Hsp70 Prevents Activation of Stress Kinases
A NOVEL PATHWAY OF CELLULAR THERMOTOLERANCE*

(Received for publication, March 14, 1997, and in revised form, April 17, 1997)

Vladimir L. Gabai Dagger §, Anatoli B. Meriin Dagger , Dick D. Mosser par , A. W. Caron par , Sophia Rits par , Victor I. Shifrin ** and Michael Y. Sherman Dagger Dagger Dagger

From the Dagger  Boston Biomedical Research Institute, Boston, Massachusetts 02114, the § Medical Radiology Research Center, 249020 Obninsk, Russia, par  Biotechnology Research Institute, Montreal H4P 2R2, Quebec, Canada, and the ** Dana Farber Cancer Institute, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Harmful conditions including heat shock, oxidative stress, UV, and so forth cause programmed cell death, whose triggering requires activation of the Jun N-terminal kinase, JNK. High levels of Hsp72, a heat-inducible member of Hsp70 family, protect cells against a variety of stresses by a mechanism that is unclear at present. Here we report that elevated levels of Hsp72 inhibit a signal transduction pathway leading to programmed cell death by preventing stress-induced activation of JNK. Stress-induced activation of another stress-kinase, p38 (HOG1), is also blocked when the level of Hsp72 is increased. Similarly, addition of a purified recombinant Hsp72 to a crude cell lysate reduced p38 kinase activation, while depletion of the whole family of Hsp70 proteins with a monoclonal antibody enhanced such activation. In addition, we have found that accumulation of abnormal proteins in cells upon incubation with amino acid analogs causes activation of JNK and p38 kinases, which can be prevented by overproduction of Hsp72. Taken together, these data suggest that, in regulation of JNK and p38 kinases, Hsp70 serves as a "sensor" of the build-up of abnormal proteins after heat shock and other stresses. The inhibitory effect of an increased level of Hsp70 on JNK appears to be a major contributor to acquired thermotolerance in mammalian cells.


INTRODUCTION

Exposure of mammalian cells to severe heat shock, strong oxidants, UV irradiation, and other stressful conditions causes activation of a family of homologous stress-activated protein kinases including JNK1 and p38 (1-3). This activation proceeds through a signal transduction pathway that involves the small GTP-binding proteins, MEK kinase, MEKK1, and dual-specificity kinases MKK3, MKK4 (SEK1), and MKK6, which in turn phosphorylate and activate JNK and p38 (4-8). JNK was recently shown to be an essential component of a signal transduction pathway which leads to programmed cell death in response to certain stimuli, including stressful conditions (9-12). In fact, overproduction of a dominant negative mutant of JNK activating kinase, SEK1, inhibits programmed cell death in response to heat shock, UV irradiation, oxidative stress (9), and certain other inducers of apoptosis (10-12).

Another protein, which when overproduced enhances cell survival following exposure to a variety of stressful conditions, is Hsp72, a heat-inducible member of Hsp70 family. Indeed, overproduction of Hsp72 either by expression under the regulation of a strong promoter on a plasmid, or physiologically, by exposure of cells to a mild heat shock, leads to a dramatic protection against severe heat shock, UV irradiation, H2O2, and other harmful conditions and factors (13-16).

Members of the Hsp70 protein family function as molecular chaperones in refolding of denatured polypeptides (17, 18). In fact, overproduction of Hsp72 was shown to reduce stress-induced denaturation and aggregation of certain proteins (19, 20) that has led to the common assumption that refolding and antiaggregating activities of Hsp72 determine its role in cellular protection against stresses (21, 22). However, under some conditions the protective action of Hsp72 appears to be unrelated to its chaperoning activity. For example, tumor necrosis factor (TNF) causes cell death by the activation of a signal transduction pathway leading to apoptosis (23). This apoptotic process can be prevented by overproduction of Hsp72 (24), which therefore seems to interfere with the apoptotic program. Similarly TNF, exposure to high temperatures, and some other stresses cause activation of the apoptosis-triggering signal transduction pathways that include activation of JNK (9-12). The protective action of Hsp72 in these circumstances may also involve direct interference with the apoptotic program. It should be noted, however, that upon exposure to extremely high temperatures, when cell death is not due to apoptosis, but to necrosis, the antiaggregating and protein refolding activities of Hsp72 may become critical for cell protection (20).

At what step can Hsp72 interfere with the apoptotic program? We hypothesized that overproduction of Hsp72 inhibits activation of JNK by harmful conditions. This idea was tested on the U937 leukemia cell line, in which JNK activation appeared to be essential for the activation of apoptosis by heat shock, UV, and oxidative stress (9), and on the PEER lymphoid cell line, in which we overproduced Hsp72 under the control of tetracycline-regulated promotor.


EXPERIMENTAL PROCEDURES

Cell Cultures

U937 and PEER human lymphoid tumor cells were grown in RPMI 1640 medium with 10% (U937) or 20% (PEER) fetal bovine serum and were used for experiments while in the mid-log phase (3-7 × 105 cells/ml). PEER cells were stably transfected with plasmids encoding Hsp72 under the control of a tetracycline-regulated transactivator. Hsp72 was inserted as a first cistron in a dicistronic expression vector that contains the green fluorescent protein (GFP) gene. In this construct synthesis of Hsp72 is repressed when grown in the presence of tetracycline, but removal of this antibiotic leads to rapid and efficient induction of Hsp72. Cells were incubated in the absence of tetracycline for 48 h in order to induce expression of Hsp72 and GFP. Cells that express GFP were selected with a cell sorter. These cells were viable and continued to grow normally when tetracycline was added to repress Hsp72 synthesis.

Cell Lysate Preparation

2 × 107 PEER cells were collected by centrifugation and washed once with phosphate-buffered saline. Cells were resuspended in 0.4 ml of a hypotonic buffer that contained 10 mM Hepes, 10 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM EDTA (pH 7.4). After 15 min of swelling on ice, cells were disrupted on ice by 60 strokes in Dounce homogenizer, and a 0.2 volume of a buffer containing 50 mM Hepes and 250 mM KCl (pH 7.4) was added. Nuclei and cell debris were removed by centrifugation at 500 × g for 5 min at 4 °C, and the lysate was returned to ice. Typically cell lysates prepared by this procedure contained 4-7 mg/ml protein.

Apoptosis Assays

For determination of cell viability, 0.5 mg/ml of MTT (Sigma) was added to 100 ml of cell suspension (0.5 × 106 cells/ml in 96-well plates) for 4 h, and the formazan formed was dissolved in acidic 2-propanol; optical density was measured using an enzyme-linked immunosorbent assay reader at 590 nm. The OD of formazan formed by control (untreated) cells was taken as 100%. PARP degradation was determined by Western blotting with anti-PARP polyclonal antibodies.

JNK and p38 Kinase Assays

JNK was assayed using glutathione S-transferase-c-Jun protein as a substrate either in an in-gel assay or after immunoprecipitation with anti-JNK antibodies (Santa Cruz Biotechnology) as described previously (25). p38 kinase activity was determined by Western blotting with polyclonal antibodies (New England Biolabs) specifically reacting with the phosphorylated (active) form of p38.


RESULTS AND DISCUSSION

We first investigated the effect of enhanced levels of Hsp72 on activation of stress kinase and apoptosis in U937 cells. To raise the level of Hsp72, U937 cells were subjected to a mild heat shock (20 min at 43 °C), followed by a 6-h recovery. Such mild heat shock caused only a weak transient activation of JNK and did not activate an apoptotic program (not shown). The second, severe, heat shock (60 min at 43 °C) was applied, and it caused death of about 70% of the cell population (Fig. 1A), as measured by the MTT assay (ability of cells' mitochondria to reduce tetrazolium salts) or by counting the number of surviving cells (not shown). Pretreatment with the mild heat shock (preheating) reduced cell death after the second heat shock by more than 90% (Fig. 1A). Cell death was preceded by an extensive cleavage of PARP (Fig. 1B) and nuclear condensation (not shown), thus indicating that it was of an apoptotic, rather than of a necrotic, nature. Both the PARP cleavage (Fig. 1B) and the nuclear condensation were strongly inhibited in the preheated cells.


Fig. 1. Preexposure of cells to a mild heat shock protects them from apoptosis and inhibits activation of JNK and p38 kinase by high temperature or ethanol treatments. A, effects of preexposure to mild heat shock on cell viability. U937 and PEER cells were exposed for 20 min at 43 °C followed by 6 h of recovery (a time period that allows an accumulation of Hsp72, not shown). Then the cells were subjected to a severe heat shock (HS, 30 or 60 min at 43 °C) or to 9% ethanol (ET) for 60 min. Cell were allowed to undergo apoptosis at 37 °C for the next 14 h, and their viability was measured by the MTT assay. B, effects of preexposure to mild heat shock on PARP cleavage. Cells were treated as in A, but instead of measuring mitochondrial respiration by the MTT assay, PARP cleavage was tested by Western blot with the anti-PARP antibody. C, control; HS, heat shock 60 min; ET, ethanol 9%, 60 min. C, effects of preexposure to mild heat shock on the activation of JNK by the second challenging stress. Cells were treated as in A, but after 60 min of the second challenging stress samples were taken, and JNK activity was tested by an in-gel assay. D, effects of preexposure to mild heat shock on the activation of p38 kinase by the second challenging stress. Cells were treated as in A, but after 60 min of the second challenging stress, samples were taken, and the amount of active p38 kinase was measured as described under "Experimental Procedures."
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Preheating of cells also led to almost complete inhibition of activation of JNK in response to the second challenge with heat shock (Fig. 1C). While JNK became activated 30 min following the heat shock and maintained its activity for 4 h, in preheated cells JNK activity was practically undetectable at any point during this time period (not shown). It is noteworthy that activation of another stress-activated kinase, p38, was also inhibited under these conditions by more than 70% (Fig. 1D). This kinase was shown to participate in induction of IL-6, activation of phospholipase A2, regulation of phosphorylation of Hsp27, and other processes (26-30). Preheating of cells also strongly reduced activation of JNK and p38 kinases in response to another stress, a challenge with 9% ethanol (Fig. 1, C and D), and inhibited ethanol-induced apoptosis (Fig. 1, A and B). Since inactivation of JNK by overexpression of a dominant negative mutant of the upstream kinase was previously shown to protect cells against heat shock and some other stresses (9), we conclude that the protective action of the mild heat shock is associated with an suppression of the JNK activation. Thus, prevention of activation of JNK in preheated cells may be a significant factor in the phenomenon of acquired thermotolerance and tolerance to ethanol. These data complement the recent observation that JNK activation in response to mild heat shock is strongly reduced in a stable thermoresistant cell line (31) that overproduced the whole set of heat shock proteins including Hsp72 (32).

Since the protective effects of the mild heat shock may be associated not only with Hsp72 but also with some other heat-inducible polypeptides, we also studied the effects of stresses on a stably transfected PEER cell line that overexpresses Hsp72 under control of a tetracycline-regulated transactivator. We first reproduced the effects of mild heat shock on this cell line, i.e. demonstrated that preheating of the cells in the presence of tetracycline (without expression of Hsp72 from a plasmid) protects against heat shock and ethanol treatments and reduces activation of JNK and p38 (Fig. 1, C and D). To study the effect of Hsp72 produced from the plasmid under normal conditions, without mild heat shock (Fig. 2A), cells were incubated in the absence of tetracycline for 24 h, followed by selection of cells containing elevated levels of Hsp72 (see "Experimental Procedures"). In these cells the level of Hsp72 was about 3-4 times higher than that of PEER cells after the mild heat shock (not shown) and was similar to the level of Hsp72 in U937 cells following the mild heat shock. The selected cells with elevated levels of Hsp72 were challenged with a severe heat shock (60 min at 43 °C) or 9% ethanol. Activation of both JNK and p38 kinases was strongly inhibited in these cells compared with the cells with the normal level of Hsp72 grown in the presence of tetracycline (Fig. 2, B and C). A strong protection against apoptosis upon heat shock and ethanol treatments was also observed in the Hsp72 overproducing cells (Fig. 2D).


Fig. 2. Overexpression of Hsp72 in PEER cells inhibits activation of JNK and p38 kinase by a number of stressful stimuli. A, the level of Hsp72 in the cells incubated without tetracycline for 48 h was measured by a Western blot with anti-Hsp72 antibody. B, JNK activity in cells overexpressing Hsp72 and control cells after heat shock (43 °C). JNK activity was measured after immunoprecipitation with anti-JNK antibodies. Cells that express tetracycline-regulated transactivator (tTA) were used as a control. C, activity p38 kinase in Hsp72 overexpressing cells after treatments with different stressful stimuli (heat shock, 43 °C; 9% ethanol; 0.4 M sorbitol; 1 mM H2O2; UV-C, 500 J/m2). D and E, effects of Hsp72 overexpression on apoptosis after treatments with heat shock (43 °C) or 9% ethanol measured by the MTT assay (D) and PARP cleavage (E).
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These results support the hypothesis that Hsp72, when overproduced, inhibits activation of JNK and p38 kinases and thus suppresses cell suicide. It should be mentioned that, in the stably transfected cells that chronically overexpress Hsp72 (13), we observed protection from apoptosis without inhibition of JNK activity.2,3 Therefore, it is possible that another mechanism of prevention of apoptosis that does not involve inhibition of JNK operates in cell lines that chronically overexpress Hsp72. On the other hand, the effect described here of the regulated acute overexpression of Hsp72 from the plasmid is identical to that of elevated levels of Hsp72 induced by the mild heat shock, indicating the physiological relevance of the observed phenomenon.

It is noteworthy that the regulated overexpression of Hsp72 in the PEER cell line not only suppressed the responses to heat shock and ethanol but also strongly inhibited activation of both JNK (not shown) and p38 (Fig. 2E) kinases caused by some other stresses, for example, osmotic shock, H2O2, and UV irradiation. Therefore, an elevated level of Hsp72 appears to interfere with transduction of signals from a variety of stressful conditions. Moreover, accumulation of Hsp72 strongly reduced activation of stress-induced kinases in response to TNF and IL-1,4 which is consistent with recent findings that the response of JNK (and potentially of p38 kinase) to UV and osmotic shock is mediated by the activation of TNF, IL-1, and epidermal growth factor receptors (33).

Does a high level of Hsp72 directly affect activation of JNK and p38 kinases or does it through induction or repression of synthesis of some other proteins? To address this question, effects of addition of a purified recombinant Hsp72 on the activation of p38 kinase in cell lysate were studied. In a crude lysate p38 kinase becomes activated upon incubation at 20 °C for 15 min (Fig. 3A). Under exposure of the lysate to 43 °C, p38 kinase was activated even further (Fig. 3B). No additional p38 activation was observed in the lysates subjected to UV irradiation (Fig. 3B). Addition of purified Hsp72 (but not bovine serum albumin, not shown) repressed the activation by about 40-70% (Fig. 3A). Importantly, the addition of a monoclonal anti-Hsp70 antibody (MA3-007, Affinity Bioreagents) raised against the conserved ATPase domain, which cross-reacts with all major species of Hsp70, had an effect opposite to that triggered by the addition of Hsp72. It led to a 1.5-3-fold stimulation of p38 kinase activity (Fig. 3B). Addition of control monoclonal anti-beta -galactosidase antibody did not cause activation of p38 (not shown). These data indicate that the level of Hsp70 in a cell may directly regulate stress-activated kinases. The observed variability in the extent of stimulation of p38 by MA3-007 antibody may be due to the presence of endogenous ADP. Indeed, addition of ADP strongly reduced the stimulatory effect of the anti-Hsp70 antibody (not shown).


Fig. 3. Effects of Hsp72 on activation of p38 kinase in cell lysate. A, effect of purified Hsp72 on the activation of p38 kinase in cell lysate. Ten µl of lysate were placed in the test tubes and incubated at 23 °C for 15 min in the presence or absence of purified Hsp72 (added at amounts of 5% of total protein). An ATP-regenerating system (20 mM creatine phosphate, 2 mM ATP, 5 IU of creatine phosphate kinase) was present in all samples. The reaction was stopped by addition of Laemmli sample buffer. 0, no incubation at 23 °C, i.e. Laemmli buffer was added to lysate kept on ice. B, effects of anti-Hsp72 antibody, incubation at high temperature, and under UV irradiation on the activation of p38 kinase in cell lysate. C0, no incubation at 23 °C; AB, lysate was preincubated for 10 min on ice with monoclonal anti-Hsp70 antibody that cross-reacts with all major species of the Hsp70 family (Affinity Bioreagents no. MA3-007) and then, after addition of the ATP-regenerating system, was transferred to 23 °C for 15 min; HS, lysate was incubated for 15 min at 43 °C; UV, lysate was irradiated with 500 J/m2 of UV-C and then was incubated at 23 °C for 15 min; C1, lysate was incubated at 23 °C for 15 min without any treatments.
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Experiments with activation of p38 kinase in a cell lysate by addition of anti-Hsp70 antibody suggested that depletion or just capturing of Hsp70 may result in activation of stress kinases in cells. Members of the Hsp70 family bind misfolded polypeptides and therefore could be captured upon a build-up of abnormal misfolded proteins in the cytosol. Therefore, to test whether accumulation of abnormal proteins cause activation of stress kinases, cells were incubated with amino acid analogs, which incorporate into newly synthesized polypeptides and prevent their proper folding. In fact, incubation of cells with the proline analog, L-azetidine carboxylate, as well as with the arginine analog, canavanine, stimulated both JNK and p38 kinases, and the overproduction of Hsp72 strongly reduced such activation (Fig. 4, A and B). A similar stimulation of stress-activated kinases was observed upon incubation of the cells with puromycin, which causes an accumulation of truncated and therefore improperly folded N-terminal fragments of proteins (not shown). Therefore, build-up of abnormal proteins activates stress kinase, while Hsp72 and probably other members of Hsp70 family suppress such activation.


Fig. 4. Amino acid analogs cause an activation of JNK and p38 kinases. A, PEER control cells and cells that overexpress Hsp72 were transferred to the media without either arginine or proline, and 10 mM of canavanine (Can) or L-azetidine carboxylate (Azc) were added. The activity of p38 kinase was tested after 2 h of incubation. B, U937 control and preheated cells (as in Fig. 1) were treated with 10 mM of Azc or heat-shocked (HS, 30 min), and JNK activity was tested after immunoprecipitation with anti-JNK antibodies.
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These data suggest a mechanism of activation of stress kinases by heat shock and some other stressful conditions. Such stresses may cause protein damage and denaturation and may lead to a build-up of abnormal proteins. In turn these abnormal proteins deplete free Hsp70, which leads to activation of stress-activated kinases (Fig. 5). The role of Hsp70 in the activation of stress kinases may be analogous to its role in activation of heat shock gene transcription (34, 35). Accordingly, we suggest that in unstressed cells Hsp70 associates with an upstream component of the kinase cascade and keeps it in an inactive form. Depletion of a free cellular Hsp70 by accumulated abnormal proteins after heat shock (21, 22) would cause dissociation of the bound Hsp70 from the upstream component of the kinase cascade, thus leading to activation of downstream kinases. Therefore, Hsp70 may function as a "stress sensor" that triggers not only transcription of the heat shock genes but also activation of stress kinases.


Fig. 5. Proposed role of Hsp70 in modulation of stress kinase activity. Various proteotoxic conditions cause depletion of free Hsp70 that leads to activation of stress kinase. In thermotolerant cells (with high level of Hsp70) such activation is prevented, thus blocking apoptosis and other kinase-dependent events.
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The results described in this report may also explain why overproduction of Hsp72 prevents activation of the phospholipase A2 by TNF (36) and prevents induction of synthesis of IL-6 by IL-1 (15). Indeed, Hsp72 may simply prevent activation by TNF or IL-1 of the p38 kinase, which was shown to activate the phospholipase A2 (27) and which is responsible for IL-6 induction (26). In summary, Hsp70 seems to play a very general role in signal transduction pathways, which lead to adaptation of cells and organisms to stressful conditions.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant RO1 (to M. Y. S.), by a Medical Foundation Grant (to M. Y. S.), and by the Yamagiva-Yoshida Grant from UICC (to V. L. G.).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.
   Contributed equally to this study.
Dagger Dagger    To whom correspondence should be addressed: Boston Biomedical Research Institute, 20 Stanford St., Boston, MA 02114. Tel.: 617-742-2010 (ext. 312); Fax: 617-523-6649; E-mail: sherman{at}bbri.harvard.edu.
1   The abbreviations used are: JNK, Jun N-terminal kinase; MTT, 3-[4,5-dimethylthiasol-2-yl]-2,5-diphenyltetrasolium bromide; PARP, poly(ADP-ribose) polymerase; TNF, tumor necrosis factor; GFP, green fluorescent protein; IL, interleukin.
2   D. D. Mosser, submitted for publication.
3   S. Rits, unpublished data.
4   V. L. Gabai, manuscript in preparation.

ACKNOWLEDGEMENTS

We thank Dr. Vladimir Volloch and Dr. Alfred Goldberg for helpful discussion.


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