INTRODUCTION

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Protein-damaging stresses, such as exposure of cells to elevated temperatures, activate an adaptive response leading to the increased synthesis of a group of proteins that regulate protein-folding processes (reviewed in reference 43). Members of the hsp70 family of molecular chaperones recognize nonnative domains that are exposed during protein translation, membrane translocation, oligomerization, and ultimately degradation. The abundant cytoplasmic and nuclear protein hsc70 is assisted in this task by the highly inducible hsp70 protein, whose synthesis is controlled by the level of nonnative protein substrates. Conditions that alter protein structure can result in the exposure of hydrophobic regions that are normally buried within the molecule, leading to their aggregation and loss of function. The ability of hsp70 to compete for binding to these hydrophobic regions coupled with an ATP-driven mechanism for release prevents protein aggregation and assists in the establishment of the native conformation (reviewed in references 4 and 18). Peptide binding is localized to a 15-kDa region near the COOH-terminal end of hsp70. Binding and release are regulated by ATP binding and hydrolysis, which occur in a 44-kDa NH₂-terminal ATPase domain. Conformational changes mediated by ATP hydrolysis stabilize a high-affinity association between hsp70 and a substrate. The rate of ATP turnover is regulated by a family of cochaperones that bind to the ATPase domain and to a COOH-terminal 10-kDa region. The COOH-terminal amino acid sequence Glu-Glu-Val-Asp, which is absolutely conserved in all eukaryotic hsc70 and hsp70 family members, is essential for association with the cochaperones hsp40 (hdj1) and Hop (8, 11). Deletion or mutation of these four residues increases the intrinsic ATPase activity and prevents stable association with peptide substrates (11).

An increased demand for the chaperoning function of hsp70 occurs at many stages in the life of a cell, as evidenced by changes in its level of expression during development and through the cell cycle. hsp70 and other stress-induced chaperones also determine the outcome of cells that are faced with death (27, 31). Elevated expression of hsp70 following exposure of cells to a mild heat stress provides an increased level of resistance to a subsequent more severe heat treatment that normally results in extensive apoptosis (36, 47). Stress-induced apoptosis is initiated by the release of cytochrome c from the mitochondrial intermembrane space (24, 70). The binding of cytochrome c, together with dATP, to the apoptotic protease-activating factor (Apaf-1) in the cytoplasm causes Apaf-1 to expose its N-terminal caspase recruitment domain, leading to association with procaspase 9 (33, 51). Recruitment of procaspase 9 into this complex, the apoptosome, results in procaspase 9 processing, and the apoptosome is then able to process downstream effector caspases, including caspases 3, 6, and 7. The inactivation and dismantling of the dying cell are brought about by the action of these proteases through cleavage of specific targets.

Elevated expression of hsp70 can block these death processes. Cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase is inhibited in hsp70-expressing cells (5, 12, 44). This is a result of reduced processing of procaspase 3 and not due to an inhibition of the activity of the processed enzyme (44). Protection by hsp70 has been reported to occur downstream of cytochrome c release (22, 29) and in some cases downstream of caspase 3 activation and cleavage of caspase 3 substrates (22). In vitro experiments, using mitochondrion-free cytoplasmic extracts, revealed that purified recombinant hsp70 can inhibit cytochrome c-dATP-mediated caspase activation (1, 29). This effect requires the peptide binding domain and C-terminal EEVD sequence of hsp70.

Stress signaling through the JNK pathway precedes apoptotic death in cells exposed to protein-damaging stresses such as heat shock and ethanol, and also occurs in response to non-protein-damaging signals such as stimulation with cytokines such as interleukin-1 and tumor necrosis factor (TNF) or following UV irradiation (reviewed in references 19 and 26). hsp70 has been shown to block JNK activation and prevent apoptosis in response to both protein-damaging and physiological stimuli (12, 39, 44, 62, 69). However, this has only been observed in cells in which hsp70 was transiently overexpressed using tetracycline-regulated expression in stable cell lines, adenovirus delivery, or a mild heat treatment (reviewed in reference 13). The ability of hsp70 to block JNK activation is not detected in cells that have been selected for constitutive hsp70 expression (5, 22, 44). Although JNK activation was no longer repressed in these cells, they were protected from apoptosis. Inhibition of JNK activation can be accomplished by an hsp70 protein lacking its ATPase domain (63). This protein with ATPase deleted has also been shown to provide protection from extreme hyperthermic exposures (30, 57, 63).

The aim of this study was to determine whether the chaperone function of hsp70 is required for the prevention of heat-induced apoptosis. Since constitutive overexpression of hsp70 is known to affect cell growth and disrupt signaling pathways (10, 44) we were concerned that forced overexpression of hsp70 could lead to the selection of variant cell lines with adaptations affecting apoptotic signaling or effector processes. We therefore used an inducible expression system to generate cell lines expressing various hsp70 domain mutants. Cell lines with tetracycline-regulated expression of hsc70, hsp70, or hsp70 lacking either the ATPase domain or the C-terminal EEVD sequence or with a replacement of the EEVD sequence with four alanines were generated using a dicistronic expression cassette that also encodes the green fluorescent protein (GFP). A cell line with tetracycline-regulated expression of both GFP and the blue fluorescent protein (BFP) was used as a control. The effect of these proteins on cell growth and on survival following heat shock was examined and compared to their ability to affect JNK signaling and apoptotic processes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Construction of plasmids. All transfected cell lines were generated using a tetracycline-regulated dicistronic expression vector derived from plasmid pTR-DC/GFP (45). Transcripts from this vector carry a gene of interest in the position of the first cistron and the GFP gene as the second cistron. This plasmid was modified by the addition of the adenovirus tripartite leader sequence and the insertion of the human hsp70 gene to create plasmid pTR5-DC/hsp70-GFP (44). This plasmid was used to create the vectors encoding the hsp70EEVD and hsp70AAAA proteins. Construction of the pTR5-DC/hsp70AAAA-GFP plasmid was described by Shi et al. (56). The pTR5-DC/hsp70EEVD-GFP plasmid was constructed using the same strategy, which involved replacing a ClaI-EcoRI fragment encoding the wild-type hsp70 sequence with a corresponding fragment from plasmid pMSHsp70EEVD (11). Plasmid pTR5-DC/GFP was modified by the addition of the hygromycin resistance gene, under the control of the thymidine kinase promoter, from pCEP4 (Invitrogen Inc., Carlsbad, Calif.) creating pTR5-DC/GFP*tk/hygro. Plasmid pTR5-DC/hsp70-GFP was also modified in the same way to create pTR5-DC/hsp70-GFP*tk/hygro. The hsp70ATPase expression plasmid was created by digesting pTR5-DC/hsp70-GFP*tk/hygro with BglII and removing the 924-bp fragment to crate an in-frame deletion of amino acids 119 to 426 comprising the ATPase domain. Plasmid pTR5-DC/hsc70-GFP*tk/hygro was created by cloning the hsc70 cDNA as a BamHI fragment from plasmid -actin-hsc70 into the unique BglII site of pTR5-DC/GFP*tk/hygro. A dicistronic expression plasmid carrying both the BFP gene and the GFP gene was constructed and used as a control. The BFP gene was amplified with BamHI ends from pQBI50-BFP (Quantum Biotech, Montreal, Quebec, Canada) and ligated into BglII-cut pTR5-DC/GFP*tk/hygro.

Generation of transfected cell lines. A PEER cell line expressing the reverse tetracycline-controlled transactivator (rtTA) was generated by transfection with plasmid pUHD172-1-neo (15). The transfected cells were selected with 400 µg of G418 (Life Technologies, Gaithersburg, Md.)/ml in complete medium, which consisted of RPMI 1640 (Mediatech Inc., Herndon, Va.) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, Utah). Following selection in bulk culture the cells were screened by transient transfection with the tetracycline-regulated GFP expression plasmid pTR/GFP and cloned as described previously (45). A single rtTA-expressing clone (PErTA) was used as the parent cell line for the generation of each of the hsp70-expressing clones. For transfection, cells (10⁷ in 0.4 ml of complete medium) were electroporated with a BTX T820 electroporator using a single pulse of 200 V at 50 ms (Genetronics Inc., San Diego, Calif.). Transfections were performed with 10 µg of linearized plasmid DNA. The pTR5-DC/hsp70AAAA-GFP and pTR5-DC/hsp70EEVD-GFP plasmids were cotransfected with plasmid ptk/hygro. Transfected cells were selected in bulk culture with 200 µg of hygromycin (Sigma Chemical Co., St. Louis, Mo.)/ml. The hsp70AAAA and hsp70EEVD cells were screened and selected by flow-cytometric sorting of GFP-positive cells after overnight incubation with 1 µg of doxycycline hydrochloride (Sigma)/ml. Clones were obtained using the AUTO-CLONE multiwell automated cell deposition system (EPICS ELITE ESP; Beckman-Coulter, Hialeah, Fla.). The other transfectants were cloned using the Quixell micromanipulator cell transfer system (Stoelting Co., Wood Dale, Ill.), which was mounted on a Leica DM-IRB inverted fluorescence microscope (Leica Canada, Montreal, Quebec, Canada). Complete details on the use of this micromanipulator can be found in Caron et al. (6). All clones were subsequently characterized by monitoring GFP expression by flow cytometry and hsp70 expression by Western blotting as described previously (44). Measurement of both hsp70 and GFP by flow cytometry was performed as described previously (44). The PETA70 cell line, in which hsp70 expression is controlled by the tetracycline-regulated transactivator, tTA, and is induced by the removal of tetracycline has been described (44).

Cell growth and viability measurements. Cell growth was measured using a flow-cytometric cell counting method. Cells in log phase growth were resuspended in fresh media with or without doxycycline (1 µg/ml) at a concentration of 1.5 × 10⁵ cells/ml, and the viable and nonviable cells were counted twice daily over a period of 4 days. Cell suspensions (490 µl) were mixed with 10 µl of a fluorescent bead suspension (Flow-Check fluorospheres; Beckman-Coulter) resulting in a bead concentration of 2 × 10⁴ beads/ml. The cell population was gated using forward and side light scattering to discriminate viable and nonviable cells on the basis of cell size and granularity. Flow-cytometric sorting of these two populations, followed by acridine orange staining and microscopic examination, confirmed that they corresponded to homogeneous populations of viable and apoptotic cells. The analysis was performed on an EPICS XL-MCL flow cytometer (Beckman-Coulter) using a 488-nm dichroic filter. The acquisition was stopped after counting 10³ beads, which permitted a constant sample volume (50 µl) to be analyzed for each sample. The total number of cells per milliliter in each map (viable and nonviable) was then calculated. The apparent growth rates (µ_app) were calculated from the time profiles of viable cells plotted against the integral of viable cells. Death rates (k_d) were calculated from the time profiles of dead cells against the integral of viable cells (49). A ratio of µ_app and k_d for the induced cells relative to the noninduced cells was calculated. This method was also used to measure the number of viable cells after heat shock.

Caspase measurements. Cell extracts were prepared for measurement of caspase activity by incubation in cell lysis buffer (ApoAlert kit; Clontech, Palo Alto, Calif.) at a concentration of 10⁶ cells/50 µl for 10 min on ice followed by centrifugation at 13,000 × g for 5 min at 4°C. The supernatants were then stored at 80°C. The protein concentration was determined using the DC protein assay kit (Bio-Rad Laboratories Canada Ltd., Mississauga, Ontario, Canada). DEVDase activity was measured by mixing 20 µl of cell extract with 50 µM N-acetyl-DEVD-7-amino-4-methylcoumarin (Ac-DEVD-AMC) (Biomol Research Laboratories, Plymouth Meeting, Pa.) in a total volume of 200 µl of reaction buffer {50 mM HEPES-KOH (pH 7.0), 10% glycerol, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 2 mM EDTA, 5 mM dithiothreitol (DTT)} and measuring the continuous liberation of AMC at 37°C using a Cytofluor multiwell plate reader (PerSeptive Biosystems, Framingham, Mass.) with excitation and emission wavelengths of 380 and 460 nm, respectively. Values for the relative fluorescence units of AMC released per minute per microgram of protein were calculated for each sample and plotted relative to the maximum value for each cell line. Procaspase processing was also measured by Western blotting as described previously (44) using an anti-caspase 3 antibody (provided by D. W. Nicholson; Merck Frosst Center for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada) and an anti-caspase 9 antibody (provided by D. R. Green; La Jolla Institute for Allergy and Immunology, San Diego, Calif.).

Analysis of JNK activation and JNK phosphatase activity. JNK activation was measured by Western blotting using a phosphorylation state-specific anti-JNK antibody (Promega Corp., Madison, Wis.). Cells (2 × 10⁶) were lysed in 50 µl of buffer C (25% glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 20 mM HEPES [pH 7.9], 1 mM DTT, 50 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 20 µg of leupeptin, 5 µg of pepstatin, and 2 µg of aprotinin/ml). The lysate was centrifuged at 16,000 rpm for 15 min at 4°C, and the supernatant was retained. A portion of each sample (20 µl) was mixed with 5 µl of 5× sodium dodecyl sulfate (SDS) sample buffer (250 mM Tris-HCl [pH 6.8], 50% glycerol, 10% SDS, 25% 2-mercaptoethanol, 0.5% bromophenol blue) and heated to 90°C for 5 min. The samples (20 µg each) were subjected to SDS-polyacrylamide gel electrophoresis (Novex, San Diego, Calif.) and transferred to Hybond-C nitrocellulose (Amersham Corp., Arlington Heights, Ill.). Membranes were blocked with 1% bovine serum albumin (BSA) in TBST (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween 20) and then incubated with a 1:5,000 dilution of the anti-ACTIVE JNK polyclonal antibody in TBST-0.1% BSA for 3 h at room temperature. The membranes were washed three times with TBST and then incubated for 1 h with a 1:10,000 dilution of a horseradish peroxidase (HRP)-conjugated sheep anti-rabbit immunoglobulin G (IgG) (Amersham). Following three washes with TBST the membranes were processed by enhanced chemiluminescence (ECL) (Amersham) and exposed to film. The blots were subsequently stripped by incubation at 50°C for 30 min in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl [pH 6.7]), washed three times with TBST, blocked in TBST-1% BSA, and then incubated overnight at 4°C with a 1:1,000 dilution of an anti-JNK antibody (New England Biolabs Inc., Beverly, Mass.). The blots were then incubated with HRP-conjugated anti-rabbit IgG and processed by ECL. This antibody detects total JNK and was used to verify that equivalent amounts of JNK were present in each sample.

Examination of cytochrome c release. Cytosolic extracts were prepared by homogenizing 2 × 10⁷ cells in an isotonic buffer [220 mM mannitol, 68 mM sucrose, 50 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 20 µg of leupeptin, 5 µg of pepstatin, and 2 µg of aprotinin/ml] using a Dounce homogenizer and a type B pestle. Cell disruption was monitored by staining an aliquot of the homogenized cells with trypan blue. The lysates were centrifuged at 800 × g for 10 min, and the resulting supernatants were clarified by centrifugation at 100,000 × g for 30 min. Equal amounts of protein from each of the 100,000 × g supernatants were analyzed by Western blotting using anti-cytochrome c (7H8.2C12; Pharmingen) and antiactin (C4; ICN) antibodies. Cell viability was assessed at the time of cell collection by counting apoptotic and viable cells after staining with acridine orange and ethidium bromide (44). Cytochrome c release was also measured in cells permeabilized with streptolysin O (66). Cells (10⁶) were washed with PBS, collected by centrifugation, and resuspended in 50 µl of StrepO buffer (20 mM HEPES-KOH [pH 7.5], 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 µM z-VAD-FMK (Biomol), 0.1 mM PMSF, and 5 µg of pepstatin, 10 µg of leupeptin, and 2 µg of aprotinin/ml) containing 60 U of streptolysin O (Sigma). After incubation at 37°C for 30 min the permeabilized cells were pelleted by centrifugation at 16,000 × g for 30 min at 4°C. The supernatant, containing cytosolic proteins, was mixed with 12.5 µl of 5× SDS sample buffer. The pellet, which contained the permeabilized cells including the mitochondria, was dissolved in 62.5 µl of 1× SDS sample buffer and sonicated. Samples were heated to 90°C for 5 min, and then equal volumes of the pellet and supernatant fractions were analyzed by Western blotting using anti-cytochrome c (7H8.2C12; Pharmingen) and anti-cytochrome oxidase subunit II (A-6404; Molecular Probes) antibodies.

	RESULTS

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Inducible expression of hsc70, hsp70, and hsp70 domain mutant proteins. A PEER cell line expressing the reverse tetracycline-controlled transactivator (PErTA) was established and used to generate each of the hsp70-expressing cell lines. The tetracycline-regulated expression cassette used to generate these cell lines encodes a dicistronic message in which the second cistron is the GFP gene (45). The first cistron encodes either hsc70, hsp70, hsp70 lacking the ATPase domain, hsp70 lacking the four C-terminal amino acids EEVD, or hsp70 in which the EEVD sequence has been replaced by AAAA (Fig. 1A). To control for possible effects of GFP expression, we generated a cell line with the dicistronic vector in which the first cistron is the BFP gene. When induced this cell line expresses both BFP and GFP. Stable cell lines were obtained, after transfection and selection for hygromycin resistance, by transient induction and selection of individual GFP-positive cells with a micromanipulator (6) or by flow-cytometric cell sorting (45). Individual clones were subsequently screened by flow cytometry, and those with similar levels of induced GFP fluorescence were selected. Figure 1B shows flow-cytometric profiles of GFP fluorescence in each of the selected clones before and after induction with doxycycline. Each of the PErTA clones was induced for 24 h except for the HSC70 and BFP-expressing cell lines, which were induced for 48 h. A single peak of GFP-positive cells was observed after induction of the HSC70, HSP70ATPase, and HSP70EEVD clones. For the other three clones the distribution was bimodal with approximately 70% of the cells showing high levels of GFP fluorescence.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Stable cell lines with tetracycline-regulated expression of hsc70, hsp70, or hsp70 domain mutant proteins. (A) Schematic representation of hsp70 functional domains. hsp70 and hsc70 have an amino-terminal ATPase domain followed by a peptide binding domain and the C-terminal sequence EEVD. (B) Flow-cytometric profiles of GFP expression in each of the clones grown in the presence (shaded profile) and absence (open profile) of doxycycline. The expression cassette is dicistronic and carries both the gene of interest and the GFP gene. In the BFP-expressing cell line the dicistronic transcript carries both the BFP and GFP genes. All of the cell lines were induced for 24 h except for the hsc70 and BFP-expressing cell lines, which were induced for 48 h. (C) Western blot analysis showing the levels of expression of the proteins encoded by the first cistron. Cells were grown with or without doxycycline as described for panel B. The upper portion shows the results with antibody N27, which recognizes both hsc70 and hsp70. The bottom portion shows the results for hsp70-specific antibody C92. Purified hsp70 (25 to 400 ng) was included to insure linearity of the signals.

Continuous expression of hsp70 affects cell growth and viability. Constitutive overexpression of hsp70 has been shown to decrease the growth rates of various transfected cell lines (21, 31, 46). This was also observed in a Drosophila melanogaster cell line in which the expression of hsp70 was regulated by the metallothionein promoter (10). In order to determine whether a specific domain of hsp70 was responsible for this growth inhibition, we compared the growth rates of each of the clones in the induced and noninduced states (Fig. 2A). Cell counts were performed by flow cytometry, which allowed us to quantitate the numbers of viable and nonviable cells in both the total cell population and the GFP-positive cells. Induced expression of hsp70 had a rapid and dramatic effect on cell growth. Differences in the rates of growth for the HSP70 cell line were evident after 24 h of induction. The µ_app of the induced cells (GFP-positive cells) was 28% of that of the noninduced cells (Fig. 2B). A similar effect on cell growth was seen in cells expressing the hsp70ATPase and to a lesser extent in the cells expressing hsp70EEVD or hsp70AAAA. The calculated µ_app decreased to 49% for hsp70ATPase-expressing cells and to 67% for hsp70EEVD- and hsp70AAAA-expressing cells. In the control cell line expressing BFP the µ_app of the induced cells was 90% of the value for the noninduced cells. The effect of hsc70 on cell growth was similar to what was observed in the BFP-expressing cell line. The µ_app of the HSC70 cell line when induced was 85% of that for the noninduced cells. However, hsc70 protein levels increased more slowly in this cell line than induced accumulation of hsp70 in each of the PErTA-HSP70 cell lines. If the analysis of growth rates is restricted to only the last 2 days of culture, the µ_app of the hsc70-expressing cells was reduced to 37% of that of the noninduced cells. These results reveal that expression of each of the hsp70 proteins reduced cell growth and that the peptide binding domain has a greater influence on growth inhibition than does the ATPase domain. Presumably this is due to multiple effects, such as titration of the cochaperones Hip or Bag1 by the ATPase domain and by interactions with rate-limiting substrates or the cochaperones Hop or hdj1 by the COOH domain.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Effect of hsc70 and hsp70 expression on cell growth. (A) Time profiles of the accumulation of viable cells (open circles), total cells (solid circles), and cell viability (diamonds) for cells grown in the absence (OFF) or presence (ON) of doxycycline. (B) µapp and kd were calculated from the data shown in panel A, and the ratios of these values for the cells grown in the presence or absence of doxycycline were calculated. The calculated values for µapp ON and µapp OFF for cells expressing the indicated proteins are, respectively, as follows: hsc70 0.0176 and 0.0206 h1; hsp70, 0.0057 and 0.0203 h1; hsp70ATPase, 0.0084 and 0.0178 h1; hsp70EEVD, 0.0155 and 0.0229 h1; hsp70AAAA, 0.0149 and 0.0222 h1; BFP, 0.0172 and 0.0191 h1. Corresponding calculated values for kd ON and kd OFF are, respectively, as follows: hsc70, 0.0071 and 0.0045 h1; hsp70, 0.0161 and 0.0034 h1; hsp70ATPase, 0.01 and 0.0041 h1; hsp70EEVD, 0.004 and 0.0022 h1; hsp70AAAA, 0.0091 and 0.0031 h1; BFP, 0.0044 and 0.0027 h1.

Prevention of heat-induced apoptosis by transient expression of hsp70 or hsc70. The cytoprotective properties of hsp70 have been well documented, although its mechanism of protection is not clear. Since the major role of hsc70 and hsp70 is in protein folding it was of interest to determine whether hsp70 protection is mediated through a chaperoning mechanism. For these experiments the cells were induced for only 24 h, except for the HSC70 and BFP-expressing cell lines, which were induced for 48 h. Induction kinetics are slower for these two cell lines, and so the additional 24 h of induction was needed in order to achieve levels of expression similar to those for the other cell lines. The levels of expression at these times are shown in Fig. 1C. The induction times that were used were also selected to minimize the effects of expression on growth inhibition or cell death. Analysis of cell cycle distribution by flow cytometry showed that at the end of the induction times there were no differences in the percentages of cells in G₁, S, and G₂ phases of the cell cycle (data not shown). The cells were extensively washed to remove doxycycline and resuspended in fresh media to shut off expression in the induced cells prior to the heat treatment.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Protection from heat-induced apoptosis by hsc70 and hsp70 proteins. (A) Demonstration that GFP fluorescence intensity correlates with levels of hsp70. Noninduced (upper panel) and induced (lower panel) cells were fixed and processed for immunocytochemical detection of hsp70 using the hsp70-specific antibody C92 and a PE-conjugated antimouse antibody. (B) Analysis of cell viability by annexin-PE staining in the hsp70-expressing cell line. Control and heat-shocked (43°C for 60 min followed by 9 h at 37°C) cells were incubated with annexin-PE and then analyzed for PE and GFP fluorescence by flow cytometry. Quadrants 1 to 4 correspond to cells that are annexin+ GFP, annexin+ GFP+, annexin GFP, and annexin GFP+, respectively. (C) Results of annexin staining for each of the cell lines after heat shock. The left panel shows the means and standard errors (n = 3) for the noninduced (OFF) and induced (ON) cells. For the induced cells the viabilities were calculated for the GFP-positive cells only. The right panel shows the means plus standard errors of the differences between the viabilities of the ON and the OFF cells. Comparison of each of these means to that of the BFP-expressing cell line shows that only hsc70 and the intact hsp70 protein provide protection against apoptosis (*, P values of a one-tailed t test are 0.0002 for the HSC70 cell line, 0.001 for the HSP70 cell line, and 0.0008 for the PETA70 cell line; all other P values are greater than 0.05). The PETA70 cell line has tTA-regulated expression of hsp70.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Cell viability after heat shock is enhanced by hsc70 and hsp70, and this requires the chaperone function of hsp70. Noninduced (OFF) and induced (ON) cells were heated at 43°C for 60 min and returned to 37°C for 24 h. Flow-cytometric cell counts were carried out before the heat shock and after the 24-h recovery period. Separate cultures of nonheated cells were also counted at the same times. Cell numbers at the end of the recovery period are plotted relative to the initial cell count for control (open bars) and heated cells (solid bars). Shown are the means plus the standard errors for three independent experiments. Only the cells expressing hsc70 or hsp70 have significantly higher numbers of viable cells after heat shock (*, P values of a one-tailed t test comparing the relative cell numbers after heat shock for the ON and OFF cells are 0.033 for the HSC70 cell line and 0.022 for the HSP70 cell line; all other P values are greater than 0.1).

View larger version (31K): [in this window] [in a new window]   FIG. 5.   At equivalent levels of expression the full-length hsp70 protein provides protection whereas the protein with ATPase deleted does not. The HSP70 (clone 8-17) and HSP70ATPase cell lines were incubated for 24 h with various doses of doxycycline ([DOX]) ranging from 100 to 1,000 ng/ml and then examined for GFP fluorescence by flow cytometry (A), expressed protein levels by Western blotting (B), and resistance to heat-induced apoptosis (D) as described for Fig. 4. Quantitation of the Western blot (C) and comparison to the percent viabilities after heat shock (E) reveal that the full-length hsp70 protein protects cells from apoptosis when expressed at a level equal to the maximum level attained in the HSP70ATPase cell line and that the protein with ATPase deleted does not protect cells at any of the expressed levels.

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Cells protected from heat-induced apoptosis by hsp70 retain their proliferative capacity. The HSP70 cell line (clone 8-17), either noninduced (solid symbols) or incubated with 200 ng of doxycycline/ml for 24 h (open symbols), was exposed to 43°C for 60 min and then returned to 37°C (diamonds). The numbers of viable cells were determined, as described for Fig. 4, over a period of 5 days and are plotted relative to the initial cell count. The growth of control non-heat-shocked cells (circles) is shown for comparison.

The chaperone function of hsp70 is not required for JNK inhibition. Since it was clear that the chaperoning function of hsp70 was necessary for prevention of apoptosis, we next examined whether this was also true for inhibition of JNK activation in heat-shocked cells. For this, each of the cell lines were heated at 43°C for 60 min and collected immediately after the heat shock and also after a return to 37°C for 1 h. The amount of activated JNK was measured by immunoblotting with an antibody that specifically recognizes the active phosphorylated forms of JNK1 and JNK2 (Fig. 7). Active JNK was not detected in either the noninduced or induced cell lines in the absence of stress. Following heat shock high levels of phosphorylated JNK1 (pJNK1) and to a lesser extent pJNK2 were seen in all of the noninduced cells. The amount of each of these phosphorylated proteins decreased during the recovery period at 37°C, except for those produced by the HSP70ATPase and -EEVD cell lines, which remained elevated. The extent of both JNK1 and JNK2 activation after heat shock was significantly reduced in all of the induced cells expressing hsc70 and hsp70 proteins, including those expressing hsp70 domain mutants. Expression of BFP had only a small effect on JNK activation. Quantitative analysis of the images revealed that the total pJNK1 and pJNK2 signal for the BFP-expressing cell line was 20% less for induced cells that were heat shocked than for noninduced cells. For the cell lines expressing hsp70 and each of the mutant hsp70 proteins the signals were approximately 70% less. In the hsc70-expressing cell line JNK2 activation was reduced by 75% and JNK1 activation was reduced by 50%. Therefore hsc70, like hsp70, is capable of inhibiting JNK activation. The inhibition of JNK activation by hsp70 occurs independently of its chaperoning activity since the ATPase mutant protein and the C-terminal regulatory domain mutant proteins were equally capable of inhibiting its activation following stress.

View larger version (38K): [in this window] [in a new window]   FIG. 7.   Inhibition of heat-induced JNK activation in hsc70- and hsp70-expressing cells. Western blot analysis showed levels of phosphorylated JNK1 and JNK2 in noninduced (OFF) and induced (ON) cells. Cells were either not treated (C), heated at 43°C for 60 min (HS), or heat shocked and returned to 37°C for 60 min (R). JNK activation was strongly inhibited in all of the induced cells lines except for the BFP-expressing cell line.

View larger version (24K): [in this window] [in a new window]   FIG. 8.   The C-terminal substitution mutant hsp70AAAA inhibits JNK activation without blocking its rate of dephosphorylation. IMR90 lung fibroblast cells (20 to 30 population doublings) were infected with an adenovirus expressing tTA (AdCMV-tTA) and an adenovirus encoding a tetracycline-regulated expression cassette encoding full-length hsp70 (+Hsp70) or hsp70 with the C-terminal four amino acids EEVD replaced with AAAA (+Hsp70-4AA). Control cells received the same total multiplicity of infection of the hsp70-encoding virus but without the Ad CMV-tTA virus (control). The infected cells were heat shocked for 30 min at 45°C and then incubated at 37°C with the protein kinase inhibitor staurosporine to block new JNK phosphorylation. Extracts were prepared from non-heat-shocked cells (Con.), heat-shocked cells (0), and heat-shocked cells that were incubated with staurosporine for 20 or 40 min at 37°C after the heat shock. Shown is an immunoblot of cytosolic extracts probed with an anti-phospho-JNK antibody.

Prevention of procaspase activation by hsp70 requires its chaperone function. We have previously shown that inhibition of stress-induced apoptosis by hsp70 occurs upstream of caspase 3 activation (44). To determine whether the cytoprotective effects of hsp70 and hsc70 overexpression are associated with an inhibition of caspase activation and whether this required the chaperone function of hsp70, we monitored DEVDase activity in extracts from heat-shocked cells using the fluorogenic substrate DEVD-AMC (Fig. 9). For each of the noninduced cell lines there was a progressive increase in DEVDase activity with time after heat shock. When induced, the HSC70 and HSP70 cell lines showed initial increases in DEVDase activity immediately after exposure to 43°C for 60 min, but this level subsequently decreased in cells that were maintained at 37°C for 6 to 9 h after the heat shock. However, in the control cell line expressing BFP and in each of the hsp70 mutant-expressing cell lines the increase in DEVDase activity was the same for both the induced and noninduced cells. This reveals that the chaperone function of hsp70 is required to prevent caspase activation.

View larger version (29K): [in this window] [in a new window]   FIG. 9.   Inhibition of heat shock-induced caspase activation in cells expressing hsc70 or hsp70 but not in cells expressing the hsp70 deletion mutant proteins. Extracts were prepared from noninduced (solid circles) and induced (open circles) cells that were heated at 43°C for 60 min and collected either immediately after the heat shock (0) or after a return to 37°C for 3, 6, or 9 h. Extracts were also prepared from control non-heat-shocked cells (C). Caspase activity was measured using a fluorometric assay with the substrate Ac-DEVD-AMC. Activities (fluorescence units per minute per microgram of protein) are plotted relative to the maximum activity obtained for each cell line.

View larger version (26K): [in this window] [in a new window]   FIG. 10.   Inhibition of procaspase 9 and procaspase 3 processing in cells expressing hsc70 or hsp70. Western blot analysis of extracts from noninduced (OFF) and induced (ON) control (C) and heat-shocked (HS; 43°C for 60 min followed by 6 h at 37°C) cells showing levels of the intact procaspase 9 protein, the intact procaspase 3 protein, and its processed large subunit (p17). Protection against loss of the proform of caspase 9 or 3 in the induced state occurs only for cells expressing hsc70 or hsp70.

Release of cytochrome c from mitochondria after heat shock is blocked in hsp70-expressing cells. Procaspase processing could be inhibited by an effect of hsp70 on apoptosome function or at the level of cytochrome c release. Purified hsp70 can block cytochrome c-dATP-mediated caspase activation in mitochondrion-free extracts (1, 29). However, this does not exclude the possibility that hsp70 might also block apoptosis upstream of apoptosome formation by preventing cytochrome c release. To examine this, cytosolic extracts were prepared by Dounce homogenization of cells in an isotonic buffer followed by ultracentrifugation. The amount of released cytochrome c present in the supernatants was assessed by Western blotting (Fig. 11A). A large increase in cytosolic cytochrome c was evident by 3 h after the heat shock for the noninduced HSP70 cell line. The amount of released cytochrome c increased further at 6 and 9 h. However, substantially less cytochrome c was released from cells that were induced to express hsp70 prior to the heat shock. The blot was probed for actin to insure that equivalent amounts of protein were present in each lane. The smaller amount of cytosolic cytochrome c in the hsp70-expressing cells correlated with the reduced number of apoptotic cells (Fig. 11B) and the reduced amount of caspase activity in the extracts (Fig. 11C).

View larger version (40K): [in this window] [in a new window]   FIG. 11.   Release of cytochrome c from mitochondria after heat shock is blocked in hsp70-expressing cells. (A) Levels of cytochrome c and actin were measured by Western blotting of cytosolic extracts prepared by Dounce homogenization of noninduced (OFF) and induced (ON) HSP70 cells (clone 8-17). Extracts were prepared from nonstressed cells (C) and cells that were exposed to 43°C for 60 min and collected either immediately after the heat shock (0) or following incubation at 37°C for 3, 6, or 9 h. (B) Cell viability was measured at the time of cell collection by counting viable and apoptotic cells after staining with acridine orange and ethidium bromide. (C) Caspase activity in the extracts was measured as described for Fig. 9.

View larger version (30K): [in this window] [in a new window]   FIG. 12.   Cytochrome c release from heat-shocked cells measured by streptolysin O permeabilization. Noninduced (OFF) and induced (ON) cells expressing hsp70 (clone 8-17) and hsp70ATPase were either not stressed (C) or heat shocked by exposure to 43°C for 60 min and then returned to 37°C for 6 h (HS). Cells were permeabilized by incubation with streptolysin O and then centrifuged to separate the cytosolic proteins (S) from the permeabilized cells (P). Equal volumes from each fraction were analyzed by Western blotting with antibodies to cytochrome c (cyt. c) and mitochondrial inner membrane protein cytochrome oxidase subunit II (COXII).

View larger version (61K): [in this window] [in a new window]   FIG. 13.   Immunocytochemical examination of cytochrome c release. The localization of cytochrome c was analyzed in noninduced (OFF) and induced (ON) HSP70 (clone 8-17) cells before (control) and after (HS) heat shock (43°C for 60 min followed by 6 h at 37°C). Cytochrome c has a punctate localization in control cells (A and E). Following a heat shock the mitochondrion-localized immunofluorescence pattern is lost and cytochrome c becomes evenly distributed throughout the cytoplasm (C). Cytochrome c remains localized to mitochondria in cells expressing hsp70 (G). Nuclei were stained with DAPI (B, D, F, and H).

View larger version (13K): [in this window] [in a new window]   FIG. 14.   The chaperone function of hsp70 is required to block cytochrome c release. Cytochrome c release was analyzed in noninduced (OFF) and induced (ON) cells expressing hsp70 (clone 8-17) and hsp70ATPase by immunocytochemistry as described for Fig. 13. The cells with mitochondrion-localized or cytoplasmic cytochrome c were counted, and the percentages of cells with mitochondrion-localized cytochrome c are plotted. Punct, punctate.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Although it is clear that hsp70 has general cytoprotective properties and can prevent stress-induced apoptosis, it is not known how this is accomplished. hsp70 has been demonstrated to affect processes regulating apoptotic signaling, effector molecule activation, and even events downstream of caspase activation. It is possible that hsp70 could affect multiple apoptotic pathways and that cell type-specific differences may account for the various points of intervention by hsp70. However, an important consideration in examining the effect of hsp70 is the manner in which it is expressed. The heat shock response is carefully regulated to ensure that, once an adequate amount of hsp70 is synthesized, transcription of the hsp70 gene is rapidly attenuated (42). This is because continuous high-level expression of hsp70 affects cell growth. In transfected Drosophila cells continuous expression of hsp70 from an inducible promoter resulted in growth inhibition and the sequestration of hsp70 into cytoplasmic granules (10). Formation of these granules occurred at a time that corresponded with a resumption of the proliferative capacity of the cells. Cell lines that constitutively overexpress hsp70 have been reported to have prolonged doubling times (21, 31, 46). Also, signaling pathways that are regulated by hsp70 overcome this level of control when hsp70 is constitutively overexpressed (5, 22, 44). In the lymphoid cell line studied here, continuous expression of hsp70 reduced growth rates by about 3.5-fold, and this was associated with an increased rate of cell death. It is likely, then, that in the generation of cell lines expressing hsp70 under the control of constitutively active promoters the isolated clones could result from the selection of variants that have defects in apoptotic signaling or effector processes.

To insure that we were examining direct effects of hsp70 and not effects that were the consequence of adaption to its continuous expression, we used an inducible expression system to generate cell lines expressing hsp70 and various domain mutant proteins. Another advantage of the approach that we have used is that by expressing hsp70 as a dicistronic message encoding the GFP reporter protein it is possible to restrict the analysis to only the GFP-positive cells. This was important because not all of the transfected cell lines express the encoded proteins in all cells after induction. Demonstration of tetracycline-regulated expression by Western blotting alone does not reveal what percentage of the induced cells are expressing the protein under study and how levels of expression vary within the population.

Continuous expression of hsp70 inhibited cell growth. The full-length protein was more inhibitory than were either of the domain mutant proteins. However, the protein with ATPase deleted inhibited growth more than either of the C-terminal mutant proteins. The protein with ATPase deleted is probably as effective at inhibiting cell growth as the full-length protein given that its level of expression was about twofold lower than that of hsp70. This suggests that inappropriate interactions between hsp70 and substrate proteins mediated by the peptide binding domain are responsible for the growth-impaired phenotype. hsc70 overexpression did not affect cell growth over 72 h of induction but was slightly growth inhibitory after this time. However, hsc70 accumulated more slowly than either hsp70 or hsp70ATPase. When the analysis was restricted to only the last 2 days, it was evident that inhibition of growth by hsc70 was greater than that of hsp70ATPase but not as severe as hsp70. Induced levels of hsc70 were less than that of hsp70 but equal to that of hsp70ATPase. Growth inhibition was associated with increased k_d. This loss of viability became apparent by 48 h of continuous hsp70 expression. The k_d, measured over the entire 96-h period, was highest for full-length hsp70. Both the ATPase mutant and the AAAA substitution mutant had elevated k_d relative to the cell line expressing BFP. However, expression of mutant hsp70EEVD did not increase the k_d above that of the BFP-expressing cell line. Therefore, the growth inhibition observed in the hsp70EEVD-expressing cell line may have been the result of the higher levels of protein expression and not due to a feature that this protein shares with the full-length protein. The absence of an effect of this protein on death rates could be because deletion of the EEVD sequence alters the conformation of the protein such that the domain responsible for this effect is no longer accessible. Bacterially expressed hsp70EEVD yielded a unique set of trypsin cleavage products compared to full-length hsp70, suggesting an altered conformation (11). Also, the fact that this protein was not recognized by hsp70-specific antibody C92, whereas hsp70AAAA was, suggests that hsp70EEVD does not fold properly when expressed in human cells.

Forced expression of hsp70 in Drosophila cells caused a reduction in growth without affecting viability (10). Recovery from growth inhibition occurred in cells where the distribution of hsp70 changed from diffuse to granular. Formation of hsp70 granules was proposed to be an adaptive response to overcome the suppressive effect that hsp70 has on cell growth. Overexpression of hsp70 in the human lymphoid cell line used in this study did not result in the formation of granules. Perhaps the inability of these cells to sequester hsp70 into cytoplasmic granules, and thereby overcome the effect that hsp70 has on growth inhibition, leads to their eventual loss of viability. The reason for the growth-suppressive effects of hsp70 are not known; however, Feder et al. (10) have suggested that hsp70 might specifically interact with cell cycle regulators. hsp70 accumulates in the nucleus during early S phase and associates with different classes of proteins in a cell cycle-dependent manner (40, 41). hsp70 plays an essential role in regulating the activity of the Wilms tumor suppressor WT1, a transcription factor that controls kidney development and that suppresses growth when overexpressed (35). Overexpression of hsp70 in HL60 cells reduced proliferation and promoted their differentiation (25). Also, the antiproliferative activity of certain prostaglandins has been attributed to their ability to induce the synthesis of hsp70 (54).

In some situations elevated expression of hsp70 has been shown to be associated with increased proliferation. hsp70 is often overexpressed in cancerous cells and can promote oncogenic transformation (20, 64). The Molt4 T-cell line has elevated basal levels of hsp70, and inhibition of its expression by an antisense oligonucleotide inhibited growth and promoted apoptosis (65). Also, in human breast cancer biopsy samples, the expression of hsp70 correlated with elevated proliferative capacity (60). Whether hsp70 will have growth-inhibiting or -promoting effects likely depends on the spectrum of cell cycle regulators that are available for modulation and their relative abundances in various cell types. A cell type-specific effect of hsp70 on growth inhibition was observed in transgenic mice expressing hsp70 under the control of the mouse H-2K promoter (28). These mice displayed severe T-cell hypoplasia with a defect in thymocyte differentiation to the CD4/CD8 double-positive stage (28). This suggests that forced overexpression of hsp70 is not