From the Department of Anatomy and Cell Biology,
Medical College of Georgia, Augusta, Georgia 30912 and
§ Department of Pathology, University of Pittsburgh School
of Medicine, Pittsburgh, Pennsylvania 15261
Received for publication, January 2, 2003, and in revised form, March 4, 2003
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ABSTRACT |
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Bcl-2 family proteins are important
regulators of apoptosis. They can be pro-apoptotic
(e.g. Bid, Bax, and Bak) or anti-apoptotic (e.g. Bcl-2 and Bcl-xL). The current
study examined Bid-induced apoptosis and its inhibition by Bcl-2.
Transfection of Bid led to apoptosis in HeLa cells. In these cells, Bid
was processed into active forms of truncated Bid or tBid. Following
processing, tBid translocated to the membrane-bound organellar
fraction. Bcl-2 co-transfection inhibited Bid-induced apoptosis but
did not prevent Bid processing or tBid translocation. On the other
hand, Bcl-2 blocked the release of mitochondrial cytochrome
c in Bid-transfected cells, suggesting actions at the
mitochondrial level. Alkaline treatment stripped off tBid from the
membrane-bound organellar fraction of Bid plus Bcl-2-co-transfected
cells, but not from cells transfected with only Bid, suggesting
inhibition of tBid insertion into mitochondrial membranes by Bcl-2.
Bcl-2 also prevented Bid-induced Bax translocation from cytosol to the
membrane-bound organellar fraction. Finally, Bcl-2 diminished
Bid-induced oligomerization of Bax and Bak within the membrane-bound
organellar fraction, shown by cross-linking experiments. In conclusion,
Bcl-2 inhibited Bid-induced apoptosis at the mitochondrial level by
blocking cytochrome c release, without suppressing Bid
processing or activation. Critical steps blocked by Bcl-2 included tBid
insertion, Bax translocation, and Bax/Bak oligomerization in the
mitochondrial membranes.
Apoptosis, also called programmed cell death, is a highly
regulated process that plays an essential role in the development and
maintenance of homeostasis within multicellular organisms (1, 2).
Dysregulation of apoptosis has been implicated in the development of
cancer, autoimmune disorder, neurodegeneration, ischemic damage, and
other devastating diseases (3-6). Whereas apoptosis regulation takes
place at multiple levels, Bcl-2 family proteins are of paramount
importance (7-10).
Bcl-2 family proteins are defined by the presence of Bcl-2 homology
(BH)1 domains (7-11). They
can be pro-apoptotic or anti-apoptotic. Specific function of individual
members is determined by the presence and organization of the BH
domains (8, 9). For example, anti-apoptotic members, like Bcl-2 and
Bcl-xL, contain four BH domains, whereas some pro-apoptotic
molecules such as Bax and Bak contain three (BH1-3), and others
contain only one, the BH3 domain (7-12).
Bid is a unique BH3-only pro-apoptotic protein (13). Unlike others, Bid
activation depends on the proteolytic processing of intact Bid into
truncated forms of tBid. tBid, thus generated, translocates to
mitochondria and leads to disruption of the organelles and the release
of apoptogenic molecules such as cytochrome c (14, 15). Bid
processing can be conducted by several proteases (16, 17); however,
caspase-8 has been shown to be the major protease responsible for Bid
cleavage during death receptor-mediated apoptosis (14, 15).
Caspase-8-mediated Bid processing therefore bridges the extrinsic death
receptor-mediated pathway of apoptosis to the intrinsic mitochondrial
pathway (14, 15, 18). This provides a mechanism to amplify the
execution signal and exacerbate the pace of cell demise.
Interactions among Bcl-2 family proteins have been documented (7-12).
Functionally, expression of anti-apoptotic Bcl-2 or Bcl-xL
suppresses cell death initiated or mediated by pro-apoptotic members.
Recent studies (19) have further suggested a sequence of Bcl-2 family
protein activation during apoptosis, where Bcl-2 is positioned to block
apoptosis at two separate steps, through inhibition of Bax/Bak and
BH3-only proteins including Bid. Despite these important observations,
it remains unclear how Bcl-2 antagonizes the pro-apoptotic action of
Bid or tBid (20, 21).
Here, by using an expression model, we have systematically analyzed
Bid-induced apoptosis and its inhibition by Bcl-2. Our results show
that Bcl-2 inhibited Bid-induced cytochrome c release and
apoptosis, without suppressing Bid processing into tBid and subsequent
tBid translocation to mitochondria. At the mitochondria, three critical
events that were blocked by Bcl-2 have been identified. First, Bcl-2
suppressed tBid insertion into mitochondrial membranes. Second, Bcl-2
inhibited Bid-induced Bax translocation from the cytosol to
mitochondria. Third, Bcl-2 diminished Bid-induced Bax/Bak oligomerization in the mitochondrial membranes. The results suggest that Bcl-2 may suppress Bid-induced apoptosis at the mitochondrial level by multiple mechanisms.
Materials--
PcDNA-Bid was prepared as described
previously (13). p Transfection--
HeLa cells were maintained in minimum
essential medium supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM
L-glutamine, and 1% antibiotics. The cells were plated at
3.0 × 105/35-mm dish to reach ~50% confluence by
the next day for transfection. Cells in each dish were transfected with
0.25 µg of empty pcDNA vectors, 0.25 µg of pcDNA-Bid, or
0.25 µg of pcDNA-Bid along with 0.25 µg of p Analysis of Apoptosis--
Apoptotic cells were identified by
their morphology as described in our previous studies (22). Typical
apoptotic morphology evaluated included cellular shrinkage and the
formation of apoptotic bodies. To verify apoptosis, nucleus was stained
with Hoechst 33342 to reveal nuclear condensation and fragmentation.
For each condition, apoptosis was monitored in five fields with ~60
cells per field. The experiments were repeated for at least four times with duplicate dishes for each condition in every experiment.
Subcellular Fractionation--
To analyze the distributions of
various proteins, cells were fractionated into cytosolic and
membrane-bound organellar fractions with low concentrations of
digitonin (23-25). Selective permeabilization of plasma membranes was
monitored by microscopy. Digitonin permeabilization has been used to
study protein redistributions within cells during apoptosis (24-27).
Briefly, cells were exposed to 0.05% digitonin in isotonic sucrose
buffer (in mM: 250 sucrose, 10 Hepes, 10 KCl, 1.5 MgCl2, 1 EDTA, and 1 EGTA; pH 7.1) for 2 min at room
temperature to collect the soluble fraction as cytosolic extracts.
Digitonin-insoluble fraction was washed with isotonic sucrose buffer
and further dissolved in 2% SDS buffer to collect the membrane-bound
organellar fraction. Because apoptotic redistribution of cytochrome
c and Bcl-2 family proteins including Bid and Bax mainly
takes place between the cytosol and mitochondria, immunoblot analysis
of the membrane-bound part is expected to reveal mitochondrial content
of the molecules.
Protein Cross-linking--
Cross-linking was conducted by
procedures modified from our previous work (25). All cross-linking
chemicals (DSP, BMH, and BSOCOES) were dissolved in Me2SO
at concentrations of 100 mM prior to experiments and
further diluted in phosphate-buffered saline to 10 mM
before using. DSP was added to the cells for 30 min of incubation at
room temperature under constant mixing. The cells were subsequently
fractionated by digitonin as described above. For BMH and BSOCOES,
cells were first fractionated to collect the membrane fraction for
cross-linking. Cross-linked samples were subjected to electrophoresis
under non-reducing conditions for immunoblot analysis.
Immunofluorescence Analysis of Cytochrome c--
Cytochrome
c immunofluorescence was examined as described in our
previous publications (23, 24). Briefly, cells were grown on
collagen-coated glass coverslips and subjected to transfection. The
cells were fixed in a modified Zamboni's fixative containing 4%
paraformaldehyde and 0.19% picric acid and permeabilized with 0.1%
SDS prior to blocking and primary antibody (mouse anti-native cytochrome c) exposure. Finally, antigenic sites within the
cells were revealed by staining with CY-3-conjugated goat anti-mouse antibodies. To examine the nucleus, Hoechst 33342 (10 µg/ml) was added to cells in phosphate-buffered saline and stained for 5 min at
room temperature.
Alkaline Treatment--
After digitonin permeabilization, the
membrane-bound organellar fraction was collected and washed once with
phosphate-buffered saline. The fraction was incubated on ice in 0.1 M Na2CO3 at pH 11.5 for 30 min and
then subjected to 1 h of centrifugation at 100,000 × g at 4 °C to collect the supernatant and pellet. The supernatant contained the proteins released during alkaline incubation, whereas the pellet contained the proteins that were resistant to the
treatment (28).
Immunoprecipitation--
Cells were extracted directly with RIPA
buffer to collect whole cell lysate, or subjected to sequential
extractions with 0.05% digitonin and RIPA buffer to collect cytosolic
and membrane-bound organellar extracts, respectively. Isotonic
digitonin buffer was described above under "Subcellular
Fractionation." The composition of RIPA buffer was (in
mM): 150 NaCl, 1 MgCl2, 1 EGTA, 10 Inhibition of Bid-induced Apoptosis by Bcl-2--
We initially
determined whether transfection of full-length Bid induced apoptosis in
HeLa cells and whether Bcl-2 co-transfection was able to suppress it.
To identify the transfected cells, a vector containing green
fluorescence protein (GFP) was co-transfected. The results are shown in
Fig. 1A. The control group
that was transfected with empty vectors had an apoptosis rate of
22.8%. Bid transfection led to a dramatic increase in apoptosis, to
81.3%. Bid-induced apoptosis was blocked by Bcl-2 co-transfection. As
a result, cells co-transfected with Bid + Bcl-2 showed an apoptosis
rate of 33.7% (Fig. 1A). The morphological observations
were confirmed by biochemical analyses. As shown in Fig. 1B,
Bid transfection led to the cleavage of lamin B into an
apoptosis-indicative fragment of 46 kDa (lane 2).
Co-transfection of Bcl-2 blocked lamin B cleavage (lane 3). Data not shown also demonstrated apoptotic cleavage of poly(ADP-ribose) polymerase in Bid-only transfected cells, which was again suppressed by
Bcl-2 co-transfection. Together, these experiments have demonstrated Bcl-2 inhibition of Bid-induced apoptosis in HeLa cells.
Bcl-2 Inhibits Bid-induced Cytochrome c Release from
Mitochondria--
A major cellular site targeted by Bcl-2 family
proteins is the mitochondrion (7-12). Disruption of this organelle
results in the release of apoptogenic molecules including cytochrome
c and may therefore underlie the pro-apoptotic actions of
Bid (20, 21). Thus, to pursue the mechanisms responsible for Bcl-2
inhibition of Bid-induced apoptosis, we examined cellular distributions
of cytochrome c. The results are shown in Fig.
2. By immunoblot analyses, the majority
of cytochrome c was detected in the mitochondrial fraction
of the control cells (Fig. 2A, lane 1). Bid
transfection led to an increase of cytochrome c in the
cytosol, accompanied by loss of the molecule from the membrane-bound
organellar fraction (lane 2), indicating cytochrome
c release from mitochondria in these cells. Significantly,
Bcl-2 co-transfection blocked Bid-induced cytochrome c
release (lane 3). The immunoblot results were confirmed by
immunofluorescence staining of cytochrome c within these
cells. As shown in Fig. 2B, Bid-transfected cells
(a, green) exhibited typical morphology of
apoptosis, assuming a round-up and fragmented configuration. In the
same cells, cytochrome c (Fig. 2B, b,
red) was released from mitochondria, resulting in whole cell
staining. In sharp contrast, cells co-transfected with Bid + Bcl-2
displayed a much healthier morphology (Fig. 2B,
c). These cells maintained cytochrome c in the
mitochondria, showing a punctated perinuclear staining (Fig.
2B, d). The results suggest that Bcl-2 inhibited Bid-induced apoptosis at the mitochondrial level by blocking cytochrome c leakage from mitochondria.
Bid Processing Is Not Prevented by Bcl-2--
The pro-apoptotic
activity of Bid depends on its processing into the active forms of tBid
(14, 15). Thus, to identify further the mechanisms responsible for
Bcl-2 inhibition, we examined Bid processing in transfected cells and
the effects of Bcl-2 co-transfection by using whole cell lysates. The
results are shown in Fig. 3. In
Bid-transfected cells, intact Bid of 22 kDa was expressed at high
levels (lane 2), compared with control transfection
(lane 1). Moreover, tBid of 15 and 13 kDa was detected,
indicating Bid processing in these cells (lane 2). Bcl-2
co-transfection did not attenuate either Bid expression or it
processing into tBid (lane 3). The results suggest that
Bcl-2 inhibited Bid-induced apoptosis without significantly
suppressing Bid expression or processing.
tBid Translocation to Mitochondria Is Not Prevented by
Bcl-2--
An important event for Bid activation and toxicity is the
targeting of mitochondria by tBid (14, 15). Thus, our subsequent experiments tested the possibility that Bcl-2 might inhibit Bid-induced apoptosis by blocking tBid translocation. For this purpose, transfected cells were fractionated into cytosolic and membrane-bound organellar fractions for immunoblot analysis of Bid/tBid. The results are shown in
Fig. 4. In Bid-only transfected cells
(lanes 1 and 3), intact Bid of 22 kDa was
detected mainly in the cytosol, whereas tBid of 15 kDa showed both
cytosolic and organellar distributions, and p13 was detected only in
the membrane-bound organellar fraction. Bcl-2 co-transfection did not
change the cellular localization of these molecules (Fig. 4,
lanes 2 and 4). The results, together with those
shown in Fig. 3, indicate that Bcl-2 inhibited Bid-induced cytochrome
c leakage and apoptosis without blocking Bid processing and
translocation.
Inhibition of Membranous Insertion of tBid by Bcl-2--
Bcl-2 did
not affect Bid processing into tBid or tBid translocation (Figs. 3 and
4). However, it inhibited Bid-induced mitochondrial disruption
including the release of cytochrome c (Fig. 2). These observations promoted us to examine the status of tBid association with
mitochondrial membranes. Specifically, we asked the following: does
Bcl-2 prevent tBid integration or insertion into the membranes? To
address this question, we utilized a classical method of alkaline treatment to examine the integration status of tBid (28). Alkaline incubation of cellular membranes at pH 11.5 leads to the dissociation of loosely attached proteins, whereas integrated proteins remain. This
approach was successfully employed to demonstrate Bax insertion into
mitochondria (29, 30). In our experiments, cells were transfected with
Bid alone or Bid + Bcl-2. Membrane-bound organellar fractions were
collected for incubation with 0.1 M NaHCO3 at
pH 11.5. Proteins stripped into the incubation solution were collected and analyzed for Bid/tBid, along with the proteins that were resistant to alkaline incubation. The results are shown in Fig.
5. In Bid-only transfected cells,
alkaline incubation stripped off intact Bid of 22 kDa from the
organellar membranes into the supernatant (lane S1).
However, tBid of 15 and 13 kDa was rather resistant to the treatment
and thus remained associated with the membranes after alkaline exposure
(lane P1), suggesting that tBid and not intact Bid had
integrated into the mitochondrial membranes in these cells. In sharp
contrast, for Bid + Bcl-2-co-transfected cells, alkaline treatment led
to the release of not only intact Bid but also a significant portion of
15-kDa tBid (lane S2). To estimate the percentage of 15-kDa
tBid that was sensitive to alkaline treatment, blots from 4 separate
experiments were analyzed by densitometry (Fig. 5B). In
cells co-transfected with Bcl-2, over 30% of 15-kDa tBid was released
during alkaline incubation; by sharp contrast, less than 2% was
released from cells transfected with Bid only. As a control, cytochrome
oxidase IV, an integral mitochondrial membrane protein, was not
stripped off from the membranes under these experimental conditions,
regardless of the presence or absence of Bcl-2 (Fig. 5A,
lower panel). The results suggest that Bcl-2 may suppress
Bid-induced mitochondrial disruption partly by prevention of tBid
insertion into the organellar membranes.
Co-immunoprecipitation of Bcl-2 with Bid but Not tBid--
Our
results suggested that Bcl-2 inhibited tBid insertion into
mitochondrial membranes (Fig. 5). However, the underlying mechanism was
unclear. One hypothesis was that Bcl-2 might directly interact with
tBid, resulting in conformational changes in this molecule that
prevented its integration into organellar membranes. To test this
possibility, we examined the interactions between Bcl-2 and Bid/tBid by
co-immunoprecipitation. In this experiment, cells were extracted either
directly with RIPA buffer to collect whole cell lysate or sequentially
with digitonin and RIPA buffer to collect the cytosolic fraction and
the membrane-bound organellar fraction including mitochondria. The
extracts were subjected to immunoprecipitation with Bcl-2 antibodies.
The immunoprecipitates were analyzed for the presence of Bid and tBid.
As shown in Fig. 6A, intact
Bid was detected in all Bcl-2 immunoprecipitates, irrespective of the
extracts utilized for immunoprecipitation. On the contrary, tBid was
not shown in any of the Bcl-2 immunoprecipitates (Fig. 6A).
To demonstrate that the extracts prior to immunoprecipitation did
contain tBid, we analyzed the extracts directly by immunoblotting (Fig.
6B). Clearly, significant amounts of 15-kDa tBid were
present in the whole cell lysates and in the membrane fraction (Fig.
6B, lanes 4 and 6). Of note, whereas
tBid of 13 and 15 kDa was detected in cell lysates extracted with
Laemmli buffer containing 2% SDS (Figs. 3 and 4), only 15-kDa tBid was
extracted by the RIPA buffer in these experiments (Fig. 6B,
lanes 4 and 6). Presumably, this was caused by
the limited extraction capacity of the RIPA buffer, which had only
0.2% SDS. Nevertheless, the results suggest that Bcl-2 did not
co-immunoprecipitate 15-kDa tBid under the experimental conditions,
although membranous insert of this molecule was suppressed by Bcl-2.
Also included in this figure was a Bcl-2 immunoblot, showing Bcl-2
expression mainly in membrane-bound fraction, with limited amounts in
the cytosol (Fig. 6C).
Bid-induced Bax Translocation and Its Inhibition by
Bcl-2--
Recent studies (19, 29, 31, 32) have documented
Bid/tBid-induced alterations of Bax and Bak, resulting in mitochondrial insertion and oligomerization of Bax and Bak in the organelles. These
observations are very important, because they suggest a sequential
cascade for the activation of Bcl-2 family proteins (32). To examine
whether Bcl-2 inhibited Bid-induced mitochondrial damage through its
actions on Bax/Bak, we first analyzed cellular distribution of these
two proteins by fractionation of the cells into the cytosolic fraction
and the membrane-bound organellar fraction including mitochondria. The
results are shown in Fig. 7. In control
cells without transfection, the majority of Bax was detected in the
cytosol (lane 1), with much weaker signals in the
membrane-bound organellar fraction (lane 4). Bid
transfection led to the loss of cytosolic Bax (lane 2),
accompanied by increases in organellar Bax (lane 5),
indicating translocation of this molecule. Bcl-2 co-transfection
significantly prevented the translocation of Bax. As a result, in these
cells Bax was detected mainly in the cytosol (lane 3), with
weaker signals in the organellar fraction (lane 6). For Bak,
a membrane-bound organellar location was always detected in control
cells, cells transfected with Bid, and cells co-transfected with Bid + Bcl-2 (Fig. 7, Bak blots in the lower panel).
Bid-induced Bax/Bak Oligomerization and Its Inhibition
by Bcl-2--
A critical event for mitochondrial disruption by
pro-apoptotic Bcl-2 family proteins seems to be the oligomerization of
these molecules within the outer membranes (19, 32-34). Moreover,
recent studies (25, 33) including ours showed that Bax oligomerization in the outer membrane of mitochondria was abolished by Bcl-2. Thus,
following the experiments demonstrating inhibition of Bax translocation
by Bcl-2, we went on to examine the oligomerization status of Bax and
Bak in the membrane-bound organellar fraction. For this purpose, cells
were subjected to cross-linking treatment to preserve protein-protein
interactions, and the membrane-bound organellar fractions were
extracted for immunoblot analysis of Bax and Bak. The results are shown
in Fig. 8. The blots in the left
panel were obtained by regular exposure, and the blots in the
right panel were subjected to overexposure to reveal Bax/Bak oligomers. In control cells, Bax oligomerization was minimal, even
after overexposure (Fig. 8A, lanes 1 and
4). Bid transfection led to the formation of Bax oligomers,
accompanied by a decrease in Bax monomers (Fig. 8A,
lanes 2 and 5). Importantly, Bcl-2
co-transfection diminished Bid-induced Bax oligomerization, as shown in
Fig. 8A, lanes 3 and 6. Similar
observations were obtained for Bak (Fig. 8B). Bak existed as
monomers in control cells (lanes 1 and 4) and
oligomerized after Bid transfection (lanes 2 and
5). Again, Bak oligomerization induced by Bid was attenuated
by Bcl-2 co-transfection (Fig. 8B, lanes 3 and
6). Together, these results suggest that, in addition to
preventing tBid insertion and Bax translocation, Bcl-2 may antagonize
Bid-induced apoptosis by blocking Bax/Bak oligomerization in the
mitochondrial membranes.
This study has examined inhibition of Bid-induced apoptosis by
Bcl-2 and the underlying mechanisms. In Bid-transfected cells, the
pro-apoptotic molecule was processed into active forms of tBid. tBid
translocated or accumulated into the mitochondria, leading to
disruption of the organelles, releasing cytochrome c.
Co-transfection of Bcl-2 inhibited cytochrome c leakage and associated apoptosis in Bid-transfected cells. On the other hand, Bcl-2
did not suppress Bid processing or tBid translocation. Three important
events that were suppressed by Bcl-2 have been identified. First, Bcl-2
suppressed organellar insertion of tBid. Second, Bcl-2 attenuated Bax
translocation from the cytosol to mitochondria. Third, Bcl-2 blocked
Bid-induced oligomerizations of Bax and Bak in the mitochondrial membranes.
Recent studies have significantly advanced our understanding of the
pro-apoptotic actions of Bid. It has been shown that Bid activation
depends on its proteolytic processing into tBid and tBid translocation
to mitochondria (14, 15). In the mitochondria, tBid may form oligomers
by itself and induces oligomerization of Bax and Bak (19, 29, 32, 34).
Although it remains unclear how tBid triggers such oligomerizations,
strong evidence has been provided to suggest that Bid-induced apoptosis
depends on the presence of Bax and Bak (19, 35). Consistent with these
observations, our experiments have demonstrated Bid processing and tBid
translocation to the membrane-bound organellar fraction including
mitochondria, which was accompanied by cytochrome c leakage
from the organelles. Significantly, Bcl-2 did not prevent either Bid
processing or tBid movement to mitochondria but blocked cytochrome
c leakage. Similar observations have been shown for
Bcl-xL in a model of tumor necrosis factor-induced
apoptosis (36). Therefore, tBid accumulation to mitochondria is an
essential but not a sufficient event leading to mitochondrial
disruption for cytochrome c leakage.
Our results suggest that, after mitochondrial association, tBid
integrates into the membranes to exert its toxicity. An important action of Bcl-2 was shown to suppress membranous insertion of tBid,
which was accompanied by the preservation of mitochondrial integrity.
Insertion of pro-apoptotic molecules into mitochondrial membranes has
been investigated for Bax. In a series of carefully controlled
experiments, Bax was shown to insert into mitochondrial membranes upon
apoptotic stimulation (30). Bax insertion depends on the unfolding of
the amino-terminal domain and the consequent exposure of the
transmembrane domain at the carboxyl terminus. Little has been learned,
however, about membrane insertion of other pro- or anti-apoptotic Bcl-2
proteins including Bid/tBid. Apparently, tBid insertion might take
place in a way that can be quite different from that of Bax. After all,
tBid does not contain a specific transmembrane domain. Nevertheless,
Bcl-2 family proteins share a conserved overall structure and
conformation. In addition, a mitochondrial targeting domain has been
identified in Bid/tBid. Proteolytic removal of the amino-terminal
fragment may increase the hydrophobicity of tBid for membrane
integration and unveil the targeting domain, leading to it
translocation to mitochondria (20, 21). Results of the current study
indicate that Bcl-2 does not inhibit Bid processing or tBid
translocation to mitochondria but suppresses tBid insertion, suggesting
that tBid translocation and membrane integration are separate events regulated by different mechanisms.
It remains unclear how Bcl-2 suppressed tBid insertion. In the
mitochondria, Bcl-2 appeared to interact with Bid but not tBid, although the latter was the predominant form associated with the organelles (Fig. 6). The results suggest that direct interaction between Bcl-2 and tBid may not be responsible for the expelling effects
of Bcl-2. On the other hand, tBid insertion may depend on the
availability of specific anchoring sites, and Bcl-2 might compete for
these targets. Alternatively, Bcl-2 expression may change the
microenvironment or composition of mitochondrial membranes and thereby
reduce the compatibility for tBid integration and the formation of
supramolecular openings in mitochondria (37). Of interest, recent
studies (38) have demonstrated Bid binding to cellular membranous
lipids at high affinities. All these possibilities remain to be tested
by further investigations.
Although our results showed co-immunoprecipitation of Bcl-2 with intact
Bid and not tBid, it remains unclear whether or not Bcl-2 interacts
with tBid in situ within the cells. Recent results from
Korsmeyer and co-workers (32) demonstrated binding of tBid with Bcl-2
by co-immunoprecipitation of prior cross-linked samples. In those
experiments, mitochondria were isolated from Bcl-2 expressing cells,
incubated with recombinant tBid, and then cross-linked with
irreversible homobifunctional cross-linkers. The mitochondria were
finally extracted with RIPA buffer to collect soluble fractions for
immunoprecipitation, showing a Bcl-2-tBid complex. Similar complexes were detected in extracts from cells undergoing tumor necrosis factor- In normal cells without transfection or overexpression, Bcl-2
associates with organellar membranes. Thus, the interaction of Bcl-2
with intact Bid in the cytosol shown in Fig. 6A was most likely a result of simultaneous overexpression of the proteins. Indeed,
portions of Bcl-2 were detected in the cytosol of the Bcl-2-transfected
cells, although the majority of the protein was shown in the membrane
fraction (Fig. 6C).
Interestingly, earlier studies (36) implied that Bcl-xL,
another important anti-apoptotic molecule, did not affect tBid insertion into mitochondrial membranes. Discrepancy between those results and our observations might be caused by specific features of
experimental models. Alternatively, Bcl-2 and Bcl-xL may
not antagonize apoptosis in exactly the same way, despite close
homology (39). Of note, in our experiments, Bcl-2 did not completely diminish tBid insertion. Significant amounts of tBid stayed integrated in mitochondrial membranes, after Bcl-2 co-transfection (Fig. 5).
Nevertheless, cytochrome c leakage as well as apoptosis in these cells was reduced close to control levels (Figs. 1 and 2). These
observations suggest that prevention of tBid insertion is an important
but not the single event responsible for the mitochondrial protective
effects of Bcl-2.
The current study has identified Bax translocation as the second action
site for Bcl-2. Consistent with previous studies (29), the majority of
Bax in control cells was detected in the cytosol, which translocated to
mitochondria upon Bid stimulation. Significantly, our results further
demonstrated that Bid-induced Bax translocation was evidently
suppressed by Bcl-2 co-transfection (Fig. 7). The signal directing Bax
translocation from the cytosol to mitochondria remains to be clarified
(7, 9). Structurally, Bax has a hydrophobic transmembrane domain at the
carboxyl terminus. In normal non-apoptotic cells, this domain is buried
by its interaction with the amino terminus of the protein. Deletion of
the amino terminus results in mitochondrial localization of Bax even in the absence of apoptotic stimuli (30). However, removal of the amino
terminus by proteolysis is not considered as a common mechanism for the
exposure of the transmembrane domain in vivo, because Bax
remains intact during apoptosis regardless of its location within the
cells. There are at least two hypotheses on the regulation of Bax
movement; each is currently supported by significant evidence. In the
first hypothesis, Bax is proposed to interact with a regulatory protein. Modifications of the interaction may lead to conformational changes in Bax and the exposure of the transmembrane domain at the
carboxyl terminus. A potential Bax interacting protein might be Bid.
During apoptosis, Bid interacts with Bax and induces conformational changes to expose the carboxyl terminus for integration of Bax into
mitochondrial membranes (29, 31, 32). Another protein regulating Bax
might be 14-3-3, as suggested by recent studies (40). In living cells,
specific isoforms of the 14-3-3 protein bind Bax. Upon apoptotic
stimulation, Bax is liberated from 14-3-3 and translocates to
mitochondria. The second hypothesis on the mechanisms of Bax
translocation emphasizes a role for alterations of the cytosolic
environment. In particular, pH changes in the cytosol might be
critical. A shift of intracellular pH toward either alkalization or
acidification has been linked to conformational changes in Bax,
followed by insertion of the molecule into mitochondrial membranes (41,
42). Apparently, these two hypotheses are not mutually exclusive. For
example, cytoplasmic alkalization as well as acidification may decrease
the interactions between Bax and its partnering proteins (40). All
these possibilities need to be tested in future investigations to
address the following questions: how did Bid trigger Bax translocation
and why was the translocation suppressed by Bcl-2?
This study has further identified Bax/Bak oligomerization in the
mitochondria as the third action site for Bcl-2. Oligomerization of
Bax/Bak has been demonstrated in the mitochondria during apoptosis (19,
29, 31, 32). Recent studies (25, 33, 43) have further suggested an
essential role for the oligomerizations in the development of
mitochondrial pathology. Results of the current study showed that, in
mitochondria of control cells, Bax and Bak existed as monomers. Bid
transfection led to oligomerization of these molecules. Significantly,
such oligomerizations were abolished by Bcl-2 expression (Fig. 8).
These results provide strong support for the scenario of sequential
activation of Bcl-2 family proteins (19, 32). In this model,
Bcl-2/Bcl-xL was proposed to sequester BH3 domain-only
proteins and prevent the formation of Bax/Bak oligomers in
mitochondria, resulting in the preservation of mitochondrial integrity.
Despite a recognized role for Bax/Bak oligomerization in apoptosis, the
mechanisms regulating such oligomerization are largely unknown. In the
present study, Bax/Bak oligomerization took place in mitochondria and
was triggered by Bid/tBid. Thus, membranous integration of tBid might
be a prerequisite for its Bax/Bak oligomerizing activity. Should this
be the case, Bcl-2 might attenuate Bax/Bak oligomerization as least in
part by preventing tBid insertion. On the other hand, tBid insertion
was not completely diminished by Bcl-2 (Fig. 5), and yet Bax/Bak
oligomerization was (Fig. 8). The results suggest that, in addition to
preventing tBid insertion, Bcl-2 may also disrupt the formation of
Bax/Bak oligomers in the mitochondrial membranes by more direct
mechanisms. These considerations are supported by recent studies (44)
showing Bax/Bak oligomerization and its prevention by Bcl-2 in the
absence of Bid activation.
One mechanism whereby Bcl-2 may directly disrupt Bax/Bak
oligomerization is through heterodimerization. Such interaction has been reported in various kinds of experimental models (45). Although
some of the heterodimerizations might be caused by the presence of
detergents during sample preparation (46), interactions between pro-
and anti-apoptotic proteins have been demonstrated by detergent-free
approaches such as yeast two-hybrid systems (47, 48). Moreover, the
lipid-rich cellular membranes including that of mitochondria may
readily provide a "detergent"-like microenvironment that favors
physical interactions between pro-apoptotic and anti-apoptotic Bcl-2
family proteins. Physical associations between Bcl-2 and Bax were shown
in mitochondria within intact cells by the technique of fluorescence
resonance energy transfer (49).
In conclusion, this study has examined Bcl-2 inhibition of Bid-induced
apoptosis in an expression model. Bcl-2 inhibited Bid-induced cytochrome c leakage from mitochondria and the associated
apoptosis, without ameliorating Bid processing or tBid translocation to
mitochondria. Three critical events that were attenuated by Bcl-2 have
been identified. First, Bcl-2 suppressed tBid insertion into
mitochondrial membranes. Second, Bcl-2 inhibited Bax translocation to
mitochondria. Third, Bcl-2 diminished Bax and Bak oligomerization in
the mitochondrial membranes. By preventing tBid integration, Bax
translocation, and Bax/Bak oligomerization, Bcl-2 may preserve the
integrity of mitochondria and abolish apoptosis in the experimental model.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
actBcl-2 was a gift from Dr. Junyin Yuan at
Harvard Medical School. pEGFP was purchased from
Clontech. Transfection reagents were purchased from
Invitrogen. Chemical cross-linkers dithiobis(succinimidyl propionate
(DSP), bismaleimidohexane (BMH), and
bis[2-(succinimidyloxycarbonyloxy)-ethyl] sulfone (BSOCOES) were
purchased from Pierce. Antibodies used in this study were from the
following sources: rabbit polyclonal anti-Bax (N-20), mouse monoclonal
anti-Bcl-2 (C-2), rabbit polyclonal anti-Bcl-2 (
C21), and goat
polyclonal anti-lamin B from Santa Cruz Biotechnology (Santa Cruz, CA);
rabbit polyclonal anti-Bak from Upstate Biotechnology, Inc. (Lake
Placid, NY); mouse monoclonal antibodies against native (6H2.B4) and
denatured (7H8.2C12) cytochrome c from Pharmingen; mouse
monoclonal 20E8 anti-cytochrome oxidase IV from Molecular Probes
(Eugene, OR); rabbit polyclonal anti-murine Bid was prepared as
described previously (13). All secondary antibodies were obtained from
Jackson ImmunoResearch (West Grove, PA).
actBcl-2. To
identify the transfectants, the same dishes were co-transfected with
pEGFP-C1, which led to the expression of green fluorescence protein in
transfected cells. Transfection was facilitated with LipofectAMINE PLUS
(Invitrogen), according to the manufacturer's instructions.
Transfection efficacy was usually over 70%. After transfection, cells
were incubated in serum-free medium for 4-5 h and then transferred to
full growth medium. Morphological and biochemical analyses were
conducted at ~17 h post-transfection.
-mercaptoethanol, 15 Tris-HCl, pH 7.4, containing 0.5% sodium
deoxycholate, 0.2% SDS, and 1% Triton X-100. Immunoprecipitation was
conducted according to our previous work (24, 25). Extracts (whole cell
extracts by RIPA, cytosolic extracts by digitonin, post-digitonin
extracts by RIPA) from ~1 × 106 cells were
pre-cleared by incubation with 1 µg of normal serum and 30 µl of
agarose protein A/G (Santa Cruz Biotechnology). The pre-cleared lysates
were subsequently incubated for 2 h with 2 µg of
immunoprecipitation antibody and 30 µl of agarose protein A/G.
Immunoprecipitates were collected by centrifugation and dissolved in
SDS sample buffer for immunoblot analysis. To detect Bcl-2 and Bid/tBid
interaction, a mouse monoclonal anti-Bcl-2 was used for
immunoprecipitation, and the resultant precipitates were subjected to
immunoblot analysis of Bid/tBid.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 1.
Inhibition of Bid-induced apoptosis by
Bcl-2. HeLa cells were transfected with Bid, Bid + Bcl-2, or empty
vectors (mock), along with pEGFP. Apoptosis of transfected cells was
identified by cell morphology, as described under "Experimental
Procedures," and calculated as a percentage of transfectants
(A). Data are expressed as the mean ± S.D.
(n = 4). To analyze lamin B breakdown, whole cell
lysates were collected for immunoblot analysis (B). The
results show that Bid transfection led to the development of apoptotic
morphology and apoptotic cleavage of lamin B. Bcl-2 co-transfection
inhibited Bid- induced apoptosis.
View larger version (31K):
[in a new window]
Fig. 2.
Bcl-2 blocks Bid-induced cytochrome
c release from mitochondria. A,
immunoblot analysis. HeLa cells transfected with Bid, Bid + Bcl-2, or
empty vectors were fractionated into cytosolic and membrane-bound
organellar fractions and analyzed for cytochrome c
(cyt. c) by immunoblotting. In mock transfection with empty
vectors, the majority of cytochrome c was in the organellar
fraction (lane 1). After Bid transfection, significant
amounts of cytochrome c appeared in the cytosol
(Cyto) (lane 2). Bcl-2 co-transfection blocked
Bid-induced release of cytochrome c (lane 3).
Mito, mitochondria. B, immunofluorescence
analysis. HeLa cells were transfected with Bid (a and
b) or Bid + Bcl-2 (c and d). pEGFP was
co-transfected to identify the transfectants. The cells were processed
for cytochrome c immunofluorescence as described under
"Experimental Procedures." The same fields were examined for
cytochrome c (red, b and
d) and transfected cells containing green fluorescence
protein (green, a and c). Bid
transfection led to apoptosis, showing cellular shrinkage and the
formation of apoptotic bodies (a). In these cells,
cytochrome c leaked into the cytosol, resulting in diffuse
cytosolic staining (b). Bcl-2 co-transfection prevented
apoptosis (c) as well as the leakage of mitochondrial
cytochrome c (d). As a result, cytochrome
c in Bcl-2-co-transfected cells was preserved in the
mitochondria, exhibiting perinuclear organellar staining
(d).
View larger version (57K):
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Fig. 3.
Bcl-2 does not prevent Bid processing into
active forms of tBid. HeLa cells were transfected with Bid, Bid + Bcl-2, or empty vectors. Whole cell lysates were extracted with Laemmli
buffer containing 2% SDS for immunoblot analysis of Bid/tBid. Clearly,
Bid of 22 kDa (p22) was processed into active forms of tBid, which
showed apparent molecular sizes of 15 and 13 kDa (lane 2, p15 and p13). Of significance, Bid processing was not inhibited by
Bcl-2 co-transfection (lane 3).
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Fig. 4.
Bcl-2 does not prevent tBid translocation to
the organellar fraction. HeLa cells transfected with Bid or Bid + Bcl-2 were fractionated into cytosolic (Cyto) and
membrane-bound (Mem) organellar fractions and analyzed for
Bid/tBid by immunoblotting. The majority of full-length Bid (p22) was
detected in the cytosol (lane 1), and tBid of
p15/p13 translocated to the organellar fraction (lane 3).
Bcl-2 co-transfection did not prevent tBid accumulation in the
organellar fraction with mitochondria (lane 4).
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Fig. 5.
Bcl-2 prevents membranous insertion of
tBid. HeLa cells were transfected with Bid only or Bid + Bcl-2.
Membrane-bound organellar fractions enriched with mitochondria were
collected from these two groups of cells and subjected to alkaline
treatment with 0.1 M NaHCO3, pH 11.5. Alkaline
incubation strips off the proteins that are not integrated into the
membranes, whereas integral proteins remain (28). A, whole
membrane fractions (lanes W1 and W2),
alkaline-released proteins (lanes S1 and S2), and
alkaline-resistant proteins (lanes P1 and P2)
were analyzed for Bid/tBid (upper panel) and cytochrome
oxidase IV (lower panel). Alkaline treatment released
a significant portion of p15 tBid from the membrane fraction of Bid + Bcl-2-co-transfected cells (lane S2); much less p15 tBid was
released from Bid-only transfected cells (lane S1).
Cytochrome oxidase IV, an integral mitochondrial protein, was
not released during alkaline treatment, regardless of the presence or
absence of Bcl-2 (lanes S2 and S1). *, a Bid
antibody-reactive band that was occasionally detected in
Bcl-2-transfected cell lysates. B, immunoblot signals from
four separate experiments were quantified by densitometry, and the p15
tBid released during alkaline incubation was expressed as a percentage
of total (mean ± S.D.; n = 4). The results
suggest that membranous integration of tBid was suppressed by
Bcl-2.
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Fig. 6.
Co-immunoprecipitation analysis of Bcl-2 and
Bid/tBid. HeLa cells were transfected with Bid + Bcl-2. One group
of cells was directly extracted with RIPA buffer to collect whole cell
lysate (W), and the other group was sequentially extracted
with digitonin and RIPA buffer to collect the cytosolic fraction
(C) and the membrane-bound organellar fraction
(M). The extracts were directly (IB), or
subjected to immunoprecipitation (IP) with a monoclonal
Bcl-2 antibody followed by detection of Bid/tBid in the precipitates by
immunoblotting (A). Intact Bid was detected in Bcl-2
immunoprecipitates from whole cell lysates, cytosolic fractions, as
well as the membrane fractions; however, tBid was not shown in any of
the precipitates (lanes 1-3). The absence of tBid was not
due to the incapability of RIPA buffer to extract the molecules. As
shown in lanes 4 and 6, p15 tBid was detected in
RIPA extracts prior to immunoprecipitation. The results suggest that
Bcl-2 did not co-immunoprecipitate p15 tBid, although membranous insert
of this molecule was suppressed by Bcl-2 (Fig. 5). The results in
C indicate that, although Bcl-2 transfection led to its
expression mainly in the membrane-bound fraction, there were limited
amounts of Bcl-2 in the cytosol.
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Fig. 7.
Suppression of Bid-induced Bax translocation
by Bcl-2. Control cells, cells transfected with Bid, and
cells transfected with Bid + Bcl-2 were fractionated into
cytosolic (Cyto) and membrane-bound organellar fractions
(Mem) for immunoblot analysis of Bax and Bak. In control
cells without gene transfection, the majority of Bax was detected in
the cytosol (lane 1), with weak signal in the membrane-bound
organellar fraction (lane 4). Bid transfection led to the
loss of cytosolic Bax (lane 2), accompanied by increases in
organellar Bax (lane 5). Bcl-2 co-transfection suppressed
the translocation of Bax. In these cells, Bax was detected mainly in
the cytosol (lane 3), with weak signal in the organellar
fraction (lane 6). Bak was always detected in the
membrane-bound organellar fractions, regardless of the
transfection of Bid or Bid + Bcl-2 (blots in the
lower panel).
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Fig. 8.
Inhibition of Bid-induced Bak and Bax
oligomerization by Bcl-2. Cells were transfected with Bid, Bid + Bcl-2, or empty vectors. The cells were subjected to cross-linking by
DSP, followed by fractionation to collect membrane-bound organellar
fractions including mitochondria. Proteins in the fractions were
resolved by electrophoresis under non-reducing conditions for
immunoblot analysis of Bax (A) and Bak (B).
Images in the left and right panels were obtained
by short and long film exposures, respectively. Bid transfection led to
oligomerization of Bax and Bak; Bcl-2 co-transfection diminished the
oligomerizations.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced apoptosis (32). In our experiments, cells
were not cross-linked before extraction. Thus, it is possible that
Bcl-2 and tBid indeed bound each other, and their interaction was
disrupted during extraction and sample preparation. Although this
possibility needs to be tested by further experiments, our results
showed that the interaction between Bcl-2 and intact Bid was not
disrupted during sample preparation under the experimental conditions
(Fig. 6A).
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ACKNOWLEDGEMENT |
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We thank Dr. M. A. Venkatachalam at the University of Texas Health Science Center, San Antonio, TX, for stimulating discussions on this work.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK58831, the American Society of Nephrology, and the National Kidney Foundation.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.
¶ Supported in part by National Institutes of Health Grants CA74885 and CA83817.
Carl W. Gottschalk Research Scholar of the American
Society of Nephrology. To whom correspondence should be addressed:
Dept. of Cellular Biology and Anatomy, Medical College of Georgia, 1459 Laney Walker Blvd., Augusta, GA 30912. Tel.: 706-721-2825; Fax: 706-721-6120; E-mail: zdong@mail.mcg.edu.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M300039200
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ABBREVIATIONS |
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The abbreviations used are: BH, Bcl-2 homology; DSP, dithiobis(succinimidyl propionate); BMH, bismaleimidohexane; BSOCOES, bis[2-(succinimidyloxycarbonyloxy)-ethyl] sulfone; GFP, green fluorescence protein.
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