From the Laboratory of Cellular Neurobiology,
Massachusetts General Hospital and Harvard Medical School, Charlestown,
Massachusetts 02129, § Department of Immunology, SmithKline
Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, and
¶ Department of Medicine and Cell Biology, University of
Massachusetts Medical Center, Worcester, Massachusetts 01655
Received for publication, August 3, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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The recruitment and cleavage of pro-caspase-8 to
produce the active form of caspase-8 is a critical biochemical event in
death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human
fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was colocalized with
cytochrome c in mitochondria and was also distributed
diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in
nuclei. Pro-caspase-8 was found in the intermembrane space, inner
membrane, and matrix of mitochondria after limited digestion of
mitochondrial fractions, and this distribution was confirmed by
immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were
treated with an inhibitor of the ADP/ATP carrier atractyloside, which
opens the mitochondria permeability transition pore. Release was
blocked by the mitochondria permeability transition pore inhibitor
cyclosporin A (CsA). After clonal striatal cells were exposed for
6 h to an apoptotic inducer tumor necrosis factor- Apoptosis (programmed cell death) originally referred to an active
form of cell death with stereotypic morphological characteristics occurring during development (1). A broad range of pathological conditions can induce apoptosis (2, 3). Unbalanced cell proliferation
and apoptosis may play a role in pathogenesis of certain types of
tumors and neurodegenerative diseases (4, 5). A family of novel
cysteine proteases, named caspase, plays an essential role in most, if
not all, forms of apoptosis (6-8). Caspases are produced as
pro-enzymes and become activated by proteolytic cleavage at internal
aspartate residues upon apoptotic stimulation (9). Two categories of
caspases important for apoptosis have been recognized: the initiators
and executioners. The initiator caspases, which include caspase-8, -9, and -10, are activated in the earlier phase of apoptosis; the
executioner caspases, which include caspase-3, -6, and-7, are activated
by initiator caspases and are responsible for dismantling cells
(10, 11). Caspase-3 activation, a convergent event in apoptosis, is
triggered by a variety of apoptotic stimuli. Caspase-3 cleaves many
cytoskeletal proteins, such as fodrin, and proteins involved in DNA
repair and fragmentation, such as poly(ADP-ribose) polymerase and DNA fragmentation factor-45 (10, 12, 13). Two pathways lead to the
activation of caspase-3 through release of cytochrome c and
cleavage of pro-caspase-9 and through ligation of death receptors by
tumor necrosis factor (TNF)1
and Fas ligand. In the pathway stimulated by TNF and Fas ligand, pro-caspase-8 is recruited and activated by the adapter molecules FADD/MORT1 (14-17). Caspase-8 then directly activates
pro-caspase-3 and cleaves BID, a member of Bcl-2 family proteins
(18-20).
The role of mitochondria in apoptosis was first appreciated in a
cell-free system when nuclear apoptotic events were induced only by
cytoplasmic fractions enriched in mitochondria (21). A large body of
evidence now suggests that many apoptotic stimuli affect the
mitochondrial permeability transition pore (MPTP) and cause the release
of pro-apoptotic molecules such as cytochrome c and Apaf-1
from mitochondria (22-26). Cytochrome c and Apaf-1 activate
pro-caspase-9 to initiate an apoptotic cascade. Recent studies
indicate that caspase-2, -3, and -9 are also released from mitochondria
during apoptosis (23, 27-29). These findings suggest that mitochondria
may be more broadly involved in apoptosis than previously thought. The
subcellular distribution of pro-caspase-8 is not clear. Since knowledge
about the localization of pro-caspase-8 could be important in
understanding its role in apoptosis, we evaluated the subcellular
localization of pro-caspase-8 in human fibroblasts and in a mouse
clonal striatal cell line. The results suggest that pro-caspase-8 is
predominantly found in the intermembrane space and inner membrane of
mitochondria and can be released upon apoptotic stimulation.
Cell Culture--
Human fibroblasts obtained from Coriell Cell
Repositories were cultured in minimum Eagle's medium (Life
Technologies, Gaithersburg, MD) supplemented with 15% fetal
bovine, 100 units/ml penicillin G/streptomycin, 0.5 µg/ml
amphotericin B, 2 mM glutamine, 2× final concentration
minimum Eagle's medium vitamins, nonessential and essential amino
acids (all from Life Technologies) at 37 °C with 5%
CO2. Clonal mouse striatal cells were cultured in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 4.5 glucose, and 50 units/ml penicillin G/streptomycin (30).
Immunofluorescence and Confocal Microscopy--
Clonal striatal
cells or human fibroblasts were cultured on
poly-L-lysine-coated microslips for 24 h.
Immunohistochemistry was performed as described previously (31-33). To
study the cellular localization of pro-caspase-8, cells were incubated
with the rabbit polyclonal antibodies against pro-caspase-8 (SK-441,
Ref. 32) or with the mouse monoclonal antibody against cytochrome
c (PharMingen, San Diego, CA) for 24 h at 4 °C.
Microslips were washed and incubated with fluorescence-conjugated goat
anti-rabbit IgG antibodies (or donkey against mouse IgG) (Molecular
Probes, Eugene, OR). To study the effects of TNF- Immunogold and Electron Microscopy--
Clonal striatal cells
were cultured in 60-mm dishes, fixed, and incubated with polyclonal
antibodies against pro-caspase-8 as described above and then with
gold-conjugated secondary antibody for 2 h at room temperature.
Cells were post-fixed in 2.5% glutaraldehyde, incubated in 1% osmium
tetroxide and 1% uranyl acetate, dehydrated in increasing grades of
alcohol, and embedded in an ethanol-soluble epon (LX112, LADD).
Embedded cells were sectioned (Ultracut E, Reichert-Jung) and examined
with a JEOL 100CX electron microscope.
Isolation of Mitochondria, Mitochondrial Fractions, and
Nuclei--
Purification of mitochondria was performed using a Percoll
gradient procedure described by Gasnier et al. (34) with
minor modifications. Clonal striatal cells were harvested and rinsed in
Hanks' balanced salt solution twice. Cells were suspended in 0.5 ml of buffer A containing 250 mM sucrose, 1 mM EDTA, 50 mM Tris-HCl, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.28 units/ml apotinin, 50 µg/ml
leupeptin, and 7 µg/ml pepstatin A (pH 7.4) and homogenized with a
glass Pyrex microhomogenizer (30 strokes). The homogenate was
centrifuged at 1000 × g at 4 °C for 10 min, and the
resultant supernatant was transferred to a new tube and centrifuged at
10,000 × g at 4 °C for 20 min to obtain the
mitochondrial pellet and supernatant. The supernatant was centrifuged
at 100,000 × g for 1 h at 4 °C to generate the
cytosolic fraction. The mitochondrial pellet was washed in buffer B
containing 250 mM sucrose, 1 mM EGTA, 10 mM Tris-HCl (pH 7.4) three times to remove lysosome and
microsome contamination. Crude mitochondria were further purified by
layering the preparation on top of a gradient consisting of 2.2 ml of
2.5 M sucrose, 6.55 ml of Percoll solution (Amersham
Pharmacia Biotech), and 12.25 ml of 10 mM Tris-HCl (pH
7.4), and 1 mM EDTA. The mixture was centrifuged at
60,000 × g for 45 min. A dense band recovered from
approximately two-thirds down the tube, corresponding to purified
mitochondria, was removed with a Pasteur pipette, diluted with 1 ml of
buffer B, and washed twice by centrifugation at 10,000 × g at 4 °C for 15 min each to remove the Percoll solution.
The purity and integrity of mitochondrial preparations were validated by electron microscopy. Protein concentration was determined with BCA
kit (Pierce).
The subfractionation of mitochondria was performed using a protocol
described by Pederson et al. (35) with minor modifications. Briefly, crude mitochondria were resuspended in H-medium containing 220 mM D-mannitol, 70 mM sucrose, 20 mM HEPES, and 0.5 mg/ml bovine serum albumin (pH 7.4). The
mitochondria suspension was mixed with equal volumes of H-medium
containing 1.2% digitonin, incubated for 15 min at 4 °C with mild
stirring, and centrifuged for 15 min at 10,000 × g
after the addition of a 2-fold volume of H-medium. The resultant
supernatant was centrifuged at 150,000 × g for 1 h at 4 °C to obtain the intermembrane space fraction (supernatant) and crude outer membrane fraction (pellet). The pellet fractions resulting from the 10,000 × g centrifugation were
washed once with H-medium, suspended in H-medium, sonicated for 2 min
on ice, and centrifuged at 150,000 × g for 1 h at
4 °C. The resultant supernatants were used as the matrix fraction,
and the pellet fractions were used as inner membrane fraction. Both
outer and inner membrane fractions were solubilized with a solution
containing 50 mM Tris-HCl, 0.5% Nonidet P-40, and 1 mM calcium chloride (TNC, pH 8.0). Protein concentrations
in all fractions were determined by the BCA kit.
For purification of nuclei, human fibroblasts were lysed in a buffer
containing 0.1% Triton X-100, 20 mM Tricine-NaOH, 250 mM KCl, 5 mM MgCl2 (pH 7.8), and
protease inhibitors tablets (Roche Molecular Biochemicals). Crude
homogenate was centrifuged at 2000 × g at 4 °C for
10 min to obtain a low speed supernatant (S1) and a crude nuclear
pellet (P1). The nuclear pellet was purified using iodixanol step
gradients according to manufacturer's instructions (Optiprep, Accurate
Chemicals, Westbury, NY).
Proteinase K Digestion of Isolated Mitochondria--
A protocol
reported by Samali et al. (36) with modifications was used.
Mitochondria were incubated in H-medium containing 0.2 µg/ml
proteinase K or in H-medium containing 1% Nonidet P-40 plus proteinase
K (0.2 µg/ml) for 3 min at room temperature. Control mitochondria
were incubated with H-medium only or H-medium with 1% Nonidet P-40.
Proteolysis was terminated by adding an equal volume of TNC buffer
containing 15 µg phenylmethylsulfonyl fluoride, 2% SDS, 20 µl
of loading buffer, and boiling for 5 min. Samples were analyzed
immediately by immunoblotting.
Determination of Pro-caspase-8 Release from Isolated
Mitochondria--
We followed the protocol of Susin et
al. (29) with minor modifications. A crude mitochondrial fraction,
prepared as described above, was suspended in a cell-free system (CFS)
containing 220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH2PO4,
0.5 mM EGTA, 2 mM MgCl2, 5 mM pyruvate, 2 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 50 µg of antipain, 1 mM dithiothreitol, 10 mM HEPES-NaOH (pH 7.4).
The suspension was equally divided into several test tubes determined
by the needs of the experiments. Release of pro-caspase-8 and
cytochrome c was induced by the ADP/ATP carrier inhibitor
atractyloside (Atr, 5 or 10 mM, Sigma). Atractyloside was
dissolved in CFS and incubated with mitochondria for 1 h at room
temperature. Separation of mitochondria and supernatant was achieved by
centrifugation (10,000 × g, 15 min). The supernatant
was then further cleared by centrifugation at 100,000 × g for 1 h. To assess whether release of pro-caspase-8 is through MPTP, cyclosporin A (CsA, 25 µM, Sigma) was
added together with Atr. After drug treatment, mitochondria suspensions
were centrifuged at 10,000 × g to pellet mitochondria.
The supernatants were centrifuged again at 100,000 × g. The levels of pro- and active caspase-8 and cytochrome
c were measured by Western blot analysis in mitochondrial
and supernatant fractions.
Determination of Pro-caspase-8 Release from Intact
Cells--
Clonal striatal cells were cultured in 60-mm dishes and
treated with TNF- Western Blot Analysis--
Protein extracts were loaded onto
4-20% SDS-polyacrylamide electrophoresis gels (Bio-Rad) and exposed
to electrophoresis for 1.5 h. Proteins were then transferred to
nitrocellulose membranes, which were blocked and incubated in
Tris-HCl-buffered saline with 0.1% Tween 20 (pH 7.4) containing 3%
nonfat dry milk. Rabbit polyclonal antibodies against pro-caspase-8
(SK-441) or active caspase-8 (SK-440) (33) or monoclonal antibodies
against cytochrome c, HSP70 (StressGen Biotechnologies
Corp., Victoria, BC, Canada) or cytochrome oxidase IV (Molecular
Probes) were applied at 4 °C overnight. Membranes were washed three
times for 5 min each with Tris-HCl-buffered saline with 0.1% Tween 20, and immunoreactivity was detected by enhanced chemiluminescence (ECL
kit, Amersham Pharmacia Biotech) and visualized by autoradiography.
Autoradiograms were analyzed by densitometry with image analysis
software (SigmaScan Pro 4.0) after capturing the digital images
with Hewlett Packard ScanJet 4C/T according to the manufacturer's
protocol. The average density and area of each band were measured. The
total values of pro-caspase-8, cytochrome c, actin, or
cytochrome oxidase were calculated by multiplying the area and average
density of each band. The values of pro-caspase-8 and cytochrome
c were then normalized to a loading control (actin for
cytosolic fractions and cytochrome oxidase for mitochondrial fractions)
and expressed as the mean ± S.E.
DNA Fragmentation Assay--
Clonal striatal cells were cultured
in 60-mm dishes and treated with TNF- Immunohistochemical and Biochemical Evidence of Mitochondrial
Localization of Pro-caspase-8--
In the cytoplasm of fibroblasts and
clonal striatal cells, pro-caspase-8 immunostaining displayed a
rod-shaped or punctate pattern with very little or no diffuse staining.
The labeling for pro-caspase-8 overlapped with the mitochondrial enzyme
cytochrome c, as revealed by double immunofluorescence
confocal microscopy (Fig. 1). There was
diffuse staining for pro-caspase-8 in some cell nuclei. The intensity
of nuclear labeling was stronger in human fibroblasts than in clonal
striatal cells (Fig. 1). The specificity of the pro-caspase-8 antibody
was fully characterized previously (33) and was tested in our studies
by omitting primary or secondary antibodies (data not shown).
To confirm the mitochondrial and nuclear localization of pro-caspase-8,
protein extracts from crude mitochondria and Percoll gradient-purified
mitochondria from clonal striatal cells and nuclei obtained from human
fibroblasts were examined by Western blot. Results showed that
pro-caspase-8 levels were strongly detected in both the crude
mitochondria and Percoll gradient-purified mitochondria (Fig.
2, A and B),
whereas the cytosolic fractions showed relatively low levels of
pro-caspase-8. As a marker for mitochondria enrichment, cytochrome
c or cytochrome oxidase was detected in the crude
mitochondrial fraction and Percoll gradient-purified mitochondrial
fraction, respectively. A similar enrichment of pro-caspase-8 was
observed in purified mitochondria from COS-1 cells (data not shown).
The purified nuclei also expressed readily detectable signal for
pro-caspase-8 in Western blot (Fig. 2C). The purity of the
nuclear preparation was verified by the enrichment of histone and the
absence of tubulin, cytochrome c, and calnexin (data not
shown).
Submitochondrial Localization of Pro-caspase-8--
To determine
where pro-caspase-8 is localized in mitochondria, crude mitochondria
were incubated with proteinase K in the absence or presence of the
detergent Nonidet P-40 (1% final concentration). Results showed that
proteinase K alone did not cause degradation of pro-caspase-8. In the
presence of 1% Nonidet P-40, however, proteinase K degraded
pro-caspase-8. In contrast, proteinase K alone caused partial
degradation of heat shock protein (HSP70), a protein previously
reported to be localized especially in the outer membrane and matrix
(38-40). When the detergent Nonidet P-40 was added, proteinase K
completely degraded HSP70. Similar to pro-caspase-8, cytochrome
c, a protein known to be localized inside mitochondria, was
degraded by proteinase K in the presence of Nonidet P-40 but not by
proteinase K alone (Fig.
3A).
The mitochondrial localization of pro-caspase-8 was further examined in
mitochondrial fractions. Western blot analysis showed that
pro-caspase-8 was detected in the intermembrane space, inner membrane,
and matrix fractions (Fig. 3B). In several independent experiments, outer membrane always contained little or no
pro-caspase-8, whereas inner membrane contained the highest levels of
pro-caspase-8. Both intermembrane space and matrix contained
intermediate levels of pro-caspase-8. Cytochrome oxidase and HSP70 were
examined for the purpose of monitoring separation of outer and inner
membranes. Consistent with the known distribution of these proteins,
cytochrome oxidase was found only in the inner membrane fraction,
whereas HSP70 was enriched in the outer membrane and in the matrix
fraction (Fig. 3B).
The localization of pro-caspase-8 inside mitochondria was also examined
with immunogold electron microscopy. Numerous gold particles were found
inside mitochondria, but none were seen on the outer mitochondrial
membrane (Fig. 4). Gold particles were absent when primary antibody was omitted (data not shown).
Release of Pro-caspase-8 from Isolated Mitochondria--
To assess
whether mitochondria release pro-caspase-8, crude mitochondria were
suspended in the cell-free system in the presence or absence of Atr (5 or 10 mM), which opens the MPTP. After incubation, mitochondria and supernatant were separated by centrifugation. Pro-caspase-8 was modestly decreased in mitochondria and robustly detected in the supernatant after Atr treatment. Quantitative analysis
of three independent experiments showed pro-caspase-8 levels in
mitochondrial fractions decreased to 86.2 ± 3% (5 mM) or 79.4 ± 3.8% (10 mM) of control
(nontreated mitochondria) and in the supernatants increased to
267.7 ± 103 (5 mM) or 351.7 ± 73 (10 mM) of control. Concurrently, cytochrome c was
markedly decreased in the mitochondrial fraction and strongly detected in the supernatant after Atr treatment. In mitochondrial fractions, cytochrome c decreased to 57.3% ± 15.1% (5 mM) or 34.8 ± 8.9% (10 mM) of control
(nontreated mitochondria), whereas in the
supernatants the levels were 246.3 ± 51.2% and 534 ± 199%, respectively (Fig. 5,
top).
To evaluate whether mitochondria release pro-caspase-8 through the
MPTP, mitochondria were treated with Atr in the presence or absence of
the MPTP inhibitor CsA (25 µM). Quantitative analysis of
results from three independent experiments showed that CsA blocked the
Atr-induced decrease in pro-caspase-8 levels in mitochondria (from
86.9 ± 9.7% of control to 113.7 ± 22% in the presence of CsA). CsA inhibited the Atr-induced increase in pro-caspase-8 levels in
the supernatants (from 693.6 ± 206 to 109.8 ± 34.8% of
control in the presence of CsA). CsA also inhibited the Atr-induced increase in cytochrome c in the supernatants (from
470.8 ± 170.5% of controls to 77.4 ± 22% in the presence
of CsA) and prevented the Atr-induced decrease in cytochrome
c in mitochondria (from 65.9 ± 5 to 99.2 ± 16.3 in the presence of CsA). In addition, after Atr treatment, the
pro-caspase-8 cleavage product (p10) was also detected in the
supernatant and was inhibited by CsA (Fig. 5,
bottom).
TNF- Release of Pro-caspase-8 from Mitochondria in Intact Cells--
To
evaluate mitochondrial release of pro-caspase-8 in intact cells under
apoptotic conditions, clonal striatal cells were treated with TNF-
To evaluate whether apoptosis was induced in clonal striatal cells
after TNF-
To evaluate whether the release of pro-caspase-8 in intact cells is
dependent on MPTP, the effect of CsA on TNF- A current model for the activation of caspase-8 in apoptosis after
stimulation by Fas and TNF- Activation of caspase-8 requires interactions with death domains in
proteins that are present in the cytosol (47, 48). Thus sequestering
pro-caspase-8 in mitochondria may be a safeguard mechanism. How
pro-caspase-8 enters mitochondria remains unclear. There is a
mitochondrial matrix-targeting signal at the N terminus of
pro-caspase-9, but other mitochondrial caspases, including pro-caspase-8, do not contain a typical mitochondria localization signal. The significance of nuclear pro-caspase-8 remains to be determined.
TNF- Isolated mitochondria released pro-caspase-8 when stimulated by Atr, an
inhibitor of the ADP/ATP carrier and a stimulus for opening of the
MPTP. The release of both pro-caspase-8 and cytochrome c was
inhibited by CsA, a widely used MPTP blocker (59, 60), suggesting that
release of pro-caspase-8 in isolated mitochondria is mediated by a
CsA-sensitive MPTP. In intact clonal striatal cells, CsA inhibited
TNF- The classic model of pro-caspase-8 activation by TNF- (TNF-
),
mitochondria immunoreactive for cytochrome c and
pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and
cytochrome c levels decreased in mitochondrial fractions
and increased, along with pro-caspase-8 cleavage products, in the
cytoplasm of the TNF-
-treated striatal cells. CsA blocked the
TNF-
-induced release of pro-caspase 8 but not cytochrome
c. Internucleosomal DNA fragmentation started at 6 h
and peaked 12 h after TNF-
treatment. These results suggest that pro-caspase-8 is predominantly localized in mitochondria and is
released upon apoptotic stimulation through a CsA-sensitive mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
on
localization of pro-caspase-8 and cytochrome c, clonal
striatal cells were treated with TNF-
(20 ng/ml) for 6 h and
then processed for immunostaining as described above. The nuclei of
these cells were stained with propidium iodide. Immunostained cells
were examined with a confocal microscope (Bio-Rad 1024) and merged in
Adobe Photoshop.
(10 or 20 ng/ml, Sigma) after cells reached
80-90% confluence. Control cells received 10 µl of
phosphate-buffered saline (vehicle for TNF-
). Cells were harvested
and washed in phosphate-buffered saline 6 h after drug
administration. To study the effect of CsA on TNF-
-induced release
of pro-caspase-8, clonal striatal cells were cultured in 60-mm dishes
and treated with TNF-
(20 ng/ml plus vehicle), CsA (10 µM), or TNF-
plus CsA. Cells were harvested 6 h
after drug administration and washed in phosphate-buffered saline.
Crude mitochondria and cytosol were prepared as described above.
Protein concentrations in cytosol and mitochondria were determined by
BCA kit. Levels of pro- and active caspase-8 and cytochrome
c were determined with Western blot analysis.
(20 ng/ml) for 3-24 h. Cells
were collected together with medium and centrifuged at 1000 × g. Cell pellets were rinsed with ice-cold phosphate-buffered
saline (pH 7.4) after removing the medium. Low molecular weight DNA
fragments were extracted according to the protocol used by Chen
et al. (37). DNA fragments were separated on 2% agarose gel
(3:1 NuSive) and visualized with an UV transilluminator after ethidium
bromide staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cellular localization of pro-caspase-8
immunoreactivity. Human fibroblasts and mouse clonal striatal
cells were double-immunostained for pro-caspase-8 and cytochrome
c and examined with confocal microscopy as described under
"Experimental Procedures." In the cytosol, pro-caspase-8
immunostaining (green) displayed punctate and rod-shaped
patterns that overlapped (yellow in merged images) with
cytochrome c immunostaining (red). Diffuse
nuclear labeling for pro-caspase-8 was more prominent in some
fibroblasts than in the clonal striatal cells.
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Fig. 2.
Subcellular localization of
pro-caspase-8. A, pro-caspase-8 in crude mitochondria
(Mit). Clonal striatal cells were processed to obtain
cytosol and crude mitochondria as described under "Experimental
Procedures." A is a representative immunoblot from three
independent experiments. Pro-caspase-8 was highly enriched in the
mitochondria but low in the cytosol. Cytochrome c was high
in the mitochondrial fraction but low in the cytosol. B,
pro-caspase-8 in purified mitochondria. Crude mitochondria were
purified with a Percoll gradient as described under "Experimental
Procedures." B is a representative immunoblot from three
independent experiments. Pro-caspase-8 was enriched in the mitochondria
but was low in the cytosol. Cytochrome oxidase was only detected in the
mitochondrial fraction but not in the cytosolic fraction. Cytochrome
c was low in mitochondria after Percoll gradient
purification; therefore cytochrome oxidase was used as a mitochondrial
marker in these studies. C, pro-caspase-8 in purified
nuclei. Human fibroblasts were processed to obtain crude homogenate,
S1, P1, and N fractions as described under "Experimental
Procedures." C is a representative immunoblot from three
independent experiments. CH is crude homogenate;
S1 is supernatant resulting from 2000 × g
centrifugation of crude homogenate. P1 is the pellet
resulting from 2000 × g centrifugation. N
is the purified nuclear fraction obtained from P1 by iodixanol step
gradients. Pro-caspase-8 is present in purified nuclei and in the P1
fraction from which nuclei were purified. 20 µg of protein were
loaded in each lane in all experiments.
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Fig. 3.
Mitochondrial localization of
pro-caspase-8. A, proteinase K degradation of
pro-caspase-8 in crude mitochondria. Crude mitochondria were isolated
as described under "Experimental Procedures." Mitochondria were
incubated in CFS in the presence or absence of proteinase K (Pro
K) or proteinase K plus Nonidet P-40. A is a
representative immunoblot from four independent experiments. Proteinase
K alone partially degraded HSP70 but had no effect on pro-caspase-8 and
cytochrome c. In the presence of Nonidet P-40, proteinase K
degraded pro-caspase-8, HSP70, and cytochrome c.
B, pro-caspase-8 distribution in mitochondrial fractions.
Mitochondria fractions were prepared as described under "Experimental
Procedures." B is a representative immunoblot from four
independent experiments. Pro-caspase-8 was found in the matrix and
intermembrane space (IMS) and at its highest levels in the
inner membrane fraction (IM). HSP70 was enriched in both
outer membrane (OM) and matrix fractions. Cytochrome oxidase
was only found in the inner membrane fraction. 20 µg of protein were
loaded in each lane.
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Fig. 4.
Immunogold electron micrographs of
pro-caspase-8 localization. Clonal striatal cells were
immunostained using polyclonal antibody against pro-caspase-8 and
gold-conjugated secondary antibody. Two representative mitochondria are
shown. Gold particles (arrows) appear inside mitochondria.
No gold particles appear in association with mitochondrial outer
membranes (arrowheads). Scale bar = 1 µm.
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Fig. 5.
Mitochondrial release of pro-caspase-8.
Crude mitochondria were prepared as described under "Experimental
Procedures." Mitochondria were suspended in CFS in the presence or
absence of Atr (5 or 10 mM). After 1 h of incubation
at room temperature, mitochondria and supernatants were separated by
centrifugation. Top, Atr-induced release of pro-caspase-8,
representative of three independent experiments. In mitochondria
(Mit), Atr reduced pro-caspase-8 (by densitometry, the
signal was decreased to 86.2 ± 3% (5 mM) or
79.4 ± 3.8% (10 mM) of control) and cytochrome
c (signal decreased to 57.3 ± 15.1% (5 mM) or 34.8 ± 8.9% (10 mM) of control).
In the supernatant (Sup), pro-caspase-8 increased to
267.7 ± 103% (5 mM) or 351 ± 73% (10 mM) of control, and cytochrome c increased to
246.3 ± 51.2% (5 mM) or 534 ± 199% (10 mM) of control. Bottom, blockade of
pro-caspase-8 release by cyclosporin A. Mitochondria were suspended in
CFS with or without Atr (5 mM). To block MPTP, cyclosporin
A (25 µM) was added. After a 1-h drug treatment,
mitochondria (Mit) and supernatants (Sup) were
separated by centrifugation. Experiment which is representative of
three independent experiments. In the supernatant, CsA inhibited the
increases in pro-caspase-8 (decreased to 109.8 ± 34.8% (Atr + CsA) from 693.6 ±206% (Atr) of control) and cytochrome c
(decreased to 77.4 ± 22% (Atr + CsA) from 470.8 ± 170.5%
(Atr) of control). In the mitochondria, CsA reduced Atr-induced
decreases in pro-caspase-8 (increased to 113.7 ± 22% (Atr + CsA)
from 86.9 ± 9.7% (Atr) of control by densitometry) and
cytochrome c (increased to 99.2 ± 16.3% (Atr + CsA)
from 65.9 ± 5% (Atr) of control). CsA also reduced pro-caspase-8
cleavage products (p10) induced by Atr in the supernatant. The total
protein loaded in each lane was 8 µg for mitochondrial
fraction and 16 µg for cytosol fraction.
Effects on Localization of Mitochondria and
Pro-caspase-8--
The effects of the apoptotic inducer TNF-
on
mitochondria localization were determined by immunofluorescence and
confocal microscopy. In untreated clonal striatal cells, cytochrome
c and pro-caspase-8 immunoreactive mitochondria were
distributed relatively evenly throughout the cytoplasm (Fig.
6, top panels, green
label). Treatment with TNF-
for 6 h induced marked
clustering, fusion, and peri-nuclear localization of mitochondria
labeled for cytochrome c or pro-caspase-8. These changes
appeared in cells with or without nuclear fragmentation, a feature of
apoptosis. In some of the cells including those that were shrunken with
fragmented nuclei, labeling for mitochondria with cytochrome
c or pro-caspase-8 was severely reduced or absent, and
diffuse staining for cytochrome c (Fig. 6, left
middle and lower panels), or pro-caspase-8 was evident
in the cytoplasm (Fig. 6, right middle and lower
panels).
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Fig. 6.
TNF- induced
alterations in localization of cytochrome c and
pro-caspase-8. Clonal striatal cells were treated with TNF-
(20 ng/ml) for 6 h and processed for immunofluorescence for cytochrome
c (green) or pro-caspase-8 (green).
Nuclei were stained with propidium iodide (red). The
top panel shows cytochrome c (left) or
pro-caspase-8 (right) immunoreactivity in untreated cells.
The middle and lower panels show TNF-
-induced
alterations in distribution of cytochrome c and
pro-caspase-8-labeled mitochondria. Mitochondria were clustered, fused,
and re-located to peri-nuclear regions. Some cells with reduced
mitochondria show diffuse staining for cytochrome c and
pro-caspase 8 in the cytoplasm. The lower left panel shows a
cell with nuclear fragmentation.
(10 or 20 ng/ml), for 6 h and then mitochondrial and cytosolic
fractions were prepared. TNF-
caused a reduction in levels of
pro-caspase-8 in mitochondria, whereas cytosolic levels of
pro-caspase-8 levels were increased (Fig.
7A). Quantitative analysis of
three independent experiments showed that in the mitochondrial fractions, pro-caspase-8 levels decreased to 78.6 ± 5.2% (10 ng/ml) or 69.4 ± 0.64% (20 ng/ml) of control (vehicle-treated
cells), whereas in the cytosol, the levels were 140.4 ± 23.7%
and 331.9 ± 34.5% of control, respectively. Cytochrome
c levels in the mitochondrial fractions were decreased to
28 ± 9.2% (10 ng/ml) or 18.1 ± 3.1% (20 ng/ml) of control
(vehicle-treated cells), whereas in the cytosol, cytochrome
c increased to 603.2 ± 362.5% (10 ng/ml) or 516.6 ± 330.5% (20 ng/ml) of control. In the mitochondrial
fraction, an active form of caspase-8 (p10) was detected in all cells
but tended to rise in cells treated with TNF-
. Active caspase-8 (p10 and p20) was almost undetectable in cytosolic fractions of control cells but readily detected in TNF-
treated cells.
View larger version (31K):
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Fig. 7.
TNF- induced release
of pro-caspase-8 and internucleosomal DNA fragmentation in clonal
striatal cells. A, clonal striatal cells were treated
with TNF-
(10 or 20 ng/ml) and harvested for isolation of
mitochondria and cytosol 6 h after drug treatment. A is
a representative immunoblot from three independent experiments. In
mitochondria (Mit), TNF-
decreased pro-caspase-8 (to
78.6 ± 5.2% (10 ng/ml) or 69.4 ± 0.64% (20 ng/ml) of
control) and cytochrome c (to 28 ± 9.2% (10 ng) or
18.1 ± 3.1% (20 ng) of control). In the cytosol
(Cyto), TNF-
increased pro-caspase-8 (increased to
140.4 ± 23.7% (10 ng/ml) or 331.9 ± 34.5% (20 ng/ml) of
control) and cytochrome c (increased to 603.2 ±362.5% (10 ng) or 516.6 ± 330.5% (20 ng) of control). Active forms of
caspase-8 (mainly p20) were detectable in the cytosol after TNF-
treatment. Protein loading per lane was 10 µg for
mitochondrial and 20 µg for the cytosol fractions. An additional band
with a molecular mass of about 60 kDa was detected in the cytosol with
the pro-caspase antibody, but the identity of this band was unknown.
B, clonal striatal cells were treated with TNF-
(20 ng/ml), and low molecular mass DNA was extracted 3-24 h after drug
administration. DNA fragments were separated on 2% agarose gel.
B is a representative DNA ladder from three independent
experiments. The density of cells at the beginning of treatment was the
same. A typical apoptotic DNA ladder appears 12-24 h after TNF-
treatment. M, DNA marker; C, control. C, effects of
cyclosporin A on TNF-
-induced release of pro-caspase-8 in striatal
clonal cells. Clonal striatal cells were treated with TNF-
(20 ng/ml) with or without CsA. Cells were harvested for isolation of
mitochondria and cytosol 6 h after drug administration.
C is a representative immunoblot from three independent
experiments. In mitochondria (Mit), CsA inhibited
TNF-
-induced decrease in pro-caspase-8 (increased to 82.3 ± 9.1% (TNF-
plus CsA) from 63.7 ± 9.8% (TNF-
) of control)
but failed to inhibit a decrease in cytochrome c (57.7 ± 11.2% (TNF-
) and 44.6 ± 11.7% (TNF-
plus CsA) of
control). In the cytosol (Cyto), CsA inhibited a
TNF-
-induced increase in pro-caspase-8 (decreased to 102.6 ± 29.6% (TNF-
plus CsA) from 209.5 ± 47.8% (TNF-
) of
control). Again, CsA was ineffective in blocking a TNF-
-induced
increase in cytochrome c (784.5 ±267.2% (TNF-
) and
644.4 ± 104.1% (TNF-
plus CsA) of control).
administration, low molecular weight DNA was extracted,
and internucleosomal DNA fragmentation was examined. Internucleosomal
DNA fragmentation was barely detectable at 6 h after TNF-
administration and clearly appeared 12-24 h after TNF-
treatment
(Fig. 7B). Since release of pro-caspase-8 and cytochrome
c occurred by 6 h, these results suggested that
mitochondrial release of pro-caspase-8 and cytochrome c
preceded the execution of apoptosis.
induced pro-caspase-8
release was examined in clonal striatal cells. These studies showed
that pro-caspase-8 and cytochrome c levels robustly increased in the cytosol and decreased in mitochondria.
Coadministration of CsA markedly attenuated TNF-
-induced increases
in the level of pro-caspase-8 in the cytosol (Fig. 7C).
Treatment with CsA alone had no effect on either cytosolic or
mitochondrial levels of pro-caspase-8 and cytochrome c (data
not shown). Quantitative analysis of three independent experiments
showed that CsA attenuated the TNF-
-induced decrease in
pro-caspase-8 levels in mitochondria from 63.7 ± 9.8% (TNF-
20 ng/ml) to 82.3 ± 9.1% (TNF-
+ CsA) of control
(vehicle-treated cells). CsA inhibited the increase in pro-caspase-8
levels in the cytosol from 209.5 ± 47.8% (TNF-
) to 102.6 ± 29.6% (TNF-
+ CsA) of control (vehicle-treated cells). However,
CsA was ineffective in blocking the effect of TNF-
on cytochrome
c. In the mitochondrial fractions, cytochrome c
levels were 57.7 ± 11.2% in TNF-
-treated cells and 44.6 ± 11.7% of control (vehicle-treated cells) in TNF-
plus
CsA-treated cells. In the cytosol, the levels of cytochrome
c were 784.5 ± 267.2% and 644.4 ± 104.1%, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
involves the recruitment of cytosolic
FADD/MORT1 and pro-caspase-8 to form a death-inducing signaling complex
(41-44). Pro-caspase-8 has the ability to activate itself. FADD/MORT1
brings into proximity two or more pro-caspase-8 molecules, which
accelerates the self-cleavage and enhances caspase-8 activation (44,
45). The subcellular localization of pro-caspase-8 has not been well
defined until now, making it unclear how pro-caspase-8 becomes
available to FADD/MORT1 during apoptosis. We showed that mitochondria
are a major site for pro-caspase-8 localization and regulation during
apoptosis. By immunofluorescent staining, immunogold labeling, and
Western blot analysis, pro-caspase-8 was located primarily inside
mitochondria. Mitochondrial levels of pro-caspase-8 and cytochrome
c were unaffected by proteinase K treatment, again consistent with localization inside mitochondria. The presence of
pro-caspase-8 in mitochondria was found in three cell lines (human
fibroblasts, monkey COS cells, mouse clonal striatal cells). Zhivotovsky et al. (46) report low levels of pro-caspase-8
in mitochondria of apoptotic Jurkat cells and no pro-caspase 8 in normal Jurkat cells by Western blot. Although only biochemical assays
were performed in their study, the discrepancy with our findings is
still hard to explain. Other caspases including pro-caspase-2, -3, -6, and-9 have also been identified in mitochondria (27-29). Similar to
pro-caspase-8, caspase-9 is enriched in mitochondria compared with
cytosol (27).
is thought to activate caspase-8 by a mechanism involving
recruitment of pro-caspase-8 by FADD/MORT1 (14-17). TNF-
and Fas
have also been shown to induce release of cytochrome c and activation of caspase-9 and -3 (49-51). In some cell types, these effects can be inhibited by Bcl-2 family proteins (52-53). Consistent with a previous report in L929 cells (54), we found that treatment of
clonal striatal cells with TNF-
altered mitochondrial localization, which can affect mitochondrial function. Concurrently, TNF-
treatment reduced pro-caspase-8 in mitochondria and increased
pro-caspase-8 and its active cleavage products in the cytoplasm. Levels
of cytochrome c were reduced in the mitochondria and
increased in the cytoplasm in the same TNF-
-treated cells. These
events preceded internucleosomal DNA fragmentation, indicating that
they occur during the initiation of apoptosis. With both Atr and
TNF-
stimulation, the decline in signal on Western blot for
pro-caspase-8 in mitochondria fractions was smaller (15-30% decrease)
than for cytochrome c (50-80% decrease). However, the
actual total protein change for pro-caspase-8 may be significant since
mitochondria fractions have much higher concentrations of pro-caspase-8
than cytosol. The role of MPTP in the transport of mitochondrial
proteins to the cytoplasm in apoptosis remains unclear. Many stimuli,
including withdrawal of nerve growth factor, UV irradiation, calcium
concentration surges, oxidative stress, and certain anti-tumor drugs
can cause collapse of mitochondrial transmembrane potential and induce
MPTP (55-57). When MPTP is induced, proteins and other molecules
participating in apoptosis can move out of mitochondria. Despite an
apparent size limitation (1500 kDa) for molecules to pass through the
MPTP (22), a molecule larger than pro-caspase-8, namely
apoptosis-inducing factor, can be released through a
CsA-sensitive MPTP (58). In addition to pro-caspase-8, mitochondria
release pro-caspases-9, -6, and -3 (27, 29); the mechanisms of release
are not clear.
-induced release of pro-caspase-8 but not cytochrome
c. The discrepancy in effects of CsA on cytochrome c release in isolated mitochondria and intact cells may
relate to differences in preparations. The effects of Atr in isolated mitochondria are directly mediated by binding to ADP/ATP carrier, whereas in intact cells, TNF-
could have direct and indirect effects
on mitochondria that cause release of pro-caspase-8 and cytochrome
c through separate mechanisms. TNF-
can release
cytochrome c in several cell types, but few studies have
shown that this effect can be blocked by CsA treatment alone (61).
Apoptotic death of mouse fibroblasts by TNF-
was inhibited by CsA in
combination with the phospholipase A2 inhibitor
aristolochic acid, indicating involvement of multiple mechanisms (62).
In our study, CsA failed to inhibit TNF-
-induced apoptosis. One
potential problem with using CsA is its toxicity in some types of cells
(63). We found that clonal striatal cells developed intense
internucleosomal DNA fragmentation 12 h after CsA administration
alone (data not shown).
and death
receptors involves recruitment of pro-caspase-8 from an unknown compartment by FADD/MORT1 through protein-protein interactions (Fig.
8). We speculate that TNF-
-induced
release of pro-caspase-8 from mitochondria could be mediated by
different signaling pathways (Fig. 8). One pathway could involve TNF
receptors through an as yet unknown mechanism. Another pathway could
involve BID. Active caspase-8 cleaves cytoplasmic BID and causes BID to
translocate to mitochondria. BID then induces release of pro-caspase-8.
These possibilities will require further study. It is unclear whether the pro-caspase-8 released from mitochondria by TNF-
is activated directly or serves to replenish the small resident cytoplasmic pool of
pro-caspase-8 depleted during apoptosis. A recent study showed that
caspase-9 is enriched in mitochondria and released by Bax and ischemic
insult (27). These findings together with our results on pro-caspase-8
suggest that mitochondria may be centrally involved in the activation
of both initiator caspases (caspase-8 and -9) and provide a major
intracellular site for integration and regulation of signaling events
mediating cell death and survival.
View larger version (18K):
[in a new window]
Fig. 8.
Proposed model of death receptor-mediated
caspase-8 activation after TNF- or Fas ligand
stimulation. The classic model of caspase-8 activation induced by
death receptors involves recruitment of pro-caspase-8 by FADD/MORT1
through protein-protein interactions. Caspase-8 activates its target
caspase, caspase-3. In this model, the source of pro-caspase-8
available to FADD/MORT1 is not known (solid arrows). Present
results suggest that pro-caspase-8 is predominantly localized in
mitochondria and is released upon TNF-
stimulation. We speculate
that an additional pathway exists (dashed arrows) in which
mitochondria are involved in death receptor-induced caspase-8
activation by releasing pro-caspase-8. It remains unclear, however, if
the pro-caspase-8 released from mitochondria directly contributes to
caspase-8 activation or replenishes a small pool of cytoplasmic
pro-caspase-8. Signals that cause mitochondria to release pro-caspase-8
in response to TNF-
remain to be determined. One mechanism could
involve BID, which can be cleaved by caspase-8 and translocate to
mitochondria. Like BAX, BID induces release of cytochrome c
from mitochondria upon its mitochondrial translocation. The other
possible mechanism may involve signals generated directly from
activation of death receptors. TNF-
-induced release of cytochrome
c can activate caspase-9, thus amplifying activation of
caspase-3.
Mitochondrial dysfunction has been implicated in certain neurological
diseases, especially Huntington's disease (64). Expanded polyglutamine
containing proteins including mutant huntingtin can recruit and
activate caspase-8 (65). Preliminary studies in our laboratory show
that the localization of mitochondria expressing pro-caspase-8
immunoreactivity is altered by increased expression of mutant
huntingtin in the cytoplasm. Alterations in mitochondrial localization
can affect the MPTP (54), thereby increasing the possibility for the
mitochondrial release of pro-caspase-8 and subsequent activation of
pro-caspase-8 by mutant huntingtin. Thus, the capacity for mitochondria
to localize and release pro-caspase-8 may have important implications
in Huntington's disease pathogenesis.
![]() |
FOOTNOTES |
---|
* This work is supported by National Institutes of Health (NIH) Grants NS 16367 and NS 35711 and a grant from the Huntington's Disease Society of America (to M. D. F.) and NIH Grant NS 38194 (to N. A).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Cellular Neurobiology, Massachusetts General Hospital and Harvard Medical School, Bldg. 149, Rm. 6604, 13th St., Charlestown, MA 02129. Tel.: 617-726-8446; Fax: 617-726-5677; E-mail:
difiglia@helix.mgh.harvard.edu.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M007028200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TNF, tumor necrosis factor; FADD, Fas-associated death domain; MPTP, mitochondrial permeability transition pore; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine; CFS, cell-free system; Atr, atractyloside; CsA, cyclosporin A; MORT, mediator of receptor-induced toxicity.
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