The induction of apoptosis in cells of the arterial wall is a critical event in the development of atheroma. 7β-Hydroxycholesterol (7β-OH) and cholesterol-5β,6β-epoxide (β-epoxide) are components of oxLDL and have previously been shown to be potent inducers of apoptosis. However, the exact mechanisms through which these oxysterols induce apoptosis remains to be fully elucidated. The specific interaction of the Fas death receptor with Fas ligand (FasL) initiates a caspase cascade culminating in apoptosis. The purpose of the present study was to determine the involvement of Fas signalling in 7β-OH- and β-epoxide-induced apoptosis. To this end we employed the Fas/FasL antagonist, Kp7-6, and examined the effect of Fas inhibition on oxysterol-induced cell death in U937 cells. Fas levels were increased following 24 h exposure to 30 μM 7β-OH while treatment with 30 μM β-epoxide had no effect. Kp7-6 reduced the Fas content of 7β-OH-treated cells to control levels and partially protected against 7β-OH-induced apoptosis. This coincided with a decrease in cytochrome c release along with a reduction in caspase-3 and caspase-8 activity. Our data implicate Fas signalling in the apoptotic pathway induced by 7β-OH and also highlight differences between apoptosis induced by 7β-OH and β-epoxide.
INTRODUCTION
The oxysterols, 7β-hydroxycholesterol (7β-OH) and cholesterol-5β, 6β-epoxide (β-epoxide) are cholesterol oxidation products. These two oxysterols are formed during oxidation of low-density lipoprotein (LDL) (Dzeletovic et al. 1995; Patel et al. 1996) and are major oxysterols in atherosclerotic plaques (Hultén et al. 1996). In addition, 7β-OH and β-epoxide are present in the diet (Tai, Chen, and Chen 1999; Przygonski, Jelen, and Wascowicz 2000; Hur, Park, and Joo 2007) and may be incorporated into plasma lipoproteins (Vine et al. 1998). Both of these oxysterols have been shown to induce apoptosis (O’Callaghan, Woods, and O’Brien 2001; Lyons, Woods, and O’Brien 2003; Ryan, O’Callaghan, and O’Brien 2004; Ryan, O’Callaghan, and O’Brien 2005; Ryan, O’Callaghan, and O’Brien 2006; Lemaire-Ewing et al. 2005; Lordan, O’Callaghan, and O’Brien 2007; Vejux et al. 2007). Given that arterial cell death appears to be a primary process in the development of atheroma (Falk 2006), the induction of apoptosis by 7β-OH and β-epoxide may be a significant contributing factor in the pathogenesis of atherosclerosis.
We have previously shown that both 7β-OH and β-epoxide are potent inducers of apoptosis in the U937 human monocytic cell line. In addition, our results thus far strongly suggest that these oxysterols stimulate different signal transduction pathways. Ryan, O’Callaghan, and O’Brien (2004) found that 7β-OH but not β-epoxide had an effect on the glutathione status of U937 cells. Additionally, the authors observed that an inhibitor of cytochrome c release (Bcl-xl BH44–23) and trolox, a water-soluble synthetic analogue of β-tocopherol, prevented 7β-OH-induced apoptosis but did not protect against cell death induced by β-epoxide. Further inspection revealed that, after 16 h, only 7β-OH-induced apoptosis was associated with a loss in mitochondrial membrane potential accompanied by cytochrome c release from the mitochondria into the cytosol (Ryan, O’Callaghan, and O’Brien 2005). A study investigating the involvement of calcium signalling in 7β-OH- and β-epoxide-induced cell death, suggested that an increase in intracellular calcium may be an important step in β-epoxide-induced apoptosis (Ryan, O’Callaghan, and O’Brien 2006), while data from our most recent study indicate that apoptosis induced by β-epoxide occurs via activation of caspase-2L (Lordan, O’Callaghan, and O’Brien 2007).
The research to date has focused mainly on the mitochondrial pathway, while much remains to be clarified at the earlier stages of oxysterol-induced apoptosis, in particular, with regard to the involvement of death receptors. Death receptors belong to a family of receptors involved in proliferation, differentiation and apoptosis called the Tumour Necrosis Factor (TNF) superfamily (Curtin and Cotter 2003). A ubiquitous death receptor in vascular cells is Fas (CD95), which is found in endothelial cells, vascular smooth muscle cells, and macrophages (Panini and Sinensky 2001). The Fas death receptor conveys apoptotic signals through binding to its cognate ligand, FasL (CD95L), resulting in the recruitment of the adaptor molecule Fas-associated death domain (FADD). FADD in turn binds to pro-caspase-8 and/or -10 through death effector domain (DED) interactions to form a complex called the death-inducing signalling complex (DISC). At the DISC, pro-caspase-8 (and/or -10) activation initiates a cascade of increasing caspase activity by cleaving and activating effector caspases (e.g. caspases-3, -6, and -7) and ultimately leading to apoptotic cell death (Houston and O’Connell 2004; Mollinedo and Gajate 2006).
Despite the lack of data there is preliminary evidence to suggest that oxysterols can affect the Fas/FasL death pathway. Lee and Chau (2001) found that 7β-OH and 25-hydroxycholesterol upregulated the expression of death mediators, p53, Fas and FasL in vascular smooth muscle cells, while Rho et al. (2005) demonstrated that 7-ketocholesterol induced apoptosis in human aortic smooth muscle cells through activation of the Fas-mediated pathway. The aim of this study was to investigate the role of Fas signalling in apoptosis induced by the two oxysterols, 7β-OH and β-epoxide. To this end, U937 cells were incubated with 7β-OH and β-epoxide in the presence of the Fas/Fas L antagonist, Kp7-6. This cell line was used as, in addition to undergoing oxysterol-induced apoptosis, increased levels of Fas have been shown to promote apoptosis in U937 cells (Liu et al. 2006; Ahmed et al. 2007). Subsequent to inhibition of Fas, we examined the effect of the oxysterols on cytochrome c release, caspase-3/7 and -8 activation and potential apoptotic cell death.
MATERIALS AND METHODS
Materials
All chemicals and cell culture reagents were obtained from the Sigma Chemical Co. (Dublin, Rep. of Ireland) unless otherwise stated. Tissue culture plastics were supplied by Greiner Bio (Frickenhausen, Germany). Fas/FasL antagonist (Kp7-6) was purchased from Calbiochem (Nottingham, UK). Information on the purity of the oxysterols (purity >95%) was obtained from Sigma. The human sCD95 (Fas) ELISA kit was purchased from Cell Sciences, Inc. (Canton, Massachusetts, USA) and the human cytochrome c immunoassay was obtained from R&D Systems Europe, Ltd. (Abingdon, UK). The Caspase-Glo 3/7 and 8 assay kits were from Promega (Wisconsin, USA). Cell lines were obtained from the European Collection of Animal Cell Cultures (Salisbury, UK).
Maintenance of Cell Lines
Human monocytic U937 cells were grown in suspension in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS) supplied by Gibco (Paisley, Scotland). The cells were grown at 37°C/5% CO2 in a humidified incubator. The cells were screened for mycoplasma contamination by the Hoechst staining method (Mowles 1990) and were cultured in the absence of antibiotics. Exponentially growing cells were used throughout.
Treatment of Cells with Kp7-6 and Oxysterols
U937 cells were adjusted to a density of 2 × 105 cells/ml in RPMI 1640 medium supplemented with 2.5% FBS. Cells were intially preincubated with 1 mM of the Fas/FasL antagonist, Kp7-6 (dissolved in distilled water), for 1 h followed by treatment with 30 μM 7β-OH or β-epoxide. Samples were then incubated for 24 h at 37°C/5% CO2. Overall, the Fas/FasL antagonist was in contact with the cells for 25 h. 7 β-OH and β-epoxide were dissolved in ethanol for delivery to cells and the final concentration of ethanol in the cultures did not exceed 0.3% (v/v). Equivalent quantities of solvent were added to control cells and incubated for up to 25 h at 37°C/5% CO2.
Measurement of Fas
Fas concentration was determined using a solid phase sandwich ELISA according to the manufacturers’ instructions. Briefly, 2 × 105 cells were added to the wells of a 96-well plate and incubated for 24 h in the presence of Kp7-6, 7β-OH and β-epoxide. The test plate was centrifuged at 1000 × g for 10 min and supernatant was added to sample wells of the ELISA plate. Biotinylated anti-CD95 was added to each well and incubated for 1 h at room temperature. At the end of the incubation period the cells were washed five times. Streptavidin-HRP was added and the cells were incubated for a further 30 min. The microwells were washed five times and the enzymatic activity was determined by the addition of chromogen TMB. The reaction was stopped after 30 min by the addition of 100 μl sulphuric acid (1.8 N). The absorbance at 450 nm was read immediately and the Fas concentration of each sample was calculated from a standard curve. Results were expressed as fold increase relative to the control.
Cytotoxicity
Cytotoxicity was measured by MTT assay using the Cell Proliferation kit I (Roche, Basel, Switzerland) according to the manufacturer’s instructions. This assay requires cells that are actively able to metabolise 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to an insoluble formazan precipitate. Briefly, 1 × 105 cells were treated with Kp7-6, 7β-OH and β-epoxide in the wells of a 96-well costar plate for 24 h. At the end of the treatment, 10 μl of MTT reagent was added. After 4 h incubation at 37°C, 100 μl of the solubilisation solution was placed in each well to dissolve the tetrazolium crystals. Following overnight incubation at 37°C/5% CO2 the absorbance at 570 nm was recorded using a plate reader.
Cell Membrane Integrity
Following a 24 h incubation period, 25 μl cells were removed for assessment of cell viability. Viability was monitored using a modification of the fluorochrome-mediated viability assay as described by Strauss (1991). Briefly, cells were mixed 1:1 (v/v) with a solution of fluorescein diacetate (FDA) and ethidium bromide (EtBr), and then incubated at 37°C for 2–5 min before being layered onto a microscope slide. Under these conditions, live cells fluoresce green, whereas dead cells fluoresce red. Dying cells have a green cytoplasm and red nucleus. Samples were examined at 200× magnification on a Nikon fluorescence microscope using blue light (450–490 nm). Cells (200) were scored from each slide and cell viability was expressed as the percentage of viable (green) cells.
Morphological Analysis of Cell Nuclei
Nuclear morphology of control and treated cells was assessed by fluorescence microscopy after staining with Hoechst 33342. Approximately 2 × 105 cells were centrifuged at 200 × g for 10 min to form a pellet. Hoechst 33342 stain (200 μl, 5 μg/ml) was added and the samples incubated at 37°C/5% CO2 for 1 h. Stained samples (25 μl) were placed on a microscope slide and examined under UV light (Nikon Labophot fluorescence microscope 400× magnification). A total number of 300 cells per sample were analysed and the percentage of fragmented and condensed nuclei was calculated. Apoptotic cells were characterised by nuclear condensation of chromatin and/or nuclear fragmentation (Dubrez et al. 1996).
Assessment of Cytochrome c Release
To assess the release of cytochrome c into the cytosol, following 24 h treatment with 30 μM oxysterols in the absence and presence of Kp7-6, U937 cells were separated into mitochondrial and cytosolic subfractions using a Cytosol/Mitochondria Fractionation Kit (Calbiochem). Briefly, 2 × 105 cells were collected by centrifugation at 800 × g for 5 min. Cells were washed in ice-cold PBS and resuspended in a cytosol extraction buffer containing DTT and protease inhibitors. Following incubation on ice for 10 min, cells were homogenised in an ice-cold dounce tissue grinder. The homogenate was transferred to a fresh microcentrifuge tube and centrifuged at 700 × g for 10 min. The supernatant was centrifuged at 10,000 × g for 30 min to obtain the cytosolic fraction. The cell pellet was resuspended in a mitochondria extraction buffer containing DTT and protease inhibitors and vortexed for 10 sec. This was saved as the mitochondrial fraction. Following dilution the samples were added to a 96-well microplate coated with a monoclonal antibody against cytochrome c and incubated for 2 h at room temperature. At the end of the incubation period the cells were washed six times. Cytochrome c conjugate was added to each well and the cells were incubated for a further 2 h. The microwells were washed six times and the enzymatic activity was determined by the addition of 200 μl of a substrate solution. After 30 min incubation at room temperature, the reaction was stopped by the addition of 50 μl sulphuric acid (2 N). The absorbance at 450 nm was recorded using a plate reader. Results were expressed as a percentage of the control (100%).
Measurement of Caspases-3/7 and -8
The Caspase-Glo 3/7 and 8 assays are homogeneous, luminescent assays that measure caspase-3/7 and -8 activities. The assay provides a proluminescent caspase-3/7 or caspase-8 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimised for caspase activity, luciferase activity, and cell lysis. The addition of the Caspase-Glo reagent results in cell lysis, followed by caspase cleavage of the substrate and generation of a luminescent signal produced by the luciferase. Luminescence is proportional to the amount of caspase present.
U937 cells (20,000 cells/well) were added to the wells of a white 96-well plate and exposed to Kp7-6, 7β-OH and β-epoxide. The contents of the buffer solution were transferred into the bottle containing the substrate to form the Caspase-Glo reagent. A volume of 50 μl of Caspase-Glo reagent was added to each well and the contents were collected using a plate shaker at 350 × g for 30 sec. The plates were then incubated at room temperature for 1 h before determination of luminescence. Results were expressed as relative luminescence units (RLU).
Statistics
All data points are the mean values (± SE) of at least 3 independent experiments. Where appropriate, data were analysed by one way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison test. The software employed for statistical analysis was GraphPad Prism, Version 4.
RESULTS
Fas Concentration
The Fas content of the cells was assessed after 24 h. A significant increase (p < .001) in Fas concentration was evident following treatment with 7β-OH, while β-epoxide had no effect on Fas levels (Figure 1). In addition, pretreatment with 1 mM Kp7-6 significantly (p < .001) reduced the Fas content of the 7β-OH-treated cells to control levels.
Effect of Kp7-6 on Oxysterol-Induced Toxicity
Following 24 h incubation, the absorbance of formazan crystals liberated over time from MTT labelling reagent was used to assess cytotoxicity. The MTT assay is used as an indicator of mitochondrial function in live cells; however, loss of activity cannot be used to distinguish compromised cells from dead cells. U937 cells were treated with 30 μM 7β-OH or β-epoxide in the absence or presence of 1 mM Kp7-6. In samples treated with 7β-OH, 1 mM of the Fas/Fas L antagonist did not completely protect against the decrease in cell function (Figure 2). Moreover, β-epoxide in the presence of Kp7-6 was significantly (p < .05) toxic to the cells.
Cell Membrane Integrity and Cell Death
Viability was assessed in U937 cells by the FDA/EtBr method and the Hoechst 33342 stain was used to examine nuclear morphology. Cell samples were treated with 30 μM of the oxysterols in the absence or presence of 1 mM Fas/Fas L antagonist. Treatment of cells with 7β-OH significantly (p < .001) decreased viability relative to the control (Table 1). While the inhibitor of Fas/Fas L did significantly (p < .01) protect against the decrease in cell viability, Kp7-6 plus 7β-OH still induced a significant (p < .001) decrease in viability compared to untreated control cells. Correspondingly, there was a significant (p < .001) increase in apoptotic nuclei following 24 h exposure to Kp7-6 and 7β-OH. In the presence of β-epoxide apoptosis was significantly (p < .001) increased, and as expected, the Fas/Fas L antagonist did not protect against β-epoxide-induced cell death.
Cytochrome c Release
The intrinsic cell death pathway involves the initiation of apoptosis as a result of a disturbance of intracellular homeostasis. In this pathway cytochrome c, normally localised in the intermembrane space of the mitochondria, is released into the cytosol initiating a cascade of events which culminates in apoptotic cell death. The release of cytochrome c in U937 cells treated with the oxysterols for 24 h was measured using an ELISA. 7β-OH has previously been shown to increase the levels of cytosolic cytochrome c in U937 cells (Ryan, O’Callaghan, and O’Brien 2005). Similarly, in the present study, there was an increase in the percentage of cytochrome c in the cytosol of both the 7β-OH and β-epoxide-treated cells (Figure 3). Moreover, Fas inhibition reduced cytochrome c release compared to the oxysterol alone.
Activity of Caspase-3/7 and -8
The activation status of caspase-3/7 and caspase-8 was measured using the Caspase-Glo Assay. After 24 h there was a significant (p < .001) increase in caspase-3/7 activity in both the 7β-OH- and β-epoxide-treated cells (Figure 4). Even though pretreatment with Kp7-6 significantly (p < .001) reduced activation, caspase-3/7 activity levels in 7β-OH-treated cells remained significantly (p < .001) higher compared to untreated control cells. In cells incubated with 7β-OH there was a significant (p < .01) increase in caspase-8 activity. While caspase-8 activity was not statistically lower following pretreatment with the Fas/Fas L antagonist, significant levels of caspase-8 activation were not generated in these cells. This suggests that Kp7-6 may be inhibiting caspase-8 activity induced by 7β-OH. Exposure to β-epoxide did not significantly increase caspase-8 activation in U937 cells.
DISCUSSION
The objective of this study was to examine the significance of Fas signalling in apoptosis induced by 7β-OH and β-epoxide in U937 cells. Numerous studies have suggested that 7β-OH, in particular, induces apoptosis via the mitochondrial pathway; however the initial stages of the signal transduction pathway have not been fully elucidated. We have previously shown that the mechanism of β-epoxide-induced apoptosis differs from 7β-OH (Ryan, O’Callaghan, and O’Brien 2004
Ryan, O’Callaghan, and O’Brien 2005
Ryan, O’Callaghan, and O’Brien 2006; Lordan, O’Callaghan, and O’Brien 2007). In this study we attempted to characterise further differences in the apoptotic signalling pathway between the two oxysterols.
Coupling of the cell surface receptor Fas with its natural ligand, FasL triggers apoptosis very efficiently in various cell types (Nagata and Golstein 1995). While few studies to date have examined the ability of oxysterols to induce Fas-mediated apoptosis, the Fas-FasL interaction appears to play a role in oxLDL-induced apoptosis. Sata and Walsh (1998) showed that oxLDL sensitised endothelial cells to death signals from the Fas receptor. Other studies have found that oxLDL increases the expression of Fas and FasL, and that subsequent treatment with a neutralising anti-Fas/FasL antibody significantly inhibited apoptosis induced by oxLDL (Li, Yang, and Mehta 1998; Napoli et al. 2000; Lee and Chau 2001; Li et al. 2006). In contrast, Fas did not appear to mediate hypochlorous acid-oxLDL-induced apoptosis in U937 cells (Vicca et al. 2003).
In the present study we initially measured the concentration of Fas following treatment with the oxysterols and assessed the capability of the Fas/FasL antagonist (Kp7-6) to inhibit the receptor. Kp7-6 is an irreversible inhibitor of Fas receptor activation. It can bind to the Fas receptor and FasL, therefore preventing FasL from forming a stable complex with the Fas receptor (Hasegawa et al. 2004). We observed that only 7β-OH caused an increase in Fas concentration, while 1 mM Kp7-6 was sufficient to block this increase and restore the Fas content to control levels. To further investigate this we examined the effect of Kp7-6 on oxysterol-induced cytotoxicity. Our results showed that Kp7-6 did protect against 7β-OH-induced apoptosis, however, even in the presence of the inhibitor the levels of apoptosis in the oxysterol-treated cells remained significantly higher compared to untreated control cells. As β-epoxide had no effect on Fas concentration, the Fas/FasL antagonist did not protect against β-epoxide-induced apoptosis.
Fas/FasL-induced apoptosis follows two major pathways; an intrinsic pathway, often referred to as a caspase-dependent mitochondrial pathway (Waring and Müllbacher 1999; Yin and Ding 2003) and an extrinsic pathway that is caspase independent and involves NF-κ B and JNK (Sharma et al. 2000). It is widely accepted that apoptosis induced by 7β-OH occurs via the mitochondrial pathway, so to further explore the role of Fas in oxysterol-induced apoptosis we looked at cytochrome c release and caspase activation. In U937 cells treated with 30 μM 7β-OH, there was an increase in release of cytochrome c into the cytosol to approximately 153% of the control, while Fas inhibition reduced this to 115%. Whilst the increase in cytochrome c release in β-epoxide-treated cells was not as extensive as in 7β-OH-treated samples, Kp7-6 had an identical effect, virtually blocking cytochrome c release. In support of this, Li et al. (2006) reported that all apoptotic events induced by oxLDL were efficiently inhibited by the dominant negative mutant Fas-associated death domain protein (DN-FADD), including the release of cytochrome c, Smac and Omi from the mitochondria to the cytosol (DN-FADD inhibits the interaction of Fas with endogenous FADD and blocks Fas signalling (Chinnaiyan et al. 1996)).
As stated previously, activation of caspase-8 occurs at formation of the DISC which signals the subsequent induction of downstream targets, such as procaspase-3, -6, and -7 (Tschopp, Irmler, and Thome 1998). A recent study using 1 mM Kp7-6 to investigate Fas-mediated pathway in acrolein-induced apoptosis in Chinese hamster ovary cells found that the antagonist effectively inhibited caspase-8 activity (Tanel and Averill-Bates 2007). Similarly, our results showed that Kp7-6 reduced the activation of caspase-8 induced by 7β-OH. In line with our Hoechst staining results, Fas inhibition lowered but did not prevent caspase-3/7 activity in the 7β-OH- and β-epoxide-treated cells.
A study by Cai et al. (1997) strongly suggests that Fas regulated apoptosis is involved in the development of advanced human atherosclerotic lesions, while Janin et al. (2002) proposed that the prevention of Fas-mediated death signalling in endothelial cells may have therapeutic implications. Moreover, it has been suggested that high FasL mRNA expression in circulating leukocytes may be a marker of high-risk for endothelial dysfunction in hyperlipidemic patients (Kotani et al. 2006). By demonstrating the involvement of Fas in 7β-OH-induced apoptosis our study supports these findings, but our results also indicate that the Fas-FasL interaction is not the driving force of this signalling pathway. The effects of Fas inhibition were exhibited at the mitochondrial level, in the lowering of cytochrome c release in both the 7β-OH and β-epoxide-treated cells. However, regardless of this alteration, levels of caspase-8, and -3/7 activities remained high and apoptosis was not fully prevented.
To our knowledge this is the first study to examine the role of Fas in oxysterol-induced apoptosis using a Fas/FasL antagonist. We conclude that the death receptor Fas-mediated pathway is at least partly responsible for 7β-OH-induced apoptosis. Fas inhibition substantially reduced apoptosis in 7β-OH-treated cells but had no effect on β-epoxide-induced apoptosis, highlighting further differences in the apoptotic pathways induced by the two oxysterols. Our observations confirm that 7β-OH-induced apoptosis occurs via the mitochondrial pathway with the involvement of caspase-8. In contrast, caspase-8 is not activated in β-epoxide-induced apoptosis with cytochrome c release and caspase-3 activation occurring to a much lesser extent.
