1 Medical Radiology Research Center, Obninsk 249020, Russia; 2 Institute of Child Health, University College London, London WC1N 1EH, United Kingdom; and 3 Department of Radiation and Cell Stress Biology, University of Groningen, 9713 AV Groningen, The Netherlands
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ABSTRACT |
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Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.
chaperone; protein aggregation; metabolic stress; ischemic tolerance
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INTRODUCTION |
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AT THE CELLULAR LEVEL, failing energy metabolism and ATP depletion are the earliest cell-damaging factors of ischemic insults. In vivo, severe depletion of ATP is a proteotoxic stress that leads to dysfunction, destabilization, and aggregation of many cellular proteins, including enzymes, ion pumps, and constituents of cytoskeletal and contractile structures (1, 9, 17, 27). Sustained lack of ATP is obviously lethal for the cell. On the contrary, a transient (reversible) drop in cellular ATP can confer tolerance to the next energy-depriving exposure, with heat shock proteins (HSPs) being involved in such an adaptive response (reviewed in Ref. 17). The HSP-involving cellular response appears to contribute to delayed ischemic tolerance (the second window of protection) found after heat or ischemic preconditioning in the myocardium. Like high temperature, cellular ATP depletion activates the heat shock transcription factor 1 (HSF1) that afterward induces HSP expression in the recovering cells (4, 36). Most HSPs are molecular chaperones stabilizing protein molecules under heat shock conditions in vitro and in vivo, and the same chaperone activity may protect HSP-enriched cells in the case of other proteotoxic stresses, e.g., ischemia or ATP-depleting treatments with inhibitors of energy metabolism. Despite the numerous reports describing HSP70-mediated cytoprotection against ischemic injury (for a review, see Refs. 5, 17, and 19), the precise mechanism of this phenomenon remains to be determined. Previously, suppression of ATP depletion-provoked protein insolubilization was found in heat-preconditioned ascites tumor cells (7, 16). Although it was speculated in those reports that such an effect of the preconditioning is due to stress-inducible HSPs (e.g., HSP70), no serious evidence was provided. Likewise, it has never been studied whether excess HSP70 in cells attenuates the proteotoxic effects of ischemia-associated ATP depletion, i.e., protects intracellular proteins from denaturation and aggregation during the stress. So far, it is also unknown how the cell viability under ischemia-like (or energy-depleting) conditions correlates with the in vivo chaperone activity of intracellular HSP70.
These two latter issues were addressed in the present study. We hypothesized that excess HSP70 in stress-preconditioned or HSP70-overexpressing cells can preserve cellular proteins from the ATP depletion-induced aggregation leading to cell death. To examine this hypothesis on an ischemia-relevant model, we used a rat embryonic heart-derived H9c2 line of myoblasts that retain some features of cardiac cells and can acquire tolerance to simulated ischemia (23) and thermal or oxidative stress (10, 34) after HSP-inducing heat pretreatments. Besides heat preconditioning, we also employed metabolic preconditioning to cause the transient (reversible) ATP depletion as an alternative HSP-inducing stress that to some extent mimics ischemic preconditioning in vivo. To evaluate contribution of the HSP induction, we treated the stress-preconditioned cells with quercetin, an inhibitor of HSF1 (13, 26). In parallel, to increase intracellular HSP70 by an HSF1-independent method, we overexpressed human inducible HSP70 in H9c2 cells using plasmid- or virus-based vectors. It was previously found that ATP depletion in cells renders many cellular proteins less extractable with a Triton X-100-containing buffer (14, 15). Such insolubilization of cellular proteins is due to their aggregation and correlates with the intensity of cell death under ATP-depleting stress (14, 17). Therefore, the stress-induced increase in the detergent insolubility of cellular proteins was quantified here to assess how the stressful preconditioning and HSP70 overexpression affect the proteotoxic impact within the energy-deprived cells.
In addition to monitoring of the total protein aggregation in the ATP-depleted myoblasts, we explored in them the catalytic activity and the solubility of a reporter enzyme, firefly (Photinus pyralis) luciferase, transiently expressed following transfection. This thermolabile enzyme is used as an in situ probe, allowing us to study protein denaturation, aggregation, and refolding in heat- or chemically stressed cells (2, 24, 25, 28, 29). Furthermore, firefly luciferase, when expressed in mammalian fibroblasts, aggregates and loses activity during cellular ATP depletion (27) and, thus, is a sensitive marker of the stress-associated proteotoxicity. We therefore introduced firefly luciferase into H9c2 myoblasts to evaluate effects of the preconditioning and the HSP70 overexpression on the proteotoxicity of ischemia-mimicking ATP depletion. Plasmids expressing recombinant forms of luciferase with either cytoplasmic or nuclear localization (25) were used to separately probe the situation within cytoplasmic and nuclear compartments of the ATP-depleted cells. To elucidate the role of inducible HSP70 alone, H9c2 cells were cotransfected with luciferase and human HSP70 simultaneously as previously employed in heat shock studies on other cell lines (24, 29).
We demonstrate here that pretreatments leading to HSP70 accumulation in the cells can reduce the protein aggregation resulting from cellular ATP loss. This attenuation of the ATP depletion-associated proteotoxicity seems to be HSP70 mediated and correlates with the elevated cell resistance to prolonged energy deprivation. Improved formation of nonaggregable (soluble) complexes between excess HSP70 and some damaged or instable proteins in the cytosol and nucleosol is suggested as a probable cause of the attenuated protein aggregation under ATP depletion in the HSP70-enriched cells. Analogous mechanisms may contribute to the phenomenon of delayed ischemic tolerance arising in the stress-preconditioned heart when the level of cardiac HSP70 is transiently increased.
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MATERIALS AND METHODS |
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Cells. The embryonic rat heart-derived H9c2 line of myoblasts was obtained from American Type Culture Collection (CRL-146; ATCC, Rockville, MD). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with sodium bicarbonate and 10% fetal bovine serum (GIBCO BRL) in a humidified atmosphere of 5% CO2 in air at 37°C. The preconfluent myoblast cultures were used for the experiments.
Stressful treatments and recovery. For heat preconditioning, dishes or tubes containing the adherent cells were plunged into a thermostatic water bath at 43°C for 30 min and then returned into a CO2 incubator at 37°C. For preconditioning by metabolic (ATP-depleting) stress, the cells were exposed to 3 h of incubation at 37°C with glucose-free DMEM containing 3% fetal bovine serum and 20 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), and then this medium was removed and the cells were washed twice with normal DMEM and added to the full growth medium. Recovery periods following either priming stress continued for 16-18 h before the challenging ATP depletion. Quercetin was added at a 30 µM concentration to some samples of the cells before the stressful preconditioning (21, 22); this drug was present in the incubation medium during the priming stress and the next 10 h, and then the drug-containing medium was replaced by the usual growth medium. To perform the challenging ATP depletion, we placed the cells in the CCCP-containing medium supplemented with 10 mM 2-deoxyglucose and incubated them at 37°C in a CO2 incubator (21, 22).
Plasmids and transfection. Plasmid pRSVLL/V encoding cytoplasmic localized firefly luciferase (cyt-luciferase) was kindly provided by Dr. S. Subramani (University of California, San Diego, CA). Construction of plasmid pRSVnlsLL/V encoding firefly luciferase fused to a nuclear localization sequence (nuc-luciferase) has been described previously (25). For in situ control of the transfection efficiency and the compartment-specific localization of the expressed products, plasmid constructs encoding cyt- or nuc-luciferase fused at their COOH termini to enhanced green fluorescent protein (EGFP) were used (constructed by Dr. E. A. A. Nollen, University of Groningen, Groningen, The Netherlands). Distribution of the EGFP-luciferases and the diamidinophenylindole (DAPI)-labeled cell nuclei was viewed on an Opton III fluorescence microscope (Karl Zeiss, Oberkochen, Germany). Plasmid pCMV70 with fragment encoding cDNA for human inducible HSP70 was constructed as previously described (24).
The preconfluent cell cultures growing in six-well plates were transiently transfected with pRSVLL/V or pRSVnlsLL/V to express cyt- or nuc-luciferase, respectively. In a part of the experiments, the cells were cotransfected with one of the EGFP-luciferase-encoding plasmids and pCMV70 to express luciferase and human inducible HSP70 simultaneously (24, 29); the coexpression of both products in the same cells was confirmed by double-label fluorescence analysis on a flow cytometer. The transfection procedure was performed with Opti-MEM (GIBCO BRL) and GenePORTER transfection reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer's protocol. In all cases, 1 µg of the plasmid DNA was carried in each well; herein the DNA quantity was equalized using pSP64 (Promega). Then, 24 h after transfection, cell culture tubes or 35-mm dishes (Nunc) were seeded with the treated cells at a density of 5 × 104 cells per tube or 3 × 105 cells per dish. Another 24 h later, these cultures were taken for experiments with the challenging ATP depletion.Infection of the cells with virus-based vectors. Human inducible HSP70 cDNA, or a gene encoding the green fluorescent protein (GFP), under the control of the cytomegalovirus promoter, were inserted into the HSV-1 genome as previously described (37). Cell culture dishes (35 mm) with equal numbers of the cells, in the state of preconfluent monolayer, were washed twice with sterile phosphate-buffered saline (PBS) from the growth medium, and then PBS was replaced with 1.5 ml of serum-free DMEM. The virus-based vectors expressing either HSP70 or GFP were added in the dishes at concentration of 30 plaque-forming units/ml and mixed into the serum-free medium. The cells were in contact with the viruses for 1 h in a humidified atmosphere with 5% CO2 at 37°C. After that exposure, the virus-containing medium was removed and replaced with normal growth medium for 24 h before to the ATP-depleting treatments (6). The efficiency of infection and expression of the products were tested by flow cytometry and Western blotting.
Measurements of luciferase activity and cellular ATP.
The luciferase transfectants adherent in tubes were instantly cooled in
ice, briefly washed with ice-cold PBS, and then lysed in 0.5 ml of
ice-cold buffer A [25 mM
H3PO4-Tris, pH 7.8, 10 mM MgCl2, 1 mM EDTA, 1% (vol/vol) Triton X-100, and 15% (vol/vol) glycerol]
containing 0.5% 2-mercaptoethanol (24, 25). The lysates
were frozen and kept at 20°C until determination of luciferase activity. The latter was measured in a luminometer for 10 s after addition of 0.1 ml of buffer A containing substrates [1.25
mM ATP and 87 µg/ml luciferin (Sigma)] to 0.15 ml of the cell
lysates (24).
Cell fractionation with Triton X-100. The ATP depletion-induced protein aggregation was assessed upon the increase in the cellular protein insolubility (14, 15) by using the method adapted for adherent cells (24, 27). Cell culture dishes (35 mm) with equal numbers of the cells were washed twice with PBS and then lysed and scraped in 0.3 ml of the ice-cold buffer A devoid of glycerol. The lysates were centrifuged at 12,000 g for 15 min at 4°C, which divided them into pellets (Triton X-100-insoluble fractions) and supernatants (Triton X-100-soluble fractions) (24, 27). The pellets were dissolved in 60 µl of 6 M urea, and the protein contents there were determined using the BCA (bicinchoninic acid) kit (Sigma) (7).
For studying the stress-induced changes in the luciferase (in)solubility, the pellets and the supernatants obtained from the cell lysates were dissolved in a Laemmli sample buffer, boiled for 5 min, and then frozen until analysis by electrophoresis and Western blotting.SDS gel electrophoresis and Western blot analysis. The samples prepared from the total cell lysates or cellular fractions were run by electrophoresis in a Laemmli system with SDS-10% polyacrylamide gel under reducing conditions. The separated proteins were electrotransferred from the gel slabs onto 0.45-µm nitrocellulose membrane (Bio-Rad). For integral detection of the endogenous HSC70 (heat shock cognate protein 70)/HSP70, monoclonal antibody N27F3-4 (StressGen) was used in a 1:2,000 dilution. The human inducible HSP70 was detected in the cell lysates with monoclonal antibody C92F3A-5 (StressGen) diluted 1:1,500. Cyt- and nuc-luciferases were detected with rabbit polyclonal antibodies (Promega) in a 1:1,500 dilution. Anti-mouse Ig- and anti-rabbit Ig-peroxidase conjugates and an ECL (enhanced chemiluminescence) kit were employed to develop the antigen band tracks on X-ray film (all from Amersham). The track images were digitized by means of a flatbed scanner (Mustek 600 II CD). The digitized images were then quantitatively analyzed using the NIH Image software.
Determination of cell death and survival. Cell death during the challenging ATP depletion was quantified by counting the number of dead cells unable to exclude the dye trypan blue as a percentage of the total number of cells. In the case of EGFP-luciferase/HSP70 cotransfection, the percentage of cells killed by ATP depletion was counted with the use of the fluorescence microscope after staining with acridine orange (AO; 3 µg/ml) and propidium iodide (PI; 10 µg/ml), as described previously (8). At least 10 microscopic fields per plate (300-500 cells) were counted, and the recounting was repeated 3-4 times.
Colony survival assays were performed after the treated and control cells were trypsinized. The cell suspensions, when collected and counted, were serially diluted and replated, in triplicate, in 10-cm2 culture dishes and stood at 37°C for 7-9 days (23). The cell cultures were then fixed with 70% ethanol and stained with 0.5% crystal violet. The number of separate colonies grown (colonies with a minimum of 50 cells) was counted. Cell survival was determined as the ratio of colonies formed by the initially plated cells and normalized to the plating efficiency (23).Metabolic 35S labeling and immunoprecipitation. Equal numbers of the cells growing in 60-mm culture dishes were preconditioned by heat or metabolic stress as described in Stressful treatments and recovery. Seven hours after the start of the preconditioning, the treated and control cells were incubated for eight hours at 37°C in methionine-free DMEM (Flow Laboratories) supplemented with 5% normal DMEM, 10% fetal bovine serum, and 40 µCi/ml [35S]methionine (produced with a specific activity of 950 mCi/mmol by Institute of Physical Energetics, Obninsk, Russia). The label-containing medium was then harvested, and the cells were washed three times with PBS and three times with the full growth medium, followed by 3 h of incubation under normal growth conditions. These radiolabeled cells were extracted with 0.6 ml of the ice-cold glycerol-free buffer A containing a protease inhibitor cocktail (Sigma), and then the Triton X-100-soluble fractions were obtained according to the technique described in Cell fractionation with Triton X-100.
The freshly prepared Triton X-100-soluble fractions were used for immunoprecipitation experiments. Each sample (0.6 ml) of the cell extracts plunged in ice was at first preincubated for 30 min with 150 µl of protein A-Sepharose (Pharmacia) to minimize a nonspecific binding. After sedimentation and removal of the Sepharose pellets, 5 µl of affinity-purified rabbit polyclonal anti-HSP70/HSC70 antibodies (produced in our laboratory against bovine HSP70/HSC70) were injected into each supernatant; in some samples, 1 mM ATP or ADP (Sigma) was added along with the antibodies. After 1 h of incubation in ice, immune complexes were caught with 150 µl of the protein A-Sepharose within 30 min. The sedimented Sepharose pellets were thoroughly washed with the ice-cold buffer A and then dissipated into 0.25 ml of a Laemmli sample buffer and boiled for 5 min. Aliquots of the eluted material were run by electrophoresis as described earlier. Tracks of 35S-labeled polypeptides were revealed by autoradiography from the dried polyacrylamide gels onto Hyperfilm-MP (Amersham). Without radiolabeling, the same protocol of immunoprecipitation was used for probing the extracts from the transfectants expressing cyt- or nuc-luciferase. The precipitated material was analyzed by electrophoresis and Western blotting.Statistical analysis. All quantitative results are expressed as means ± SE of 4-6 separate experiments. Statistical differences between compared groups of the cells were analyzed using ANOVA (a multiway analysis of variance or covariance). ANOVA significance was applied when P < 0.05 and confirmed with the F-test.
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RESULTS |
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Elevated resistance to ATP depletion-induced cell death in the
stress-preconditioned and HSP70-overexpressing cells.
Fluctuations in the ATP levels in H9c2 cells during and after heat
preconditioning (43°C for 30 min) were insignificant (data not
shown). Preconditioning of the cells by metabolic stress (glucose starvation + uncoupling of oxidative phosphorylation with CCCP) caused severe but reversible decrease in cellular ATP (Fig.
1A). Both preconditioning
treatments followed by a 16- to 18-h period of recovery resulted in an
approximately fourfold increase in the intracellular content of
HSC70/HSP70 (Fig. 2A, Table
1). Probably, the content of
other HSPs also increases in the cells following either stressful
preconditioning, but their expression was not analyzed in the present
study.
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Reduced aggregation of endogenous cellular proteins during ATP
depletion in the tolerant (stress-preconditioned or
HSP70-overexpressing) cells.
Severe depletion of cellular ATP evokes massive damage and aggregation
of proteins within the stressed cells as revealed by the accumulation
of many initially soluble proteins in the Triton X-100-insoluble
cellular fraction (1, 14-16, 27). Here, the stress-induced aggregation of endogenous proteins was quantified by
comparing the relative content of protein in the Triton X-100-insoluble fractions from the ATP-depleted cells with that from the unstressed ones (7, 16). The basal level of total Triton
X-100-insoluble protein in the cells following the delayed stressful
preconditioning or virus infection, or the quercetin treatment, was the
same as in the nonpretreated control cells (not shown). The data
presented in Table 2 demonstrate that the
detergent-insoluble protein component does increase during cellular ATP
depletion. The extent of this insolubilization was significantly
reduced in the stress-preconditioned and HSP70-overexpressing cells
compared with control (the nonpreconditioned cells and the
GFP-overexpressing cells, respectively).
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Stressful preconditioning and HSP70 overexpression diminish the
rate of inactivation and extent of insolubility of cyt- and
nuc-luciferase in the tolerant ATP-depleted cells.
Firefly luciferase is an ATP-dependent reporter enzyme that, when
expressed in mammalian cells, is inactivated and insolubilized during
heat or chemical stress (2, 24, 25, 28, 29) or depletion
of cellular ATP (27). For probing of the situation within
the cytoplasm and the nucleus separately, we transfected the cells with
plasmid constructs encoding luciferases engineered for expression in
either the cytoplasm (cyt-luciferase) or the nucleus (nuc-luciferase)
(25). This specific compartmentalization of the expressed
products and the transfection efficiency were checked by microscopic
analysis of the distribution of EGFP-cyt- and EGFP-nuc-luciferases in
the cells with DAPI-labeled nuclei (Fig.
5). The transfection usually yielded up
to 20-25% positive cells (see Fig. 5), and its procedure did not
affect the time course of the subsequent challenging ATP depletion (not
shown).
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Tolerant (stress-preconditioned or HSP70-overexpressing) cells,
when depleted of ATP, have larger amounts of soluble HSP70 and its
complexes with other proteins.
To prove the direct involvement of HSP70 in the described effects, we
compared the behavior of this chaperone during ATP depletion in the
control and tolerant cells. It was previously shown in various cell
lines that a major part of HSC70/HSP70 is insolubilized in response to
ATP depletion (14, 15, 27), but this effect was never
explored in cells rendered tolerant. Figure
8 demonstrates that, whereas H9c2
myoblasts are deprived of ATP, HSC70/HSP70 also accumulates into the
Triton X-100-insoluble cellular fraction. There were not significant
differences in the levels of the insolubilized HSP70 between the
control and tolerant cells (Fig. 8). However, compared with control,
both the stress-preconditioned and HSP70-overexpressing cells contain
much more HSP70 in the Triton X-100-soluble fractions isolated at
different times of the ATP-depleting exposure. In other words, the
tolerant cells, being HSP70-enriched before ATP depletion, retain the
soluble pool of this chaperone longer during sustained ATP depletion
(Fig. 8).
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DISCUSSION |
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Preconditioning of H9c2 cells by nonlethal heat or metabolic stress transiently enhances the HSP70 expression. In parallel, cell resistance to the cytotoxic and proteotoxic effects of the challenging ATP depletion is transiently acquired, thus suggesting a casual link between these events. Here we discuss molecular mechanisms of the in vivo ATP depletion-associated proteotoxicity, its link to the stress-induced cell death, and the role of HSP70 in protection from the protein aggregation in energy-deprived cells.
In 1994, Nguyen and Bensaude (27) described two reporter
enzymes, Eschericia coli -galactosidase and firefly
luciferase, that, both being Triton X-100-soluble in unstressed cells,
are insolubilized during ATP depletion. Although those foreign enzymes were artificially expressed in mammalian cells, their insolubilization appears to reflect the in situ response of many endogenous cellular proteins to the stress. The data of Table 2 reveal that rather a large
percentage of total cellular protein lost solubility because of ATP
depletion. Such reaction implies at least two processes that may occur
in parallel: some initially soluble proteins 1) form
aggregates that precipitate in Triton X-100-containing solutions and/or
2) undergo translocation to the detergent-resistant cellular compartments such as the cytoskeleton, chromatin, and nuclear matrix.
Probably, both the former and latter are caused by structural changes
in some instable protein molecules resulting from ATP depletion per se
or its harmful consequences (e.g., ionic imbalance including acidosis).
These structural changes promote undesirable intermolecular
interactions leading to aggregation and abnormal translocation of a
number of affected intracellular proteins. This may result in
dysfunction of the involved proteins through the loss of their native
conformations and through the inability of aggregated or abnormally
localized proteins to interact with their natural substrates and cofactors.
The question arises, which intracellular proteins aggregate and are
insolubilized in response to ATP depletion? Among mature cytosolic
proteins, this feature is seen for the 68-kDa double-stranded RNA-dependent protein kinase (17, 27) and a major
glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase
(1). The latter, being an abundant cytosolic enzyme in
unstressed cardiomyocytes, associates with myofibrils as a result of
ischemia-provoked ATP depletion (1). Within the
nuclear compartment, proteins of the chromatin and/or nuclear matrix
may coaggregate with the stress-sensitive nucleosolic enzymes;
otherwise, the latter may aggregate with each other without sticking to
the originally insoluble structures. As for nonenzymatic proteins, the
cytosolic pools of cytoskeletal proteins such as G-actin, myosin,
vinculin, and -actinin can also supply material for aggregating in
ATP-depleted cells (9, 12, 14-17). Despite the fact
that the preexisting cytoskeleton undergoes disintegration in
ATP-depleted cells, its debris remains insoluble and may entrap
surrounding proteins, thus aggravating the total aggregation
(17).
Importantly, the proteotoxic effects of ATP depletion correlate with its cytotoxicity as indicated by comparison of the data of Tables 2 and 3 and Figs. 3, 4, 6, and 7. The same suggestion came from the previous studies performed on murine tumor cells (7, 14, 16). Quercetin completely abolished both the HSP70-inducing and the cytoprotective and anti-proteotoxic effects of the stressful preconditioning, suggesting that the inducible HSP(s) can alleviate injury of ATP-depleted cells by reducing the protein aggregation. Although quercetin could exert other effects besides the HSF1 inhibition, we found that, in our model, this drug did not affect the time course of in vivo ATP depletion/replenishment, the basal level of the insoluble cellular protein, or the cell viability and protein aggregation under the challenging ATP depletion in the cells of control groups (non-stress-preconditioned or GFP-overexpressing H9c2 cells). While the HSF1-independent (i.e., plasmid or viral vector induced) overexpression of inducible HSP70 was already sufficient to moderate the cytotoxic and proteotoxic influence of ATP depletion in H2c9 cells, the quercetin treatment was not able to prevent these HSP70 overexpression-mediated effects. Taken together, these results allow us to attribute the cytoprotective and anti-proteotoxic effects of the stressful preconditioning to stress-induced HSP(s) (e.g., HSP70). Although other inducible HSPs besides HSP70 can also be involved in cellular defense/repair from injurious consequences of ATP depletion, HSP70 appears to play a major role in the attenuation of the ATP depletion-induced protein aggregation revealed after the stressful preconditioning. Such a conclusion follows from the experiments with overexpression of HSP70 alone, which yielded both the intracellular HSP70 accumulation and the impairment of the ATP depletion-associated proteotoxicity quite comparable with those in the stress-preconditioned cells.
Intriguingly, HSP70 is known as an ATP-dependent chaperone, and, at first glance, it seems unclear how it could combat the proteotoxicity in the case of lack of ATP. However, previous heat shock studies (20, 33) showed that overexpression of the deletion mutant of human HSP70 devoid of the ATP-binding domain confers thermoresistance and reduced intranuclear protein aggregation in heat-shocked Rat-1 cells. Because the inability of the mutant to bind and hydrolyze ATP did not yet abolish its protective effects, the authors suggested that the mutant is still able to form complexes with heat-denatured cellular proteins, thus reducing protein aggregation during heat shock (20, 33). This heat shock model with the deletion mutant of HSP70 rather closely mimics the present situation with ATP depletion in HSP70-enriched H9c2 cells. Taking into account that HSP70 binds ADP much stronger than ATP (11, 31, 32) and the ATP/ADP ratio obviously declines in energy-deprived cells (15, 18), it seems likely that under severe depletion of ATP in vivo, most (if not all) HSC70/HSP70 molecules are in the "ADP state." The latter is characterized as having a slower on-rate but also slower off-rate of substrate than HSP70 in the "ATP state." Therefore, excess HSP70 in the ADP state within the ATP-depleted tolerant cells may form stable complexes with proteins affected by the stress. In favor of this idea, we find 1) the longer sojourn of excess HSP70 in the soluble fractions of the tolerant cells undergoing ATP depletion, 2) the larger amounts of soluble protein material coimmunoprecipitating with excess HSP70 from the tolerant cells deprived of ATP, and 3) the dissociating effect of exogenous ATP on the soluble HSP70-protein complexes formed for lack of cellular ATP (see Figs. 8 and 9). Apparently, the proteins bound to HSP70 are thereby protected from aggregation with each other and/or sticking to the insoluble cytoskeletal/nuclear structures or debris despite the fact that the chaperone cannot undergo an ATP-ADP cycle in the ATP-deprived cells. As for the stress-damaged proteins, their stable association with HSP70 may retain them in a soluble, folding-competent state that should save these proteins from the aggregation during cellular ATP depletion. Besides the stress-damaged mature proteins, the preexisting pool of nascent polypeptide chains can recruit some part of HSP70 in ATP-depleted cells (3), and the chains bound to the chaperone may be better preserved from the stress-induced aggregation. Very similar mechanisms appear to act in the luciferase model: during ATP depletion, the stress-sensitive enzyme can be either involved in the aggregation cascade (inactivation and insolubilization) or trapped by HSP70 (protection). We suppose that during ATP depletion in the HSP70-enriched cells, the large part of inactivated luciferase still remains in a folding-competent state being bound to HSP70, and then, under cell lysis into the ATP-containing buffer, the instant ATP-mediated dissociation of the enzyme-chaperone complexes occurs, with released luciferase being detected as catalytically active in the assay. The immunoprecipitation experiments demonstrating the improved HSP70-luciferase complex formation in the tolerant cells depleted of ATP as well as the complex-disrupting effect of exogenous ATP (Fig. 9B) provide strong arguments in support of such a supposition.
Although the protection of two model enzymes within the HSP70-enriched cells cannot be directly linked to what happens with the cells' vital proteins, our data do indicate that excess HSP70 can diminish the ATP depletion-induced protein aggregation in both the cytoplasm and the nucleus. Moreover, evident correlation is observed between the cell viability under the challenging ATP depletion and the in situ chaperone potential of HSP70 toward cyt- and nuc-luciferase during the stress (see Figs. 6 and 7 and Table 3). Extrapolating the luciferase data to endogenous proteins of the ATP-depleted cells (see Table 2), we suggest that HSP70 can protect stress-sensitive cytoplasmic and nuclear proteins that otherwise are involved in the detrimental aggregation leading to necrotic cell death. The fact of partial migration of cytosolic HSP70 into the nuclei of ischemia-stressed rat cardiomyocytes (35) supports the suggestion that nuclear proteins can also become the chaperone targets under the energy-depriving stress. It seems likely that in the case of ischemia in vivo, excess HSP70 would also be able to minimize the proteotoxic impact of cellular ATP depletion, thereby allowing HSP70-enriched cells to better tolerate an acute phase of ischemic insults. Such speculations generate an additional reason for development of clinically applicable ways enabling to increase the HSP70 level in ischemia-attacked tissues of patients.
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ACKNOWLEDGEMENTS |
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We thank Drs. A. A. Michels, E. A. A. Nollen, B. Kanon, and J. F. Brunsting for providing the plasmid samples and technical assistance, and Dr. B. Brar for helpful discussions.
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FOOTNOTES |
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This study was supported by grants from the Netherlands Organization for Scientific Research and the Wellcome Trust (062891).
Address for reprint requests and other correspondence: A. E. Kabakov, Medical Radiology Research Center, 4 Korolev St., Obninsk 249020, Russia (E-mail: aekabakov{at}hotmail.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 27, 2002;10.1152/ajpcell.00503.2001
Received 19 October 2001; accepted in final form 24 March 2002.
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