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Keywords:

  • apoptosis;
  • ceramide;
  • oxidative stress;
  • photoreceptor;
  • retina;
  • sphingomyelinase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Nitric oxide and reactive oxygen species play a critical role in photoreceptor apoptosis. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. Here, we demonstrate that the sphingolipid ceramide is the key mediator of oxidative stress-induced apoptosis in 661W retinal photoreceptor cells. Treatment of 661W cells with the nitric oxide donor, sodium nitroprusside, activates acid sphingomyelinase. As a result, sphingomyelin is hydrolysed, which leads to an increase in the concentration of ceramide. We also show that ceramide is responsible for the activation of the mitochondrial apoptotic pathway in 661W photoreceptor cells and subsequent activation of the caspase cascade. Furthermore, we show for the first time that ceramide is responsible for the increased Ca2+ levels in the mitochondria and cytosol that precedes activation of the calpain-mediated apoptotic pathway. Additionally, we provide evidence that ceramide also activates the endolysosomal protease cathepsin D pathway. In summary, our findings show that ceramide controls the cell death decisions in photoreceptor cells and highlight the relevance of acid sphingomyelinase as a potential therapeutic target for the treatment of retinal pathologies.

Abbreviations used
Ac-DEVD-pNA

acetyl-Asp-Glu-Val-Asp-p-nitroanilide

Ac-LEHD-pNA

acetyl-Leu-Glu-His-Asp-p-NitroAniline

BSA

bovine serum albumin

BSTFA

(bis-(trimethylsilyl)trifluoroacetamide

C6-NBD-SM

N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl}-d-erythro-sphingosylphosphorylcholine sphingomyelin

ΔΨm

mitochondrial membrane potential

DMSO

dimethyl sulfoxide

DHE

hydroethidine

GC-MS

gas chromatography-mass spectrometry

JC-1

5,5′,6,6′-tetrachloro-1,1′,3,3′-tetrabenzimidazolecarbocyanine iodide

3-OMS

(3-O-methylsphingomyelin)

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PI

propidium iodide

ROS

reactive oxygen species

RP

retinitis pigmentosa

SDS

sodium dodecyl sulfate

SMase

sphingomyelinase

SNP

sodium nitroprusside

TBS/T

Tris-buffered saline/Tween-20

Photoreceptors in the vertebrate retina are the cells responsible for vision. Photoreceptors capture light and convert it into an electrical signal. Numerous retinal pathologies, such as retinitis pigmentosa (RP) and glaucoma, are characterized by degeneration of photoreceptors, resulting in loss of vision. Despite the diversity of retinal degeneration disorders, apoptosis of photoreceptors seems to be a feature common to all (Chang et al. 1993; Dunaief et al. 2002; Carella 2003). Apoptosis is a form of cell suicide in which the cell directly participates in its own demise (Ferri and Kroemer 2001; Kaufmann and Hengartner 2001). The signalling pathways of apoptosis in photoreceptor cell death are still not fully understood. For example, it has largely been accepted that a series of caspase enzymes plays a key role in both the initiation and execution pathways of apoptosis (Thornberry and Lazebnik 1998; Nicholson 1999). However, the involvement of caspases does not seem to be so clear-cut in retinal cell death (Carmody and Cotter 2000; Donovan and Cotter 2002; Doonan et al. 2003). Therefore, the mechanisms operating in photoreceptor death may involve calpains rather than caspases as the executing enzymes. On the other hand, several studies have demonstrated that the eye is particularly sensitive to oxidative damage due to its high oxygen consumption and its constant exposure to light (Liang and Godley 2003). For this reason, modifications of the cellular redox state of the eye are believed to contribute to the pathogenesis of many diseases (Neufeld et al. 1999; Gao and Talalay 2004; Osborne et al. 2004). In this context, recent work from our laboratory has demonstrated that nitric oxide and reactive oxygen species (ROS) are key signalling molecules in driving apoptosis both in in vitro and in vivo models of retinal disease (Donovan et al. 2001; Sanvicens et al. 2004). Nevertheless, further work is necessary to understand fully the exact mechanisms of action of these radicals in retinal apoptosis.

Ceramide is an endogenous sphingolipid that has been related to apoptosis (Obeid et al. 1993; Jarvis et al. 1994). Emerging evidence indicates that oxidative stress and ceramide are intimately connected (Andrieu-Abadie et al. 2001). For example, ceramide can stimulate ROS production (Garcia-Ruiz et al. 1997; Quillet-Mary et al. 1997), and a connection between nitric oxide and ceramide to mediate cell death has been established (Huwiler et al. 1999; Takeda et al. 1999). Very little is known about the role ceramide plays in retinal degeneration, and the mechanisms by which ceramide mediates apoptosis in the eye remain undefined. Only recently, it was demonstrated for the first time that the sphingolipid pathway is involved in photoreceptor apoptosis in Drosophila (Acharya et al. 2003), and that there is a direct link between sphingolipid-mediated apoptosis and retinal degeneration in RP (Tuson et al. 2004). Studies aimed to elucidate apoptotic pathways in the eye have often been complex because the retina is a non-homogeneous tissue composed of several different cell types. The production and characterization of the photoreceptor cell line, 661W, has greatly facilitated the work and has proven useful for in vitro studies investigating photoreceptor apoptosis (Al-Ubaidi et al. 1992; Krishnamoorthy et al. 1999; Sanvicens et al. 2004; Tan et al. 2004; Gomez-Vicente et al. 2005; Tanito et al. 2005). In the present study, we demonstrate that acid sphingomyelinase (SMase)-derived ceramide is the key mediator of oxidative stress-induced apoptosis in retina-derived 661W cells, and show how ceramide orchestrates the activation of multiple apoptotic pathways. Therefore, our results highlight acid SMase as a potential therapeutic target in eye pathologies associated with oxidative stress.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Drugs, reagents and antibodies

Sodium nitroprusside (SNP), pepstatin A and desipramine were obtained from Sigma Chemical Co (Dublin, Ireland). Imipramine was acquired from Calbiochem (Nottingham, UK) and 3-O-methylsphingomyelin (3-OMS) from Biomol (Exeter, UK). N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl}-d-erythro-sphingosylphosphorylcholine (C6-NBD-SM) was obtained from Matreya (Pleasant Gap, PA, USA). Cell Signalling Technology (Beverly, MA, USA) provided caspase 3 (#9662), caspase 9 (#9504) and cytochrome c (#4272) antibodies. Calpain-2 (#208755) antibody was from Calbiochem and Bid antibody from R & D Systems (Oxon, UK). Cathepsin D antibody (#sc-6486) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and calpastatin and anti-α-tubulin from Sigma. Secondary antibodies antirabbit, goat or mouse, peroxidase-conjugated were from Dako (Glostrup, Denmark). Alexis Biochemicals (Läufefingen, Switzerland) provided the caspase 3 and 9 substrates, Ac-DEVD-pNA (Acetyl-Asp-Glu-Val-Asp-p-NitroAniline) and Ac-LEHD-pNA (acetyl-Leu-Glu-His-Asp-p-NitroAniline), respectively.

Cell culture

The 661W photoreceptor cell line was generously provided by Dr Muayyad Al-Ubaidi (Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA). As described elsewhere (Krishnamoorthy et al. 1999), these cells were routinely grown in Dubelcco's modified Eagle's medium, supplemented with 10% heat-inactivated fetal calf serum (both from Sigma) and 1% penicillin/streptomycin, at 37°C in a humidified atmosphere with 5% CO2. To induce apoptosis, 75 000 cells were seeded in tissue culture six-well plates (NalgeNUNC International, Hereford, UK) and allowed to attach for 24 h. Subsequently, cells were treated with 0.3 mm SNP for 24 h, unless otherwise indicated. Pre-treatment of 661W photoreceptors with pepstatin A, imipramine or desipramine was carried out for 1 h at 37°C in the conditions described above before SNP exposure. Then, cells were detached with a trypsin/EDTA solution (Sigma) and, together with their supernatant fluids, washed once with ice-cold phosphate-buffered saline (PBS, pH 7.4).

Assays for acid SMase activity

Acid SMase activity was indirectly measured in 661W cells as previously described using the Amplex Red Sphingomyelinase Assay Kit (Molecular Probes, Leiden, the Netherlands) (Zhang et al. 2001) and C6-NBD-SM (Taguchi et al. 2004). Acid SMase activity was determined in cell homogenates (50 µg) in a two-step reaction system following the protocol provided with the Amplex Red Sphingomyelinase Assay kit. Fluorescence was measured at 610 nm (excitation at 560 nm) with a FlexStation II instrument (Molecular Devices, Palo Alto, CA, USA) at 37°C. For the C6-NBD-SM assay, cell homogenates were incubated for 3 h at 37°C with C6-NBD-SM (10 µm) in 50 µL 0.1 m sodium acetate buffer (pH 4.7) containing 0.1% Triton X-100. Lipids were extracted (Bligh and Dyer 1959) and fluorescence was measured at 530 nm (excitation at 465 nm).

Measurement of cellular ceramide levels

Cells were treated with 0.3 mm SNP in the presence or absence of desipramine (1 µm) for 24 h and subsequently collected as described above. Lipids were extracted according to the method of Bligh and Dyer (1959). Lipid extracts were dissolved in chloroform and applied to solid phase extraction cartridges (50 mg Extract-Clean Silica; Alltech, Lancashire, UK). Ceramides were separated from neutral lipids as previously described (Schmid et al. 1997; Zhang et al. 2001). The ceramide fraction was acid-hydrolysed (5% v/v HCl : methanol, 80°C, 4 h) and free sphingosine was extracted with chloroform in the presence of the internal standard, C2 dihydrosphingosine (0.1 µg; Sigma). Subsequently, free sphingosine was derivatized by adding bis-(trimethylsilyl)trifluoroacetamide (BSTFA; 4 µL/ml) and incubated for 4 h at room temperature (20°C). Samples were injected into the gas chromatography-mass spectrometry (GC-MS) Hewlett Packard 5890 series II gas chromatograph coupled to a Hewlett Packard (Alltech) 5989A mass spectrometer. Injections (2 µL) were splitless with solvent delay of 7 min. An HP-5 (cross-linked (5%-Phenyl)) (PH ME) siloxane; 1 µm film thickness; 60 m × 0.25 mm) column was used. The ion source temperature was set at 220°C and He was the carrier gas employed at 1 mL/min. GC conditions were as follows: temperature programme 120°C (3 min), 120–300°C and 300°C (10 min); injector and detector temperatures were 230 and 280°C, respectively. For the SCAN mode, the mass range explored was 50/550. For the SIM mode, the ions m/z 73 (trimethylsilyl peak) and m/z 90 (trimethylsilanol peak) were monitored.

Cell death measurement

Quantification of cell death was performed with propidium iodide (PI; Sigma). Following treatments, cells were collected as described above, washed once with ice-cold PBS buffer and resuspended to a final concentration of 1 × 105 cells/mL. PI was added to a final concentration of 50 µg/mL and samples were immediately analysed by flow cytometry. Fluorescence was measured in fluorescence channel 2 (FL-2) (590 nm) on a Becton-Dickinson (Franklin Lakes, Dublin, Ireland) FACScan flow cytometer.

Analysis of mitochondrial membrane depolarization

Mitochondrial membrane depolarization was analysed using the probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetrabenzimidazolecarbocyanine iodide (JC-1; Molecular Probes). Cells were washed twice with ice-cold PBS before incubation with JC-1 (5 µg/mL) in darkness for 15 min at room temperature (20°C). Fluorescence was measured in FL-2 (590 nm).

Analysis of intracellular ROS generation

Superoxide anion levels were measured as previously described (Gorman et al. 1997). Briefly, cells were loaded with 10 µm hydroethidine (DHE; Molecular Probes) for 15 min at 37°C. Superoxide anion levels were monitored in FL-2 with excitation and emission settings of 488 and 590 nm, respectively.

Measurement of mitochondrial and cytosolic free Ca2+ levels

Cytosolic and mitochondrial free Ca2+ levels were determined using the Ca2+ probes Fluo-3-AM (acetoxymethyl ester) and Rhod-2-AM, respectively (Molecular Probes). Cells were collected and subsequently washed once with PBS. Samples were split in two, resuspended in fresh buffer and washed once more. Half of the samples were incubated in darkness (room temperature (20°C) for 15 min) with Fluo-3-AM (1 µm) and the corresponding half with Rhod-2-AM (1 µm) under the same conditions. Fluorescence was measured in FL-1 (530 nm) for Fluo-3-AM and in FL-2 (590 nm) for Rhod-2-AM. CellQuest (Becton-Dickinson) software was used for flow cytometry data analysis and at least 10 000 events/sample were acquired.

Western blot analysis

Cells (8 × 105/flask) were plated in 75 cm2 flasks (Sarstedt AG & Co., Nümbrecht, Germany) and allowed to attach overnight. After exposure to drug, whole cell extracts were obtained and resolved by denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Briefly, cells were scraped and, together with the supernatant fluid, washed twice with ice-cold PBS followed by resuspension in cell lysis buffer (50 mm Tris-HCl pH 7.4, 150 mm NaCl, 1 mm Na3VO4, 1 mm NaF, 1 mm EGTA, 1% NP40, 0.25% sodium deoxycholate) containing the cocktail of protein inhibitors described above. After incubation on ice for 20 min, debris was recovered by a 10 min centrifugation (10 000 g) at 4°C and protein concentration was normalized. Proteins (20–40 µg) were diluted in 2× sample buffer [10% SDS, 100 mm dithiothreitol (DTT), glycerol, bromophenol blue, Tris-HCl] and resolved on 8–15% SDS–PAGE gels. Then, proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell, Whatman, Dassel, Germany) and blots were blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline/0.1% Tween-20 (TBS/T), for 1 h at room temperature (20°C). Membranes were incubated at 4°C overnight with the appropriate dilution of primary antibody (1 : 400 anti-cathepsin D; 1 : 1000 all others). After three 5 min washes with TBS/T, blots were incubated with the corresponding peroxidase-conjugated secondary antibody (dilution 1 : 1000) for 1 h at room temperature (20°C). Then, they were washed again three times with TBS/T, rinsed briefly with PBS and developed with the enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Buckinghamshire, UK). Detection of α-tubulin (1 : 5000) was used as control for equal loading of protein.

Immunofluorescence imaging

661W cells were cultured on glass coverslips and treated with 0.3 mm SNP for 4 h in the presence or absence of desipramine as described above. Then, Mitotracker red or Lysotracker red (Molecular Probes) was added to the medium at a final concentration of 50 nm and incubated for 10 min at 37°C. Subsequently, cells were washed three times with PBS and fixed in 3% buffered paraformaldehyde (Sigma) for 10 min at room temperature (20°C). Quenching was performed for 10 min with 50 mm NH4Cl. Cells were then permeabilized with 0.05% buffered saponin (Sigma) for 5 min at room temperature (20°C). After the cells had been washed three times with PBS and blocked for 1 h with a 1% BSA buffered solution, the corresponding primary antibody was added (cathepsin D and cytochrome c diluted 1 : 40 and 1 : 100, respectively, in 1% BSA) and incubated for 2 h at room temperature (20°C) in the dark in a humidified chamber. Following incubation, cells were washed three times in PBS and incubated in the dark with the appropriate secondary antibody (anti-goat FITC, anti-rabbit FITC; 1 : 100 in 1% BSA; Jackson ImmunoResearch, Soham, UK) and Hoechst (to counterstain nuclei; Sigma) for 1 h at room temperature (20°C). Following three washes with PBS, the coverslips were mounted in mowiol (Calbiochem). Indirect immunofluorescence was analysed under 100× magnification using a Leica DM LB2 fluorescent microscope (Laboratory Instruments and Supplies, Ashbourne, Meath, Ireland) and photographed with a Nikon DXM1200 digital camera (The Micron Optical Company, Wexford, Ireland) using the software ACT-1 (The Micron Optical Company).

Caspase activity assay

661W cells were pre-incubated with the cathepsin D inhibitor pepstatin A (100 µm) for 1 h prior to exposure to 0.3 mm SNP. Untreated and 0.3 mm SNP-treated 661W cells were used as negative and positive controls, respectively. After a 24 h incubation, cells were collected and centrifuged at 500 g for 5 min. Subsequently, pellets were resuspended in 50 µL ice-cold lysis buffer {50 mm HEPES, pH 7.4, 100 mm NaCl, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 m DTT, 100 µm EDTA and 0.1% NP-40)} and incubated in ice for 10 min. Following a 20 s sonication, cell lysates were centrifuged for 10 min (10 000 g) at 4°C. Assay buffer (as lysis buffer, minus 1% NP-40) was added to 50 µg total protein to make a final volume of 100 µL. Lysates were incubated with 0.2 mm caspase 3, 9 substrates (Ac-DEVD-pNA and Ac-LEHD-pNA, respectively) at 37°C for 20 h. Cleavage of peptide substrates was monitored by liberation of the chromogenic pNA in a SpectraMax-340 plate reader (Molecular Devices) by measuring absorption at 405 nm.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

SNP activates SMase and induces ceramide production in 661W photoreceptor cells

Ceramide has been identified as an intracellular mediator of stress signalling that leads to cell death (Andrieu-Abadie et al. 2001). In this context, a growing body of evidence indicates that ceramide and NO are related in cell death (Andrieu-Abadie et al. 2001; Mimeault 2002). For example, NO can up-regulate ceramide levels by activation of acid SMase, which, in turn, triggers the apoptotic cascade (Matsumoto et al. 2003; Pilane and LaBelle 2004). Recent work from our laboratory demonstrated that NO is a key signalling molecule in driving apoptosis, both in in vitro and in vivo models of retinal disease (Donovan et al. 2001; Sanvicens et al. 2004). To investigate whether SMase-derived ceramide is involved in photoreceptor apoptosis, acid SMase activity was quantified in retina-derived 661W cells after treatment with the NO donor, SNP, using the Amplex Red SMase Assay Kit (Zhang et al. 2001) and C6-NBD-SM (Taguchi et al. 2004). As shown in Fig. 1a(i and ii), acid SMase activity is significantly increased in SNP-treated cells. Pre-treatment of 661W cells with the acid SMase inhibitor, desipramine (Hurwitz et al. 1994; Arena et al. 2002; Hundal et al. 2003; Erdreich-Epstein et al. 2005), before SNP exposure completely restored activity levels of acid SMase to normal conditions. Next, we examined whether acid SMase activation by SNP led to ceramide over-production. Figure 1(b) illustrates the GC-MS scan chromatograms of untreated, SNP-treated and desipramine-pre-treated cells (i, ii and iii, respectively). As can be observed, oxidative stress induced by SNP increases ceramide levels in 661W cells (Fig. 1bii). On the other hand, formation of ceramide is notably reduced in those cells pre-treated with desipramine. Figure 1b(iii) illustrates a twofold decrease in the area of the ceramide peak at 14.6 min in those samples incubated with desipramine before SNP treatment; ceramide peaks at 20.9 and 23.3 min are no longer detectable. Nevertheless, the presence of ceramide in samples treated with desipramine suggested that additional pathways of ceramide production could not be disregarded in SNP-treated 661W cells. To investigate whether the de novo synthesis pathway was also involved in ceramide production, we assessed the effect of the serine palmitoyltransferase inhibitor, myriocin, on SNP-induced apoptosis in the photoreceptor cell line. Cell death measurements demonstrated that myriocin did not prevent apoptosis (data not shown), which suggests that the de novoroute does not participate in ceramide generation in SNP-treated 661W cells. Additional experiments were performed in order to determine whether neutral SMase was a source of ceramide in our system. Cell death was quantified by flow cytometry analysis. Photoreceptor cells were stained with PI, a fluorescent indicator of plasma membrane integrity. Treatment with 0.3 mm SNP for 24 h induced 53 ± 4% cell death. Figure 1(c) shows that 3-OMS, an inhibitor of neutral SMase, reduced cell death to 39%. Nevertheless, the effect of 3-OMS on apoptosis was not as significant as that of desipramine or imipramine. Remarkably, desipramine conferred very high protection against SNP-induced apoptosis (12 ± 1%; Fig. 1c), while imipramine completely prevented cell death. In summary, these results suggest that acid SMase rather than neutral SMase-derived ceramide plays the mayor role in oxidative stress-induced apoptosis in 661W cells.

image

Figure 1.  Acid SMase activation increases ceramide levels in SNP-treated 661W cells. (a) SNP induces acid SMase activity. (i) Cells were treated with SNP (0.3–0.4 mm; 24 h) in the presence or absence of desipramine (1 µm). After lysis of cells, acid SMase activity was measured in supernatant fractions using the Amplex Red Sphingomyelinase Assay Kit. (ii) Cells were exposure to SNP for 24 and 48 h in the presence or absence of desipramine. C6-NBD-SM was used to determine acid SMase activity. Values are mean ± SD of three independent experiments run in triplicate. (b) Ceramide levels are increased after SNP exposure. Cells were treated with SNP (0.3 mm; 24 h) with or without desipramine. Then, lipids were extracted and ceramide analysis was performed by GC-MS. GC-MS scan chromatograms of (i) untreated, (ii) SNP-treated and (iii) desipramine-pre-treated cells. C2 dihydrosphingosine was used as internal standard (0.1 µg; 10.6 min). The presence of the ion fragments m/z 311, 132 and 103 in peaks at 14.7, 20.9 and 23.3 min was consistent with ceramide species (Raith et al. 2000). Additionally, trimethylsilyl and trimethylsilanol peaks were detected at m/z 73 and 90, respectively. Peaks at 14.7, 20.9 and 23.3 min correspond to ceramide species. Results are representative of three independent experiments. (c) Cell death was detected by flow cytometric analysis using PI. Figures are representative of three experiments carried out in duplicate.

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Ceramide activates cathepsin D in SNP-treated 661W cells

The endolysosomal protease, cathepsin D, is a mediator of oxidative stress-induced apoptosis in different systems (Roberg et al. 1999; Kagedal et al. 2001; Mathiasen and Jaattela 2002). Cathepsin D has been identified as a direct target of acid SMase-derived ceramide. In particular, it has been demonstrated that specific binding of ceramide derived from acid SMase results in autocatalytic activation of cathepsin D from its 52 kDa inactive form to its 32 kDa mature isoform (Heinrich et al. 1999). To determine whether ceramide induces cathepsin D activation in our system, 661W cells were treated with 0.3 mm SNP over 24 and 48 h, prior to western blot analysis of cathepsin D. Figure 2(a) shows the presence of the active 32 kDa isoform of cathepsin D at 24 h. The level of this fragment was further increased at 48 h. Additionally, an increase in the pre-pro-mature and the pro-mature forms of cathepsin D (52 and 48 kDa fragments, respectively) was observed. Heinrich and co-workers suggested that this augmentation was the result of an enhanced re-synthesis of cathepsin D precursors as a consequence of the increased production of the 32 kDa mature isoform (Heinrich et al. 1999). Activation of cathepsin D was prevented by pre-treatment with pepstatin A, a specific cathepsin D inhibitor (Fig. 2b). Desipramine also completely reduced the overall amount of active cathepsin D. Cell death quantification by flow cytometry showed that cathepsin D inhibition reduced apoptosis to 26 ± 2%, further supporting the suggestion that cathepsin D participates in oxidative stress-induced apoptosis in 661W cells (Fig. 2c). Lysosomal cathepsins have been shown to translocate to the cytosol to mediate apoptosis in response to various stimuli (Turk et al. 2002). The influence of ceramide on the intracellular location of cathepsin D was investigated by indirect immunofluorescence analysis. Staining of cathepsin D in untreated 661W cells showed a punctuated pattern of distribution. In contrast, 661W cells treated with SNP displayed a diffuse distribution of cathepsin D, indicating translocation to the cytosol (Fig. 2d). In accordance with the previous result, inhibition of ceramide production in 661W cells prevented cathepsin D processing and its subsequent release to the cytosol.

image

Figure 2.  Ceramide mediates cathepsin D activation in 661W cells. (a) Cell lysates were taken 24 and 48 h after treatment with 0.3 mm SNP. Untreated cells were used as negative control. Cathepsin D activation is indicated by the presence of a 32 kDa band in the blot. (b) Pre-treatment with pepstatin A and desipramine prevents cathepsin D activation. 661W cells were treated with SNP (0.3 mm; 24 h). Pepstatin A- and desipramine-pre-treated cells were incubated for 1 h (100 and 1 µm, respectively) before SNP exposure. Detection of α-tubulin was used as loading control. A representative result of three independent experiments is shown in all immunoblots. (c) Pepstatin A reduces cell death in 661 W cells. Cell death measurements were made by flow cytometry using PI. Results are representative of three independent experiments carried out in duplicate. (d) Ceramide induces translocation of cathepsin D to the cytosol and can be prevented by desipramine. Cells were left untreated or treated with 0.3 mm SNP (with and without desipramine) for 4 h before indirect immunofluorescence staining of cathepsin D with an anti-goat FITC antibody (upper panels). (Lower panels) Lysosome and nuclei staining with Lysotracker red and Hoechst, respectively. The experiment was performed at least three times with identical results.

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Ceramide, but not cathepsin D, is the key mediator in the activation of the mitochondrial apoptotic pathway in SNP-treated 661W photoreceptors

Work performed by our group has demonstrated that photoreceptors are capable of caspase-dependent and -independent cell death, with the initial insult and ageing shaping the cellular response (Donovan and Cotter 2002; Doonan et al. 2003; Sanvicens et al. 2004; Gomez-Vicente et al. 2005). The retina of newborn animals still expresses caspases, and this explains why the caspase machinery is activated in 661W photoreceptor cells (661W is a cell line derived from the retina of 8-day-old post-natal mice) after exposure to SNP (Sanvicens et al. 2004). Cathepsin D has been described as being able to activate the caspase machinery (Roberg et al. 1999; Johansson et al. 2003). Although no direct activation of caspases by cathepsin D has been found, several studies have identified Bid, one of the pro-apoptotic proteins of the Bcl-2 family, as a cathepsin D target (Kagedal et al. 2001; Cirman et al. 2004; Heinrich et al. 2004). These studies exemplify how cleavage of Bid by cathepsin D facilitates cytochrome c release from the mitochondria which, in turn, leads to the activation of caspase 9. To evaluate the potential role of cathepsin D in the molecular mechanism of caspase activation in our system, we first investigated Bid as a cathepsin D target in SNP-treated 661W cells. Bid cleavage (full-length form 21 kDa) was observed after 24 h of treatment with 0.3 mm SNP (Fig. 3a). Generation of truncated Bid (a band of approximately 15 kDa) was completely prevented by pepstatin A, suggesting that cathepsin D is responsible for Bid cleavage.

image

Figure 3.  Inhibition of ceramide production prevents caspase activation in 661W cells. (a) Cathepsin D mediates Bid cleavage in SNP-treated cells, which can be prevented by pepstatin A. Cells were incubated with pepstatin A (100 µm; 1 h) before SNP exposure (0.3 mm; 24 h). Bid activation is demonstrated by the presence of its cleaved fragment (15 kDa). (b) In vitro caspase 3 and 9 activities were measured with pNA-conjugated substrates (Abs405) in 661W lysates of UT, SNP-, desipramine-, imipramine- and pepstatin A-treated samples. Results are expressed in arbitrary units. Error bars correspond to SD of three independent experiments run in duplicate. (c) Inhibition of ceramide production prevents caspase 9 and 3 activation. Cells were incubated with desipramine (1 µm; 1 h) before SNP exposure (0.3 mm) and left for 24 h. Pro-caspase 9 and 3 processing was verified by the presence of the 39/37 and 17 kDa active subunits, respectively. Detection of α-tubulin was used as loading control. Figures 3a and 3c are representative of at least three independent experiments.

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To investigate whether Bid cleavage by cathepsin D was required for the activation of the mitochondrial apoptotic pathway, we evaluated caspase 9 and 3 activities by cleavage of their colorimetric substrates, Ac-LEHD-pNA and Ac-DEVD-pNA, respectively, in 661W cells pre-treated with pepstatin A prior to exposure to SNP. Figure 3(b) shows that substrates were hydrolysed, therefore revealing caspase activity, in both SNP-treated 661W and in those cells pre-treated with the cathepsin D inhibitor pepstatin A. This result indicates that cathepsin D inhibition failed to prevent the activation of the caspase machinery, suggesting that Bid cleavage is not essential for the activation of caspases. On the other hand, cells pre-treated with desipramine and imipramine showed no caspase activity, suggesting that ceramide itself may have a role in the activation of the caspase cascade in SNP-treated 661W cells. To verify this result, the activation state of caspase 3 and 9 in cells treated with SNP, and in those pre-treated with desipramine before SNP exposure, was analysed by western blot. Immunoblotting analysis of caspase 9 and 3 revealed caspase 9 (45 kDa) processing into its 39/37 kDa active fragments in SNP-treated cells (Fig. 3c). Accordingly, caspase 3 underwent cleavage into its 17 kDa active form. In contrast, pre-treatment of 661W cells with desipramine completely prevented caspase 9 and 3 activation. No active bands for caspase 9 and 3 were present in those cells in which ceramide production had been inhibited. These results indicate that ceramide is responsible for the initiation of the caspase cascade in oxidative stress-induced apoptosis in 661W cells, and that Bid cleavage by cathepsin D might only play a secondary role in the mechanism.

SMase inhibition prevents mitochondrial membrane potential (ΔΨm) collapse, increased ROS production and cytochrome c release from the mitochondria in SNP-treated 661W cells

Lack of caspase activity in desipramine-treated cells suggested an interaction between ceramide and mitochondria in oxidative stress-induced apoptosis in 661W cells. Numerous studies have identified mitochondria as a ceramide target in ceramide-mediated apoptosis (Andrieu-Abadie et al. 2001; Mimeault 2002; Pettus et al. 2002). Interaction between ceramide and mitochondria results in a disruption of the mitochondrial functions, which include inhibition of the respiratory chain, induction of the decrease of the mitochondrial membrane potential and ROS overproduction (Garcia-Ruiz et al. 1997; Quillet-Mary et al. 1997). Taking into consideration all these precedents, we investigated whether ceramide was responsible for mitochondrial dysfunction in SNP-treated 661W cells. The lipophilic probe, JC-1, was used to study the effect of SNP treatment on the mitochondrial membrane potential. JC-1 forms J-aggregates when the ΔΨm is intact, the fluorescence of which can be detected at 590 nm. Therefore, a reduction in fluorescence emission (FLH-2, 590 nm) can be interpreted as a reduction in ΔΨm. Figure 4(a) illustrates how SNP induces depolarization of the mitochondrial membrane. Treatment with increasing concentrations of the NO donor produces a shift in the fluorescence emission to lower FLH-2 values, indicating a decrease in ΔΨm that can be prevented by pre-treatment of 661W cells with desipramine. Alteration of mitochondrial membrane potential can lead to ROS generation. Previous work from our group has demonstrated that ROS are key signalling molecules for driving apoptosis in both in vitro and in vivo models of retinal disease (Donovan et al. 2001; Sanvicens et al. 2004). Therefore, superoxide anion formation was monitored using the probe, hydroethidine (DHE). Superoxide anions oxidize DHE intracellularly to produce ethidium bromide, which fluoresces upon interaction with DNA at 590 nm. Figure 4(b) shows that superoxide anion levels are highly increased in SNP-treated 661W cells, and that inhibition of acid SMase with desipramine and imipramine restores levels to basal conditions. Finally, we determined whether ceramide mediates cytochrome c release from the mitochondrion in SNP-treated 661W cells. As shown in Fig. 4(c), inhibition of ceramide production prevents translocation of cytochrome c to the cytosol. Taken together, these results indicate that ceramide is responsible for the mitochondrial dysfunctions, observed in SNP-treated 661W cells, that lead to the activation of the caspase cascade.

image

Figure 4.  Ceramide mediates ΔΨm collapse, ROS production and cytochrome c release from the mitochondria in 661W cells. (a) ΔΨm was measured by flow cytometry using the fluorescent probe JC-1. In untreated cells (0 mm SNP, continuous line), JC-1 aggregates can be detected in the FL-2 channel. After 4 h of treatment with dose-dependent concentrations of SNP (0.1–0.5, dotted line in histograms on the left), reduced ΔΨ could be detected in 661W cells. In desipramine-pre-treated cells (dotted line in histograms on the right), ΔΨ levels remained practically unaltered. Results are representative of three experiments carried out in duplicate. (b) Superoxide levels were detected with DHE prior to SNP exposure (untreated cells, continuous line) and after exposure to SNP (0.3 mm; 24 h). Increased fluorescence in the FL-2 channel indicates increased levels of superoxide. Desipramine and imipramine pre-treatment (1 h; 1 and 25 µm, respectively) reduced superoxide production. (c) Ceramide is responsible for cytochrome c release from the mitochondria. Cells were left untreated or treated with 0.3 mm SNP (with and without desipramine) for 4 h before indirect immunofluorescence staining of cytochrome c with an anti-rabbit FITC antibody (upper panels). (Lower panels) Mitochondria and nuclei stain with Mitotracker red and Hoechst, respectively. The experiment was performed at least three times with identical results.

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Ceramide is responsible for Ca2+ homeostasis deregulation and subsequent activation of the calpain-mediated apoptotic pathway in SNP-treated 661W cells

We have previously shown that cytosolic Ca2+ levels are increased in SNP-treated 661W cells (Sanvicens et al. 2004). Pinton et al. (2001) reported that ceramide can cause Ca2+ release directly from the endoplasmic reticulum, which leads to an increase in cytosolic Ca2+ levels. In this manner, pathological elevations in cytosolic Ca2+ can then induce mitochondrial membrane permeabilization and subsequent release of cytochrome c to the cytosol (Green and Reed 1998; Jacotot et al. 1999). In view of these precedents, we suggested two possible mechanisms for ceramide-induced mitochondrial dysfunction in SNP-treated 661W cells. Ceramide could either be affecting mitochondrion stability through an indirect mechanism mediated by increased concentrations of cytosolic Ca2+, or ceramide could be interacting directly with the mitochondria (Gudz et al. 1997; Degli Esposti and McLennan 1998). To clarify the mechanism of action of ceramide in our system, we investigated whether mitochondrial Ca2+ deregulation preceded the increase in cytosolic Ca2+ level. To establish the sequence of events, we compared the time courses of mitochondrial and cytosolic Ca2+ for 1 h after treatment with 0.3 mm SNP. Cytosolic free Ca2+ levels were determined using the fluorescent probe, Fluo-3-AM, while mitochondrial free Ca2+ was measured with Rhod-2-AM. Figure 5(a) shows that the concentration of mitochondrial Ca2+ was significantly increased within 15 min after SNP exposure and continued to rise throughout the hour. However, no increase in cytosolic Ca2+ concentration was observed during the first 30 min of the study, and only a slight shift in the Ca2+ peak was detectable after 1 h. This observation suggested that SMase-derived ceramide itself caused the alterations in the mitochondrial functions, including the increase in the mitochondrial Ca2+ levels. In order to confirm this result, mitochondrial and cytosolic Ca2+ concentrations in those cells pre-treated with desipramine or imipramine were compared with control SNP-treated 661W cells. Figure 5(b) shows that changes in Ca2+ concentrations can be prevented by inhibiting ceramide production in 661W cells. This result supports the idea that the alteration in Ca2+ level is due to ceramide activity in SNP-treated 661W cells.

image

Figure 5.  Ceramide is responsible for the alteration of Ca2+ homeostasis and the activation of m-calpain in SNP-treated 661W cells. (a) Analysis of the concentration of mitochondrial and cytosolic Ca2+. Mitochondrial and cytosolic free Ca2+ levels were monitored using the Rhod-2-AM and Fluo-3-AM probes, respectively, during a 1 h after exposure to SNP (untreated cell, continuous line; SNP-treated cells dotted line). (b) Effect of desipramine and imipramine on increased mitochondrial and cytosolic Ca2+ levels. Figures 5a and 5b are representative of three independent experiments carried out in duplicate. (c) Ceramide mediates the activation of the calpain–apoptotic pathway in 661W cells. Immunoblotting analysis of m-calpain and its inhibitor, calpastatin, in control cells and in those pre-treated with 1 µm desipramine, was performed using equivalent quantities of total protein 24 h after SNP exposure (0.3 mm). The results show that m-calpain activation is prevented by desipramine. Results are representative of three experiments.

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Our previous work demonstrated a role for calpains in oxidative stress-induced apoptosis in 661W cells (Sanvicens et al. 2004). We showed that Ca2+ overload activated both m- and µ-calpain. These data are in agreement with other studies carried out in our laboratory with animal models, which provided additional evidence of the contribution of calpains to photoreceptor apoptosis (Donovan and Cotter 2002; Doonan et al. 2005). The observation that desipramine and imipramine pre-treatment prevented Ca2+ deregulation in SNP-treated cells led us to investigate whether ceramide inhibition could prevent Ca2+-mediated calpain activation as well. Control cells treated with 0.3 mm SNP for 24 h were compared with those incubated with desipramine by western blot analysis. Figure 5(c) shows that inhibition of ceramide production prevents m-calpain activation. Levels of the 80 kDa calpain band are completely restored in SNP-treated 661W cells incubated with desipramine. To corroborate this result, we studied the extent of the cleavage of the calpain endogenous inhibitor, calpastatin, in cells pre-treated with desipramine before SNP exposure. As shown in the blot, ceramide inhibition can partially attenuate calpastatin cleavage. Therefore, these results provide evidence that ceramide is also responsible for the activation of the calpain-mediated apoptotic pathway in 661W cells.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The present work establishes a critical role for the sphingolipid ceramide in an in vitro model of photoreceptor apoptosis. We show how acid SMase-derived ceramide orchestrates the activation of different apoptotic pathways during oxidative stress-induced apoptosis in 661W cells. Ceramide mediates the activation of cathepsin D and the mitochondrial apoptotic pathway. Furthermore, ceramide is responsible for the deregulation of Ca2+ homeostasis and the consequent activation of the calpain-mediated apoptotic pathway (Fig. 6).

image

Figure 6.  Schematic representation of the events that take place distal to treatment of 661W cells with SNP. SNP activates acid SMase, which leads to an increase in the concentration of ceramide. Ceramide promotes the maturation of cathepsin D which, in turn, cleaves Bid. Ceramide is also responsible for the activation of the mitochondrial apoptotic pathway. Thus, ceramide induces ΔΨm collapse, ROS overproduction, cytochrome c release from the mitochondria and subsequent activation of the caspase cascade. Furthermore, ceramide is responsible for the alteration of Ca2+ homeostasis that precedes activation of the calpain-mediated apoptotic pathway. Together, these events lead to the apoptotic death of 661W cells.

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It has been reported that oxidative stress promotes activation of SMase (Andrieu-Abadie et al. 2001). This study provides evidence that SNP treatment of 661W cells leads to the activation of acid SMase (Fig. 1a) which, in turn, increases ceramide concentration in the cells (Fig. 1b) and triggers the apoptotic machinery (Fig. 1c). Inhibition of acid SMase with desipramine and imipramine completely prevented cell death, which further corroborates a role for acid SMase-derived ceramide in oxidative stress-induced photoreceptor apoptosis. Nevertheless, additional pathways of ceramide production might also be involved. Notably, the neutral SMase inhibitor 3-OMS rendered a slight protection in SNP-induced apoptosis in 661 W cells (Fig. 1c).

The aspartic protease, cathepsin D, has previously been implicated in oxidative stress-induced apoptosis (Roberg and Ollinger 1998; Roberg et al. 1999; Ollinger 2000; Kagedal et al. 2001). Furthermore, it has been reported that acid SMase-derived ceramide can mediate cathepsin D activation directly (Heinrich et al. 1999). In accordance with this, the current study demonstrates that ceramide induces cathepsin D activation and translocation to the cytosol, which can be prevented by inhibition of acid SMase (Fig. 2). Inhibition of cathepsin D with pepstatin A resulted in a reduction in apoptosis, suggesting a role for cathepsin D in our model (Fig. 2b and c). Thus, we show that cathepsin D is responsible for the processing of Bid to its active form (Fig. 3a). However, Bid activation does not seem to be indispensable for the activation of caspases in SNP-treated 661W cells (Fig. 3b and c). Therefore, further studies will be required to determine the exact role that cathepsin D plays in photoreceptor apoptosis, and whether other members of the cathepsin family are released to the cytosol in response to oxidative damage.

Ceramide can modulate mitochondrial functions and in this context, two different mechanisms have been proposed regarding the interaction between ceramide and mitochondria. Ceramide can indirectly alter the expression levels of pro- and anti-apoptotic proteins of the Bcl-2 family, such as Bax and Bcl-2 (Ruvolo et al. 1999; Sawada et al. 2000; Sawai et al. 2004), and by so doing can indirectly affect the mitochondrial functions. The effect of ceramide on Bax and Bcl-2 levels in SNP-treated 661W cells was investigated, but no change was observed in the expression of the proteins in comparison with control cells (data not shown). On the other hand, recent studies have demonstrated that ceramide can also interact directly with mitochondria (Dai et al. 2004; Novgorodov et al. 2005). It has been reported that ceramide can be transferred intracellularly between different organelles by different mechanisms, such as a vesicle- and non-vesicle-mediated transport (Van Meer and Holthuis 2000; Ardail et al. 2003). Additionally, a ceramide transfer protein (CERT) has been isolated and characterized (Hanada et al. 2003). Therefore, it is reasonable to suppose that ceramide produced in the lysosomes in SNP-treated 661W cells may end up in the mitochondrial membrane, where it is required essentially for ΔΨm collapse, increased ROS production and cytochrome c release, through any of the mechanisms described above. In this manner, inhibition of ceramide production abolishes mitochondrial disruption (Fig. 4a–c) which, in turn, completely prevents the activation of the caspase cascade in oxidative stress-induced apoptosis in 661W cells (Fig. 3b).

Work performed in our laboratory indicated that the mechanisms operating in photoreceptor death may involve the Ca2+-dependent proteases, calpains, rather than caspases, as the executing enzymes (Donovan and Cotter 2002; Doonan et al. 2005; Gomez-Vicente et al. 2005). In a previous study, we showed that oxidative stress induces an increase in cytosolic Ca2+ levels during apoptosis in 661W cells that leads to the activation of calpains (Sanvicens et al. 2004). The current work provides evidence that ceramide is responsible for the alteration in Ca2+ homeostasis and the subsequent activation of the calpain apoptotic pathway. Ceramide overproduction induces deregulation of mitochondrial Ca2+ homeostasis, which precedes the increase of Ca2+ levels observed in the cytosol (Fig. 5a). The increase in Ca2+ concentration both in the mitochondria and in the cytosol is prevented by inhibition of acid SMase (Fig. 5b); this is reflected in m-calpain inactivation in desipramine and imipramine pre-treated 661W cells (Fig. 5c). To our knowledge, this is the first demonstration that the endogenous sphingolipid, ceramide, is responsible for deregulation of Ca2+ homeostasis in apoptosis and the subsequent activation of the Ca2+-dependant proteases, calpains.

Recent work from our laboratory using the rd mouse demonstrated that photoreceptor apoptosis is a complex process involving multiple pathways (Doonan et al. 2005). The present study shows that cathepsin D, calpains and caspases participate in an in vitro model of oxidative stress-induced photoreceptor apoptosis (Fig. 6). Our results demonstrate a critical role for acid SMase-derived ceramide in the activation of the death pathways, and highlight acid SMase as a potential therapeutic target in eye pathologies associated with oxidative stress. Further work will be required to investigate the role of ceramide in in vivo models of photoreceptor cell death.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by the Health Research Board of Ireland. We thank Dr Muayyad Al-Ubaidi for providing the 661W photoreceptor cell line. We also acknowledge Dr Maryanne Donovan and Dr Violeta Gómez-Vicente for useful discussions, and Ms Sarah Reilley and Ms Noreen Casey for helping with GC-MS.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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