 |
INTRODUCTION |
Sphingolipid ceramide has been recognized as one of
important molecules to mediate proapoptotic signaling in cell death
induced by a diverse array of stresses (1, 2). Oxidative damage caused
by reactive oxygen species
(ROS)1 was shown to increase
ceramide generation to induce apoptosis (3, 4). In contrast, the relief
of oxidative damage by the ROS scavenging system consisting of
N-acetylcysteine, glutathione (GSH), catalase, and
glutathione peroxidase (GPx) was reported to inhibit ceramide
generation in tumor necrosis factor-
, interleukin-1
, hypoxia, and
daunorubicin-induced apoptosis (5-10). Activation of a cysteine
protease, called caspase-3, is indispensable for ceramide generation by
nitric oxide and H2O2 to induce apoptosis (11,
12), and it is known that ROS generation in mitochondria activates
caspase-3 via cooperation of cytochrome c, Apaf-1, and caspase-9 (13, 14) and increases ceramide generation through sphingomyelinase in the cell-reconstruction system (11). Therefore, these results suggest that oxidative damage caused by ROS increases ceramide generation through caspase-3 activation for the induction of apoptosis.
On the other hand, ceramide was reported to induce oxidative damage by
increasing ROS generation (15-21) or by inhibiting ROS scavenger
glutathione (22, 23), and addition of exogenous N-acetylsphingosine (C2-ceramide) and generation
of intracellular ceramide by tumor necrosis factor-
and
lipopolysaccharide were shown to increase oxidative damage in
mitochondria to induce apoptosis (21, 23). In addition, treatment with
C2-ceramide increased H2O2
generation in U937 cell apoptosis, and ceramide-induced apoptosis was
inhibited by the treatment with antioxidants such as
N-acetylcysteine, pyrolidine dithiocarbamate, GSH,
glutathione peroxidase-1, catalase, and CuZn-superoxide dismutase (9,
10, 24-26). Moreover, it was reported that ceramide activated
caspase-3 downstream of caspase-8 by releasing cytochrome c
from mitochondria through dysregulation of mitochondrial
respiratory chain (23, 27, 28), and many other reports (29-33) suggest
the intimate involvement of caspase-3 downstream of oxidative damage in
induction of apoptosis. Thus, as it is reported that oxidative damage
increases ceramide generation, and at the same time, ceramide can
increase oxidative damage, these results may suggest the existence of
an up-regulation mechanism between ceramide and oxidative damage
through activation of caspase-3.
However, although we suggested previously (11) that
ROS-activated caspase-3 increased ceramide through sphingomyelinase activation, the mechanism through which ceramide-activated caspase-3 increases ROS generation is still unknown. We showed recently (34) that
ceramide-induced oxidative damage through catalase depletion was
involved in vesnarinone-induced apoptosis and that inhibition by
insulin-like growth factor-1 of ceramide-induced apoptosis was because
of restoring catalase activity in a caspase-3-dependent manner (35). These results showed the intimate relation between ceramide-activated caspase-3 and catalase function in apoptosis. Therefore, it was here examined how apoptosis because of oxidative damage was enhanced by a low concentration of
H2O2 in ceramide-pretreated cells and
investigated through which mechanisms ceramide-activated caspase-3
depleted catalase function to induce oxidative damage and
apoptosis. The results suggest that a low concentration of H2O2 enhances oxidative damage and apoptosis
because of a decrease of ROS scavenging ability through
ceramide-depleted catalase function, because ceramide inhibits catalase
activity by increasing a proteolysis of catalase protein in a
caspase-3-dependent manner.
 |
MATERIALS AND METHODS |
Chemicals and Reagents--
C2-ceramide,
C2-dihydroceramide, and sphingosine were from Matreya
(Pleasant Gap, PA). 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI) and 3-amino-1h-1,2,4-triazole (ATZ) were from Nacarai Tesque (Kyoto, Japan). Acetyl-Asp-Met-Gln-Asp-aldehyde (DMQD-CHO) and acetyl-Asp-Glu-Val-Asp (DEVD-CHO) were purchased from the
Peptide Institute (Osaka, Japan), dissolved at 10 mM in
Me2SO, and stored at
80 °C. 2',7'-Dichlorofluorescin
diacetate (DCFH-DA) and thiobarbituric acid-reactive substances (TBARS)
were from Pierce and Nakarai Tesque (Kyoto, Japan), respectively. ECL
Western blotting kit and [32P]dCTP (6000 Ci/mmol) were
purchased from Amersham Biosciences. Purified and active
caspase-3, -6, -8, and -9 were obtained from Peptide Institute (Osaka,
Japan). Other chemicals, if not mentioned, were purchased from Sigma.
Cell Culture--
Human myeloid leukemic cell line HL-60 was
kindly provided by Dr. Saito (National Cancer Institute, Tokyo, Japan).
The cells were grown in RPMI 1640 medium (Sigma) containing 10%
heat-inactivated fetal bovine serum, 1 µM sodium
selenite, and kanamycin (80 µg/ml) at 37 °C in a humidified 5%
CO2 atmosphere. A few hours prior to each experiment, the
cells at an initial concentration of 1-2.5 × 105/ml
were replaced into fresh media containing 2% fetal bovine serum.
Measurement of ROS--
Production of ROS was measured with
DCFH-DA, which is cell-permeable and oxidized inside the cells to
fluorescent dichlorofluorescin in the presence of
H2O2 (36, 37). Briefly, the cells treated with
or without C2-ceramide were washed with phosphate-buffered saline (PBS) twice, resuspended in 1 ml of PBS, and incubated with 5 µM DCFH-DA for 15 min at 37 °C. Then the cells were
washed in PBS, and fluorescence was determined by using FACScan (BD
Biosciences) with an excitation wavelength of 488 nm and an emission
wavelength of 530 nm. Generally, 10,000 events were monitored, and data
analysis was performed by using Cell Quest software (BD Biosciences).
Determination of TBARS--
Oxidative damage was determined as
the production of TBARS, according to the modified method of Ohkawa
et al. (38). Briefly, a 200-µl homogenate of the cells was
supplemented with a 650-µl mixture of TBARS and 150 µl of acetic
acid buffer, vortexed on ice for 1 h and boiled at 100 °C for
1 h. The reactants were then supplemented with 1 ml of the mixture
of n-butanol and pyridine, vortexed vigorously, and
centrifuged for 10 min at 650 × g. Absorbance was
measured at 532 nm on spectrophotometer, and the results were expressed
as nmol TBARS per mg protein.
Detection of Apoptosis--
Apoptosis was identified by staining
the cells with DAPI or by May-Giemsa staining. The cells were washed,
fixed with 1% glutaraldehyde for 30 min, and then labeled with 2 µg/ml DAPI. After labeling, apoptotic cells were visualized under a
fluorescent microscope (BX60-34FFB1; Olympus). Morphological changes of
apoptotic cells were also determined with May-Giemsa staining
under microscope with a ×400 magnificent force. The cells with
condensed, or fragmented nuclei were scored as apoptotic cells. At
least 200 cells were counted in each experiment. The data show the
means of at least three independent experiments.
Immunoprecipitation--
After treatment, the cells (1 × 107) were lysed in 700 µl of lysing buffer (50 mM Tris-HCl, pH 7.6, 0.5% Triton X-100, 300 mM
NaCl, 5 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate). Endogenous caspase-3 was
immunoprecipitated by 2 h of incubation with 10 µg of
anti-caspase-3 (Santa Cruz Biotechnology, Inc.), anti-catalase
(Calbiochem) at 4 °C, followed with 2 h of incubation with 50 µl of protein A-Sepharose (Amersham Biosciences) at 4 °C.
Endogenous catalase was immunoprecipitated by 2 h of incubation with 10 µg of anti-catalase antibody (Polysciences, Inc.) at 4 °C,
followed with 50 µl of protein A-Sepharose (Amersham Biosciences) at
4 °C. Immune complexes were washed five times with lysis buffer, boiled, and subjected to SDS-PAGE.
Immunoprecipitated Catalase and Myeloperoxidase (MPO)
Activity Assay--
The cells at the concentration of 1 × 106/ml were washed with PBS twice, and after homogenization
catalase was immunoprecipitated by anti-catalase or anti-MPO antibody
(Calbiochem). Catalase activity was assayed by measuring the kinetic
loss of H2O2 as described previously (39). For
catalase activity, immunoprecipitated protein was mixed with
H2O2 in PBS, and loss of
H2O2 monitored at 240 nm by spectrophotometer
was detected as catalase activity. For MPO activity, immunoprecipitated
protein was measured as described elsewhere (40).
Western Blotting--
The cells treated with or
without C2-ceramide were lysed in a solubilizing buffer
containing 20 mM Tris, pH 7.4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, and
0.15 units/ml aprotinin for 40 min at 4 °C. The cell lysate was
centrifuged at 12,000 × g for 15 min to remove
insoluble materials. Twenty µg per lane of whole cell lysate was
denatured by boiling and resolved by the SDS-PAGE method. Following
electrophoresis, the gels were transferred to Immobilon-P membrane
(Millipore) by electroblotting. The membranes were blotted in 5% skim
milk overnight and then probed with anti-catalase antibody. After
subsequent incubation with horseradish peroxidase-conjugated second
antibody, expressions of protein were visualized by using the ECL kit
(Amersham Biosciences) according to the manufacturer's recommended protocol.
RNA Preparation and Northern Blotting--
Total RNA
was prepared using TRIZOL reagent (Invitrogen) according to the
manufacturer's protocol, and 20 µg of total RNA was used for the
Northern blotting analysis as described previously (41). Briefly,
oligonucleotide probes for human catalase was labeled with
[
-32P]dCTP using a multiprime labeling kit (Amersham
Biosciences). After electrophoresis and transferring RNA to Immobilon-S
membrane, hybridizations with labeled catalase cDNA probes were
performed at 42 °C for 24 h, and the membranes were washed in
2× SSC/0.1% SDS (1× SSC containing 0.15 M NaCl and 15 mM sodium citrate) at room temperature for 30 min and at
50 °C for 20 min. The membranes were exposed to Fuji x-ray films
with the intensifying screens at
80 °C and calculated by a
BAS-III image analyzer. Equal loading of RNA was confirmed by
the amount of
-actin mRNA detected by the above method in
each-III sample.
Fluorometric Assay of Caspase-3 Activity--
The
cells after each treatment 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-7-amino-4-methyl-coumarin),
which was followed by incubation at 25 °C for 60 min. Fluorescence
was measured by a microplate reader (MTP-100F; Corona Electric)
using 360-nm excitation and 450-nm emission filters. Concentrations of
7-amino-4-methyl-coumarin liberated as a result of cleavage were
calculated compared with standard 7-amino-4-methyl-coumarin solutions.
Affinity Labeling of Active Caspases--
Caspase-3, -4, -6, -7, and -8 were purified as described (42). Purified caspases were
incubated with indicated concentrations of
N-(acetyltyrosinylvalinyl-N
-biotinyllysyl)
aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone for 5 min at
37 °C. The samples were run on 16% SDS-PAGE gels, transferred to
nitrocellulose membranes, and stained with horseradish
peroxidase-conjugated streptavidin as described (43).
In Vitro Detection of Catalase Activity--
Purified catalase
(5000 units/mg protein) obtained from Calbiochem was incubated with
various concentrations of purified and active caspase-3 for 2, 4, and
6 h. Then the samples in the tubes were supplemented with
phosphate buffer and H2O2, and loss of H2O2 was monitored at 240 nm using a
spectrophotometer as described elsewhere (39).
Detection of Catalase Cleavage by Caspase-3 on
SDS-PAGE--
Purified catalase (1 µg) obtained from Calbiochem was
incubated with 2 or 5 units of purified and active caspase-3 (Peptide Institute, Osaka, Japan) for 4 h and then the reaction mixture was
denatured by boiling in Laemmli buffer for 5 min and resolved by the
SDS-PAGE method using 12.5% running gel. Following electrophoresis, the gels were silver-stained.
 |
RESULTS |
Enhancement of Apoptosis by Addition of
H2O2 in Ceramide-pretreated Cells--
After
pretreatment with various concentrations of C2-ceramide for
12 h, 20 µM H2O2 was added
and then apoptosis was examined by the DAPI method 1 or 12 h after
addition of H2O2. As shown in Fig.
1A, the treatment with 20 µM H2O2 alone for 12 h
slightly increased apoptosis from 4 to 8%. When the pretreatment with
2, 5, and 8 µM C2-ceramide alone for 24 h increased apoptosis from 4% to 12, 14, and 19%, respectively,
addition of 20 µM H2O2 after 12 h of pretreatment with 2, 5, and 8 µM
C2-ceramide increased apoptosis from 8% to 22, 34, and
48%, respectively, 24 h after treatment with
C2-ceramide. The results showed that ceramide-induced apoptosis was significantly enhanced by addition of
H2O2 (p < 0.01). Because the
simultaneous treatment with 5 µM C2-ceramide and 20 µM H2O2 did not show the
enhancement of apoptosis by H2O2 (Fig.
2B), we next examined how long
the pretreatment with C2-ceramide was required for
enhancing apoptosis by H2O2. The results showed that the pretreatment with C2-ceramide for 12 h was,
at least, required and that for 6 h was not enough for enhancement
of ceramide-induced apoptosis by H2O2 (Fig.
1B).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Enhancement of apoptosis by
H2O2 in C2-ceramide-pretreated
cells. HL-60 cells were pretreated with various
concentrations of C2-ceramide (A, 0, 2, 5, and 8 µM; B, 5 µM) for the indicated
times (A, 12 h; B, 0, 6, 12, and 24 h)
and were then treated for 12 h with or without 20 µM
H2O2. The percentage of apoptotic cells was
assessed by DAPI staining as described under "Materials and
Methods." The data from three independent experiments (B)
are shown. The bars mean one standard deviation.
Statistical significance of apoptosis was performed by analysis of
variance between treatment with C2-ceramide alone and with
C2-ceramide and H2O2
(A).
|
|

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 2.
Enhancement of ROS generation and TBARS
production by H2O2 in
C2-ceramide-pretreated cells. HL-60 cells were
pretreated with various concentrations of C2-ceramide (0, 2, 5, and 8 µM) for 12 h and then incubated for
1 h (A) or 12 h (B) in the presence or
absence of 20 µM H2O2. Then, the
fluorescent 2',7'-dichlorofluorescin and oxidative damage were
determined by flow cytometry (A) and TBARS production
(B), respectively, as described under "Materials and
Methods." Obtained data were analyzed by Cell Quest software, and the
representatives from three independent experiments are shown in
panel A. The average of three independent experiments are
shown, and the bars mean one standard deviation
(B). Statistical significance of TBARS production was
performed by analysis of variance between treatment with
C2-ceramide alone and with C2-ceramide and
H2O2 (B).
|
|
Enhancement of ROS Generation and TBARS Production by
H2O2 in Ceramide-pretreated Cells--
Amount
of intracellular ROS was measured by the DCFH-DA method, which is
reported to be sensitive to H2O2 generation
(36, 37). As shown in Fig. 2A, the treatment with
C2-ceramide alone for 13 h and 20 µM
H2O2 for 1 h showed little increase of ROS generation (Fig. 2A, a), although a higher
concentration of 100 µM H2O2
showed a prominent increase of ROS (data not shown). After the cells
were pretreated with various concentrations of C2-ceramide for 12 h, ROS generation was dose-dependently
increased 1 h after addition of 20 µM
H2O2 (Fig. 2A,
a-d). By addition of 20 µM
H2O2 for 1 h after 12 h of
pretreatment with 5 µM C2-ceramide, the amount of ROS generation was increased ~2-fold of no
H2O2-added control level (Fig. 2A,
c).
In addition, it was examined whether C2-ceramide-induced
oxidative damage as judged by TBARS production was increased in the presence of H2O2. The treatments with 2, 5, and
8 µM C2-ceramide alone for 24 h showed
the increases of TBARS production from 0.1 to 0.6, 0.7, and 0.8 nmol/mg
protein, respectively (Fig. 2B). The same concentrations of
C2-ceramide increased TBARS production from 0.1 to 0.8, 1.3, and 2.2 nmol/mg protein 12 h after treatment in the presence
of 20 µM H2O2 for 12 h
(p < 0.05 for 2 µM
C2-ceramide, and p < 0.01 for 5 and 8 µM C2-ceramide; see Fig. 2B).
Although the increase of ROS generation and TBARS production by 20 µM H2O2 alone were not
significantly detected 12 h after treatment, respectively, ceramide-induced ROS generation and TBARS production were enhanced by
20 µM H2O2 (Fig. 2B).
Therefore, 20 µM H2O2 seemed to
increase oxidative damage in ceramide-pretreated cells, because
ceramide abrogated the ROS scavenging system before
H2O2 addition.
Effects of ATZ and Purified Catalase on Enhancement of Apoptosis,
ROS Generation, and TBARS Production by H2O2 in
Ceramide-pretreated Cells--
As shown in Figs. 1 and 2,
apoptosis and oxidative damage were enhanced by 20 µM H2O2 in 5 µM
C2-ceramide-pretreated cells. In these conditions, we
examined whether ATZ (40 mM) or purified catalase (100 units/ml) modulated the enhancement by H2O2 of
apoptosis, ROS generation, and TBARS production in ceramide-pretreated
cells. The treatment with 40 mM ATZ further increased the
enhancement of apoptosis by 20 µM
H2O2 from 35 to 60% in the cells pretreated with 5 µM C2-ceramide whereas 100 units/ml
purified catalase decreased apoptosis from 35 to 7% (Fig.
3A). Similarly, ROS generation
and TBARS production enhanced by H2O2 in
C2-ceramide-pretreated cells were increased approximately
from a 2-fold increase to a 4-fold increase of the control level and
from 1.2 to 1.7 nmol/mg protein, respectively, by ATZ (see Fig. 3,
B (a and d) and C). In
contrast, those were inhibited from a 2-fold increase to the control
level and from 1.2 to 0.2 nmol/mg protein, respectively, by purified catalase (see Fig. 3, B (a and c) and
C). In addition, ATZ, which inhibits catalase activity,
seemed to mimic the effect of ceramide, because 20 µM
H2O2 alone enhanced apoptosis and oxidative
damage in ATZ-pretreated cells (Fig. 3). Therefore, these results
suggested that ceramide inhibited the ROS scavenging system, probably
catalase.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3.
Effects of purified catalase and ATZ on
enhancement by H2O2 of apoptosis,
ROS generation, and TBARS production in
C2-ceramide-pretreated cells. HL-60 cells were
pretreated with or without 5 µM C2-ceramide
for 12 h and incubated with 20 µM
H2O2 in the presence of 200 units/ml catalase
or 40 mM ATZ. Apoptosis judged by the DAPI method
(A), ROS generation judged by the DCFH method
(B), and oxidative damage judged by TBARS production
(C) were, respectively, performed 12, 1, and 12 h after
addition of H2O2 as described under
"Materials and Methods." Results were obtained from more than three
different experiments, and the bars mean 1 S.D.
(A and C). Obtained data were analyzed by Cell
Quest software, and the representatives from three independent
experiments are shown in panel B.
|
|
Effects of C2-ceramide on Immunoprecipitated Catalase
and Myeloperoxidase Activity--
The data shown in Figs. 1-3 and our
recent reports (34, 35) suggest that catalase activity is regulated
downstream of proapoptotic ceramide action. We, therefore, examined
whether and through which mechanisms ceramide affects the function of
catalase. The activities of catalase, which was immunoprecipitated by
anti-catalase antibody, were time-dependently inhibited by
the treatment with C2-ceramide (Fig.
4A). By the treatment with 5 µM C2-ceramide for 12 h, catalase activity was significantly decreased from 79 units/mg protein to 70 units/mg protein (p < 0.05) and more drastically
inhibited to 36 units/mg protein 36 h after treatment. These
results suggested that catalase activity started to be inhibited by
ceramide before the timing of H2O2 addition,
which enhanced apoptosis and oxidative damage after pretreatment with
ceramide at least for 12 h (see Figs. 1 and 2). In addition,
immunoprecipitated MPO, which has a possibility to reduce the amount of
intracellular H2O2 inhibited by ATZ, was not
affected by the treatment with 5 µM
C2-ceramide for 24 h (Fig.
4B).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Inhibitory effects of C2-ceramide
on activities of catalase and MPO. HL-60 cells were pretreated
with or without 200 µM DMQD-CHO for 1 h before
incubation with 5 µM C2-ceramide for the
indicated times (A, 0, 6, 12, 18, 24, and 36 h;
B, 0 and 24 h). Then cell extracts were
immunoprecipitated by anti-catalase or anti-MPO antibody, and catalase
(A) and MPO (B) activity were analyzed as
described under "Materials and Methods." Results were obtained from
more than six different experiments (A) and from three
different experiments (B). The bars mean 1 S.D.
Statistical significance of catalase activity was performed by analysis
of variance between treatment with and without C2-ceramide
(A).
|
|
Inhibition of Catalase by C2-ceramide at mRNA and
Protein Levels--
To know through which mechanisms ceramide inhibits
catalase function, we examined whether catalase was depleted at
mRNA or protein level by C2-ceramide. The treatment
with 5 µM C2-ceramide inhibited mRNA
levels of catalase in a dose- and time-dependent manner but
not those of
-actin (Fig.
5A). Significant decrease of
mRNA level was observed 36 h after treatment with 5 µM C2-ceramide, although decrease of catalase
activity by ceramide was also detected at least 12 h after
treatment (Fig. 4A). In contrast, as shown in Fig.
5B, the protein levels of catalase were also decreased by 5 µM C2-ceramide in a
time-dependent manner, and decrease of catalase protein by
5 µM ceramide was already found 12 h after treatment, which was earlier than that at mRNA level (Fig. 5, A and B), suggesting that ceramide-induced
inhibition of catalase at protein level was not mediated by the
decrease of catalase at mRNA level.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibitory effects of
C2-ceramide on mRNA and protein levels of
catalase. HL-60 cells were incubated with the various
concentrations (0, 2, 5, and 8 µM) of
C2-ceramide for the indicated times (A, 0, 12, 24, 36, and 48 h; B, 0, 12, 18, 24, and 36 h). The
mRNA and protein levels of catalase and -actin were examined by
Northern (A) and Western (B) blotting analysis,
respectively, as described under "Materials and Methods." The
representatives from three (A) or six (B)
independent experiments were shown, and the average of ratios of
catalase density to -actin density in Northern and Western blotting
analysis was calculated and shown at the bottom of each
panel.
|
|
Inhibition of Ceramide-induced Apoptosis, TBARS Production, and
Catalase Depletion by DMQD-CHO--
We next examined whether caspase-3
was involved in the enhancement by H2O2 of
apoptosis and oxidative damage in ceramide-pretreated cells. In the
presence of 200 µM DMQD-CHO, which was designed from a
cleavage site of protein kinase
by caspase-3 and shown to be a
caspase-3-dominant inhibitor (Table
I) (34, 35, 44), H2O2 did not enhance apoptosis and TBARS
production despite the pretreatment with C2-ceramide for
12 h (Fig. 6, A and
B). The results suggested that the mechanism through which
ceramide induced apoptosis and oxidative damage was
caspase-3-dependent.
View this table:
[in this window]
[in a new window]
|
Table I
Inhibition of caspases by DMQD-CHO in vitro
Purified His-tagged caspase-3 (215 pg) and E. coli lysates
caspase-4, 6, 7, and 8 (0.7-1.2 µg of total protein) were
preincubated with DMQD-CHO at the indicated concentrations for 15 min
in 10 µl of mixture before labeling with 10 µM
YV(bio)KD-aomk. Cleaving ability was measured by densitometry after
SDS-PAGE gel analysis. Results were obtained from more than three
different experiments.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Inhibition of ceramide-induced apoptosis,
TBARS production, and catalase depletion by DMQD-CHO. HL-60 cells
were treated with 5 µM C2-ceramide for
12 h and incubated for another 12 h in the presence (A and B) or absence
(C and D) of 20 µM
H2O2. Apoptosis judged by DAPI method
(A), oxidative damage judged by TBARS production
(B), and catalase function (activity, C; protein
level by Western blotting analysis, D) were examined as
described under "Materials and Methods." Results were obtained from
more than three different experiments. The bars mean 1 S.D.
The average of ratios of catalase density to -actin density in
Western blotting analysis was calculated and shown at the bottom of
panel D.
|
|
As shown in Fig. 6C, addition of 200 µM DMQD-CHO restored 5 µM
C2-ceramide-inhibited catalase activity from 70 to 78 units/mg protein and from 36 to 68 units/mg protein 12 and 36 h
after treatment, respectively, suggesting that caspase-3 was upstream
of ceramide-induced inhibition of catalase. Judging from the time
course of decrease of catalase message, protein, and activity,
regulation of catalase by ceramide might occur at protein level (Fig.
5). We, therefore, examined whether caspase-3 was involved in the
depletion of catalase at protein level by ceramide. When 5 µM C2-ceramide inhibited catalase at activity
and protein level in a similar time-dependent manner (see
Fig. 4A and Fig. 5B), the pretreatment with 200 µM DMQD-CHO restored inhibition by ceramide of catalase
at activity and protein level (Fig. 6, C and D).
These results clearly suggested that caspase-3 was closely involved in
ceramide-induced oxidative damage and apoptosis through the regulation
of catalase at protein level rather than at mRNA level.
Colocalization of Caspase-3 and Catalase in the Cells
and in Vitro Inhibition of Catalase Activity through
Caspase-3-dependent Proteolysis--
We further
investigated whether ceramide-activated caspase-3 directly regulates
catalase at protein level, because a decrease of catalase at mRNA
level does not seem to be the cause of catalase depletion. To know the
possibility of direct interaction between caspase-3 and catalase in the
cells, their colocalization was first examined by immunoprecipitation
using antibodies for caspase-3 and catalase. When the cells were
treated with 5 µM C2-ceramide for 48 h,
anti-caspase-3 antibody coimmunoprecipitated catalase protein and
vice versa, and the amount of coimmunoprecipitated catalase
protein was reduced despite the equal amount of immunoprecipitated caspase-3 (Fig. 7A). As this
decrease of the amount of precipitated-catalase might be because
caspase-3 directly cleaved catalase protein, we next examined the
effect of purified caspase-3 on the activity of catalase in
vitro. Approximately 5000 units/ml of catalase activity was
decreased to 2200 units/ml by the presence of 2 units of caspase-3 for
4 h but was not decreased by purified caspase-6, -8, or -9 at the
same experimental conditions (Fig. 7B). The inhibition of
catalase activity by purified and active caspase-3 in vitro was in a dose- and time-dependent manner (Fig.
7C), and the inhibition of catalase activity by caspase-3
was almost completely restored by the pretreatment with 200 µM DMQD-CHO (Fig. 7D). As shown in Fig.
7E catalase protein was degraded in the presence of
purified caspase-3, and DMQD-CHO blocked the direct proteolysis of
catalase by caspase-3 (Fig. 7F). These results suggested
that the proteolysis of catalase is involved in the mechanism of
caspase-3 to inhibit catalase activity in ceramide-induced
apoptosis.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
Colocalization of caspase-3 and
catalase protein in the cells and in vitro inhibition
of catalase activity through its proteolysis by caspase-3. HL-60
cells were treated with or without 5 µM
C2-ceramide for 48 h. Then cell extracts were
immunoprecipitated with anti-caspase-3 or anti-catalase antibody. After
the samples were separated by SDS-PAGE, precipitated catalase and
caspase-3 were analyzed by Western blotting analysis using
antibodies for them as described under "Materials and Methods"
(A). After in vitro incubation simultaneously
with purified catalase (5000 units/mg) and 2 units of various kinds of
purified and active caspases (caspase-3, -6, -8, and -9) for 2 h,
catalase activity was measured (B). Catalase activity
in vitro was also measured in the presence of various
concentrations of caspase-3 (0, 0.5, 1, 2, and 5 units) for the
indicated times (2, 4, and 6 h) (C) or in the presence
of 5 units of caspase-3 for 4 h after pretreatment with 200 µM DMQD-CHO or DEVD-CHO for 1 h (D).
Proteolysis of catalase protein (1 µg) on SDS-PAGE was examined in
the presence of 2 or 5 units of caspase-3 for 4 h (E)
or in the presence of 5 units of caspase-3 for 4 h after 1 h
of pretreatment with 200 µM DMQD-CHO (F) as
described under "Materials and Methods." The representatives from
three independent experiments are shown in panels A,
E, and F. Results were obtained from three
different experiments, and the bars mean 1 S.D.
(B, C, and D).
|
|
 |
DISCUSSION |
Ceramide has been recognized as one of key molecules to mediate
apoptosis in many cell systems (1, 2, 45). As downstream signals of
ceramide action, caspase-3 and oxidative damage caused by ROS
generation are known to play an important role in inducing apoptosis
(23, 25, 27, 41, 46). We reported recently (34) that an inotropic
agent, vesnarinone, induced ceramide generation and apoptosis through
caspase-3-increased lipid peroxidation and that insulin-like growth
factor-1 inhibited ceramide-induced apoptosis by suppressing oxidative
damage in a phosphatidylinositol 3-kinase- or caspase-3 dependent
manner (35). Oxidative damage was reported to increase ceramide content
through activation of caspase-3 (11-14), but in contrast, the
mechanistic connection of caspase-3 with oxidative damage downstream of
ceramide action is still unknown. Therefore, we examined whether and
through which mechanism ceramide increased oxidative damage and
apoptosis in a caspase-3-dependent manner.
ROS generation and oxidative damage were judged by the conversion of
DCFH-DA to dichlorofluorescin and by the detection of TBARS production, respectively. As shown in Fig. 1, 20 µM H2O2 enhanced oxidative
damage after pretreatment with 5 µM
C2-ceramide for at least 12 h. As the treatment with
20 µM H2O2 or 5 µM
C2-ceramide alone showed little increase of ROS generation
(Fig. 2A, a), it was clear that
H2O2 was accumulated in the cells by the
pretreatment with C2-ceramide (Fig. 2A). Similar
enhancement by H2O2 of oxidative damage as
judged by TBARS production was also found in ceramide-pretreated cells
(Fig. 2B). Thus, these results suggest that ceramide
increases oxidative damage by suppression of the ROS scavenging system
rather than by a direct increase of ROS generation.
In general, the scavenging system for
H2O2 consists of catalase, GSH, and GPx (47,
48). The treatment with catalase, GSH, or GPx was reported to inhibit
ceramide-induced apoptosis by the relief of ROS-caused oxidative damage
(6, 9, 26, 49, 50), and in contrast, ceramide was found to reduce
GSH/GPx function in apoptosis (10, 22, 23). In addition to these
results, as we showed here that the enhancement by
H2O2 of ceramide-induced apoptosis, ROS
generation, and TBARS production were increased by a catalase
inhibitor, ATZ, and inhibited by purified catalase (Fig. 3), the
pretreatment with ceramide is suggested to inhibit catalase function to
increase oxidative damage and apoptosis. This notion was confirmed by
the fact that, like ceramide, ATZ increased apoptosis and oxidative
damage in the presence of H2O2 alone (Fig. 3).
However, it is so far poorly understood through which mechanisms
ceramide affects the function of catalase. As shown in Fig.
4A, 5 µM C2-ceramide inhibited the
activity of catalase, which was immunoprecipitated by anti-catalase
antibody, in a time-dependent manner. To eliminate the
possible contamination of MPO activity during catalase assay (35), we
here measured a specific catalase activity by using immunoprecipitation
(Fig. 4A) and also confirmed that the activity of
immunoprecipitated MPO was not affected by C2-ceramide
(Fig. 4B). Although it was reported previously (51, 52) that
catalase activity was decreased during HL-60 cell differentiation toward myeloid lineage a couple days after treatment, we found that
ceramide decreased catalase activity at least 12 h after treatment. Furthermore, as ceramide inhibited catalase activity in
Jurkat T cells, as well as in HL-60 cells (data not shown), it is sure
that the inhibition of catalase activity was not the result of myeloid differentiation.
The activation of caspase-3 is well known to be
indispensable for DNA fragmentation and morphological changes of nuclei
in ceramide-induced apoptosis (53, 54). As shown in Fig. 6 the enhancement by H2O2 of ceramide-induced
oxidative damage and apoptosis was restored by a caspase-3 dominant
inhibitor, DMQD-CHO (44). Although DMQD-CHO also inhibited caspase-8
activity weekly as compared with other caspases in vitro
(Table I), C2-ceramide did not affect caspase-8 activity in
HL-60 cells (data not shown), suggesting that ceramide inhibited a ROS
scavenger catalase through caspase-3 activation. However, it still
remains to be clarified through which mechanisms ceramide-activated
caspase-3 depletes catalase function. In this work, we therefore
examined whether catalase function was inhibited by ceramide at
mRNA or protein synthesis levels in a
caspase-3-dependent manner. As shown in Fig. 5A,
C2-ceramide inhibited catalase at mRNA level 36 h
after treatment but already decreased its protein level 12 h after
treatment (Fig. 5B). In addition, as shown Fig. 6,
C and D, ceramide-inhibited catalase at activity
and protein levels was restored by DMQD-CHO. As ceramide-induced
inhibition of catalase and increase of apoptosis and TBARS production
occurred at least 12 and 24 h after treatment, respectively, the
results suggested that ceramide-activated caspase-3 increased oxidative
damage through the inhibition of catalase at protein levels.
Even if a decrease of catalase message by ceramide may not be involved
in early increase of oxidative damage, it is interesting to know how
caspase-3 inhibits catalase at mRNA level. Recently NRF2, which
belongs to the NF-E2 family of basic region leucine zipper
transcription factors and induces ROS scavenging enzymes, was reported
to be cleaved by caspase-3 (55). In addition, SP1- and
CCAAT-recognizing factors (56) and hepatocarcinogenesis-related negative regulatory factor (57) were suggested to be candidates for
transcriptional regulation of catalase gene by caspase-3. Therefore,
although the involvement of some transcription factor may be plausible,
it remains to be clarified in the future how caspase-3 regulates
catalase function through its transcriptional regulation in
ceramide-induced apoptosis.
In terms of the mechanism through which ceramide decreases the levels
of catalase protein, we showed that caspase-3 was colocalized with
catalase by immunoprecipitation assay (Fig. 7A). As shown in
Fig. 7A, after the treatment with C2-ceramide
the amount of catalase coimmunoprecipitated with anti-caspase-3
antibody was decreased as compared with the control level, suggesting
the direct interaction of caspase-3 with catalase to regulate catalase
function. Indeed, the in vitro catalase activity was
inhibited by the addition of purified caspase-3 in a time- and
dose-dependent manner but not by other caspases such as
caspase-6, -8, and -9 (Fig. 7, B and C).
Specificity of caspase-3 for catalase inhibition was also confirmed by
the data showing the restoration of caspase-3-inhibited catalase by
caspase-3 inhibitors, DMQD-CHO and DEVD-CHO, in vitro (Fig.
7D) (34, 35, 44). Finally, we investigated whether caspase-3
has the ability to cleave catalase protein in vitro. Although a DXXD-like site for cleavage by caspase-3 does not
exist in the amino acid sequence of catalase, the results showed that the amount of catalase protein was decreased and cleaved to small segments in the presence of active and purified caspase-3 (Fig. 7E). In addition, this cleavage was shown to be inhibited by
DMQD-CHO (Fig. 7F). Caspase-3 was reported to cleave the
protein having no DXXD sequence (58), but the mechanism of
catalase cleavage by caspase-3 is, at present, not clear.
In summary, we showed here that ceramide-activated caspase-3 inhibited
catalase activity because of its proteolysis but not because of
inhibition at mRNA level and that increased oxidative damage by
this catalase depletion is closely related to ceramide-induced apoptosis. It is also suggested that ceramide efficiently induces apoptosis not only by enhancing pro-apoptotic signal caspase-3 but
also by depleting anti-apoptotic molecule catalase through caspase-3.