(Received for publication, September 3, 1996, and in revised form, December 24, 1996)
From the The accumulation of misfolded proteins in the
cytosol leads to increased expression of heat-shock proteins, while
accumulation of such proteins in the endoplasmic reticulum (ER)
stimulates the expression of many ER resident proteins, most of which
function as molecular chaperones. Recently, inhibitors of the
proteasome have been identified that can block the rapid degradation of
abnormal cytosolic and ER-associated proteins. We therefore tested
whether these agents, by causing the accumulation of abnormal proteins, might stimulate the expression of cytosolic heat-shock proteins and/or
ER molecular chaperones and thereby induce thermotolerance. Exposure of
Madin-Darby canine kidney cells to various proteasome inhibitors,
including the peptide aldehydes (MG132, MG115,
N-acetyl-leucyl-leucyl-norleucinal) and lactacystin,
inhibited the degradation of short-lived proteins and increased
markedly the levels of mRNAs encoding cytosolic heat-shock proteins
(Hsp70, polyubiquitin) and ER chaperones (BiP, Grp94, ERp72), as shown
by Northern blot analysis. However, inhibitors of cysteine proteases
(E64), serine proteases (leupeptin), or metalloproteases
(1,10-phenanthroline) had no effect on the levels of these mRNAs.
The relative efficacies of the peptide aldehyde inhibitors in inducing
these mRNAs correlated with their potencies against the proteasome.
Furthermore, reduction of the aldehyde group of MG132 decreased its
inhibitory effect on proteolysis and largely prevented the induction of
these mRNAs. Although treatment with the proteasome inhibitors
caused rapid increases in mRNA levels (as early as 2 h after
treatment with MG132), the inhibitors did not detectably affect total
protein synthesis, total protein secretion, ER morphology, or the
retention of ER-lumenal proteins, even after 18 h of treatment.
Together, the findings suggest that inhibition of proteasome function
induces heat-shock proteins and ER chaperones due to the accumulation
of sufficient amounts of abnormal proteins and/or the inhibition of
degradation of a key regulatory factor (e.g. heat-shock
factor). Since expression of heat-shock proteins can protect cells from
thermal injury, we tested whether the proteasome inhibitors might also
confer thermotolerance. Treatment of cells with MG132 for as little as 2 h, markedly increased the survival of cells subjected to high temperatures (up to 46 °C). Thus, these agents may have applications in protecting against cell injury.
The cellular "heat-shock response," manifested by increased
expression of heat-shock proteins, represents a basic defense mechanism
employed by cells to protect themselves against high temperature and
various other injurious conditions (1, 2). Most of the major heat-shock
proteins function as molecular chaperones involved in the folding,
assembly, and/or degradation of proteins and therefore appear to
prevent the accumulation of aggregated, misfolded, or damaged proteins
in the affected cell (1, 3, 4). In the cytosol, heat-shock or other
harsh conditions cause increased transcription and translation of a
group of chaperones (e.g. Hsp70) and polyubiquitin (a
cofactor in intracellular protein degradation). In the endoplasmic
reticulum (ER),1 harsh growth conditions
(e.g. glucose deprivation) induce the increased production
of ER molecular chaperones, i.e. the glucose-regulated proteins (e.g. Grp78/BiP, Grp94, and ERp72) (2-5). It is
now well established in bacterial (6) and animal (1, 7) cells that the
signal for the induction of heat-shock proteins is the accumulation of
abnormal proteins in the cytosol. Similarly, conditions that perturb
folding or glycosylation of surface or secreted proteins cause a
buildup of misfolded proteins in the ER, which appears to be a common
signal, at least indirectly, for the increased expression of the ER
chaperones (8, 9).
Abnormal proteins in the cytosol and nucleus are degraded primarily
through the ubiquitin-proteasome pathway (10-14). The proteasome is
the major neutral proteolytic apparatus in the cell and is an essential
component of the ATP-dependent degradative pathway (10, 15,
16). Recent studies with inhibitors in lymphoblasts suggest that the
proteasome is responsible not only for the degradation of many rapidly
turned-over proteins, but also for the bulk of other proteins (17). The
degradation of misfolded proteins transiting the ER is less well
understood, although at least in the case of one transmembrane protein,
the cystic fibrosis transmembrane conductance regulator, the proteasome
appears to be involved (18, 19).
Recently a variety of reversible (17) and irreversible (20) inhibitors
of the 20 S proteasome have been identified that can enter mammalian
cells and inhibit degradation of proteins by the ubiquitin-proteasome
pathway. One group of such inhibitors are peptide aldehydes
(e.g. carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132))
which reversibly bind to active sites and inhibit cleavage of
hydrophobic or acidic substrates (17). A more specific inhibitor is the
naturally occurring bacterial compound, lactacystin, which covalently
modifies threonine residues in the proteasome's active site and does
not seem to affect any other known protease (20). Such agents can
inhibit protein degradation (17-19) and major histocompatibility class
I antigen presentation (17) in a variety of mammalian cells and have
been widely used to probe the physiological function of the
ubiquitin-proteasome pathway.
Since the blockage of protein breakdown in the cell (e.g. by
inhibition of proteasomes) should lead to an accumulation in cells of
proteins otherwise targeted for degradation, it seemed likely that
these agents might also signal the induction of the cytosolic and/or ER
chaperones. To test this possibility, we examined the ability of
several types of protease inhibitors to increase the levels of mRNA
encoding the cytosolic heat-shock proteins (i.e. Hsp70 and
polyubiquitin) and/or the ER chaperones (i.e. BiP, Grp94,
and ERp72). In addition to specific inhibitors of the proteasome, we
also studied the effects of an inhibitor of cysteine proteases (such as
lysosomal cathepsins B, H, and L and calpains) and a metal chelating
agent that inhibits metalloproteases. These other inhibitors have also
been reported to affect the degradation of certain ER proteins
(21-24). However, in our studies, only those agents which have been
reported to inhibit the activity of the proteasome (17, 20) were able
to consistently stimulate expression of mRNAs for heat-shock
proteins and ER chaperones. Furthermore, proteasome inhibition was
found to protect cells from subsequent thermal injury, a finding that
may have broad implications, not only for the interpretation of
experimental studies using these inhibitors, but possibly for
therapeutic use of these highly specific inhibitors.
cDNA probes were kindly provided
by the following investigators: ERp72 and Grp94 by Michael Green (St.
Louis University); BiP by Mary-Jane Gething (Melbourne University,
Australia). The cDNA probe for Hsp70 was from ATCC (Rockville, MD).
Carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) and
carbobenzoxyl-leucinyl-leucinyl-norvaline (MG115) were kindly provided
by ProScript (formerly Myogenics (Cambridge, MA)). Lactacystin was
kindly provided by S. Omura (The Kitasato Institute, Tokyo, Japan).
Tunicamycin was from Boehringer Mannheim. N-Acetyl-leucyl-leucyl-norleucinal (aLLN), E64,
1,10-phenanthroline, and leupeptin were from Sigma.
Anti-BiP antibody was from Affinity Bioreagents (Neshanic Station, NJ),
and fluorescein isothiocyanate-labeled secondary antibodies were from
Jackson Immunologicals (West Grove, PA).
Confluent
monolayers of Madin-Darby canine kidney (MDCK) cells were incubated for
up to 18 h in the absence or presence of either tunicamycin (a
potent inducer of ER stress proteins) or protease inhibitors in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal
calf serum (FCS). Total RNA was isolated from the cells by
phenol-chloroform extraction, electrophoresed on 1%
formamide/formaldehyde-agarose gels, and transferred to nitrocellulose as described previously (5, 25). The nitrocellulose blots were then hybridized with random primed 32P-labeled
cDNA overnight at 42 °C, washed with SSC, and exposed to
autoradiographic film (26).
To examine the degradation of
short-lived proteins, MDCK cells were incubated at 37 °C for 1 h in tyrosine-free DMEM supplemented with 5% FCS to which 5 µCi/ml
[3H]tyrosine had been added, followed by thorough washing
in Dulbecco's modified phosphate-buffered saline (DPBS) and a chase
for 2 h at 37 °C in 200 µl of complete DMEM (104 µg/ml
tyrosine·2Na·2H2O), 5% FCS containing the inhibitors.
Chloroquine (20 µM) was added to all samples to block the
lysosomal protein degradation (17). At the end of the chase period,
trichloroacetic acid was added to a final concentration of 10%, and
the medium was collected and centrifuged at maximum speed for 30 min in
an Eppendorf microcentrifuge. The total acid-soluble radioactivity in
clarified supernatants was determined in a scintillation counter. To
measure degradation of long-lived proteins, MDCK cells were incubated
for 18 h at 37 °C in tyrosine-free DMEM, 5% FCS supplemented
with 5 µCi/ml [3H]tyrosine, then washed in DPBS
and chased for 1 h in normal DMEM, 5% FCS (104 µg/ml
tyrosine·2Na·2H2O; 20 µM chloroquine was
added for the final 30 min of this chase). After this chase, the medium was replaced with fresh unlabeled DMEM, 5% FCS containing inhibitors and 20 µM chloroquine and chased for an additional
2 h at 37 °C. Determination of total acid-soluble radioactivity
was performed at the end of this chase as described above.
Since it is the aldehyde group of the
peptide aldehyde inhibitors that is thought to interact with the
proteasome and thus interfere with its ability to degrade proteins (34,
35), inactivation of this aldehyde group should ameliorate the effects
of these inhibitors. Inactivation of the aldehyde group was
accomplished by reduction of this group to an alcohol. DPBS with or
without MG132 (final concentration 0.7 mM) was treated with
0.1 mM sodium borohydride for 1 h at room temperature.
The reaction was then stopped by acidification of the samples to pH 5 with glacial acetic acid to inactivate the sodium borohydride. The
entire solution was then refrigerated for at least 1 h to ensure
complete inactivation of the sodium borohydride. Confluent monolayers
of MDCK cells were then incubated in DMEM, 5% FCS in the absence or
presence of tunicamycin, MG132, borohydride-reduced MG132, or an
equivalent volume of borohydride-treated DPBS. After 18 h of
incubation, total RNA was collected, and Northern blot analysis was
performed as described above.
Confluent monolayers of
MDCK cells were treated with inhibitors as described above. A time
course of incorporation of 35S-labeled methionine into
cellular and secreted proteins was performed. Cells were pulsed with
35S for 30 min in methionine-free DMEM, 5% FCS following
either 0, 3, 6, 9, 12, or 15 h of growth in normal DMEM, 5% FCS
(30 µg/ml methionine) containing protease inhibitors. The cells were
then chased in nonradioactive normal DMEM, 5% FCS (30 µg/ml
methionine) containing protease inhibitors for an additional 2.5 h. Cells and media were collected separately and labeled proteins were precipitated with 10% trichloroacetic acid. Acid-precipitated proteins
were collected on glass microfiber filters (Whatman), washed with
ice-cold 5% trichloroacetic acid, ice-cold absolute acetone, and the
total acid-precipitable radioactivity (representing the total
incorporation of 35S-labeled methionine into cellular or
secreted proteins) was measured in a scintillation counter.
Confluent monolayers of MDCK cells
growing on coverslips were treated with the inhibitors for 12-18 h.
They were then fixed by plunging into Following 12-18 h of growth in
the presence of protease inhibitors, the medium was collected,
trichloroacetic acid was added, and the acid-precipitated material was
solubilized in SDS-polyacrylamide gel electrophoresis sample buffer.
Equivalent amounts of the conditioned medium were electrophoresed on a
10% SDS-polyacrylamide gel. Western blots and probing of
nitrocellulose membranes with primary antisera were performed as
described previously (27-29). Immunoblots were developed using the ECL
chemiluminescent system (Amersham Corp.) with horseradish
peroxidase-conjugated secondary antisera.
Confluent monolayers of MDCK cells were
incubated at 37 °C for 2 h in the absence or presence of the
inhibitors. Following this "pretreatment," cells were washed three
times (15 min each wash) in fresh media to remove the inhibitors. After
the final wash, the cells were incubated at 37 °C in fresh medium
for an additional 3 h. The culture media was then changed a final
time, and the cells were incubated in a separate CO2
incubator maintained at 46 °C for an additional 4-5 h. The cells
were then rinsed in PBS and exposed for 5 min to 0.1% trypan blue in
PBS (supplemented with physiological levels of Ca2+ and
Mg2+), and the percentage of nonviable cells was
determined.
Inhibitors of proteasome function can block the degradation of
most short- and long-lived proteins (17), and therefore treatment with
these agents is likely to cause the accumulation of such proteins not
only in the cytosol, but possibly also in the ER, since a
proteasome-mediated degradative pathway has been reported to function
in the quality control of some ER-associated proteins (18, 19). To test
whether treatment of cells with proteasome inhibitors may cause the
induction of the Hsps and ER chaperones, we examined the ability of
several inhibitors of the proteasome to increase the content of the
mRNAs for the cytoplasmic Hsps, Hsp70 and polyubiquitin, and/or ER
chaperones, BiP, ERp72, and Grp94 (2, 5, 28-30), in MDCK cells, a
widely studied polarized epithelial cell line.
When cells were incubated for 12-16 h with the peptide aldehyde
proteasome inhibitor, MG132, the mRNA levels of both the cytosolic Hsps and the ER chaperones increased severalfold (Fig.
1). Another aldehyde proteasome inhibitor, aLLN (at
concentrations greater than 10 µM), was also able to
induce consistent increases in both groups of mRNAs (Fig.
2A). In the case of MG132 (10 µM), the most potent inhibitor of the proteasome, the
levels of BiP and Grp94 mRNA were roughly comparable with those
achieved with tunicamycin (10 µg/ml) treatment (Fig. 1), one of the
most potent inducers of ER chaperones described (31-33). Two other
protease inhibitors, which have been reported to retard the degradation
of certain proteins transiting the ER, including the specific inhibitor
of cysteine proteases, E64 (22), and the metalloprotease inhibitor, 1,10-phenanthroline (23, 24), were found to have little or no effect on
these mRNAs (Fig. 1). In addition, leupeptin, a potent inhibitor of
certain serine proteases and many thiol proteases (e.g. the
lysosomal cathepsins and calpain), which blocks most lysosomal
proteolysis, but does not affect degradation of short-lived or abnormal
proteins, also had no effect on these mRNA levels (Fig. 1). Thus,
only those agents that inhibited the function of the proteasome (17)
were found to increase mRNAs for cytosolic heat-shock proteins or
ER chaperones (Figs. 1 and 2A), both of which are generally
believed to be induced by the accumulation of abnormally folded protein
(1, 3, 4, 6-9). To confirm that these effects were due to a reduction
in proteolysis, the rate of breakdown of short- and long-lived proteins
was measured in control and treated cells. Although measurement of
protein degradation following treatment of cells with either MG132 or aLLN showed that both MG132 and aLLN were capable of blocking the
degradation of proteins, MG132 (particularly at the concentrations used
to induce the chaperones (< 10 µM)), was a more potent
inhibitor of the degradation of short-lived proteins (Fig.
3A). Treatment of cells with as little as 1 µM MG132 reduced the degradation of short-lived proteins
up to 60-70% of control, while this level of inhibition was achieved
only after treatment with 30 µM aLLN. Moreover,
MG132 was found to inhibit the degradation of long-lived proteins with
much greater efficacy than aLLN at all concentrations tested (Fig.
3B). Furthermore, even a concentration of 30 µM aLLN was only able to achieve roughly half the
inhibition of long-lived protein degradation as 1 µM
MG132 (Fig. 3B). By contrast, borohydride-reduced MG132 (see
below) was much less effective blocking protein degradation (<30%
inhibition of proteolysis) (Fig. 3C). Moreover, as expected, tunicamycin, an inhibitor of N-linked glycosylation, which
also induces BiP expression, had no effect of total proteolysis (data not shown).
To determine whether the observed increases in the mRNAs for the
Hsps and ER chaperones were in fact due to the inhibition of the
proteasome and not the result of nonspecific effects, cells were
treated with an inactive form of MG132 incapable of inhibiting proteolytic activity of the proteasome. The aldehyde group of peptide
aldehydes, such as MG132, forms a transition state hemiacetyl complex
with the threonine active site of the proteasome and interferes with
its ability to degrade proteins (34, 35). Thus, if this aldehyde group
were altered (e.g. reduced to an alcohol), the ability of
MG132 to inhibit the proteasome should largely be lost. We therefore
inactivated MG132 by reducing its aldehyde group with sodium
borohydride. Incubation of MDCK cells with the reduced MG132 had no
effect on the mRNA levels for Hsp70, polyubiquitin, or the ER
chaperones, even after 16 h of incubation (Fig. 2B). Together with the finding that the borohydride-reduced MG132 was ineffective in blocking protein degradation (see above; Fig. 3), these
experiments indicate that the increases in the message levels for the
Hsps and ER chaperones observed with active peptide aldehyde inhibitors
are most likely due to inhibition of the ubiquitin-proteasome proteolytic pathway and the accumulation of undegraded proteins and/or
inhibition of a key regulatory factor (e.g. heat-shock factor).
Additional support for this hypothesis was obtained when cells were
treated with the less potent peptide aldehyde,
carbobenzoxyl-leucinyl-leucinyl-norvaline (MG115), or the irreversible
natural product, lactacystin. MG115 (like MG132) is a hydrophobic
peptide aldehyde that also inhibits the proteasome (17). Lactacystin,
which reacts with the proteasome active site threonine on the
To test if the induction of Hsps and ER chaperones could also
conceivably be a consequence of cell toxicity, we studied what effect
the proteasome inhibitors had on the overall ability of cells to
synthesize and secrete proteins. Fig. 6A
shows that treatment with the protease inhibitors MG132, lactacystin,
or aLLN caused no inhibition of total protein synthesis in MDCK cells,
even after 18 h of growth in medium containing the inhibitors
(Fig. 6A). Likewise, the total amount of protein secreted by
MDCK cells remained unaffected following treatment with the inhibitors
(Fig. 6B). Thus, the observed increases in the mRNA
encoding the ER chaperones are not likely to be due to an accumulation
of proteins in the ER resulting from a generalized blockage of the
secretory pathway. While it is possible that the secretion of specific
proteins may be affected by these inhibitors, the profile of secreted
proteins analyzed by standard SDS-polyacrylamide gel electrophoresis
from 35S-labeled cells showed no obvious differences (data
not shown).
Accumulation of undegraded proteins could conceivably lead to
alterations in cellular and/or ER morphology. However, indirect immunofluorescence with antibodies against the ER chaperone, BiP, revealed that the morphology of the ER was not affected at this level
of analysis, even after 12-16 h of treatment with any of the
proteasome inhibitors (Fig. 7B). Moreover,
the retention mechanism of ER lumenal proteins, which has been reported
to be affected by other perturbations, such as Ca2+
ionophores (36), remained intact. Thus, Western blot analysis of the
media from cells treated with the proteasome inhibitors demonstrated
that the cells did not nonspecifically release the ER resident lumenal
chaperones BiP and Grp94 (Fig. 7A). Together, these results
suggest that inhibition of the proteasome has minimal (if any) effects
on the processing of normal proteins, at least in MDCK cells,
admittedly a "hardy" cell line. Since several highly sensitive
measures of cellular and ER well-being appear unaffected, it is likely
that the observed induction of the Hsps and ER chaperones is due to a
specific effect of inhibition on the proteasome.
The observed increases in mRNA
expression induced by proteasome inhibition will very likely lead to
increased cellular content of Hsps and ER stress proteins. Induction of
the heat-shock proteins, especially Hsp70, such as occurs during
exposure of cells to high temperature, protects cells against the
lethal effects of subsequent exposure to very high temperature or other
toxic insults (1, 37-41). To determine if the proteasome inhibitors
induced this protective response, we investigated whether exposure of
MDCK cells to these agents could protect them against high temperature (thermotolerance), as occurs upon induction of Hsps by other
mechanisms. Incubation of MDCK cells at 37 °C for 2 h with 1 µM MG132 was found to dramatically increase cell survival
after subsequent exposure to temperatures as high as 46 °C for
4 h (Fig. 8). Moreover, cellular survival was
increased also upon exposure for 5 h at temperatures as high as
50 °C (data not shown). Treatment with 1 µM MG132 for
this period appeared to maximally enhance MDCK cell survival at the
higher temperature. In contrast, however, treatment with 5 µM and 10 µM MG132 appeared to reduce cell
survival at high temperature, while treatment with 0.1 µM
and 0.01 µM MG132 had little or no effect on survival
(data not shown). In addition, the survival rate of MDCK cells
"pretreated" longer (>4 h) or with intermediate concentrations of
MG132 (
Potent inhibitors of the proteasome, including the peptide
aldehydes MG132, MG115, as well as the highly specific inhibitor, lactacystin, represent novel tools to study the importance of this
cellular degradative pathway. In this study, we examined the ability of
these inhibitors, as well as aLLN (which was initially described as an
inhibitor of calpain, but has since also been shown to inhibit the
ubiquitin-proteasome degradation pathway (17)), to induce the Hsps and
ER chaperones. Included in our analysis were other protease inhibitors
(E64 and 1,10-phenanthroline) that have been suggested to interfere
with the degradation of certain proteins in the ER (Refs. 21-24;
reviewed in Ref. 42), as well as leupeptin (a potent inhibitor of
lysosomal proteases).
We observed that inhibition of the proteasome with the peptide
aldehydes, aLLN, and lactacystin resulted in increases in the mRNAs
encoding both the cytoplasmic Hsps and the ER chaperones (Figs. 1, 2,
and 4). Although peptide aldehyde proteasome inhibitors (MG132, MG115,
and aLLN) can also inhibit calpains and lysosomal proteases in
vitro, the higher potency of these agents in blocking proteasome-mediated degradation (Fig. 3; Ref. 17), together with the
fact that other calpain inhibitors (such as E64) showed no effect on
the mRNA levels of either Hsps or ER chaperones (Fig. 1) and the
finding that lactacystin (the most selective proteasome inhibitor
currently known (20)) also caused marked increases in mRNAs for
both the Hsps and the ER chaperones (Fig. 4), indicates that these
increases are the result of an effect of the inhibitors on the
proteasome.
One surprising finding is the rapidity with which the transcription of
the heat-shock proteins and ER chaperones rise upon addition of MG132.
Under conditions where intracellular protein breakdown is inhibited by
about 70% (Fig. 3), the mRNAs for Hsp70 and BiP clearly increased
within 2 h (Fig. 5), presumably due to the accumulation of
abnormal proteins or various short-lived, normal regulatory proteins.
In Escherichia coli, inhibitors of the cell's major
ATP-dependent proteases can also trigger the heat-shock
response (6). These findings support the notion that saturation of the
cell's proteolytic capacity can trigger the expression of the
heat-shock genes, most of which encode either molecular chaperones or
components of the proteolytic pathway (e.g. polyubiquitin).
These proteins together can promote the refolding or the destruction of
damaged polypeptides whose accumulation could be toxic. Molecular
chaperones appear to be important in the cellular mechanism monitoring
the buildup of such proteins. The accumulation of such unfolded
proteins in E. coli leads to a stabilization of the normally
short-lived, positive regulatory factor, 32 Although this mechanism can explain the increases in Hsp70 mRNA by
proteasome inhibitors, the mechanism for increases in the mRNA
encoding the ER chaperones remains unclear. It is possible that the ER
chaperones are induced by the same signal as the Hsps. This is not
without precedent, as it has been shown in yeast that KAR2 (the yeast
BiP homolog) is up-regulated in response to heat-shock along with Hsp70
(46, 47). However, recent evidence suggests that the increases in ER
chaperone mRNA we observed with proteasome inhibitors might be the
result of a more direct effect on the ER. For example, the degradation
of several membrane proteins have been found to require ubiquitination
and the proteasome in yeast ER (48, 49). The degradation of the cystic
fibrosis transmembrane conductance regulator is also blocked by the use of inhibitors of the proteasome, suggesting that the proteasome is also
involved in the ER degradation of some mammalian membrane-associated proteins (18, 19). How the cytosolic proteasome may function in the
degradation of proteins contained within an apparently inaccessible
membrane-bound organelle such as the ER remains unclear and constitutes
an important area for future study. However, the proteasome (or a
proteasome-like structure) has been detected in the microsomal
fractions of various cells, although it is believed to be in
association with the cytosolic surface of the membrane (16, 50).
Although the proteasome would appear to be in a strategic location to
assist in the degradation of transmembrane ER proteins with extensive
cytosolic domains, it remains unclear how the proteasome localized to
the cytosolic face of the ER would affect the degradation of proteins
contained entirely within the lumen of the ER, if indeed it does.
However, if a coordinated degradative mechanism were to exist, such
that the lumenal, transmembrane, and cytosolic domains of transmembrane
proteins were degraded simultaneously (as has been suggested (reviewed
in Ref. 42)), it may be sufficient to inhibit the degradation of such
transmembrane proteins alone to elicit an ER stress response. In
addition, it is also possible that at least some proteins transiting
the ER (transmembrane and secretory), which are targeted for
degradation, are extruded from the ER into the cytosol where they are
then degraded by the proteasome. Regardless of the mechanism involved, our data suggest that inhibition of the proteasome somehow results in a
"backup" in the degradative pathway and accumulation of abnormal proteins (presumably within the ER) with consequent increases in the
expression of ER lumenal chaperones.
Treatment with MG132 was also found to impart thermotolerance to
mammalian cells (Fig. 8), although the window of efficacy may be
somewhat narrow, even for MDCK cells, a relatively hardy cell line.
Similar findings have been obtained upon treatment of yeast with these
proteasome inhibitors, which become resistant to several insults,
including high temperature, anoxia, and alcohol following treatment
with proteasome inhibitors.2 Together these
findings suggest novel uses for the proteasome inhibitors in the
protection of cells from injury. Previous studies have demonstrated
that preinduction of the heat-shock proteins by mild stress can protect
cells from a variety of subsequent, more severe stresses, including
heat, H2O2, anoxia, alcohol, heavy metals, and
others (1, 37-41). Most likely, prior heat-shock induces the Hsps by
causing the misfolding of cellular protein that is mild or reversible
enough for the cell to withstand subsequent greater injury, presumably
due to increased cytosolic chaperone synthesis and enhanced folding
and/or degradative capacity. Nevertheless, heat-shock and many other
stresses that induce cytosolic chaperones are likely to affect a wide
variety of cellular functions, and finer tools are obviously necessary
for use in therapeutic settings. The observed increases in the messages
for cytosolic and ER stress response proteins following treatment with
highly specific proteasome inhibitors may be due to the accumulation of
abnormal protein targeted for degradation in these cellular
compartments or alternatively to the inhibition of the degradation of a
key factor responsible for the heat-shock response (e.g.
heat-shock factor; see above) without any major effect on the
processing of normal protein (Figs. 5, 6). Since exposure to these
inhibitors can induce thermotolerance, they may have investigative or
perhaps therapeutic applications in protecting against cell injury.
Renal Division,
Reagents and Chemicals
80 °C methanol and processed
for indirect immunofluorescence as described previously (27).
Fig. 1.
Treatment with proteasome inhibitors leads to
increased levels of mRNAs for Hsps and ER stress proteins.
Northern blot analysis of total RNA isolated from MDCK cells grown for
12-16 h in the absence (control (lane 1)) or presence of
protease inhibitors: 10 µM MG132 (lane 2), 1 µM E64 (lane 3), 10 µM E64
(lane 4), 100 µM 1,10-phenanthroline
(lane 5), 10 µM aLLN (lane 6), 10 µM leupeptin (lane 7). Tunicamycin (2 µg/ml
(lane 8) and 10 µg/ml (lane 9)), a potent
inducer of ER chaperones, was used as a positive control. Blots were
probed with 32P-labeled cDNA probes for the gene
products indicated at the left. Ethidium bromide staining of the 28 S
rRNA was used to indicate that equal amounts of RNA were loaded on the
gel.
[View Larger Version of this Image (128K GIF file)]
Fig. 2.
Increases in mRNAs for Hsp70 and BiP
require specific inhibition of proteasome. Northern blot analysis
of total RNA from MDCK cells grown for 12-16 h in the absence (control
(A, lane 1; B, lane 1)) or presence of either 10 µg/ml tunicamycin (positive control (A, lane 2; B,
lane 2)) or proteasome inhibitors as described under
"Experimental Procedures": A, 15 µM aLLN
(lane 3), 7.5 µM aLLN (lane 4), 3.8 µM aLLN (lane 5), and 1.9 µM
aLLN (lane 6); B, 10 µM MG132
(lane 3), 10 µM sodium borohydride-reduced MG132 (lane 4), and sodium borohydride treated PBS
(Lane 5). Blots were probed with 32P-labeled
cDNA for BiP and Hsp70, as indicated. Ethidium bromide staining of
the 28 S rRNA was used to indicate the equivalence of the gel
load.
[View Larger Version of this Image (39K GIF file)]
Fig. 3.
Proteasome inhibitors block degradation of
short-lived and long-lived proteins in MDCK cells. Graph showing
the effects of tunicamycin, MG132, sodium borohydride-reduced MG132, or
aLLN on the degradation of short- and long-lived proteins in MDCK
cells. A, for short-lived proteins, MDCK cells were labeled
for 1 h with [3H]tyrosine, washed and chased for
2 h in fresh medium with or without 1, 10, or 30 µM
MG132 or 1, 10, or 30 µM aLLN. B, for long-lived proteins, MDCK cells were labeled for 18 h with
[3H]tyrosine, washed, and chased for 1 h in fresh
complete medium. The cells were then chased for an additional 2 h
in complete medium with or without 1, 10, or 30 µM MG132
or 1, 10, or 30 µM aLLN. C, reduction of
the aldehyde group of MG132 attenuates the inhibitory effects of MG132
on protein degradation.
[View Larger Version of this Image (18K GIF file)]
subunit, appears to be totally selective for the proteasome (20).
Northern blot analysis revealed that treatment of cells with either of
these inhibitors of the proteasome (both at 10 µM) caused
increases in the mRNA levels for both Hsp70 and BiP (Fig.
4A). The similar results obtained upon
treatment with MG115 (as well as the other peptide aldehyde inhibitors,
MG132 and aLLN) and lactacystin, which inhibit the proteasome by very
different mechanisms, argues that the mRNA increases directly
result from specific inhibition of the proteasome (probably due to an
accumulation of undegraded protein). Consistent with this idea, the
relative potency of the peptide aldehyde inhibitors in inducing the
Hsps and ER chaperones correlated with their reported potencies in
inhibiting the proteasome and intracellular proteolysis: MG132 > MG115 > aLLN (17) (Figs. 1, 2, 3, 4). Furthermore, the time course of
the induction of various chaperone message levels revealed differences
between the proteasome inhibitors. For example, MG132 was found to
increase mRNA levels for BiP and Hsp70 by 2 h after treatment,
and this response reached apparently maximal levels after 6 h
(Fig. 5). In comparison, lactacystin increased these
mRNAs after 3 h (with levels comparable with MG132 at 6 h), while aLLN did not cause any significant increases in either BiP or
Hsp70 mRNAs after even 6 h of treatment (Fig. 5) (although these increases were seen after 12 h (Figs. 1 and
2A).
Fig. 4.
Additional inhibitors of the proteasome also
induce the ER molecular chaperones. Northern blot analysis of
total RNA from MDCK cells grown for 12-16 h in the absence (control
(lanes 1 and 5) or presence of either 10 µg/ml
tunicamycin (positive control (lanes 2 and 6)),
10 µM MG132 (lanes 3 and 7), 10 µM MG115 (lane 4), or 10 µM
lactacystin (lane 8) as described under "Experimental Procedures." Blots were probed with 32P-labeled probe for
BiP and Hsp70. Ethidium bromide staining of the 28 S rRNA was used to
indicate the equivalence of the load.
[View Larger Version of this Image (49K GIF file)]
Fig. 5.
Hsp70 and BiP are induced rapidly upon
incubation with proteasome inhibitors. Northern blot analysis of
total RNA from MDCK cells grown for up to 6 h in the absence
(control (lane A) or presence of either 10 µg/ml
tunicamycin) positive control (lane B)), 10 µM
MG132 (lane C), 10 µM aLLN (lane
D), or 10 µM lactacystin (lane E). Total
RNA was collected 1, 2, 3, and 6 h after incubation with the
agents (as indicated on the left), and Northern analysis was
performed using the 32P-labeled probe for BiP or the
32P-labeled probe for Hsp70.
[View Larger Version of this Image (54K GIF file)]
Fig. 6.
Neither protein synthesis nor cellular
secretion is affected by treatment with proteasome inhibitors.
A, graph showing effects of MG132, lactacystin, or aLLN on
the rate of protein synthesis in MDCK cells. 35S-Labeled
cells grown in the absence (control) or presence of the inhibitors (10 µM MG132, 15 µM aLLN, 10 µM
lactacystin) were collected and solubilized every 3 h for 18 h, and the amount of radioactive protein was measured. Protein
synthetic rate of MDCK cells is apparently unaffected, even after
18 h of incubation in the protease inhibitors. B, graph
showing effects of MG132, lactacystin, or aLLN on the secretion rate of
MDCK cells. The growth medium from radiolabeled MDCK cells incubated in
the absence or presence of either 10 µM MG132, 10 µM lactacystin, or 15 µM aLLN was collected
every 3 h, and the level of radiation was measured. Even after
18 h of incubation the protease inhibitors have little or no
effect on the secretory rate of MDCK cells.
[View Larger Version of this Image (33K GIF file)]
Fig. 7.
Treatment of MDCK cells with proteasome
inhibitors does not detectably alter ER morphology by
immunofluorescence or cause release of ER molecular chaperones, BiP and
Grp94. A, Western blot analysis of levels of BiP and Grp94
proteins in MDCK cells (Cells) and growth medium
(Media) following 16-20 h of growth in the absence
(Control) or presence proteasome inhibitors (10 µM MG132 (MG132), 15 µM aLLN
(ALLN), 10 µM lactacystin
(Lactacystin)). In addition, cells were also treated with
tunicamycin (10 µg/ml) as a positive control. In all cases, little or
no protein was detected in the medium. B, MDCK cells grown
overnight in the absence (Control) or presence of proteasome
inhibitors (10 µM MG132 (MG132), 15 µM aLLN (ALLN), 10 µM
lactacystin (Lactacystin)) were fixed and processed for
immunofluorescent localization of the ER chaperone BiP. Examination of
cells revealed little apparent alteration in distribution of this ER
protein following treatment with the inhibitors.
[View Larger Version of this Image (62K GIF file)]
2.5 µM) was similar to that of control cells
(data not shown). Thus, there appears to be a relatively narrow window
for enhancement of thermotolerance by MG132 in MDCK cells, which, in
our experience, are more resistant to environmental stress than many
cell lines. Treatment of cells with other protease inhibitors,
including aLLN, borohydride-reduced MG132, E64, and
1,10-phenanthroline, was found to have little if any effect on the
survival of cells at 46 °C for 4-5 h (Fig. 8). On the contrary,
some of these nonproteasome inhibitors appeared to reduce cell survival
at high temperature (Fig. 8). The fact that treatment of cells with
aLLN had no significant effect on the survival of cells at high
temperatures is probably due to the much lower potency of this drug and
the longer time required for any observable increase in the mRNAs
for the Hsps (Fig. 5). The effects of lactacystin could not be examined
in such experiments as it irreversibly binds to the proteasome and
cannot be washed out from the cells. Thus, of all the inhibitors
tested, only MG132 proteasome inhibition appears to confer
thermotolerance to MDCK cells.
Fig. 8.
Treatment with MG132 confers thermotolerance
to MDCK cells. Graph showing effects of protease inhibitors on the
survival of MDCK cells at 46 °C. Confluent monolayers of MDCK cells
were incubated for 2 h at 37 °C in the absence (control
(column 1)) or presence of either 1 µM MG132
(column 2), 10 µM borohydride-reduced MG132
(column 3), 15 µM aLLN (column 4),
10 µM E64 (column 5), 100 µM
1,10-phenanthroline (column 6), or 10 µM
leupeptin (column 7). Cells were then washed to remove
inhibitors, followed by an additional 3 h of incubation at
37 °C in fresh medium. The cells were then incubated at 46 °C for
4 h. At this time the cells were rinsed in PBS and exposed to
0.1% trypan blue in PBS for 5 min, rinsed, and the number of nonviable
(blue) cells was determined.
[View Larger Version of this Image (37K GIF file)]
, the
specific subunit of RNA polymerase necessary for transcription of
heat-shock genes. The rapid degradation of 32
requires,
as cofactors, the bacterial molecular chaperone, DnaK (the Hsp70
homolog) and its cofactors, DnaJ and GrpE, as well as the
ATP-dependent protease (43). Apparently, the accumulation of unfolded proteins saturates DnaK and DnaJ proteins, and thus prevents degradation of 32
and leads to increased
transcription of Hsps. In eukaryotic cells, a distinct, but related,
mechanism involving molecular chaperones seems to function in
regulating heat-shock proteins (44). The heat-shock factor is normally
associated with Hsp70, which inhibits the expression of heat-shock
genes (45). The buildup of abnormal proteins saturates the chaperone
and competitively blocks this inhibition of heat-shock factor by Hsp70,
leading to expression of heat-shock proteins. The findings presented
here would be consistent with such models and provide a new
experimental system for its analysis.
*
This work was supported by grants from the NIDDK (to
S. K. N.) and the NIGMS (to A. L. G.), National Institutes of
Health, the Human Frontier Program (to A. L. G.), and Proscript, Inc. (to A. L. G.). This work was done during the tenure of an American Heart Association Established Investigatorship (to S. K. N.).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: Renal Division,
Brigham and Women's Hospital, Harvard Medical School, 75 Francis St.,
Boston, MA 02115. Tel.: 617-278-0436 Fax: 617-732-6392; E-mail: sknigam{at}bics.bwh.harvard.edu.
1
The abbreviations used are: ER, endoplasmic
reticulum; MDCK, Madin-Darby canine kidney; BiP, immunoglobulin-binding
protein; Grp94, 94-kDa glucose-regulated protein; ERp72, 72-kDa
endoplasmic reticulum protein; Hsp, heat-shock protein; aLLN,
N-acetyl-leucyl-leucyl-norleucinal; MG132,
carbobenzoxyl-leucinyl-leucinyl-leucinal; MG115,
carbobenzoxyl-leucinyl-leucinyl-norvaline.
2
D. H. Lee and A. L. Goldberg, manuscript in
preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.