Pretreatment with inducers of ER molecular chaperones protects
epithelial cells subjected to ATP depletion
Kevin T.
Bush1,
Sathish K.
George1,
Ping L.
Zhang2, and
Sanjay K.
Nigam1
1 Renal Division, Department of
Medicine, Brigham and Women's Hospital and Harvard Medical School and
2 Department of Pathology, Beth
Israel Hospital and Harvard Medical School, Boston, Massachusetts
02115
 |
ABSTRACT |
We have investigated the potential cytoprotective role of
endoplasmic reticulum (ER) molecular chaperones in a cultured cell model of renal ischemia. Madin-Darby canine kidney (MDCK) cells were pretreated with tunicamycin (an inducer of ER but not cytosolic molecular chaperones) for 12-16 h, followed by 6 h of ATP
depletion. A rapid and severe depletion of cellular ATP was noted in
both control and tunicamycin-treated cells. Trypan blue exclusion
assays indicated that pretreatment of MDCK cells with tunicamycin
reduced ATP depletion-induced cell damage by ~80% compared with
nonpretreated controls. This apparent cytoprotective effect was also
found following pretreatment with another inducer of ER molecular
chaperones (i.e., A23187). For example, A23187 was found to reduce
lactate dehydrogenase release by ~50% compared with untreated
controls, whereas E-64, a cysteine protease inhibitor which may affect
degradation of some proteins in the ER, had little or no effect on cell
injury. Moreover, a fluorescent assay confirmed the marked reduction in cell damage following ATP depletion (up to 80% reduction in
tunicamycin-pretreated cells). Together, these findings are consistent
with the notion that induction of ER molecular chaperones leads to the
acquisition of cytoprotection in the face of ATP depletion. However,
inhibition of protein translation by cycloheximide was found to only
partially attenuate the observed cytoprotective effect, raising the
possibility that other, as yet to be identified, nonprotein
synthesis-dependent mechanisms may also play a role in the observed cytoprotection.
cytoprotection; glucose-regulated proteins; antimycin A; BiP; grp94; ERp72; Hsp70; endoplasmic reticulum
 |
INTRODUCTION |
HEAT-SHOCK PROTEINS (Hsps) were originally identified
as a group of cellular proteins whose transcription was induced
following the exposure of cells to high temperature (14, 38). A variety of cell "stresses" (e.g., hypoxia, alcohol, heavy metals, and anoxia), which appear to cause the accumulation of misfolded or abnormal protein, can also induce the Hsps. Many Hsps, particularly of
the Hsp70 family of proteins, are thought to function as molecular chaperones involved in the normal folding, assembly, and/or degradation of cellular proteins (2, 6, 10, 38). It is this chaperone function of
the Hsps that appears to be important to the cellular response to
stress. Hsps are believed to bind to misfolded or abnormal proteins and
prevent their aggregation, either by rescuing such proteins from
irreversible damage (2, 6, 10, 12, 14, 32, 38) or by increasing their
susceptibility to proteolytic attack (12). Regardless, increased levels
of Hsps in response to stresses that damage and/or denature cellular
protein or perturb protein biosynthetic processes appear to be
important for cell survival.
Although a wealth of data support an important role for cytosolic Hsps,
in particular Hsp70, in cell survival after stress (reviewed in Ref.
32), the role of analogous proteins found in other cellular
compartments, including the endoplasmic reticulum (ER), remains
unclear. In a recent study, whole kidney ischemia and
reperfusion as well as ATP depletion of cultured renal and thyroid
epithelial cells was found to increase not only the expression of
cytosolic Hsps (e.g., Hsp70), but also the expression of ER molecular
chaperones (e.g., BiP, grp94, and ERp72) (17). Because the
accumulation of unfolded or misfolded proteins in the ER is associated
with the induction of the ER molecular chaperones (5, 20, 28) and
because proper folding in the ER requires ATP, one major effect of ATP
depletion, and perhaps whole organ ischemia as well, is likely
to be the perturbation of normal protein processing within the ER
leading to the induction of ER molecular chaperones. Furthermore,
because a number of proteins whose functions are thought to be
disrupted by ischemia (i.e., cell adhesion molecules, intercellular junction proteins, integrins, extracellular matrix proteins) are folded and assembled in the ER and molecular chaperones of the ER are believed to bind nascent polypeptides and catalyze the
folding and/or assembly of proteins transiting this organelle (10, 14,
29, 38), the interesting possibility exists that ER molecular
chaperones could play an important role in both the cellular response
to ischemia and recovery from an ischemic insult.
Therefore, we investigated the possible cytoprotective role of ER
molecular chaperones in protecting cells against damage due to ATP
depletion. Pretreatment of Madin-Darby canine kidney (MDCK) cells with
agents that selectively induce ER molecular chaperones (but do not
affect the expression of cytosolic Hsp70) was found to significantly
increase cell viability [i.e., reduced cell membrane damage
(4)] following ATP depletion.
 |
EXPERIMENTAL PROCEDURES |
Reagents and chemicals.
The cDNA probe for BiP was kindly provided by Dr. Mary-Jane Gething
(Melbourne University). A cDNA probe which contains the entire coding
sequence for a human heat-shock protein of 70 kDa was from ATCC
(Rockville, MD). Tunicamycin, A23187, cycloheximide, and E-64 were from
Calbiochem (La Jolla, CA). Antimycin A was from Sigma (St. Louis, MO).
The lactate dehydrogenase (LDH) cytotoxicity detection kit was from
Boehringer Mannheim (Indianapolis, IN). The fluorescent Live/Dead
viability-cytotoxicity kit was from Molecular Probes (Eugene, OR).
Pretreatment with inducers of ER molecular chaperones.
Confluent monolayers of MDCK cells growing in Dulbecco's modified
Eagle's medium (DMEM) containing 5% heat-inactivated fetal calf serum
(FCS) and 1× concentration of a penicillin-streptomycin mixture were
rinsed twice in Dulbecco's phosphate-buffered saline (DPBS) and then
incubated for 3-16 h in fresh DMEM with or without various
chemical agents (i.e., tunicamycin, A23187, E-64). At the end of this
incubation period, the cells were thoroughly rinsed in DPBS and
incubated for an additional 2-3 h in normal DMEM-5% FCS (i.e.,
"recovery" period).
ATP depletion.
After the so-called 2-3 h recovery period, the pretreated cells
were rinsed twice in DPBS and then incubated for 0-6 h in the
absence or presence of 10 µM antimycin A in DPBS supplemented with
1.5 mM CaCl2 and 2 mM
MgCl2 to deplete cellular ATP
(ATP-depleting conditions). Intracellular ATP
concentrations were determined from separate dishes of cells using a
modified luciferase assay (Sigma).
Northern blot analysis.
Confluent monolayers of MDCK cells growing in
10-cm2 tissue culture dishes were
incubated for 0-16 h in DMEM-5% FCS with or without tunicamycin,
A23187, or E-64. The cells were then rinsed twice in DPBS, scraped into
a microfuge tube, and centrifuged at 4°C for 5 min at 3,000 rpm.
The supernatant was removed, and total RNA was then isolated from the
cell pellet by phenol-chloroform extraction. In the case of Northern
blot analysis following ATP depletion, pretreated and nonpretreated
cells were subjected to 2 h of ATP depletion followed by 4 h of ATP
repletion in normal DMEM, after which total RNA was isolated. The RNA
was electrophoresed on 1% formamide-formaldehyde agarose gels and
transferred to nitrocellulose as previously described (3, 17). The
nitrocellulose blots were then hybridized with random primed
[32P]cDNA for either
BiP or Hsp70 overnight at 42°C, washed with saline-sodium citrate
and exposed to autoradiographic film.
Trypan blue exclusion assay.
After preinduction of ER chaperones and 6 h of ATP depletion as
previously described, confluent monolayers of MDCK cells growing in
12-well tissue culture dishes were carefully rinsed twice in PBS and
exposed for 5 min to 0.1% trypan blue in PBS supplemented with 1.5 mM
CaCl2 and 2 mM
MgCl2. The cells were examined
using an inverted Nikon Diaphot microscope, and the number of viable and nonviable cells was determined.
LDH assay.
Confluent monolayers of MDCK cells growing in 96-well titer plates were
incubated with or without ER chaperone-inducing agents for 12-16 h
and subjected to ATP depletion for 6 h as described. A total of 200 µl of ATP-depleting medium was added to each well. To compare total
cellular LDH with LDH released into the medium due to cell death, 10%
Triton X-100 was added to final concentration of 0.1% to one-half of
the wells before LDH determination. The plate was centrifuged at 1,000 rpm for 10 min at 4°C, and 100 µl of supernatant were removed
from each well to a separate 96-well titer plate. To each well, 100 µl of LDH-colorimetric determination solution (Boehringer-Mannheim)
were added. The plates were incubated in subdued light for 10 min at
room temperature. Determination of LDH content was done on a microplate
reader at 490 nm. The amount of released LDH to total cellular LDH was calculated.
Fluorescent cell death assay.
MDCK cells growing on glass coverslips were incubated for 12-16 h
with the various agents and subjected to 6 h of ATP depletion as
previously described. The cells were rinsed in DPBS and
incubated for 45 min at room temperature in the Live/Dead fluorescent
dyes (Molecular Probes) diluted in PBS supplemented with 1.5 mM
CaCl2 and 2 mM
MgCl2. Calcein-acetoxymethyl
ester, a dye indicating viable cells, is transported across the plasma
membrane in the form of a cleavable ester. The cleavage of the ester
occurs only in viable cells and activates the fluorescence at a
wavelength comparable to that of FITC. Ethidium homodimer, which
indicates nonviable cells, enters cells in which the integrity of the
plasma membrane is lost and binds to nuclear DNA. The DNA binding
activates the fluorescent dye at a wavelength comparable to that of
tetramethylrhodamine B isothiocyanate. Determination of
viable versus nonviable cells was done using a Nikon Labphot microscope
equipped with epifluorescence. The ratio of nonviable cells versus the
total number of cells was determined.
Cycloheximide treatment.
Confluent monolayers of MDCK cells growing in 96-well titer plates were
rinsed in DPBS and incubated for 12-14 h in the absence or
presence of 1 µM tunicamycin and 50 µM cycloheximide. After this
pretreatment period, the cells were rinsed in DPBS and allowed to
recover in fresh DMEM-5% FCS for 2-3 h prior to depletion of ATP
with antimycin A as previously described. Cellular viability (i.e.,
plasma membrane integrity) was measured using the LDH release assay.
 |
RESULTS AND DISCUSSION |
Over the past several years, a number of studies have demonstrated that
overexpression of cytosolic Hsps can protect cells and tissues
subjected to ischemia or ATP depletion. For example, heat shock
treatment and overexpression of Hsps have been correlated with
increased cellular survival following ischemia and reperfusion in whole tissues, including heart, retina, neurons-brain, and kidney
(1, 7, 16, 21, 25, 27, 31, 32). Furthermore, preinduction of Hsp70 by
mild heat shock protects cultured cells from injury due to ATP
depletion by antimycin A (9, 15, 37). In addition, preinduction of both
Hsp70 and ER molecular chaperones with inhibitors of the cytosolic
proteasome protects MDCK cells from thermal injury (2). Although it is
unclear how the overexpression of Hsps protects cells from damage, the
enhanced viability is thought to be due, at least in part, to an
increase in the "chaperoning" capacity of the cells. Thus,
overexpression of Hsps presumably protects the cells by providing more
chaperones involved in the renaturation, refolding, or degradation of
denatured, misfolded, or aggregated proteins, which accumulate during
ischemia and reperfusion, and other kinds of injury (2, 6, 12,
14, 32, 35, 38).
We have recently shown that whole kidney ischemia or ATP
depletion of cultured renal and thyroid epithelial cells induces ER
molecular chaperones (i.e., an ER stress response), in addition to a
cytosolic stress response (17). In cultured cells, messages coding for
the ER molecular chaperones can also be increased by treatment with
agents that cause the accumulation of unfolded proteins in the ER
(i.e., an ER stress response) (2, 5, 18, 20, 28). For example,
treatment of animal cells with tunicamycin, a nucleoside antibiotic
that blocks N-linked glycosylation of secretory and transmembrane
proteins in the ER, can induce the transcription of several ER
molecular chaperones including BiP, grp94, ERp72, and FKBP13 (3, 17).
Thus, as expected, treatment of MDCK cells for 12-16 h with
0.1-10 µM tunicamycin elicited an ER stress response as
indicated by the pronounced rise in the levels of mRNA encoding BiP
(Fig.1), one of many ER chaperones induced
under these conditions. Because a concomitant induction of the
cytosolic Hsps was not observed, we wondered if this agent might prove
useful in evaluating the potential cytoprotective properties of ER
chaperones in the ATP-depletion model for ischemic injury, particularly
as a preliminary study had already raised this possibility (40).

View larger version (70K):
[in this window]
[in a new window]
|
Fig. 1.
Tunicamycin treatment causes selective increases in levels of mRNA
encoding the endoplasmic reticulum (ER) molecular chaperone BiP.
Northern blot analysis of total RNA collected from Madin-Darby canine
kidney (MDCK) cells grown in DMEM-5% FCS for 12-16 h in the
absence (control, lanes 1 and
7) or presence of various
concentrations of tunicamycin (lane 2,
10 µM; lane 3, 1 µM;
lane 4, 0.1 µM;
lane 5, 10 nM; lane
6, 1 nM). Blot was probed with
[32P]cDNA probes for
gene products indicated at left. Ethidium bromide staining of 28s rRNA
was used to indicate that equal amounts of RNA were loaded on the gels.
This figure is representative of at least 3 separate experiments.
|
|
Consequently, mitochondrial oxidative phosphorylation was inhibited in
MDCK cells (both control and preinduced) with antimycin A (17, 35).
Treatment of MDCK cells with 10 µM antimycin A caused a rapid
decrease in the intracellular level of ATP in both control cells and
those that had been treated for 12-16 h with tunicamycin (Fig.
2). Despite the fact that the intracellular levels of ATP for both control and tunicamycin-pretreated groups were
essentially zero for the majority of the experiment, the initial rates
of decline in intracellular ATP levels were somewhat slower in
tunicamycin-pretreated cells (Fig. 2). However, by 60 min the levels
for both groups were
5% of control levels, and by 120 min the levels
of ATP for both groups were below the limit of detection (Fig. 2).
These intracellular levels of ATP remained at this low level for the
entire time that the cells were exposed to antimycin A (i.e., up to the
6-h time point when cell injury was measured). Because it has been
shown that cellular injury and stress response are dependent on the
level and duration of ATP depletion (36), it is possible that the
differences in the initial intracellular ATP decline between the two
groups could have had an effect on the viability of the cells. However,
the finding that intracellular ATP levels of ~50% were found to be the threshold for the initiation of a stress response (36), together
with the fact that the ATP level for each treatment group was
essentially zero for more than 5 h, the relatively minor difference in
the rate of decline would argue against such a possibility. Thus it
seems reasonable to conclude that pretreatment with tunicamycin did not
cause the cells to retain high levels of ATP in the face of long-term
antimycin A treatment and that the observed cytoprotection is due to
its other effect(s), presumably on the ER.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Treatment with antimycin A depletes cellular ATP levels. Graph showing
effects of treatment of MDCK cells with 10 µM antimycin A on the
intracellular level of ATP in control and tunicamycin-pretreated cells.
|
|
Northern blot analysis demonstrated that treatment of MDCK cells with
10 µM antimycin A followed by recovery for 4 h caused both an ER and
a cytosolic stress response as indicated by the marked increases in the
mRNA encoding both Hsp70 and BiP (Fig. 3).
Interestingly, ATP depletion of tunicamycin-pretreated MDCK cells led
to additional increases in the mRNA level of BiP (compared with cells
not pretreated with tunicamycin), but not Hsp70 mRNAs (compare
lanes 2 and
4, Fig. 3). This suggests that the
capacity of the cells to induce ER molecular chaperones is not
maximized by 12-16 h of treatment with tunicamycin and that the
two stresses (tunicamycin and ATP depletion) have additive effects on
the levels of this ER molecular chaperone. These results also confirm
that tunicamycin treatment does not elicit a cytosolic stress response (i.e., induction of the Hsps), nor does pretreatment interfere with the
cell's ability to mount such a response in the face of ATP depletion.

View larger version (87K):
[in this window]
[in a new window]
|
Fig. 3.
ATP depletion causes increases in both BiP and Hsp70 mRNA levels. MDCK
cells grown for 12-16 h in DMEM-5% FCS with
(lanes 3 and
4) or without
(lanes 1 and
2) 1 µM tunicamycin were incubated
for 90 min in the absence (control, lanes
1 and 3) or presence
of 10 µM antimycin A (lanes 2 and
4) followed by 4 h of growth in
fresh normal DMEM. Total RNA was isolated, and Northern blot analysis
was performed by probing blots with
[32P]cDNA probes for
gene products indicated at left. Ethidium bromide staining of 28s rRNA
was used to indicate equivalent loading of RNA on the gels. This figure
is representative of at least 3 separate experiments.
|
|
To determine if preinduction of ER molecular chaperones was associated
with an effect on cell damage, MDCK cells (control and tunicamycin
pretreated) were subjected to ATP depletion and the extent of cellular
injury (i.e., plasma membrane integrity) to the cells was determined
and compared. Using trypan blue exclusion, a well-characterized and
much utilized measure of cell viability (4), we found a significant
decrease in the level of cell injury (~80%) induced by ATP depletion
in MDCK cells pretreated with tunicamycin (Fig.
4), suggesting that ER molecular chaperones preinduced by tunicamycin might play a cytoprotective role in cells
subjected to ATP depletion. This phenomenon was investigated further by
examining the ability of other agents known to induce ER molecular
chaperones (such as the calcium ionophore A23187) to affect MDCK cell
injury following ATP depletion.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 4.
Pretreatment of MDCK cells with tunicamycin reduces cell damage
following ATP depletion. Graph showing effects of pretreatment of MDCK
cells with 2 µM tunicamycin on cell injury. Trypan blue exclusion
assays were performed on MDCK cells pretreated in the absence (control,
1) or presence of 2 µM tunicamycin (2) after 6 h of ATP depletion.
Data are expressed as means ± SE;
n = 8. * P 0.01.
|
|
As with tunicamycin, treatment of MDCK cells for 12-16 h with
A23187 selectively caused an ER stress response as indicated by the
increased transcription of BiP, but not cytosolic Hsp70 (Fig.
5, lane
3). In contrast, the cysteine protease inhibitor E-64, which enters cells and has been reported to interfere with the
degradation of some proteins in the ER (39), did not cause increases in
the mRNA levels of either ER (BiP) or cytosolic (Hsp70) chaperones
(Fig. 5, lane 4), and was used as a
negative control in the experiments. Based on the ability of A23187 to
selectively induce the ER molecular chaperones, we investigated its
potential cytoprotective effects in ATP-depleted MDCK cells. However,
because calcium ionophores also disrupt intracellular calcium
homeostasis, thereby potentially complicating interpretation, the
subsequent experiments were performed in tandem with tunicamycin.

View larger version (93K):
[in this window]
[in a new window]
|
Fig. 5.
Agents that deplete ER calcium stores selectively induce BiP. Northern
blot analysis of total RNA isolated from MDCK cells treated for
12-16 h in the absence (control, lane
1) or presence of either 1 µM tunicamycin
(lane 2), 1 µM A23187
(lane 3), or 100 mM E-64
(lane 4). Blots were probed with
[32P]cDNA probes for
gene products indicated at left. Ethidium bromide staining of 28s rRNA
was used to indicate that equal amounts of RNA were loaded on the gels.
This figure is representative of at least 3 separate experiments.
|
|
Two different measures of cell injury were employed: LDH release and a
fluorescent dye-based assay (see Experimental
Procedures). Cellular LDH is only released from cells
when the plasma membrane is damaged, ultimately leading to cell death.
Thus LDH release (detectable in the growth medium) is a sensitive
measure of cell viability (i.e., plasma membrane damage) (4), and
increases in LDH in the culture medium are indicative of the presence
of dead and/or dying cells. Preinduction of ER molecular chaperones with tunicamycin or A23187 significantly reduced LDH release from ATP-depleted MDCK cells (Fig. 6). For
example, pretreatment with tunicamycin or A23187 caused a reduction in
LDH release approaching 60%, indicating a significant reduction in the
level of cell injury after ATP depletion (Fig. 6). In contrast, E-64
did not induce ER chaperones or alter the level of LDH release (Fig.
6), suggesting that induction of an ER stress response (i.e., induction
of ER molecular chaperones) is necessary for the observed
cytoprotective effect.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Pretreatment with agents that elicit an ER stress response decrease
cell damage caused by ATP depletion. Graph of MDCK cell death as
measured by lactate dehydrogenase (LDH) release assay following 6 h of
ATP depletion in medium containing 10 µM antimycin A. Cells were
pretreated in absence (control) or presence of either 1 µM
tunicamycin, 1 µM A23187, or 100 mM E-64. Data are expressed as means ± SE; n = 8. * P 0.001.
|
|
A fluorescent assay for cell viability provided additional support for
this hypothesis. Microscopic examination of MDCK cells grown on
coverslips showed a marked decrease in the number of nonviable cells in
tunicamycin-pretreated cells (compare Fig. 7, C and
D). Treatment of MDCK cells with
tunicamycin reduced the level of cell injury by almost 80%, compared
with uninduced controls (Fig. 7E).
This reduction in plasma membrane damage following tunicamycin
pretreatment was comparable to that seen using the trypan blue
exclusion assay (compare Figs. 4 and
7E). A23187 also reduced cell death,
although not to the same level as tunicamycin pretreatment (Fig.
7E). However, whereas the calcium
ionophore reduced cell injury by ~60%, E-64 appeared to accelerate
cell injury in this assay (Fig. 7E).

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 7.
Inducers of ER molecular chaperones increase cell survival following
ATP depletion.
A-D:
fluorescent analysis of MDCK cells grown for 12-16 h in absence
(control, A and
C) or presence of 1 µM tunicamycin
(B and
D) followed by 6 h of incubation in
ATP-depletion medium. A and
B: calcein-acetoxymethyl ester
fluorescent analysis of living cells.
C and
D: ethidium homodimer analysis of dead
cells. E: graph of MDCK cell death as
measured by fluorescent Live/Dead (Molecular Probes) kit after 6 h of
ATP depletion. Cells were pretreated in the absence (control) or
presence of either 1 µM tunicamycin, 1 µM A23187, or 100 mM E-64.
Data are expressed as means ± SE;
n = 3. * P 0.05. ** P 0.001.
|
|
Thus all three independent measures of cell injury suggest that agents
that cause a selective induction of ER molecular chaperones protect
cells from damage due to ATP depletion. To strengthen the temporal
association between ER-chaperone induction and cytoprotection, MDCK
cells were pretreated with tunicamycin for varying time periods (3-14 h) prior to being subjected to ATP depletion. The results demonstrate that as little as 3 h of treatment with tunicamycin, which
coincides with the point at which the first demonstrable evidence for
BiP mRNA induction is noted (Fig.
8A,
lane 3), also coincides with the
onset of cytoprotection in the face of ATP depletion (Fig.
8B). Despite increasing levels of
BiP induction with time, near maximal protection is achieved at the 3-h
time point. This may represent a threshold phenomenon where a marginal increase from the constitutive level of BiP production is adequate to
be cytoprotective or may imply that BiP transcription does not
adequately reflect that of the other potential chaperones involved. In
addition, because ATP depletion was performed following a 2- to 3-h
recovery period following treatment with either tunicamycin or A23187,
it is possible that this is a sufficient period of time to
"rev-up" the level of ER chaperones to a cytoprotective level.
Thus the lack of a robust increase of BiP mRNA on the Northern blot
(which was performed immediately following the treatment with either
tunicamycin or A23187) might not be truly indicative of the amount of
chaperone protein at the time of ATP depletion. Nevertheless, the
possibility that other nonchaperone-dependent mechanisms of
cytoprotection are also activated by treatment with agents that induce
ER molecular chaperones cannot be completely excluded.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 8.
Time course of ER chaperone induction correlates with cytoprotection.
A: Northern blot analysis of total RNA
collected from MDCK cells grown in DMEM-5% FCS for 12-16 h in the
absence (control, lane 1) or
presence of 1 µM tunicamycin for 1 h (lane
2), 3 h (lane 3),
6 h (lane 4), and 14 h
(lane 5), respectively. The blot was
probed with a
[32P]cDNA probe for
BiP, and ethidium bromide staining of 28S rRNA was used to indicate the
amounts of RNA that were loaded on the gels. This figure is
representative of 2 experiments. B:
graph of MDCK cell death as measured by LDH release assay after 6 h of
ATP depletion. Cells were treated as previously described before being
ATP depleted and assayed for LDH release. Data are expressed as means ± SE; n = 8. P < 0.004 for all groups vs.
control.
|
|
As stated above, the data demonstrate a correlation between ER
chaperone induction and cytoprotection. However, they do not prove that
the induction of ER chaperones per se directly leads to cytoprotection.
For example, A23187 has other cellular effects (i.e., perturbation of
cellular calcium homeostasis) and the possibility that the decreases in
cell injury were due to something other than overexpression of ER
molecular chaperones remained. Although this seems less likely in the
case of tunicamycin, believed to be a specific inducer of ER
chaperones, we nevertheless examined the effect of cycloheximide
treatment on the acquisition of cytoprotection following pretreatment
with tunicamycin. The premise was that if synthesis of new chaperone
protein (as opposed to a mechanism not involving new synthesis, such as
alteration of cell metabolite levels) mediates the observed
cytoprotection, inhibiting message translation should at least
attenuate the observed cytoprotective effect. Simultaneous treatment of
MDCK cells with 1 µM tunicamycin and 50 µM cycloheximide attenuated
the protective effect of tunicamycin (Fig
9). Pretreatment with tunicamycin alone
reduced the level of LDH release by ~80%, whereas the combination of
tunicamycin and cycloheximide reduced LDH release by ~50%. Although
this suggests that cytoprotection is, at least in part, a
protein-mediated effect, the inability to completely abolish
tunicamycin-induced cytoprotection at this concentration of
cycloheximide raises the possibility that other mechanisms, as yet
unidentified, unrelated to chaperone induction possibly contribute to
the totality of this effect. However, it should be noted that higher
concentrations of cycloheximide, in combination with tunicamycin
treatment and ATP depletion were too toxic and contributed to greatly
increased levels of cell death (data not shown). Thus it is possible
that the concentration of cycloheximide used was inadequate to
completely abolish new protein synthesis. Furthermore, as discussed
below, it is also possible that BiP induction does not accurately
reflect the contribution of other ER chaperones to cytoprotection.
Nevertheless, although the data suggest that multiple mechanisms may be
involved in the acquisition of cytoprotection, it is likely that ER
molecular chaperones play a major role.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 9.
Cycloheximide-induced protein translational arrest attenuates
tunicamycin-mediated cytoprotection. Graph of MDCK cell death as
measured by LDH release after 6 h of ATP depletion. MDCK cells were
pretreated in the absence (1, solid bar, control, no tunicamycin or
cycloheximide; hatched bar, 50 µM cycloheximide) or presence of 1 µM tunicamycin (2, open bar, 1 µM tunicamycin alone without
cycloheximide; hatched bar, 1 µM tunicamycin and 50 µM
cycloheximide) for 12-14 h. Data are expressed as means ± SE;
n = 24. P < 0.001 compared with control.
|
|
Although the data presented suggest that ER chaperone synthesis
mediates at least a component of the observed cytoprotection, especially given the high specificity of tunicamycin, direct
implication of the ER chaperones may require using other protocols to
overexpress the individual chaperones such as BiP (e.g., transgenic
mice or transfected cells) or necessitate using antisense technology to block their synthesis, as has been done for various Hsps (22, 34). It
is important to emphasize that tunicamycin and A23187 induce a variety
of ER luminal proteins (BiP, grp94, ERp72, and FKBP13) with chaperone
function and that it is likely that multiple ER chaperones participate
in the folding of membrane and secreted proteins (2, 3, 5, 17-20,
28). Given their potential overlapping specificities and structural
homologies, it therefore seems quite plausible that affecting the
expression of a single chaperone may not sufficiently affect the
folding environment of the ER (especially in the face of concomitant
high expression of other chaperones) to significantly alter cell
survival as measured here. Nevertheless, it will be important to pursue
some of these approaches, primarily to try to characterize the
individual contribution of each ER chaperone (i.e., BiP, grp94, ERp72,
calreticulin, protein disulfide isomerase, etc.) to the observed
cytoprotective effect against ATP depletion.
The data presented here provide evidence that pretreating cells with
agents that induce ER molecular chaperones results in cytoprotection in
the face of ATP depletion. This is, at least in part, a
protein-mediated effect, and, much like the case of cytosolic Hsps, the
precise mechanism of protection remains to be elucidated. One may
speculate that aside from the likely positive effect on the folding
environment, increased levels of chaperones ameliorate severe cellular
stress by binding potentially toxic or malformed proteins within the
ER, which could potentially be toxic to the cell. Another mechanistic
possibility centers on the demonstrated ability of ER chaperones to
function as calcium-binding proteins (23, 29, 30). As the ER also
serves as a major intracellular reservoir for this cation, which has
been implicated in epithelial cell injury, it is possible that
increased levels of ER chaperones serve as a "sink" for otherwise
toxic calcium concentrations that develop following ATP depletion and
thereby protect the cells from further injury. In support of this
hypothesis, recent studies have shown that ER chaperones play an
important role in enabling the cell to resist oxidative damage by
buffering the subsequent rise in intracellular calcium (24). This
feature may also be instrumental in stabilizing the ER calcium pool,
preventing apoptosis from being triggered (13) and thereby bestowing
the cell with a marked survival benefit.
Given our postulate, what physiological role might ER molecular
chaperones play in the setting of ischemic injury and recovery? Epithelial cells establish and maintain their characteristic phenotype, at least in part, through the action of a number of specialized cellular structures and processes (i.e., adherens and tight junctions, and cell-cell substratum attachments). ATP depletion or
ischemia has been shown to disrupt these structures and
processes, leading to loss of the permeability barrier and
apical-basolateral polarity, as well as impaired cell-substratum
interactions (8, 11, 26). Because the folding and assembly of the
integral membrane and secreted proteins that comprise these structures
occur in the ER and are mediated by ER molecular chaperones, the
ability to recover from ischemic insults would be expected to depend on the ability of the cell to replace or repair these proteins. Thus preinduction of the ER molecular chaperones could enhance cell survival
by increasing the chaperoning capacity of the ER, for example, for
junctional proteins, cell adhesion molecules, and integrins, thereby
increasing the rate at which that recovery of cell function occurs.
Strategies to induce ER chaperones could conceivably be useful in
clinical settings, either as preemptive measures or to enhance recovery
after injury.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a National Institute of Diabetes
and Digestive and Kidney Diseases Grant to S. K. Nigam and by a
National Research Service Award to S. K. George. K. T. Bush is
supported by an American Heart Association Scientist Development Grant.
 |
FOOTNOTES |
This work was done during tenure as an Established Investigator of the
American Heart Association (S. K. Nigam).
Address for reprint requests and other correspondence: S. K. Nigam,
Renal Division, Brigham and Women's Hospital, Harvard Medical School,
77 Ave. Louis Pasteur, Boston, MA 02115 (E-mail:
snigam{at}rics.bwh.harvard.edu).
Received 5 March 1997; accepted in final form 12 April 1999.
 |
REFERENCES |
1.
Barbe, M.,
M. Tytell,
D. Gower,
and
W. Welch.
Hyperthermia protects against light damage in the rat retina.
Science
241:
1817-1820,
1988[Medline].
2.
Bush, K. T.,
A. L. Goldberg,
and
S. K. Nigam.
Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance.
J. Biol. Chem.
272:
9086-9092,
1997[Abstract/Free Full Text].
3.
Bush, K. T.,
B. A. Hendrickson,
and
S. K. Nigam.
Induction of the FK506-binding protein, FKBP13, under conditions which misfold proteins in the endoplasmic reticulum
Biochem. J.
303:
705-708,
1994[Medline]. (Erratum, Biochem J. 305: 1031, 1995).
4.
Cook, J. A.,
and
J. B. Mitchell.
Viability measurements in mammalian cell systems.
Anal. Biochem.
179:
1-7,
1989[Medline].
5.
Cox, J.,
C. Shamu,
and
P. Walter.
Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase.
Cell
73:
1197-1206,
1993[Medline].
6.
Craig, E.,
J. Weissman,
and
A. Horwich.
Heat shock proteins and molecular chaperones: mediators of protein conformation and turnover in the cell.
Cell
78:
365-372,
1994[Medline].
7.
Emami, A.,
J. Schwartz,
and
S. Borkan.
Transient ischemia or heat stress induces a cytoprotectant protein in the rat kidney.
Am. J. Physiol.
260 (Renal Fluid Electrolyte Physiol. 29):
F479-F485,
1991[Abstract/Free Full Text].
8.
Fish, E. M.,
and
B. A. Molitoris.
Alterations in epithelial polarity and the pathogenesis of disease states.
N. Engl. J. Med.
330:
1580-1587,
1994[Free Full Text].
9.
Gabai, V.,
and
A. Kabakov.
Rise in heat-shock protein level confers thermotolerance to energy deprivation.
FEBS Lett.
327:
247-250,
1993[Medline].
10.
Gething, M. J.,
and
J. Sambrook.
Protein folding in the cell.
Nature
355:
33-45,
1992[Medline].
11.
Goligorsky, M.,
W. Lieberthal,
L. Racusen,
and
E. Simon.
Integrin receptors in renal tubular epithelium: new insights into pathophysiology of acute renal failure.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F1-F8,
1993[Abstract/Free Full Text].
12.
Hayes, S.,
and
J. Dice.
Role of molecular chaperones in protein degradation.
J. Cell Biol.
132:
255-258,
1996[Medline].
13.
He, H.,
M. Lam,
T. S. McCormick,
and
C. W. Distelhorst.
Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2.
J. Cell Biol.
138:
1219-1228,
1997[Abstract/Free Full Text].
14.
Hightower, L.
Heat shock, stress proteins and proteotoxicity.
Cell
66:
191-197,
1991[Medline].
15.
Kabakov, A. E.,
and
V. L. Gabai.
Heat shock-induced accumulation of 70-kDa stress protein (HSP70) can protect ATP-depleted tumor cells from necrosis.
Exp. Cell Res.
217:
15-21,
1995[Medline].
16.
Knowlton, A. A.
The role of heat shock proteins in the heart.
J. Mol. Cell. Cardiol.
27:
121-131,
1995[Medline].
17.
Kuznetsov, G.,
K. Bush,
P. Zhang,
and
S. Nigam.
Pertubations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.
Proc. Natl. Acad. Sci. USA
93:
8584-8589,
1996[Abstract/Free Full Text].
18.
Kuznetsov, G.,
L. Chen,
and
S. Nigam.
Multiple molecular chaperones complex with misfolded large oligomeric glycoproteins in the endoplasmic reticulum.
J. Biol. Chem.
272:
3057-3062,
1997[Abstract/Free Full Text].
19.
Kuznetsov, G.,
L. B. Chen,
and
S. K. Nigam.
Several endoplasmic reticulum stress proteins, including ERp72, interact with thyroglobulin during its maturation.
J. Biol. Chem.
269:
22990-22995,
1994[Abstract/Free Full Text].
20.
Kuznetsov, G.,
and
S. K. Nigam.
Folding of secretory and membrane proteins.
N. Engl. J. Med.
339:
1688-1695,
1998[Free Full Text].
21.
Li, Y.,
M. Chopp,
Z. G. Zhang,
R. L. Zhang,
and
J. H. Garcia.
Neuronal survival is associated with 72-kDa heat shock protein expression after transient middle cerebral artery occlusion in the rat.
J. Neurol. Sci.
120:
187-194,
1993[Medline].
22.
Li, Y.,
and
R. A. Kloner.
Does protein kinase C play a role in ischemic preconditioning in rat heart?
Am. J. Physiol.
268 (Heart Circ. Physiol. 37):
H426-H431,
1995[Abstract/Free Full Text].
23.
Lievremont, J. P.,
R. Rizzuto,
L. Hendershot,
and
J. Meldolesi.
BiP, a major chaperone protein of the endoplasmic reticulum lumen, plays a direct and important role in the storage of the rapidly exchanging pool of Ca2+.
J. Biol. Chem.
272:
30873-30879,
1997[Abstract/Free Full Text].
24.
Liu, H.,
E. Miller,
B. van de Water,
and
J. L. Stevens.
Endoplasmic reticulum stress proteins block oxidant-induced Ca2+ increases and cell death.
J. Biol. Chem.
273:
12858-12862,
1998[Abstract/Free Full Text].
25.
Mailhos, C.,
M. K. Howard,
and
D. S. Latchman.
Heat shock protects neuronal cells from programmed cell death by apoptosis.
Neuroscience
55:
621-627,
1993[Medline].
26.
Mandel, L. J.,
R. Bacallao,
and
G. Zampighi.
Uncoupling of the molecular "fence" and paracellular "gate" functions in epithelial tight junctions.
Nature
361:
552-555,
1993[Medline].
27.
Mestril, R.,
and
W. H. Dillmann.
Heat shock proteins and protection against myocardial ischemia.
J. Mol. Cell. Cardiol.
27:
45-52,
1995[Medline].
28.
Mori, K.,
W. Ma,
M. J. Gething,
and
J. Sambrook.
A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus.
Cell
74:
743-756,
1993[Medline].
29.
Nigam, S. K.,
A. L. Goldberg,
S. Ho,
M. F. Rohde,
K. T. Bush,
and
M. Sherman.
A set of endoplasmic reticulum proteins possessing properties of molecular chaperones includes Ca(2+)-binding proteins and members of the thioredoxin superfamily.
J. Biol. Chem.
269:
1744-1749,
1994[Abstract/Free Full Text].
30.
Nigam, S. K.,
and
T. Towers.
Subcellular distribution of calcium-binding proteins and a calcium-ATPase in canine pancreas
J. Cell Biol.
111:
197-200,
1990[Abstract]. (Erratum, J. Cell Biol. 111: 1726, 1990).
31.
Perdrizet, G. A.,
H. Kaneko,
T. M. Buckley,
M. A. Fishman,
and
R. T. Schweizer.
Heat shock protects pig kidneys against warm ischemic injury.
Transplant. Proc.
22:
460-461,
1990[Medline].
32.
Plumier, J. C.,
and
R. W. Currie.
Heat shock-induced myocardial protection against ischemic injury: a role for Hsp70?
Cell Stress Chaperones
1:
13-17,
1996.[Medline]
34.
Steinhoff, U.,
U. Zugel,
A. Wand-Wurttenberger,
H. Hengel,
R. Rosch,
M. E. Munk,
and
S. H. Kaufmann.
Prevention of autoimmune lysis by T cells with specificity for a heat shock protein by antisense oligonucleotide treatment.
Proc. Natl. Acad. Sci. USA
91:
5085-5088,
1994[Abstract].
35.
Tsukamoto, T.,
and
S. K. Nigam.
Tight junction proteins form large complexes and associate with the cytoskeleton in an ATP depletion model for reversible junction assembly.
J. Biol. Chem.
272:
16133-16139,
1997[Abstract/Free Full Text].
36.
Van Why, S. K.,
A. S. Mann,
G. Thulin,
X. H. Zhu,
M. Kashgarian,
and
N. J. Siegel.
Activation of heat-shock transcription factor by graded reductions in renal ATP, in vivo, in the rat.
J. Clin. Invest.
94:
1518-1523,
1994[Medline].
37.
Wang, Y. H.,
and
S. C. Borkan.
Prior heat stress enhances survival of renal epithelial cells after ATP depletion.
Am. J. Physiol.
270 (Renal Physiol. 39):
F1057-F1065,
1996[Abstract/Free Full Text].
38.
Welch, W.
Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease.
Physiol. Rev.
72:
1063-1081,
1992[Free Full Text].
39.
Wileman, T.,
L. P. Kane,
and
C. Terhorst.
Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.
Cell Regul.
2:
753-765,
1991[Medline].
40.
Zhang, P. L.,
K. T. Bush,
and
S. K. Nigam.
Involvement of endoplasmic reticulum molecular chaperones (stress proteins) in recovery from epithelial ischemia (Abstract).
J. Am. Soc. Nephrol.
6:
993,
1995.
Am J Physiol Renal Physiol 277(2):F211-F218
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society