAM exposure from necrosis to apoptosis. Using this model, we also show that prior ER stress, but not heat shock, prevented IDAM-induced apoptosis. Finally, the data suggest that functionally distinct ER signaling pathways are involved in cytoprotection from apoptosis and necrosis after IDAM treatment.

Materials and Methods Materials. Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from GIBCO/BRL (Grand Island, NY). LLC-PK1 cells, a renal epithelial cell line with proximal tubular epithelial cell characteristics (50, 51), were obtained from American Type Culture Collection (Rockville, MD) at passage 195 and were used from passage 205 to 215. N,N′-Diphenyl-p-phenylenediamine (DPPD) was obtained from Kodak (Rochester, NY). The acetoxy methyl ester of EGTA (EGTA/AM) was obtained from Molecular Probes (Eugene, OR).

van de Water et al. Desferroxamine mesylate (DFOM), cisplatin, oxidized dithiothreitol (DTTox), dithiothreitol (DTT), and IDAM were from Sigma (St. Louis, MO). [35S]Methionine and [35S]cysteine with varying specific activities were purchased as a mixture from NEN Life Science Products and diluted in culture medium (see below). zVAD.fmk was from Enzyme System Products (Dublin, CA). All other chemicals were obtained from commercial sources. Cell Cultures. LLC-PK1 cells were cultured as described previously (24, 52) and maintained in DMEM supplemented with 10% FBS (complete medium). LLC-PK1 cells expressing an antisense grp78 transcript have been previously described (18). Selected clones of LLC-PK1 cells that contained the pASgrp78 construct (pkASgrp78 clones 5, 8, and 10) and control cells (pkNEO clones 1, 9, and 10) carrying only the neomycin resistance marker were used. Both pkASgrp78 and pkNEO cells were routinely maintained in 400 µg/mL G418 in complete medium but were plated in complete medium without G418 prior to each experiment. Cell Treatments. Prior to treatment with IDAM, cells were washed once with Earle’s Balanced Salt Solution (EBSS). Thereafter, cells were incubated with freshly prepared IDAM in EBSS for 15, 30, 45, or 60 min at the indicated concentration. Following IDAM treatment, cells were washed once with EBSS and allowed to recover in complete medium. Where appropriate, the cells were incubated with the lipophilic antioxidant DPPD (20 µM), prepared as a 20 mM stock in DMSO, during the treatment period as well as the recovery period. To induce ER stress proteins, cells were treated with DTTox (10 mM) for 3 h in EBSS followed by recovery for 16 h in complete medium (17). Cells were also pretreated with 300 nM thapsigargin (Thaps) or 1.5 µg/mL tunicamycin (Tun) in complete medium for 16 h, to induce ER stress proteins, and then used without recovery. For heat shock treatment, confluent cells were incubated for 30, 60, or 120 min in complete medium in a humidified incubator at 46 °C and then returned to complete medium at 37 °C and allowed to recover for 16 h at 37 °C. Determination of Cell Death. The extent of total cell death was determined by measuring the rate of release of LDH from the cells into the medium as described previously (24). Apoptotic cell death was assessed by morphological analysis of the nucleus after staining with Hoechst 33258 and DNA-ladder formation as described previously (48, 49) or by cell cycle analysis after staining with propidium iodide. Cell cycle analysis was performed using a Coulter Epics XL instrument (Coulter Corp., Miami, FL). Floating cells were collected by washing in EBSS. Attached cells were released from the culture surface with trypsin. The floating and trypsinized cells were combined and fixed in 70% ethanol before staining with propidium iodide (Sigma). Cell cycle distribution was analyzed, and the cell populations, including subG0/G1 cells, were identified using the Multicycle application of the Multiplus software supplied by Phoenix Flow Systems (Tucson, AZ). We also measured the activation of caspase-3-like protease activity in cell extracts using acetyl-Asp-Glu-Val-Asp(DEVD)-p-nitroanaline (Enzyme System Products) as the substrate (53). Western Blotting and Analysis of Newly Synthesized Proteins. For protein analysis, cells were washed once after treatment with ice-cold PBS (pH 7.4) and once with ice-cold Tris/ sucrose buffer [10 mM Tris-HCl, 250 mM sucrose, and 1 mM EGTA (pH 7.4)]. Cells were scraped in ice-cold Tris/sucrose containing PMSF (1 mM), leupeptin (10 µg/mL), aprotonin (10 µg/mL), and DTT (1 mM) and disrupted by sonicating three times with 5 s bursts. Protein concentrations were determined by the Bio-Rad methods using IgG as a standard. The level of new protein synthesis was determined by incubating cells with [35S]methionine/cysteine (50 µCi/mL) in complete medium for the indicated periods of time. Equal amounts of cell protein were separated by SDS-PAGE, and newly synthesized proteins were detected by autoradiography. Western blotting was performed on equal amounts of cellular proteins that had been separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked

Endoplasmic Reticulum and Cell Death

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Figure 1. Effect of DPPD on IDAM-induced cytotoxicity. LLC-PK1 cells were treated with 100 µM IDAM for 15, 30, or 45 min in the presence or absence of 20 µM DPPD. Thereafter, cells were allowed to recover in complete medium in the presence or absence of DPPD. (A) After 24 h, phase contrast pictures were taken. Note the attached cells with pyknotic nuclei when IDAM was administered alone and the presence of shrunken and refractile floating cells when IDAM was administered with DPPD (IDAM/DPPD). (B) The extent of LDH release was determined at various times after treatment with IDAM/DPPD, and the data were expressed as the percentage of cell death. The data are the mean ( the standard deviation from triplicate samples collected in a single experiment that is representative of two or three independent experiments; standard deviations were omitted from the figure for clarity, but were typically less than 10% of the mean. (C) After 24 h, low-molecular mass DNA was isolated from cells treated with IDAM plus DPPD as described for panel B, and low-molecular mass DNA was analyzed by agarose gel electrophoresis. (D) After 24 h, control cells and cells treated with IDAM/DPPD for 45 min were fixed with 3.7% formaldehyde, and DNA was stained with Hoechst 33258. Note the fragmentation of the nucleus after IDAM/DPPD treatment. with 5% nonfat dry milk. Western blot analysis for stressinducible Hsp70 (Hsp72) (1:5000 dilution; Amersham) and Hsp90 (1:1000 dilution: StressGen) was carried out using monoclonal antibodies. PARP was detected using a monoclonal antibody (Enzyme System Products). The presence of caspase-3 and caspase-3 fragments was confirmed using a polyclonal rabbit antibody generously provided by D. Nicholson (Merck Frosst). Proteins containing the ER retention sequence, KDEL, were detected using an anti-KDEL antibody (Affinity Bioreagents). Statistical Analysis. A Student’s t test was used to determine if there was a significant difference between two means (p < 0.5). When multiple means were compared, significance was determined by a one-way analysis of variance (ANOVA; p < 0.5). For ANOVA, letter designations are used to indicate significant differences. Means with a common letter designation are not different; those with a different letter designation are significantly different from all other means with different letter designations. For example, a mean designated as A is significantly different from a mean designated B, but neither is different from a mean designated AB.

Results Antioxidants Block IDAM-Induced Necrosis but Not Apoptosis or Caspase Activation. Lipid peroxidation causes plasma membrane rupture and is the final event in the necrotic cell death pathway after IDAM treatment (18, 23). Although DPPD blocks membrane rupture after IDAM exposure for 15 min, it did not block cell death, as indicated by LDH release, after longer periods of exposure (Figure 1A,B). In the presence of the antioxidant DPPD, IDAM-exposed cells died 12-24 h after treatment with morphological and biochemical characteristics of apoptosis, including cell shrinkage and detachment, nuclear blebbing, and nucleosomal fragmentation (Figure 1A-D). Cell cycle analysis confirmed the presence of cells with a DNA content that was lower than the 2N content in G0/G1 cells (subG0/G1) population (Figure 2). Typically, no more than 30% of the cells were detected in the subG0/G1 population, in agreement with

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Figure 2. Cell cycle analysis of IDAM/DPPD-treated cells. A representative cell cycle analysis profile of propidium iodidestained control cells treated with 20 µM DPPD alone (left) and 24 h after treatment with 100 µM IDAM in the presence of DPPD (right). The percentages indicated over the peaks from left to right in each panel are the fraction of the cells in the subG0/G1 (apoptotic), G0/G1, and G2/M populations.

the LDH release data. Detached and floating cells always had an apoptotic morphology and constituted the majority of the subG0/G1 population. Initially, apoptotic cells were impermeable to trypan blue, but eventually became trypan blue positive, accounting for the leakage of LDH, a process termed secondary necrosis (20). Therefore, the extent of LDH release was used as a measure of the level of total cell death, while apoptosis was monitored by following DNA-ladder formation, morphological changes, and the appearance of subG0/G1 cell populations. Caspases, particularly caspase-3 (YAMA/Apopain/ CPP32), are central to the execution phase of apoptosis (54, 55). IDAM treatment in the presence of DPPD also increased caspase-3-like activity in total cell lysates from 166 ( 15 pmol (mg of protein)-1 min-1, in cells treated with DPPD alone, to 326 ( 28 pmol min-1 (mg of protein)-1 in cells treated with 200 µM IDAM (n ) 2). The majority of the caspase activity was found in the detached apoptotic cells. No increase in caspase-1 (ICE)

van de Water et al.

activity was detected (data not shown). PARP and caspase-3 cleavage were also detected largely in the detached apoptotic cells (Figure 3C). The fact that there is relatively less 85 kDa PARP cleavage fragment in the floating cells after 100 µM treatment (100F) relative to that after 50 µM treatment (50F) may reflect an increased level of degradation of cleavage products with increasing injury. In addition, the general caspase inhibitor, zVAD, blocked DNA-ladder formation and the appearance of subG0/G1 cells; however, it did not inhibit cell rupture as determined by the extent of LDH release (Figure 3A,B). Taken together, these data indicate that antioxidants prevent the loss of membrane integrity and necrotic cell death, but the cells go on to die by apoptosis via a caspase-3-mediated mechanism. Although caspase inhibition blocks IDAM-induced apoptosis, it is not sufficient to prevent cell death. Loss of GSH, oxidation of protein thiols, increased intracellular calcium levels, and formation of reactive oxygen species precede lipid peroxidation, plasma membrane rupture, and necrosis following IDAM treatment (18, 23, 24). Reducing agents, such as DTT, the iron chelator desferroxamine, and the cell permeable calcium chelator EGTA/AM block necrotic cell death after IDAM exposure (18, 23). Apoptosis and LDH release induced by IDAM were also blocked by dithiothreitol (DTT) and desferroxamine (Figure 4). However, unlike necrosis, apoptosis was not blocked significantly by EGTA/AM. Similar results were obtained by cell cycle analysis (data not shown). Thus, protein thiol oxidation and reactive oxygen species, but not an increase in the level of intracellular free calcium, are important biochemical events in IDAM-induced apoptosis. ER Stress Provides Tolerance to Cell Death. IDAM treatment activated expression of stress proteins of the Hsp and Grp families. Within 6 h of an IDAM challenge, there was a clear increase in the extent of

Figure 3. Caspase activation and apoptosis after IDAM/DPPD treatment. Cells were treated with IDAM (50 or 100 µM for 60 min) and 20 µM DPPD or with DPPD alone as the control (cont) in the presence or absence of zVAD (100 µM) and were then returned to complete medium containing DPPD (20 µM). Samples of medium, cells, and cell protein were collected 24 h after IDAM treatment. (A) The percentage of cell death in the presence and absence of zVAD was determined by either LDH release (black bars) or analysis of subG0/G1 populations by cell cycle analysis (striped bars). The percentage of cell death and subG0/G1 data are the mean ( the standard deviation of data from five (n ) 5) and three (n ) 3) experiments, respectively. (B) DNA-ladder formation in cells treated with IDAM with or without zVAD; a representative experiment typical of three. (C) Cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3 (Casp-3) in a representative set of samples. Attached (designated A) and floating (designated F) cells were collected separately and the proteins used for Western blotting. There were no floating cells in the control cultures (designated 0). The top arrows in each panel show the position of uncleaved PARP (116 kDa) or caspase-3 (32 kDa) and a fragment (lower arrow) of each, 85 and 17 kDa, respectively. Note that appreciable cleavage of either caspase-3 or PARP was only detected in the floating (apoptotic) cells.


Figure 4. Effect of DTT, desferoxamine (DFOM), and EGTA/ AM on IDAM/DPPD-induced cell death. LLC-PK1 cells were treated with 100 µM IDAM and 20 µM DPPD for 45 min in the presence or absence of 50 µM EGTA/AM or 1 mM DFOM. Thereafter, cells were allowed to recover in complete medium containing DPPD in the presence or absence of 1 mM DFOM or 50 µM EGTA/AM. When cells were treated with DTT, they were first challenged with IDAM and then treated with 10 mM DTT in EBSS for 30 min followed by a recovery period in complete medium with DPPD. (A) After 24 h, low-molecular mass DNA was isolated and DNA-ladder formation was analyzed by agarose gel electrophoresis. (B) After 24 h, the percentage of cell death was determined by LDH release as described in Materials and Methods. The data are presented as the mean ( the standard error of the mean from three or four independent experiments. Significant differences were determined by ANOVA as described in Materials and Methods. Means with a different letter designation are significantly different (p < 0.05).

synthesis of Grp78 and Grp94 as well as Hsp70 and Hsp90 (Figure 5). Prior ER stress, but not heat shock, renders cells tolerant to IDAM-induced necrosis (18, 19). Treating cells with the oxidized form of DTT (DTTox), thapsigargin, or tunicamycin, all of which increase the level of expression of ER stress proteins in LLC-PK1 cells (17, 18), prior to IDAM exposure protected against apoptosis (Figure 6A,B) and prevented nuclear fragmentation and DNA-ladder formation (Figure 6) as well as the appearance of subG0/G1 cell populations (data not shown). Heat shock increases the level of expression of Hsp70, as expected (data not shown and ref 24), but did not block IDAM-induced cell death to the extent observed with pre-ER stress (Table 1). Thus, pre-ER stress, but not heat shock, is able to protect LLC-PK1 cells from both necrotic (18) and apoptotic cell death caused by exposure to IDAM. Distinct ER Signaling Pathways Prevent Apoptosis and Necrosis after IDAM Treatment. Grp78 is the prototypical stress inducible ER chaperone (5, 56, 57). Expression of a 0.5 kb antisense grp78 fragment in LLCPK1 cells (pkASgrp78 cells) results in defective upregulation of Grp78 after ER stress. Consequently, pkASgrp78 cells do not develop tolerance to IDAM in response to pre-ER stress and are more sensitive to


Figure 5. Induction of stress protein after IDAM treatment. LLC-PK1 cells were treated with 100 µM IDAM and 20 µM DPPD for 45 min. Thereafter, cells were allowed to recover in complete medium with DPPD for 6 h in the presence of [35S]methionine/cysteine so newly synthesized proteins which were visualized by autoradiography after separation by SDS-PAGE could be labeled. The autoradiogram is representative of data collected in three independent experiments. The positions of Grp94, Hsp90, Grp78, and Hsp72/73 are shown.

induction of necrosis by both IDAM and tert-butyl hydroperoxide challenge (18, 19). Surprisingly, the pkASgrp78 cells were less sensitive to IDAM-induced apoptosis (Figure 7). We also determined if pkASgrp78 cells were resistant to apoptosis caused by treating cells with thapsigargin to deplete the ER calcium pool (Table 2). Neither pkNEO nor pkASgrp78 cells underwent apoptosis when they were treated with thapsigargin alone, but in the presence of cycloheximide and thapsigargin, there was a significant increase in the level of apoptosis in pkNEO cells. By contrast, there was no significant increase in the level of cell death in the pkASgrp78 cells treated with thapsigargin and cycloheximide compared to the level in those treated with cycloheximide alone. It should be noted that the pkASgrp78 cells were more sensitive to cycloheximide alone, but this does not account for the lack of an effect with thapsigargin. Thus, the data suggest that the pkASgrp78 cells were more also resistant to thapsigargininduced apoptosis. In preliminary experiments, we found that when cycloheximide was added after IDAM treatment, the tolerance imparted by pre-ER stress was reversed, suggesting that tolerance depends on both prior stress protein synthesis and general protein synthesis after IDAM challenge (data not shown). To determine if pkASgrp78 cells were capable of undergoing apoptosis in response to IDAM, we determined if cycloheximide reversed tolerance to IDAM-induced apoptosis in pkASgrp78 cells. Tolerance to IDAM-induced apoptosis was almost completely blocked in pkASgrp78 cells by adding cycloheximide during the recovery period (Figure 7). Thus, the defect in pkASgrp78 cells is not due to an

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Figure 6. Effect of pre-ER stress on IDAM/DPPD-induced cell death. LLC-PK1 cells were subjected to pre-ER stress by pretreating them with DTTox, thapsigargin (THAPS), or tunicamycin (TUN). Thereafter, cells were treated with 100 µM IDAM and 20 µM DPPD, indicated as IDAM or IDAM/DPPD in the figure, for 45 min followed by recovery in complete medium with DPPD. (A) Phase contrast micrograph of the cell 24 h after treating with IDAM and DPPD only (IDAM/DPPD) or with DTTox pretreatment (IDAM/ DPPD + DTTox). (B) The extent of LDH release into the medium was also determined 24 h after treatment and expressed as the percentage of cell death. The data presented are the mean ( the standard deviation of three to five independent experiments. Significant differences were determined by ANOVA as described in Materials and Methods. Means with a different letter designation are significantly different (p < 0.05). (C) Low-molecular mass DNA was isolated from cells treated with IDAM as described for panel B and analyzed by agarose gel electrophoresis. (D) Cells treated with IDAM with DPPD and IDAM with DTTox, as described for panel A, were fixed with 3.7% formaldehyde, and DNA was stained with Hoechst 33258. Note the fragmentation of the nucleus after IDAM/DPPD treatment but not in the cell treated with IDAM/DPPD after DTTox pretreatment in panel D. Also note that the cells pretreated with DTTox (A) exhibit normal morphology after IDAM challenge (compare to Figure 1). Table 1. Effect of Pre-Heat Shock on IDAM-Induced Cell Deatha treatment

% cell death

treatment

% cell death

none pre-HS

0.9 ( 0.1A 0.7 ( 0.7A

IDAM pre-HS/IDAM

54 ( 13B 31 ( 14B

a Cells either were heat shocked for 2 h at 46 °C followed by recovery for 16 h at 37 °C in complete medium or were left at 37 °C. Thereafter, cells were treated with 100 µM IDAM for 45 min in EBSS and recovered in complete medium. After 24 h, the percentage of cell death was determined by measuring the amount of LDH released into the medium. The data are presented as the mean ( the standard error of the mean of data collected in two experiments. Significant differences were determined by ANOVA as described in Materials and Methods. Means with a different letter designation are significantly different (p < 0.05). Although heat shock did not have a statistically significant effect on cell death, there was a reduction in the extent of LDH release in both experiments.

inability to initiate apoptosis since the cells remain responsive to IDAM-induced apoptosis when protein synthesis is blocked. Perturbing grp78 expression with antisense or expressing ATPase dead mutants of Grp78 causes ER stress (9, 16, 58); therefore, we determined if expression of other ER stress proteins, such as Grp94, was induced as an indication of constitutive ER stress in the pkASgrp78 cells. Indeed, Grp94 protein levels were increased markedly in the pkASgrp78 clones compared to those in control pkNEO clones. The level of a ∼50 kDa protein that

reacted with an antibody against the KDEL sequence, found in many ER resident proteins, also increased but not to the extent observed with Grp94 (Figure 8). However, the level of another ER stress protein, calreticulin, was not altered appreciably (Figure 8). Thus, blocking Grp78 biosynthesis with antisense grp78 apparently disrupted ER function, causing increase expression of other ER stress proteins. Activation of these ER stress pathways may prevent apoptotic cell death following IDAM treatment.

Discussion Recently, we showed that induction of ER stress proteins protects LLC-PK1 cells from necrosis induced by IDAM, nephrotoxic cysteine conjugates, and tert-butyl hydroperoxide (17-19). In addition, we demonstrated that regulating plasma membrane integrity is an important determinant of whether renal epithelial cells die by apoptosis or necrosis after exposure to chemical toxicants (48, 49). In this study, we combined these models to investigate the role of ER stress proteins in protection against apoptosis and necrosis using a single agent, IDAM. Several important conclusions can be drawn from the results. First, as with DCVC (48, 49), antioxidants can protect against IDAM-induced necrosis, but actually have a permissive effect on apoptotic cell death. Second, the biochemical events that trigger IDAM-induced cell



Figure 7. IDAM/DPPD-induced apoptosis in pkASgrp78 cells. LLC-PK1 cells expressing an antisense grp78 construct (pkASgrp78) or NEO-resistant control (pkNEO) were treated with 100 µM IDAM and 20 µM DPPD, indicated as IDAM in the figure, for 45 min in EBSS. Thereafter, cells were allowed to recover in complete medium containing DPPD in the presence or absence of 10 µM cycloheximide (CHX). (A) After 24 h, the amount of LDH released into the medium was determined and expressed as the percentage of cell death. The data are the mean ( the standard deviation of data collected from three independent pkNEO and pkASgrp78 clones. Significant differences were determined by ANOVA as described in Materials and Methods. Means with a different letter designation are significantly different (p < 0.05). (B) After 24 h, low-molecular mass DNA was isolated from cells treated with IDAM for 45 min and analyzed by agarose gel electrophoresis. DNA ladders for pkNEO clone 10 and pkASgrp78 clone 10 are shown and are representative of the patterns observed in the three clones of pkNEO and pkASgrp78 cells that were tested in two independent experiments. Table 2. Thapsigargin-Induced Cell Death in pkAsgrp78 Cellsa treatment pkNEO pkNEO pkNEO pkNEO pkASgrp78 pkASgrp78 pkASgrp78 pkASgrp78

% cell death control Thaps CHX Thaps/CHX control Thaps CHX Thaps/CHX

4.2 ( 2.9A 4.2 ( 2.9A 9.9 ( 3.9B 30.0 ( 5.2C 2.7 ( 1.2A 6.3 ( 2.9A 15.5 ( 3.5B,D 22.9 ( 3.0D

a LLC-PK1 cells expressing an antisense grp78 construct (pkASgrp78) or NEO-resistant control (pkNEO) were treated with thapsigargin (Thaps; 300 nM) with or without cycloheximide (CHX; 10 µM) in complete medium. After 24 h, the percentage of cell death was determined by measuring the extent of LDH release into the medium. Data are the means ( the standard error of the mean for cell death determined from three independent pkNEO and pkASgrp78 clones in three independent experiments. Significant differences were determined by ANOVA as described in Materials and Methods. Means with a different letter designation are significantly different (p < 0.05).

death by necrosis and apoptosis are similar in that both require the loss of (non)protein thiols and formation of reactive oxygen species. However, unlike IDAM-induced necrosis, apoptosis apparently does not require an uncontrolled rise in the level of intracellular calcium. Third, pre-ER stress protected against apoptosis and necrosis after IDAM treatment, yet the mechanisms through which the ER regulates protection against these distinct pathways of cell death are different. To our knowledge, it has not been shown that functionally distinct pathways emanating from the ER regulate necrosis versus apoptosis. Preliminary studies indicate that ER stress also prevents nephrotoxicant-induced cell death in primary cultures of proximal tubule epithelial cells and in the kidney in vivo, supporting a more general role for ER stress in protecting renal epithelial cells from injury.2 Our observations are also in general agreement with the

Figure 8. Expression of Grp94, calreticulin, and KDELcontaining proteins in pkASgrp78 and pkNEO cells. The amounts of total cellular protein from confluent pkASgrp78 and pkNEO monolayers were separated by SDS-PAGE and immunoblotted for Grp94, calreticulin (CRT), and KDEL-containing proteins. Note that pkASgrp78 cells contain relatively more Grp94 and proteins containing the ER retention signal KDEL. The data are from one pkNEO clone and one pkASgrp78 clone but are representative of all the pkASgrp78 and pkNEO clones used to generate the data depicted in Figure 7.

observation that induction of ER stress proteins is important in protection against apoptosis caused directly by ER stress (45). Heat shock proteins have been shown to play a role in protecting HeLa cells from heat-induced apoptosis (59). However, in LLC-PK1 cells, the protection afforded by prior heat shock against IDAM-induced cell death was only slight, and indeed insignificant, while pre-ER stress completely blocked apoptosis. Similar effects were observed in IDAM-induced necrosis (18). However, we are not suggesting that Hsps are not important for survival, but rather that they may play a distinct and complementary role in the cellular response to stress. For example, protein thiol oxidation strongly activates Hsp transcription and is an early event in both apoptotic and necrotic cell death after IDAM exposure (Figure 2 and refs 18 and 24). It seems likely that protein thiol oxidation causes an immediate cellular crisis, such as the release of ER calcium or the loss of protein synthesis, which is checked by an ER stress response. Yet, repair of the oxidized and denatured proteins must also occur if the cells are to survive, a repair process that is likely to involve Hsps (1-3). If the kinetics of the protein repair process are slow, relative to the time course of cell killing, then Hsp induction could not serve as a primary line of defense against cell death. However, the heat shock response would play a later role in general protein repair once the cell has survived the immediate crisis by increasing the level of ER stress protein synthesis. Thus, Grp and Hsp induction may both be integral components 2

B. van de Water and J. L. Stevens, unpublished results.

950


of the cellular response to stress, but act in different aspects of the repair response. Although ER stress proteins prevent both necrosis and apoptosis after IDAM treatment, several lines of evidence suggest that the role of the individual ER stress proteins and the signaling pathways that are responsible are functionally distinct. In LLC-PK1 cells, perturbing Grp78 synthesis with an antisense grp78 construct rendered cells more sensitive to IDAM- and tert-butyl hydroperoxide-induced necrosis but resistant to IDAM-induced apoptosis (18, 19). Apparently, these cells adapted to the presence of the antisense grp78 RNA by increasing the levels of expression of other ER stress proteins, including Grp94 and another KDEL protein (Figure 8). This adaptive response must involve proteins or combinations of proteins that are different from those that protect against necrosis since pkASgrp78 cells are less sensitive to IDAM-induced apoptosis (18). In addition, LLC-PK1 cells that overexpress calreticulin are tolerant to ionomycin, IDAM, and tert-butyl hydroperoxide-induced necrosis but are not resistant to IDAM-induced apoptosis.3 Thus, it seems clear that distinct ER signaling pathways and/or subsets of ER stress proteins mediate protection against apoptosis and necrosis and by functionally distinct mechanisms. Differences in the role of calcium in necrosis and apoptosis may underlie the differences seen in the ability of ER stress and different ER stress proteins to prevent these two forms of cell death (18). For example, DPPD blocks the uncontrolled rise in the level of calcium seen after IDAM treatment (18) but does not prevent apoptosis, indicating that increasing the cytosolic level of free calcium is not a signal for apoptosis in this model. In addition, EGTA/AM did not protect against apoptosis but prevents necrosis because it buffers the increase in the level of cellular calcium (18, 19). Although these data are not consistent with a role for increased cellular calcium levels in apoptosis, they do not exclude a role for depletion of ER calcium as a signal. Indeed, thapsigargin, which depletes ER calcium by blocking the ER Ca2+-ATPase, can induce apoptosis in these cells (data not shown) and others (44, 60, 61), supporting a role for ER calcium depletion in apoptosis. In conclusion, pre-ER stress can protect against both necrosis and apoptosis, yet the mechanisms by which ER stress signals and/or subsets of stress proteins control necrosis and apoptosis are different. Future investigations should delineate the roles of specific ER stress proteins in controlling either type of cell death.

Acknowledgment. These studies were supported by a Colgate Palmolive Fellowship to B.v.d.W., a Talent Stipend from the Dutch Organization for Scientific Research to B.v.d.W., and by Grants DK46267 and ES05670 to J.L.S. We also thank Don Nicholson for providing reagents. Special thanks go to Steve Goodrich and Russel Bowes and other members of the laboratory for technical support and for comments on the manuscript. We also appreciate the assistance of Dr. JoEllen Welsh and Ms. Betty Nolan with the analysis of apoptosis by flow cytometry. 3 S. Asmellash, B. van de Water, and J. L. Stevens, unpublished results.

van de Water et al.

References (1) Georgopoulos, C., and Welch, W. J. (1993) Role of the major heat shock proteins as molecular chaperones. Annu. Rev. Cell Biol. 9, 601-634. (2) Hendrick, J. P., and Hartl, F. U. (1993) Molecular chaperone functions of heat-shock proteins. Annu. Rev. Biochem. 62, 349384. (3) Hightower, L. E. (1991) Heat shock, stress proteins, chaperones and proteotoxicity. Cell 66, 191-197. (4) 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. (5) Lee, A. S. (1992) Mammalian stress response: induction of the glucose-regulated protein family. Curr. Opin. Cell Biol. 4, 267273. (6) Gething, M.-J., and Sambrook, J. (1992) Protein folding in the cell. Nature 355, 33-45. (7) Helenius, A., Trombetta, E. S., Hebert, D. N., and Simons, J. F. (1997) Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol. 7, 193-200. (8) Brostrom, M. A., Cade, C., Prostko, C. R., Gmitter-Yellen, D., and Brostrom, C. O. (1990) Accommodation of protein synthesis to chronic deprivation of intracellular sequestered calcium. A putative role for GRP78. J. Biol. Chem. 265, 20539-20546. (9) Li, L.-J., Li, X., Ferrario, A., Rucker, N., Liu, E. S., Wong, S., Gomer, C. J., and Lee, A. S. (1992) Establishment of a Chinese hamster ovary cell line that expresses grp78 antisense transcripts and suppresses A23187 induction of both GRP78 and GRP94. J. Cell Physiol. 153, 575-582. (10) Little, E., and Lee, A. S. (1995) Generation of a mammalian cell line deficient in glucose-regulated protein stress induction through a targeted ribozyme driven by a stress-inducible promoter. J. Biol. Chem. 270, 9526-9534. (11) Gomer, C. J., Ferrario, A., Rucker, N., Wong, S., and Lee, A. S. (1991) Glucose regulated protein induction and cellular resistance to oxidative stress mediated by porphyrin photosensitization. Cancer Res. 51, 6574-6579. (12) Brostrom, C. O., Prostko, C. R., Kaufman, R. J., and Brostrom, M. A. (1996) Inhibition of translational initiation by activators of the glucose-regulated stress protein and heat shock protein stress response systems. Role of the interferon-inducible double-stranded RNA-activated eukaryotic initiation factor 2R-kinase. J. Biol. Chem. 271, 24995-25002. (13) Hughes, C. S., Shen, J. W., and Subjeck, J. R. (1989) Resistance to etoposide induced by three glucose-regulated stresses in Chinese hamster ovary cells. Cancer Res. 49, 4452-4454. (14) Shen, J., Hughes, C., Chao, C., Cai, J., Bartels, C., Gessner, T., and Subjeck, J. (1987) Coinduction of glucose-regulated proteins and doxorubicin resistance in Chinese hamster cells. Proc. Natl. Acad. Sci. U.S.A. 84, 3278-3282. (15) Sugawara, S., Takeda, K., Lee, A., and Dennert, G. (1993) Suppression of stress protein GRP78 induction in tumor-B/C10ME eliminates resistance to cell mediated cytotoxicity. Cancer Res. 53, 6001-6005. (16) Morris, J. A., Dorner, A. J., Edwards, C. A., Hendershot, L. M., and Kaufman, R. J. (1997) Immunoglobulin binding protein (BiP) function is required to protect cells from endoplasmic reticulum stress but is not required for the secretion of selective proteins. J. Biol. Chem. 272, 4327-4334. (17) Halleck, M. M., Liu, H., North, J., and Stevens, J. L. (1997) Reduction of trans-4,5-dihydroxy-1,2-dithiane (DTTox) by cellular oxidoreductases activates gadd153 and grp78 transcription and induces cellular tolerance in kidney epithelial cells. J. Biol. Chem. 272, 21760-21766. (18) Liu, H., Bowes, R. C., van de Water, B., Sillence, C., Nagelkerke, J. F., and Stevens, J. L. (1997) Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances and cell death in renal epithelial cells. J. Biol. Chem. 272, 21751-21759. (19) Liu, H., Miller, E., van de Water, B., and Stevens, J. L. (1998) Endoplasmic reticulum stress proteins block oxidant-induced Ca2+ increases and cell death. J. Biol. Chem. 274, 12858-12862. (20) Majno, G., and Joris, I. (1995) Apoptosis, oncosis and necrosis. An overview of cell death. Am. J. Pathol. 146, 3-15. (21) Goldstein, R. S., and Schnellmann, R. G. (1996) Toxic responses of the kidney. In Toxicology (Klaassen, C. D., Ed.) pp 417-442, McGraw-Hill, New York. (22) Chen, Q., Yu, K., and Stevens, J. L. (1992) Regulation of the cellular stress response by reactive electrophiles: The role of covalent binding and cellular thiols in transcriptional activation of the 70 kDa heat shock protein gene by nephrotoxic cysteine conjugates. J. Biol. Chem. 267, 24322-24327.

Endoplasmic Reticulum and Cell Death (23) Chen, Q., and Stevens, J. L. (1991) Inhibition of iodoacetamide and tert-butylhydroperoxide toxicity in LLC-PK1 cells by antioxidants: A role for lipid peroxidation in alkylation induced cytotoxicity. Arch. Biochem. Biophys. 284, 422-430. (24) Liu, H., Lightfoot, D. L., and Stevens, J. L. (1996) Activation of heat shock factor (HSF) by alkylating agents is triggered by glutathione depletion and oxidation of protein thiols. J. Biol. Chem. 271, 4805-4812. (25) Chen, Q., Yu, K., Holbrook, N. J., and Stevens, J. L. (1992) Activation of the growth arrest and DNA damage-inducible gene gadd153 by nephrotoxic cysteine conjugates and dithiothreitol. J. Biol. Chem. 267, 8207-8212. (26) Mayeux, P. R., and Shah, S. V. (1993) Intracellular Calcium Mediates the Cytotoxicity of Lipid-A in LLC-PK(1) Cells. J. Pharmacol. Exp. Ther. 266, 47-51. (27) Ueda, N., and Shah, S. V. (1992) Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells. J. Clin. Invest. 90, 2593-2597. (28) Soler, A. P., Mullin, J. M., Knudsen, K. A., and Marano, C. W. (1996) Tissue remodeling during tumor necrosis factor-induced apoptosis in LLC-PK1 renal epithelial cells. Am. J. Physiol. 270, F869-F879. (29) Nicotera, P., Bellomo, G., and Orrenius, S. (1990) The role of Ca2+ in cell killing. Chem. Res. Toxicol. 3, 484-494. (30) Trump, B. F., and Berezesky, I. K. (1995) Calcium-mediated cell injury and cell death. FASEB J. 9, 219-228. (31) Reed, D. J. (1990) Review of the current status of calcium and thiols in cellular injury. Chem. Res. Toxicol. 3, 495-502. (32) Farber, J. L. (1990) The role of calcium in lethal cell injury. Chem. Res. Toxicol. 3, 503-508. (33) van de Water, B., Zoeteweij, J. P., de Bont, H. J. G. M., Mulder, G. J., and Nagelkerke, J. F. (1994) Role of mitochondrial calcium in the oxidative stress-induced dissipation of the mitochondrial membrane potential: Studies in isolated proximal tubular cells using the nephrotoxin 1,2-dichlorovinyl-cysteine. J. Biol. Chem. 269, 14546-14552. (34) Pozzan, T., Rizzuto, R., Volpe, P., and Meldolesi, J. (1994) Molecular and cellular physiology of intracellular calcium stores. Physiol. Rev. 74, 595-636. (35) Brostrom, C. O., and Brostrom, M. A. (1990) Calcium-dependent regulation of protein sythesis in intact mammalian cells. Annu. Rev. Physiol. 52, 577-590. (36) Sambrook, J. F. (1990) The involvement of calcium in transport of secretory proteins from the endoplasmic reticulum. Cell 61, 197-199. (37) Brostrom, C. O., and Brostrom, M. A. (1997) Regulation of translational initiation during cellular responses to stress. Prog. Nucleic Acid Res. Mol. Biol. 58, 79-125. (38) Macer, D. R. J., and Koch, G. L. E. (1988) Identification of a set of calcium-binding proteins in reticuloplasm, the luminal content of the endoplasmic reticulum. J. Cell Sci. 91, 61-70. (39) Villa, A., Podini, P., Clegg, D. O., Pozzan, T., and Meldolesi, J. (1991) Intracellular Ca2+ stores in chicken purkinje neurons: Differential distribution of the low affinity-high capacity Ca2+ binding protein, calsequestrin, of Ca2+ ATPase and of the ER lumenal protein, BiP. J. Cell Biol. 113, 779-791. (40) Michalak, M., Milner, R. E., Burns, K., and Opas, M. (1992) Careticulin. Biochem. J. 285, 681-692. (41) Lievremont, J.-P., Rizzuto, R., Hendershot, L., and Meldolesi, J. (1997) BiP, a major chaperone protein of the endoplasmic reticulum lumen, plays a direct and important role in the storage of the rapidly exchanging pool of Ca2+. J. Biol. Chem. 272, 3087330879. (42) Carlberg, M., Dricu, A., Blegen, H., Kass, G. E., Orrenius, S., and Larsson, O. (1996) Short exposures to tunicamycin induce apoptosis in SV40-transformed but not in normal human fibroblasts. Carcinogenesis 17, 2589-2596. (43) Zinszner, H., Kuroda, M., Wang, X., Batchvarova, N., Lightfoot, R. T., Remotti, H., Stevens, J. L., and Ron, D. (1998) CHOP is


(44) (45)

(46)

(47) (48)

(49) (50) (51)

(52)

(53)

(54) (55) (56)

(57)

(58)

(59)

(60) (61)

implicated in programmed cell death in response to impared function of the endoplasmic reticulum. Genes Dev. 12, 982-995. He, H., Lam, M., McCormick, T. S., and Distelhorst, C. W. (1997) Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2. J. Cell Biol. 138, 1219-1228. McCormick, T. S., McColl, K. S., and Distelhorst, C. W. (1997) Mouse lymphoma cells destined to undergo apoptosis in response to thapsigargin treatment fail to generate a calcium-mediated grp78/grp94 stress response. J. Biol. Chem. 272, 6087-6092. Baffy, G., Miyashita, T., Williamson, J. R., and Reed, J. C. (1993) Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production. J. Biol. Chem. 268, 6511-6519. Reynolds, J. E., and Eastman, A. (1996) Intracellular calcium stores are not required for Bcl-2-mediated protection from apoptosis. J. Biol. Chem. 271, 27739-27743. van de Water, B., Kruidering, M., and Nagelkerke, J. F. (1996) Role of F-actin damage and cell detachment in apoptotic cell death of cultured proximal tubular cells. Am. J. Physiol. 270, F593F603. Zhan, Y., Cleveland, J. L., and Stevens, J. L. (1997) A role for c-myc in chemically induced renal-cell death. Mol. Cell. Biol. 17, 6755-6764. Hull, R. N., Cherry, W. R., and Weaver, G. W. (1976) The origin and characteristics of a pig kidney cell strain, LLC-PK1. In Vitro 12, 670-677. Stevens, J., Hayden, P., and Taylor, G. (1986) The role of glutathione conjugate metabolism and cysteine conjugate β-lyase in the mechanism of S-cysteine conjugate toxicity in LLC-PK1 cells. J. Biol. Chem. 261, 3325-3332. Chen, Q., Jones, T. W., Brown, P. C., and Stevens, J. L. (1990) The mechanism of cysteine conjugate cytotoxicity in renal epithelial cells. Covalent binding leads to thiol depletion and lipid peroxidation. J. Biol. Chem. 265, 21603-21611. Datta, R., Kojima, H., Banach, D., Bump, N. J., Talanian, R. V., Alnemri, E. S., Weichselbaum, R. R., Wong, W. W., and Kufe, D. W. (1997) Activation of a CrmA-insensitive, p35-sensitive pathway in ionizing radiation-induced apoptosis. J. Biol. Chem. 272, 19651969. Nicholson, D. W., and Thornberry, N. A. (1997) Caspases: killer proteases. Trends Biochem. Sci. 22, 299-306. Cohen, G. M. (1997) Caspases: the executioners of apoptosis. Biochem. J. 326, 1-16. Li, W. W., Sistonen, L., Morimoto, R. I., and Lee, A. S. (1994) Stress induction of the mammalian GRP78/BiP protein gene: In vivo genomic footprinting and identification of p70CORE from human nuclear extract as a DNA-binding component specific to the stress regulatory element. Mol. Cell. Biol. 14, 5533-5546. Li, W. W., Hsiung, Y., Zhou, Y., Roy, B., and Lee, A. S. (1997) Induction of the mammalian GRP78/BiP gene by Ca2+ depletion and formation of aberrant proteins: Activation of the conserved stress-inducible grp core promoter element by the human nuclear factor YY1. Mol. Cell. Biol. 17, 54-60. Hendershot, L. M., Wei, J. Y., Gaut, J. R., Lawson, B., Freiden, P. J., and Murti, K. G. (1995) In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum. Mol. Biol. Cell 6, 283-296. Mosser, D. D., Caron, A. W., Bourget, L., Denis-Larose, C., and Massie, B. (1997) Role of human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol. Cell. Biol. 17, 5317-5327. McConkey, D. J., and Orrenius, S. (1996) The role of calcium in the regulation of apoptosis. J. Leukocyte Biol. 59, 775-783. Distelhorst, C. W., Lam, M., and McCormick, T. S. (1996) Bcl-2 inhibits hydrogen peroxide-induced ER Ca2+ pool depletion. Oncogene 12, 2051-2055.

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