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,¶
*
Research Center and
Nephrology Service, Notre-Dame Hospital, Center hospitalier universitaire de l’Université de Montréal, University of Montreal, Montreal, Canada;
Montreal General Hospital, Montreal, Canada;
Institute of Molecular Virology, St. Louis University Medical Center, St. Louis, MO 63110; and
¶ Department of Surgery, McGill University, Montreal, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
, and c-Jun proteins, occurs via the proteasome pathway
(4, 5, 6, 7, 8, 9, 10, 11); transacting NF-B matures after cotranslational
processing of its precursor peptide by proteasomes (12).
Selective ubiquitination of proteins is an important mechanism
controlling the discriminative nature of protein degradation via
proteasomes (1). Apoptosis is an essential cellular event in organ development and tissue remodeling. In the immune system, apoptosis is required for positive and negative selection of T and B cells, and for maintaining homeostasis of peripheral lymphocytes. It is also a mechanism for lymphocytes to kill their target cells. Abnormal apoptosis could result in pathological conditions such as autoimmune diseases and failure to terminate infectious diseases, whereas properly induced apoptosis might be useful in controlling undesirable immune responses. Because proteasomes degrade 70–90% of cellular proteins (3), it is conceivable that many cellular events, including apoptosis, are directly or indirectly controlled by them. A specific proteasome inhibitor, lactacystin (LAC),6 can repress three major peptidase activities of proteasomes (i.e., chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide-hydrolyzing activities), but does not affect other proteases, such as calpain, cathepsin B, chymotrypsin, trypsin, and papain (13). By using this inhibitor, we have demonstrated previously that proteasome inhibition results in apoptosis of cycling T cells (14).
In this study, we further investigated the underlying mechanisms in Jurkat T cells (JC) and Namalwa B cells (NC). Our results suggest that proteasomes play a critical role in maintaining the balance between pro- and anti-apoptotic factors and in maintaining proper functions of the mitochondria (Mito). These are critical events that decide the fate of cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640, FCS, penicillin-streptomycin, and
L-glutamine were purchased from Life Technologies
(Burlington, Ontario, Canada). LAC was obtained from Dr. E. J.
Corey (Ref. 13; Harvard University, Boston, MA). The caspase inhibitor
benzyloxycarbonyl-Val-Ala-Asp(Ome) fluoromethyl ketone (zVAD.fmk) was
purchased from Enzyme Systems Products (Livermore, CA). The fluorogenic
caspase substrate Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin was
purchased from Bachem Bioscience (King of Prussia, PA). Mouse mAb
(clone 7H812C12) against cytochrome c (CytC) was obtained
from BD PharMingen (San Diego, CA). Rabbit Abs against Bax, Bak, and
Bad, normal rabbit IgG, and goat Ab against Bik were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Swine anti-goat Ab was
obtained from Caltag (San Francisco, CA). Mouse mAb against
hemagglutinin (HA) and rabbit Ab against poly(ADP-ribose) polymerase
(PARP) were purchased from Roche Diagnostics (Laval, Quebec). Digitonin
(Dig), succinate (Suc), antimycin A (Anti A),
tetramethyl-p-phenylenediamine (TMPD), ascorbate (Asc),
carbonyl cyanide m-chlorophenylhydrazone (CCCP), carbonyl
p-trifluoromethoxyphenylhydrazone (FCCP), and camptothecin
(Campt) were obtained from Sigma (Oakville, Ontario, Canada).
5,5',6,6'-Tetrachloro-1,1'3,3'-tetraethyl-benzimidazolocarbocyanine
iodide (JC-1) was purchased from Molecular Probes (Eugene, OR). ECL
kits and protein G-agarose were obtained from Amersham (Oakville,
Ontario, Canada). [-32P]dCTP (3000
µCi/mmol), Tran-35S-label (1175 Ci/mmol), and
Biotrans nylon membranes were obtained from ICN Pharmaceuticals
(Mississauga, Ontario, Canada).
Cell culture
JC and NC were cultured in RPMI 1640 supplemented with 10% FCS, L-glutamine, and antibiotics as described elsewhere (15).
Electron microscopy
JC were examined by electron microscopy as described by Tsao and Duguid (16).
DNA laddering assay
The assay was performed according to a protocol by Liu et al. (17) with some modifications (14).
The DNA filter elution assay
JC or NC were labeled with [14C]thymidine (0.02 µCi/ml)for 24 h and chased in isotope-free medium overnight before drug treatment. After drug treatment, the samples were processed as described earlier (18). DNA fragmentation was determined by liquid scintillation as the fraction of fragmented DNA relative to total DNA. Background DNA fragmentation (in untreated cells) was deducted in the final results presented according to the formula: [(F - F0)/(1 - F0)] x 100, where F and F0 represent DNA fragmentation in treated and untreated cells, respectively.
Caspase 3-like activity (DEVDase) assay
JC were washed twice with ice-cold PBS and lysed at 4°C at a density of 1.0 x 107 cells/ml in lysis buffer containing 10 mM HEPES, 20 mM NaCl, 80 mM KCl, 5 mM MgCl2, 5 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 0.15 U/ml aprotinin, 10% glycerol, and 0.1% Nonidet P-40, pH 7.4. After incubation at 4°C for 10 min with gentle agitation, the samples were centrifuged at 4°C at 10,000 x g for 10 min to remove cell debris. The supernatants were used as cytosolic extracts. DEVDase activities in the cytosolic extracts were measured by kinetic assay in which fluorescence generated by DEVDase-specific fluorogenic substrates was monitored continuously at 37°C (19).
Preparation of Mito
A protocol adapted from Krippner et al. (20) with some modifications was used to prepare Mito from JC and NC. One hundred million cells were resuspended in a buffer containing 300 mM sucrose, 1 mM EGTA, 20 mM MOPS, and 0.1% BSA at pH 7.4. Dig was added to give a final concentration of 0.01% (w/v), and the samples were incubated on ice for 10 min. All of the following steps were conducted at 4°C. The cell suspensions were centrifuged at 10,000 x g for 10 min. This step transformed them into the form of solid pellets so that the cells could be broken more easily by homogenization. The pellets were homogenized by 20 strokes with a homogenizer (Wheaton, Wheaton Millville, NJ), and the resulting suspensions were centrifuged at 2,500 x g for 15 min to remove unbroken cells and cellular debris. The supernatants were further centrifuged at 10,000 x g for 30 min. The pellets from these centrifugations represented mitochondrial fractions, and the supernatants were used as cytosolic fractions. The mitochondrial but not the cytosolic fraction thus prepared could respire (data not shown). In some experiments, the mitochondrial fractions were lysed by a buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 50 mM NaCl, and 1% Nonidet P-40 and centrifuged at 10,000 x g for 15 min to pellet the debris. The supernatants were used as sources of mitochondrial protein in the immunoblotting.
Mito from rat kidney proximal tubules (RKM) served as positive controls for normal respiratory functions. They were isolated by differential centrifugation after homogenization in a buffer containing 250 mM sucrose, 10 mM HEPES-Tris (pH 7.5), and 250 µM EDTA. Cell debris was removed by centrifugation at 2,500 x g for 15 min, and the Mito were precipitated by centrifugation at 10,000 x g for 30 min. They were finally washed with the same buffer in the absence of EDTA. Protein concentrations of the mitochondrial suspensions were measured after solubilizing the Mito in 0.1% SDS.
The purity of the mitochondrial and cytosolic fractions was determined by the activities of glutamate dehydrogenase and lactate dehydrogenase in each fraction. Contamination of cytosolic protein in the mitochondrial fraction was <35%, whereas contamination of mitochondrial protein in the cytosolic fraction was <15% as described previously (21).
Immunoblotting
Immunoblotting was used to evaluate PARP cleavage and the protein levels of Bik, Bax, Bak, Bad, Bcl-xL, and CytC. The general protocol can be found in our previous publication (14). For PARP, Bax, Bak, and Bad detection, the membranes were hybridized with specific rabbit Abs at dilutions suggested by the manufacturers. For CytC and HA-Bcl-xL detection, the membranes were hybridized with mouse mAb against pigeon CytC or mouse mAb against the HA tag, respectively. The signals on the membranes were detected by ECL. For the detection of Bik, goat anti-Bik Ab was used as the first Ab in immunoblotting. The membranes were then either hybridized with a swine anti-goat Ab followed by [125I]-protein A or ECL for signal detection.
Northern blot analysis
The method is described in our previous publication (22). Briefly, total cellular RNA of JC and NC was extracted with the guanidine/CsCl method and used in the Northern blots. A 402-bp Bik cDNA lacking the transmembrane domain was labeled by 32P with random primers and was served as a probe. Bik cDNA has been described in our previous publication (23).
Metabolic labeling of Bik
JC were labeled with Tran-35S (1,175 Ci/mmol, 0.9 mCi/20 x 106 cells/5 ml; ICN Pharmaceuticals) for 20 min, then cultured in normal RPMI 1640 medium with 10% normal FCS in the absence or presence of 8 µM LAC for 6 h. The cells of each treatment (20 x 106 cells/treatment) were lysed with 0.5 ml of lysis buffer in the presence of protease inhibitors (24) for 1 h on ice. After spinning, the supernatants (0.7 mg protein/treatment) were precleared for 1 h at 4°C with 10 µl of normal goat serum and 20 µl of protein G-agarose. The supernatants were then incubated with goat anti-Bik Ab overnight at 4°C. Immune complexes were precipitated with 50 µl of protein G-agarose, resolved in 4–20% gradient SDS-PAGE, and transferred to polyvinylidene difluoride. Signals were visualized by autoradiography, and band intensities were determined by densitometry.
Immunoprecipitation
Bcl-xL-transfected NC were lysed in 1% Brij lysis buffer containing proteinase inhibitors as described previously (24). The cleared lysate (400 µg protein/sample) was incubated with mouse anti-HA mAb (10 µg) on ice for 1 h. To recover the immune complexes formed, 5 µg of rabbit anti-mouse IgG and 30 µl of Pansorbin (formalin-fixed Staphylococcus aureus Cowan I) were added to the lysate, and the samples were kept at 4°C with rotation for 2 h. Pansorbin was washed four times with PBS, and the immune complexes were eluted with an SDS-PAGE sample buffer followed by electrophoresis on 7.5% SDS-PAGE. Proteins were then transferred onto polyvinylidene difluoride membranes and blotted sequentially with goat anti-Bik Ab and a mAb against the HA tag.
Transient transfection
JC and NC were transfected by electroporation in serum-free RPMI 1640 medium. Ten million cells/0.8 ml medium/10 µg plasmid/cuvette were electroporated at room temperature. The electroporation settings were 270 V/960 µF for NC and 360 V/960 µF for JC.
Measurement of mitochondrial membrane potential
(
m)
m was measured with JC-1
(25). JC-1 (0.1 µM) uptake by Mito in live cells
(Dig-permeabilized JC and NC) as well as by purified Mito from rat
kidney, JC, and NC at 37°C was monitored continuously with a
spectrofluorometer (Photon Technology International, South Brunswick,
NJ). The excitation wavelength was 490 nm (slit width, 2 nm) and the
emission wavelength was 590 nm (slit width, 4 nm). The signals were
recorded by Felix (Version 1.1) software (Photo Technology
International, South Brunswick, NJ). The incubation buffer, substrates,
and inhibitors for measurement of m were
identical with those used in the respiration assays (as described
below). For each determination, 0.5 x 106
cells or purified Mito equivalent to 50 µg of protein from JC and NC
were used.
Respiration assay
Electron transport in Mito was assessed according to oxygen consumption during respiration. JC (30 x 106 cells/ml) or RKM (0.5 mg protein/ml) were incubated in a 1-ml thermojacketed chamber at 37°C in respiration buffer (200 mM sucrose, 5 mM MgCl2, 5 mM KH2PO4, and 30 mM HEPES-Tris, pH 7.5). Various reagents (0.005% Dig, 10 mM Suc, 1 mM Asc, 0.4 mM TMPD, 1 µM CCCP, 1 µM FCCP, 0.1 µM rotenone, 50 nM antimycin A, 1 mM KCN, and 100 µM CytC) were added during the assay. The respiration rate was measured polarographically with a Clarke oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH). Oxygen concentration was calibrated with air-saturated buffer in a hypoxanthine-xanthine oxidase/catalase system (chemical zero). Oxygen consumption was registered continuously by a MacLab/8 multichannel recorder (Worx, Dover, NH) connected to a Macintosh SE computer (Apple Computer, Cupertino, CA) and was analyzed with MacLab Chart software (version 3.3.4; Worx). The oxygen consumption rates are expressed as nanograms of atoms of oxygen per minute.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown previously that proteasome inhibition results in the
death of cycling T cells (14). The mode of cell death
induced by LAC was investigated here in fast-growing JC. According to
electron microscopy (Fig. 1
, A
and B), there was apparent nuclear condensation in JC
treated with LAC at 6 µM for 24 h. DNA laddering in the cells
could be detected as early as 6 h after LAC treatment (Fig. 1C). Thus, blocking proteasome activity induces apoptosis in
these cells.
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C). Moreover, from 2 µM and up, LAC further enhanced the
apoptosis induced by PHA. Because the LAC concentrations used here
covered the full spectrum from complete inhibition to no inhibition of
proteasome activity according to our previous study (29),
we conclude that proteasome inhibition only promotes apoptosis but has
no antiapoptotic effect in our model.
To assess more quantitatively the apoptosis induced by proteasome
inhibition, we used a DNA filter elution assay.
[14C]Thymidine-labeled JC were treated with LAC
at different concentrations (from 0.75 to 10.0 µM) for 6 or 24
h, and DNA fragmentation was measured according to small DNA fragments
released from the nuclei. The results showed that the DNA fragmentation
of JC increased dose- and time-dependently after LAC treatment (Fig. 1D). Twenty-four-hour treatment at concentrations above 6
µM caused 
90% DNA fragmentation.
DEVDase is activated in apoptosis induced after proteasome inhibition
In most known pathways of apoptosis, various caspases are
implicated in the initiation or execution phases. Is this also the case
in apoptosis induced by proteasome inhibition? A broad-spectrum caspase
inhibitor, zVAD.fmk, could effectively inhibit LAC-induced DNA
fragmentation in JC (Fig. 2A).
Inhibition was dose-dependent, whereas zVAD.fmk by itself had no
effect. This result shows that caspase activation is required in
LAC-induced DNA fragmentation.
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B) after LAC treatment. The 24-kDa band was a
characteristic product after DEVDase activation (32) and
appeared as early as 5 h after the blockage of proteasome
activity. An enzyme assay with a fluorogenic substrate specific for
DEVDase was used to further confirm DEVDase activation ex vivo. As
shown in Fig. 2C, DEVDase was indeed augmented in JC after
5 h of proteasome inhibition. Taken together, the above results
show DEVDase activation in these cells after proteasome inhibition. Proteasome inhibition results in accumulation of Bik protein but not several other pro-apoptotic members of the Bcl-2 family
The function of proteasomes is to degrade proteins, and logically,
a simple mechanism by which a proteasome inhibitor induces apoptosis
could be to differentially block the degradation of proapoptotic
factors and thus shift the balance between antiapoptotic and
proapoptotic factors in favor of the latter, hence apoptosis. This
mechanism is applicable to members of the Bcl-2 family, some of which
are antiapoptotic, and the others are proapoptotic (33).
Therefore, we studied the effect of LAC on the protein levels of
several proapoptotic Bcl-2 family members. As shown in Fig. 3A, Bik was present mainly in
Mito and was hardly detectable in the cytosol of JC. After 4–6 h of
LAC treatment, the Bik level increased 70–100% in the mitochondrial
fraction (Fig. 3, A and B), and became detectable
in the cytosol (Fig. 3A). Several other proapoptotic members
such as Bax, Bak, and Bad were also present in the mitochondrial
fraction, but their levels remained unchanged in this fraction (Fig. 3B) as well as in the cytosolic fraction (data not shown)
after LAC treatment.
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, A and B).
Like LAC, a protein kinase inhibitor, staurosporine (STS), could also
induce apoptosis in JC and NC (data not shown), but it did not augment
Bik levels (STS). The membranes in Figs. 3 and 4 were probed more than
once with different Abs to rule out artifacts caused by uneven protein
loading. Even protein loading was also assured by bands recognized by
anti-cytochrome oxygengase IV appearing in the immunoblots (Fig. 4, bottom).
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A). This result suggests that
the accumulation of Bik protein after LAC treatment is unlikely
attributable to enhanced protein synthesis. We further confirmed with
metabolic labeling that LAC inhibited Bik protein turnover (Figs. 5B), and this was quantified by densitometry (Fig. 5C). Thus, the accumulation of Bik was due to its reduced
degradation.
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One way to test our hypothesis that the imbalance between Bik and antiapoptotic Bcl-2 family members results in apoptosis was to ascertain whether overexpression of antiapoptotic factors would overcome the effect of LAC and tip the balance in favor of cell survival. Thus, NC were stably transfected with pCEP4-HA-Bcl-xL to express Bcl-xL fused with a HA tag sequence (19).
We then tested whether Bcl-xL-transfected NC were resistant
to apoptosis induced by LAC. Wild-type NC underwent DNA fragmentation
when treated with a DNA topoisomerase I inhibitor Campt (1 µM) or LAC
(1.5 µM) for 24 h, according to a DNA laddering assay (Fig. 6A), and the degree of
laddering was reduced in Bcl-xL-transfected cells (Fig. 6B). The protective role of Bcl-xL in this model
was assayed quantitatively by DNA filter elution assay. Wild-type and
Bcl-xL-transfected NC were treated with LAC of different
concentrations (0.75–10 µM) for 0–96 h. Under all the conditions
tested, Bcl-xL-transfected cells had significantly reduced
DNA fragmentation (Fig. 6C). This indicates that
Bcl-xL overexpression protects these cells from apoptosis
induced by proteasome inhibition, although the protection is not
complete.
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D, the immune complexes precipitated by anti-HA, which
binds tags in overexpressed exogenous Bcl-xL, contained Bik
signals. The same membrane when hybridized with anti-HA mAb
revealed Bcl-xL signals, indicating that exogenous
Bcl-xL was indeed expressed in the transfected NC. This
result suggests that Bik and Bcl-xL interact with each
other in these cells. Expression of exogenous Bik leads to apoptosis of lymphocytes
Does augmentation of the Bik level, as seen after proteasome
inhibition, suffice for the induction of apoptosis in lymphocytes? To
answer this question, we transiently transfected NC and JC with a Bik
expression construct pCDNA3-HA-Bik, which has been described in a
previous publication (23), and apoptosis of these cells
underwent DNA filter elution assay at 24, 48, and 72 h after
transfection. As shown in Fig. 7, electroporation caused 15–20% and 45% DNA fragmentation,
respectively, in NC and JC transfected with empty vectors (blank
columns). Above such a background, the Bik construct-transfected NC and
JC (dotted columns, first and last panels) had
significantly augmented DNA fragmentation, which was more pronounced
and occurred earlier in JC. This indicates that augmentation of Bik
expression alone is indeed sufficient to cause apoptosis in
lymphocytes. An additional experiment showed that exogenous Bik
expression in Bcl-xL-protected cells did not lead to
augmented apoptosis (Fig. 7, middle), indicating that the
apoptosis induced by pCDNA3-HA-Bik transfection was not attributable to
nonspecific cytotoxicity of the plasmid.
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m after proteasome inhibition and its
prevention by Bcl-xL
Because Bik is mainly located on the outer membrane of Mito, these
organelles are then implicated in apoptosis induction. This notion is
supported by observations from other studies (reviewed in Ref.
34). Cells undergoing apoptosis show an early
reduction of m. Permanent
collapse of m marks a point-of-no-return
during apoptosis induction in most cases, although it is not known
whether m collapse per se is responsible
for triggering apoptosis.
To evaluate the involvement of Mito in LAC-induced apoptosis, we used
JC-1 fluorometric assay to measure m driven
by Suc. In the presence of Suc as a substrate, the generation of
m is maximal and depends only on electron
transport chain function and permeability of the inner mitochondrial
membrane, and the function of the electron transport chain is not
limited by the availability of and/or access to endogenous substrates
in permeabilized cells. Under such an extreme condition, potential
defects were easy to be identified. Moreover, this experimental
condition was identical with those of the respiration studies, and the
results of m and respiration could thus be
compared.
In untreated JC permeabilized by Dig, the addition of Suc led to JC-1
accumulation, which reached the maximum at 400 s (Fig. 8A, control (Cont)). JC-1
accumulation under this condition was due to generation of
m, as inhibition of the respiratory chain
by Anti A and uncoupler FCCP, which are known to dissipate
m, resulted in decreased JC-1 fluorescence.
Fig. 8A shows that treatment of JC with 6 µM LAC for 2, 4,
or 8 h progressively diminished the capacity of Mito to generate
m in permeabilized cells.
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B
shows the significant reduction of m
generation by Mito purified from LAC-treated JC. The
m of Mito purified from STS-treated cells
was used as a positive control and was also diminished dramatically
(Fig. 8B). Due to the reduced diffusion time of purified
Mito, their responses to Anti A and FCCP were significantly faster than
those of whole cells, yet both experimental approaches produced
comparable results.
A similar compromise in m generation
was also found in Mito from NC. m
generation was significantly reduced in Mito purified from wild-type NC
treated for 6 h with LAC or STS (Fig. 8C). In contrast,
Mito isolated from Bcl-xL-transfected NC treated with
either LAC or STS were able to generate m
similar to that of untreated cells (Fig. 8D). Thus,
Bcl-xL overexpression protects NC from diminished
m generation during LAC-induced
apoptosis.
In a separate experiment, we found that the caspase inhibitor zVAD.fmk
could not prevent the LAC-induced compromise of
m (data not shown). This indicates that
this mitochondrial event is upstream of caspase activation.
The electron transport chain is defective after complex III following proteasome inhibition, and the defect can be reversed by exogenous CytC
Generation of m primarily depends on
efficient function of the electron transport chain and the
integrity of the inner mitochondrial membrane. Thus, we next
asked whether there is a functional defect in the electron transport
chain in Mito of LAC-treated JC.
The function of the electron transport chain was studied by respiration
assay (Fig. 9) with Dig-permeabilized JC
under conditions similar to those of the membrane potential assay (Fig. 8A). In these experiments, purified RKM were used as a
control. Respiration of RKM on endogenous substrates was blocked by
rotenone (inhibitor of NADH-dehydrogenase or complex I), resumed by Suc
(substrate of Suc-dehydrogenase or complex II), and reached its maximum
after dissipation of the m by CCCP (Fig. 9A). Respiration of Mito in Dig-permeabilized JC with or
without LAC treatment for 2 h was similar to that of RKM (Fig. 9A). However, the treatment of JC with LAC for 4 h
(LAC, Fig. 9B) significantly diminished Suc-driven
respiration and prevented additional respiration stimulated by CCCP,
when compared with the respiration of untreated JC (Cont). The addition
of purified normal RKM into this experiment resumed oxygen consumption,
indicating that the assay system was functional. These results suggest
defective function of the mitochondrial electron transport chain
somewhere between complex II and IV.
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C). The addition of CCCP further accelerated electron flow
(Fig. 9C). The respiration of Mito from Dig-permeabilized JC
with or without LAC treatment for 2 h (Fig. 9C, curves
LAC and Cont) functioned as normal RKM. Respiration was accelerated by
Asc plus TMPD, further boosted by CCCP, and completely blocked by KCN
(inhibitor of complex IV). However, treatment of JC with LAC for 4
h (Fig. 9D, LAC curve) led to a significant decrease of
electron flow through complex IV, and this flow failed to be stimulated
by CCCP, when compared with respiration of Mito from untreated JC (Fig. 9D, Cont curve). The addition of purified normal RKM in this
experiment resumed oxygen consumption (Fig. 9D, LAC curve).
These results show that after 4 h of proteasome inhibition, there
is a defect between complex III and IV.
CytC is a low-m.w. soluble protein localized in mitochondrial
intramembrane spaces and functions as an electron transport protein
shuttling between complex III and IV. In most models of apoptosis, CytC
leaks out of Mito in the early stage of apoptosis (35, 36). Therefore, we tested whether exogenous CytC could correct
the defect in the respiration chain in LAC-treated JC. As shown in Fig. 9E, the addition of exogenous CytC had no effect on
Asc-TMPD-driven and FCCP-accelerated respiration of Dig-treated normal
RKM (RKM curve), or Dig-permeabilized untreated JC (Cont curve).
However, the exogenous CytC resulted in acceleration of respiration
with restoration of electron flow in JC treated with LAC for
4 h.
The results of this section show that after proteasome inhibition, the electron transport chain is compromised after complex III, and the defect likely involves CytC, which either leaks out or is functionally defective.
Bcl-xL overexpression prevents CytC leakage out of Mito after proteasome inhibition
Was the defect in the electron transport chain attributable to
leakage of CytC out of Mito? We assessed CytC concentration in the Mito
of JC and NC. STS is known to induce apoptosis and cause leakage of
CytC from the Mito (36). STS treatment resulted in a
decrease of CytC in the Mito prepared from both JC and NC (Fig. 10, A and B) as
expected. When these cells were treated with LAC (6 µM) for 6 h
(Fig. 10, A and B), the level of CytC in the Mito
also was decreased compared with that of untreated cells. A nonspecific
band of 75 kDa recognized by anti-CytC mAb had similar intensity in
all the samples, and this band served as internal controls for protein
loading in immunoblotting.
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C) in LAC-treated cells (6 h), while before the treatment
(0 h), CytC was not detectable in the cytosol. This indicates that
proteasome inhibition triggers the leakage of CytC from the Mito to the
cytosol.
Could Bcl-xL prevent CytC leakage after proteasome
inhibition? Bcl-xL overexpression in the Mito and cytosol
of Bcl-xL-transfected NC was evident according to 32.7-kDa
bands recognized by a mAb against the HA tag of the fusion protein
(Fig. 10D), whereas in wild-type NC, it was not detectable,
as expected (Fig. 10C). In Bcl-xL-transfected
cells, there was no decrease of CytC in Mito, nor did CytC become
detectable in the cytosol after 6 h of LAC treatment. These
results show that Bcl-xL prevents CytC from leaking out
of Mito.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m. Third, we have extended current
knowledge on the protective role of Bcl-xL to LAC-induced
apoptosis and diminished m and showed that
this is likely achieved by direct interaction between Bik and
Bcl-xL.
Bik was found to be accumulated in Mito and cytosol after proteasome
inhibition, whereas several other members, such as Bax, Bak, and Bad,
were not affected. Bik/NBK/Blk, BID, Hrk, NIP3,
BimL/BOD, and Bad belong to a so-called
"BH3-only" proapoptotic Bcl-2 subgroup (37). They are
more potent in their apoptosis-inducing activity than other members
that contain additional BH1 and BH2 domains (38). BID is
able to cause disruption of the outer mitochondrial membrane, because
recombinant BID, especially truncated BID added to purified Mito, could
lead to CytC release (38). In a cell-free
Xenopus oocyte system, the BH3 domain alone can induce CytC
release and activation of caspases (39). In keeping with
these features of BH-3-only molecules, Bik overexpression results in
decrease of m (40), and
recombinant Bik induces CytC release from purified Mito
(41). Moreover, expression of exogenous Bik suffices to
induce apoptosis as demonstrated in leukemic cells in our study and in
other types of cells (23, 42, 43). Based on these results,
we believe that Bik accumulation after proteasome inhibition plays an
important role in apoptosis induction in our model.
Our observation that proteasome activity is required to prevent apoptosis in leukemic cells is supported by another study in which Imajoh-Ohmi et al. reported that LAC induces apoptosis in U937 cells (44). However, blocking proteasomes seems to prevent apoptosis under certain circumstances, such as dexamethasone-treated thymocytes (26) and neurons deprived of nerve growth factor (28). Is this due to different degrees of inhibition? We have covered the full spectrum of LAC concentrations and no antiapoptotic effect was observed in our model. The function of the proteasome is the same in all these cells, i.e., to degrade proteins. Why then do different cells respond to proteasome inhibitors differently? It is to be noted that the protein levels of not only proapoptotic but also antiapoptotic factors could be proteasome dependent. We speculate that under normal circumstances proteasomes maintain a dynamic equilibrium between antiapoptotic and proapoptotic factors. If this is correct, we could think of at least two scenarios in which proteasome inhibitors can cause opposite effects in apoptosis. First, it is possible that different types of cells or cells at different differentiation stages are using different antiapoptotic or proapoptotic factors (45, 46, 47). For example, it has been reported that in thymocytes, degradation of inhibitor of apoptosis is necessary for glucocorticoid-induced apoptosis, and the degradation is a proteasome-mediated process (48). If inhibitor of apoptosis has a significant weight in the equilibrium, this can then explain quite well why in thymocytes, proteasome inhibitors prevent but not induce apoptosis. Second, even the same factor might have different rates of synthesis and proteasome-dependent degradation in different cells. We have shown that the levels of proapoptotic factors Bak and Bax are not changed in JC and NC after proteasome inhibition, whereas in a pancreatic tumor cell line BxPC-3 both Bak and Bax are accumulated after LAC treatment, as reported in our recent publication (49). This suggests that the same factors, such as Bak and Bax, have different rates of proteasome-dependent protein degradation in different cells. Therefore, in either of these scenarios, one of the two opponent groups (i.e., antiapoptotic vs proapoptotic) might have an upper hand in their overall effects. The final balance decides whether the proteasome inhibitor promotes or represses apoptosis.
To better understand the mechanism of how the relative levels of Bik and antiapoptotic Bcl-2 members decide the fate of leukemic cells, we tested the interaction between Bik and Bcl-xL and the consequences of such interaction. We found that Bik coprecipitates with antiapoptotic Bcl-xL in Bcl-xL-transfected NC, and this is a physical basis for them to counteract each other’s effect. Functionally, Bcl-xL overexpression protected apoptosis induced by both proteasome inhibition and by expression of exogenous Bik, suggesting that Bcl-xL and Bik likely interact in the cells. It is conceivable that in wild-type lymphocytes the Bik accumulated after proteasome inhibition will trap more endogenous Bcl-xL, which could normally prevent CytC release from Mito. The Mito will then be prone to CytC escape and the freed CytC will form apoptosomes, which activate the downstream caspase pathway reported previously (37, 50). In our model, we do have additional supporting evidence that caspase-3 was activated.
Although the pan-caspase inhibitor zVAD.fmk could inhibit DNA fragmentation, LAC-treated cells eventually died in the presence of zVAD-fmk (data not shown), as has been reported in other apoptosis models (51). This indicates that LAC caused irreversible damage to the cells and the damage was upstream of caspase activation. In all likelihood, the damage is at the mitochondrial level, according to our findings.
Although we have proposed that the augmented level of Bik is sufficient
in LAC-induced apoptosis, we are clearly aware that proteasomes can
degrade a large number of proteins, some of whichmight also contribute
significantly to the process of apoptosis. There are 6 antiapoptotic
and 12 proapoptotic members in the mammalian Bcl-2 family documented so
far (33, 52). Conceivably, the levels of some other
members could also be controlled by proteasomes. The degradation of
IB, an inhibitor of NF-B, is mediated by proteasomes
(10), and IB prevents NF-B translocation to the
nucleus. Failure of such translocation during T cell activation results
in the apoptosis of T cells (53). We believe that pathways
of apoptosis induction, such as that via Bik, IB, or other so far
unknown ones, do not have to be mutually exclusive but could
coexist.
We have analyzed in detail the effect of proteasomes on the
mitochondrial electron transport chain and for the first time
showed that respiration in Mito depends on functioning
proteasomes. It is well-known that m
generation is electron transport chain dependent. Thus, not
surprisingly, when the function of the electron transport chain is
compromised by LAC, m is diminished.
Defective generation or maintenance of m is
a hallmark of apoptosis induction, and we have shown that proteasomes
can regulate this event, although the relevance of such altered
m to apoptosis is not clear at present.
According to the information obtained from our study, we propose the following pathway of apoptosis induction. Proteasome inhibition leads to accumulation of proapoptotic Bik, which in turn results in CytoC leakage out of Mito. Antiapoptotic Bcl-xL can directly bind to Bik and counteract the CytC-releasing effect of Bik. CytC leakage on the one hand causes compromise of the electron transport chain, which in turn diminishes the capacity ofMito to generate Suc-stimulated membrane potential on the inner membrane. On the other hand, the released CytC in the cytosol triggers caspase-3 activation and, consequently, nuclear condensation as well as DNA fragmentation, hence apoptosis of the cells. We reiterate that this pathway is by no means exclusive and other pathways might coexist.
In summary, our study shows that proteasomes can modulate apoptosis of lymphocytes by affecting the half-life of Bcl-2 family members.
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Acknowledgments |
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Footnotes |
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2 V.M. and X.W. contributed equally to this work.
3 Current address: Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Boston, MA 02129-2020.
4 Current address: Novartis Pharmaceuticals, 556 Morris Avenue, Summit, NJ 07901-1398.
5 Address correspondence and reprint requests to Dr. Jiangping Wu, Laboratory of Transplantation Immunology, Pavilion J. A. DeSève, Research Center, Center hospitalier universitaire de l’Université de Montréal, Notre-Dame Hospital, 1560 Sherbrooke Street East, Room Y-5616, Montreal, Quebec, H2L 4M1, Canada.
6 Abbreviations used in this paper: LAC, lactacystin; JC, Jurkat cells; NC, Namalwa B cells; Mito, mitochondria; zVAD.fmk, benzyloxycarbonyl-Val-Ala-Asp(Ome) fluoromethyl ketone; CytC, cytochrome c; HA, hemagglutinin; PARP, poly(ADP-ribose) polymerase; Dig, digitonin; Suc, succinate; Anti A, antimycin A; TMPD, tetramethyl-p-phenylenediamine; Asc, ascorbate; CCCP, carbonyl cyanide m-chlorophenylhydrazone; FCCP, carbonyl p-trifluoromethoxyphenylhydrazone; Campt, camptothecin; JC-1, 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethyl-benzimidazolocarbocyanine iodide; DEVDase, caspase-3-like activities; RKM, rat kidney mitochondria; m, mitochondrial membrane potential; STS, staurosporine; Cont, control.
Received for publication July 27, 2000. Accepted for publication December 22, 2000.
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