Hsp70 Inhibits Heat-induced Apoptosis Upstream of Mitochondria by Preventing Bax Translocation*

Hsp70 overexpression can protect cells from stress-induced apoptosis. Our previous observation that Hsp70 inhibits cytochrome c release in heat-stressed cells led us to examine events occurring upstream of mitochondrial disruption. In this study we examined the effects of heat shock on the proapoptotic Bcl-2 family member Bax because of its central role in regulating cytochrome c release in stressed cells. We found that heat shock caused a conformational change in Bax that leads to its translocation to mitochondria, stable membrane association, and oligomerization. All of these events were inhibited in cells that had elevated levels of Hsp70. Hsp70 did not physically interact with Bax in control or heat-shocked cells, indicating that Hsp70 acts to suppress signals leading to Bax activation. Hsp70 inhibited stress-induced JNK activation and inhibition of JNK with SP600125 or by expression of a dominant negative mutant of JNK-blocked Bax translocation as effectively as Hsp70 overexpression. Hsp70 did not protect cells expressing a mutant form of Bax that has constitutive membrane insertion capability or cells treated with a small molecule activator of apoptosome formation, indicating that it is unable to prevent cell death after mitochondrial disruption and caspase activation have occurred. These results indicate that Hsp70 blocks heat-induced apoptosis primarily by inhibiting Bax activation and thereby preventing the release of proapoptotic factors from mitochondria. Hsp70, therefore, inhibits events leading up to mitochondrial membrane permeabilization in heat-stressed cells and thereby controls the decision to die but does not interfere with cell death after this event has occurred.

Regulated cell death is essential for the proper functioning of all multicellular organisms. Cells that have been damaged by stress have the option of actively engaging the intrinsic apoptotic pathway leading to their self-destruction (1,2). Dismantling of the dying cell is carried out by the caspase family of cysteine proteases, which normally lie dormant in healthy cells (3). Their activation requires the formation of the apoptosome, an oligomeric complex containing an active initiator caspase (caspase-9), which processes and activates effector caspases (caspases 3, 6, and 7). Formation of the apoptosome occurs through a cytochrome c-mediated conformational change in the cytosolic protein Apaf-1 5 that facilitates its oligomerization and pro-caspase-9 recruitment (4). Ultimately, the process of cell death is controlled by the regulated release of cytochrome c and other proapoptogenic factors such as the apoptosis inducing factor from mitochondria (5). This step is under the control of the Bcl-2 family of pro-apoptotic and anti-apoptotic proteins (6). Each of the anti-apoptotic members of this family contains 3 or 4 conserved Bcl-2 homology (BH) domains. Their ability to suppress apoptosis is prevented by interactions with pro-apoptotic BH3-only proteins. It is believed that discrete stress signals are conveyed to the anti-apoptotic Bcl-2 family members by diverse BH3-only proteins (6). Inhibition of the prosurvival function of these proteins is essential for the activation of the proapoptotic BH1-3 family members Bax and Bak, although some BH3-only members can directly activate Bax (7). The exact mechanism by which stress leads to Bax activation is not completely understood. Once activated, the normally cytoplasmic Bax assumes a conformation that exposes its C-terminal membrane insertion domain allowing it to integrate into the outer mitochondrial membrane where its oligomerization leads to membrane permeabilization (8 -11).
Another option that is available to cells under stress is to suppress the apoptotic program and attempt to repair the damage. This can be accomplished by members of the heat shock protein family of molecular chaperones. These proteins play essential roles in regulating protein conformation by preventing protein misfolding and aggregation as well as assisting in the productive folding of proteins to their native state (12). When cells are exposed to protein-damaging stresses, the increased burden of non-native protein conformations triggers the transcriptional induction of heat shock protein genes (13). Cells recovering from a transient exposure to mild hyperthermia contain elevated levels of heat shock proteins and consequently possess an increased ability to tolerate normally lethal exposures to elevated temperatures, a phenomenon known as induced thermotolerance (14). This thermotolerant state is in part attributable to the ability of heat shock proteins to inhibit apoptosis (15). The induced synthesis of heat shock proteins represents an adaptive response that allows organisms to cope with fluctuating environmental conditions. The ability to suppress apoptosis must be tightly regulated to prevent the inappropriate survival of damaged cells. Deregulated apoptosis is an essential step in tumor progression (16). Tumor cells often contain elevated levels of antiapoptotic proteins or express reduced levels or defective forms of proapoptotic proteins including Bax (17). Several of the heat shock proteins are also overexpressed in tumors, and the selective prosurvival advantage that they provide could contribute to the process of tumorigenesis (18,19). Consequently, targeting heat shock protein function in tumor cells is currently assessed as a potential anticancer therapy (20). Clearly, an understanding of how heat shock proteins prevent apoptosis will be needed to effectively judge whether they will serve as useful therapeutic targets.
The heat shock proteins appear to be pleiotropic inhibitors of apoptotic pathways. Hsp70, the major stress-inducible heat shock protein, has been shown to inhibit the stress-induced JNK signaling pathway (21,22), cytochrome c release (23,24), apoptosome formation (25,26), and caspase activation (21,27) as well as events that are independent of caspase activation, including prevention of lysosomal disruption (28) and nuclear uptake of apoptosis inducing factor (29). Whether Hsp70 acts directly to suppress all of these effects to maintain survival under diverse forms of stress-induced apoptosis is not clear. Two studies have reported an ability of Hsp70 to prevent apoptosis without inhibiting cytochrome c release (30,31). As well, a number of reports indicate that inhibition of JNK activation is not universally observed in cells overexpressing Hsp70 (21,27,30). Recently, the ability of Hsp70 to prevent the cytochrome c/dATP-mediated activation of Apaf-1 in vitro has been questioned (28,32).
Much of the focus on the antiapoptotic function of Hsp70 has been on events that occur after mitochondrial disruption. In contrast there is very little information on the effects of hyperthermia on the activity of pro-apoptotic Bcl-2 family members and how heat shock proteins might regulate their activities. In this study we examined the effects of heat shock on the pro-apoptotic protein Bax in cells with tetracyclineregulated expression of Hsp70. An important question was whether or not Hsp70 could prevent apoptosis after Bax translocation and apoptosome formation had occurred. Our results suggest that inhibition of Bax translocation to mitochondria by Hsp70 is the critical determining factor in the cellular decision to either initiate or suppress heat-induced apoptosis.
Cells and Treatments-The human acute lymphoblastic T cell line, PEER, with tetracycline-regulated expression of Hsp70 (PErTA70) and Hsp70 lacking the ATPase domain (PErTA70⌬ATPase) have been described previously (24). PErTA70 cells were maintained at 37°C in a 5% CO 2 humidified incubator in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum (Invitrogen). Cells were heat-shocked by placing log-phase cultures into 15-ml centrifuge tubes and immersion in a circulating water bath maintained at 43 Ϯ 0.1°C. After the hyperthermic treatments, the cells were either diluted in fresh 37°C medium, transferred to a culture flask and returned to the 37°C incubator, or placed directly on ice and washed in cold PBS for collection.
A HeLa cell line with tetracycline-regulated expression of a Bax-yellow fluorescent protein (YFP) fusion protein (HeLa-rtTA/Bax:YFP) was generated by stable transfection of HeLa-rtTA cells with the plasmid pBI-EYFP:Bax*tk/hygro. The plasmid was constructed by first replacing the GFP sequence in pBI-EGFP (BD Biosciences Clontech, Palo Alto, CA) with YFP from pEYFP-N1 (Clontech). The hygromycin resistance gene, under control of the thymidine kinase promoter (from pCEP4, Invitrogen) was then added to this plasmid. The Bax sequence with an N-terminal linker from the plasmid GFP-Bax (10) (obtained from R. J. Youle, National Institutes of Health) was ligated in-frame with the YFP sequence using the unique BsrGI site present in the sequence of both GFP and YFP to create the plasmid pBI-EYFP:Bax*tk/hygro. HeLa-rtTA cells were transfected by calcium phosphate precipitation. Stably transfected cells were selected with hygromycin B (Invitrogen Inc.) and cloned by limiting dilution. Clones with minimal fluorescence in the non-induced state and highly fluorescent when induced were selected. The HeLa-rtTA and HeLa-rtTA/Bax:YFP cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.
To inhibit the activation of ERK or JNK, cells were pretreated with PD98059 (#EI-360 Biomol, Plymouth Meeting, PA) or SP600125 (Anthra [1,9-cd]prazol-6(2H)-one, #EI-305 Biomol) before exposure to hyperthermia. Preliminary experiments were carried out to determine the minimal concentration of each compound that was required to inhibit the activation of each kinase by monitoring ERK and c-Jun phosphorylation by Western blotting with phospho-specific antibodies (p-ERK #9102 Cell Signaling; p-c-Jun KM-1 Santa Cruz). Cells treated with these inhibitors were collected for Western blotting and for analysis of Bax translocation by immunofluorescence staining. Both compounds were dissolved in Me 2 SO and stored in aliquots at Ϫ80°C. The concentration of Me 2 SO in the treated cells was 0.1% or less (except for cells treated with 150 M PD98059 which received 0.15% Me 2 SO). Control cells were treated with an equivalent amount of Me 2 SO. JNK1 was also inhibited by transient overexpression of a dominant negative JNK1 mutant (JNK1 and dnJNK1 expression plasmids were obtained from R. J. Davis, University of Massachusetts) (33). HeLa-rtTA/Bax:YFP cells were transiently transfected with plasmids to express CFP (pECFP-N1), Hsp70 fused to CFP, JNK1, or dnJNK1. Transfections with JNK1 and dnJNK1 also included pECFP-N1 (Clontech) to identify the transfected cells. The pECFP-N1:Hsp70 plasmid was created by inserting a PCR-amplified Hsp70 sequence from pH2.2 (34) that included ScaI and AgeI restriction enzyme sites into the SmaI and AgeI sites in pECFP-N1, which places ECFP at the C terminus of Hsp70. Expression of Bax-YFP was induced 24 h after transfection. After an additional 24 h the transfected cells were exposed to 45°C for 60 min followed by a return to 37°C for 1 h before observation. Bax-YFP translocation in the CFP-positive cells was scored with an inverted fluorescence microscope (IRE2, Leica Microsystems, Richmond Hill, ON) equipped with a CCD camera (Orca ER, Hamamatsu), motorized external filter wheels and stage (Ludl Electronic Products, Hawthorne, NY), and controlled by Openlab software (Improvision, Coventry UK). CFP fluorescence was visualized with the Leica internal filter cube (excitation, 436/20; emission, 480/40). YFP fluorescence in these cells was visualized using external excitation (495/10) and emission (540/50) filters. This was necessary to eliminate CFP fluorescence in the YFP filter channel. Cells were scored in 20 equally distributed regions that were selected by the X-Y stage module of the Openlab software. Viability after hyperthermia was scored in HeLa-rtTA cells that were transfected as described for the Bax-YFP cell line. Viability of the CFP-positive cells was scored as judged by their morphology and attachment to the culture dish.
The ability of Hsp70 to prevent cell death in cells expressing a constitutively active form of Bax was examined in HeLa-rtTA cells that were transiently transfected with plasmids to express CFP, Hsp70-CFP, or Bcl-2 together with either GFP-tagged wild-type Bax or mutant Bax S184V (10). Each of the Bax expression plasmids is under the control of the tetracycline-regulated transactivator. The tet-inducible Bax plasmids were constructed by inserting BsrGI/XbaI Bax-GFP fragments (plasmids obtained from RJ Youle) into BsrGI/XbaI cut pBI-EGFP (Clontech). The test plasmid to Bax plasmid ratio was 5:1. Bcl-2-transfected cells received a 1:1 mixture of mitochondrial-targeted Bcl-2 (prc-CMVbclact) and ER-targeted Bcl-2 (pcrCMVbcl2cb5) plasmids (obtained from DW Andrews, McMaster University, CA) (35). Viability of the GFP-positive cells was assessed by counting the attached flattened cells and rounded detached cells 48 h after transfection.
Apoptosome activation was induced by treating cells with apoptosis activator compound 2 (AA2, 1-[(3,4-dichlorophenyl)methyl]-1H-indole-2,3-dione, #2098, Tocris, Ellisville, MO) (36). Cells were incubated with various concentrations of AA2 at 37°C for 6 h or with 20 M AA2 for various periods of time and then collected for analysis of caspase activation by measuring DEVDase and LEHDase activity and by Western blotting. Apoptosis was also monitored in AA2-treated cells by staining with acridine orange and ethidium bromide (4 g/ml each) and counting cells with normal or apoptotic nuclei using a fluorescence microscope (21).
Western Blotting and Immunoprecipitation-Western blotting was carried out as described previously (24). For immunoprecipitation 5 ϫ 10 6 cells were collected by centrifugation, washed twice with PBS, and lysed in IP lysis buffer (20 mM Tris-HCl (pH 7.4) 137 mM NaCl, 2 mM EDTA, 2% CHAPS) containing protease inhibitors (2 g/ml each of pepstatin, leupeptin, and aprotinin) and phosphatase inhibitors (50 mM sodium fluoride, 1 mM sodium vanadate) for 30 min on ice. Lysates were centrifuged at 20,000 ϫ g for 10 min at 4°C, and the supernatants were collected. Protein concentration in the supernatants was determined using the Pierce BCA protein assay kit and adjusted to 1 mg/ml with lysis buffer. Samples containing 300 g of protein were incubated with 1 g of anti-Hsp70, anti-Bax (BD Pharmingen #554104), anti-Bak, or normal rabbit serum (Jackson ImmunoResearch, West Grove, PA) overnight at 4°C. Immunoprecipitates were collected by the addition of 10 l of protein A (for anti-Bax, anti-Bak, and normal rabbit serum) or protein G (for anti-Hsp70) EZview Affinity Gel (Sigma-Aldrich) and incubation for 1 h at 4°C. Complexes were collected by centrifugation and washed 4ϫ with lysis buffer. Finally the pelleted beads were resuspended in 1ϫ SDS sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% ␤-mercaptoethanol) and heated at 95°C for 5 min before analysis by SDS-PAGE and Western blotting. In some experiments the cells were lysed in either 2% CHAPS lysis buffer or 2% CHAPS buffer supplemented with 1% Triton X-100. The remaining steps were carried out in the same buffer that was used for cell lysis.
Immunofluorescence-For immunofluorescence analysis, cells (5 ϫ 10 4 ) were collected onto glass slides in a cytocentrifuge (Cytospin 4, Shandon Products Inc.), air-dried, and fixed in 4% paraformaldehyde for 10 min at room temperature. After washing in PBS the cells were permeabilized with 0.2% Triton X-100 for 2 min, washed twice with PBS, and then blocked with 3% bovine serum albumin in PBS overnight at 4°C. This was followed by a 1-h incubation at room temperature with an anti-Bax antibody (YTH-6A7; Trevigen). The cells were then washed 2ϫ with PBS and incubated with a fluorescein-conjugated goat antimouse IgG (Molecular Probes) for 1 h at room temperature. Nuclei were stained with 4Ј,6-diamidino-2-phenyl-indole (Sigma-Aldrich), which was included at a concentration of 1 g/ml with the anti-mouse antibody. After 3 washes with PBS, a fluorescence mounting medium containing anti-fading compounds (DakoCytomation; Carpinteria, CA) was added, and the cells were observed using a Leica IRE-2 fluorescence microscope. Images were acquired using a Hamamatsu ORCA ER digital CCD camera and Openlab software (Improvision; Coventry, England).
Preparation of Cytosolic and Membrane Fractions-Subcellular fractions were prepared by digitonin lysis as described by Mikhailov et al. (37). Cells (1 ϫ 10 7 /ml) were lysed for 2 min at room temperature in digitonin lysis buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% digitonin, protease, and phosphatase inhibitors). Lysis was monitored by trypan blue exclusion. The lysates were centrifuged at 15,000 ϫ g for 10 min at 4°C, and the supernatant, containing soluble proteins, was collected. The pellets were resuspended in the same buffer except that digitonin was replaced with 1% Nonidet P-40 and was incubated on ice for 60 min followed by centrifugation at 15,000 ϫ g for 10 min at 4°C. The supernatant, containing the membrane fraction was collected, and the pellet was resuspended in 1ϫ SDS sample buffer. The soluble and membrane fractions were mixed with 2ϫ SDS sample buffer. Equivalent volumes of each fraction were loaded onto SDS-PAGE gels (12%), and proteins were transferred to polyvinylidene difluoride membranes for detection of Bax. Efficiency of separating membrane and soluble fractions was confirmed by probing for the mitochondrial membrane protein cytochrome oxidase subunit II using antihuman cytochrome oxidase subunit II mouse monoclonal antibody (clone 12C4-F12, #A-6404, Molecular Probes).
Chemical Cross-linking-Cell extracts were prepared by lysing 5 ϫ 10 6 cells on ice for 30 min in CHAPS lysis buffer (10 mM HEPES (pH 7.4), 137 mM NaCl, 2 mM EDTA, 2% CHAPS, protease, phosphatase inhibitors). Lysates were clarified by centrifugation at 15,000 ϫ g for 10 min at 4°C, and the protein concentrations in the supernatants were determined. Fifty g of each sample in a total volume of 50 l was treated with 1 mM ethylene glycolbis(succinimidylsuccinate) (16.1 Å spacer arm) or DST (disuccinimidyl tartrate, 6.4 Å spacer arm) (Pierce). The cross-linkers were freshly dissolved in Me 2 SO before use. Control extracts received Me 2 SO alone. The extracts were incubated for 30 min at room temperature, and then the reactions were terminated by the addition of Tris/HCl (pH 7.4) to a final concentration of 20 mM. The samples were then mixed with one volume of 2ϫ SDS sample buffer (100 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 10% ␤-mercaptoethanol), separated on a 12% SDS-PAGE gel, and transferred to polyvinylidene difluoride membranes. Bax was detected using a rabbit polyclonal antibody against amino acids 11-30 of human Bax (Bax N20: sc-493; Santa Cruz Biotechnology).
Gel Filtration Chromatography-Cell extracts were prepared by lysing 10 ϫ 10 6 cells on ice for 30 min in CHAPS buffer containing 20 mM Tris-HCl (pH 7.4) in place of HEPES. After lysis the extracts were clarified by centrifugation at 20,000 ϫ g for 20 min at 4°C. The protein concentrations of the supernatants were determined using the Pierce BCA protein assay kit. Samples containing 500 g of protein were sep-arated through Sephacryl 300 (Amersham Biosciences) packed in a 1-cm ϫ 50-cm Econo-column (Bio-Rad) equilibrated in 2% CHAPS buffer. The column was calibrated with gel filtration standards (Bio-Rad). Fractions (0.5 ml) were collected with a Bio-Rad Biologic Work station at 4°C, and 0.3 ml of every other fraction were precipitated with 3 volumes of acetone at Ϫ20°C overnight. Precipitated proteins were redissolved in 1ϫ SDS sample buffer and separated by SDS-PAGE followed by Western blotting for the detection of Bax (N20 rabbit polyclonal antibody).
Caspase Activity Assays-Cell extracts were prepared for measurement of caspase activity by incubation in cell lysis buffer (50 mM HEPES (pH 7.9), 0.1% CHAPS, 0.1 mM EDTA) at a concentration of 1.5 ϫ 10 6 cells/50 l for 30 min on ice followed by centrifugation at 12,000 rpm for 10 min at 4°C. The protein concentration was determined using the BCA protein assay kit (Pierce). Caspase activity was measured by mixing

Heat-induced Bax Translocation Is Inhibited in Cells Expressing
Elevated Levels of Hsp70-Our previous observation that Hsp70 blocks stress-induced cytochrome c release led us to examine whether this was the result of an ability of Hsp70 to inhibit the translocation of Bax from the cytosol to mitochondria. Bax translocation was examined in cells with tetracycline-regulated expression of Hsp70. After induction for 24 h the induced and non-induced PErTA70 cells were exposed to a 43°C heat shock for 60 min and either collected immediately or returned to 37°C for 6 h. Immunofluorescence analysis was performed using a conformation specific anti-Bax (6A7) antibody that detects only the form that is competent for membrane insertion (38). Exposure of the non-induced cells to heat shock resulted initially in a slight increase in the number of Bax positive cells; however, nearly 50% of these cells were Bax positive by 6 h after the heat shock ( Fig. 1A and B). In cells that were induced to express Hsp70 only about 20% of the heat-shocked cells showed anti-Bax 6A7 signals (Fig. 1). Cells that were Bax-positive also showed typical apoptotic nuclear morphology. Therefore, as reported previously, tet-regulated overexpression of Hsp70 inhibited heat-induced cell death (21,24). Because inhibition of caspase activation and cytochrome c release required the chaperone function of Hsp70 (24), we examined whether inhibition of Bax translocation was also dependent upon this activity by examining heat-induced Bax translocation in cells expressing a mutant version of Hsp70 lacking the ATPase domain. Expression of Hsp70⌬ATPase did not prevent the translocation of Bax in heat-shocked cells (Fig. 1), indicating that the chaperone function of Hsp70 is required for the inhibition of Bax translocation.
Cellular stress can in some circumstances result in increased production of the Bax protein and its subsequent translocation to mitochondria. However, this did not occur during heat-induced apoptosis, since we did not detect any change in the cellular content of Bax in the heatshocked cells (Fig. 1C). This was the case for non-induced or induced cells that were exposed to 43°C for either 60 or 90 min before returning to 37°C for 6 h. Caspase-3 activation was clearly evident in the noninduced cells but greatly reduced in the induced cells that were exposed to hyperthermia. Caspase activation was not required for Bax translocation since we did not observe any effect of the caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone on Bax translocation (data not shown). Heat-stressed cells were exposed to 43°C for 60 min and then returned to 37°C for 6 h. Nuclei are stained with 4Ј,6-diamidino-2-phenylindole dihydrochloride. B, Bax-positive cells were counted in control cultures and in cultures that were heat-shocked and either analyzed immediately (HS/0) or after a 6-h incubation at 37°C (HS/6). At least 400 cells were counted for each sample. C, heat shock does not alter Bax protein levels. Cells were exposed to 43°C for either 60 or 90 min and then returned to 37°C for 6 h. Western blotting was performed using the indicated antibodies (p17 represents the processed fragment detected with the anti-caspase 3 antibody). D, association of Bax with intracellular membranes in heat-shocked cells. Soluble (S), membrane (M), and pellet (P) fractions were obtained by differential detergent lysis and centrifugation. Bax is primarily soluble in control cells (C) but becomes membrane-associated after exposure to heat shock (60 or 90 min at 43°C followed by 6 h at 37°C). This translocation from the cytosol to membrane fraction is inhibited in the Hsp70-expressing cells (ON). Cytochrome oxidase subunit II (COXII) reactivity demonstrates the purity of the membrane and soluble fractions.
To confirm the immunofluorescence results we next examined the association of Bax with mitochondrial membranes by Western blotting of soluble and membrane fractions obtained by digitonin lysis (37). Bax was found predominantly in the soluble fraction of control cells before heat shock (Fig. 1D). In cells that were exposed to heat shock the majority of Bax was now recovered in the membrane fraction for the noninduced cells. However, Bax remained associated with the soluble fraction after heat shock in cells that had been induced to express Hsp70. Therefore, Hsp70 appears to prevent stress-induced apoptosis by inhibiting the regulated movement of Bax from the cytosol to the mitochondrial membrane. This required the chaperone function of Hsp70 since expression of Hsp70 lacking the ATPase domain did not prevent this movement of Bax to the membrane fraction (data not shown).
Hsp70 Prevents Bax Oligomerization-Bax oligomerization is required for mitochondrial outer membrane disruption and cytochrome c release. Because inhibition of oligomerization by Hsp70 could prevent stable association of Bax with mitochondria we next examined the oligomeric structure of Bax in the non-induced and induced cells before and after heat shock. Cell extracts in CHAPS lysis buffer were treated with chemical cross-linkers before separation by SDS-PAGE and Western blotting ( Fig. 2A). Bax was monomeric in non-stressed cells; however, cross-linked dimers were the predominant forms detected in non-induced heat-shocked cells. Higher molecular weight Bax multimers could not be equivocally discerned due to the presence of cross-reacting proteins detected by this antibody. Cells expressing Hsp70 contained a greatly reduced amount of cross-linked Bax dimers after heat-shock relative to the non-induced cells, indicating that oligomerization was inhibited by Hsp70.
We next used gel filtration chromatography to examine higher molecular weight Bax complexes (Fig. 2B). Non-induced and induced cells were exposed to 43°C for 60 min and either collected immediately after the heat shock (HS/0) or after a 6-h incubation at 37°C (HS/6). Extracts prepared in 2% CHAPS buffer were fractionated on a Sephacryl 300 column and analyzed by Western blotting. Bax existed primarily as a monomer in control cells. Some higher molecular weight forms were detected; however, these likely were derived from the small percentage of apoptotic cells in control cultures. High molecular weight complexes containing Bax were produced in cells exposed to heat shock. These complexes of ϳ100 kDa could be detected immediately after the heat shock and increased in size after the cells were incubated at 37°C for 6 h. The major size classes (ϳ100 and ϳ250 kDa) are similar to what was observed in apoptotic HeLa cells (39). In contrast to the non-induced cells, the formation of Bax oligomers was diminished in cells expressing Hsp70 immediately after heat shock and were nearly undetectable in the heat-shocked cells that were returned to 37°C for 6 h. Thus, the inhibition of cytochrome c release in cells expressing Hsp70 can be attributed to the ability of Hsp70 to prevent the oligomerization of Bax into poreforming channels.
Hsp70 Prevents Bax from Acquiring a Membrane-insertion Competent Conformation but Does Not Directly Interact with Bax-The preceding results suggest that Hsp70 acts at some step before the conformational change in Bax that allows it to insert into mitochondrial membranes. We tested this by performing immunoprecipitation experiments with a conformation-specific anti-Bax antibody (N20) that recognizes an epitope in the N-terminal domain of Bax that is exposed only after Bax acquires a membrane-insertion competent conformation (9). Bax could not be immunoprecipitated from control cells with this antibody (Fig. 3A). However, in extracts from cells collected immediately after exposure to heat-shock or 1 h later, some of the Bax had acquired a conformation that allowed antibody binding. Cells collected 6 h after the heat shock had a much higher level of Bax in this conformation. In cells expressing Hsp70, far less Bax acquired the membrane-insertion competent conformation after the heat shock treatment. Immunoprecipitation of the same extracts with an antibody that recognizes an epitope within residues 43-61 of Bax pulled down an equivalent amount of Bax from each extract.
Because Hsp70 was able to prevent monomeric Bax from attaining the membrane-insertion pore-forming conformation, we next asked whether this is accomplished through a direct interaction between Hsp70 and Bax. For this we immunoprecipitated Hsp70 or Bax from extracts of control and heat-shocked cells and probed for the presence of co-precipitated proteins by Western blotting (Fig. 3B). Bax was not detected in Hsp70 precipitates, and Hsp70 was not detected in Bax precipitates from either control or heat-shocked cells. A small amount of Hsp70 was seen with the Bax immunoprecipitated from induced cells that had been exposed to heat-shock and returned to 37°C for 6 h. However, a similar amount of Hsp70 was detected when normal rabbit serum was used in place of the anti-Bax antibody for the immunoprecipitation. The inability to detect Hsp70 and Bax in co-precipitates was not because the conditions were not conducive for the detection of co-precipitating proteins since we were able to detect Bax in anti-Bak precipitates (Fig. 3C) and Hsp40 in anti-Hsp70 precipitates (Fig. 3D). Bax was only found associated with Bak in extracts from non-induced cells collected 6 h after heat shock, when the majority of the cells were apoptotic. Bak was not associated with Bax in extracts prepared from induced cells that were exposed to heat shock, indicating that Hsp70 prevents the formation of Bak-Bax oligomers.
We also attempted to detect an association of Hsp70 with Bax in vitro using conditions that alter Bax conformation. Cells induced to express Hsp70 were lysed in either 2% CHAPS buffer or the same buffer supplemented with 1% Triton X-100. The nonionic detergents Nonidet P-40 and Triton X-100 can induce the conformational change and oligomerization of Bax (37, 40). The conformation-specific antibody N20 immunoprecipitated Bax from extracts lysed in Triton X-100 but not from FIGURE 2. Heat-induced oligomerization of Bax is prevented by Hsp70. A, Bax dimerization was examined by chemical cross-linking with ethylene glycolbis(succinimidylsuccinate (EGS) or DST in extracts from non-induced and induced cells that were maintained at 37°C (C) or exposed to 43°C for 60 min followed by 6 h at 37°C (HS). Western blotting with anti-Bax antibody shows dimer formation (*) after heat shock in the noninduced cells with much reduced dimer formation in the cells expressing Hsp70. NT, treated with Me 2 SO only. B, Bax oligomerization was examined by gel filtration chromatography of extracts lysed in 2% CHAPS buffer. Non-induced (OFF) and Hsp70 expressing (ON) cells were either not treated (C) or exposed to 43°C for 60 min and collected immediately (HS/0) or after 6 h of incubation at 37°C (HS/6). The migration position in the column of calibration standards is shown above the figure (kDa).
CHAPS extracts, whereas the polyclonal antibody raised against amino acids 43-61 of Bax immunoprecipitated both inactive (CHAPS lysate (C)) and active (Triton X-100 lysate (T)) conformations of Bax (Fig. 3E). Hsp70 was not detected in the Bax immunoprecipitates from either CHAPS or Triton X-100-containing lysates. The minor amount of Hsp70 present in these immunoprecipitates is equivalent to what is obtained using normal rabbit serum and, therefore, reflects a nonspecific association of Hsp70 with the antibody. Similarly, Bax was not detected in either of the Hsp70 immunoprecipitates. Therefore, Hsp70 not only does not associate with Bax in vivo, it also does not bind to Bax that has been activated by detergent treatment in vitro, ruling out a direct interaction with Bax as a possible mechanism for Hsp70 ability to prevent Bax translocation to mitochondria.
Hsp70 Prevents the Heat-induced Activation of ERK and JNK-Cellular stresses including hyperthermia activate signaling pathways that control the decision to proliferate or die (19,(41)(42)(43). Members of the Bcl-2 family of apoptosis regulators have been shown in some situations to be targets of the kinases that are activated in stressed cells (6). Because the ERK and JNK pathways have been shown to be negatively regulated by Hsp70, we wished to determine whether their activation was required for Bax translocation in heat-shocked cells. Fig. 4A shows the extent of JNK phosphorylation in PErTA70 cells exposed to 43°C for periods of time ranging from 30 min to 2 h and the nearly complete inhibition of phosphorylation in cells expressing Hsp70. JNK activation can be prevented by treatment with the specific inhibitor SP600125 (44). Inhibition of JNK was monitored by examining the extent of c-Jun phosphorylation (Fig. 4B). SP600125 treatment inhibited JNK activation as efficiently as Hsp70 expression and was as effective as Hsp70 in blocking Bax translocation (Fig. 4C). Cells expressing Hsp70 did not display significantly increased protection by SP600125 treatment, indicating that Hsp70 may act primarily through suppression of JNK.
Further evidence of a role for JNK activation in heat-induced Bax translocation was obtained by transient expression of a dominant negative mutant form of JNK1. For these experiments, we transiently transfected HeLa cells with plasmids to express either JNK1 or dnJNK1 (each together with pECFP-N1), Hsp70 fused to CFP, or CFP alone. One day after transfection the cells were exposed to hyperthermia (45°C for 60 min) and either collected immediately to examine JNK phosphorylation status or returned to 37°C for 16 h to assess viability. Expression of either Hsp70-CFP or dnJNK1 reduced JNK1 activation and increased the number of viable cells after hyperthermic exposure compared with CFP-transfected cells (Fig. 5, A and B). Cells expressing the wild-type JNK1 were slightly more sensitive to heat shock. We next examined the effects of JNK1 suppression on Bax translocation by performing the same set of transfections in a HeLa cell line with tetracycline-regulated expression of Bax fused to YFP (Bax-YFP). Expression of Bax-YFP was induced by the addition of doxycycline at the time of transfection. Doxycycline was washed out 24 h after transfection and before the hyperthermic exposure. Cells were exposed to 45°C for 60 min and returned to FIGURE 3. Hsp70 and Bax do not physically interact. A, immunoprecipitation (IP) of activated Bax from heat-shocked PErTA70 cells using a conformation specific antibody (N20). Cells were exposed to 43°C for 60 min and then either collected immediately (0) or after a 1 h (1) or 6 h (6) incubation at 37°C. Extracts prepared in 2% CHAPS lysis buffer were immunoprecipitated with either N20, which detects the activated form of Bax, or with an antibody raised against amino acids 43-61, which detects Bax irrespective of its conformation. Western blotting (N20) shows that activated Bax can be detected immediately after heat shock but is more abundant after 6 h of incubation at 37°C. Hsp70 reduced the amount of activated Bax in heat-shocked cells. Immunoprecipitation with the non-conformation-specific antibody shows that equivalent amounts of total Bax were immunoprecipitated from each sample. B, immunoprecipitation of Bax or Hsp70 from 2% CHAPS lysates of control cells (C) and cells that were heat-shocked at 43°C for 1 h and either collected immediately (HS/0) or after a 6-h incubation at 37°C (HS/6). Immunoprecipitation experiments were performed using either normal rabbit serum (RS) as a control for nonspecific interactions, the Hsp70-specific antibody C92 (HSP70), or the polyclonal anti-Bax antibody raised against amino acids 43-61 (Bax). Washed immunoprecipitates were analyzed by Western blotting (WB) with antibodies to Hsp70 (C92) and Bax (N20). C, Bax is co-precipitated with Bak in anti-Bak immunoprecipitates. D, Hsp40 is co-precipitated with Hsp70 in anti-Hsp70 immunoprecipitates of extracts from induced cells. E, Hsp70 does not interact with Bax that has undergone the activating conformational change in vitro. Immunoprecipitations were carried out using extracts from ON cells prepared in either 2% CHAPS lysis buffer (C) or CHAPS buffer supplemented with 1% Triton X-100 (T). Exposure to the non-ionic detergent Triton X-100 induces Bax to undergo a conformational change, as evidenced by its immunoprecipitation with the conformation specific antibody N20.

FIGURE 4. Role of JNK activation in Bax translocation. A, non-induced (OFF) and
Hsp70 expressing (ON) PErTA70 cells were exposed to 43°C for 30, 60, 90, or 120 min and collected immediately. Western blotting was performed to examine the levels of phosphorylated JNK (p-JNK) and total JNK. B, inhibition of JNK activation in cells treated with 10 M SP600125. Cells were incubated with the JNK inhibitor for 30 min before exposure to 43°C for 60 min (HS). Expression of Hsp70 was induced by the addition of doxycycline (ϩDOX) 24 h before SP600125 treatment. Cells were collected immediately after the heat shock and levels of p-JNK, p-c-Jun and actin were examined by Western blotting. C, control cells. C, Bax translocation was measured by immunofluorescence staining of control (Me 2 SO (DMSO)) and SP600125-treated cells that were either maintained at 37°C (C) or exposed to 43°C for 1 h followed by 6 h at 37°C (HS) (n ϭ 3).
37°C for 1 h. Cellular localization of Bax-YFP was scored in the CFPexpressing cells using an inverted fluorescence microscope (Fig. 5C). Expression of dnJNK1 was as effective as Hsp70 in suppressing the heat-induced translocation of Bax-YFP (Fig. 5D). Western blots of extracts from these cells shows the levels of expression of Hsp70-CFP, JNK1, dnJNK1, and Bax-YFP (Fig. 5E). Together these results show that inhibition of JNK activation by expression of a dominant-negative JNK1 mutant protein prevents heat-induced Bax translocation and protects cells from the lethal effects of hyperthermia to a similar extent as overexpression of Hsp70.
In addition to JNK activation, heat shock treatment also led to an increased level of ERK phosphorylation, which was also inhibited in cells expressing Hsp70 (Fig. 6A). Cells were treated with the ERK inhibitor PD98059, and the effectiveness of inhibition was evaluated by monitoring the level of ERK phosphorylation (Fig. 6B). Heat-induced activation of ERK was inhibited more efficiently by PD98059 than by Hsp70 expression; however, it was only moderately effective in blocking Bax translocation (Fig. 6C). As well, treatment of Hsp70-expressing cells with PD98059 resulted in a further reduction in the extent of ERK activation but did not reduce the extent of Bax translocation further than what was achieved by expression of Hsp70 alone. These results suggest that ERK activation contributes to Bax translocation in heat-stressed cells but that inhibition of ERK on its own is not sufficient to provide significant levels of protection. As well, the inhibition of ERK by Hsp70 is unlikely to play a major role in the inhibition of Bax translocation. In support of this suggestion, we treated cells with the potent ERK activator phorbol 12-myristate 13-acetate, which results in an ϳ10-fold higher level of phosphorylated ERK than does heat shock treatment, and found no difference in viability or the amount of Bax translocation after heat shock in these cells (data not shown). Altogether, our results suggest that Hsp70 prevents apoptosis in heat-stressed cells by inhibiting the stress-induced activation of JNK, which prevents the events leading to conversion of Bax to the membrane-insertion competent state.
Hsp70 Does Not Prevent Cell Death after Bax Translocation or Apoptosome Activation Has Occurred-We next asked whether Hsp70 could prevent cell death in cells after Bax had acquired the membrane-insertion competent state. For these experiments we transfected HeLa-rtTA cells with either wild-type Bax fused to GFP or a mutant form of Bax fused to GFP in which a single amino acid substitution (S184V) within the Bax C-terminal transmembrane domain converts Bax to a form that exists only in the membrane-insertion competent state (10). The expression of Bax-GFP and Bax S184V -GFP is under the control of the tetracycline-regulated promoter. Cells were co-transfected with the Bax-GFP plasmids together with CFP, Hsp70-CFP, or Bcl-2 expression plasmids. Expression of Bax-GFP and Bax S184V -GFP was induced by the addition of doxycycline 24 h after transfection. The viability of Bax-GFP and Bax S184V -GFP-expressing cells was assessed 24 h later by determining the percentage of floating and attached GFP-positive cells. In the control (CFP-transfected) cells overexpression of wild-type Bax and FIGURE 6. Role of ERK activation in Bax translocation. A, non-induced (OFF) and Hsp70 expressing (ON) PErTA70 cells were exposed to 43°C for 30, 60, 90, or 120 min and collected immediately. Western blotting was performed to examine the levels of phosphorylated ERK (p-ERK) and total ERK. B, Western blot of extracts prepared from cells treated with the ERK inhibitor PD98059 (150 M). Cells were pretreated with the inhibitor for 2 h before exposure to heat shock (HS). C, control cells. C, Bax translocation was measured by immunofluorescence staining of control (Me 2 SO (DMSO)) and PD98059treated cells that were either maintained at 37°C (C) or exposed to 43°C for 1 h followed by 6 h at 37°C (HS) (n ϭ 3). Bax S184V resulted in a loss of viability with higher levels of cell death in cells expressing Bax S184V (Fig. 7A). The percentage of non-viable cells when wild-type Bax or Bax S184V was expressed was the same in Hsp70-CFP-expressing cells as in CFP-expressing cells. In contrast, expression of Bcl-2 effectively reduced the percentage of non-viable cells expressing either the wild-type or mutant Bax proteins. Approximately equivalent levels of Bax-GFP were expressed in each of the groups of transfected cells (Fig. 7B). Therefore, although Bcl-2 reduced cell death in cells expressing the membrane-insertion competent form of Bax, Hsp70 did not, indicating that Hsp70 likely prevents cell death by acting before the Bax conformational change occurs.
Several recent reports have indicated that Hsp70 can prevent stressinduced apoptosis at various points downstream of mitochondria, including a role in blocking apoptosome formation, caspase-9 recruitment to the apoptosome, caspase-3 activation, and inhibition of cell death in cells with active caspase-3 (25,26,30,31). We examined whether Hsp70 could provide resistance after engagement of the apoptosome by treating cells with a compound that was recently identified in a screen for small molecule activators of apoptosis. This cell-permeable indoledione compound (AA2) was shown to trigger apoptosis in a variety of tumor cell lines by directly promoting the oligomerization of Apaf-1 into a functional apoptosome (36). We treated non-induced and induced PErTA70 cells with various concentrations of this compound for 6 h and measured caspase activation by monitoring LEHD-AMC (caspase-9 substrate) and DEVD-AMC (caspase-3 substrate) cleavage activity in cell extracts (Fig. 8, A and B). Whereas Hsp70 expression inhibited caspase activity in heat-shocked cells, it did not reduce the level of activated caspases in AA2-treated cells. The activity assay results are supported by Western blots showing the level of caspase-9 and caspase-3 cleavage in heat-shocked and AA2-treated cells (Fig. 8C). A concentration of 20 M AA2 resulted in a similar level of caspase-9 activity and caspase-9 processing as did exposure to heat shock, indicating that at this concentration of the compound an equivalent number of apoptosomes were formed. A time course experiment with AA2 (20 M)-treated cells shows that similar kinetics of caspase activation occurred in the non-induced and induced cells (Fig. 8D). As well, similar numbers of apoptotic cells accumulated over time in the non-induced and induced cells. Bax translocation occurred in the AA2-treated cells; however, the percentage of cells with punctate Bax immunofluorescence was far less than in heat-shocked cells and likely represents a late event in the dying cells (Fig. 8E). In summary, these results reveal that Hsp70 is unable to prevent apoptosome formation in cells treated with AA2 and that Hsp70 does not inhibit apoptosis after apoptosome activation has occurred.

DISCUSSION
In an attempt to identify how Hsp70 prevents heat-induced apoptosis, we examined events occurring upstream of cytochrome c release primarily because our previous results showed that this step was efficiently impaired by Hsp70 in heat-shocked cells (24). We focused on the proapoptotic Bcl-2 family member Bax because of its central role in regulating cytochrome c release in stressed cells. We found that heat shock resulted in (i) a conformational change in Bax, (ii) its translocation to mitochondria, (iii) stable membrane association, and (iv) oligomerization. All of these events were inhibited in cells that had elevated levels of Hsp70. However, Hsp70 did not protect cells expressing a mutant form of Bax that has constitutive membrane insertion capa-  bility or cells treated with a small molecule activator of apoptosome formation. These results indicate that Hsp70 blocks heat-induced apoptosis primarily by inhibiting Bax activation and thereby preventing the release of proapoptotic factors from the mitochondrial intermembrane space.
Bax-dependent mitochondrial outer membrane permeabilization is regulated by interactions between proapoptotic and antiapoptotic members of the Bcl-2 family (6). These interactions occur between the hydrophobic face of the BH3 ␣ helix of the proapoptotic BH3-only proteins and a hydrophobic groove formed by the BH1, BH2, and BH3 domains of the anti-apoptotic members of the Bcl-2 protein family. Several of the Bcl-2 homologues contain a C-terminal hydrophobic helical domain that is essential for stable membrane insertion. This transmembrane domain is buried within the BH 1/2/3 hydrophobic groove of monomeric Bax (45). Bax activation is associated with exposure of the C-terminal transmembrane region by an unknown mechanism. Single amino acid substitutions in the C terminus of Bax can convert it from the monomeric cytoplasmic form to a conformation that is capable of constitutively localizing to mitochondria in the absence of stress (10). This activation step, which could be associated with transient exposure of hydrophobic domains, might be expected to provide an ideal target for interaction with molecular chaperones. The peptide binding domain of Hsp70 has a high affinity for short stretches of hydrophobic amino acids (12). Binding of Hsp70 to activated Bax could potentially prevent its stable integration into membranes. It is for this reason that we attempted to determine whether complexes containing Bax and Hsp70 could be detected in control or heat-shocked cells by performing coimmunoprecipitation experiments. However, we were unable to detect any significant interaction between these proteins in either Hsp70 or Bax immunoprecipitates, thus ruling out a direct effect of Hsp70 on Bax activity. This suggests that the conformational change that occurs during Bax activation is regulated such that exposure of hydrophobic regions are transient or locally confined and consequently do not provide a substrate for interaction with Hsp70. Additionally, the fact that Hsp70 did not prevent the mitochondrial localization of Bax S184V or prevent cell death in cells expressing this membrane-insertion competent mutant form of Bax also argues against a role for Hsp70 in preventing the insertion of Bax into mitochondria by binding to the hydrophobic C-terminal tail of activated Bax.
Recently, Gotoh et al. (46) showed that Bax translocation in nitric oxide-treated cells could be prevented by overexpression of Hsp70 together with either of the cochaperones hdj-1 or hdj-2. They also found that Bax could be co-immunoprecipitated with Hsp70, Hsc70, hdj-1, and hdj-2 in both control and heat-shocked cells. However, we were unable to detect an interaction between Bax and Hsp70 in cells with tet-regulated expression of Hsp70 (Fig. 4) or in cells that were given a thermotolerance-inducing heat shock treatment (43°C for 30 min followed by 6 h 37°C) that resulted in increased expression of Hsp70 and Hsp40 (results not shown). Therefore, our inability to co-immunoprecipitate Bax and Hsp70 is not due to a requirement for both Hsp70 and an Hsp40 cochaperone.
The inability of Hsp70 to prevent apoptosis in cells expressing Bax S184V also suggests that Hsp70 is unable to prevent cell death after mitochondrial disruption and release of proapoptotic factors has occurred. Previous studies have implicated Hsp70 in apoptosis repression downstream of mitochondrial membrane permeabilization. For example, Hsp70 has been shown to interact with Apaf-1 and prevent cytochrome c-mediated apoptosome formation and caspase-9 recruitment in vitro (25,26). A recent report, however, has questioned these in vitro studies (32). We tested the ability of Hsp70 to affect apoptosome formation in living cells by using a recently identified small molecule (AA2) that efficiently triggers Apaf-1 oligomerization in cell extracts and in cells (36). This allowed us to examine whether Hsp70 can inhibit apoptosome formation in living cells in the absence of mitochondrial outer membrane permeabilization and to assess whether Hsp70 can protect cells from apoptosis downstream of this event. Treatment of PErTA70 cells with AA2 led to increased DEVDase activity and cell death with kinetics similar to heat-induced apoptosis. We did not observe any effect of Hsp70 on either caspase-3 or caspase-9 activation or cell survival in AA2-treated cells. Thermotolerant HeLa cells were also no more resistant to AA2 treatment then were control cells (data not shown). Taken together, the lack of protection by Hsp70 in cells expressing Bax S184V or after treatment with AA2 suggests that Hsp70 is unable to block apoptosis after mitochondrial outer membrane permeabilization and release of proapoptotic factors has occurred.
Because Hsp70 does not interact with Bax, it must instead prevent Bax translocation by inhibiting signaling events that lead to its activation. The ERK and JNK signaling pathways are known regulators of Bcl-2 family proteins. For example, Bax translocation and cytochrome c release are impaired in Jnk1 Ϫ/Ϫ Jnk2 Ϫ/Ϫ cells (47). Also, Bax Ϫ/Ϫ Bak Ϫ/Ϫ fibroblasts resist apoptosis induced by expression of a constitutively active JNK (48). JNK can phosphorylate and activate the proapoptotic BH3-only protein Bim leading to cell death, which is dependent upon Bax expression (49,50). Cytoplasmic localization of Bax has been shown to be mediated by interaction with 14-3-3 proteins, with phosphorylation of 14-3-3 by JNK leading to Bax release and mitochondrial translocation (51). Other targets of JNK in the apoptotic pathway include Bcl-2 and Bad. Phosphorylation inactivated the antiapoptotic activity of Bcl-2 (52) and the proapoptotic activity of Bad (53). Whereas JNK activation is proapoptotic in most settings, ERK activation is generally protective. In the presence of growth factors ERK phosphorylates Bim, inhibiting its ability to associate with and activate Bax (54). Heat shock activates the JNK and ERK kinase pathways, both of which are suppressed in cells overexpressing Hsp70 (21,22,55,56).
To determine the contribution of these kinase pathways to heatinduced apoptosis we examined Bax translocation after heat shock in cells that were treated with the JNK inhibitor SP600125 or the ERK inhibitor PD98059. JNK inhibition provided resistance to heat-induced Bax translocation that was equivalent to what was observed in Hsp70 overexpressing cells. This was observed both in SP600125-treated cells and in cells expressing a dominant negative JNK protein. The addition of SP600125 to cells expressing Hsp70 did not provide any additional level of protection, indicating that Hsp70 and SP600125 provide resistance through a common mechanism. Inhibition of ERK activation did not increase heat sensitivity as was expected but instead provided a partial level of resistance as seen by a 10% reduction in the percentage of cells with translocated Bax. A role for ERK activation in promoting apoptosis has been observed in cisplatin-induced cell death (57). ERK activation does not likely play a major role in signaling cell death in heat-stressed cells since the level of ERK activation by heat shock was minor compared with treatment with phorbol 12-myristate 13-acetate, which had no effect on heat-induced Bax translocation or survival (data not shown). Also, inhibition of ERK activation by Hsp70 is likely not a major factor in limiting Bax translocation since complete inhibition of ERK activation in Hsp70-expressing cells treated with PD98059 did not result in higher levels of resistance than were seen in Hsp70-expressing cells without PD98059 treatment. Therefore, our results suggest that the major role of Hsp70 in blocking heat-induced Bax translocation is through its ability to suppress JNK activation. Although we have not identified the target of JNK in heat-induced apoptosis, our model is that Hsp70 prevents apoptosis by limiting the JNK-mediated phosphorylation of a Bcl-2 family protein that regulates Bax translocation. Currently it is not known which of the BH3-only members of the Bcl-2 family contribute to heat-stress induced apoptosis.
Suppression of apoptosis by Hsp70 is believed to be a contributing factor in tumor development (18,19). Tumor cells often express Hsp70 as well as other heat shock proteins at abnormally elevated levels, and this heightened expression has been correlated with poor patient outcome. In addition to these tumor biopsy studies it has been shown that experimental overexpression of Hsp70 in transformed rodent cell lines can increase their tumorigenicity when implanted into syngenic mice (58). As well, malignant T-cell lymphomas were found to develop when Hsp70 was overexpressed in transgenic mice (59). Furthermore, recent studies have demonstrated that continued expression of Hsp70 is required to sustain tumor cell viability since siRNA or antisense RNAtargeted reduction of Hsp70 levels in tumor cell lines inhibits cell growth and triggers apoptosis (60 -62). Non-transformed cell lines were not affected by this treatment. If Hsp70 contributes to tumor development by inhibiting apoptosis, then one would expect that disruption of the apoptotic regulators that are affected by Hsp70 would also contribute to cancer development. Although the Bcl-2 family members have been demonstrated to play a role in tumorigenesis, neither Apaf-1 nor caspase-9 has this ability (63)(64)(65). This might suggest that Hsp70 contributes to tumorigenesis by preventing the initiation of the Bcl-2 regulated signaling events that lead to mitochondrial membrane permeabilization rather than by directly preventing caspase activation. Targeting apoptosome or caspase activation in tumor cells using small molecule activators should provide an efficient means to treat cancer cells that express Hsp70 at elevated levels. Hsp70 could also contribute to tumorigenesis by suppressing caspase-independent death pathways, which would represent the predominate form of cell death in cells in which apoptosis is deregulated (28,29,62,66).
Molecular chaperones have been reported to intervene at nearly every step in the apoptotic pathway (19,67). This lack of specificity might reflect the general role that these proteins play in regulating protein-protein interactions and protein trafficking. Many of the apoptotic control points are regulated by such mechanisms. However, a regulatory system that controls cell survival must include precise safeguards to prevent the accidental elimination of cells that may be capable of repairing stress-induced damage. Stress-induced apoptotic pathways also possess effective means to amplify the death signal to ensure that apoptosis is rapidly and efficiently executed. Proper control over cell death would require tight regulation of the decision to die without interfering with the machinery of cell destruction in cells that are committed to die. The ability to block both the initiation and execution of apoptosis is a dangerous property for a single class of proteins to possess. This might also be expected to be a difficult task for one protein to achieve since it would need to effectively inhibit many different apoptotic mediators that act through a variety of mechanisms. If Hsp70 were to prevent apoptosis primarily by acting after mitochondrial outer membrane permeabilization, it would need to inhibit the activity of caspases, apoptosis-inducing factor, endonuclease G, and perhaps other mediators of caspase-independent cell death. However, by acting upstream of mitochondria to prevent outer membrane permeabilization, Hsp70 would participate in the decision to either repair the damage and prevent apoptosis or allow commitment to apoptosis when the extent of cellular damage becomes overwhelming. In this way Hsp70 would not interfere with the apoptotic process after commitment had occurred. Our results suggest that this is precisely the role of Hsp70 in heat-stress induced apoptosis, since it is capable of preventing the events that lead up to membrane permeabilization but does not interfere with cell death after this event has occurred. These results do not exclude the possibility that Hsp70 could suppress cell death that is not dependent upon JNK activation or which proceeds through an alternative cell death pathway that does not involve mitochondrial disruption and apoptosome formation.