From the Boston Biomedical Research Institute,
Boston, Massachusetts 02114, § Institute of Influenza, St.
Petersburg, Russia 197022, ¶ Medical Radiology Research Center,
Obninsk, Russia 249020, and
Dana Farber Cancer Institute,
Boston, Massachusetts 02115
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ABSTRACT |
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Inhibition of the major cytosolic protease,
proteasome, has been reported to induce programmed cell death in
several cell lines, while with other lines, similar inhibition blocked
apoptosis triggered by a variety of harmful treatments. To elucidate
the mechanism of pro- and antiapoptotic action of proteasome
inhibitors, their effects on U937 lymphoid and 293 kidney human tumor
cells were tested. Treatment with peptidyl aldehyde MG132 and other proteasome inhibitors led to a steady increase in activity of c-Jun
N-terminal kinase, JNK1, which is known to initiate the apoptotic
program in response to certain stresses. Dose dependence of
MG132-induced JNK activation was parallel with that of apoptosis. Furthermore, inhibition of the JNK signaling pathway strongly suppressed MG132-induced apoptosis. These data indicate that JNK is
critical for the cell death caused by proteasome inhibitors. An
antiapoptotic action of proteasome inhibitors could be revealed by a
short incubation of cells with MG132 followed by its withdrawal. Under
these conditions, the major heat shock protein Hsp72 accumulated in
cells and caused suppression of JNK activation in response to certain
stresses. Accordingly, pretreatment with MG132 reduced JNK-dependent apoptosis caused by heat shock or ethanol,
but it was unable to block JNK-independent apoptosis induced by TNF. Therefore, proteasome inhibitors activate JNK, which initiates an
apoptotic program, and simultaneously they induce Hsp72, which suppresses JNK-dependent apoptosis. A balance between these
two effects might define the fate of cells exposed to the
inhibitors.
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INTRODUCTION |
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Mammalian cells respond to various stressful conditions by activation of stress kinase signaling cascades. Activation of a stress-kinase, c-Jun N-terminal kinase (JNK),1 by strong oxidants, UV irradiation, and some other stressful conditions proceeds through a signal transduction pathway that involves small GTP-binding proteins and a cascade of kinases (1, 2). Prolonged activation of JNK is harmful, since it initiates a special program that leads to cell death, apoptosis (3-6). Indeed, overproduction of dominant negative mutants of JNK or other components of the kinase cascade inhibits programmed cell death in response to heat shock, UV irradiation, oxidative stress (5), and certain other inductors (3, 4, 6). However, there are certain types of apoptosis (e.g. Fas- or TNF-induced) that are JNK-independent (7, 8).
Many stressful conditions also induce heat shock proteins, including Hsp72, which increases a cell's tolerance to stresses. Therefore, the cell's decision to die or to survive is determined by the fine balance of these two systems. In cells affected by stresses, Hsp72 binds to damaged and misfolded polypeptides and can either facilitate their repair or target nonreparable polypeptides for degradation by the ubiquitin- and proteasome-dependent pathway (see Refs. 9 and 10 for review). Our recent findings demonstrate, however, that the cell-protective action of Hsp72 may be unrelated to its activity in protein refolding or degradation. In fact, Hsp72 interferes with the activation of stress kinase JNK, and thus prevents apoptotic signaling during certain stresses (11). This suppression of JNK appears to be responsible for Hsp72-mediated protection of cells from apoptosis induced by heat shock, ethanol, and some other stresses (11).2
Recently, inhibition of major cellular protease, proteasome, was shown to activate a cell death program (12-18). In fact, incubation of cells with potent proteasome inhibitors, namely peptide aldehydes and lactocystin, either facilitated apoptosis caused by TNF or Fas-ligand (14) or was alone sufficient to induce apoptosis (13, 15-18). Since many important regulatory proteins, e.g. p53, are the substrates of proteasome, it was suggested that inhibition of the breakdown of these proteins could result in their accumulation, thereby leading to apoptosis (16, 17). On the contrary, other laboratories reported that treatment with inhibitors of proteasome-dependent protein breakdown protected cells from apoptosis triggered by growth factor withdrawal, ionizing irradiation, exposure to glucocorticoids, or phorbol ester (19, 20). Such protection was observed with quiescent cells, while induction of apoptosis by proteasome inhibitors was demonstrated mainly with proliferating cultures. This led to the idea that inhibition of protein degradation has much more severe effects on cells progressing through the cell cycle. However, there are some examples where exposure of quiescent cells to inhibitors also activated apoptosis (16, 20).
Effects of the inhibition of proteasome-dependent proteolysis on apoptosis could provide new insights into the mechanisms triggering programmed cell death. Furthermore, information about the nature of the inhibitor's cytotoxicity could also be helpful in drug development, since some of the proteasome inhibitors are currently in use as lead compounds for drug design. In this work, we have studied effects of proteasome inhibitors on the activation of JNK and the role of this kinase in cytotoxicity of such inhibitors. We also investigated the role of induction of Hsp72 in the antiapoptotic effects of the proteasome inhibitors.
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EXPERIMENTAL PROCEDURES |
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Materials and Cell Culture-- U937 human lymphoid tumor cells were grown in RPMI 1640 medium with 10% fetal bovine serum and were used for experiments while in the midlog phase (3-7 × 105 cells/ml). 293 human cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and were used in transient transfection experiments while at 40% of confluence. MG132 was purchased from Biomol Research Laboratory, Inc. N-Acetyl-leucinyl-leucinyl-norleucinal (aLLN) was purchased from Sigma. MG132 analogs were synthesized and purified by high pressure liquid chromatography. Anti-hsp72 antibody (SPA810) was from StressGene Biotechnologies Corp. (Canada), and anti-p53 antibody (PAb1801) was from Santa Cruz Biotechnology, Inc.
JNK and p38 Assay--
JNK activity was assayed using GST-c-Jun
protein as a substrate after immunoprecipitation with anti-JNK1
antibodies (sc-474, Santa Cruz Biotechnology) or anti-HA antibody for
transfected JNK (Berkeley Antibody Company, Richmond, CA). Briefly,
cells were lysed in a buffer containing 20 mM Tris-HCl, pH
7.4, 50 mM NaCl, 2 mM EDTA, 1% Triton X-100,
25 mM -glycerophosphate, 10 mM NaF, 1 mM Na3VO4, and protease inhibitors
(1 mM phenylmethylsulfonyl fluoride and a 25 µg/ml
concentration each of aprotenin, pepstatin, and leupeptin). After
immunoprecipitation with anti-JNK1 or anti-HA antibody and protein
A-Sepharose for 2 h at 4 °C and washing, a kinase reaction was
carried out in a buffer containing 25 mM HEPES, 10 mM MgCl2, 2 mM dithiothreitol, 25 mM
-glycerophosphate, 2 mM
Na3VO4, and 25 µCi of
[
-32P]ATP at 37 °C for 10 min. Then the samples
were subjected to SDS-polyacrylamide gel electrophoresis followed by
transfer to a nitrocellulose membrane and autoradiography. This
membrane was later used for immunoblot with anti-JNK1 antibody to
ensure that equal amounts of the kinase were immunoprecipitated. p38
kinase activity was determined by Western blot with polyclonal antibody (New England Biolabs) specifically recognizing the phosphorylated (active) form of p38. Later the same blot was treated with anti-p38 antibody (New England Biolabs) to ensure equal loading of the kinase on
different lanes.
Apoptosis Assays-- Poly(ADP-ribose) polymerase (PARP) degradation was followed by Western blotting with anti-PARP monoclonal antibodies (C2-10; G. Poirier, Montreal, Canada). Apoptosis of 293 cells was detected by cleavage of a truncated form of U1 70-kDa protein d12 (21). Cells were transfected by calcium phosphate method with a plasmid encoding d12 tagged with the T7 epitope alone or together with a dominant-negative mutant form of SEK1 (K/R) or c-Jun (TAM67). After 48 h, cells were either treated with 10 µM MG132 or subjected to heat shock at 45 °C for 15 min. After an additional 24 h, cells were lysed, and cleavage of d12 was detected by Western blot with anti-T7 epitope antibody.
Protein Degradation Assays--
U937 cells were washed once with
PBS, resuspended in RPMI 1640 without leucine and with dialyzed fetal
bovine serum, and incubated for 40 min. Proteins were then labeled for
1 h with 10 mCi/ml of [14C]leucine. Cells were
washed once and chased in regular RPMI 1640 media with 0.2 mg/ml
unlabeled leucine, 150 mM cycloheximide (to inhibit protein
synthesis and reincorporation of a labeled leucine), and 20 mM chloroquine (to inhibit lysosomal protein degradation) in the presence of different concentrations of MG132 (preincubation of
cells with MG132 for 3 h did not change the level of inhibition of
degradation). At the end of the chase period, 10% trichloroacetic acid
was added, and samples were left for 30 min on ice. Then samples were
spun down in Eppendorf centrifuge for 5 min, and the radioactivity in
the supernatant was measured. A percentage of degradation was
calculated as a ratio of trichloroacetic acid-soluble and total
radioactivity in a sample. To assay for inhibition of I-B
degradation by MG132, cells were incubated for 45 min with different
concentrations of the inhibitor, and then TNF
(5 ng/ml) was added.
Samples were collected 10 min later and subjected to polyacrylamide gel
electrophoresis followed by Western blot with anti-I-
B antibody
(sc-847, Santa Cruz Biotechnology) and quantitated with a laser
scanner.
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RESULTS |
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Proapoptotic Effects of the Proteasome Inhibitors-- To dissect the mechanisms of induction of apoptosis by proteasome inhibitors, we tested their effects on activation of JNK, a stress kinase required for initiation of the apoptotic program in response to a variety of stressful conditions. Effects of a very potent proteasome inhibitor, benzyloxycarbonyl-leucinyl-leucinyl-leucinal (MG132), on the U937 cell line were studied (Fig. 1A). The degree of apoptosis was followed by the cleavage of PARP and the appearance of an 86-kDa fragment of PARP, which is the hallmark of this process. A fraction of cleaved PARP correlated with a fraction of cells that underwent shrinkage and nuclear condensation (not shown). The first signs of apoptosis were seen at 4-5 h of incubation with 50 µM of MG132, and after 7 h of incubation, more than 50% of cells underwent apoptosis. Prolonged exposure to the inhibitor (20 h) caused programmed cell death in almost 100% of the cell population. Similar results (100% apoptosis) were observed upon titration of MG132 down to 1.5 µM (Fig. 2A). Therefore, dose dependence of apoptosis on MG132 covered a narrow range of concentrations between 0.2 and 1.5 µM.
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Antiapoptotic Effects of the Proteasome Inhibitors--
Recent
publications indicate that under certain conditions, inhibition of
proteasome-dependent protein breakdown prevents apoptosis
triggered by harmful stimuli (19, 20). We suggested that proteasome
inhibition may cause both pro- and antiapoptotic effects, and depending
on which effect dominates, cells either undergo apoptosis or become
protected from apoptosis caused by another stress. As shown above,
proapoptotic effects of the proteasome inhibitors are associated with
the activation of the JNK pathway. What could be the nature of
antiapoptotic action of the inhibitors? It was described previously
that exposure to proteasome inhibitors induces the 70-kDa heat shock
protein, Hsp72 (12, 29). Recently, we and others reported that Hsp72
accumulation confers resistance to apoptosis (11, 30-33). Therefore,
we suggested that antiapoptotic effects of proteasome inhibitors could
be mediated by accumulation of Hsp72. In our experiments, incubation of
U937 cells with MG132 (Fig.
7A), lactacystin--lacton,
NIP-L3VS, or aLLN (not shown) was found to dramatically
increase the levels of Hsp72. Hsp40 and Hsp27 also appeared to be
induced by incubation with MG132 (not shown). By contrast, treatment
with even high concentrations of less active and inactive MG132
analogs, benzyloxycarbonyl-leucinyl-leucinyl-leucine, benzyloxycarbonyl-leucinyl-leucinyl-norvaline, or
benzyloxycarbonyl-leucinyl-leucinyl-leucinyl-amide, did not lead
to any significant induction of Hsp72. These data indicate that
induction of Hsp72 was caused by inhibition of the proteasome-dependent protein breakdown.
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DISCUSSION |
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Recently, proteasome inhibitors were demonstrated to cause very complex effects on programmed cell death. With some exceptions, it appeared that dividing cells respond to the inhibitors by activation of apoptosis, while in nondividing cells such inhibitors showed antiapoptotic effects. Therefore, the common assumption was that this difference is attributed to difference in the cell types or in proliferating activity. Here we demonstrated that both pro- and antiapoptotic effects of proteasome inhibitors could be seen with the same cell line, U937, depending on the conditions of treatment (i.e. prolonged treatment resulted in apoptosis, while shorter exposure did not kill cells; it induced Hsp72 that caused protective effects). Therefore, we suggested that in cell types where JNK could not be highly activated by proteasome inhibitors, they do not manifest a proapoptotic effect (in fact, we observed that in the COS-7 cell line where MG132 does not activate JNK, it is unable to induce apoptosis). Then, accumulation of Hsp72 in response to proteasome inhibitors would render cells protected from certain types of apoptosis. In prior publications that reported antiapoptotic effects of proteasome inhibitors, these inhibitors were applied simultaneously with the apoptotic stimuli (19, 20). Probably MG132 is a poor activator of JNK in cell lines used in these experiments. Fast Hsp72 accumulation may also contribute to prevention of commitment of these cells to apoptosis.
Proteasome inhibitor-induced apoptosis was dependent on the stress kinase JNK, as with heat shock and certain other stresses. In fact, these inhibitors caused very strong activation of JNK, and dose dependence of such activation closely correlated with the dose dependence of induction of apoptosis (see Fig. 2). Moreover, specific suppression of the JNK signaling pathway by either expression of a dominant-negative form of JNK activator SEK1 or of a dominant-negative form of c-Jun (TAM67) rendered cells substantially less sensitive to MG132-induced apoptosis.
MG132 apparently causes JNK activation through the inhibition of the
proteasome. In fact, proteasome inhibitors other than MG132 with
different mechanisms of action, like lactacystin--lacton and aLLN,
displayed similar cytotoxic and JNK activating properties, indicating
that all of these compounds act through their common ability to inhibit
proteasome. Furthermore, three inactive MG132 analogs did not cause
apoptosis, even when added to cells at high concentrations.
It is noteworthy that MG132 activated JNK in concentrations that did
not significantly affect the breakdown of a total bulk of short lived
proteins. Nevertheless, certain short lived proteins (e.g.
I-B, which we used as a model) are strongly stabilized even at these
low concentrations of MG132. Therefore, exposure to proteasome
inhibitors may specifically block degradation of a certain short lived
protein(s) that upon its accumulation directly or indirectly
up-regulates JNK, resulting in apoptosis. An alternative possibility is
that inhibition of the proteasome by itself could somehow signal JNK
and induce apoptosis, analogously to specific activation of JNK and
induction of apoptosis by inhibition of ribosome (34). This
possibility, however, seems unlikely because inhibition of protein
degradation by suppression of ubiquitination without affecting
proteasome also causes
apoptosis.4
What could be the reason for specific inhibition by MG132 of the breakdown of certain proteins without affecting the degradation of bulk of short lived polypeptides? One possibility is that, while for the majority of short lived proteins the rate-limiting step in degradation pathway is ubiquitination, for certain regulatory proteins the rate-limiting step could be cleavage by the proteasome. Hence, the inhibition of proteasome activity to a certain extent would have a much stronger impact on the breakdown of such proteins than on the degradation of the rest of polypeptides. Alternatively, the rate-limiting step in degradation of some proteins could be cleavage at the hydrophobic site of the proteasome, which is known to be the target of MG132, while the rate-limiting step in degradation of the bulk of short lived proteins could be cleavage at some other site(s).
It was previously reported that the apoptosis induced by the proteasome inhibitors is dependent on p53 (16). However, we did not detect any accumulation of p53 in U937 cells upon exposure to MG132, even at concentrations that induce 100% apoptosis, indicating that in this cell line such apoptosis is p53-independent. Therefore, the p53 dependence of apoptosis induced by the proteasome inhibitors is indeed cell type-specific.
Findings that quiescent cells are less sensitive to proteasome inhibitor-induced apoptosis led to the suggestion by several authors that accumulation of a certain protein involved in the cell cycle causes apoptosis (13, 15). The fact that MG132 caused 100% apoptosis in asynchronous U937 cell culture in less than 16 h, while the cell cycle of U937 cells takes about 24 h, practically rules out this suggestion. Furthermore, maximal activation of JNK was seen within 7 h after the addition of MG132, indicating that critical events of the apoptotic process initiate even earlier. Therefore, apoptosis-inducing action of the proteasome inhibitors, at least in U937 cells, is independent upon the passing of a certain point of the cell cycle. Furthermore, MG132 could not cause apoptosis in rapidly proliferating COS-7 cells (while activating expression of Hsp72), which suggests that correlation between the cell division and proapoptotic action of the proteasome inhibitors is not universal. It does not exclude, however, the possibility that certain rapidly proliferating cells are more sensitive to exposure to the proteasome inhibitors, because a protein regulator of JNK may accumulate in such cells more efficiently, for example, due to a faster synthesis.
What could be the mechanism of another cellular response to inhibition of proteasome synthesis of Hsp72? It is well established that accumulation of misfolded proteins induces Hsp72 (for a review, see Ref. 35). Therefore, we originally suggested that the primary signal for induction of Hsp72 by the proteasome inhibitors is accumulation of newly synthesized polypeptides, which for certain reasons cannot acquire a proper conformation. However, our data rule out this possibility, because MG132 was able to dramatically induce Hsp72 at concentrations that did not affect the breakdown of the bulk of short lived proteins. Therefore, it appears that accumulation of a specific regulatory polypeptide(s) causes induction of Hsp72. Although we have not identified the putative short lived protein inducer of Hsp72, probably it is different from the putative activator of JNK. In fact, although dose dependence of Hsp72 induction closely correlated with that of JNK activation in U937 cells, in primary fibroblast culture the dose dependences of these two events were clearly different (not shown). Furthermore, we demonstrated that in COS-7 cells MG132 did not activate JNK but still caused accumulation of Hsp72 (not shown).
Previously, we demonstrated that overproduction of Hsp72 prevents
apoptosis in response to a variety of stresses by suppression of
activation of JNK (11). Accordingly, in this work MG132-induced accumulation of Hsp72 suppressed activation of JNK in response to
severe heat shock and blocked apoptosis (Fig. 8, A and
B). These data suggested that the protective effect of the
proteasome inhibitors is specific to the apoptotic pathway that
involves JNK activation. By contrast, JNK-independent apoptosis was
expected not to be sensitive to preincubation of cells with MG132.
Although TNF is a strong activator of JNK, TNF
-induced apoptosis
was reported to be independent of JNK (7, 8). Accordingly, we observed
that cells pretreated with MG132 suppressed TNF
-activation of JNK
but did not reduce TNF
-induced apoptosis (Fig. 8, B and C). Thus, the protective effects of the proteasome
inhibitors indeed appeared to be limited to the
JNK-dependent apoptotic pathway. Previously, it was
reported that MG132 protects cells from apoptosis induced by the nerve
growth factor withdrawal,
-irradiation, and phorbol ester (19, 20).
Although there are no data on the JNK-dependence of phorbol
ester-induced apoptosis, it has been shown that JNK is essential
for apoptosis induced by both nerve growth factor withdrawal and
-irradiation (3, 4).
Therefore, the inhibition of proteasome activates the apoptotic pathway dependent on the stress-kinase JNK and simultaneously induces synthesis of the protective protein Hsp72, which suppresses JNK. We suggest that the balance between these two activities could define whether pro- or anti-apoptotic action of the inhibitors would dominate.
The ability of the proteasome inhibitors to induce Hsp72 and to suppress JNK and p38 kinases could be very useful in the design of new drugs. In fact, JNK-mediated apoptosis appears to be involved in the pathology of myocardial ischemia (36), while p38 is critical for a variety of inflammatory responses (37). Therefore, MG132 could be used as a prototype of therapeutics against certain myocardial and inflammatory pathologies.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Avruch and Dr. R. Davis for kindly providing the plasmids used in this work.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant RO1 (to M. Y. S.) and by a Medical Foundation grant (to M. Y. S.).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.
** To whom correspondence should be addressed. Tel.: 617-742-2010 (ext. 312); Fax: 617-523-6649; E-mail: sherman{at}bbri.harvard.edu.
1 The abbreviations used are: JNK, c-Jun N-terminal kinase; TNF, tumor necrosis factor; PARP, poly(ADP-ribose) polymerase; aLLN, N-acetyl-leucinyl-leucinyl-norleucinal.
2 V. L. Gabai, J. Yaglom, A. B. Meriin, and M. Y. Sherman, manuscript in preparation.
3 J. Yaglom, unpublished data.
4 L. Monney, personal communication.
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REFERENCES |
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