The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action

Abstract

The mechanism of action of the anti-apoptotic oncogene Bcl-2 is still largely obscure. We have recently shown that the overexpression of Bcl-2 in HeLa cells reduces the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) by increasing the passive Ca2+ leak from the organelle. To investigate whether this Ca2+ depletion is part of the mechanism of action of Bcl-2, we mimicked the Bcl-2 effect on [Ca2+]er by different pharmacological and molecular approaches. All conditions that lowered [Ca2+]er protected HeLa cells from ceramide, a Bcl-2-sensitive apoptotic stimulus, while treatments that increased [Ca2+]er had the opposite effect. Surprisingly, ceramide itself caused the release of Ca2+ from the endoplasmic reticulum and thus [Ca2+] increased both in the cytosol and in the mitochondrial matrix, paralleled by marked alterations in mitochondria morphology. The reduction of [Ca2+]er levels, as well as the buffering of cytoplasmic [Ca2+] changes, prevented mitochondrial damage and protected cells from apoptosis. It is therefore concluded that the Bcl-2-dependent reduction of [Ca2+]er is an important component of the anti-apoptotic program controlled by this oncogene.

Fig. 1. C₂ ceramide induces apoptotic cell death. (A) HeLa cells were maintained in KRB supplemented with 1 mM Ca²⁺ (1 mM Ca²⁺/KRB), and challenged with 10 µM ceramide (+ Cer). The number of viable cells was determined by phase contrast microscopy after 16 h. The percentage of living cells is reported in the upper right corner. Ceramide induces caspase activation in HeLa cells (B). Cells were incubated with ceramide for 16 h, and caspase-3-like activity of cell lysates was measured as detailed in Materials and methods, and expressed as fluorescence arbitrary units. The caspase inhibitor Ac-DEVD-CHO (inh.) has also been used to show specificity of the caspase-3-like activity. Bcl-2 overexpression protects cells from C₂ ceramide-induced cell death (C). HeLa cells were cotransfected with Bcl-2 and with mtGFP expression plasmids. Thirty-six hours after transfection, cells were challenged with increasing ceramide concentrations (from 0 to 20 µM) and viability was assessed by microscope count of living GFP-expressing cells (see text for details). mtGFP alone does not affect cell viability. In order to eliminate possible errors due to the detachment of dead cells during the transfer of the coverslip to the chamber of the fluorescent microscope, the effect of Bcl-2 expression on cell survival was evaluated, and expressed as the percentage of fluorescent cells in the microscope field. Data are averages ± SD of triplicate determinations from experiments repeated at least five times.

Fig. 2. C₂ ceramide-induced cell death is dependent on [Ca²⁺]_e (A and B). Cells were incubated in KRB supplemented with different [Ca²⁺]_e and treated with 10 µM C₂ ceramide. (A) Representative microscopic fields. The inset in the upper left and lower right corners report the [Ca²⁺]_er and [Ca²⁺]_e of each condition, respectively. (B) Average values of cell viability obtained from analyzing >50 fields (including >500 cells) in five independent experiments. (C) tBuBHQ mimics the effect of [Ca²⁺]_e reduction on cell viability. Cells were incubated in 1 mM Ca²⁺/KRB and treated with different [tBuBHQ]. Cell viability was evaluated as in (B).

Fig. 3. C₂ ceramide induces chromatin condensation, and nuclear shrinkage in cells maintained in 1 mM Ca²⁺/KRB but not in 50 µM Ca²⁺/KRB. (A) Control; (B) 1 mM Ca²⁺/KRB + Cer; (C) 50 µM Ca²⁺/KRB + Cer. The figure shows a representative microscopic field taken 16 h after the addition of 10 µM ceramide. Cells were permeabilized with 100 µM digitonin and nuclei were stained with 1 µM propidium iodide, as specified in Materials and methods.

Fig. 4. PMCA overexpression increases while SERCA overexpression reduces cell viability. HeLa cells were transfected with expression plasmids driving either recombinant PMCA (A) or SERCA (B). Transfected cells were identified by visualizing co-expressed mtGFP as specified in Figure 1C. Increasing ceramide concentrations (from 0 to 20 µM) were added and cell death was evaluated after 16 h. Data are expressed as in Figure 1C. Experiments were repeated at least five times.

Fig. 5. HeLa cells overexpressing calreticulin are more sensitive to ceramide-induced cell death. Cells were transfected with a calreticulin-expressing plasmid as detailed in Materials and methods. Transfected cells were identified by visualizing co-expressed mtGFP as specified in Figure 1C. Ceramide concentrations were as in Figure 1. Data are expressed as in Figure 1C. Experiments were repeated at least five times.

Fig. 6. C₂ ceramide causes a time-dependent elevation in the [Ca²⁺]_c. HeLa cells were loaded with the Ca²⁺ indicator Fura-2/AM and [Ca²⁺]_c changes were measured as detailed in Materials and methods. The coverslips with the cells were maintained in 1 mM Ca²⁺/KRB (A and B) or in Ca²⁺-free 0.5 mM EGTA/KRB (C and D). Where indicated, the cells were challenged with 10 µM ceramide (+ Cer) (A and C) or 10 µM di-hydroceramide (DH-Cer) (B and D). The traces show the calibrated [Ca²⁺]_c values {Δ[Ca²⁺] are 212 ± 35 nM (A), 20 ± 15 nM (B), 40 ± 12 nM (C), 6 ± 4 nM (D)}. The experiment shown is representative of at least five similar trials. Ionomycin (Iono).

Fig. 7. C₂ ceramide induces a rise in [Ca²⁺]_m. HeLa cells were transfected with a mtAEQ expression plasmid and analyzed 36 h after transfection. Detection of aequorin luminescence and calibration into [Ca²⁺] values were carried out as described in Materials and methods. The trace shows the calibrated [Ca²⁺]_m values (Δ[Ca²⁺] is 0.48 ± 0.12 µM). Where indicated, the cells were challenged with 100 µM C₂ ceramide (a higher concentration was employed because we observed that perfusion through plastic tubing is very inefficient, and a markedly higher ceramide concentration is needed to elicit the biological effect, as verified by the monitoring of cytosolic [Ca²⁺] changes and of the apoptotic efficacy of the perfusion effluent). The experiment shown is representative of at least five similar trials.

Fig. 8. C₂ ceramide induces early morphological changes in the mitochondrial network, which are inhibited by lowering extracellular Ca²⁺, or chelating cytosolic Ca²⁺. HeLa cells were transfected with mtGFP, and treated with 10 µM ceramide for 1 h. Mitochondrial structure was evaluated by visualizing mtGFP with a high-resolution digital imaging system, as specified in Materials and methods. A larger magnification of the images is presented in the insets, to allow a better appreciation of mitochondrial structure. (A and A′) Control; (B and B′) 1 mM Ca²⁺/KRB + Cer; (C and C′) 40 µM Ca²⁺/KRB + cer; (D and D′) BAPTA + Cer. (A–D) Time 0; (A′–D′) 1 h after C₂ ceramide addition.

Fig. 8. C₂ ceramide induces early morphological changes in the mitochondrial network, which are inhibited by lowering extracellular Ca²⁺, or chelating cytosolic Ca²⁺. HeLa cells were transfected with mtGFP, and treated with 10 µM ceramide for 1 h. Mitochondrial structure was evaluated by visualizing mtGFP with a high-resolution digital imaging system, as specified in Materials and methods. A larger magnification of the images is presented in the insets, to allow a better appreciation of mitochondrial structure. (A and A′) Control; (B and B′) 1 mM Ca²⁺/KRB + Cer; (C and C′) 40 µM Ca²⁺/KRB + cer; (D and D′) BAPTA + Cer. (A–D) Time 0; (A′–D′) 1 h after C₂ ceramide addition.