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INTRODUCTION |
Nitric oxide is a highly reactive and unstable free radical gas
that can cross cell membranes easily by diffusion without depending on
any release or uptake mechanisms. NO is involved in several signaling
pathways related to a diverse array of cell functions. Low levels of NO
constitutively produced by an endothelial nitric-oxide synthase play a
physiological role such as in regulation of vasodilatation (1) and
platelet aggregation (2). On the other hand, high levels of NO produced
by an inducible NO synthase mainly in macrophages and neutrophils
mediate cytotoxicity as the first line of self-defense against invading
microorganisms (3) or tumor cells (4). Recently, NO-mediated apoptosis was reported in macrophages (5, 6), a pancreatic beta cell line (7),
and mouse thymocytes (8). NO-generating compounds such as sodium
nitroprusside (SNP),1
S-nitroso-N-acetylpenicillamine, and
S-nitroglutathione have been reported to induce apoptosis in
human leukemia HL-60 and U937 cells (9-11). Although the mechanisms of
NO-mediated cytotoxicity are still controversial, several possible
systems described as follows have been proposed; 1) formation of
iron-nitrosyl complexes with FeS-containing critical enzymes, which
would cause an impairment of mitochondrial function and an energy
depletion (12); 2) direct DNA damage by deamination and cross-linking
of DNA, which increase mutagenesis (13); 3) generation of peroxynitrite
by reaction of NO with superoxide, which may play a significant role in
the cytotoxic process (14, 15); and 4) inactivation of several antioxidant enzymes, including catalase, glutathione peroxidase, and
superoxide dismutases (15, 16).
Sphingolipid metabolites including ceramide have been implicated as
potential regulatory molecules in signal transductions involving cell
growth, differentiation, and death. Many stresses against cell
viability such as tumor necrosis factor-
, anti-Fas antibody,
ionizing radiation, and serum deprivation (17, 18); anti-cancer drugs
(19, 20); heat shock (21); and hydrogen peroxide (22) were reported to
be accompanied by an increase in intracellular ceramide. As downstream
targets of ceramide, ceramide-activated protein phosphatase (23),
protein kinase C
(24), a ceramide-activated protein kinase
(25), and the interleukin-1
-converting enzyme family of proteases
called caspases (26) have been suggested. We have also demonstrated the
requirement of the transcription factor component AP-1 and cytosolic
translocation of protein kinases C
and -
from the membrane
fraction for ceramide-mediated apoptosis (27, 28).
Recently, a family of proteases known as caspases has been implicated
as a common executioner of a variety of death signals. Caspase-dependent ceramide generation has been proposed in
several apoptosis models (29-31), whereas Mizushima et al.
(32) reported that cell-permeable ceramide induced the cleavage and
activation of caspase-3. These results may indicate the activation of
caspases both upstream and downstream of ceramide production, but the
precise relation between the caspase cascade and ceramide generation in apoptosis remains to be clarified. NO was also reported to increase caspase-3 to induce apoptosis (33).
Here, the relation between ceramide generation and caspase-3
in apoptosis induced by NO in HL-60 cells has been investigated. We demonstrated that SNP (a NO donor) increased ceramide generation via
activation of magnesium-dependent neutral sphingomyelinase (N-SMase), but not via magnesium-independent and acid SMases. The
ceramide generation seemed to be mediated upstream of
NO-activated caspase because the inhibition of caspase by
acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO) prevented NO-induced
apoptosis, activation of magnesium-dependent N-SMase, and
ceramide formation. Moreover, recombinant purified caspase-3
could increase N-SMase activity in a cell-free system. As far as
we know, this is the first report suggesting the effect of
caspase-3 on magnesium-dependent N-SMase.
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MATERIALS AND METHODS |
Cells and Reagents--
Human leukemia HL-60 cells were kindly
provided by Dr. M. Saito (National Cancer Institute, Tokyo, Japan).
C2-ceramide was purchased from Matreya, Inc.
[
-32P]ATP (6000 Ci/mmol) was purchased from Amersham
Pharmacia Biotech. Diacylglycerol (DAG) kinase was kindly provided by
Dr. Y. Hannun (Duke University). Sodium nitroprusside was purchased
from E. Merck (Darmstadt, Germany).
Acetyl-Asp-Glu-Val-Asp-
-(4-methylcoumaryl-7-amide) (DEVD-MCA) and
DEVD-CHO were purchased from the Peptide Institute (Osaka, Japan),
dissolved at 10 mM in Me2SO, and stored at
80 °C. Recombinant purified caspase-3 and -6 were prepared as
described (34). Other chemicals, if not mentioned, were obtained from Sigma.
Cell Culture--
Human myelogenous leukemia HL-60 cells were
maintained in RPMI 1640 medium containing 10% fetal bovine serum at
37 °C in a 5% CO2 incubator. HL-60 cells in
exponentially growing phase were washed in RPMI 1640 medium,
resuspended in 2% serum-containing medium at a concentration of 2 × 105 cells/ml overnight, and then treated, if not
described otherwise below. Viable cell numbers were assessed by the
0.025% trypan blue dye exclusion method under microscopic observation.
Cell numbers and survival rates were also measured by the WST-1 assay (cell counting kit, Dojindo, Kumamoto, Japan) using a 96-well microplate reader. Reagents were applied 30 min before adding SNP in
the culture medium, if not described otherwise below.
Analysis of DNA Fragmentation--
DNA was isolated using a
GENOME kit (Bio 101, Inc., Vista, CA), electrophoresed through a 3%
NuSieve agarose minigel (FMC Corp. BioProducts) in 40 mM
Tris acetate and 1 mM EDTA at 50 V for 3 h, and
visualized under UV light after ethidium bromide staining.
Flow Cytometry--
Flow cytometric DNA analysis was performed
for quantification of cell death by apoptosis. Due to DNA degeneration
and subsequent leakage from cells (35), apoptotic cells can be detected
by diminished staining with DNA-specific fluorochromes. In brief, 2 × 106 cells were harvested, washed with
phosphate-buffered saline, and resuspended in phosphate-buffered saline
containing 0.5% paraformaldehyde and 0.5% saponin for fixation of
cells (36). The cells were then washed and resuspended in fluorochrome
solution (50 µg/ml propidium iodide and 1 mg/ml RNase (Bachem
California, Torrance, CA)). Red fluorescence was measured with a
FACScan (Becton Dickinson Advance Cellular Biology, San Jose, CA). We
could assess the number of hypodiploid cells (apoptotic cells) and
cells with more than diploid DNA content (non-apoptotic cells).
Ceramide Measurement--
Extraction of cellular lipids by the
Bligh-Dyer method (54) and ceramide measurement using DAG kinase were
performed as described (37, 38). The solvent system used to separate
ceramide phosphate was chloroform/acetone/methanol/acetic
acid/H2O (10:4:3:2:1). We confirmed that DAG kinase
activity was not increased by SNP because C2-ceramide did
not change as an internal standard during the procedure, and the
amounts of phospholipid phosphate corresponded to the viable cell
numbers (data not shown).
Nitrite Assay--
NO undergoes a series of reactions with
several molecules present in biological fluids. The final products of
NO in vivo are nitrite and nitrate. The sum of nitrite and
nitrate can be the index of total NO production. Nitrite, a stable NO
oxidation product, was determined using the Griess reaction
(nitrate/nitrite assay kit, Cayman Chemical Co., Inc., Ann Arbor, MI).
Phenol red-free Dulbeccos' modified Eagle's medium was harvested
after treatment. First, nitrate was converted to nitrite utilizing
nitrate reductase, and then Griess reagents were added to convert
nitrite into a deep-purple azo compound. The absorbance of the azo
chromophore was measured to determine the nitrite concentration at 540 nm using the plate reader.
Sphingomyelin (SM) Quantitation--
The cells were washed with
phosphate-buffered saline, seeded at 5 × 105 cells/ml
in RPMI 1640 medium containing 2% fetal calf serum, and labeled with
[14C]choline chloride (0.1 µCi/ml) at 37 °C in 5%
CO2 for 36 h. The labeled cells were treated with 1 mM SNP for the indicated times. After harvesting the cells,
the lipids were extracted by the Bligh-Dyer method (54) and applied to
a Silica Gel 60 TLC plate (Whatman). Inorganic phosphate in the extract
was measured to calculate phospholipid content. The TLC plate was
developed in solvent containing chloroform/methanol/acetic acid/H2O (50:30:8:5), and the bands corresponding to SM
were detected with a Fuji BAS system.
Assay Procedure for SMases--
HL-60 cells (1 × 107) were harvested; washed twice with ice-cold
phosphate-buffered saline; and homogenized in 0.5 ml of lysis buffer
containing 10 mM Tris- HCl (pH 7.5), 1 mM EDTA,
and 0.1% Triton X-100 after each treatment. The homogenate was
centrifuged at 100,000 × g for 1 h at 4 °C.
The supernatant was used as an enzyme source. The assay mixture for the
measurement of magnesium-dependent N-SMase contained 0.1 M Tris-HCl (pH 7.5), 60 nmol of
[methyl-14C]sphingomyelin (specific
activity = 1.74 GBq/mmol; Amersham Pharmacia Biotech), 10 mM MgCl2, 0.1% Triton X-100, and 50-300 µg
of enzyme in a final volume of 0.1 ml. For magnesium-independent
N-SMase, MgCl2 was removed from the reaction mixture. For
acid SMase, 0.1 M sodium acetate (pH 5.0) was used instead
of Tris-HCl. Incubation was carried out at 37 °C for 30 min. The
reaction was stopped by adding 1.5 ml of chloroform/methanol (2:1).
Then, 0.2 ml of double-distilled water was added to the tubes and
vortexed. The tubes were centrifuged at 1000 × g for 5 min to separate the two phases. The clear upper phase (0.4 ml) was
removed, placed in a glass scintillation vial, and counted with a
scintillation counter (Packard Instrument Co.). Protein concentrations
were determined using a Bio-Rad protein assay kit.
Fluorometric Assay of DEVD-MCA Cleavage Activity--
After each
treatment, the cells were homogenized in lysis buffer containing 10 mM HEPES/KOH (pH 7.4), 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 100 µM pepstatin, 0.15 units/ml aprotinin, and
50 µg/ml leupeptin and centrifuged at 10,000 × g for
10 min. The supernatant was collected as an enzyme source and added to
the reaction mixture (10% sucrose, 10 mM HEPES/KOH (pH
7.4), 5 mM dithiothreitol, 0.1% CHAPS, and 10 µM DEVD-MCA), followed by incubation at 25 °C for 60 min. Fluorescence was measured with a microplate reader (MTP-100F,
Corona Electric, Tokyo, Japan) using 360-nm excitation and 450-nm
emission filters. Concentrations of 7-amino-4-methylcoumarin liberated
as a result of cleavage were calculated comparing with standard
7-amino-4-methylcoumarin solutions.
Preparation of Cell Extracts for Assay of SMase in a Cell-free
System--
The cells were suspended in lysis buffer containing 10 mM HEPES/KOH (pH 7.4), 2 mM EDTA, 0.1% CHAPS,
5 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 100 µM pepstatin, 0.15 units/ml aprotinin, and
50 µg/ml leupeptin; left on ice for 20 min; passed through a 27-gauge
needle; and then centrifuged at 10,000 × g for 15 min.
Protein concentrations were determined with the Bio-Rad assay.
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RESULTS |
SNP-induced Apoptosis in HL-60 Cells--
SNP showed a time- and
dose-dependent induction of apoptosis in HL-60 cells (Fig.
1, A and B). Three
h after treatment with 1 mM SNP, HL-60 cells showed
morphological changes (blebbing, shrinkage, and chromatin condensation)
and DNA fragmentation characteristic for apoptosis (Fig.
1C). The percentage of apoptotic cells measured by flow
cytometric analysis increased from 8.6 to 30.5% 4 h after treatment with 1 mM SNP. At higher concentrations, the
number of apoptotic cells did not show any more increase, and necrosis was observed judging from trypan blue dye staining. After 24 h, the cell numbers decreased to ~20% of the control level (data not
shown). Potassium hexacyanoferrate, which is structurally similar to
SNP except for the absence of a nitroso group, did not affect cell
growth at the same concentration as SNP (data not shown), suggesting
that the effects of SNP on cell growth and apoptosis were due to NO
generation, but not to cyanoid effects.

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Fig. 1.
SNP-induced apoptosis in human leukemia HL-60
cells. A, time dependence of apoptotic induction by
SNP. Cells (2 × 105/ml) were treated with 1 mM SNP and harvested at the indicated times. Apoptotic
cells were determined by fluorescence-activated cell sorter analysis
using the propidium iodide staining method as described under
"Materials and Methods." B, dose dependence of
apoptotic induction by SNP. The cells were treated with the
indicated concentrations of SNP for 4 h. The results were obtained
from at least three different experiments. The error bars
indicate 1 S.D. C, effects of SNP on morphological changes
and DNA fragmentation. The left panels show the
morphological changes in the cells without (upper) and with
(lower) 1 mM SNP for 3 h. The cells were
applied to uncoated glass microscope slides using cytocentrifugation
and then stained with May-Giemsa stain. The right panel
shows SNP-induced DNA fragmentation 0, 90, and 180 min after treatment
with 1 mM SNP. Analysis of DNA fragmentation on agarose gel
was performed as described under "Materials and Methods." The
results are representative of three different experiments.
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Increase in Intracellular Ceramide Induced by SNP--
To date,
our studies (37, 38) and others (17, 18) have demonstrated that
ceramide, a lipid second messenger, plays an important role in
regulating cell growth, differentiation, and death. To investigate the
interrelation between NO and ceramide generation, we measured nitrite
concentration in the culture medium after addition of
C2-ceramide. We could not detect any change within 6 h, whereas SNP increased nitrite production in a
time-dependent manner (Fig.
2). Although ceramide was reported to
enhance the expression of inducible NO synthase in rat astrocytes (39), it may be that ceramide does not increase NO generation in HL-60 cells.
To examine the possible involvement of the ceramide signaling pathway
in NO-induced cell stress, we measured intracellular ceramide levels
after addition of SNP. Ceramide generation measured by the DAG kinase
assay method began to increase at 90 min after addition of 1 mM SNP and reached a maximum level, which was ~160% of
the control level, 4 h after treatment (Fig.
3A). To justify ceramide
measurement by the DAG kinase assay, we confirmed that the DAG kinase
and phospholipid phosphate activities of the same numbers of cells did
not change during treatment with SNP, as described under "Materials
and Methods." Concentrations higher than 1 mM SNP did not
increase ceramide levels more than 1 mM SNP, probably
due to the induction of necrosis (Fig. 3B).

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Fig. 2.
Changes in nitrite concentration in culture
medium after treatment with SNP or C2-ceramide. The
nitrite concentration was determined by the Griess reaction method as
described under "Materials and Methods." Although nitrite increased
after treatment with 1 mM SNP as a positive control, no
change was observed after treatment with 5 µM
C2-ceramide. The error bars indicate 1 S.D.
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Fig. 3.
Ceramide generation, decrease in
sphingomyelin, and increase in magnesium-dependent N-SMase
activity induced by SNP. A,
time-dependent increase in ceramide induced by SNP. Lipids
were extracted at the indicated times, and ceramide contents were
measured by the DAG kinase method as described under "Materials and
Methods." The results were obtained from three different experiments.
The error bars indicate 1 S.D. B,
dose-dependent increase in ceramide induced by SNP. The
cells were exposed to the indicated concentrations of SNP for 4 h.
The results were obtained from at least three different experiments.
The error bars indicate 1 S.D. C, decrease in
sphingomyelin induced by SNP. The cells prelabeled with
[14C]choline chloride were treated with 1 mM
SNP for the indicated times. Lipids were extracted, and labeled
sphingomyelin was measured as described under "Materials and
Methods." D, SNP-induced activation of
magnesium-dependent N-SMase. The cells were treated with 1 mM SNP for the indicated times, and the activities of
SMases were determined as described under "Materials and Methods."
The results were obtained from three different experiments. The
error bars indicate 1 S.D.
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Sphingomyelin Hydrolysis through Magnesium-dependent
N-SMase by SNP--
Since there are several possible metabolic
pathways as a mechanism of ceramide generation, we, first of all,
measured changes in labeled SM contents caused by SNP to examine the
possible involvement of SMase. SM levels decreased to 70% of the
control level 2 h after treatment with 1 mM SNP and
then returned to the control level by 6 h (Fig. 3C). We
examined the activities of three different types of SMases, which have
been reported to be involved in SM hydrolysis (37). As shown in Fig.
3D, magnesium-dependent N-SMase (basal specific
activity = 1.08 nmol/mg of protein/h) increased to 156 ± 18% of the control level 2 h after treatment with SNP and
returned to the control level by 6 h. The activity of
magnesium-independent N-SMase (basal specific activity = 0.65 nmol/mg of protein/h) did not change following treatment with SNP up to
6 h. The activity of acid SMase (basal specific activity = 9.72 nmol/mg of protein/h) slightly decreased, but did not increase
after treatment with SNP. The biological meaning of this decrease is
unclear at present. These results suggest that ceramide generation
induced by SNP results from SM hydrolysis via the increase in
magnesium-dependent N-SMase activity.
Increase in DEVD-MCA Cleavage Activity and Its Involvement in
SNP-induced Apoptosis--
Since many stresses are reported to
activate caspase-3 as an executioner of apoptosis, we investigated
whether SNP-generated NO activates caspase-3. The activity of caspase-3
was assessed by measuring the proteolytic cleavage of DEVD-MCA, a
fluorogenic substrate of caspase-3, and it increased after treatment
with SNP (Fig. 4A). The
activities 4 h after treatment with 0.5 and 1 mM SNP
were 88 and 134 pmol/mg of protein/min, respectively, compared with the
control level of 30 pmol/mg of protein/min. SNP increased the caspase-3
activities in a time-dependent manner. DEVD-CHO (200 µM), an inhibitor of caspase-3, completely inhibited the
increase in DEVD-MCA cleavage activity induced by SNP (Fig. 4B). Moreover, SNP-induced apoptotic cells markedly
decreased from 31 to 11% upon addition of DEVD-CHO (Fig.
4C). These results suggest that the activation of caspase-3
is required to induce HL-60 cell apoptosis by SNP.

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Fig. 4.
SNP-induced activation of caspase and
prevention of its activation and apoptosis by DEVD-CHO.
A, induction of DEVD-MCA cleavage activity after treatment
with SNP. After the indicated periods of incubation with 0.5 or 1 mM SNP, cleavage activity was measured as described under
"Materials and Methods." B, inhibition of SNP-induced
caspase by DEVD-CHO. After preincubation for 1 h in the presence
or absence of 200 µM DEVD-CHO, the cells were treated
with or without 1 mM SNP for 4 h. C,
inhibition of SNP-induced apoptosis by DEVD-CHO. After preincubation
for 1 h in the absence or presence of 200 µM
DEVD-CHO, the cells were treated with or without 1 mM SNP
for 4 h. Panel a, control; panel b, SNP;
panel c, DEVD-CHO; panel d, DEVD-CHO + SNP.
Apoptotic cells were determined by fluorescence-activated cell
sorter analysis. The results are representative of three different
experiments.
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Inhibitory Effects of DEVD-CHO on Ceramide Generation and
Activation of Magnesium-dependent N-SMase--
We
investigated whether ceramide generation by
magnesium-dependent N-SMase was upstream or downstream of
caspase-3 in SNP-induced apoptosis. We examined the effects of DEVD-CHO
on ceramide generation and the increase in
magnesium-dependent N-SMase activity induced by SNP. The
SNP-induced increase in intracellular ceramide, which showed the
maximum 4 h after treatment, was completely inhibited by
preincubation with 200 µM DEVD-CHO, as shown in Fig.
5A. By the same procedure, the
activation of magnesium-dependent N-SMase (146 ± 16%
of the control level 4 h after treatment with SNP) was also
completely inhibited (Fig. 5B). These results show that the
protease including DEVDase is activated upstream of ceramide generation
by magnesium-dependent N-SMase for NO-induced
apoptosis.

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Fig. 5.
Prevention of SNP-induced ceramide generation
and magnesium-dependent N-SMase activity by DEVD-CHO.
A, cells were preincubated for 1 h in the absence or
presence of 200 µM DEVD-CHO and treated for 4 h with
or without 1 mM SNP. Lipids were extracted, and ceramide
contents were determined by the DAG kinase assay as described under
"Materials and Methods." B, after 1 h of
preincubation in the absence or presence 200 µM DEVD-CHO,
the cells were incubated with or without 1 mM SNP for
4 h. The activity of magnesium-dependent N-SMase was
measured as described under "Materials and Methods." The results
were obtained from three different experiments. The error
bars indicate 1 S.D.
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Activation of Magnesium-dependent N-SMase by Purified
Caspase-3 in a Cell-free System--
It became clear that the caspase
inhibitor DEVD-CHO inhibits ceramide generation and
magnesium-dependent N-SMase activity in the process of
NO-induced apoptosis. Although it is the nature of things that SMase
can generate ceramide through hydrolysis of SM, there was no direct
evidence that caspase-3 could increase the activity of
magnesium-dependent N-SMase. Therefore, we used recombinant
purified caspase-3 from Escherichia coli transfected with
caspase-3 cDNA, which did not have any types of SMase activity in
itself (data not shown), but which could increase
magnesium-dependent N-SMase activity with the cell
extracts. Addition of recombinant purified caspase-3 (600 ng) to the
cell extracts (250 µg) induced a 3-fold increase in
magnesium-dependent N-SMase activity (Fig. 6). Since we previously showed that
caspase-3 activates caspase-6 in Fas-induced apoptosis (40), we
examined whether caspase-6 increased magnesium-dependent
N-SMase activity, but this activity did not increase even in the
presence of the cell extracts (data not shown). Our results suggest the
direct role of caspase-3 in activating magnesiumdependent
N-SMase and ceramide generation in NO-induced apoptosis.

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Fig. 6.
Activation of magnesium-dependent
N-SMase by recombinant purified caspase-3 in a cell-free system.
In the presence or absence of the cell extract (250 µg), the assay
for magnesium-dependent N-SMase was performed with or
without the indicated doses of purified caspase-3. The basal specific
activity of SMase was 1.29 nmol/mg/h. The results were obtained from
three different experiments. The error bars indicate 1 S.D.
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 |
DISCUSSION |
The sphingomyelin cycle and ceramide generation were first
discovered in cell differentiation of HL-60 cells in response to 1
,25-dihydroxyvitamin D3 (37, 38), and recent studies
have shown that many cell types respond to diverse stresses with
ceramide generation (17, 18). Recently, NO has been reported to be related to induction of apoptosis in various cell lines, including human leukemia HL-60 cells (5-11). In this report, we have
investigated whether and how NO is related to ceramide generation in
the caspase/apoptotic death pathway in HL-60 cells.
First of all, we showed that SNP induced growth inhibition and
apoptosis in a time- and dose-dependent manner (Fig. 1). We investigated whether ceramide increased NO generation and vice versa.
We found that ceramide had no effect on NO generation in HL-60 cells by
measuring nitrite in the culture medium (Fig. 2). This result agreed
with previous reports showing that ceramide itself does not affect NO
synthase in isolated pancreatic islets and astrocytes, even though
ceramide enhances lipopolysaccharide-induced NO synthase activation
(39, 41). When apoptosis was induced by NO, intracellular levels of
ceramide increased to ~160% of the control level 4 h after
treatment. Since there were several possible mechanisms to generate
ceramide metabolically, the changes in SM levels and three different
types of SMase were investigated. As shown in Fig. 3C, SM
levels decreased 2-4 h after treatment with SNP and then returned to
the control level, corresponding reciprocally to the time course of
ceramide generation. Although acid SMase was reported to be involved in
tumor necrosis factor-
-induced apoptosis (42), the activities of
acid SMase and magnesium-independent N-SMase did not increase in
NO-induced apoptosis (Fig. 3D). These findings show that NO
increases ceramide generation by activating magnesium-dependent N-SMase.
Although well characterized downstream signals of NO are guanylate
cyclase (43), adenyl cyclase (44, 45), and protein kinase C (46), it is
controversial whether protein kinases G, A, and C are closely involved
in NO-induced apoptosis (47-49). We therefore examined the effects of
several kinases, such as protein kinase A, G, or C, on NO-induced
ceramide generation by using activators and inhibitors of these
kinases. The results demonstrated that neither activators nor
inhibitors of protein kinases G, A, and C had any significant effect on
ceramide generation (data not shown), suggesting that NO induced
ceramide generation, independently of protein kinase A-, G-, or
C-related signaling.
In this work, we suggested that ceramide might be a mediator in the NO
signaling pathway and is generated from SM hydrolysis through the
activation of magnesium-dependent N-SMase. The
membrane-associated, magnesium-dependent neutral SMase was
purified and characterized very recently (50). In this study, N-SMase
was activated by caspase-3 because DEVD-CHO, a caspase-3 inhibitor,
blocked both SNP-induced SMase activation and ceramide generation
(Figs. 4 and 5). To confirm the more direct effect of caspase-3 on
SMase, recombinant purified caspase-3 was added to the cell extracts. In this cell-free system, we could detect the increase in
magnesium-dependent N-SMase activity in the presence of
recombinant caspase-3 (Fig. 6), whereas recombinant caspase-6 could not
increase SMase activity (data not shown). Judging from the present
data, the NO-generated ceramide signal is the downstream target of
caspase-3. However, at present, in terms of the mode of action of
caspases on SMase, it is unknown whether caspase-3 cleaved plausible
pro-SMase, cleaved and inactivated the SMase inhibitor, or activated
the SMase activator. As far as we know, this is the first evidence
showing the activation of magnesium-dependent N-SMase via
caspase-3, whereas activation of acid SMase activity has been shown to
occur in response to caspases (51). In terms of the relation of the
caspase cascade and ceramide in induction of apoptosis, many previous
reports suggest that ceramide is upstream of the caspase cascade (26). Our data and others related to REAPER in Drosophila (29),
which showed that caspase could enhance ceramide generation in
vivo, may suggest a new function of ceramide as a modulator of the
caspase cascade. At present, unfortunately neither this idea nor the
indispensability of caspase-generated ceramide in NO-induced apoptosis
can be demonstrated because we do not have any biochemical tool to
inhibit directly the activity of N-SMase to generate ceramide.
In contrast to our data, exposure to NO donors such as
S-nitroso-N-acetylpenicillamine and SNP or
activation of NO synthase was reported to inhibit apoptosis in T
lymphocytes and human umbilical vein endothelial cells (52, 53) because
NO induced cGMP-dependent or direct inhibition of caspase-3
through protein S-nitrosylation. These data seem
inconsistent with our data showing that NO could induce apoptosis by
increasing ceramide generation through caspase-3 activation. The
discrepancy in the NO effect on apoptosis may be due to differences in
intensity and duration of NO exposure and kinds of cells. Indeed, we
observed that low concentrations of SNP <100 µM showed a
protective effect against serum deprivation-induced apoptosis in HL-60
cells. Induction of apoptosis and an increase in intracellular ceramide
were generally observed upon treatment with >250 µM SNP.
Caspase-3 activity measured by cleavage of DEVD-MCA was also enhanced
at higher (but not lower) concentrations of SNP.2
NO could generate peroxynitrite by reacting with superoxide anion. NO
could also modulate endogenous antioxidant enzymes such as catalase,
glutathione peroxidase, and superoxide dismutases (15, 16). Since
intracellular ceramide was reported to be increased by hydrogen
peroxide (22), changing of redox status may be another one of the
mechanisms regulating apoptotic signals between NO and ceramide
generation. Finally, it remains to be elucidated in the future how NO
activates caspase-3 and what are the mechanisms of activation of
magnesiumdependent N-SMase to understand the biochemical and
physiological implications of the ceramide signal in NO-induced apoptosis.