Ceramide Increases Oxidative Damage Due to Inhibition of Catalase by Caspase-3-dependent Proteolysis in HL-60 Cell Apoptosis*

Kazuya IwaiDagger , Tadakazu KondoDagger , Mitsumasa WatanabeDagger , Takeshi YabuDagger , Toshiyuki KitanoDagger , Yoshimitu TaguchiDagger §, Hisanori Umehara, Atsushi TakahashiDagger , Takashi UchiyamaDagger , and Toshiro OkazakiDagger ||

From the Dagger  Departments of Hematology and Oncology and  Clinical Immunology, Graduate School of Medicine, Kyoto University, 54 Syogoin-Kawaramachi, Sakyo-ku, Kyoto 606-8507 and the § Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan

Received for publication, February 25, 2002, and in revised form, October 11, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated through which mechanisms ceramide increased oxidative damage to induce leukemia HL-60 cell apoptosis. When 5 µM N-acetylsphingosine (C2-ceramide) or 20 µM H2O2 alone induced little increase of reactive oxygen species (ROS) generation as judged by the 2'-7'-dichlorofluorescin diacetate method, 20 µM H2O2 enhanced oxidative damage as judged by ROS accumulation, and thiobarbituric acid-reactive substance production after pretreatment with 5 µM C2-ceramide at least for 12 h. The treatment with a catalase inhibitor, 3-amino-1h-1,2,4-triazole, increased oxidative damage and apoptosis induced by H2O2, and in contrast, purified catalase inhibited the enhancement of oxidative damage by H2O2 in ceramide-pretreated cells, suggesting that the oxidative effect of ceramide is involved in catalase regulation. Indeed, C2-ceramide inhibited the activity of immunoprecipitated catalase and decreased the levels of catalase protein in a time-dependent manner. Moreover, acetyl-Asp-Met-Gln-Asp-aldehyde, which dominantly inhibited caspase-3 and blocked the increase of oxidative damage and apoptosis due to C2-ceramide-induced catalase depletion at protein and activity levels. In vitro, active and purified caspase-3, but not caspase-6, -8, and -9, inhibited catalase activity and induced the proteolysis of catalase protein whereas these in vitro effects of caspase-3 were blocked by acetyl-Asp-Met-Gln-Asp-aldehyde. Taken together, it is suggested that H2O2 enhances apoptosis in ceramide-pretreated cells, because ceramide increases oxidative damage by inhibition of ROS scavenging ability through caspase-3-dependent proteolysis of catalase.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha , interleukin-1beta , 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-alpha 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
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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 [alpha -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 beta -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-Nepsilon -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.

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ABSTRACT
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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).


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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).


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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.


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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).


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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 beta -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.


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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 beta -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 beta -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 delta  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.

                              
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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.


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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 beta -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.


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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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel./Fax: 81-75-751-3154; E-mail: toshiroo@kuhp.kyoto-u.ac.jp.

Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M201867200

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; GSH, glutathione; GPx, glutathione peroxidase; C2-ceramide, N-acetylsphingosine; DAPI, 4',6-diamidine-2'-phenylindole dihydrochloride; ATZ, 3-amino-1h-1,2,4-triazole; DCF-DA, 2',7'-dichlorofluorescin diacetate; TBARS, thiobarbituric acid-reactive substances; PBS, phosphate-buffered saline; MPO, myeloperoxidase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DMQD, acetyl-Asp-Met-Gln-Asp-aldehyde; DEVD-CHO, acetyl-Asp-Glu-Val-Asp-aldehyde.

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DISCUSSION
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