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Ceramide-induced apoptosis: role of catalase and hepatocyte growth factor
Free Radical Biology & Medicine, Vol. 37, No. 2, pp. 166 – 175, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2004.04.011
Original Contribution CERAMIDE-INDUCED APOPTOSIS: ROLE OF CATALASE AND HEPATOCYTE GROWTH FACTOR RAM KANNAN,*,y,z MANLIN JIN,*,§ MARIA-ANDREEA GAMULESCU, y and DAVID R. HINTON*,y,z,§ z
*The Arnold and Mabel Beckman Macular Research Center; y Doheny Eye Institute; § Department of Pathology; and Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USA (Received 30 October 2003; Revised 26 March 2004; Accepted 8 April 2004) Available online 6 May 2004
Abstract—The aim of this study was to elucidate cellular mechanisms involved in ceramide-induced apoptosis and its attenuation by hepatocyte growth factor (HGF). Human retinal pigmented epithelial cells (RPE) incubated with C2 ceramide accumulated reactive oxygen species (ROS) in mitochondria and underwent apoptosis in a dose-dependent manner. Ceramide-treated cells showed increased caspase-3 activation and an increase in mitochondrial membrane permeability transition (MPT). Low doses of H2O2 (100 AM) alone induced negligible apoptosis; however, ceramideinduced apoptosis was significantly enhanced by co-incubation with H2O2 (100 AM). Furthermore, ceramide treatment significantly decreased catalase enzymatic activity and protein expression. HGF pretreatment (20 ng/ml) significantly inhibited ceramide-induced apoptosis and reduced the accumulation of ROS, the activation of caspase-3, and the increase in MPT and prevented the reduction in catalase activity and expression. Together, the data suggest that ceramide induces apoptosis in RPE cells by increasing ROS production, MPT, and caspase-3 activation. The ceramide effect is potentiated by H2O2 and associated with a reduction in catalase activity, suggesting that catalase plays a central role in regulating this apoptotic response. The ability of HGF to attenuate these effects demonstrates its effectiveness as an antioxidant growth factor. D 2004 Elsevier Inc. All rights reserved. Keywords—Ceramide, Oxidant stress, Mitochondria, Apoptosis, Catalase, Hepatocyte growth factor, Free radicals
factors such as NFnB [3 –9]. It is not known whether ceramide plays a role in the stimulation of other forms of stress-induced apoptosis. Thus, although the involvement of reactive oxygen intermediates in cellular signaling pathways via plasma membrane-anchored receptors and enzymes is established, it is not clear whether the process is mediated by ceramide as a second messenger. The close link between reactive oxygen species (ROS) generation, sphingolipid metabolism, and ceramide production has been investigated in several cell types. ROS have been implicated in the generation of ceramide and in the induction of apoptosis by ceramide [10,11]. In other studies, ceramide was reported to induce oxidative damage by increasing ROS generation or by inhibiting the ROS scavenger glutathione [12 – 16]. Mitochondria are one of the most important cellular sources of ROS due to their quantitative consumption of molecular oxygen. The effects of ceramide on mitochondrial function were examined in recent studies using
INTRODUCTION
Ceramide is a membrane sphingolipid that has recently emerged as a second messenger involved in the induction of apoptosis [1]. Ceramide can be generated by the de novo biosynthetic pathway, which is catalyzed by ceramide synthase [1]. Ceramide can also be generated as a result of sphingomyelin hydrolysis by sphingomyelinases in the hydrolytic pathway, which is the major source for ceramide in cellular responses to extracellular signaling [2,3]. It has been shown that ceramide plays a critical role in apoptosis, proliferation, cellular senescence, and gene regulation through modulation of a variety of protein kinases and phosphatases and activation of transcription Address correspondence to: David R. Hinton, Department of Pathology, Keck School of Medicine of the University of Southern California, 2011 Zonal Avenue, HMR 209, Los Angeles, CA 90033, USA; Fax: (323) 442-6688; E-mail: [email protected]. 166
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lung epithelial cells and hepatocytes [16,17]. It was found that ceramide, generated as a result of tumor necrosis factor a signaling, targets the mitochondrial electron transport chain, leading to overproduction of H2O2 and ROS [17]. Apoptosis plays a critical role in the pathogenesis of many blinding disorders of the retina, including agerelated macular degeneration (AMD), retinitis pigmentosa (RP), and retinal detachment [18 –20]. Although there is growing evidence supporting a role for ceramide in apoptosis in neurons, lung epithelial cells, hepatocytes, and HL-60 cells [15 – 17,21,22], little is known about the role of ceramides in retinal cells or disease. In vitro studies using retinal pigmented epithelium (RPE) cell lines show that ceramide can inhibit calcium-activated K+ current and induce apoptosis in these cells [23,24]. The RPE plays an important role in the maintenance of photoreceptor function and survival and RPE cell apoptosis has been identified in tissue samples from patients with AMD [18,25]. In Drosophila, targeted expression of neutral ceramidase has been shown to decrease ceramide levels and rescue retinal degeneration in arrestin and phospholipase C mutants, suggesting a pathogenic role for ceramide in the apoptotic death of retinal cells [26]. Most recently, a mutation in a novel human ceramide kinase gene, CERKL, was shown to be responsible for an autosomal recessive form of RP, providing a direct link between human retinal degeneration and altered sphingolipid metabolism [27]. Thus, manipulating the sphingolipid metabolic pathway may represent a novel therapeutic approach for management of some forms of retinal degeneration and disease. Hepatocyte growth factor (HGF) is a glycoprotein that induces proliferation, survival, dissociation, motility, and invasiveness of epithelial and endothelial cells [28]. Its biologic actions are mediated by c-Met, a transmembrane tyrosine kinase receptor [28]. Our laboratory has shown that RPE cells express c-Met and respond chemotactically to HGF [29]. Overexpression of HGF in RPE promotes survival of reactive RPE and photoreceptors in vivo [30]. In other epithelial cell types, HGF protects cells from apoptosis induced by the loss of contact with the substratum, DNA damage, or treatment with interferon-g, Adriamycin, X rays, or UV light [31 – 35]. In contrast, a sarcoma cell line showed growth suppression when treated with HGF [36]. Recently, Zhang et al. [37] found that the oxidant menadione induced apoptosis in RPE cells through release of apoptosis-inducing factor and that HGF abrogated this effect. In the present study, we have elucidated the intracellular pathways mediating ceramide-induced apoptosis in early passage human RPE and the protective effect of HGF on these cells.
167 EXPERIMENTAL PROCEDURES
Cell culture and treatment The Institutional Review Board of the University of Southern California approved our use of cultured human RPE cells. RPE cells were isolated from human eyes obtained from Advanced Bioscience Resources, Inc. (Alameda, CA, USA) and cultured in Dulbecco’s minimal Eagle’s medium (DMEM; Fisher Scientific, Pittsburgh, PA, USA) with 2 mM L-glutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin (Sigma, St. Louis, MO, USA), and 10% heat-inactivated fetal bovine serum (FBS; Irvine Scientific, Santa Ana, CA, USA) as previously described [38]. Third to fourth passage cells grown to confluence were used for these experiments. Cells were changed to DMEM (Invitrogen, Carlsbad, CA, USA) containing 1% FBS for 24 h, pretreated 24 h with HGF (20 ng/ml; R&D Systems) in 1% FBS, and then treated continuously with the C2 or C6 analogs of ceramide or the corresponding dihydroceramides (Biomol, Plymouth Meeting, PA, USA). Apoptosis assays DNA cleavage, which characteristically occurs in apoptosis, was measured by TdT – mediated dUTP nick-end labeling (TUNEL; In Situ Cell Death Detection Kit, Fluorescein; Roche Molecular Biochemicals). Briefly, after treatment, floating and adherent cells (released by trypsin) were collected and fixed with freshly prepared 4% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature and then permeabilized with 0.1% Triton X100 in 0.1% sodium citrate for 2 min on ice. To label DNA strand breaks, cells were incubated with 50 Al TUNEL reaction mixture containing TdT and fluorescein– dUTP in the binding buffer and incubated for 1 h at 37jC in a humidified chamber. Cells were then washed and analyzed by flow cytometry. In some experiments, cells were pretreated with caspase inhibitor z-VAD-fmk or Ac-DEVDCHO (Promega) before incubation with C2 ceramide. In experiments designed to assess the contribution of ceramide biosynthetic pathways to the HGF-mediated inhibition of ceramide-induced apoptosis, confluent RPE cells were pretreated for 2 h with 25 AM fumonisin B1, a ceramide synthase inhibitor [39], or 5 AM desipramine, an acidic sphingomyelinase inhibitor [40,41] (Sigma Biochemicals). These cells were then incubated with C2 ceramide for 24 h, with or without HGF pretreatment, and were then evaluated for apoptosis as described above. Attached cells and floating cells from untreated and treated RPE from various experiments were collected and pooled together for viability assays. Cell viability was assessed using the LIVE/DEAD viability assay kit (Molecular Probes, Eugene, OR, USA) [42].
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ROS determination Detection of ROS accumulation in RPE cells was achieved using a carboxy-H2-DCFDA staining method (Molecular Probes). This assay is based on the principle that the nonpolar, nonionic H2-DCFDA crosses cell membranes, where it is hydrolyzed by intracellular esterases to nonfluorescent H2-DCF [43]. In the presence of ROS, H2-DCF is rapidly oxidized to become highly fluorescent DCF. In these experiments, cells were incubated for 1 h at 37jC with 5 AM carboxy-H2-DCFDA dissolved in the culture medium. Cells were examined using a laser scanning confocal microscope (LSM510, Zeiss, Thornwood, NY, USA), and ROS were identified by the presence of green fluorescence. To determine the compartmentalized accumulation of ROS, mitochondria were labeled by a cell-permeable, mitochondria-specific red fluorescent dye, CMXRos (Molecular Probes; 500 nM for 30 min), and rapidly evaluated by confocal microscopy. A yellow color is observed when accumulated ROS (green) are colocalized in the mitochondria (red). Measurement of mitochondrial membrane permeability transition (MPT) MPT was measured using the fluorescent lipophilic cationic dye tetramethyl rhodamine methyl ester (TMRM; 250 nM; Molecular Probes), which accumulates within mitochondrial membranes. After treatment with C2 ceramide for 24 h, cells were loaded with TMRM for 15 min, and red fluorescence was measured by flow cytometry, using the FL-2 setting. In this assay, the induction of MPT results in loss of fluorescence. In each analysis, 10,000 events were recorded. Caspase activation assay Caspase activation was determined by using a CaspACE FITC-VAD-fmk In Situ Marker kit (Promega, Madison, WI, USA). This assay utilizes a cell-permeable caspase inhibitor (VAD-fmk) conjugated to fluoroisothiocyanate (FITC) that binds irreversibly to activated caspase-3. Cells were collected and incubated with labeled VAD-fmk for 60 min and fluorescence was measured by flow cytometry, using the FL-1 setting. In each analysis, 10,000 events were recorded. Catalase activity assay RPE cells (106/ml) were washed twice with PBS, homogenized, and immunoprecipitated with anti-catalase antibody (Calbiochem, Carlsbad, CA, USA) [44]. Catalase activity was measured using a Catalase Assay Kit (Calbiochem) following the manufacturer’s protocol. Briefly, catalase protein samples were incubated in the presence of a known concentration of H2O2. After 1 min incubation, the reaction was quenched with sodium azide.
Catalase activity was determined by a spectrophotometric assay (520 nm) that measures the rate of dismutation of H2O2. Western blot analysis for catalase protein Equal amounts of protein from lysed RPE cell cultures were resolved on Tris – HCl polyacrylamide gels (120 V, Ready Gel; Bio-Rad, Hercules, CA, USA) and transferred to PVDF blotting membranes (Millipore, Bedford, MA, USA). Membranes were probed with rabbit polyclonal anti-caspase-3 antibody (PharMingen), rabbit polyclonal anti-catalase antibody (Calbiochem), or rabbit polyclonal anti-h-actin antibody (Sigma), followed by chemiluminescence detection (Amersham Pharmacia Biotech, Cleveland, OH, USA). Statistical analysis Results are expressed as means F SEM. An unpaired, two-tailed Student t test was used to determine statistical difference between two group means. Differences were considered statistically significant when p < .05. RESULTS
Concentration-dependent induction of apoptosis by ceramide Figure 1 shows the development of apoptosis by TUNEL assay as a function of treatment of RPE with C2 ceramide in the concentration range 10– 50 AM. The percentage of apoptotic cells increased with increasing C2 ceramide concentration, reaching f63% with 50 AM C2 (24 h). When higher concentrations (75 and 100 AM) were used, the cells began to detach and viability (determined by LIVE/DEAD cell viability assay) decreased to 40– 50% (results not shown). On the other hand, 50 AM C2 dihydroceramide, used as a negative control, did not cause significant apoptosis. In parallel experiments, RPE cells incubated for 24 h with C6 ceramide (10 – 50 AM) did not show a dose-dependent increase in apoptosis. C6 ceramide induced significantly less apoptosis ( p < .01 vs. corresponding C2 ceramide concentration) and at the highest concentration tested resulted in only 7% apoptosis (data not shown). Therefore, all subsequent studies were carried out with the C2 analog. Inhibition of ceramide-induced apoptosis by HGF Figure 2 shows the effects of pretreatment of RPE with 20 ng/ml HGF on the induction of apoptosis by either 30 or 40 AM C2 ceramide. Pretreatment with HGF (24 h) inhibited ceramide-induced apoptosis by 52 and 59% respectively ( p < .01 vs. untreated control for both 30 and 40 AM C2 ceramide). Although higher concen-
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were additionally treated with catalase (200 U/ml), there was a significant inhibition of apoptosis compared to ceramide alone (32%, p < .01) or ceramide + H2O2 (24%, p < .001). Apoptosis was increased in RPE treated with the catalase inhibitor ATZ (40 AM) or ATZ + H2O2 (100 AM), compared to their respective controls ( p < .01). C2 ceramide treatment and accumulation of ROS
Fig. 1. Dose dependency of RPE cells undergoing apoptosis as determined by TUNEL assay and measured by FACS analysis. Confluent RPE in 1% serum containing DMEM were incubated for 24 h at 37jC with varying concentrations of ethanolic solutions of C2 ceramide. Dihydro-C2 ceramide (DC2) was used as a negative control. Control represents RPE cells treated with vehicle alone. Data are means F SEM from three or four determinations from three different donors per group. Asterisk indicates significant difference ( p < .01) vs. control.
trations of HGF (30 and 50 ng/ml) decreased apoptosis in ceramide-treated RPE, the protection due to HGF was maximal at 20 ng/ml. Cell viability in the control, C2 ceramide, and C2 ceramide plus HGF groups ranged from 85 to 95% and was not significantly different among the three groups. In separate experiments, co-incubation of C2-ceramide-treated cells with the caspase-3 inhibitor z-VAD-fmk (10 AM) decreased the percentage of apoptotic cells at 24 h (mean of 50 F 6.8% in C2-treated cells to a mean of 20 F 4.5% in caspase-3 inhibitor-treated cells, p < .01). Similar results were found for Ac-DEVD-CHO (20 AM). Experiments also were performed to determine whether apoptosis induced by exogenous ceramide was mediated through its action on the endogenous ceramide generation pathway. Pretreatment of RPE for 2 h with fumonisin B1, a ceramide synthase inhibitor, did not significantly alter the extent of apoptosis from C2 ceramide, either in the presence or in the absence of HGF ( p = .1). Similarly, the acidic sphingomyelinase inhibitor desipramine did not affect C2-ceramide-induced apoptosis either in the presence or in the absence of HGF ( p = .2).
Figure 4 shows the ceramide-induced accumulation of intracellular ROS in RPE, their predominant localization to mitochondria, and the prevention of ROS accumulation with pretreatment of cells with HGF. Preliminary studies revealed the dose dependency of ROS accumulation after 24 h of C2 ceramide (data not shown). Untreated control cells (Figs. 4A –4C) showed only minimal accumulation of ROS. Treatment with 40 AM C2 ceramide resulted in pronounced intracellular accumulation of ROS that was predominantly localized to mitochondria as demonstrated by colocalization with a mitochondrial label (Figs. 4D – 4F). Preincubation of RPE with 20 ng/ml HGF markedly decreased the ceramide-induced ROS accumulation (Figs. 4G –4I). Effects of ceramide and HGF on mitochondrial MPT Figure 5 shows the accumulation of the fluorescent mitochondrial lipophilic dye TMRM in RPE and its loss after induction of MPT by treatment with C2 ceramide (40 AM, p < .01). Pretreatment of RPE with HGF (20 ng/ ml) significantly inhibited this effect ( p < .01). Effect of ceramide on caspase-3 activation Figure 6 shows that in control cultures, caspase-3 activity is infrequent (9.8%); however, the number of
Effects of H2O2 on ceramide-induced apoptosis As shown in Fig. 3, incubation of RPE with low concentrations of H2O2 (100 AM, 12 h) resulted in minimal apoptosis (2%); however, RPE pretreated with C2 ceramide (40 AM, 8 h) + H2O2 (100 AM, 12 h) showed increased apoptosis (62%) compared to C2 ceramide pretreatment alone (46%, p < .01). When cells
Fig. 2. Attenuation of C2 ceramide-induced apoptosis in RPE cells by HGF. Cells were pretreated with HGF (20 ng/ml) for 24 h followed by exposure to either 30 or 40 AM C2 ceramide for an additional 24 h. Apoptosis was monitored by TUNEL assay and flow cytometry as in Fig. 1. Data are means F SEM (n = 3). Asterisk indicates statistical difference ( p < .01) compared to the corresponding C2-treated group.
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Fig. 3. Potentiation of ceramide-induced apoptosis by H2O2 and effects of purified catalase and ATZ. Confluent RPE cells were treated with 40 AM C2 ceramide in DMEM containing 1% serum and incubated with 100 AM H2O2 for an additional 12 h. Effects on apoptosis of treatments with ATZ (30 AM), catalase (200 U/ml), and their combination with C2 or H2O2 are also shown. *p < .01 vs. control; **p < .01 vs. C2-treated group; ***p < .01 vs. C2 + H2O2 group.
cells expressing active caspase-3 after C2 ceramide treatment (40 AM) is markedly increased (89.6%, p < .001). Pretreatment with HGF significantly decreased
ceramide-induced caspase-3 activation (48.5%, p < .01 vs. ceramide-treated RPE). Western blots confirmed the ceramide-induced conversion of procaspase-3 to
Fig. 4. (D – F) Accumulation of ROS in mitochondria of RPE cells treated with C2 ceramide and (G – I) effects of HGF. (E) Confocal microscopy demonstrates the intracellular accumulation of ROS after 24 h treatment with 40 AM C2 ceramide (green fluorescence). (A, D, G) ROS accumulated predominantly in labeled mitochondria (red fluorescence) after ceramide treatment as shown by (F) colocalization of merged fluorescence images (red + green labels seen as yellow fluorescence). (H) HGF pretreatment results in decreased accumulation of ROS after ceramide treatment. Bar, 10 Am.
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Fig. 5. Effects of ceramide and HGF on mitochondrial membrane permeability transition (MPT). RPE cells were treated with 40 AM C2 ceramide, with or without overnight preincubation with 20 ng/ml HGF. Cells were loaded with the mitochondrial membrane dye TMRM, and MPT results in loss of fluorescence. Flow cytometry histograms show (A) cells without fluorescent dye (negative control), (B) control (non-ceramide-treated) cells with fluorescent dye, (C) C2-treated cells with fluorescent dye, and (D) HGF pretreated, C2treated cells with fluorescent dye.
active caspase-3 and the attenuation of this effect by HGF. Effect of C2 ceramide and HGF on catalase activity and expression Table 1A shows the enzyme activity of catalase that had been immunoprecipitated from RPE cell lysates. Incubation of RPE with 40 AM C2 ceramide caused a
33 and 77% decrease in catalase enzyme activity at 12 and 24 h compared with the corresponding untreated controls ( p < .05). HGF treatment did not significantly alter basal catalase enzyme activity of control RPE cells. When ceramide-treated RPE were pretreated with HGF, the decrease in catalase activity was significantly attenuated ( p < .01 for 12 and 24 h). Consistent with these findings, Fig. 7 shows that the amount of catalase protein
Fig. 6. Ceramide treatment increases caspase-3 activation. RPE cells were treated with 40 AM C2 ceramide for 24 h and active caspase-3 was detected by flow cytometry after incubation with a cell-permeable FITC-labeled caspase inhibitor that binds irreversibly to active caspase-3. Western blot analysis (bottom) was performed using a caspase-3-specific polyclonal antibody. Untreated controls show only procaspase-3 (32 kDa), whereas after ceramide treatment a band representing active caspase-3 (16 – 18 kDa) is also seen.
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(A) Control C2 C2 + HGF (B) Control C2 C2 + z-VAD-fmk
32.5 29.3 26.5 38.9 36.3 35.1
F F F F F F
12 h 1.2 0.8 1.1 3.2 0.8 2.4
29.8 20.1 27.3 37.5 24.1 31.2
F F F F F F
3.9 5.8* 2.3** 3.9 5.8 4.5***
24 h 33.1 7.6 20.1 36.1 10.6 23.4
F F F F F F
5.6 2.3* 4.1** 5.6 6.3 3.6***
In (A), the effects of pretreatment with HGF (20 ng/ml, 24 h) on catalase activity were determined in ceramide-treated RPE (12 and 24 h). In (B), this experiment was repeated in the presence of the caspase inhibitor z-VAD-fmk (10 AM); caspase activity was reduced by 72.1% in z-VAD-fmk-treated cells compared to those treated for 24 h with ceramide alone. Catalase was immunoprecipitated and enzyme activity was measured as described under Experimental Procedures. * p < .05 control vs. C2. ** p< .01 C2 vs. C2+HGF. *** p < .01 C2 vs C2 + z-VAD-fmk.
detected by Western blot decreased progressively from 12 to 24 h after ceramide treatment. When HGF (20 ng/ ml)-pretreated cells were exposed to C2 ceramide, the expression of catalase protein remained at levels similar to those of untreated controls. In order to determine whether caspase-3 activation was required to elicit the ceramide-induced decrease in catalase activity, cells were incubated with ceramide in the presence of the caspase inhibitor z-VAD-fmk (10 AM). At 12 h, catalase levels in the ceramide + z-VADfmk-treated cells remained equal to those of untreated controls (Table 1B). At 24 h, catalase levels were now reduced from untreated control levels but they were significantly higher than those found in the ceramidetreated controls ( p < .01; Table 1B). Caspase-3 activation was reduced by 72.1% when cells were co-incubated for 24 h with z-VAD-fmk, compared to treatment with ceramide alone.
be both stimulus- and cell-type-specific. Studies examining the effects of exogenous ceramides of varying acyl chain lengths, from the synthetic short-chain C2 analog to the physiologically more relevant C16 ceramide (C2-C16), in breast cancer and mouse macrophage cell lines suggest that chain length is inversely proportional to cytotoxic activity, with C6 being the most active [45]. Other studies have used short-chain, easily cell-permeable C2 and C6 ceramide analogs as inducers of apoptosis in several cell types and have concluded that C2 ceramide caused more apoptosis than the C6 analog [22,24]. Our findings in low-passage human RPE are consistent with the latter view and may be explained by the more efficient solubility of C2 ceramide at room temperature in the vehicle used. A dosedependent increase in percentage of apoptotic cells was found with 24 h C2 treatment up to 50 AM. At concentrations above 50 AM, there was a 50 – 60% loss of cell viability. It was recently proposed that ceramide is both a signaling product of oxidative stress and a mediator of the production of reactive oxidants in the mitochondria [17,21,46]. This implies a bidirectional relationship between oxidative stress and ceramide production in mitochondria. ROS and activated caspase-3 have both been implicated as downstream mediators of ceramide-induced apoptosis [15,17,21,47 –49]. In our study, ROS accumulated preferentially in mitochondria, consistent with the importance of the mitochondrial respiratory chain as a site of ROS production [50,51]. Future studies should determine the exact step of the mitochondrial respiratory chain that is altered by ceramide. The subsequent ceramide-mediated induction of MPT in RPE further points to the mitochondria as a critical target of ceramide action. Oxidative damage has been shown to increase ceramide content through activation of caspase-3 [49]. In the present study, ceramide treatment increased levels of active caspase-3, whereas the addition of
DISCUSSION
We demonstrate that C2 ceramide induces apoptosis in RPE in a dose-response manner, with mitochondrial accumulation of ROS and caspase-3 activation, whereas pretreatment with HGF significantly inhibits these effects. Furthermore, the apoptosis-inducing effect of ceramide treatment is potentiated by co-incubation with low doses of H2O2, suggesting the importance of antioxidant scavengers in this process. Consistent with this hypothesis, we showed that ceramide treatment decreases catalase activity and protein and that this decrease is mediated through activation of caspase-3. The mechanism by which ceramide elicits apoptosis has not been fully elucidated, and the pathways seem to
Fig. 7. Effects of ceramide on catalase protein expression. RPE cells were treated with C2 for 12 and 24 h in the presence or absence of HGF. The protein levels of catalase and h-actin were examined by Western blot of RPE cell lysates. A representative blot from three independent experiments is shown.
Ceramide-induced apoptosis
caspase-3-specific inhibitors blocked ceramide-induced apoptosis. These findings are consistent with the hypothesis that ceramide-induced apoptosis in RPE is mediated, at least in part, through caspase-3 [47]. Recently, ROS generation has also been shown to lead to apoptosis by caspase-independent pathways [52]. Zhang et al. [37] showed that menadione-induced cell death differed between U937 cells and ARPE-19 cells in that the former exhibited caspase-3 activation, whereas in the latter, apoptosis-inducing factor and not caspase-3 was involved in cell death. It would be of interest to further evaluate the contribution of a caspase-independent mechanism(s) of ceramide-induced apoptosis in RPE. Our study revealed that ceramide-induced apoptosis was enhanced by treatment with low concentrations of H2O2. The scavenging system for H2O2 includes catalase, glutathione, and glutathione peroxidase [53]. Our observation of increased oxidative damage and increased apoptosis induced by H2O2 along with the catalase inhibitor ATZ suggests a critical role for catalase in this process. The enhancement of apoptosis by H2O2 in ceramide-treated cells was accompanied by a decrease in catalase enzyme activity and protein expression. Accordingly, preincubation with exogenous catalase decreased apoptosis in RPE treated with ceramide and/or H2O2. Previous work has shown that the protection of mononuclear cells from ceramide-induced apoptosis by exogenous catalase is likely the result of scavenging of cytosolic peroxides that diffuse out of the cells [54]. Internalization of exogenous catalase can be excluded as a mechanism because cells exposed to exogenous catalase do not show an increase in intracellular catalase content [55,56]. Our data support the involvement of active caspase-3 in the regulation of catalase activity. Inhibition of caspase-3 activity not only attenuated the development of apoptosis, but also significantly protected cells from the ceramide-induced loss of catalase activity and protein. Recent work in ceramide-treated HL-60 cells suggests that active caspase-3 proteolytically cleaves catalase, thus reducing its level of expression and activity [44]. Our demonstration that HGF treatment attenuates both the ceramide-induced reduction in catalase and the activation of caspase-3 supports the contention that HGF-mediated protection against catalase loss is a result of decreased caspase-3 activation; however, additional direct effects on catalase synthesis cannot be ruled out. It is well established that glutathione (GSH) is an effective antioxidant defense against accumulation of ROS and downstream events leading to apoptosis. We found that treatment with either C2 or C6 ceramide at 40 AM did not cause significant changes in the
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intracellular GSH pool in RPE cells compared to untreated controls (Kannan et al., unpublished observations). This finding is consistent with a recent, similar observation in Molt-4 leukemia cells [57]. However, exogenous GSH supplementation is known to decrease mitochondrial ROS levels and attenuate apoptosis in several cell types. Accordingly, we also observed that incubation of ceramide-treated RPE with GSH monoethyl ester reduced ROS levels, inhibited activation of caspase-3, and reduced the extent of apoptosis (data not shown). Our data support the hypothesis that in RPE, exogenous ceramide promotes the accumulation of mitochondrial ROS, induces MPT, activates caspase-3, and stimulates subsequent apoptosis. These effects are accentuated by the degradation of catalase by activated caspase-3, with loss of peroxide scavenger activity and further accumulation of mitochondrial ROS. The ability of HGF to protect RPE from ceramide-induced apoptosis and to inhibit ceramide-induced alterations at each step of this cascade, including the preservation of catalase activity, suggests that its major effect results from attenuation of mitochondrial ROS accumulation. Further studies will determine if there is a direct effect of HGF on multiple stages of the cascade and if other antioxidant enzymes and signaling molecules are involved [58,59]. In contrast to our work, a previous study showed that HGF induced growth suppression in the sarcoma 180 cell line and that this effect was mediated through an increase in ROS with activation of caspase-3 [36]. In the sarcoma cells, treatment with an intracellular free radical scavenger (N-acetylcysteine) blocked the growth suppression, whereas addition of exogenous catalase had no effect. The opposing effects of HGF in sarcoma cells, compared to cultured RPE, are likely a result of dysregulated growth factor signaling in the transformed cells [36]. Sarcoma 180 cells show constitutive activation of the HGF receptor c-Met, with only a modest increase in receptor phosphorylation with HGF treatment [36]. On the other hand, we have shown that cultured RPE demonstrate regulated c-Met activation with strong and rapid induction of c-Met phosphorylation after exposure to HGF [29]. The present work may have relevance in designing therapeutic approaches for the prevention of apoptotic injury to the retina and the RPE. The recognition of ceramide as an important endogenous molecule mediating proapoptotic signaling, and the link between alterations in ceramide metabolism and photoreceptor degeneration [26,27], suggests that approaches aimed at modulating the sphingolipid biosynthetic or degradative pathways should be explored as potential treatments for retinal degenerative disorders.
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Acknowledgments — This work was supported in part by Grants EY02061 and EY03040 from the National Institutes of Health, a grant from the Arnold and Mabel Beckman Foundation, a grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc. (New York, NY), and a grant from the Deutsche Akademie der Naturforscher Leopoldina (BMBF-LPD 9901/8-52), Germany. We thank Christine Spee and Ernesto Barron for their technical support in this project. REFERENCES [1] Hannun, Y. A. The sphingomyelin cycle and the second messenger function of ceramide. J. Biol. Chem. 269:3125 – 3128; 1994. [2] Perry, D. K. Ceramide and apoptosis. Biochem. Soc. Trans. 27:399 – 404; 1999. [3] Obeid, L. M.; Linardic, C. M.; Karolak, L. A.; Hannun, Y. A. Programmed cell death induced by ceramide. Science 259: 1769 – 1771; 1993. [4] Jayadev, S. B.; Liu, A. E.; Lee, J. Y.; Nazaire, F.; Pushkareva, M. Y.; Obeid, L. M.; Hannun, Y. A. Role of ceramide in cell cycle arrest. J. Biol. Chem. 270:2047 – 2052; 1995. [5] Spiegel, S.; Merrill, A. H., Jr. Sphingolipid metabolism and growth retardation. FASEB J. 10:1388 – 1397; 1996. [6] Schutze, S.; Potthoff, K.; Machleidt, T.; Berkovic, D.; Wiegmann, K.; Kronke, M. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced ‘‘acidic’’ sphingomyelin breakdown. Cell 71:756 – 776; 1992. [7] Wiegmann, K.; Schutze, S.; Machleidt, T.; Witte, D.; Kronke, M. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78:1005 – 1015; 1994. [8] Ruvolo, P. P. Intracellular signal transduction pathways activated by ceramide and its metabolites. Pharm. Res. 47:383 – 392; 2003. [9] Kajimoto, T.; Shirai, Y.; Sakai, N.; Yamamoto, T.; Matzuzaki, H.; Kikkawa, U.; Saito, N. Ceramide-induced apoptosis by translocation, phosphorylation and activation of protein kinase Cdelta at Golgi complex. J. Biol. Chem. 279:12668 – 12676; 2004. [10] Mansat-de Mas, V.; Bezombes, C.; Quillet-Mary, A.; Bettaieb, A.; D’Orgeix, A. D.; Laurent, G.; Jeffrezou, J. P. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol. 56:867 – 874; 1999. [11] Cabrero, A.; Algret, M.; Sanchez, R. M.; Adzet, T.; Laguna, J.C.; Carrera, M. V. Increased reactive oxygen species production down-regulates peroxisome proliferator-activated alpha pathway in C2C12 skeletal muscle cells. J. Biol. Chem. 277: 10100 – 10107; 2002. [12] Pedersen, J. Z.; Marcocci, L.; Rossi, L.; Mavelli, I.; Rotilio, G. Generation of daunomycin radicals on the outer side of the erythrocyte membrane. Biochem. Biophys. Res. Commun. 168: 240 – 247; 1990. [13] Wang, J. H.; Redmond, H. P.; Watson, R. W.; Bouchier Hayes, D. Induction of human endothelial cell apoptosis requires both heat shock and oxidative stress responses. Am. J. Physiol. 272: C1543 – C1551; 1997. [14] Kondo, T.; Matsuda, T.; Tashima, M.; Umehara, H.; Domae, N.; Yokoyama, K.; Uchiyama, T.; Okazaki, T. Suppression of heat shock protein-70 by ceramide in heat shock induced HL-60 cell apoptosis. J. Biol. Chem. 275:8872 – 8879; 2000. [15] Kondo, T.; Matsuda, T.; Kitano, T.; Takahashi, A.; Tashima, M.; Ishikura, H.; Umehara, H.; Domae, N.; Uchiyama, T.; Okazaki, T. Role of c-jun expression increased by heat-shock- and ceramideactivated caspase-3 in HL-60 cell apoptosis. Possible involvement of ceramide in heat shock-induced apoptosis. J. Biol. Chem. 275:7668 – 7676; 2000. [16] Lavrentiadou, S. N.; Chan, C.; Kawcak, T.; Ravid, T.; Tsaba, A.; van der Vliet, A.; Rasooly, R.; Goldkorn, T. Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione. Am. J. Respir. Cell. Mol. Biol. 25:676 – 684; 2001. [17] Garcia-Ruiz, C.; Colell, A.; Mari, M.; Morales, A.; FernandezCheca, J. C. Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen
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doi:10.1016/j.freeradbiomed.2004.04.011
Original Contribution CERAMIDE-INDUCED APOPTOSIS: ROLE OF CATALASE AND HEPATOCYTE GROWTH FACTOR RAM KANNAN,*,y,z MANLIN JIN,*,§ MARIA-ANDREEA GAMULESCU, y and DAVID R. HINTON*,y,z,§ z
*The Arnold and Mabel Beckman Macular Research Center; y Doheny Eye Institute; § Department of Pathology; and Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USA (Received 30 October 2003; Revised 26 March 2004; Accepted 8 April 2004) Available online 6 May 2004
Abstract—The aim of this study was to elucidate cellular mechanisms involved in ceramide-induced apoptosis and its attenuation by hepatocyte growth factor (HGF). Human retinal pigmented epithelial cells (RPE) incubated with C2 ceramide accumulated reactive oxygen species (ROS) in mitochondria and underwent apoptosis in a dose-dependent manner. Ceramide-treated cells showed increased caspase-3 activation and an increase in mitochondrial membrane permeability transition (MPT). Low doses of H2O2 (100 AM) alone induced negligible apoptosis; however, ceramideinduced apoptosis was significantly enhanced by co-incubation with H2O2 (100 AM). Furthermore, ceramide treatment significantly decreased catalase enzymatic activity and protein expression. HGF pretreatment (20 ng/ml) significantly inhibited ceramide-induced apoptosis and reduced the accumulation of ROS, the activation of caspase-3, and the increase in MPT and prevented the reduction in catalase activity and expression. Together, the data suggest that ceramide induces apoptosis in RPE cells by increasing ROS production, MPT, and caspase-3 activation. The ceramide effect is potentiated by H2O2 and associated with a reduction in catalase activity, suggesting that catalase plays a central role in regulating this apoptotic response. The ability of HGF to attenuate these effects demonstrates its effectiveness as an antioxidant growth factor. D 2004 Elsevier Inc. All rights reserved. Keywords—Ceramide, Oxidant stress, Mitochondria, Apoptosis, Catalase, Hepatocyte growth factor, Free radicals
factors such as NFnB [3 –9]. It is not known whether ceramide plays a role in the stimulation of other forms of stress-induced apoptosis. Thus, although the involvement of reactive oxygen intermediates in cellular signaling pathways via plasma membrane-anchored receptors and enzymes is established, it is not clear whether the process is mediated by ceramide as a second messenger. The close link between reactive oxygen species (ROS) generation, sphingolipid metabolism, and ceramide production has been investigated in several cell types. ROS have been implicated in the generation of ceramide and in the induction of apoptosis by ceramide [10,11]. In other studies, ceramide was reported to induce oxidative damage by increasing ROS generation or by inhibiting the ROS scavenger glutathione [12 – 16]. Mitochondria are one of the most important cellular sources of ROS due to their quantitative consumption of molecular oxygen. The effects of ceramide on mitochondrial function were examined in recent studies using
INTRODUCTION
Ceramide is a membrane sphingolipid that has recently emerged as a second messenger involved in the induction of apoptosis [1]. Ceramide can be generated by the de novo biosynthetic pathway, which is catalyzed by ceramide synthase [1]. Ceramide can also be generated as a result of sphingomyelin hydrolysis by sphingomyelinases in the hydrolytic pathway, which is the major source for ceramide in cellular responses to extracellular signaling [2,3]. It has been shown that ceramide plays a critical role in apoptosis, proliferation, cellular senescence, and gene regulation through modulation of a variety of protein kinases and phosphatases and activation of transcription Address correspondence to: David R. Hinton, Department of Pathology, Keck School of Medicine of the University of Southern California, 2011 Zonal Avenue, HMR 209, Los Angeles, CA 90033, USA; Fax: (323) 442-6688; E-mail: [email protected]. 166
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lung epithelial cells and hepatocytes [16,17]. It was found that ceramide, generated as a result of tumor necrosis factor a signaling, targets the mitochondrial electron transport chain, leading to overproduction of H2O2 and ROS [17]. Apoptosis plays a critical role in the pathogenesis of many blinding disorders of the retina, including agerelated macular degeneration (AMD), retinitis pigmentosa (RP), and retinal detachment [18 –20]. Although there is growing evidence supporting a role for ceramide in apoptosis in neurons, lung epithelial cells, hepatocytes, and HL-60 cells [15 – 17,21,22], little is known about the role of ceramides in retinal cells or disease. In vitro studies using retinal pigmented epithelium (RPE) cell lines show that ceramide can inhibit calcium-activated K+ current and induce apoptosis in these cells [23,24]. The RPE plays an important role in the maintenance of photoreceptor function and survival and RPE cell apoptosis has been identified in tissue samples from patients with AMD [18,25]. In Drosophila, targeted expression of neutral ceramidase has been shown to decrease ceramide levels and rescue retinal degeneration in arrestin and phospholipase C mutants, suggesting a pathogenic role for ceramide in the apoptotic death of retinal cells [26]. Most recently, a mutation in a novel human ceramide kinase gene, CERKL, was shown to be responsible for an autosomal recessive form of RP, providing a direct link between human retinal degeneration and altered sphingolipid metabolism [27]. Thus, manipulating the sphingolipid metabolic pathway may represent a novel therapeutic approach for management of some forms of retinal degeneration and disease. Hepatocyte growth factor (HGF) is a glycoprotein that induces proliferation, survival, dissociation, motility, and invasiveness of epithelial and endothelial cells [28]. Its biologic actions are mediated by c-Met, a transmembrane tyrosine kinase receptor [28]. Our laboratory has shown that RPE cells express c-Met and respond chemotactically to HGF [29]. Overexpression of HGF in RPE promotes survival of reactive RPE and photoreceptors in vivo [30]. In other epithelial cell types, HGF protects cells from apoptosis induced by the loss of contact with the substratum, DNA damage, or treatment with interferon-g, Adriamycin, X rays, or UV light [31 – 35]. In contrast, a sarcoma cell line showed growth suppression when treated with HGF [36]. Recently, Zhang et al. [37] found that the oxidant menadione induced apoptosis in RPE cells through release of apoptosis-inducing factor and that HGF abrogated this effect. In the present study, we have elucidated the intracellular pathways mediating ceramide-induced apoptosis in early passage human RPE and the protective effect of HGF on these cells.
167 EXPERIMENTAL PROCEDURES
Cell culture and treatment The Institutional Review Board of the University of Southern California approved our use of cultured human RPE cells. RPE cells were isolated from human eyes obtained from Advanced Bioscience Resources, Inc. (Alameda, CA, USA) and cultured in Dulbecco’s minimal Eagle’s medium (DMEM; Fisher Scientific, Pittsburgh, PA, USA) with 2 mM L-glutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin (Sigma, St. Louis, MO, USA), and 10% heat-inactivated fetal bovine serum (FBS; Irvine Scientific, Santa Ana, CA, USA) as previously described [38]. Third to fourth passage cells grown to confluence were used for these experiments. Cells were changed to DMEM (Invitrogen, Carlsbad, CA, USA) containing 1% FBS for 24 h, pretreated 24 h with HGF (20 ng/ml; R&D Systems) in 1% FBS, and then treated continuously with the C2 or C6 analogs of ceramide or the corresponding dihydroceramides (Biomol, Plymouth Meeting, PA, USA). Apoptosis assays DNA cleavage, which characteristically occurs in apoptosis, was measured by TdT – mediated dUTP nick-end labeling (TUNEL; In Situ Cell Death Detection Kit, Fluorescein; Roche Molecular Biochemicals). Briefly, after treatment, floating and adherent cells (released by trypsin) were collected and fixed with freshly prepared 4% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature and then permeabilized with 0.1% Triton X100 in 0.1% sodium citrate for 2 min on ice. To label DNA strand breaks, cells were incubated with 50 Al TUNEL reaction mixture containing TdT and fluorescein– dUTP in the binding buffer and incubated for 1 h at 37jC in a humidified chamber. Cells were then washed and analyzed by flow cytometry. In some experiments, cells were pretreated with caspase inhibitor z-VAD-fmk or Ac-DEVDCHO (Promega) before incubation with C2 ceramide. In experiments designed to assess the contribution of ceramide biosynthetic pathways to the HGF-mediated inhibition of ceramide-induced apoptosis, confluent RPE cells were pretreated for 2 h with 25 AM fumonisin B1, a ceramide synthase inhibitor [39], or 5 AM desipramine, an acidic sphingomyelinase inhibitor [40,41] (Sigma Biochemicals). These cells were then incubated with C2 ceramide for 24 h, with or without HGF pretreatment, and were then evaluated for apoptosis as described above. Attached cells and floating cells from untreated and treated RPE from various experiments were collected and pooled together for viability assays. Cell viability was assessed using the LIVE/DEAD viability assay kit (Molecular Probes, Eugene, OR, USA) [42].
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ROS determination Detection of ROS accumulation in RPE cells was achieved using a carboxy-H2-DCFDA staining method (Molecular Probes). This assay is based on the principle that the nonpolar, nonionic H2-DCFDA crosses cell membranes, where it is hydrolyzed by intracellular esterases to nonfluorescent H2-DCF [43]. In the presence of ROS, H2-DCF is rapidly oxidized to become highly fluorescent DCF. In these experiments, cells were incubated for 1 h at 37jC with 5 AM carboxy-H2-DCFDA dissolved in the culture medium. Cells were examined using a laser scanning confocal microscope (LSM510, Zeiss, Thornwood, NY, USA), and ROS were identified by the presence of green fluorescence. To determine the compartmentalized accumulation of ROS, mitochondria were labeled by a cell-permeable, mitochondria-specific red fluorescent dye, CMXRos (Molecular Probes; 500 nM for 30 min), and rapidly evaluated by confocal microscopy. A yellow color is observed when accumulated ROS (green) are colocalized in the mitochondria (red). Measurement of mitochondrial membrane permeability transition (MPT) MPT was measured using the fluorescent lipophilic cationic dye tetramethyl rhodamine methyl ester (TMRM; 250 nM; Molecular Probes), which accumulates within mitochondrial membranes. After treatment with C2 ceramide for 24 h, cells were loaded with TMRM for 15 min, and red fluorescence was measured by flow cytometry, using the FL-2 setting. In this assay, the induction of MPT results in loss of fluorescence. In each analysis, 10,000 events were recorded. Caspase activation assay Caspase activation was determined by using a CaspACE FITC-VAD-fmk In Situ Marker kit (Promega, Madison, WI, USA). This assay utilizes a cell-permeable caspase inhibitor (VAD-fmk) conjugated to fluoroisothiocyanate (FITC) that binds irreversibly to activated caspase-3. Cells were collected and incubated with labeled VAD-fmk for 60 min and fluorescence was measured by flow cytometry, using the FL-1 setting. In each analysis, 10,000 events were recorded. Catalase activity assay RPE cells (106/ml) were washed twice with PBS, homogenized, and immunoprecipitated with anti-catalase antibody (Calbiochem, Carlsbad, CA, USA) [44]. Catalase activity was measured using a Catalase Assay Kit (Calbiochem) following the manufacturer’s protocol. Briefly, catalase protein samples were incubated in the presence of a known concentration of H2O2. After 1 min incubation, the reaction was quenched with sodium azide.
Catalase activity was determined by a spectrophotometric assay (520 nm) that measures the rate of dismutation of H2O2. Western blot analysis for catalase protein Equal amounts of protein from lysed RPE cell cultures were resolved on Tris – HCl polyacrylamide gels (120 V, Ready Gel; Bio-Rad, Hercules, CA, USA) and transferred to PVDF blotting membranes (Millipore, Bedford, MA, USA). Membranes were probed with rabbit polyclonal anti-caspase-3 antibody (PharMingen), rabbit polyclonal anti-catalase antibody (Calbiochem), or rabbit polyclonal anti-h-actin antibody (Sigma), followed by chemiluminescence detection (Amersham Pharmacia Biotech, Cleveland, OH, USA). Statistical analysis Results are expressed as means F SEM. An unpaired, two-tailed Student t test was used to determine statistical difference between two group means. Differences were considered statistically significant when p < .05. RESULTS
Concentration-dependent induction of apoptosis by ceramide Figure 1 shows the development of apoptosis by TUNEL assay as a function of treatment of RPE with C2 ceramide in the concentration range 10– 50 AM. The percentage of apoptotic cells increased with increasing C2 ceramide concentration, reaching f63% with 50 AM C2 (24 h). When higher concentrations (75 and 100 AM) were used, the cells began to detach and viability (determined by LIVE/DEAD cell viability assay) decreased to 40– 50% (results not shown). On the other hand, 50 AM C2 dihydroceramide, used as a negative control, did not cause significant apoptosis. In parallel experiments, RPE cells incubated for 24 h with C6 ceramide (10 – 50 AM) did not show a dose-dependent increase in apoptosis. C6 ceramide induced significantly less apoptosis ( p < .01 vs. corresponding C2 ceramide concentration) and at the highest concentration tested resulted in only 7% apoptosis (data not shown). Therefore, all subsequent studies were carried out with the C2 analog. Inhibition of ceramide-induced apoptosis by HGF Figure 2 shows the effects of pretreatment of RPE with 20 ng/ml HGF on the induction of apoptosis by either 30 or 40 AM C2 ceramide. Pretreatment with HGF (24 h) inhibited ceramide-induced apoptosis by 52 and 59% respectively ( p < .01 vs. untreated control for both 30 and 40 AM C2 ceramide). Although higher concen-
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were additionally treated with catalase (200 U/ml), there was a significant inhibition of apoptosis compared to ceramide alone (32%, p < .01) or ceramide + H2O2 (24%, p < .001). Apoptosis was increased in RPE treated with the catalase inhibitor ATZ (40 AM) or ATZ + H2O2 (100 AM), compared to their respective controls ( p < .01). C2 ceramide treatment and accumulation of ROS
Fig. 1. Dose dependency of RPE cells undergoing apoptosis as determined by TUNEL assay and measured by FACS analysis. Confluent RPE in 1% serum containing DMEM were incubated for 24 h at 37jC with varying concentrations of ethanolic solutions of C2 ceramide. Dihydro-C2 ceramide (DC2) was used as a negative control. Control represents RPE cells treated with vehicle alone. Data are means F SEM from three or four determinations from three different donors per group. Asterisk indicates significant difference ( p < .01) vs. control.
trations of HGF (30 and 50 ng/ml) decreased apoptosis in ceramide-treated RPE, the protection due to HGF was maximal at 20 ng/ml. Cell viability in the control, C2 ceramide, and C2 ceramide plus HGF groups ranged from 85 to 95% and was not significantly different among the three groups. In separate experiments, co-incubation of C2-ceramide-treated cells with the caspase-3 inhibitor z-VAD-fmk (10 AM) decreased the percentage of apoptotic cells at 24 h (mean of 50 F 6.8% in C2-treated cells to a mean of 20 F 4.5% in caspase-3 inhibitor-treated cells, p < .01). Similar results were found for Ac-DEVD-CHO (20 AM). Experiments also were performed to determine whether apoptosis induced by exogenous ceramide was mediated through its action on the endogenous ceramide generation pathway. Pretreatment of RPE for 2 h with fumonisin B1, a ceramide synthase inhibitor, did not significantly alter the extent of apoptosis from C2 ceramide, either in the presence or in the absence of HGF ( p = .1). Similarly, the acidic sphingomyelinase inhibitor desipramine did not affect C2-ceramide-induced apoptosis either in the presence or in the absence of HGF ( p = .2).
Figure 4 shows the ceramide-induced accumulation of intracellular ROS in RPE, their predominant localization to mitochondria, and the prevention of ROS accumulation with pretreatment of cells with HGF. Preliminary studies revealed the dose dependency of ROS accumulation after 24 h of C2 ceramide (data not shown). Untreated control cells (Figs. 4A –4C) showed only minimal accumulation of ROS. Treatment with 40 AM C2 ceramide resulted in pronounced intracellular accumulation of ROS that was predominantly localized to mitochondria as demonstrated by colocalization with a mitochondrial label (Figs. 4D – 4F). Preincubation of RPE with 20 ng/ml HGF markedly decreased the ceramide-induced ROS accumulation (Figs. 4G –4I). Effects of ceramide and HGF on mitochondrial MPT Figure 5 shows the accumulation of the fluorescent mitochondrial lipophilic dye TMRM in RPE and its loss after induction of MPT by treatment with C2 ceramide (40 AM, p < .01). Pretreatment of RPE with HGF (20 ng/ ml) significantly inhibited this effect ( p < .01). Effect of ceramide on caspase-3 activation Figure 6 shows that in control cultures, caspase-3 activity is infrequent (9.8%); however, the number of
Effects of H2O2 on ceramide-induced apoptosis As shown in Fig. 3, incubation of RPE with low concentrations of H2O2 (100 AM, 12 h) resulted in minimal apoptosis (2%); however, RPE pretreated with C2 ceramide (40 AM, 8 h) + H2O2 (100 AM, 12 h) showed increased apoptosis (62%) compared to C2 ceramide pretreatment alone (46%, p < .01). When cells
Fig. 2. Attenuation of C2 ceramide-induced apoptosis in RPE cells by HGF. Cells were pretreated with HGF (20 ng/ml) for 24 h followed by exposure to either 30 or 40 AM C2 ceramide for an additional 24 h. Apoptosis was monitored by TUNEL assay and flow cytometry as in Fig. 1. Data are means F SEM (n = 3). Asterisk indicates statistical difference ( p < .01) compared to the corresponding C2-treated group.
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Fig. 3. Potentiation of ceramide-induced apoptosis by H2O2 and effects of purified catalase and ATZ. Confluent RPE cells were treated with 40 AM C2 ceramide in DMEM containing 1% serum and incubated with 100 AM H2O2 for an additional 12 h. Effects on apoptosis of treatments with ATZ (30 AM), catalase (200 U/ml), and their combination with C2 or H2O2 are also shown. *p < .01 vs. control; **p < .01 vs. C2-treated group; ***p < .01 vs. C2 + H2O2 group.
cells expressing active caspase-3 after C2 ceramide treatment (40 AM) is markedly increased (89.6%, p < .001). Pretreatment with HGF significantly decreased
ceramide-induced caspase-3 activation (48.5%, p < .01 vs. ceramide-treated RPE). Western blots confirmed the ceramide-induced conversion of procaspase-3 to
Fig. 4. (D – F) Accumulation of ROS in mitochondria of RPE cells treated with C2 ceramide and (G – I) effects of HGF. (E) Confocal microscopy demonstrates the intracellular accumulation of ROS after 24 h treatment with 40 AM C2 ceramide (green fluorescence). (A, D, G) ROS accumulated predominantly in labeled mitochondria (red fluorescence) after ceramide treatment as shown by (F) colocalization of merged fluorescence images (red + green labels seen as yellow fluorescence). (H) HGF pretreatment results in decreased accumulation of ROS after ceramide treatment. Bar, 10 Am.
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Fig. 5. Effects of ceramide and HGF on mitochondrial membrane permeability transition (MPT). RPE cells were treated with 40 AM C2 ceramide, with or without overnight preincubation with 20 ng/ml HGF. Cells were loaded with the mitochondrial membrane dye TMRM, and MPT results in loss of fluorescence. Flow cytometry histograms show (A) cells without fluorescent dye (negative control), (B) control (non-ceramide-treated) cells with fluorescent dye, (C) C2-treated cells with fluorescent dye, and (D) HGF pretreated, C2treated cells with fluorescent dye.
active caspase-3 and the attenuation of this effect by HGF. Effect of C2 ceramide and HGF on catalase activity and expression Table 1A shows the enzyme activity of catalase that had been immunoprecipitated from RPE cell lysates. Incubation of RPE with 40 AM C2 ceramide caused a
33 and 77% decrease in catalase enzyme activity at 12 and 24 h compared with the corresponding untreated controls ( p < .05). HGF treatment did not significantly alter basal catalase enzyme activity of control RPE cells. When ceramide-treated RPE were pretreated with HGF, the decrease in catalase activity was significantly attenuated ( p < .01 for 12 and 24 h). Consistent with these findings, Fig. 7 shows that the amount of catalase protein
Fig. 6. Ceramide treatment increases caspase-3 activation. RPE cells were treated with 40 AM C2 ceramide for 24 h and active caspase-3 was detected by flow cytometry after incubation with a cell-permeable FITC-labeled caspase inhibitor that binds irreversibly to active caspase-3. Western blot analysis (bottom) was performed using a caspase-3-specific polyclonal antibody. Untreated controls show only procaspase-3 (32 kDa), whereas after ceramide treatment a band representing active caspase-3 (16 – 18 kDa) is also seen.
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(A) Control C2 C2 + HGF (B) Control C2 C2 + z-VAD-fmk
32.5 29.3 26.5 38.9 36.3 35.1
F F F F F F
12 h 1.2 0.8 1.1 3.2 0.8 2.4
29.8 20.1 27.3 37.5 24.1 31.2
F F F F F F
3.9 5.8* 2.3** 3.9 5.8 4.5***
24 h 33.1 7.6 20.1 36.1 10.6 23.4
F F F F F F
5.6 2.3* 4.1** 5.6 6.3 3.6***
In (A), the effects of pretreatment with HGF (20 ng/ml, 24 h) on catalase activity were determined in ceramide-treated RPE (12 and 24 h). In (B), this experiment was repeated in the presence of the caspase inhibitor z-VAD-fmk (10 AM); caspase activity was reduced by 72.1% in z-VAD-fmk-treated cells compared to those treated for 24 h with ceramide alone. Catalase was immunoprecipitated and enzyme activity was measured as described under Experimental Procedures. * p < .05 control vs. C2. ** p< .01 C2 vs. C2+HGF. *** p < .01 C2 vs C2 + z-VAD-fmk.
detected by Western blot decreased progressively from 12 to 24 h after ceramide treatment. When HGF (20 ng/ ml)-pretreated cells were exposed to C2 ceramide, the expression of catalase protein remained at levels similar to those of untreated controls. In order to determine whether caspase-3 activation was required to elicit the ceramide-induced decrease in catalase activity, cells were incubated with ceramide in the presence of the caspase inhibitor z-VAD-fmk (10 AM). At 12 h, catalase levels in the ceramide + z-VADfmk-treated cells remained equal to those of untreated controls (Table 1B). At 24 h, catalase levels were now reduced from untreated control levels but they were significantly higher than those found in the ceramidetreated controls ( p < .01; Table 1B). Caspase-3 activation was reduced by 72.1% when cells were co-incubated for 24 h with z-VAD-fmk, compared to treatment with ceramide alone.
be both stimulus- and cell-type-specific. Studies examining the effects of exogenous ceramides of varying acyl chain lengths, from the synthetic short-chain C2 analog to the physiologically more relevant C16 ceramide (C2-C16), in breast cancer and mouse macrophage cell lines suggest that chain length is inversely proportional to cytotoxic activity, with C6 being the most active [45]. Other studies have used short-chain, easily cell-permeable C2 and C6 ceramide analogs as inducers of apoptosis in several cell types and have concluded that C2 ceramide caused more apoptosis than the C6 analog [22,24]. Our findings in low-passage human RPE are consistent with the latter view and may be explained by the more efficient solubility of C2 ceramide at room temperature in the vehicle used. A dosedependent increase in percentage of apoptotic cells was found with 24 h C2 treatment up to 50 AM. At concentrations above 50 AM, there was a 50 – 60% loss of cell viability. It was recently proposed that ceramide is both a signaling product of oxidative stress and a mediator of the production of reactive oxidants in the mitochondria [17,21,46]. This implies a bidirectional relationship between oxidative stress and ceramide production in mitochondria. ROS and activated caspase-3 have both been implicated as downstream mediators of ceramide-induced apoptosis [15,17,21,47 –49]. In our study, ROS accumulated preferentially in mitochondria, consistent with the importance of the mitochondrial respiratory chain as a site of ROS production [50,51]. Future studies should determine the exact step of the mitochondrial respiratory chain that is altered by ceramide. The subsequent ceramide-mediated induction of MPT in RPE further points to the mitochondria as a critical target of ceramide action. Oxidative damage has been shown to increase ceramide content through activation of caspase-3 [49]. In the present study, ceramide treatment increased levels of active caspase-3, whereas the addition of
DISCUSSION
We demonstrate that C2 ceramide induces apoptosis in RPE in a dose-response manner, with mitochondrial accumulation of ROS and caspase-3 activation, whereas pretreatment with HGF significantly inhibits these effects. Furthermore, the apoptosis-inducing effect of ceramide treatment is potentiated by co-incubation with low doses of H2O2, suggesting the importance of antioxidant scavengers in this process. Consistent with this hypothesis, we showed that ceramide treatment decreases catalase activity and protein and that this decrease is mediated through activation of caspase-3. The mechanism by which ceramide elicits apoptosis has not been fully elucidated, and the pathways seem to
Fig. 7. Effects of ceramide on catalase protein expression. RPE cells were treated with C2 for 12 and 24 h in the presence or absence of HGF. The protein levels of catalase and h-actin were examined by Western blot of RPE cell lysates. A representative blot from three independent experiments is shown.
Ceramide-induced apoptosis
caspase-3-specific inhibitors blocked ceramide-induced apoptosis. These findings are consistent with the hypothesis that ceramide-induced apoptosis in RPE is mediated, at least in part, through caspase-3 [47]. Recently, ROS generation has also been shown to lead to apoptosis by caspase-independent pathways [52]. Zhang et al. [37] showed that menadione-induced cell death differed between U937 cells and ARPE-19 cells in that the former exhibited caspase-3 activation, whereas in the latter, apoptosis-inducing factor and not caspase-3 was involved in cell death. It would be of interest to further evaluate the contribution of a caspase-independent mechanism(s) of ceramide-induced apoptosis in RPE. Our study revealed that ceramide-induced apoptosis was enhanced by treatment with low concentrations of H2O2. The scavenging system for H2O2 includes catalase, glutathione, and glutathione peroxidase [53]. Our observation of increased oxidative damage and increased apoptosis induced by H2O2 along with the catalase inhibitor ATZ suggests a critical role for catalase in this process. The enhancement of apoptosis by H2O2 in ceramide-treated cells was accompanied by a decrease in catalase enzyme activity and protein expression. Accordingly, preincubation with exogenous catalase decreased apoptosis in RPE treated with ceramide and/or H2O2. Previous work has shown that the protection of mononuclear cells from ceramide-induced apoptosis by exogenous catalase is likely the result of scavenging of cytosolic peroxides that diffuse out of the cells [54]. Internalization of exogenous catalase can be excluded as a mechanism because cells exposed to exogenous catalase do not show an increase in intracellular catalase content [55,56]. Our data support the involvement of active caspase-3 in the regulation of catalase activity. Inhibition of caspase-3 activity not only attenuated the development of apoptosis, but also significantly protected cells from the ceramide-induced loss of catalase activity and protein. Recent work in ceramide-treated HL-60 cells suggests that active caspase-3 proteolytically cleaves catalase, thus reducing its level of expression and activity [44]. Our demonstration that HGF treatment attenuates both the ceramide-induced reduction in catalase and the activation of caspase-3 supports the contention that HGF-mediated protection against catalase loss is a result of decreased caspase-3 activation; however, additional direct effects on catalase synthesis cannot be ruled out. It is well established that glutathione (GSH) is an effective antioxidant defense against accumulation of ROS and downstream events leading to apoptosis. We found that treatment with either C2 or C6 ceramide at 40 AM did not cause significant changes in the
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intracellular GSH pool in RPE cells compared to untreated controls (Kannan et al., unpublished observations). This finding is consistent with a recent, similar observation in Molt-4 leukemia cells [57]. However, exogenous GSH supplementation is known to decrease mitochondrial ROS levels and attenuate apoptosis in several cell types. Accordingly, we also observed that incubation of ceramide-treated RPE with GSH monoethyl ester reduced ROS levels, inhibited activation of caspase-3, and reduced the extent of apoptosis (data not shown). Our data support the hypothesis that in RPE, exogenous ceramide promotes the accumulation of mitochondrial ROS, induces MPT, activates caspase-3, and stimulates subsequent apoptosis. These effects are accentuated by the degradation of catalase by activated caspase-3, with loss of peroxide scavenger activity and further accumulation of mitochondrial ROS. The ability of HGF to protect RPE from ceramide-induced apoptosis and to inhibit ceramide-induced alterations at each step of this cascade, including the preservation of catalase activity, suggests that its major effect results from attenuation of mitochondrial ROS accumulation. Further studies will determine if there is a direct effect of HGF on multiple stages of the cascade and if other antioxidant enzymes and signaling molecules are involved [58,59]. In contrast to our work, a previous study showed that HGF induced growth suppression in the sarcoma 180 cell line and that this effect was mediated through an increase in ROS with activation of caspase-3 [36]. In the sarcoma cells, treatment with an intracellular free radical scavenger (N-acetylcysteine) blocked the growth suppression, whereas addition of exogenous catalase had no effect. The opposing effects of HGF in sarcoma cells, compared to cultured RPE, are likely a result of dysregulated growth factor signaling in the transformed cells [36]. Sarcoma 180 cells show constitutive activation of the HGF receptor c-Met, with only a modest increase in receptor phosphorylation with HGF treatment [36]. On the other hand, we have shown that cultured RPE demonstrate regulated c-Met activation with strong and rapid induction of c-Met phosphorylation after exposure to HGF [29]. The present work may have relevance in designing therapeutic approaches for the prevention of apoptotic injury to the retina and the RPE. The recognition of ceramide as an important endogenous molecule mediating proapoptotic signaling, and the link between alterations in ceramide metabolism and photoreceptor degeneration [26,27], suggests that approaches aimed at modulating the sphingolipid biosynthetic or degradative pathways should be explored as potential treatments for retinal degenerative disorders.
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Acknowledgments — This work was supported in part by Grants EY02061 and EY03040 from the National Institutes of Health, a grant from the Arnold and Mabel Beckman Foundation, a grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc. (New York, NY), and a grant from the Deutsche Akademie der Naturforscher Leopoldina (BMBF-LPD 9901/8-52), Germany. We thank Christine Spee and Ernesto Barron for their technical support in this project. REFERENCES [1] Hannun, Y. A. The sphingomyelin cycle and the second messenger function of ceramide. J. Biol. Chem. 269:3125 – 3128; 1994. [2] Perry, D. K. Ceramide and apoptosis. Biochem. Soc. Trans. 27:399 – 404; 1999. [3] Obeid, L. M.; Linardic, C. M.; Karolak, L. A.; Hannun, Y. A. Programmed cell death induced by ceramide. Science 259: 1769 – 1771; 1993. [4] Jayadev, S. B.; Liu, A. E.; Lee, J. Y.; Nazaire, F.; Pushkareva, M. Y.; Obeid, L. M.; Hannun, Y. A. Role of ceramide in cell cycle arrest. J. Biol. Chem. 270:2047 – 2052; 1995. [5] Spiegel, S.; Merrill, A. H., Jr. Sphingolipid metabolism and growth retardation. FASEB J. 10:1388 – 1397; 1996. [6] Schutze, S.; Potthoff, K.; Machleidt, T.; Berkovic, D.; Wiegmann, K.; Kronke, M. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced ‘‘acidic’’ sphingomyelin breakdown. Cell 71:756 – 776; 1992. [7] Wiegmann, K.; Schutze, S.; Machleidt, T.; Witte, D.; Kronke, M. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78:1005 – 1015; 1994. [8] Ruvolo, P. P. Intracellular signal transduction pathways activated by ceramide and its metabolites. Pharm. Res. 47:383 – 392; 2003. [9] Kajimoto, T.; Shirai, Y.; Sakai, N.; Yamamoto, T.; Matzuzaki, H.; Kikkawa, U.; Saito, N. Ceramide-induced apoptosis by translocation, phosphorylation and activation of protein kinase Cdelta at Golgi complex. J. Biol. Chem. 279:12668 – 12676; 2004. [10] Mansat-de Mas, V.; Bezombes, C.; Quillet-Mary, A.; Bettaieb, A.; D’Orgeix, A. D.; Laurent, G.; Jeffrezou, J. P. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol. 56:867 – 874; 1999. [11] Cabrero, A.; Algret, M.; Sanchez, R. M.; Adzet, T.; Laguna, J.C.; Carrera, M. V. Increased reactive oxygen species production down-regulates peroxisome proliferator-activated alpha pathway in C2C12 skeletal muscle cells. J. Biol. Chem. 277: 10100 – 10107; 2002. [12] Pedersen, J. Z.; Marcocci, L.; Rossi, L.; Mavelli, I.; Rotilio, G. Generation of daunomycin radicals on the outer side of the erythrocyte membrane. Biochem. Biophys. Res. Commun. 168: 240 – 247; 1990. [13] Wang, J. H.; Redmond, H. P.; Watson, R. W.; Bouchier Hayes, D. Induction of human endothelial cell apoptosis requires both heat shock and oxidative stress responses. Am. J. Physiol. 272: C1543 – C1551; 1997. [14] Kondo, T.; Matsuda, T.; Tashima, M.; Umehara, H.; Domae, N.; Yokoyama, K.; Uchiyama, T.; Okazaki, T. Suppression of heat shock protein-70 by ceramide in heat shock induced HL-60 cell apoptosis. J. Biol. Chem. 275:8872 – 8879; 2000. [15] Kondo, T.; Matsuda, T.; Kitano, T.; Takahashi, A.; Tashima, M.; Ishikura, H.; Umehara, H.; Domae, N.; Uchiyama, T.; Okazaki, T. Role of c-jun expression increased by heat-shock- and ceramideactivated caspase-3 in HL-60 cell apoptosis. Possible involvement of ceramide in heat shock-induced apoptosis. J. Biol. Chem. 275:7668 – 7676; 2000. [16] Lavrentiadou, S. N.; Chan, C.; Kawcak, T.; Ravid, T.; Tsaba, A.; van der Vliet, A.; Rasooly, R.; Goldkorn, T. Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione. Am. J. Respir. Cell. Mol. Biol. 25:676 – 684; 2001. [17] Garcia-Ruiz, C.; Colell, A.; Mari, M.; Morales, A.; FernandezCheca, J. C. Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen
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