Involvement of Calcium in 7 β -Hydroxycholesterol and Cholesterol-5 β,6 β -Epoxide-Induced Apoptosis

Abstract

Oxidized low-density lipoprotein (oxLDL) is believed to play a central role in the development of atherosclerosis. 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. The exact mechanism through which these oxysterols induce apoptosis remains to be fully elucidated. A perturbation of intra-cellular calcium homeostasis has been found to trigger apoptosis in many experimental systems. The aim of the present study was to determine the involvement of calcium signaling in 7β-OH and β-epoxide–induced apoptosis. To this end, the authors employed the calcium channel blockers verapamil and nifedipine and inhibitors of calpain activation, ALLM and ALLN. Verapamil protected against the decrease in viability induced by 7β-OH whereas nifedipine had a protective effect in both 7β-OH and β-epoxide–treated cells, though these compounds did not restore viability to control levels. Verapamil, nifedipine, and ALLM prevented apoptosis induced by β-epoxide. None of the compounds employed in the current study protected against 7β-OH–induced apoptosis. Our results implicate calcium signaling in the apoptotic pathway induced by β-epoxide and also highlight differences between apoptosis induced by 7β-OH and β-epoxide.

Oxidized low-density lipoprotein (oxLDL) is involved in the initiation and acceleration of atherosclerotic lesions. The induction of apoptosis in cells of the arterial wall is a primary process in the development of atheroma (Leonarduzzi, Sottero, and Poli 2002). Numerous studies have shown that oxLDL is capable of inducing apoptosis in a variety of cell lines (Kinscherf et al. 1998; Sata and Walsh 1998; Lee and Chau 2001; Vicca et al. 2003; Chen et al. 2004). The ability of oxLDL to induce apoptosis has been attributed to some of its bioactive lipid components, such as oxysterols. Oxysterols are 27-carbon compounds formed through the enzymatic or nonenzymatic oxidation of cholesterol and known to induce apoptosis in cells of the arterial wall (Lizard et al. 1999; O’Callaghan, Woods, and O’Brien 2001). Though the subject of intense research over the last couple of decades, the exact mechanism of oxysterol-induced apoptosis has yet to be fully elucidated.

Apoptosis, also know as programmed cell death, is a complex process characterized by nuclear and cytoplasmic condensation, membrane budding, formation of apoptotic bodies, and DNA fragmentation. Certain oxysterols have been shown to induce apoptosis via generation of an oxidative stress (Lizard et al. 1998; Ryan, O’Callaghan, and O’Brien 2004a), loss of mitochondrial transmembrane potential (Lizard et al. 2000), release of cytochrome c (Miguet et al. 2001), activation of caspase-9 leading to subsequent activation of caspase-3 (Agrawal et al. 2002; Biasi et al. 2004), poly(ADP-ribose), a polymerase (PARP) degradation (Miguet-Alfonsi et al. 2002), and DNA fragmentation (O’Callaghan, Woods, and O’Brien 2002). Much remains to be clarified at the earlier stages of the oxysterol-induced apoptotic pathway, prior to mitochondrial dysfunction and caspase activation.

Alterations in intracellular calcium homeostasis have been implicated in the induction of cell death in many experimental systems. Fluctuations in calcium levels resulting in a sustained increase in calcium may activatate calcium-dependent endonucleases, resulting in apoptosis, or calcium-dependent proteases involved in necrosis. Therefore disruptions in calcium homeostasis may be a trigger for both apoptosis and necrosis. Some studies have suggested that an increase in intracellular calcium may be an initial step in the apoptotic pathway induced by certain oxysterols. Ares et al. (1997) showed that an influx of extracellular calcium was crucial for the induction of apoptosis in human aortic smooth muscle cells incubated with 25-hydroxycholesterol (25-OH). Cell death induced by 25-OH was prevented when cells were co-incubated with calcium channel blockers, verapamil or nifedipine. A further study conducted by Ares et al. (2000) found that 7β-OH–induced apoptosis, in human aortic smooth muscle cells, was preceded by sustained intracellular calcium oscillations. More recently Berthier et al. (2004) showed that apoptosis induced by 7-ketocholesterol (7-keto) in THP-1 cells was associated with an increase in cytosolic-free calcium.

Individual oxysterols may induce apoptosis via distinct mechanisms. We have previously shown that, in contrast to 7β-OH, β-epoxide–induced apoptosis does not involve the generation of an oxidative stress, opening of the mitochondrial permeability transition pore or release of cytochrome c from the mitochondria (Ryan, O’Callaghan, and O’Brien 2004b, 2005). The objective of this study was to examine the importance of calcium in apoptosis induced by these two oxysterols, 7β-OH and β-epoxide. To this end, U937 cells, which are known to undergo oxysterol-induced apoptosis (O’Callaghan, Woods, and O’Brien 2001; Ryan, O’Callaghan, and O’Brien 2004b), were incubated with 7β-OH or β-epoxide in the presence or absence of the calcium channel blockers verapamil and nifedipine. Cal-pains are cytosolic cysteine proteases that are activated by a rise in intracellular calcium and believed to function in calcium signaling. We examined the ability of calpain inhibitors, ALLN (also known as calapain inhibitor I) and ALLM (also known as calpain inhibitor II), to protect against oxysterol-induced apoptosis. Etoposide, a semisynthetic derivative of podophyllotoxin and an effective antitumor agent was used as a positive control as its mode of apoptosis is well characterized and known to proceed via the mitochondrial route.

MATERIALS AND METHODS

Materials

All chemicals and cell culture reagents were obtained from the Sigma Chemical (Poole, UK) unless otherwise stated. Tissue culture plastics were supplied by Costar (Cambridge, UK). Information on the purity of the oxysterols (purity > 95%) was obtained from Sigma. The inhibitors verapamil, nifedipine, ALLM, and ALLN were obtained from Calbiochem (Nottingham, UK). 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) foetal bovine serum (FBS). 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 (O’Callaghan, Woods, and O’Brien 1999) and were cultured in the absence of antibiotics. Exponentially growing cells were used throughout.

Treatment of Cells

U937 cells were adjusted to a density of 2 × 10⁵ cells/ml in RPMI-1640, supplemented with 2.5% FBS, and pretreated for 1 h with 100 μM verapamil, 100 μM nifedipine, 50 μM ALLM, or 2 μM ALLN. Following preincubation, cells were treated with 10 μM etoposide, 30 μM 7β-OH, or 30 μM β-epoxide. Samples were incubated for a further 24 h at 37°C/5% CO₂. 7β -OH, β-epoxide, ALLM, and ALLM were dissolved in ethanol for delivery to cells and the final concentration of ethanol in the cultures did not exceed 0.3% (v/v). Nifedipine and etoposide were dissolved in DMSO. Verapamil was dissolved in distilled H₂O. Equivalent quantities of ethanol and DMSO were added to control cells.

Cell Viability

Following a 24-h incubation, 25 μl of 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), then incubated at 37°C for 2 to 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 to 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 etoposide-, 7β-OH–, and β-epoxide–treated cells was assessed by fluorescence microscopy following staining with Hoechst 33342 (O’Callaghan, Woods, and O’Brien 1999). Approximately 4 × 10⁵ cells were centrifuged at 200 × g for 10 min to form a pellet. Hoechst 33342 stain (200 μl, 5μg/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 ultraviolet (UV) light (Nikon Labophot fluorescence microscope, 400× magnification). A total of 300 cells per sample were analyzed and the percentage of fragmented and condensed nuclei was calculated. Slides were numbered and viewed in random order. Apoptotic cells were characterized by nuclear condensation of chromatin and/or nuclear fragmentation (Dubrez et al. 1996).

Statistical Analysis

All data points are the mean values (±SE) of at least three independent experiments. Where appropriate, data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test. The software employed for statistical analysis was Prism.

RESULTS

Effect of Ca^{2 +} Channel Blockers and Calpain Inhibitors on Cell Membrane Integrity

Viability was assessed in U937 cells by the FDA/EtBr method. Cell samples were treated with 30 μM 7β-OH, 30 μM β-epoxide or 10 μM etoposide in the presence or absence of 100 μM verapamil, 100 μM nifedipine, 50 μM ALLM, or 2 μM ALLN. A dose response for each compound was completed before commencing viability or apoptosis studies. IC₅₀ values for the compounds were determined and we selected a subtoxic concentration for each compound. Verapamil significantly (p < .05) protected against the decrease in viability induced by 7β-OH but had no effect in β-epoxide–treated cells (Table 1). In cells treated with 7β-OH or β-epoxide, in the presence of nifedipine, the Ca²⁺ channel blocker, significantly (p < .05) increased cell viability compared to cells treated with 7β-OH or β-epoxide alone, though pre-treatment with this compound did not restore cell viability to control levels (Table 1). Verapamil and nifedipine did not protect against etoposide-induced cell death. The calpain inhibitors, ALLM and ALLN, did not protect against the decrease in viability in cells treated with 7β-OH, β-epoxide, or etoposide (Table 1) and ALLM seemed to exacerbate the toxicity of these compounds, though this finding was not significant (p < .05).

Effect of Ca^{2 +} Channel Blockers and Calpain Inhibitors on Oxysterol-Induced Cell Death

Morphological examination of cell nuclei was accomplished by staining cells with Hoechst 33342 and examination by fluorescence microscopy. Nuclei that had undergone blebbing, fragmentation, chromatin marginalization, and condensation were identified as cells that were most likely to be apoptotic. The level of these nuclei did not exceed 4% in untreated samples of U937 cells. Though 7β-OH decreased viability in U937 cells to approximately 50%, between 30% and 40% of U937 cells were apoptotic, suggesting that a certain number of cells were also dying by necrosis. In cells pretreated with verapamil or nifedipine followed by incubation with 7β-OH, viability was increased to approximately 80% (Table 1) and the number of apoptotic nuclei was approximately 20% (Table 2). In U937 cells incubated with β-epoxide, approximately 30% of the cells were apoptotic. Pretreatment with verapamil, nifedipine, and ALLM, prior to incubation with β-epoxide, significantly (p < 0.05) decreased the number of apoptotic cells compared to cells treated with β-epoxide alone (Table 2). ALLN did not protect against oxysterol-induced apoptosis (Table 2). Etoposide is a known potent inducer of apoptosis. The signaling pathway of etoposide-induced cell death is thought to proceed via depolarization of mitochondrial membrane potential, rupture of the outer membrane followed by cytochrome c release, and subsequent caspase-9 and caspase-3 activation (Custodio et al. 2002). None of the inhibitors were significant in protecting against apoptosis induced by etoposide.

DISCUSSION

The objective of the present study was to examine the importance of calcium in apoptosis induced by 7β-OH and β-epoxide. Numerous studies have suggested that 7β-OH induces apoptosis via the mitochondrial pathway; however, the initial stages of the signal transduction pathway induced by 7β-OH have not been fully elucidated. We have previously shown that the mechanism of β-epoxide–induced apoptosis differs from 7β-OH and does not involve the generation of an oxidative stress or cytochrome c release from the mitochondria (Ryan et al. 2004b). In this study we attempted to characterize further differences in the apoptotic signaling pathway between the two oxysterols.

Certain apoptotic processes have been associated with a sustained increase in cytosolic free Ca²⁺ levels, a depletion of intracellular Ca²⁺ stores or a disruption of mitochondrial function and subsequent oxidative stress that leads to inhibition of the Ca²⁺ transport systems located in the plasma membrane, endoplasmic reticulum, or mitochondria (Salvayre et al. 2002). A sustained elevation of Ca²⁺ can activate degradative enzymes such as Ca²⁺-dependent proteases (calpain) and endonucleases responsible for DNA fragmentation (Pinton et al. 2002). Calcium is transported into mitochondria through uniporters, specific calcium channels that open once cytoplasmic Ca²⁺ concentrations rise (Ermak and Davies 2001).

In the present study we investigated the ability of the Ca²⁺ channel blockers verapamil and nifedipine to prevent apoptosis induced by 7β-OH and β-epoxide. Both of the Ca²⁺ channel blockers increased viability in cells treated with 7β-OH but did not protect against 7β-OH–induced apoptosis. Cytoplasmic calcium influx, exceeding the tolerated physiologic threshold in cell signaling events, can induce either apoptosis or necrosis depending on its final concentration (Burek et al. 2003). Verapamil and nifedipine decreased 7β-OH–induced cytotoxicity compared to cells treated with 7β-OH alone. These results suggest that calcium may play a role in necrosis induced by 7β-OH rather than apoptosis. In U937 cells, treated with β-epoxide in the presence of the Ca²⁺ channel blockers, there was a significant (p < 0.05) increase in viability and a significant (p < 0.05) decrease in the percentage of apoptotic nuclei compared to cells treated with the oxysterol alone. These findings indicate that an increase in Ca²⁺ may play a role in the apoptotic pathway induced by β-epoxide.

To further probe the involvement of calcium in oxysterol-induced apoptosis, we employed two calpain inhibitors, ALLM (calpain inhibitor II) and ALLN (calpain inhibitor I), routinely used in the study of calpain inhibition (Logie et al. 2005; Lokuta, Nuzzi, and Huttenlocher 2003; Orzechowski et al. 2003). Cal-pains are cytosolic cysteine proteases that are activated by a rise in intracellular Ca²⁺, and are believed to function in stimulating Ca²⁺ signaling on cell activation, leading the cell to differentiation, proliferation, and death. Inhibition of calpain activation has previously been shown to protect against apoptosis in certain systems (Ding, Shen, and Ong 2002). Pretreatment with ALLM did not protect against β-epoxide–induced cytotoxicity but did significantly (p < .05) decrease the number of apoptotic nuclei compared to cells treated with β-epoxide alone. This finding suggests that calpain may be involved in β-epoxide–induced apoptosis as prevention of its activation encouraged cells to die by necrosis. Calpain activation does not seem to be a factor in 7β-OH–induced cell death. Though both 7β-OH and β-epoxide are potent inducers of apoptosis in the U937 cell line, their structural differences may dictate the mechanism through which apoptosis is induced.

The role of calcium in oxysterol-induced apoptosis is relatively specific to the type of oxysterol. The present study highlights a role for calcium in the apoptotic signaling pathway induced by β-epoxide. Studies by Ares et al. (1997) and Rusinol et al. (2000) showed that apoptosis induced by 25-OH was prevented by the calcium channel blocker nifedipine. The cellular uptake of calcium does not appear to be an important step in apoptosis induced by 7β-OH. Gregorio-King and colleagues (2004) demonstrated similar results when examining the importance of calcium in apoptosis induced by 25-OH, 7β-OH, and 7-keto in HL-60 cells. Their study showed that 25-OH–induced apoptosis, in contrast to 7β-OH and 7-keto, occurred by a calcium dependent mechanism.

In conclusion, we present further evidence that the oxysterols 7β-OH and β-epoxide induce apoptosis in U937 cells by different mechanisms. Our data suggest that an increase in intracellular calcium may be an important step in β-epoxide–induced apoptosis, whereas it is unlikely that calcium has a significant role in 7β-OH–induced apoptosis.