In these experiments, we investigated the role of calcium as a second messenger in the apoptotic activation of cytosolic phospholipase A2 (cPLA2). As our model, we used a murine fibroblast cell line (C3HA) that was induced to undergo apoptosis by a combination of TNF and cycloheximide. Using fura 2 Ca2+ imaging, we found strong evidence for an intracellular calcium response after 1 h of treatment, which correlated with the onset of phosphatidylserine externalization, but preceded effector procaspase processing by several hours. The response was strongest in the perinuclear region, where mean levels rose 83% (144 ± 14 nM in untreated cells vs 264 ± 39 nM in treated), while cells displaying morphological evidence of apoptosis had the highest levels of calcium (250–1000 nM). Verapamil blocked this response, indicating an extracellular source for the calcium. Fluorescence microscopy revealed a pattern of nuclear translocation of cPLA2 during apoptosis, which was also blocked by verapamil, indicating an important role for calcium in this process. In addition, we found that verapamil prevented the release of [3H]arachidonic acid from C3HA cells induced to undergo apoptosis by the chemotherapeutic agents vinblastine, melphalan, and cis-platinum. Together, these data suggest that calcium is important for cPLA2 activation by diverse apoptotic stimuli.

Tumor necrosis factor is a 17-kDa inflammatory cytokine that mediates a broad range of inflammatory responses (1). In this study, we focus on the ability of TNF to induce apoptosis. Although most normal and transformed cells are resistant to lysis, TNF can cause the death of certain tumor-derived cell lines (2). Susceptibility to TNF can also be induced by infection with certain viruses (3, 4, 5, 6, 7), intracellular bacteria (8), or treatment with inhibitors of transcription or translation (9, 10). In resistant cells, TNF activates NF-κB that induces the expression of resistance gene products (11, 12), and sensitivity arises when this process is inhibited (11). The TNF-induced death of sensitized, normal cells has been implicated in a number of pathologies, including HIV dementia (13), type I diabetes (14), and hepatitis (15).

Phospholipase A2 (PLA2) 3 enzymes catalyze the hydrolysis of acyl groups, at the sn-2 position, from membrane glycerophospholipids. These enzymes are organized into groups within three families known as cytosolic PLA2 (cPLA2), secretory PLA2, and Ca2+-independent PLA2 (16). Of the three isozymes within the cPLA2 family, cytosolic phospholipase A2-α, referred to in this work as cPLA2, is the most well characterized. This 85-kDa enzyme is unique among other PLA2 enzymes in that it is highly selective for arachidonic acid at the sn-2 position (17, 18). The activity of cPLA2 is tightly regulated by phosphorylation (19, 20, 21, 22) and/or intracellular calcium (23, 24), and interplay between cPLA2 and secretory PLA2 enzymes has been shown, in some systems, to contribute to its activation (25, 26). The activity of cPLA2 is important during inflammation because arachidonic acid is the substrate for the production of leukotrienes and PGs (27, 28). cPLA2 has also been shown to be necessary for TNF to induce apoptosis. We have reported previously that both adenovirus (29) and metabolic inhibitors (30) sensitize human and murine cells to TNF in a cPLA2-dependent fashion. cPLA2 is also required for TNF-induced apoptosis of macrophages infected with Mycobacterium tuberculosis (31) and a variety of other tumor-derived cell lines (32, 33, 34). The role cPLA2 and arachidonic acid play in apoptosis is, however, controversial. One study suggests that arachidonic acid activates one or more sphingomyelinases that in turn induce apoptosis through ceramide production (35). More recently, arachidonic acid was found to act directly on the mitochondria to induce the permeability transition leading to cytochrome c release and cell death (36).

The regulation of cPLA2 by phosphorylation and intracellular calcium has been well documented (19, 20, 21, 22, 23, 24). Phosphorylation of cPLA2 results from the activation of agonist-induced mitogen-activated protein kinase signaling cascades, while Ca2+ binds the N-terminal C2 domain, allowing for the translocation and efficient membrane binding of cPLA2. Our lab has been interested in identifying the signals necessary for activation of cPLA2 during TNF-induced apoptosis. Our previous data have shown that phosphorylation of cPLA2 is likely to be important because sustained phosphorylation of cPLA2 leading to arachidonic acid release and cell death occurred only in sensitized cells (37). We also postulated a second signal because the phosphorylation of cPLA2 preceded arachidonic acid release. Because verapamil blocked arachidonic acid release and cell death, we proposed that calcium functioned as a second signal for cPLA2 activation during TNF-induced apoptosis.

In this study, we attempted to obtain direct evidence of a calcium response that precedes the activation of cPLA2 during TNF-induced apoptosis. As our model, we used a murine fibroblastic cell line (C3HA) that was rendered sensitive to TNF with cycloheximide (CHI). Our results show that a sustained rise in the intracellular free calcium concentration ([Ca2+]i) does indeed occur before cPLA2 activation. Calcium was also linked to the release of arachidonic acid from cells triggered to undergo apoptosis by various chemotherapeutic agents, suggesting that calcium is important to many cPLA2-dependent, apoptotic responses.

C3HA is a 3T3-like murine fibroblast cell line kindly provided by L. Gooding (Emory University, Atlanta, GA). Cells were cultured in DMEM supplemented with 10% FBS and maintained at 37°C in 8% CO2. Media and reagents, unless otherwise indicated, were purchased from Sigma-Aldrich (St. Louis, MO). CHI and verapamil were purchased from Calbiochem (La Jolla, CA). Fura 2-acetoxymethyl ester (fura 2-AM) was obtained from Molecular Probes (Eugene, OR). [3H]Arachidonic acid (5,6,8,9,11,12,14,15-[3H](N)) was purchased from PerkinElmer Life Sciences (Boston MA). Protein assay kits were purchased from Pierce (Rockford, IL).

A total of 1 × 105 cells was plated into 12-well flat-bottom tissue culture plates (Fisher Scientific, Pittsburgh, PA) and labeled overnight with 0.1 μCi/ml 3[H]arachidonic acid. The following morning, the cells were washed twice with HBSS, allowed to recover for an additional 2 h, and washed again before treatment. Cells were treated with TNF (20 ng/ml) and/or CHI (25 μg/ml), vinblastine (1 μM), melphalan (200 μg/ml), or cis-platinum (30 μM) with or without the presence of verapamil (10 μM) in a total of 600 μl of medium. At indicated time points after treatment, 300-μl aliquots of medium were removed from the wells and centrifuged to remove debris. A total of 200 μl of the supernatant was removed for scintillation counting (Beckman model LS 5801; Beckman Coulter, Fullerton, CA), and total [3H]arachidonic acid release was calculated by multiplying by a factor of 3. Each point was performed in triplicate, and maximum radiolabel incorporation was determined by lysing untreated controls with 0.01% SDS and counting the total volume.

Cells were plated on chambered coverglass slides (Nalge Nunc International, Naperville, IL) and incubated overnight. For 1-h treatments, the C3HA cultures were washed with HBSS and loaded with 4 μM of fura 2-AM in serum-free DMEM for 1 h at 37°C in the dark. The dye was then removed, and the cells were washed two more times before stimulation with the appropriate reagents. In experiments in which verapamil was used, 10 μM of verapamil was added during dye loading as well as throughout the duration of the experiment. The slide was then mounted on a Zeiss Axiovert microscope (Zeiss, Oberkochen, Germany) and cells were viewed through a Fluar ×40/1.3 numerical aperture, oil immersion objective lens (Zeiss). For 3-h treatments, cells were washed and then treated with TNF and CHI for 3 h. Cells were loaded with fura 2-AM during the last hour of treatment (38).

Dual-excitation wavelengths of 340 and 380 nm stimulated indicator-loaded cells, and emitted light was monitored at 510 nm. Fluorescence and differential interference contrast (DIC) images were captured by a charge-coupled device (CCD) camera (Princeton Instruments, Trenton, NJ) and processed using Metafluor imaging software (Universal Imaging, West Chester, PA). After background fluorescence was subtracted, user defined regions generated within Metamorph imaging software (Universal Imaging) were drawn to enclose an entire cell or in small, 2-μm2 areas within the indicated cellular compartment. Nuclear measurements were made at the center of the nucleus, perinuclear measurements within 2–3 μm of the nuclear membrane, and cytosolic measurements halfway between the nuclear and plasma membranes. The ratio of the 340–380 nm average pixel intensities was converted to [Ca2+]i using a calibration curve (Molecular Probes), as described by Grynkiewicz (39), after adjustments for the in situ vs in vitro fura 2 dissociation constant were made (40).

Cells were plated in 8-well glass chamberslides (Nalge Nunc International), incubated overnight, and then treated with the appropriate reagents. To identify the subcellular localization of cPLA2, cells were washed twice with PBS and fixed with 10% formaldehyde in PBS for 30 min at 37°C. Cells were probed with a 1° monoclonal mouse IgG2b anti-cPLA2-α Ab (2 μg/ml) (sc-454; Santa Cruz Biotechnology, Santa Cruz, CA) diluted in PBS containing 20% normal goat serum and saponin (100 μg/ml) for 30 min at 37°C, followed identically by a 2° rhodamine-conjugated goat anti-mouse IgG Ab (1/100 dilution) (Sigma-Aldrich). Cells were washed twice and mounted in 10% glycerol. Microscopy was conducted on a Zeiss Axioscop, and images were captured and processed by a Spot CCD camera and software (Diagnostic Instruments, Sterling Heights, MI). The nuclear pore complex Ab was obtained from Abcam (Cambridge, U.K.).

PS exposure was analyzed using a modification of a protocol, designed for adherent cell cultures, as described previously (41). Briefly, cells were plated in 8-well glass chamberslides (Nalge Nunc International), incubated overnight, and then treated with TNF (20 ng/ml) and CHI (10 μg/ml). Following treatment for the indicated times, cells were washed twice with binding buffer (10 mM of HEPES, 140 mM of NaCl, and 2.5 mM of CaCl2, pH 7.4, 37°C) and stained with FITC annexin V (diluted in binding buffer) (Molecular Probes) for 15 min at room temperature. The FITC annexin V stain was then removed, and the slide was mounted in 10% glycerol (diluted in binding buffer). Microscopy was conducted on a Zeiss Axioscop 2 Plus, and images were captured and processed by a Spot CCD camera and software (Diagnostic Instruments).

Total cellular protein lysates were prepared by scraping and resuspending cell cultures in lysis buffer (50 mM of HEPES (pH 7.4), 1 mM of EGTA, 1 mM of EDTA, 0.2 mM of sodium orthovanadate, 0.5% SDS, and freshly added protease inhibitors). Protein concentrations for each sample were determined with a SmartSpec 3000 spectrophotometer (Bio-Rad, Hercules, CA) using BSA standards. Protein samples (35 μg) were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were probed with a polyclonal Ab raised against either the full-length human recombinant human procaspase-3 or procaspase-7 (StressGen Biotechnologies, Victoria, British Columbia, Canada). HRP secondary Abs were purchased from Sigma-Aldrich, and bands were visualized using the SuperSignal West Pico chemiluminescent substrate (Pierce).

Statistical analysis was performed using the Student’s t test (standard two-tailed t procedure). Values of p < 0.05, p < 0.01, or p < 0.001 were considered the thresholds for defining statistical significance where indicated.

C3HA cells are murine, 3T3-like fibroblasts that are normally resistant to TNF, but can be rendered sensitive by treatment with inhibitors of transcription or translation such as actinomycin D or CHI (42). The TNF-induced lysis of sensitized C3HA cells is accompanied by the release of arachidonic acid. We have shown previously that this release (and apoptosis) is dependent on the activity of cPLA2 because this effect can be suppressed with antisense oligonucleotides specific for cPLA2 (29, 30). As shown in Fig. 1, verapamil, a calcium channel antagonist, suppressed the release of [3H]arachidonic acid from CHI-sensitized C3HA cells by ∼50%, suggesting that calcium is important for cPLA2 activation during apoptosis. Verapamil also significantly suppressed the release of [3H]arachidonic acid release following treatment with 1 μM of vinblastine, 200 μg/ml melphalan, or 30 μM of cis-platinum, concentrations that we found readily induced apoptosis in agreement with published observations (43, 44, 45). Taken together, these data suggest that calcium is an important second messenger for the activation of cPLA2 during multiple apoptotic responses.

FIGURE 1.

Verapamil abrogates agonist-induced [3H]arachidonic acid release from C3HA fibroblasts. C3HA fibroblasts were labeled overnight with [3H]arachidonic acid, and then treated with 20 ng/ml TNF and 25 μg/ml CHI, 1 μM of vinblastine, 200 μg/ml melphalan, 30 μM of cis-platinum, alone or in combination with 10 μM of verapamil. [3H]Arachidonic acid release was measured after a 6-h treatment with TNF and CHI, vinblastine, and melphalan, and after a 24-h treatment with cis-platinum. All points were performed in triplicate, and the experiment shown is representative of three repeats. The results are presented as the percentage of increase (mean ± SEM) over the spontaneous release from untreated cells. ∗, p < 0.05; ∗∗, p < 0.01.

FIGURE 1.

Verapamil abrogates agonist-induced [3H]arachidonic acid release from C3HA fibroblasts. C3HA fibroblasts were labeled overnight with [3H]arachidonic acid, and then treated with 20 ng/ml TNF and 25 μg/ml CHI, 1 μM of vinblastine, 200 μg/ml melphalan, 30 μM of cis-platinum, alone or in combination with 10 μM of verapamil. [3H]Arachidonic acid release was measured after a 6-h treatment with TNF and CHI, vinblastine, and melphalan, and after a 24-h treatment with cis-platinum. All points were performed in triplicate, and the experiment shown is representative of three repeats. The results are presented as the percentage of increase (mean ± SEM) over the spontaneous release from untreated cells. ∗, p < 0.05; ∗∗, p < 0.01.

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To confirm that calcium was indeed important for cPLA2 activation during apoptosis, we sought direct visualization using the ratio imaging dye fura 2-AM. We chose treatment with TNF and CHI as our method to induce apoptosis because we have characterized this form of cell death extensively (42, 46, 47, 48) and selected a time point early in the apoptotic response, preceding large-scale release of [3H]arachidonic acid into the culture medium. Fig. 2 shows the results of a typical time-course experiment in which the release of [3H]arachidonic acid into the culture medium is measured following treatment with TNF (20 ng/ml) and/or CHI (25 μg/ml). TNF itself typically triggers a rapid, 20–30% increase in [3H]arachidonic acid release, which ceases after ∼2 h. This treatment does not induce apoptosis (42) and, as shown in Table I, was not correlated with increased levels of intracellular calcium. Treatment with CHI itself also does not induce apoptosis (46), and has the opposite effect, causing a rapid 40–50% reduction in [3H]arachidonic acid release (Fig. 2), which gradually returns to control levels after 4–5 h. Again, this treatment was not accompanied by significant changes in levels of intracellular calcium (Table I). Rapid suppression of [3H]arachidonic acid release is also seen with the apoptosis-inducing combination of TNF and CHI (Fig. 2). In this case, however, levels of [3H]arachidonic acid rapidly rebound and a near linear pattern of release is seen over the next 12 h. As shown in Table I, we found that this treatment was indeed associated with significantly increased levels of intracellular calcium. Mean whole cell levels rose 57% to 159 ± 17 nM, perinuclear levels rose 87% to 264 ± 39 nM., and nuclear levels rose 79% to 193 ± 23 nM. Overall, at 1 h, the percentage of cells that had levels of [Ca2+]i higher than mean control values was 79, 50, 79, and 83%, respectively, for whole cell, cytosol, perinuclear, and nuclear regions. However, as shown in Fig. 3, we found that the increase in calcium levels was not uniform in the population. Several cells showed very high levels of intracellular calcium, with levels greater than 400 nM in the perinuclear and nuclear regions. Finally, to confirm that the increased levels of calcium we were monitoring were indeed derived from an extracellular source, we examined the effects of verapamil. Verapamil itself caused a 34% decrease in [Ca2+]i in the perinuclear region. Verapmil also strongly inhibited the increases seen with TNF and CHI treatment in all cellular compartments (Table I), and the effects of verapamil were uniform in the population (Fig. 3). Taken together, these results strongly indicate that an influx of extracellular Ca2+ is necessary for the apoptotic activation of cPLA2 following treatment with TNF and CHI.

FIGURE 2.

The kinetics of [3H]arachidonic acid release from C3HA fibroblasts treated with TNF and CHI. Cell cultures were labeled overnight with [3H]arachidonic acid and treated with TNF (20 ng/ml), CHI (25 μg/ml), or both compounds. [3H]Arachidonic acid release was measured at indicated time points, as described in Materials and Methods. All points were performed in triplicate and values shown are means ± SEM. Where not shown, error bars are less than symbol size. The experiment shown is representative of numerous repeats.

FIGURE 2.

The kinetics of [3H]arachidonic acid release from C3HA fibroblasts treated with TNF and CHI. Cell cultures were labeled overnight with [3H]arachidonic acid and treated with TNF (20 ng/ml), CHI (25 μg/ml), or both compounds. [3H]Arachidonic acid release was measured at indicated time points, as described in Materials and Methods. All points were performed in triplicate and values shown are means ± SEM. Where not shown, error bars are less than symbol size. The experiment shown is representative of numerous repeats.

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Table I.

Mean [Ca2+]i within whole cells and subcellular compartments following treatmenta

Treatment[Ca2+]i Within Region (nM)
Whole CellCytoplasmicPerinuclearNuclear
Untreated 101 ± 6 88 ± 5 144 ± 14 108 ± 8 
TNF 87 ± 8 68 ± 7 118 ± 13 107 ± 9 
CHI 97 ± 5 73 ± 5 130 ± 9 120 ± 8 
TNF/CHI 159 ± 17b 120 ± 16 264 ± 39b 193 ± 23b 
Verapamil 81 ± 6 73 ± 7 95 ± 8b 72 ± 7b 
TNF/CHI+ Verapamil 78 ± 4b 66 ± 3 100 ± 6b 67 ± 4b 
Treatment[Ca2+]i Within Region (nM)
Whole CellCytoplasmicPerinuclearNuclear
Untreated 101 ± 6 88 ± 5 144 ± 14 108 ± 8 
TNF 87 ± 8 68 ± 7 118 ± 13 107 ± 9 
CHI 97 ± 5 73 ± 5 130 ± 9 120 ± 8 
TNF/CHI 159 ± 17b 120 ± 16 264 ± 39b 193 ± 23b 
Verapamil 81 ± 6 73 ± 7 95 ± 8b 72 ± 7b 
TNF/CHI+ Verapamil 78 ± 4b 66 ± 3 100 ± 6b 67 ± 4b 
a

[Ca2+]i was measured within whole cells, and subcellular regions within C3HA fibroblasts. Cells were pretreated with or without verapamil following a 1-h treatment with TNF, CHI, or both, as described in Materials and Methods. Data are expressed as the mean ± SEM of 24 cells that were taken from three separate experiments.

b

, p < 0.01 (compared with untreated cells).

FIGURE 3.

Population measurements of elevated [Ca2+]i. C3HA fibroblasts were loaded with fura 2-AM and left untreated (A) or treated with TNF and CHI for 1 h (B), verapamil (C), or verapamil, TNF, and CHI (D). [Ca2+]i measurements were taken from areas corresponding to the whole cell (W), cytosolic (C), perinuclear (P), and nuclear (N) regions of individual cells. Each data point represents [Ca2+]i within a subcellular region of an individual cell (n = 24 from three experiments). Statistical analysis of mean [Ca2+]i differences between treated and untreated cells is presented in Table I.

FIGURE 3.

Population measurements of elevated [Ca2+]i. C3HA fibroblasts were loaded with fura 2-AM and left untreated (A) or treated with TNF and CHI for 1 h (B), verapamil (C), or verapamil, TNF, and CHI (D). [Ca2+]i measurements were taken from areas corresponding to the whole cell (W), cytosolic (C), perinuclear (P), and nuclear (N) regions of individual cells. Each data point represents [Ca2+]i within a subcellular region of an individual cell (n = 24 from three experiments). Statistical analysis of mean [Ca2+]i differences between treated and untreated cells is presented in Table I.

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As shown in Fig. 3, a range of responses is seen following treatment with TNF and CHI. Further analysis of this population revealed that specific levels of intracellular calcium were correlated with specific stages of apoptotic morphology. A typical DIC image of an untreated cell is shown in Fig. 4,A, while Fig. 4,B shows the ratiometric image of the same cell in pseudocolor. Fig. 4,C shows a cell displaying an elevated and pronounced nucleus, which is one of the earliest discernible changes in the morphology of C3HA cells as they undergo apoptosis (47). Cells of this type contained enhanced [Ca2+]i, depicted by the light blue color (250–350 nM), that localized to the perinuclear region (Fig. 4,D). Cells further in the apoptotic process, i.e., those with small blebs on their surfaces, yet still attached to the substrate (Fig. 4,E), contained the highest levels of [Ca2+]i (green color) that ranged from 350 to 900 nM (Fig. 4,F). In addition, [Ca2+]i was elevated in both cytosolic and nuclear compartments within these cells. Fig. 4,G shows a cell later in the apoptotic process, which has undergone considerable cytoplasmic shrinkage and lost most of its adhesiveness. This cell also shows elevated levels of [Ca2+]i (Fig. 4,H). Finally, Fig. 4,I shows a cell in the end stages of apoptosis that was completely overcome with large membrane blebs and was no longer attached to the substrate. Again, levels of [Ca2+]i are clearly elevated (Fig. 4 J). Overall, we examined 21 cells with characteristic apoptotic morphology, and all of these displayed enhanced [Ca2+]i, with one exception. Because this particular cell was at the end stages of apoptosis, it is likely that calcium and fura 2-AM were released from the cell due to the rupture of the plasma membrane during secondary necrosis.

FIGURE 4.

Paired single cell DIC and pseudocolor ratiometric images of [Ca2+]i. C3HA fibroblasts were loaded with fura 2-AM, then either left untreated (A and B) or treated with 20 ng/ml TNF and 25 μg/ml CHI for 1 h (C–F) or 2 h (G-H). In the ratio images, relative [Ca2+]i levels are indicated as dark blue (<250 nM), light blue (250–350 nM), and green (>350 nM). Images are representative of cells from three experiments.

FIGURE 4.

Paired single cell DIC and pseudocolor ratiometric images of [Ca2+]i. C3HA fibroblasts were loaded with fura 2-AM, then either left untreated (A and B) or treated with 20 ng/ml TNF and 25 μg/ml CHI for 1 h (C–F) or 2 h (G-H). In the ratio images, relative [Ca2+]i levels are indicated as dark blue (<250 nM), light blue (250–350 nM), and green (>350 nM). Images are representative of cells from three experiments.

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The translocation of cPLA2 to an intracellular membrane is necessary for it to release arachidonic acid, and in this study we tested whether calcium is important to this process. Fluorescence microscopy, using a mAb to cPLA2, was used to monitor its intracellular position. As shown in Fig. 5,A, in normal untreated C3HA cells, we found cPLA2 to be distributed in a diffuse manner throughout the cytoplasm with some areas of punctate staining. A similar pattern with both diffuse and punctate staining was observed following treatment with TNF or CHI independently (data not shown). In contrast, we found that treatment with TNF and CHI changed that distribution pattern. Fig. 5,B shows a group of cells early in the apoptotic process, after 1 h of treatment with TNF and CHI, at the stage in which the nuclei have become pronounced. We found that cPLA2 was lost from the peripheral cytosol, and both perinuclear and intranuclear punctate staining was observed. Fig. 5,C shows cells later in the apoptotic process, after 2 h of treatment, in which we observed only intranuclear staining. Intranuclear staining was also observed in apoptotic cells with large membrane blebs extending from their surfaces (Fig. 5,D). As shown in Fig. 5,E, we found that verapamil blocked the perinuclear and intranuclear nuclear accumulation of cPLA2 (although some aggregation in the cytosol did occur), indicating an important role for extracellular calcium in the translocation process. For reference, Fig. 5,F shows C3HA cells stained with an Ab to the nuclear pore complex, and Fig. 5 G shows C3HA cells stained only with secondary Ab.

FIGURE 5.

Verapamil inhibits the translocation of cPLA2 during apoptosis. C3HA fibroblasts were left untreated (A) or treated with TNF (20 ng/ml) and CHI (25 μg/ml) for 1 h (B) or 2 h (C and D). E, Treatment with verapamil preceded 2-h treatment with TNF and CHI. Cell cultures were fixed and probed with a α-cPLA2 mAb, followed by a 2° rhodamine-conjugated goat anti-mouse Ab. Staining of untreated cells with a nuclear pore complex Ab (F) or the secondary Ab alone (G) was performed as controls. Representative images are shown from nine independent experiments.

FIGURE 5.

Verapamil inhibits the translocation of cPLA2 during apoptosis. C3HA fibroblasts were left untreated (A) or treated with TNF (20 ng/ml) and CHI (25 μg/ml) for 1 h (B) or 2 h (C and D). E, Treatment with verapamil preceded 2-h treatment with TNF and CHI. Cell cultures were fixed and probed with a α-cPLA2 mAb, followed by a 2° rhodamine-conjugated goat anti-mouse Ab. Staining of untreated cells with a nuclear pore complex Ab (F) or the secondary Ab alone (G) was performed as controls. Representative images are shown from nine independent experiments.

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Finally, we sought to establish the position of the calcium response on the timeline of biochemical events that are used to characterize apoptosis. We examined two events, the exposure of phosphatidylserine (PS) on the exterior surface of the plasma membrane and the cleavage of effector caspases. As shown in Fig. 6, B and C, increased annexin staining was first detected 1 h after treatment with TNF and CHI, and was pronounced on cells with membrane blebs at the 2-h time point. Annexin-positive cells were common in the culture by the 2-h time point (Fig. 6,C). In contrast, cleavage of caspase-3 and caspase-7 occurred later in the apoptotic process. As shown in Fig. 6, D and E, loss of procaspases did not occur until 4 h after treatment was initiated. Taken together, these data and the results of our morphological analysis suggest that levels of intracellular calcium are rising early in the apoptotic process coincident with, if not preceding, exposure of PS on the plasma membrane.

FIGURE 6.

TNF and CHI induce PS externalization and cleavage of procaspase-3 and procaspase-7 in C3HA fibroblasts. C3HA cells were either left untreated (A) or treated for 1 h (B) or 2 h (C) with TNF (20 ng/ml) and CHI (10 μg/ml). Cells were then washed and stained with FITC annexin V, as described in Materials and Methods. D and E, C3HA cells were treated with TNF and CHI for the indicated times. Following treatment, protein was extracted and analyzed by SDS-PAGE and Western blot with anti-caspase-3 (D) and anti-caspase-7 (E) Abs.

FIGURE 6.

TNF and CHI induce PS externalization and cleavage of procaspase-3 and procaspase-7 in C3HA fibroblasts. C3HA cells were either left untreated (A) or treated for 1 h (B) or 2 h (C) with TNF (20 ng/ml) and CHI (10 μg/ml). Cells were then washed and stained with FITC annexin V, as described in Materials and Methods. D and E, C3HA cells were treated with TNF and CHI for the indicated times. Following treatment, protein was extracted and analyzed by SDS-PAGE and Western blot with anti-caspase-3 (D) and anti-caspase-7 (E) Abs.

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In this study, we examined the role of calcium in cPLA2 activation during TNF-induced apoptosis. We performed these experiments using a murine fibroblast cell line that was rendered sensitive to TNF by CHI. We found that a calcium increase, resulting from an influx of extracellular calcium, occurred early in the apoptotic process and was responsible for the translocation of cPLA2. We noted that this response only occurred following treatment with TNF and CHI, not when either compound was used independently. The role of CHI in these experiments is to inhibit the expression of TNF-induced resistance gene products. The results of our experiments suggest that one of these gene products acts by preventing TNF-induced increases in [Ca2+]i. Finally, we also linked a calcium response to apoptosis induced by chemotherapeutic agents, suggesting that calcium may be important for many apoptotic processes that involve cPLA2.

Calcium first gained attention as a possible second messenger during apoptosis when it was discovered that the death of glucocorticoid-treated thymocytes was dependent on an influx of extracellular calcium (49). It is now clear that, in thymocytes and mature T cells, a calcium response occurs following a number of apoptotic stimuli, including γ-irradiation (50), TCR and Fas ligation (51, 52), toxic metals (53), as well as the removal of specific growth factors from culture medium (54). Calcium is believed to activate a number of calcium-binding proteins during apoptosis (55, 56, 57, 58) as well as calcium-dependent endonucleases that mediate DNA fragmentation (59, 60, 61). The loss of calcium from the endoplasmic reticulum, and subsequent increase in the cytosol and mitochondria, has also been identified as a trigger to initiate apoptosis (62).

In contrast to the well-established role for calcium during T cell apoptosis, little is known about the involvement of calcium during TNF-induced apoptosis. Two studies, in L929 (63) and BT-20 (38) cell lines (which are spontaneously susceptible to TNF), demonstrated that cellular calcium levels were elevated after treatment. The increase in calcium was gradual and spread to the peripheral cytosol, but initially localized in and around the nucleus, where levels remained most pronounced. In addition, apoptosis was mediated by an influx of extracellular calcium because the cell death was abrogated by verapamil or by chelating calcium from incubation medium. In contrast, other investigations have failed to find evidence for a calcium response during TNF-induced apoptosis. Calcium levels were unmodified within TNF treated in U937 monocytes (64), as well as KYM-1 and HeLa cells that overexpress the p75 TNF receptor (65).

The results of our experiments were similar to the findings of Bellomo et al. (38) and Kong et al. (63) with BT-20 and L929 cell lines. We found that a large and sustained increase in [Ca2+]i accompanied the death of C3HA cells treated with TNF and CHI. We sought to position this response within the time line of apoptosis in two ways, morphologically and biochemically. Morphologically, we found that the appearance of a pronounced nucleus correlated with the onset of elevated [Ca2+]i, which rose even higher within cells that began forming membrane blebs, yet remained spread and attached to the substrate. We also observed that levels of [Ca2+]i remained elevated within cells during the late stages of apoptosis. Biochemically, we found that elevated levels of [Ca2+]i appeared coincidentally with the exposure of PS, in agreement with previous studies suggesting that exposure of PS during apoptosis is calcium dependent (66). In contrast, cleavage of the effector caspase-3 and caspase-7 occurred much later in the apoptotic process. Together, these data suggest that the calcium response is an early event in the apoptotic response.

A major thrust of our investigations was to define the role of calcium in the activation of cPLA2 during TNF-induced apoptosis. We have shown previously that cPLA2 is rapidly phosphorylated and activated during TNF-induced apoptosis, but that the release of arachidonic acid is delayed (37). Based on the inhibitory effects of verapamil, we proposed that calcium might act as a second signal for the activation of cPLA2 (37). The results of these experiments are consistent with that hypothesis. We found that the calcium response occurred 1–2 h before the onset of arachidonic acid release and that verapamil blocked the intracellular translocation of cPLA2, suggesting that calcium is required for cPLA2 translocation to its intracellular target membrane. The rapid nature of the calcium response also raises the possibility that additional signals are necessary for cPLA2 activation and release of arachidonic acid during TNF-induced apoptosis.

Our studies revealed an interesting pattern of perinuclear and intranuclear cPLA2 translocation during apoptosis. This pattern of cPLA2 translocation has been noted previously (67, 68) and raises several possibilities for the role that cPLA2 plays during apoptosis. For example, arachidonic acid can be a substantial component of nuclear membranes (69, 70), which may be the source of the arachidonic acid released by cPLA2 during apoptosis. Alternatively, arachidonic acid may be released from cytosolic membranes, and nuclear cPLA2 is playing an as yet undescribed role in apoptosis. In a recent study of apoptosis induced by serum starvation, cPLA2 was found to interact with a nuclear protein (phospholipase-interacting protein) (68), a splice variant of the transcriptional regulator, Tip60. It is possible, therefore, that nuclear cPLA2 functions during apoptosis by interacting with one or more nuclear proteins. Finally, the nuclear cPLA2 we have observed may not be critical to apoptosis and results from the prolonged, high levels of calcium in apoptotic cells (71).

In summary, our studies suggest that calcium is important for the activation of cPLA2 during TNF-induced apoptosis. These results may explain why verapamil has a therapeutic effect on the pathology associated with autoimmune disorders, such as experimental allergic neuritis (72), cyclosporine nephrotoxicity (73), and hepatitis (74), all of which may involve TNF (74, 75, 76). Further research into the effects of inhibitors that block calcium mobilization will determine whether these compounds may be generally useful for reducing tissue damage that is associated with TNF.

We acknowledge Nina Allen and the support of the personnel in the North Carolina State University Cellular and Molecular Imaging Facility.

1

This work was supported by Grant CA-59032 (to S.M.L.) from the National Institutes of Health and by Project 06333 from the North Carolina Agricultural Research Service.

3

Abbreviations used in this paper: PLA2, phospholipase A2; [Ca2+]i, intracellular calcium concentration; CCD, charge-coupled device; CHI, cycloheximide; cPLA2, cytosolic PLA2; DIC, differential interference contrast; fura 2-AM, fura 2-acetoxymethyl ester; PS, phosphatidylserine.

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