From the Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba R3E 0V9, Canada
Received for publication, September 14, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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Many cell death pathways converge at the
mitochondria to induce release of apoptogenic proteins and permeability
transition, resulting in the activation of effector caspases
responsible for the biochemical and morphological alterations of
apoptosis. The death receptor pathway has been described as a triphasic
process initiated by the activation of apical caspases, a mitochondrial phase, and then the final phase of effector caspase activation. Granzyme B (GrB) activates apical and effector caspases as well as
promotes cytochrome c (cyt c) release and loss
of mitochondrial membrane potential. We investigated how GrB affects
mitochondria utilizing an in vitro cell-free system and
determined that cyt c release and permeability transition
are initiated by distinct mechanisms. The cleavage of cytosolic BID by
GrB results in truncated BID, initiating mitochondrial cyt
c release. BID is the sole cytosolic protein responsible
for this phenomenon in vitro, yet caspases were found to
participate in cyt c release in some cells. On the other
hand, GrB acts directly on mitochondria in the absence of cytosolic
S100 proteins to open the permeability transition pore and to disrupt
the proton electrochemical gradient. We suggest that GrB acts by two
distinct mechanisms on mitochondria that ultimately lead to
mitochondrial dysfunction and cellular demise.
Granzyme B (GrB)1 is an
aspartyl serine protease located in the granules of cytotoxic T
lymphocytes and natural killer cells that induces apoptosis (1, 2).
Upon cytotoxic T lymphocyte degranulation at the interface with a
target cell, GrB crosses the plasma membrane and enters the cytoplasm
aided by perforin (3, 4). Execution of apoptosis by GrB requires the
participation of the caspase family of proteases (5, 6), which are
central regulators of the apoptotic pathway for multiple cell death
signals (7, 8). Recent work suggests that mitochondria play an
important if not key role in the regulation of cell death in mammalian
cells (9-13). Apoptotic signals induce mitochondria to release
cytochrome c (cyt c), which interacts with
APAF-1 in an ATP-dependent manner, and caspase 9 (14, 15) to form an active holoenzyme that processes and activates
downstream caspase 3 (16). Cells deficient in APAF-1 or caspase 9 are
resistant to some apoptotic signals that are thought to require
mitochondrial cyt c release (17, 18).
The signals that induce cyt c release are often propagated
through members of the BCL-2 family of pro-apoptotic proteins. Translocation from the cytoplasm to mitochondria during induction of
apoptosis has been reported for BID (19, 20), BAX (21), BAK (22), BAD
(23, 24), BIM (25, 26), and NOXA (27). These molecules can be regulated
by phosphorylation, dimerization, or proteolytic cleavage (10, 24, 28,
29). For example, BAX, BAK, and BIM are held inactive in the cytoplasm
and are translocated to the mitochondria after a cell death signal.
Following Fas ligation and recruitment of FADD to the trimeric receptor
complex, caspase-8 is activated and then cleaves cytoplasmic BID,
producing a truncated fragment (tBID) that is able to translocate to
mitochondria to induce cyt c release (19, 20). BID is also
cleaved by GrB, although the function of the cleaved form has not yet
been defined (19, 30).
In response to stress signals such as Ca2+ or reactive
oxygen species as well as most apoptosis signals, the mitochondrial
membrane opens to allow solutes and water to enter the matrix in a
phenomenon termed permeability transition (PT) (12, 31). It has been proposed that this is a result of the opening of a multiprotein complex
spanning the inner and outer membranes of mitochondria known as the PT
pore complex (12, 13, 31). The most abundant components of the PT pore
are the voltage-dependent anion channel/porin and adenine
nucleotide translocator, plus proteins such as cyclophilin D, creatine
kinase, and others. Upon PT pore opening, mitochondria lose their
mitochondrial membrane potential ( Although one model of GrB action suggests that it directly cleaves and
activates the downstream caspase-3 (6, 36) and would thus mediate
apoptosis directly without the need for mitochondria, in earlier work,
we found that GrB and perforin rapidly induce cyt c release
and decrease In this study, we demonstrate that both APAF-1 and caspase 9, and
therefore, a mitochondrial cyt c pathway, are required for efficient GrB-induced apoptosis of murine embryonic fibroblast (MEF)
cells. GrB mediates cyt c release from isolated mitochondria in vitro through an S100 protein that we identify as the
BCL-2 family member BID. Furthermore, GrB in the absence of BID or
other S100 proteins can open the mitochondrial PT pore with the loss of
Cell Lines and Reagents--
HeLa cells were cultured in
Dulbecco's minimal essential medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum (Cansera). MEFs deficient in
APAF-1 or caspase 9 were cultured as described previously (17). Rat GrB
and perforin were isolated from RNK-1 cells as described previously
(2). Recombinant GrB (rGrB) and mutant inactive GrB (S203A) were a gift
of Dr. Tim Ley (Washington University Medical School, St. Louis, MO).
Yeast-derived rGrB has been shown to have the same proteolytic activity
and specificity and response to inhibitors as purified wild-type GrB (38). In addition, rGrB can replace the lost apoptotic activity of
cells deficient in
GrA
The fluorophores calceinAM, tetramethylrhodamine (TMRM), rhodamine 123, and MitoTrackerTM Green TM were obtained from Molecular
Probes, Inc. (Eugene, OR). The caspase inhibitors
benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-fmk),
acetyl-Asp-Glu-Val-Asp-fmk and acetyl-Tyr-Val-Ala-Asp-fmk were from
Peptides International Inc. (Louisville, KY), and the control peptide
Z-Phe-Ala-fmk was from Enzyme System Products (Livermore, CA).
Cyclosporin A (CsA) was purchased from Sigma.
Mitochondrial Isolation and S100 Preparation--
For each
experiment, fresh mitochondria were isolated from mouse liver with six
strokes of a Dounce homogenizer before purifying on a Percoll gradient
as described previously (40). The mitochondria were kept on ice in
HB (300 mM sucrose, 5 mM TES, and 0.2 mM EGTA, pH 7.2) and used within 4 h of isolation.
Murine liver S100 was prepared by collecting the supernatant at the
8740 × g step and centrifuging it twice at 14,400 × g for 10 min before a final centrifugation at
100,000 × g for 1 h at 4 °C.
S100 proteins from HeLa cells were prepared by harvesting cells at the
log phase of growth and washing four times with Hanks' balanced salt
solution and 10 mM HEPES, pH 7.2, before resuspension at
108 cells/ml in HB plus 0.2% bovine serum albumin. The
cells were lysed by 2-4-s bursts of a Polytron homogenizer (setting
6), and the cell debris was removed by centrifugation at 792 × g for 10 min, followed by centrifugation of the supernatant
twice at 14,400 × g for 15 min and finally at
100,000 × g for 1 h at 4 °C.
Cytochrome c and AIF Release Assay--
Freshly isolated
mouse liver mitochondria (1 mg/ml) were incubated with GrB and mouse
liver S100 (100 µg/ml), partially purified BID, or rBID in a V-bottom
96-well plate in a total volume of 60 µl of Mitochondrial (M)
buffer (220 mM sucrose, 68 mM mannitol, 10 mM KCl, 5 mM KH2PO4, 2 mM MgCl2, 0.5 mM EGTA, 5 mM succinate, 2 mM rotenone, and 10 mM HEPES, pH 7.2) at 37 °C for 2 h. After centrifugation at 760 × g for 5 min, 30 µl of the
supernatant was resolved on a 15% SDS-polyacrylamide gel as previously
described (37) and Western-blotted for cyt c, AIF, and BID.
The lanes labeled M in the figures represent the
maximum cyt c release and were prepared using an aliquot of
mitochondria in 60 µl of M buffer. Mouse monoclonal anti-cyt
c antibody (Pharmingen, Mississauga, Ontario, Canada) and
rat anti-BID antibody (19) were used with goat anti-mouse and goat
anti-rat peroxidase conjugates (Sigma), respectively, to develop the blots.
FPLC Purification of Cytochrome c-releasing Factor--
All
chromatography steps were carried out using an FPLC system (Amersham
Pharmacia Biotech), and samples were maintained on ice throughout.
Mouse liver S100 (30 ml from three livers) was applied to a self-packed
hydroxyapatite column (10-ml bed volume; HA-Ultrogel, BioSepra Inc.,
Marlborough, MA) equilibrated with buffer A (2 mM
phosphate, pH 7.0, with protease inhibitors (0.5 µg/ml aprotinin, 0.5 µM leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride)). The column was washed with 5 column volumes of buffer A and
eluted with a linear gradient of buffer B (buffer A plus 2 M NaCl). The fractions with GrB-mediated cyt
c-releasing activity were pooled. Solid
(NH4)2SO4 was directly added to the
active fraction (0.5 M final concentration). After
centrifugation at 10,000 × g for 10 min, the pool was
applied to a phenyl-Sepharose 6 column (HiPrep 16/10 phenyl (high
sub), Amersham Pharmacia Biotech). The column was equilibrated
with buffer C (buffer B plus 0.5 M (NH4)2SO4) and eluted with a linear
gradient of buffer D (10 mM phosphate, pH 7.5, with the
protease inhibitors listed above). The pooled active fractions were
concentrated to 2 ml with a Centricon-10 microconcentrator (Millipore
Corp., Bedford, MA) and diluted 10 times with buffer D before
application to a Mono Q column (HR 5/5, Amersham Pharmacia
Biotechnology), followed by elution with a linear gradient of buffer E
(buffer D plus 2 M NaCl). The highest activity fractions
were diluted 10 times with buffer D and applied to a second Mono Q
column, again eluting with 0-50% buffer E.
Immunofluorescent Microscopy--
Cytochrome c was
detected in HeLa cells with the mouse monoclonal anti-cyt c
antibody and a Cy3-conjugated goat anti-mouse secondary antibody
(Chemicon International, Inc., Don Mills, Ontario) as previously
described (37). When required, cells were pretreated for 15 min with 20 µM Z-VAD-fmk before GrB and perforin treatment. Image
analysis was performed with Northern Eclipse Version 5.0 software
(Empix Inc., Toronto, Ontario) as described (41).
Transmembrane Potential of Isolated Mitochondria Determined
by Fluorometry--
We used an in vitro cell-free
assay to examine the GrB effect on mitochondria (37, 42). Freshly
isolated mitochondria were added to a 96-well plate in M buffer in the
presence or absence of the S100 fraction and GrB and then incubated at
37 °C for the indicated times. Finally, 5 µM rhodamine
123 (excitation at 485 nm and emission at 538 nm) or 150 µM TMRM (excitation at 544 nm and emission at 590 nm) was
added, and the samples were read on a Titertek Fluoroskan II
fluorometer (MTX Labsystems, Vienna, VA).
PT Pore Opening and
Whole cells were grown on coverslips for 2 days and then washed and
incubated in Hanks' balanced salt solution, 10 mM HEPES, and 4 mM NaH2CO3, pH 7.4, for 90 min with GrB and sublytic amounts of perforin. When required,
cells were pretreated with 20 µM CsA for 30 min. The
cells were stained with 1 µM calceinAM and 5 mM CoCl2 for 15 min at 23 °C.
CoCl2 quenches the cytoplasmic calcein fluorescence and
allows visualization of mitochondria (43). Cells were washed four times
and resuspended in Hanks' balanced salt solution and 10 mM
HEPES, and calcein images were captured on an Olympus IX70 inverted
confocal laser microscope using Fluoview 2.0 software and a band bypass
filter of 488 nm, with Nomarski optics for transmitted light
images of the same cells. The fluorescence of individual cells was
determined using Northern Eclipse Version 5.0 software.
Enhanced Resistance of apaf-1 Granzyme B-induced Cytochrome c Release in Vitro--
As
APAF-1/caspase 9-directed apoptosis is dependent on cyt c
(15), we established an in vitro cell-free system using
purified murine liver mitochondria and S100 to further examine GrB
regulation of cyt c release (see "Experimental
Procedures"). We found that GrB in the presence of S100 initiated cyt
c release from isolated mitochondria within 30 min (Fig.
2A) and in a
dose-dependent manner (Fig. 2B), whereas GrB or
S100 alone had no effect (Fig. 2A). Thus, we assume that GrB
must act through a factor(s) in S100 that exerts its effect on
mitochondria. To determine whether caspases are important for the
release of cyt c, the peptide caspase inhibitors DEVD-fmk, YVAD-fmk, Z-VAD-fmk, and control Z-FA-fmk at concentrations previously shown to inhibit caspase activity (37) were mixed with S100
before the addition of GrB. None of the inhibitors was able to block
cyt c release (Fig. 2C). We also investigated
whether another mitochondrial intermembrane protein, AIF, was released along with cyt c and determined that it was not (Fig.
2D), suggesting that cyt c was released in a
restricted manner and was not part of the general permeabilization of
the outer mitochondrial membrane.
Previously, we had demonstrated in HeLa cells that GrB-mediated cyt
c release was significantly inhibited by the pan caspase inhibitor Z-VAD-fmk (37), whereas our current in vitro data suggested that GrB was acting by a caspase-independent mechanism. To
determine whether the requirement for caspases differs between cell
types or between intact cells and the in vitro cell-free assay, we compared the caspase dependence of GrB-induced cyt
c release in whole HeLa cells with the in vitro
assay using liver mitochondria and HeLa cell S100 over a dose range and
time course. We found that Z-VAD-fmk significantly blocked cyt
c release from mitochondria in intact HeLa cells following
GrB and perforin treatment over a wide dose range and up to 8 h of
treatment (Fig. 2, E and F). The most pronounced
effects were seen at 2 h with GrB doses between 400 and 800 ng/ml,
but inhibition at other doses and times was always >50% of that of
the untreated cells. In contrast, Z-VAD-fmk had little effect on cyt
c release in vitro either in a HeLa S100 dose
response or over an extended time course (Fig. 2, G and
H).
Granzyme B Cleaves BID Protein--
We then started to purify the
protein in mouse liver S100 that mediates cyt c release by
passing S100 through sequential FPLC columns, hydroxyapatite,
phenyl-Sepharose, and Mono Q (see "Experimental Procedures").
Positive fractions from the hydroxyapatite column were pooled and
loaded on the phenyl-Sepharose column, and then the positive eluted
fractions were loaded on the Mono Q column (Fig.
3A). We analyzed the Mono Q
fractions for GrB-induced cyt c release and BID, a
pro-apoptotic protein and GrB substrate known to mediate Fas signaling
to the mitochondria through caspase 8. The peak activity fractions
coincided exactly with the elution of BID (Fig. 3A).
It has been reported that GrB can cleave BID (19, 30). We confirmed
these data by showing that BID in S100 was rapidly cleaved to a p15
truncated form (tBID) by rat GrB or mouse rGrB in a time- and
dose-dependent manner (Fig. 3B). rGrB cleaved
BID and induced cyt c release at similar doses using either
BID-containing Mono Q fractions or whole S100 (Fig. 3C). GrB
proteolytic activity was required for BID cleavage, as rGrB with an
inactivating mutation (S203A) failed to cleave BID and to induce cyt
c release (Fig. 3C).
Granzyme B Uses BID to Induce Cytochrome c Release in Isolated
Mitochondria--
To determine whether BID protein in S100 is required
for GrB-induced cyt c release, we immunoprecipitated BID
with rat antiserum. BID was highly depleted from S100 by the rat
antiserum, but not by normal rat serum (Fig.
4A). The treated sera were
then serially diluted and added to mitochondria with a constant dose of
GrB, and cyt c release was measured. The BID-depleted serum
was unable to support GrB-induced cyt c release compared
with controls (Fig. 4B).
The experiments to this point supported the hypothesis that the S100
protein BID mediates cyt c release. If this is correct, then
we would predict that BID protein alone can replace S100. Thus, we next
examined the effect of purified rBID protein on cyt c
release in the presence and absence of GrB. GrB induced mitochondria to
release cyt c in the presence of BID protein, and the effect
was dependent on both GrB and BID concentration (Fig. 4, C
and D). We found that BID protein at higher levels induced
cyt c release in the absence of GrB; however, at BID doses that were ineffective alone, a GrB-dependent BID-mediated
cyt c release was observed. The cyt c release in
the absence of GrB is not due to contaminating caspases, as it has been
shown previously that higher doses of full-length BID can also cause
cyt c release by an unknown mechanism (19, 20). However,
tBID is 100 times more efficient at inducing the release of cyt
c compared with full-length BID (19).
Granzyme B Induces Permeability Transition and
GrB could induce the loss of
We next determined whether GrB-induced opening of the PT pore in cells
was the same as that observed in isolated mitochondria (Fig.
6C). GrB plus perforin treatment of HeLa cells induced a decrease in mitochondrial calcein fluorescence in comparison with either of the untreated controls (Fig. 6D), GrB alone, or
perforin alone (data not shown). The addition of CsA blocked the GrB-
and perforin-induced calcein release from mitochondria (Fig.
6D). Using image analysis software, we were able to
determine the relative fluorescent intensity of individual cells and
the number of cells with high, medium, and low mitochondrial
fluorescence. The percentage of cells with low calcein
fluorescence (calceinlo) increased from 9 to 71% following
GrB and perforin treatment and was decreased to 18% by CsA.
Corresponding shifts in populations with medium
(calceinmed) and high (calceinhi) calcein
fluorescence levels were observed with these treatment conditions. This
experiment was repeated three times with similar results, and one
example is shown in Fig. 6D.
We observed that GrB acts on mitochondria directly to cause
We have examined how GrB mediates apoptosis by disrupting
mitochondrial function and have determined that GrB acts through two
distinct mechanisms: (i) cleaving the cytosolic protein BID, resulting
in the release of cyt c, and (ii) acting directly on mitochondria to cause PT and loss of Candidate proteins mediating the regulation of cyt c release
come from the BCL-2 family, which contains both anti-apoptotic (BCL-2 and BCL-XL) and pro-apoptotic (BAX, BAK, BID, etc.)
proteins that either directly or indirectly affect mitochondrial
function through heterodimerization (for reviews, see Refs. 52-55). In
our search for the proteins involved in regulating GrB-induced cyt c release in vitro, we determined that BID was
the only cytosolic protein required. BID has been shown to interact via
its BH3 domain with the BCL-2 and BCL-XL domains and the
BAX BH1 domain (56-58). Precedence for BID in mitochondrial signaling
comes from the Fas/CD95 death pathway, in which caspase 8-cleaved BID
is responsible for cyt c release as well as the subsequent
apoptotic cellular changes resulting from holoenzyme formation and
activation of downstream caspases (19, 20, 59, 60). In vitro
BID BH3 mutants and BID-depleted cell extracts have reduced capability
to release cyt c (20, 56). Our observations that GrB-induced
cyt c release requires BID and that APAF-1 and caspase 9 are
necessary for efficient apoptosis support the idea that GrB may use a
mitochondrial pathway similar to Fas/CD95 in which GrB cleavage of BID
leads to the activation of downstream caspases.
In the cell-free assay, we demonstrated that caspases were not
required, as GrB and rBID induced cyt c release in the
absence of any other S100 proteins. Also, caspase inhibitors failed to block cyt c release when S100 was present, supporting a
model in which BID is directly cleaved by GrB. This is in agreement with previously published data in Jurkat cells, where cyt c
release following GrB and perforin treatment was found to be
caspase-independent (44), and BID was cleaved within 60 min in the
presence of Z-VAD-fmk (30). However, we have also demonstrated that
Z-VAD-fmk, while efficiently inhibiting cyt c release in
HeLa cells, fails to block release in vitro using S100 from
HeLa cells and liver mitochondria (37). This indicates that in some
cells, GrB operates primarily through a caspase-dependent
mechanism, although in others such as Jurkat, it primarily targets
BID. Why the caspase dependence of cyt c release in HeLa
cells is not duplicated in the in vitro assay is not clear,
but suggests that in a disrupted cell, GrB prefers BID as a substrate
or that the caspase targeted by GrB is not present in the S100
fractions of the HeLa cells. We were also unable to determine whether
the participation of caspases in cyt c release in HeLa cells
is a result of direct processing of a caspase by GrB or is secondary to
GrB-induced BID processing in a feed-forward amplification from the
mitochondrial activation of downstream caspases. Whether in the
presence or absence of active caspases, it appears that BID is a
pivotal protein in GrB-mediated apoptotic cellular changes.
The mechanism of cyt c release from mitochondria has not
been determined; however, several theoretical models have been
suggested such as the release through the PT pore (34) or a cyt
c-specific channel (61) or a nonspecific release from
rupturing of the outer mitochondrial membrane due to swelling from PT
(62). GrB-induced cyt c release through the PT pore is
unlikely, as the release is not blocked by PT pore inhibitors CsA and
bongkrekic acid.2 The
mechanism is also not due to outer membrane rupture, as we did not see
a coordinated release of cyt c and other intermembrane proteins such as AIF. This suggests that BID must be acting on some
other protein associated with the mitochondria. BID is a cytosolic
protein until a death signal results in its cleavage to the truncated
form (tBID) that is translocated to the mitochondria. Using rBID, we
found that it is cleaved directly by GrB as predicted since BID
contains a single GrB cleavage site (72IEPDS76
for murine and 72IEADS76 for humans) and has
been reported to be directly cleaved by GrB at this site (19, 63) and
during GrB-mediated apoptosis (30). The current model suggests that
mitochondrial membrane-bound death antagonists BCL-2 and
BCL-XL and agonist BAX can function independently of each
other, but compete for tBID as a ligand to block or induce cyt
c release (57, 63-65). When bound by tBID, BAX undergoes a
conformational change enabling its integration into the mitochondrial membrane, resulting in cyt c release (64). Furthermore,
BAX-induced cyt c release has been shown to be independent
of the PT pore (66), similar to our observations with GrB-induced cyt
c release. Another possibility for the action of BID can be
found in its tertiary structure, which resembles BAX and the
pore-forming domains found in the bacterial toxins diphtheria and
colicin (63). It has been demonstrated that BID can form pores in
membranes under low pH conditions (67), so perhaps BID is sufficient to
induce cyt c release by itself under certain conditions.
It is noteworthy that cytochrome c release and PT,
accompanied by loss of Although our in vitro assays indicate that S100 (and
therefore, any BID it contains) is not required for GrB to suppress
We can now view GrB-induced apoptosis as a three-phase process similar
to other death signals in which the pre-mitochondrial phase is followed
by convergence at the mitochondria, resulting in the release of
pro-apoptotic factors that activate the post-mitochondrial effector
phase. Although cell death pathways exist that can bypass the
mitochondria such as in the Fas/CD95 type I apoptotic program, disruption of mitochondrial function through an amplification feedback
loop is still observed. Downstream caspase 3 can activate upstream
caspase 8, which leads to efficient BID cleavage and mitochondrial
dysfunction (68). We do not yet know if GrB can bypass mitochondria and
then feedback in a similar manner.
GrB acts on many substrates in the cell, including several caspases,
BID, ICAD, and PARP, suggesting that GrB could potentially act
on all simultaneously, inducing death by multiple mechanisms to
guarantee the cells' demise. However, there appear to be dominant pathways such as the caspase- and mitochondrial-dependent
pathways; and if blocked, then cell death could proceed more slowly via secondary pathways. For example, if neither the mitochondrial nor
caspase pathway is utilized, then GrB could inactivate ICAD directly to
cause CAD activation, ensuring DNA fragmentation, albeit in a
delayed and less efficient manner (39). The presence of the dominant
mitochondrial pathways mediates rapid and efficient apoptosis; but
clearly, there are other apoptosis mechanisms that come into play when
the mitochondrial pathway is rendered inactive. In
conclusion, we have demonstrated that GrB disrupts the mitochondria through two very distinct mechanisms: cleavage of the cytosolic protein
BID, resulting in release of cyt c; and the induction of PT
and loss of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m) across the inner
membrane, which is reported to be an early response to many apoptotic
signals (32). BCL-2 family pro-apoptotic proteins that translocate to
the outer mitochondrial membrane such as BAX are reported in some
models to physically interact with the voltage-dependent anion channel and adenine nucleotide translocator, and it has been
proposed that this results in PT pore opening and cyt c
release (33-35). However, the exact mechanism of cyt c
release remains controversial and unresolved.
m before or coincident with apoptosis (37).
Furthermore, in an in vitro cell-free system, the addition
of mitochondria to S100 increases GrB-induced nuclear chromatin
condensation 15-fold, indicating that mitochondria have a powerful
amplification effect on GrB function (37). This activity requires
proteolytically active GrB. These observations suggest a role for
mitochondria in the GrB apoptotic response.
m. Thus, we have identified two mechanisms by which GrB
disrupts mitochondrial function and promotes cell death.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
GrB
/
(39).
Purified recombinant BID (rBID) protein and rat anti-BID antiserum were
a gift of Dr. Junying Yuan and have been described previously (19).
m Determined by Confocal
Laser Imaging in Cells and Purified Mitochondria--
Freshly isolated
mitochondria were centrifuged onto coverslips at 1475 × g for 5 min at 4 °C, washed, resuspended in M buffer, and
then stained for 15 min with 100 nM tetramethylrhodamine
and 8 µM calceinAM. After four washes, the mitochondria
were resuspended in M buffer, and GrB was added. When required,
mitochondria were pretreated with CsA 30 min before GrB treatment. The
mitochondria were imaged, and total fluorescence was determined on an
Olympus IX70 inverted confocal laser microscope using Fluoview 2.0 software (Carson Group Inc., Markham, Ontario).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
and caspase
9
/
MEFs to Granzyme B-induced Apoptosis--
GrB can
induce mitochondrial cyt c release and loss of
m (30, 37, 44), and mitochondria can amplify nuclear apoptotic responses to GrB in vitro (37). Although this
suggests that mitochondria may be important regulators of GrB-induced
apoptosis, it is possible that the observed mitochondrial dysregulation
is secondary to caspase activation by GrB and is not responsible for
the induction of apoptosis (6). To test this hypothesis, we examined
apaf-1
/
or caspase
9
/
MEF cells, which are highly resistant to
many apoptotic death signals except Fas/CD95, which can bypass
mitochondria and directly activate downstream caspases (17, 18). If GrB
were able to bypass mitochondria, then a deficiency of either protein
would have no effect on GrB apoptosis. We therefore treated
apaf-1
/
or caspase
9
/
MEFs with GrB and perforin and measured
apoptosis by Hoechst staining of condensed chromatin. Fig.
1 (A and B)
illustrates that both apaf-1
/
and caspase 9
/
MEFs were more
resistant to GrB compared with normal MEFs over a wide dose range.
Adriamycin was also unable to initiate apoptosis in the deficient cells
(data not shown) (18), thus confirming an earlier report (17). The MEFs
were not completely resistant to higher doses of GrB, indicating that
part of the GrB apoptotic activity did not require either APAF-1 or
caspase 9. When examining the kinetics of GrB-induced nuclear changes
(Fig. 1B), nuclear condensation of the wild-type MEFs
occurred rapidly, as almost 60% were apoptotic at 2 h, and a
plateau of 80% apoptosis was reached by 4 h of treatment. In
contrast, apoptosis of the
apaf-1
/
and caspase
9
/
MEFs was 15% of the wild-type levels in
the first hours of the assay and then slowly increased, so by 24 h, it was ~40% of the wild-type MEF levels. Clearly, GrB-induced
nuclear apoptosis is most efficient when utilizing APAF-1 and caspase 9 on the mitochondrial-dependent pathway.
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Fig. 1.
Granzyme B-induced apo ptosis of
apaf-1 /
and caspase
9
/
MEFs. A, MEF cells from
apaf-1
/
(
), caspase
9
/
(
), and parental (wild-type;
)
mice were incubated with GrB at the indicated doses plus perforin (0.12 µg/ml) or with perforin (Pf) alone for 2 h and then
stained with Hoechst dye, and apoptotic cells were enumerated. The data
represent means ± S.E. from three experiments. B, MEFs
were treated and analyzed as described for A using GrB (2 µg/ml) plus perforin (0.12 µg/ml) over a 24-h time course. The
closed symbols are the same as described for A,
and the open symbols represent MEFs treated with perforin
only. The data represent means ± S.E. from three
experiments.
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Fig. 2.
Granzyme B-induced cytochrome c
release in an in vitro cell-free system.
A, purified mouse liver mitochondria were incubated with GrB
(0.7 µg/ml) and mouse liver S100 (100 µg/ml) for the indicated
times. The supernatant was recovered after centrifugation and
Western-blotted for cyt c. M is a control from
untreated mitochondria (in A, C, G,
and H). B, GrB at the indicated doses was
incubated with purified mitochondria and S100 for 2 h and then
analyzed as described for A. C, peptide caspase
inhibitors (50 µM) were preincubated with S100 for 15 min
before adding GrB and mitochondria for 2 h, and then cyt
c release was analyzed as described for A. D, mitochondria were treated as described for B
and Western-blotted for AIF. E, HeLa cells were
preincubated with ( ) and without (
) 50 µM Z-VAD-fmk
for 30 min before treatment with perforin (0.2 µg/ml) and GrB at
various doses. Cyt c release was detected by immunostaining
cells with anti-cyt c antibody plus the Cy3-conjugated
secondary antibody. Cells were imaged by fluorescent microscopy, and
the number of cells staining positively for cyt c were
counted. The data represent means ± S.E. from three experiments.
F, HeLa cells were treated and analyzed as described for
E using GrB (1 µg/ml) plus perforin (0.2 µg/ml) (
) or
perforin only (
) over an 8-h time course. The open
symbols represent cells that receive a 50 µM
Z-VAD-fmk treatment for 30 min. The data represent means ± S.E.
from three experiments. G, purified mouse liver mitochondria
and serially diluted HeLa S100 were preincubated, separately, with 50 µM Z-VAD-fmk for 15 min before adding GrB (0.7 µg/ml)
for 2 h. Cyt c release was analyzed as described for
A. H, purified mouse liver mitochondria and HeLa
S100 were preincubated, separately, with 50 µM Z-VAD-fmk
for 15 min before adding GrB (0.7 µg/ml) over a 3-h time course. Cyt
c release was analyzed as described for A. A
Western blot for actin was performed as a loading control.
View larger version (20K):
[in a new window]
Fig. 3.
Identification of BID in S100 protein as the
mediator of granzyme B-induced cytochrome c
release. A, BID in mouse liver S100 copurifies
with granzyme B-induced cyt c releasing activity. Mouse
liver S100 was sequentially passed through hydroxyapatite,
phenyl-Sepharose and two Mono Q FPLC columns (see "Experimental
Procedures"). Fractions able to support cyt c release in
the presence of GrB (heavy bars) were pooled and loaded on
the next column. Mono Q fractions were analyzed for BID protein and
GrB-induced mitochondrial cyt c release by Western blotting.
B is the sample before loading, and C is the
sample not binding to the column. B, shown are the results
from GrB hydrolysis of BID protein in S100. GrB (0.7 µg/ml) was
incubated with mouse liver S100 and then Western-blotted for BID.
C, the partially purified Mono Q fractions shown in
A or murine liver S100 was added to mitochondria and rGrB or
mutant inactive rGrB (S203A) at the indicated doses for 2 h, and
then the supernatant was Western-blotted for cyt c or BID.
M is a control from untreated mitochondria.
View larger version (26K):
[in a new window]
Fig. 4.
BID is necessary and sufficient for granzyme
B-induced cytochrome c release from isolated
mitochondria. A, mouse liver S100
(Control) was incubated with rat anti-BID antiserum or
normal rat serum and immunoprecipitated. S100 was then Western-blotted
for BID. B, GrB (0.7 µg/ml) was added to mitochondria and
serially diluted S100 fractions treated as described for A
for 2 h, and then supernatants were Western-blotted for cyt
c. M is a control from untreated mitochondria (in
B and D). C, rBID was added to
serially diluted GrB plus mitochondria for 2 h, and the
supernatants were Western-blotted for cyt c. D,
serially diluted rBID was added to mitochondria in the presence or
absence of GrB and incubated for 2 h before Western blot analysis
of supernatants for cyt c.
m
Suppression--
PT and suppression of
m occur as a
result of GrB and perforin treatment of cells (37, 44). To determine
the proteins required for
m loss, we utilized a
fluorescent in vitro cell-free assay in which we could
combine mitochondria with GrB in the presence or absence of the murine
liver S100 fraction. We observed a significant suppression of
m whether or not S100 was present, indicating that no
cytosolic protein was required (Fig.
5A). The response was
dependent on the dose of GrB, with a maximum effect at 3-4 µg/ml
(Fig. 5B). A minor but inconsistent inhibitory effect of
S100 on
m was observed at some concentrations. The
earliest detectable
m dissipation was observed at 30 min
after the start of GrB (3 µg/ml) treatment and was complete by
90-120 min (Fig. 5C).
View larger version (15K):
[in a new window]
Fig. 5.
Granzyme B acts directly on isolated
mitochondrial to suppress
m. A, freshly
isolated mitochondria were treated with GrB, and
m was
determined by measuring rhodamine 123 fluorescence.
m
suppression occurred in the presence (white bars) or absence
(black bars) of the S100 fraction after a 1-h treatment with
GrB. B, shows is the effect of GrB concentration on
m after a 1-h incubation with mitochondria in the absence
of S100. C, shown is a time course of GrB (3 µg/ml)-mediated
m suppression in the absence of
S100.
m by opening the PT pore. To
examine the state of the PT pore, we used the fluorescent dye
calceinAM (45). CalceinAM is freely permeable across cellular membranes, but becomes fluorescent and impermeable upon cleavage by
intracellular esterases, which prevents its exit from mitochondria until the PT pore is opened. Isolated mitochondria were loaded with
calceinAM and simultaneously stained with the potentiometric dye TMRM
to follow
m. The mitochondria were then treated with GrB
and analyzed by confocal laser microscopy at increasing time periods
(Fig. 6, A and B).
Within 20 min, calcein and TMRM fluorescence was reduced to ~50% of
control levels, eventually falling to almost zero fluorescence by 40 min. To verify that the loss of fluorescence was due to opening of the
PT pore, the potent pore inhibitor cyclosporin A was added under the
same treatment conditions. CsA maintained calcein and TMRM fluorescence
near the control maximum levels well beyond 60 min of incubation (Fig. 6, A and B).
View larger version (56K):
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Fig. 6.
Granzyme B causes permeability transition in
mitochondria. A, freshly isolated murine
mitochondria were pretreated with or without 20 µM CsA
for 30 min and then stained with 8 µM calceinAM for
permeability transition and 100 nM tetramethylrhodamine
(TMRM) for m, before treatment with GrB (3 µg/ml) for the indicated times. The images and fluorescence levels of
treated mitochondria were determined by confocal laser microscopy.
B, shown is the quantitation of the confocal images shown in
A. The CsA-treated samples are the open symbols
for calceinAM (
) and TMRM (
). C, HeLa cells were
treated with 3 µg/ml GrB and 50 ng/ml perforin for 75 min and then
stained with 1 µM calceinAM and 5 mM
CoCl2 for 15 min before visualizing cells by confocal
microscopy (left panels) and Nomarski optics (right
panels). When required, cells were pretreated with 20 µM CsA 30 min prior to GrB treatment. D,
calcein fluorescence in cells from C was quantitated by
image analysis, and the percentage of cells measured as low
(CalceinLO), intermediate
(CalceinMED), or high (CalceinHI)
average fluorescence units (AFU) per cell is shown. The
experiment was repeated three times with similar results.
analysis
was carried out: p
0.001 for the comparison of GrB + perforin (black bars) versus the control
(white bars) and CsA + GrB + perforin (gray
bars).
m loss in the absence of any other cytosolic protein; however, it has been reported that the addition of BID can produce the
same effect (19, 30). We examined the effect of rBID on the relative
level of
m in isolated mitochondria treated with GrB
using TMRM fluorescence. We found that both GrB and rBID suppressed
m in a dose-dependent manner and that the combination of rBID and GrB reduced
m in an additive manner (Fig. 7, A and
B).
View larger version (18K):
[in a new window]
Fig. 7.
BID increases granzyme B-induced
m dissipation.
A, serially diluted GrB was added to freshly isolated mouse
mitochondria with or without rBID (100 ng/ml) for 2 h, and
m was determined by measuring rhodamine 123 fluorescence
levels.
, GrB alone;
, GrB + rBID. B, same as
in A, but serially diluted rBID was add to mitochondria with
or without GrB (1.5 µg/ml).
, rBID alone;
, rBID + GrB.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m. Since
mitochondria appear to be dysregulated in GrB-mediated apoptosis,
we examined whether GrB acts directly on the mitochondria or indirectly
through other proteins to perturb mitochondrial function. One
regulatory mechanism that is important for apoptosis is the activation
of the downstream effector caspase cascade via activation of caspase 9 during integration into the holoenzyme (15, 9, 46). Caspase 3 is
generally activated by caspase 9 in a
mitochondrial-dependent pathway; however, it has been
suggested that GrB can directly activate caspase 3 (36, 47, 48). In
this study, we show that the GrB mitochondrial-dependent
pathway is the dominant apoptotic pathway using
apaf-1
/
and caspase
9
/
MEF cells. Other death signals delivered
to apaf-1
/
and
caspase 9
/
MEFs have been shown
to reduce caspase 3 activation and to increase survival (18, 49). As
APAF-1 and caspase 9 require cyt c to form an active
holoenzyme for caspase 3 processing (50, 51), this suggests that cyt
c release from mitochondria may be a key event in
GrB-induced apoptosis. We were able to determine from the kinetics of
the nuclear changes induced by GrB in
apaf-1
/
and caspase
9
/
MEFs that only ~15-20% of the
apoptotic signal of wild-type cells is seen in the first few hours of
GrB and perforin treatment. Apoptosis occurs much more slowly; and by
24 h, apaf-1
/
and
caspase 9
/
MEF cell apoptosis
increases to ~40% of that seen in wild-type cells. These
observations led us to conclude that mitochondria are the major
regulators of GrB-induced apoptosis in MEFs. Whether the mitochondrial
pathway will be as important in other cell types remains to be determined.
m, were independent of one another
even though both were detected at similar times in response to GrB in
the in vitro cell-free assay. Both PT and
m
suppression occur in the absence of any cytosolic S100 protein,
indicating that caspases are not required and that GrB acts directly on
the mitochondrial outer membrane. The caspase independence of the GrB-mediated
m loss mechanism in a cell-free system is
similar to our findings in intact HeLa cells (37), which was confirmed
by Heibein et al. (44) in Jurkat cells. As CsA inhibits PT, we may assume that GrB acts on the PT pore or some other
protein associated with the pore. The PT pore acts as a gated
channel regulating mitochondrial matrix Ca2+, pH, volume,
and
m (33). Opening of the 1.5-kDa PT pore results
in PT and the equilibration of solutes across the inner membrane. This
can lead to uncoupling of the electron transport chain, loss of
m, inhibition of ATP synthesis, mitochondrial swelling,
and an increased reactive oxygen species production. The general serine
protease inhibitor phenylmethylsulfonyl fluoride can block GrB-mediated
PT and
m loss; therefore, we determined whether the PT
pore components voltage-dependent anion channel and adenine
nucleotide translocator as well as some of the proteins shown to bind
to or regulate the pore (BAX and BCL-2) were cleaved by GrB using
Western analysis. However, we were unable to demonstrate that GrB
cleaved any of these proteins (data not shown).
m, we found that rBID increases the loss of
m. This suggests either that the concentration of BID in
S100 is too low to act on the PT pore after GrB processing or that S100
contains another protein(s) that interferes with tBID-induced
suppression of
m. We are unable to determine whether or
not rBID and GrB are acting at the same or distinct sites on the
mitochondrial outer membrane.
m, which ultimately lead to the disruption of
mitochondrial function, assuring the death of the cell.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Elizabeth Henson, John Rutherford,
and Dr. Guangming Zhong (University of Manitoba) for assistance with
the fluorescent microscopy. We thank Drs. Junying Yuan and Alexi
Degertev for the gift of rBID protein and antiserum. We are grateful to
Dr. Tim Ley for rGrB and to Drs. R. Hakem and Tak Mak for the
apaf-1/
and caspase
9
/
MEF cells.
![]() |
FOOTNOTES |
---|
* This work is supported by the National Cancer Institute of Canada, the Canadian Institutes for Health Research, the Manitoba Health Research Council, and the George H. Sellers 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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Manitoba Inst. of Cell Biology, University of Manitoba, 675 McDermot Ave., Winnipeg, MB R3E 0V9, Canada. Tel.: 204-787-2112; Fax: 204-787-2190; E-mail: agreenb@cc.umanitoba.ca.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M008444200
2 L. Shi, J. B. Alimonti, and A. H. Greenberg, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GrB, granzyme B;
rGrB, recombinant granzyme B;
cyt c, cytochrome
c;
tBID, truncated BID;
rBID, recombinant BID;
PT, permeability transition;
m, mitochondrial membrane
potential;
MEF, murine embryonic fibroblast;
Z, benzyloxycarbonyl;
fmk, fluoromethyl ketone;
CsA, cyclosporin A;
TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid;
FPLC, fast protein liquid chromatography;
AIF, apoptosis-inducing
factor;
CAD, caspase-activated DNase;
ICAD, inhibitor of CAD;
PARP, polyCADP-ribose) polymerase;
TMRM, tetramethylrhodamine, Rh123,
rhodamine 123.
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