From the Department of Surgery, School of Medicine,
University of Pittsburgh, Pittsburgh, Pennsylvania 15261 and the
¶ Department of Molecular and Cellular Biochemistry, School of
Medicine, Kanwon National University, Chunchon, Kangwon-do, Korea
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ABSTRACT |
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Nitric oxide (NO) is a potent inhibitor of
apoptosis in many cell types, including hepatocytes. We and others have
described NO-dependent decreases in caspase activity in
cells undergoing apoptosis. However, previous work has not determined
whether NO disrupts the proteolytic processing and thus the activation
of pro-caspases. Here we report that NO suppresses proteolytic
processing and activation of multiple pro-caspases in intact cells,
including caspase-3 and caspase-8. We found that both exogenous NO as
well as endogenously produced NO via adenoviral inducible NO synthase gene transfer protected hepatocytes from tumor necrosid factor (TNF)
Apoptosis, or the programmed cell death, is essential to the
normal development of multicellular organisms as well as physiologic cell turnover (reviewed in Refs. 1-3). In pathologic states, while a
failure to undergo apoptosis may cause abnormal cell overgrowth and
malignancy, excessive apoptosis may contribute to organ injury. The
signaling pathways leading to apoptosis are now known to involve the
sequential activation of cysteine proteases known as caspases (4), in
association with changes in mitochondrial membrane potential and
release of cytochrome c (5). Caspases are constitutively present in cells as zymogens and require proteolytic cleavage into the
catalytic active heterodimer. All activated caspases are comprised of a
17-20-kDa large subunit that contains a redox-sensitive cysteine at
the active site and a small subunit of approximately 10 kDa. To date,
14 mammalian caspases have been identified (4, 6, 7). Based on a number
of genetic and biochemical studies, the functions of various caspases
appear different and these caspases can be tentatively grouped into
three categories: caspases that function primarily in cytokine
maturation (e.g. caspase-1, -4, and -5), effector proteases
in the execution phase of apoptosis (caspase-3, -6, and -7), and
initiator caspases involved in the early steps of apoptotic signaling
(e.g. caspase-8, -9, and -10). Several synthetic peptide
derivatives that mimic the cleavage sites of natural substrates of
caspases have been used to inactivate these caspases. For example,
Ac-DEVD-CHO is a caspase-3-like protease inhibitor, Ac-YVAD-CHO a
caspase-1-like protease inhibitor, and z-VAD-fmk an irreversible
inhibitor for all caspases. Studies using these peptide inhibitors,
together with mutational and genetic analysis, unequivocally
established the central role of caspases in apoptosis in numerous cell
systems (4). While some members of the caspase family are indispensable
during mammalian development others seem to be redundant or have
limited roles in programmed cell death. However, the specific role of
an individual caspase may vary between cell types or apoptotic stimuli
(8).
Recent evidence indicates that mitochondria play an essential role in
apoptotic cell death induced by various stimuli. Activation of upstream
caspases has been shown to lead to the degradation of Bcl-2 family
members (9, 10), which promotes the release of cytochrome c.
This occurs in association with the loss of the inner mitochondrial
membrane potential in many situations (11, 12). Released cytochrome
c subsequently binds to Apaf-1 in the presence of dATP and
activates caspase-9 which then activates downstream effector caspases,
such as caspase-3 (13).
Although nitric oxide (NO)1
is cytotoxic in a number of cell types, it suppresses apoptosis in
others. Cell types shown to be protected from apoptosis by NO include
lymphocytes (14, 15), endothelial cells (16), eosinophils (17),
splenocytes (18), multiple cell lines (19, 20), certain neurons (21,
22), ovarian follicles (23), and hepatocytes (24, 25). One mechanism for the inhibition of apoptosis by NO is through the suppression of
caspase enzymatic activity. NO can directly inhibit caspase activity
through S-nitrosylation of the active cysteine conserved in
all caspases (16, 26). However, it is not clear whether NO interferes
only with activated caspases or whether NO blocks pro-caspase
processing and activation. In this study we examined this question by
determining the number of caspases activated in TNF Materials--
Williams Medium E, penicillin, streptomycin,
L-glutamine, Opti-MEM, and Hepes were purchased from Life
Technologies Inc.
N-Acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA), caspase inhibitors
N-acetyl-DEVD-aldehyde (Ac-DEVD-CHO) and
benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (z-VAD-fmk) were from
Alexis Co. (San Diego, CA). Stock solutions were prepared at 100 mM in dimethyl sulfoxide.
N-Acetyl-Ile-Glu-Thr-Asp-p-nitroanilide (Ac-IETD-pNA), caspase-8 inhibitor z-IETD-fmk, and
biotin-conjugated VAD-fmk were from Calbiochem (San Diego, CA).
S-Nitroso-N-acetylpenicillamine (SNAP) was synthesized as
described (27). Oxidized SNAP (oxi-SNAP) was prepared by incubating
SNAP aqueous solution at room temperature for 2 days to completely
liberate NO. Mouse recombinant TNF Preparation of Primary Hepatocytes and Cell Culture--
Primary
rat hepatocytes were isolated and purified from male Sprague-Dawley
rats and cultured as described previously (28). Highly purified
hepatocytes (>98% purity and >98% viability by trypan blue
exclusion) were suspended in Williams medium E supplemented with 10%
calf serum, 1 µM insulin, 2 mM
L-glutamine, 15 mM HEPES (pH 7.4), 100 units/ml
penicillin, and 100 µg/ml streptomycin. The cells were plated on
collagen-coated tissue culture plates at a density of 2 × 105 cells/well in 12-well plates for cell viability
analysis or 5 × 106 cells/100-mm dish for Western
blot and enzyme assays. After 18 h preculture, the cells were
washed and further cultured with fresh medium containing 5% calf
serum. Apoptosis was induced by incubating the hepatocytes in the
culture medium containing 2000 units/ml TNF Enzyme Activity Assay--
Caspase activity was evaluated by
measuring proteolytic cleavage of chromogenic substrate
Ac-DEVD-pNA as described previously (26).
Ac-DEVD-pNA was used as the substrate for caspase-3-like proteases and Ac-IETD-pNA as caspase-8 substrate. Briefly,
cell lysate (100 µg of protein) was added into buffer A containing 200 µM Ac-DEVD-pNA or 100 µM
Ac-IETD-pNA in a final volume of 100 µl. The reaction
mixture was incubated at 37 °C for 1 h. The increase in
absorbance of enzymatically released pNA was measured at 405 nm in a microplate reader.
Adenoviral iNOS Gene Transfer--
Modified adenoviral vectors
carrying the human iNOS or bacterial Affinity Labeling of Active Caspases--
Aliquots of hepatocyte
lysate (50 µg of protein) were incubated with 1 µM
biotin-VAD-fmk for 1 h at room temperature in buffer A in a final
volume of 50 µl. The reaction was stopped by addition of 1/2 volume of 2 × SDS sample buffer followed by heating at 95 °C
for 5 min. Alternatively, hepatocytes (5 × 106) were
resuspended in 250 µl of buffer A containing 10 µg/ml cytochalasin B and 2 µM biotin-VAD-fmk, and incubated for 15 min at
37 °C. Following 3 cycles of freezing-thawing the cell lysate was
prepared as above. Proteins were resolved by 15% SDS-PAGE and
transferred to nitrocellulose membranes. Nonspecific binding was
blocked with TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% non-fat milk for
1 h at room temperature. After washing two times with TBS-T, the
membrane was probed with horseradish peroxidase-linked streptavidin
(1:2000 dilution in TBS-T with 1% bovine serum albumin) for 1 h,
and washed for four times with TBS-T. Labeled proteins were then
visualized by enhanced chemiluminescence (SupersignalTM)
according to the manufacturer's instructions.
For two-dimensional electrophoresis of affinity labeled active
caspases, after incubation with biotin-VAD-fmk, cell lysates were
diluted in dSDS (0.3% SDS, 1% Measurement of Immunoblotting Analysis--
Cytosol fractions were prepared
from 5 × 106 hepatocytes by homogenization and
differential centrifugation in buffer A containing 250 mM
sucrose as described previously (33). Cytosolic proteins were used for
evaluating cytochrome c release. Cell lysates and whole
cells were used for immunoblotting analysis of caspases and PARP,
respectively. Thirty µg of protein was separated on 14 or 8%
SDS-PAGE and transferred onto a nitrocellulose membrane. Nonspecific
binding was blocked with TBS-T containing 5% non-fat milk for 1 h
at room temperature. Anti-caspase-8 monoclonal antibody C15 was diluted
1:20 in TBS-T containing 1% bovine serum albumin, anti-caspase-3
polyclonal antibody H277 was diluted 1:500, anti-PARP antibody 1:500
and anti-cytochrome c antibody 1:1000 in TBS-T containing
1% non-fat milk. After a 1-h incubation at room temperature with
agitation, membranes were washed three times with TBS-T. The secondary
antibody was incubated at 1:5000 dilution for 1 h. Following 4-5
washes with TBS-T, the protein bands were visualized with
SupersignalTM according to the manufacturer's instructions.
NO Inhibits TNF Caspase-3 Is Processed and Activated in TNF
We have shown that NO inhibits increases in caspase-3-like activity in
TNF Endogenously Produced NO Inhibits Apoptosis and Pro-caspase-3
Processing/Activation--
To assure that the effects of the exogenous
NO donor also occur when NO was generated by endogenous enzyme,
adenoviral iNOS (AdiNOS) and its control partner adenoviral LacZ
(AdLacZ) were used to transfect hepatocytes. iNOS expression in
AdiNOS-transfected cells was confirmed by immunoblotting analysis and
NO production was monitored by measuring
NO2
Exposure to TNF
In many cell systems apoptosis is accompanied by the proteolytic
cleavage of poly(ADP-ribose) polymerase (PARP) from a 116-kDa polypeptide to a 85-kDa fragment. In primary hepatocytes, PARP, an
intracellular substrate of caspase-3 and -3-like proteases, was cleaved
during TNF NO Inhibits Processing/Activation of Multiple Caspases in Primary
Hepatocytes Induced to Undergo Apoptosis--
Numerous caspases may be
activated in the apoptosis cascade. To determine whether multiple
caspases are activated in hepatocytes treated with TNF
Affinity labeling of active caspases with biotin-VAD-fmk was carried
out under conditions where more than 95% of caspase-3-like activity
was inactivated in lysates from TNF
Since the large subunits of all known caspases have similar molecular
weights, one-dimensional SDS-gel electrophoresis may not adequately
distinguish various active caspases. Therefore, we used two-dimensional
isoelectric focusing/SDS-gel electrophoresis to better resolve the
biotin-VAD-fmk-labeled active caspases. The two-dimensional isoelectric
focusing/SDS-gel electrophoresis analysis at the 7-h time point
revealed at least four newly labeled spots present in
TNF Involvement of Mitochondria in TNF
We next investigated whether translocation of cytochrome c
to cytoplasm occurs in TNF NO Inhibits Caspase-8 Activation--
It is well described that
the most proximal caspase activated following interaction of TNF This study was undertaken to determine if NO inhibited caspase
activation and the associated mitochondrial changes in
TNF Participation of caspases has been demonstrated in hepatocyte apoptosis
induced by TNF Substantial evidence is emerging that mitochondria participate in both
the initiation and the execution phases of apoptosis. Release of
cytochrome c or apoptosis inducing factor from mitochondria to cytoplasm induces activation of effector caspases and nuclear apoptosis in a cell-free system (13, 44). Microinjection of cytochrome
c into cells has been shown to initiate the apoptotic cascade (45). The mechanism by which cytochrome c is
translocated from the mitochondrial intermembranous space to the
cytoplasm remains elusive. Mitochondrial permeability transition has
been implicated in cytochrome c release, probably through
the rupture of the mitochondrial outer membrane. Onset of permeability
transition results in membrane depolarization, uncoupling of oxidative
phosphorylation, and mitochondrial swelling. Disruption of
mitochondrial transmembrane potential ( Inhibition of iNOS during endotoxemia results in increased hepatic
apoptosis and this effect is inhibited by the simultaneous infusion of
a NO generator (48, 49). Likewise, infusion of a NO donor blocks TNF The anti-apoptotic function of NO is not unique to hepatocytes, neither
is it restricted only to death receptor- or growth factor
withdrawal-mediated apoptosis. Endogenous NO synthesis or exposure to
low level NO donors has been shown previously to prevent apoptosis in
human B lymphocytes (15), splenocytes (18), eosinophil (17), ovarian
follicles (24), and endothelial cells (16). Various mechanisms for the
anti-apoptotic effect of NO have been proposed in addition to
S-nitrosylation of caspases. They include, NO-induced
up-regulation of protective proteins such as heat shock protein 70 (Hsp70) (27) and Bcl-2 (18), NO-dependent disruption of JNK
activation (17), and increases in cGMP levels (22, 24). Results in the
present study revealed that NO inhibited TNF plus actinomycin D (TNF
/ActD)-induced apoptosis. Affinity labeling with biotin-VAD-fmk of all active caspase species in TNF
-mediated apoptosis identified four newly labeled spots
(activated caspases) present exclusively in TNF
/ActD-treated cells.
Both NO and the caspase inhibitor, Ac-DEVD-CHO, prevented the
appearance of the four newly labeled spots or active caspases.
Immunoanalysis of affinity labeled caspases demonstrated that caspase-3
was the major effector caspase. Western blot analysis also identified the activation of caspase-8 in the TNF
/ActD-treated cells, and the
activation was suppressed by NO. Furthermore, NO inhibited several
other events associated with caspase activation in cells, including
release of cytochrome c from mitochondria, decrease in
mitochondrial transmembrane potential, and cleavage of poly(ADP-ribose) polymerase in TNF
/ActD-treated cells. These findings indicate the
involvement of multiple caspases in TNF
-mediated apoptosis in
hepatocytes and establish the capacity of NO to inhibit not only active
caspases but also caspase activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
plus actinomycin
(TNF
/ActD)-treated hepatocytes and assessing the effect of NO on
caspase activation. Here we provide evidence that NO prevented the
proteolytic activation of all involved caspases, including the most
apical caspase, caspase-8. NO also prevented other key events
associated with the activation of caspases including the loss of
mitochondrial transmembrane potential and release of cytochrome
c.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was obtained from R & D Systems
(Minneapolis, MN). Antibodies used in this study were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, for caspase-3, iNOS, and
Hsp70), Oncogene Research Product (Cambridge, MA, for PARP), and
Pharmingen (San Diego, CA, for cytochrome c). Caspase-8
monoclonal antibody was kindly provided by Dr. P. H. Krammer
(German Cancer Research Center, Germany). Horseradish peroxidase-linked
streptavidin and SupersignalTM chemiluminescence detection
reagents were from Pierce Chemical Co. (Rockford, IL). Unless indicated
otherwise, all other chemicals were from Sigma.
and 0.2 µg/ml
actinomycin D for various time points as specified in the figure
legends. Cells were then scraped off the plates and centrifuged, washed
twice with cold phosphate-buffered saline and resuspended in a 5-fold
volume of hypotonic buffer A (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mm MgCl2, 1 mM EGTA, 1 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride,
5 µg/ml aprotinin, 5 µg/ml pepstatin, and 10 µg/ml leupeptin).
After three to four cycles of freezing and thawing, cell debris was
removed by centrifugation at 13,000 × g at 4 °C for
20 min and the supernatant used as cell lysate. Protein concentration
was determined with the BCA assay (Pierce) with bovine serum albumin as
standard. Cell viability was determined by the crystal violet method as
described previously (27). In brief, cells were stained with 0.5%
crystal violet in 30% ethanol and 3% formaldehyde for 10 min at room
temperature. Plates were then washed 4 times with tap water. Cells were
solubilized with 1% sodium dodecyl sulfate solution and dye uptake was
measured at 550 nm using a microplate reader.
-galactosidase cDNA were
prepared as described previously (29). After 18 h of preculture,
hepatocytes (5 × 106/10-cm plate) were washed with
Hanks' buffered saline and incubated with adenoviral vector containing
either the human iNOS or bacterial
-galactosidase (LacZ) cDNA at
multiplicity of infection of 1 in a volume of 2 ml of Opti-MEM.
Following a 2-h infection, the medium was changed to fresh Williams
medium E containing 5% calf serum in the presence or absence of 1 mM of the NOS inhibitor NG-monomethyl-L-arginine. The
infected hepatocytes were recovered overnight prior to changing to
fresh medium and subjecting to induction of apoptosis.
-mercaptoethanol, 5 mM
Tris, pH 8.0) and boiled for 5 min (30). The samples were then snap frozen with liquid nitrogen and vacuum dried. The pellets were solubilized in 40 µl of urea buffer (9.0 M urea, 4%
(v/v) Nonidet P-40, 2% (v/v)
-mercaptoethanol, 20% Bio-Lyte pH
3-10) and stored at
20 °C. Isoelectric focusing electrophoresis
(IEF) was performed using a Hoefer Mighty Small II SE 260 tube gel
adaptor system (Hoefer Scientific Instruments, CA). Aliquots of samples
(25 µg of protein) were separated in a 3% acrylamide-IEF (Bio-Rad
Ampholyte pH 5-7) according to the instruction from Heofer (31). For
the second dimensional electrophoresis, the tube gels were transferred to the top of 15% SDS-polyacrylamide gel (14 × 16 cm, where each slab gel contained two tube gels) and subjected to SDS-PAGE for 4 h at 60 mA. Proteins were then transferred to nitrocellulose and probed
with horseradish peroxidase-linked streptavidin as described above.
m by Flow Cytometry--
For mitochondrial
inner transmembrane potential (
m) measurements, hepatocytes were
incubated with DiOC6(3) (80 nM, Molecular Probes Inc.) for 30 min in culture medium at 37 °C, 5%
CO2. DiOC6(3) is a fluorescent dye that is
incorporated into mitochondria in a
m-dependent
manner. As a positive control for
m loss, some hepatocytes were
incubated with the uncoupling agent CCCP (50 µM), a
protonophore that disrupts
m. The cells were then washed twice
with ice-cold phosphate-buffered saline and collected by centrifugation
at 200 × g. Incorporation of DiOC6(3) by
hepatocytes was determined by flow cytometry (Beckton Dickinson) using
excitation of a single 488 nm argon laser. Loss of nuclear DNA
(hypodiploidy) was determined by propidium iodine staining of
ethanol-fixed cells as described previously (32).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced Apoptosis in Primary
Hepatocytes--
Consistent with previous reports (34, 27), TNF
at
2000 units/ml in the presence of actinomycin D (TNF
/ActD) induced massive apoptosis in hepatocytes in a time-dependent
manner. The appearance of apoptotic morphology, including cell
shrinkage, membrane blebbing, and apoptotic body formation, was seen as
early as 6 h, and evident in the majority of cells (>70%) by
8 h (data not shown). Hepatocytes treated with TNF
/ActD for
12 h showed 40-50% decline in cell viability as compared with
control cells (Fig. 1A).
Consistent with our previous report (24), co-incubation with the NO
donor, SNAP, prevented the loss of cell viability in a
concentration-dependent manner, whereas SNAP alone had no effect on cell morphology or viability at the concentrations used (Fig.
1A). To confirm that the cell death induced by TNF
/ActD was in fact apoptosis, we analyzed the hypodiploidy of cellular DNA
with propidium iodide staining and flow cytometry analysis. In
agreement with the cell viability data, SNAP suppressed
TNF
/ActD-induced formation of hypodiploidy (Fig. 1B). A
caspase-3-like protease inhibitor, Ac-DEVD-CHO, also blocked
TNF
/ActD-mediated apoptosis, indicating the involvement of
caspase-3-like proteases in TNF
/ActD-induced apoptosis in
hepatocytes (Fig. 1B).
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Fig. 1.
Nitric oxide inhibits
TNF /ActD-induced apoptosis in cultured primary
rat hepatocytes. A, hepatocytes were treated with or
without 2000 units/ml TNF
plus 0.2 µg of ActD (TNF
/ActD) in the
absence or presence of increasing concentrations of SNAP for 12 h.
Cell viability was measured by crystal violet staining (mean ± S.D., n = 4). B, cells were treated with or
without TNF
/ActD as above in the presence of SNAP or 200 µM Ac-DEVD-CHO for 10 h. Cells were then labeled
with propidium iodine and the percentage of cells exhibiting
hypodiploidy was determined in duplicate by flow cytometry as described
under "Experimental Procedures." Results are representative of
three separated experiments with similar results.
/ActD-induced
Apoptosis--
Although we have previously shown that caspase-3-like
activity increases in TNF
/ActD-treated hepatocytes, it has not been established that pro-caspase-3 cleavage/activation takes place. Fig.
2 demonstrates that the appearance of the
p17 caspase-3 cleavage product coincides with measurable increases in
caspase-3-like activity. The earliest increase of caspase-3-like enzyme
activity was observed around 3 h while the p17 band was detected
at 4 h by immunoblotting analysis (Fig. 2, A and
B).
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Fig. 2.
Activation of caspase-3 in
TNF /ActD-induced apoptosis and its inhibition
by NO and caspase inhibitor Ac-DEVD-CHO. A,
time-dependent activation of caspase-3-like activity in
apoptotic hepatocytes. Cells were treated with or without TNF
/ActD
(2000 units/ml and 0.2 µg/ml, respectively). At indicated time
points, cells were collected, washed with ice-cold phosphate-buffered
saline, and cell lysate was prepared. Caspase-3-like activity was
determined using a colorimetric assay with Ac-DEVD-pNA. B,
immunoblotting analysis of caspase-3 activation. Thirty µg of cell
lysates obtained as above was loaded into each lane, separated on 14%
SDS-PAGE, transferred onto a nitrocellulose membrane, and visualized as
described under "Experimental Procedures." C and
D, hepatocytes were treated with TNF
/ActD in the presence
of 800 µM SNAP or 200 µM Ac-DEVD-CHO for
8 h. Caspase-3-like activity (mean ± S.D, n = 3) (C) and caspase-3 Western blot (D) was
carried out as above. Results are representative of three separate
experiments with similar results.
/ActD-treated hepatocytes (24). To determine if NO blocked
pro-caspase-3 processing we exposed TNF
/ActD-treated cells to SNAP
and performed Western blotting analysis for the p17 cleavage product.
Co-incubation with SNAP prevented the rise of caspase-3-like activity
in cells treated with TNF
/ActD (Fig. 2C). As shown in
Fig. 2D, SNAP blocked the cleavage/activation of
pro-caspase-3, indicating that NO acts either at the level of
pro-caspase-3 or disrupts the upstream signal that leads to caspase-3 activation.
accumulation in the medium (Fig.
3A). NO was produced only in hepatocytes transfected with AdiNOS. The NOS inhibitor, NIO, completely inhibited NO production. It should be noted that in the presence of
TNF
/ActD the NO production was only slightly decreased, suggesting that even in the presence of the transcription inhibitor, ActD, enough
iNOS protein persists to produce NO throughout the experimental period.
This was further substantiated by immunoblotting analysis for iNOS
protein (Fig. 3A, inset).
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Fig. 3.
AdiNOS gene transfer suppresses
TNF /ActD-induced apoptosis. Primary
hepatocytes were infected with AdLacZ or AdiNOS and then cultured with
or without TNF
/ActD in the absence or presence of 1 mM
NIO for 7 h. A, NO production.
NO2
levels were measured in the
culture media by Greiss reaction (mean ± S.D., n = 3). Inset is a Western blot for iNOS for the same groups.
B, phase-contrast photomicrographs showing cellular
morphology of control untreated cells (a), AdLacZ
transfected, TNF
/ActD-treated cells (b), AdiNOS
transfected, TNF
/ActD-treated cells (c), and AdiNOS
transfected, TNF
/ActD plus NIO-treated cells (d).
Photomicrographs were taken at magnification of × 20. C, caspase-3-like activity; and D, Western blots
of caspase-3 and PARP of the same groups. Results are representative of
at least three independent experiments with similar results.
/ActD resulted in apoptosis in control cells and
AdLacZ-transfected cells (Fig. 3B). In contrast, cells
transfected with AdiNOS were protected significantly from apoptosis and
this protection was completely reversed by the NOS inhibitor NIO (Fig. 3B). Likewise, caspase-3-like activity was inhibited only in
AdiNOS-transfected cells but not in the AdLacZ cells upon TNF
/ActD
exposure. When NO synthesis was inhibited by NIO, the caspase-3-like
activity was brought back to the level similar to that detected in the AdLacZ group (Fig. 3C). Furthermore, processing and
activation of pro-caspase-3 was nearly completely blocked by NO
produced by iNOS (Fig. 3D).
/ActD-mediated apoptosis to its characteristic 85-kDa
fragment (Fig. 3D). In agreement with the decreased
caspase-3-like activity and suppressed pro-caspase-3 activation in
AdiNOS transfected cells upon TNF
/ActD treatment, PARP cleavage was
suppressed by NO (Fig. 3D).
/ActD, we used
the biotin-linked caspase inhibitor, biotin-VAD-fmk, to label all the
active caspase species present in hepatocytes undergoing apoptosis.
Z-VAD-fmk is an irreversible caspase inhibitor that covalently binds to
the active cavity of a wide spectrum of active caspases. Z-VAD-fmk (100 µM) effectively prevented TNF
/ActD-induced apoptosis
in cultured primary hepatocytes (data not shown). When incubated with
cell lysates prepared from TNF
/ActD-treated hepatocytes,
biotin-VAD-fmk inhibited caspase-3-like activity in a
dose-dependent manner (Fig.
4A). Thus, biotin-VAD-fmk interacts directly with activated caspases, establishing the basis for
affinity labeling of all the active caspases.
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Fig. 4.
NO suppresses the activation of multiple
caspases. A, aliquots (50 µg of protein) of cell
lysate from hepatocytes treated with TNF /ActD for 7 h were
incubated with increasing concentrations of biotin-VAD-fmk for 1 h
at room temperature. Caspase-3-like activity in the aliquots was then
determined by the colorimetric assay. B, affinity labeling
blot of active caspases. Hepatocytes were induced to undergo apoptosis
with TNF
/ActD in the absence and presence of 100 µM
z-VAD-fmk (+V) for indicated times or for 7 h. Cell
lysates were prepared and active caspases were labeled with 1 µM biotin-VAD-fmk. The labeled caspases were resolved on
SDS-PAGE, transferred to nitrocellulose membrane, and visualized as
described under "Experimental Procedures." To determine the
specificity of the labeling, cell lysate (50 µg of protein) from
cells treated with TNF
/ActD for 7 h was incubated with
biotin-VAD-fmk as above in the presence of 10-fold excess of
Ac-DEVD-CHO in the cell-free system (last lane).
C, biotin-VAD-fmk affinity labeling of cell lysate prepared
from hepatocytes treated with indicated reagents for 7 h.
D, two-dimensional electrophoresis analysis of
biotin-VAD-fmk labeling of active caspases. Hepatocytes were treated
under specified conditions for 7 h. Cell lysate was labeled with
biotin-VAD-fmk and resolved by two-dimensional electrophoresis as
described under "Experimental Procedures." a, control
cells without treatment; b, TNF
alone; c, ActD
alone; d, TNF
/ActD (arrow and
arrowheads indicate new spots, black arrow points
the p17 subunit of caspase-3); e, 800 µM SNAP
alone; f, TNF
/ActD plus 800 µM SNAP;
g,, TNF
/ActD plus 800 µM Oxidized SANP
(OxiSNAP); h, TNF
/ActD plus 200 µM
Ac-DEVD-CHO, and i, overexposed image of blot d
to better demonstrate the faint new spots. Results are representative
of two to four similar experiments.
/ActD-treated cells. Fig.
4B shows labeled active caspases in hepatocytes treated with TNF
/ActD for various periods of time. No active caspases were detected when cell lysate from control cells was incubated with biotin-VAD-fmk. In contrast, 3 h after treatment with TNF
/ActD a double band representing active caspases was detected with the biotin
probe. The intensity of the labeling increased with time in a manner
similar to caspase-3-like activity (see above Fig. 2A). The
presence of a 10-fold excess of Ac-DEVD-CHO abolished the ability of
biotin-VAD-fmk to label caspases (last lane in Fig.
4B), demonstrating the labeling specificity for active
caspases. Furthermore, there was no labeling of active caspases if
cells were previously exposed to TNF
/ActD in the presence of
z-VAD-fmk, providing further support for the conclusion that these new
bands represent active caspases. As shown in Fig. 4C, cell
treatment with SNAP effectively prevented the formation of active
caspases whereas oxidized SNAP had no effect.
/ActD-treated cells that were absent in the control cells and in
cells treated with TNF
/ActD in the presence of Ac-DEVD-CHO. Spots
for two of the active caspases were of much higher intensity (Fig.
4D). The major spot (black arrow, Fig. 4D) was identified as the large subunit of caspase-3 by
immunoblotting analysis of the same blot (data not shown). In agreement
with our above one-dimensional electrophoresis results, NO nearly
completely blocked the activation of all the caspases including
caspase-3 when cells were treated with TNF
/ActD and SNAP (Fig.
4D). In contrast, oxidized SNAP had no effect on the
processing of these caspases. No active caspases were detected in cells
treated with ActD or TNF
alone.
/ActD-mediated Apoptosis and
the Effect of NO--
The above data indicate that NO prevents the
activation of multiple caspases. Caspase activation is thought to
contribute to changes in mitochondrial function with release of
cytochrome c. These mitochondrial events can also lead to
activation of downstream caspases. To determine the involvement of
mitochondria in hepatocyte apoptosis, we first examined the changes of
mitochondrial transmembrane potential upon induction of apoptosis with
TNF
/ActD. Mitochondrial inner transmembrane potential (
m) was
monitored by incorporated fluorescence of the cationic lipophilic dye
DiOC6(3), a potential-sensitive dye. The pattern of the
fluorescence of DiOC6(3) taken up by control cultured
hepatocytes revealed two cell populations, the major one with higher
fluorescence representing cells with intact high
m and a second
smaller population with low
m (Fig.
5A). The presence of a
population with low
m was not surprising since there was a basal
level of apoptosis in these cultures of primary cells as revealed by
the propidium iodine staining of DNA (Fig. 1B) and by
Annexin V labeling analysis (data not shown). When cells were labeled
with DiOC6(3) in the presence of 50 µM CCCP, a protonophore that disrupts
m, the population of cells with higher
m was converted to the low
m cells (Fig.
5A), confirming that the dye was sensitive to mitochondrial
transmembrane depolarization. The amount of DiOC6(3) dye
taken up by cells remained unaltered for at least 6 h after
TNF
/ActD treatment (data not shown). However, a reduction in
m
was detected 8 h after exposure to TNF
/ActD. Coincubation of
SNAP with TNF
/ActD suppressed the disruption of
m, whereas
oxidized SNAP had no effect on the alteration of
m in response to
TNF
/ActD-induced apoptosis. The caspase-3-like protease inhibitor
Ac-DEVD-CHO also abolished the mitochondrial depolarization, indicating
that the activation of the Ac-DEVD-CHO-inhibitable proteases occurred
prior to mitochondrial depolarization in primary hepatocytes.
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Fig. 5.
NO inhibits mitochondrial membrane potential
changes and cytochrome c release. A:
left panel, hepatocytes were loaded with 80 nM
DiOC6(3) in the absence and presence of 50 µM
CCCP for 30 min at 37 °C. Incorporation of the dye was evaluated
with flow cytometry. Right panel, hepatocytes were cultured
in the absence and presence of 2000 units/ml TNF plus 0.2 µg/ml
ActD supplemented with or without 800 µM SNAP, 800 µM Oxi-SNAP, or 200 µM Ac-DEVD-CHO as
indicated for 7.5 h prior to addition of DiOC6(3).
Cells were labeled for 30 min and mitochondrial membrane potential was
then determined by flow cytometry. B and C,
immunoblots for cytosolic cytochrome c. Cells were cultured
with TNF
(2000 units/ml) plus ActD (0.2 µg/ml) in the absence and
presence of 800 µM SNAP for the indicated times
(B) or for 8 h (C). Cytosolic extract was
obtained by centrifugation at 100,000 × g for 1 h
at 4 °C after homogenization of cells in buffer A containing 250 mM sucrose. Proteins were resolved by SDS-PAGE, transferred
to nitrocellulose, and detected with anti-cytochrome c
antibody as detailed under "Experimental Procedures." Results are
representative of two similar experiments.
/ActD-treated hepatocytes. Release of cytochrome c to cytoplasm in apoptotic cells was detected by
immunoanalysis. As shown in Fig. 5B, cytosol from untreated
cells contained negligible cytochrome c whereas cytochrome
c started to accumulate in the cytosol as early as 4 h
in cells exposed to TNF
/ActD with additional increases through
8 h. The level of cytochrome c in cytosol of hepatocytes treated with TNF
/ActD for 8 h was brought back down to the control levels by co-treatment of cells with SNAP, demonstrating that NO acts at or above the level of cytochrome c release
(Fig. 5C).
with its receptor is caspase-8 (35). Activation of caspase-8 can lead
to direct activation of other caspases or to the release of cytochrome
c from mitochondria (9, 10, 36). The observation that NO
inhibits the proteolytic activation of all caspases as well as
apoptosis-associated mitochondrial changes suggests that activation of
caspase-8 was inhibited by NO. To further determine whether activation
of caspase-8 occurs and whether it is in fact inhibited by NO, we first
examined the time course for caspase-8 activity in cytosolic extracts
from TNF
/ActD-treated hepatocytes using the caspase-8-specific
substrate IETD-pNA. IETD-pNA is preferentially
cleaved by caspase-8, with a
kcat/Km value around 60-fold
higher when compared with that of caspase-3 (37). Caspase-8 activity
was increased in TNF
/ActD-treated cells in a
time-dependent manner (Fig.
6A) and was significantly
inhibited by SNAP (Fig. 6B). Like z-VAD-fmk, the caspase-8
inhibitor z-IETD-fmk (50 µM, Ref. 38) inhibited the
caspase-8 activity and rescued the cells from TNF
/ActD-induced cell
death, providing further supports for the involvement of caspase-8 and
the role for NO-dependent caspase-8 inhibition. Furthermore, caspase-8 activity was suppressed in cells transfected with iNOS and subsequently exposed to TNF
/ActD, a suppression completely reversed by the iNOS inhibitor NIO (Fig. 6C).
Finally, we performed immunoblotting analysis specifically for the
large subunit of caspase-8. Using a monoclonal antibody that recognizes the large subunit of caspase-8, we found that a
18-kDa polypeptide was detectable as early as 3 h after cell exposure to TNF
/ActD, and that its intensity increased in a time-dependent manner
(Fig. 7A). The catalytically
active subunit of caspase-8 was seen only in apoptotic cells, but not
in the control cells. When cells were treated with TNF
/ActD in the
presence of SNAP or z-VAD-fmk, processing of caspase-8 was inhibited
(Fig. 7A). Likewise, AdiNOS transfection significantly
suppressed the appearance of this proteolytic fragment whereas addition
of NIO reversed this suppression (Fig. 7B), demonstrating that in TNF
/ActD-treated hepatocytes caspase-8 was indeed activated and its proteolytic processing was markedly inhibited by NO.
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Fig. 6.
NO inhibits caspase-8 activation.
A, time-dependent activation of caspase-8 in
apoptotic hepatocytes. Cellular proteins isolated from hepatocytes
exposed to TNF /ActD for indicated times were analyzed for
Ac-IETD-pNA cleavage activity. B, cells were treated with or
without TNF
/ActD (2000 units/ml and 0.2 µg/ml, respectively) in
the presence of SNAP (800 µM), z-IETD-fmk (50 µM), or z-VAD-fmk (50 µM) for 8 h and
cell lysate were prepared. Casaspe-8 activity was determined using a
colorimetric assay with Ac-IETD-pNA (100 µM).
C, hepatocytes were infected with AdLacZ or AdiNOS and then
cultured with or without TNF
/ActD in the absence or presence of 1 mM NIO for 6 h. Caspase-8 activity in the cell lysates
was measured as above. Results are one representative of two
independent experiments measured in duplicates.
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Fig. 7.
NO suppresses caspase-8 activation.
A, time-dependent activation of caspase-8 in
apoptotic hepatocytes. Cellular proteins isolated from hepatocytes
treated with TNF /ActD for indicated times or treated in the presence
of 800 µM SNAP or 100 µM z-VAD-fmk for
8 h were separated on SDS-PAGE and transferred onto a
nitrocellulose membrane. The catalytic subunit p18 of caspase-8 was
visualized by immunoblotting analysis. B,
NO-dependent suppression of caspase-8 activation.
Hepatocytes were infected with AdLacZ or AdiNOS as described in the
legend to Fig. 3 and under "Experimental Procedures." The large
subunit of caspase-8 was visualized by immunoblotting analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ActD-induced apoptosis. We first identified the number of
caspases activated in hepatocytes. We show that at least four caspases
were activated including caspase-3 and caspase-8. Furthermore, caspase
activation occurred in association with loss of mitochondrial membrane
potential and accumulation of cytochrome c in the cytosol.
Our results demonstrate that exposure to NO, either via a NO donor or
through expression of iNOS, markedly suppresses activation of all of
the caspases and prevents loss of mitochondrial membrane potential and
release of cytochrome c. Thus, NO effectively inhibits not
just the activity of active caspases but also the proteolytic
cleavage/activation of pro-caspases in cells.
(24, 25), staurosporine (39), transforming growth
factor-
(39), aging in culture and Fas (40). We (24, 26) and others
(39-41) have previously shown that caspase-3-like protease activity
increases in hepatocytes undergoing spontaneous apoptosis or apoptosis
in response to transforming growth factor-
, TNF
or Fas, while
caspase-1-like activity does not. The caspase-3-like family of
proteases is defined by a specificity for the DEVD substrate sequence
and includes caspases-3 and -7 as well as caspase-8 due to the overlap
in substrate specificity (26, 42, 43). The identity of specific
caspases activated under these conditions has not been well
established. Here two-dimensional electrophoresis analysis of affinity
labeled activated caspases in TNF
/ActD-treated cells revealed at
least four different active caspase species. Immunoblotting analysis of
the two-dimensional gel electrophoresis identified one major spot as
caspase-3. The identity of the other active caspases detected by this
approach is uncertain and currently under investigation in our
laboratory. Based on the fact that TNF
/ActD-induced hepatocyte
apoptosis requires the death receptor TNFR1 and current knowledge on
death receptor-mediated signaling pathway, it is tempting to speculate that at least one of the active caspase species labeled by
biotin-VAD-fmk is caspase-8, the most apical caspase in the cascade.
This is in line with our immunoblotting analysis with a monoclonal
antibody that recognizes the large subunit of caspase-8 (Fig. 7), where pro-caspase-8 was processed/activated to p18 in TNF
/ActD-treated hepatocytes but not in control cells. Two-dimensional electrophoresis analysis of biotin-VAD-fmk-labeled recombinant human caspase-8 revealed
three active species with the same sizes but slightly different
charges. Two of the three caspase-8 species closely co-migrated with
two of the four labeled active caspases in the TNF
/ActD-treated rat
hepatocytes (same IEF positions but migrated slightly slower in
SDS-PAGE, probably due to species difference) (data not shown),
indicative of the identity of two labeled spots as caspase-8.
m) has been reported to
precede caspase-3 activation and the final execution stage of apoptosis
in many situations, but not all (46). In TNF
/ActD-induced hepatocyte
apoptosis, changes in mitochondrial
m were not evident until the
late phase of apoptosis (>8 h following apoptosis induction) when
caspase-3 was fully activated (Fig. 5A). The
TNF
/ActD-induced disruption of
m was blocked by the caspase
inhibitor Ac-DEVD-CHO, suggesting that mitochondrial depolarization
requires caspase activation. In contrast, mitochondrial cytochrome
c was released by 4 h into cytoplasm in
TNF
/ActD-treated hepatocytes and paralleled caspase-3-like protease
activation (Fig. 5B). Most recently Fas-mediated activation of caspase-8 has been shown to cleave Bid, a Bcl-2 interacting protein,
resulting in the translocation of cleaved Bid to the mitochondria where
it triggers cytochrome c release (9, 10). The effect of
caspase-8 may be amplified by cytochrome
c-dependent downstream caspase activation,
leading to an amplification loop between caspases and mitochondria (36,
47). Loss of cytochrome c could uncouple oxidative
phosphorylation and thus result in disruption in
m. The capacity
of NO to suppress all of these events could be explained by the
inhibition of caspase activation by NO.
+ N-galactosamine-induced hepatic injury and apoptosis
in vivo (48). Thus, NO is a potent endogenous inhibitor of
apoptosis in the liver. Previous mechanistic studies have shown that NO
directly inactivates active caspases via S-nitrosylation of
the catalytic cysteine residue (26). In the present study, we
demonstrate that NO prevents the proteolytic activation of multiple
pro-caspases in intact cells. This includes the inhibition of the most
proximal protease, caspase-8 as well as the executioner caspase-3. Two
nonexclusive mechanisms have been implicated for the propagation of the
apoptotic signaling cascade from caspase-8 to caspase-3 in
TNF
-mediated cell death. These include the direct activation of
caspase-3 by caspase-8 and the cytochrome
c-dependent activation of caspase-9. According
to current understanding, death receptors (TNFR1 and Fas) induce the
recruitment of FADD (Fas-associated death domain protein) and
pro-caspase-8, thus forming a membrane anchored death-inducing signal
complex (35). Protein-protein interaction between the death effector
domain of pro-caspase-8 and FADD results in the cleavage/activation of
pro-caspase-8. It has been shown in a cell-free system that at higher
concentrations active caspase-8 can directly activate downstream
caspases such as caspase-3 and thereby promotes the apoptotic cascade.
However, at lower concentrations of caspase-8, the mitochondrial
release of cytochrome c is necessary for death signal
amplification and a complete engagement of the apoptotic machinery
(36). It is also possible that direct activation of caspase-3 by an
upstream caspase can lead to mitochondrial changes and release of
cytochrome c through the capacity of caspase-3-like
proteases to cleave the Bcl-2 family members (50, 51). This inhibition
of cytochrome c release and loss of mitochondrial
m by
NO found in this study could have been due to the inhibition of
caspase-8 activation, thus preventing the downstream events.
Furthermore, NO does not appear to interrupt the death signal
transduced from Fas receptor to FADD/pro-caspase-8, since NO was still
able to suppress apoptosis induced by overexpression of FADD or
caspase-8 in Jurkat cells (20). It is not clear at present how NO
inhibits the processing of pro-caspase-8. In the previous studies
showing inactivation of caspase enzymes via S-nitrosylation,
including caspase-8 (20, 26), only processed and activated caspases
were used. It is likely that in intact cells such as the system used in
this study, a limited amount of caspase-8 was activated upon the
formation of the death-inducing signal complex but subsequently
inactivated by NO through S-nitrosylation. As a result,
further activation of caspase-8 and other effector caspases was
diminished. This possibility is supported by our previous work (24)
showing that the reducing agent dithiothreitol partially reversed the
NO-mediated suppression of caspase activity in cells. Alternatively, NO
may S-nitrosylate the essential cysteine residue of the
pro-caspase-8 and thus prevent the zymogen from undergoing proteolytic
activation. Despite evidence for NO-dependent
S-nitrosylation of activated caspases, it remains unclear
whether NO can effectively S-nitrosylate pro-caspases and
whether this S-nitrosylation has any effect on pro-caspase processing.
/ActD-induced apoptosis
even when transcription was inhibited, demonstrating an interaction of
NO or products of NO with the death cascade. Furthermore, levels of
Hsp70 and Bcl-2 were not altered in TNF
/ActD-treated hepatocytes
upon simultaneous exposure to NO donor or to NO produced from iNOS in
the cells.2 In addition, we
have previously shown that the NO-mediated protection in hepatocytes is
only partially due to increases in cGMP levels (24). Even though much
of the protection by NO is cGMP-independent, it is interesting to note
that high concentrations of cGMP analogues effectively inhibit
apoptosis in hepatocytes (24). Finally, NO has no effect on the
activation of JNK kinase induced by TNF
/ActD.2 Taken
together, our data demonstrate that the anti-apoptotic effect of NO is
a result of the capacity of NO to prevent caspase cleavage/activation
into catalytic active form and consequently the apoptosis resulting
from caspase activation. Considering the central roles of caspases in
apoptosis and the wide distribution of NO synthase in tissues, one
might speculate that the regulation of apoptosis by NO by modulating
caspase activation could be of great significance.
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ACKNOWLEDGEMENT |
---|
We thank Drs. P. H. Krammer for providing caspase-8 antibody and M. Kibbe for advice on adenoviral gene transfer and the artwork. We thank D. L. Williams and A. Green for technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM-44100 (to T. R. B.).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.
§ Recipient of Postdoctoral Fellowship GM19866 from United States Department of Public Health Service.
Recipient of Korea Science and Engineering Foundation Grant
981-0714-100-2.
** To whom correspondence should be addressed: A1010 PUH, Dept. of Surgery, University of Pittsburgh, Pittsburgh, PA 15213. Tel.: 412-648-9862; Fax: 412-648-1033; E-mail: billiartr{at}msx.upmc.edu.
2 J. Li and T. R. Billiar, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
NO, nitric oxide;
Ac-, acetyl-;
ActD, actinomycin D;
CHO, aldehyde;
pNA, p-nitroanilide;
SNAP, S-nitroso-N-acetylpenicillamine;
Oxi-SNAP, oxidized SNAP;
NIO, N-iminoethyl-L-ornithine;
iNOS, inducible nitric oxide synthase;
CCCP, carbonyl cyanide
m-chlorophenylhydrazone;
PI, propidium iodide;
z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone;
TNF, tumor
necrosis factor
;
PARP, poly(ADP-ribose) polymerase;
PAGE, polyacrylamide gel electrophoresis;
IEF, isoelectric focusing;
m, mitochondrial inner transmembrane potential;
AdiNOS, adenoviral iNOS;
LacZ,
-galactosidase;
AdLacZ, adenoviral LacZ, FADD, Fas-associated
death domain protein.
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REFERENCES |
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