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J. Biol. Chem., Vol. 277, Issue 16, 13812-13820, April 19, 2002

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Sustained ER Ca²⁺ Depletion Suppresses Protein Synthesis and Induces Activation-enhanced Cell Death in Mast Cells^*

Jonathan Soboloff and Stuart A. Berger

From the Arthritis and Immune Disorder Research Centre, University Health Network and the Department of Immunology, University of Toronto, Toronto, Ontario M5G 2M9, Canada

Received for publication, December 19, 2001, and in revised form, January 3, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Depletion of Ca²⁺ from the endoplasmic reticulum (ER) induces large increases in cytoplasmic Ca²⁺, mitochondrial Ca²⁺ loading, protein synthesis inhibition, and cell death. To clarify the connections among these events, we have evaluated the effect of Ca²⁺ mobilizing agents thapsigargin (Tg), econazole (Ec), and the growth factor Steel Factor (SLF) on bone marrow-derived mast cells (BMMCs). BMMC Ca²⁺ stores were found to consist of a Tg-sensitive ER compartment, the Tg-insensitive SIC store, and mitochondrial stores. Low levels of Ec interfered with Tg-stimulated mitochondrial loading while promoting progressive leakage of Ca²⁺ from the ER. Low levels of Ec completely reversed Tg toxicity while higher levels blocked store-operated influx and induced cell death in a SLF-enhanced manner. Both Ec and Tg inhibited protein synthesis, however, only SLF plus Tg or very high levels of Ec were able to significantly stimulate EIF-2 phosphorylation. Cycloheximide only partially protected BMMCs from Tg toxicity yet strongly synergized with Ec to induce cell death. These results therefore indicate that although both Tg and Ec deplete ER Ca²⁺ levels, Ec-induced cell death results from sustained protein synthesis inhibition while Tg toxicity results primarily from mitochondrial Ca²⁺ overload and secondarily from ER stress associated with Ca²⁺ depletion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In non-excitable cells, ligand-receptor interactions that activate phospholipase C stimulate increases in cytoplasmic Ca²⁺ ([Ca²⁺]_c)¹ through the release of Ca²⁺ from the endoplasmic reticulum (ER) (1). ER Ca²⁺ release stimulates the opening of store-operated Ca²⁺ channels (SOCs) in the plasma membrane (2), leading to sustained increases in [Ca²⁺]_c which can last for several minutes after the initiating signal. In parallel, Ca²⁺-ATPases located on the ER (SERCA) and plasma membrane actively remove Ca²⁺ from the cytosol, promoting the return of [Ca²⁺]_c to resting levels. In addition, mitochondrial uptake of Ca²⁺ can decrease peak [Ca²⁺]_c. As Ca²⁺-ATPase-induced efflux of Ca²⁺ decreases [Ca²⁺]_c, mitochondrial Ca²⁺ is released, thereby extending the total time of the Ca²⁺ transient (3, 4). As such, Ca²⁺ signals are regulated through the coordinated activity of elements in the plasma membrane, ER, and mitochondria.

The ER consists of physiologically distinct Ca²⁺ stores that have been classified by their sensitivity to pharmacological inhibition. The inositol 1,4,5-trisphosphate-sensitive ER compartment can be emptied by exposure to thapsigargin (Tg), which is an irreversible SERCA inhibitor (5). Inositol 1,4,5-trisphosphate-insensitive compartments of the ER can be further distinguished into ryanodine (ryanodine)/caffeine-sensitive stores and ryanodine/caffeine-insensitive Ca²⁺ stores (SIC store) (6).

Tg is toxic to a variety of cell types and its mechanism of toxicity has been the subject of considerable investigation. One model proposes that the large increase in [Ca²⁺]_c stimulated by Tg is primarily responsible for its toxicity (7). Increased cytosolic Ca²⁺ content leading to concomitant increases in mitochondrial [Ca²⁺] can stimulate the mitochondrial permeability transition, initiation of the caspase cascade, and apoptosis (7). An alternate model proposes that depletion of ER Ca²⁺ content is a major contributing factor in Tg toxicity. Ca²⁺-dependent ER-resident chaperones are required to promote protein folding. ER Ca²⁺ depletion therefore produces unfolded proteins, which tend to self-aggregate, leading to the immobilization of the ER-Golgi transport system (8). It has been documented that Tg-mediated ER Ca²⁺ depletion can suppress protein synthesis as part of the unfolded protein response. This response is effected through the kinases PKR and PERK which phosphorylate translation initiation factor EIF2 (8-10). Shutdown of protein synthesis may lead to the induction of apoptosis (11, 12). However, a recent study by Harding et al. (13) suggested that the inhibition of protein synthesis observed following ER stress was protective under conditions of ER Ca²⁺ depletion. Similarly, PKR-induced EIF-2 phosphorylation is well accepted as a viral defense mechanism (14), supporting the concept that suppression of protein synthesis can be beneficial to the cell. The precise role of protein synthesis regulation in mediating Ca²⁺ depletion-associated stress and anti-viral responses therefore remains undetermined.

Since many biological agonists stimulate Ca²⁺ release from the ER, such signals may also modulate ER stress due to Ca²⁺ depletion. To clarify the connection between ER Ca²⁺ depletion, protein synthesis, and cell viability, we have evaluated the effect of Ca²⁺ mobilizing agent Tg, SOC antagonist Ec, and the growth factor Steel Factor (SLF) on bone marrow-derived mast cells (BMMCs). Econazole, like Tg, depletes ER Ca²⁺ (15), but unlike Tg also blocks SOC-mediated Ca²⁺ influx (16). The ability to decouple ER depletion from influx therefore makes Ec a useful pharmacological tool. BMMCs are ex vivo non-adhering mast cells morphologically similar to mucosal mast cells. Ca²⁺ signals play important roles in a variety of mast cell functions including degranulation (17, 18), cytokine synthesis (1, 19, 20), cell survival and proliferation (21, 22). SLF, the ligand for the c-Kit receptor tyrosine kinase, is one of the primary regulators of mast cell survival, growth, and differentiation (23). As with many growth factors and their receptors, one of the key signal transduction events resulting from the binding of SLF to c-Kit is activation of phospholipase C- leading to the production of inositol 1,4,5-trisphosphate and the mobilization of intracellular Ca²⁺ (24). Using the 32D murine myelomonocytic cell line model, we recently demonstrated that phospholipase C- activation was essential for the membrane-bound form of SLF to support c-Kit-positive 32D cells in vitro and in vivo (22). Moreover, we have shown that inhibition of store-operated Ca²⁺ channels (SOCs) using Ec, ketotifen, or Ni²⁺, while concurrently stimulating cells with SLF, results in the induction of activation enhanced cell death (21) in both BMMCs and 32D-Kit leukemia cells. In the current study, we have established the physiological identities of BMMC Ca²⁺ stores and characterized Ec-induced changes in ER Ca²⁺ content and mitochondrial loading. We further show that in BMMCs depleted of ER Ca²⁺, suppression of protein synthesis can be either partially protective or profoundly lethal, depending upon the extent and the duration of suppression.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- BMMCs were generated from C57/Bl6 mice as previously described (25). Cells were cultured in Opti-MEM (Invitrogen, Grand Island, NY) supplemented with 5% heat-inactivated fetal bovine serum and 4% WEHI-3 supernatant (source of IL-3) for 5 to 10 weeks prior to use in these experiments. All cultures contained 55 µmol/liters -mercaptoethanol and antibiotics (Sigma).

Clonogenicity Assays-- The clonogenicity of treated cells was determined by measuring colony forming ability in semi-solid medium. BMMCs (5 × 10⁴) were incubated in 100 µl of ITS-RPMI in 96-well plates in the presence of treatments for the indicated time periods. At the end of the culture period, BMMCs (10⁴ cells) were transferred into RPMI containing fetal bovine serum (10%), IL-3 (4% WEHI-3 supernatant), SLF (1 µg/ml; produced as previously described (21)), IL-4 (1.5 ng/ml; R&D Systems, MI), and 0.9% methylcellulose (4000 centipoises; Stem Cell Technologies, Vancouver) for 8 to 10 days. At the end of the incubation period, the number of colony forming units was determined.

Specrofluorimetry-- [Ca²⁺]_c measurements were performed by flow cytometry. BMMCs (5 × 10⁵ cells/ml) were growth factor-deprived for ~2 h in Tyrode's buffer (HEPES (10 mM), NaCl (100 mM), KCl (5 mM), CaCl₂ (1.4 mM), MgCl₂ (1 mM), glucose (5.6 mM), and BSA (0.05%)). Subsequently, cells were incubated in loading buffer (30 min; 5 µM indo-1AM, 0.03% pluronic F-127 in Tyrode's buffer), washed (2 times), and incubated (greater than 15 min; 4 °C) to allow for the complete removal and/or conversion of indo-1AM to Ca²⁺-sensitive indo-1. Measurements were performed using a laser tuned to 338 nm while monitoring emissions at 405 and 450 nm. The concentration of intracellular free Ca²⁺ was calculated according to the following formula (26).

(Eq. 1)

Where R is the ratio of the fluorescence intensities measured at 405 and 450 nm during the experiments and F is the fluorescence intensity measured at 450 nm. R_min, R_max, F_min, and F_max were determined from in situ calibration of unlysed cells using 4 µM ionomycin in the absence (R_min and F_min; 10 mM EGTA) and presence (R_max and F_max) of Ca²⁺. K_d (250 nM) is the dissociation constant for indo-1 at 37 °C. R_min, R_max, F_min, and F_max varied depending upon settings and were determined at the beginning of each experimental procedure.

Detection of the SIC Store-- To quantify the Ca²⁺ content of the SIC store, indo-1-loaded BMMCs were treated with FCCP and Tg to deplete the Ca²⁺ content of the mitochondria and ER, respectively, in the presence of Ni²⁺, an inhibitor of transmembrane Ca²⁺ influx (EGTA caused excessive nonspecific loss of Ca²⁺ in BMMCs over extended incubation; data not shown). The Ca²⁺ ionophore ionomycin was then added in the presence of EGTA, which will release all of the remaining Ca²⁺ from the cell. Based upon the findings of Pizzo et al. (6), this Ca²⁺ comes from the SIC store. The magnitude of the ionomycin-induced Ca²⁺ transient was compared qualitatively between BMMCs under different conditions.

EIF-2 Phosphorylation-- Assays for EIF-2 phosphorylation were performed in BMMC cells in the presence of SLF (500 ng/ml) or IL-3 (10% WEHI). Cells were treated with Ec (0, 4, 8, and 12 µM) in the absence or presence of Tg (30 nM) for 30 min or 6 h. After centrifugation (1250 rpm), the cells were lysed in chilled RIPA buffer (Nonidet P-40 (1% v/v), sodium deoxycholate (0.05% w/v), SDS (0.1% w/v)) containing phenylmethylsulfonyl fluoride (10 µg/ml), and protease inhibitor mixture I (Sigma)) by sonication (30 s) followed by an incubation period (30 min; 4 °C). The supernatants (18,000 × g; 20 min; 4 °C) were stored in aliquots (20 °C) for subsequent analysis. Proteins were resolved by 15% SDS-PAGE and then electroblotted to nitrocellulose membranes. After transfer, the gels were stained with Coomassie Blue to verify even sample loading.

After transfer, the nitrocellulose membranes were blocked (1 h, room temperature in Tris-buffered saline-Tween 20 (TBST; Tris (10 mM), NaCl (150 mM), Tween 20 (0.1%), pH 8.0)) containing powdered skim milk (2%; Carnation) and subsequently incubated (overnight, 4 °C) with rabbit anti-mouse EIF-2 (phosphoserine 51 specific; BIOSOURCE International) in 2% blocking solution (1:1000). Membranes were then washed (2 × 7 min) in TBST and incubated with secondary antibody (30 min; goat anti-rabbit IgG conjugated to horseradish peroxidase; 1:2500 in 2% blocking solution). Membranes were then washed in TBST (3 × 5 min) followed by Tris-buffered saline (TBS; 1 × 5 min; Tris (10 mM), NaCl (150 mM), pH 8.0). Peroxidase activity was visualized using the ECL kit as per the manufacturer's instructions.

Protein Synthesis Measurement-- Cells (5 × 10⁵/sample) were collected, washed with phosphate-buffered saline, and then re-suspended in ITS-RPMI (RPMI supplemented with insulin (10 µg/ml)-transferrin (5.5 µg/ml)-selenite (5 ng/ml; Sigma) and fatty acid-free BSA (0.05%; Sigma)). Cells were treated with Ec (0, 4, 8, and 12 µM) and/or Tg (30 nM) for 15 min or 5.5 h. After centrifugation (2500 rpm; 5 min), cells were pulse-labeled with [³H]leucine (50 µCi/ml) for 10 min (37 °C; 5% CO₂) in leucine-free ITS-RPMI. After two washes in RPMI, pellets were lysed with Triton (50 µl; 0.5% in phosphate-buffered saline) followed by trichloroacetic acid (50 µl, 10% w/v; 4 °C). Samples were washed in trichloroacetic acid (5% w/v), and the protein pellets were re-suspended in microscintillant (Packard, Meriden, CT) and measured using a microplate scintillation counter (Packard).

Statistical Analysis-- Colony data was analyzed by two-way analysis of variance, with differences between individual means determined by Bonferroni's post-tests. Data were expressed as mean ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ca²⁺ Stores in BMMCs-- [Ca²⁺]_c was monitored in indo-1-loaded BMMCs by flow cytometry. We found that basal [Ca²⁺]_c in BMMCs was 93 ± 5.8 nM (n = 25). Preliminary studies were performed to assess the identity of ER Ca²⁺ stores in BMMCs. Exposure to ryanodine did not result in significant increases in [Ca²⁺]_c (Fig. 1A), suggesting that no ryanodine-sensitive Ca²⁺ stores are present in this cell type. In contrast, exposure of BMMCs to Tg resulted in a large increase in [Ca²⁺]_c even in the absence of extracellular Ca²⁺ (Fig. 1B), consistent with the presence of Tg-sensitive Ca²⁺ stores. The SIC store is not fully characterized but can be detected by treating cells with Ca²⁺ ionophore after other stores (i.e. mitochondria and Tg-sensitive stores) have been depleted. We therefore pretreated cells with FCCP and Tg to release Ca²⁺ from mitochondria and Tg-sensitive stores, respectively, and also incubated cells in Ni²⁺ to block influx. As shown in Fig. 1B, exposure of these cells to ionomycin/EGTA reveals a pool of Ca²⁺ indicating the existence of the SIC store (Fig. 1B). Consequently, BMMCs contain three intracellular Ca²⁺ stores, the mitochondria and two functionally distinguishable ER compartments based upon Tg sensitivity.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Physiologically and pharmacologically distinct intracellular Ca2+ stores in BMMCs. A, BMMCs were treated with ryanodine (1 µM). B, BMMCs pretreated with Ni2+ were exposed to FCCP (1 µM), Tg (2 µM), and then ionomycin (4 µM)/EGTA (3 mM) to reveal the presence of the SIC store. As a control (Ctl), cells were treated with ionomycin (4 µM)/EGTA (3 mM) alone, to compare the amount of Ca2+ released from the SIC store with total intracellular Ca2+ content. Data are representative of three separate experiments.

Econazole-induced Mobilization of Intracellular Ca²⁺-- To determine the direct effects of Ec on Ca²⁺ stores in BMMCs, a preliminary dose response was performed to determine the effective concentration. In the presence of 0.05% BSA, we observed instantaneous Ec-induced increases in [Ca²⁺]_c at concentrations above 30 µM. Based upon the findings of Gamberucci et al. (27) and our own observations, the presence of BSA decreases free Ec concentrations, thereby accounting for the difference in dose requirement compared with previous studies in Madin-Darby canine kidney cells (15). Under the conditions of the current study, 115 ± 8.4 nM (n = 8) increases in [Ca²⁺]_c which maintained themselves for several minutes after the addition of Ec were observed (Fig. 2A). Moreover, the subsequent addition of Tg (2 µM) failed to stimulate further increases in [Ca²⁺]_c (n = 3), suggesting that Ec induces the release of Ca²⁺ from Tg-sensitive stores. To determine whether the SIC store is similarly emptied by Ec, residual Ca²⁺ content following exposure to FCCP and Ec (30 µM) was measured using ionomycin/EGTA. As shown in Fig. 2B, the SIC store was not emptied following Ec exposure (n = 3), suggesting that Tg-sensitive stores are the primary Ec targets.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Econazole-induced mobilization of Ca2+ empties Tg-sensitive intracellular Ca2+ stores. A, BMMC were exposed to econazole (30 µM) without pretreatment, followed by Tg (2 µM). B, BMMCs were exposed to FCCP (1 µM), Ec (30 µM), Tg (2 µM), and then ionomycin (4 µM)/EGTA (3 mM) to demonstrate the effect of Ec on the SIC store. Data are representative of three separate experiments.

SLF-enhanced, Econazole-induced Blockage of SOC and Depletion of Tg-sensitive Ca²⁺ Stores-- Although no instantaneous Ec-induced changes in [Ca²⁺]_c were observed at concentrations less than 30 µM, the imidazole has potent effects on BMMC viability at much lower concentrations. However, this may be the result of cumulative effects over longer incubation. To assess this possibility, cells were incubated for 3 h with Ec (0, 4, 8, 12, and 15 µM) in the presence of SLF, a Ca²⁺-mobilizing growth factor or IL-3, a growth-promoting cytokine that does not mobilize Ca²⁺. Tg was used to release ER Ca²⁺. We observed that Tg-induced changes in [Ca²⁺]_c were increased in SLF-stimulated cells (Fig. 3A) in comparison with IL-3-stimulated cells (Fig. 3B). However, this difference was lost in the presence of Ec at concentrations greater than 8 µM. Second, the peak Tg-induced [Ca²⁺]_c increased as Ec concentrations increased up to 8 µM, after which point Tg-induced [Ca²⁺]_c began to decrease, with highly significant attenuation of Tg-induced Ca²⁺ release observed at 15 µM Ec (Fig. 3C). Finally, while Tg-induced changes in [Ca²⁺]_c normally stabilize at a [Ca²⁺]_c well above normal, Ec dose dependently decreased steady state Tg-induced [Ca²⁺]_c between 8 and 15 µM, at which point Tg-induced [Ca²⁺]_c returned to basal levels indicating inhibition of SOC-mediated influx. Two-way ANOVA analysis of the data in Fig. 3C revealed that the differences observed between SLF and IL-3 were significant (p < 0.05), as was the variation due to Ec (p < 0.0001).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   The effect of econazole on thapsigargin-sensitive Ca2+ stores. Cells were incubated in the presence of econazole (0, 4, 8, 12 or 15 µM) and either SLF (500 ng/ml; panels A, C, and D) or IL-3 (10 ng/ml; panels B, E, and F) for 3 h at 37 °C. BMMCs were exposed to thapsigargin (Tg; 2 µM) in the absence (panels A-C) or presence of Ni2+ (5 mM; panels D-F). Panels A, B, D, and E depict representative experiments, while panels C and F show the average change in peak Tg-induced [Ca2+]c (n = 3).

In the presence of Ni²⁺, Tg-induced increases in [Ca²⁺]_c come only from internal Ca²⁺ stores. Consequently, comparison of the amount of Tg-releasable Ca²⁺ can be used to estimate the relative amount of Ca²⁺ in the Tg-sensitive Ca²⁺ store. In this case, an acceleration in the rate of [Ca²⁺]_c increase in the presence of SLF was noted although no overall difference in total Tg releasable Ca²⁺ was observed between SLF (Fig. 3D) and IL-3 (Fig. 3E). A linear dose-dependent decrease in ER Ca²⁺ content was observed for Ec between 4 and 15 µM (Fig. 3F). Therefore, Ec induces dose-dependent decreases in ER Ca²⁺ content which led to enhanced Ca²⁺ release after the addition of Tg and was enhanced by the presence of SLF. Finally, at the highest doses studied (between 12 and 15 µM) Ec-induced inhibition of Ca²⁺ influx was observed.

Ec Suppresses Mitochondrial Ca²⁺ Uptake-- BMMCs treated with 2 µM Tg typically displayed a two-step increase in [Ca²⁺]_c (see Fig. 3, A and B, insets). The first step is characterized by a slow, ~2-fold increase in [Ca²⁺]_c, and is followed by a more rapid and extensive second step with [Ca²⁺]_c peaking between 600 and 1,000 nM. We observed that Ec concentrations as low as 4 µM eliminated the first step of the Tg-induced Ca²⁺ transient. We also observed that FCCP, the protonophore that collapses the mitochondrial membrane potential preventing Ca²⁺ entry into the mitochondria, also eliminated the first step of the Tg-induced Ca²⁺ rise (not shown). These observations therefore suggested that the slower nature of the first step of the Ca²⁺ rise was due to mitochondrial buffering, and that Ec could interfere with this process. To test this possibility, we pretreated cells with Ec, exposed them to Tg, and then probed mitochondrial Ca²⁺ content by adding FCCP, which releases mitochondrial Ca²⁺ into the cytoplasm. As shown in Fig. 4, in the absence of Ec, Tg pretreatment followed by FCCP results in a large increase in [Ca²⁺]_c. In contrast, cells pretreated with Ec failed to release any Ca²⁺ into the cytoplasm following FCCP treatment. These results therefore suggest that Ec pretreatment interferes with the ability of mitochondria to load Ca²⁺ following release from Tg-sensitive stores. This conclusion is also consistent with the observed rise in peak Tg-induced [Ca²⁺]_c in the presence of 4 or 8 µM Ec (Fig. 3), since Ca²⁺ that would normally load the mitochondria remains in the cytoplasm.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   The effect of econazole on thapsigargin-induced loading of mitochondrial Ca2+ stores. Cells were preincubated with Ec (0, 4, and 15 µM) for 3 h at 37 °C. One hour immediately prior to the beginning of the experiment, thapsigargin (2 µM) was added to induce Ca2+ overload. As depicted in the figure, BMMCs were exposed to FCCP (1 µM) while monitoring their response by flow cytometry. The data is representative of three separate experiments.

Econazole and Thapsigargin Regulate BMMC Colony Forming Ability in a Biphasic Manner-- To study the effect of ER Ca²⁺ depletion on mast cell viability, we incubated BMMCs with Tg and Ec and measured their effect on clonogenicity. Preliminary studies showed that incubation of BMMCs with as little as 30 nM Tg was sufficient to reduce clonogenicity by 1 order of magnitude. Surprisingly, we observed that in the presence of SLF, Ec (2 to 4 µM) completely protected mast cells from 30 nM Tg, resulting in an approximate 10-fold increase in colony forming ability (Fig. 5A; p < 0.001). This protection was lost when 8 µM Ec was used, while 12 µM Ec reduced BMMC clonogenicity by greater than 3 orders of magnitude. When BMMCs were treated with Tg (30 nM) and maintained by IL-3 (10% WEHI-3 conditioned medium), protection from Tg toxicity was also observed from 2 to 8 µM Ec (p < 0.001). In addition, colonies (2) were observed in the presence of 12 µM Ec alone, but not in the presence of Tg (Fig. 5B), suggesting that at high concentrations, this antagonism is reversed. Therefore, Ec protected cells from Tg at concentrations where loss of Ca²⁺ from the ER occurs and mitochondria are blocked from filling with Ca²⁺, but these compounds synergized to promote cell death at concentrations where Ec also blocks Ca²⁺ influx. Furthermore, cell death at high Ec concentrations was enhanced by SLF compared with IL-3.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Ec-thapsigargin interactions decrease BMMC colony forming ability in a biphasic manner. BMMCs (5 × 105 cells/ml) were incubated in the presence or absence of econazole (0, 4, 8, or 12 µM) and/or thapsigargin (30 nM) in the presence of either SLF (1 µg/ml; panel A) or IL-3 (10% WEHI; panel B). After a 24-h incubation period, 104 cells were transferred into methylcellulose (0.9%) containing IL-3 (10% WEHI), SLF (1 µg/ml), and IL-4 (1.5 ng/ml) incubated for 7 to 9 days. Data are expressed as the number of colony forming units per plate (CFU/104 cells). Significant differences from the untreated samples are indicated within the graphs with: *, p < 0.05; **, p < 0.01; or ***, p < 0.001.

SLF Depletes Tg-sensitive Ca²⁺ Stores in the ER-- As demonstrated above, Tg becomes highly toxic to BMMCs at concentrations which are considerably lower than those typically used to probe ER [Ca²⁺]. However, at 30 nM, Tg-induced increases in [Ca²⁺]_c (Fig. 6A) were similar in magnitude to Tg (2 µM; Fig. 3)-induced [Ca²⁺]_c, although the rate of [Ca²⁺]_c increase was significantly slower at the lower concentration. Moreover, unlike in response to 2 µM Tg, there was little decline in peak [Ca²⁺]_c over the time course of the measurement. Since the clonogenicity studies demonstrated that SLF enhanced cell death in the presence of high Ec concentrations, we measured Tg (30 nM)-induced Ca²⁺ release in the presence of SLF (Fig. 6A). This resulted in an extremely rapid increase in [Ca²⁺]_c which reached a magnitude higher than in the absence of the growth factor, followed by a slow decline until typical Tg (30 nM)-induced [Ca²⁺]_c was reached. To assess the ER [Ca²⁺] independent of influx, these experiments were repeated in the presence of Ni²⁺ which blocks all Ca²⁺ influx (Fig. 6B). Unlike in response to 2 µM Tg, [Ca²⁺]_c did not return to normal even 30 min after challenge with the 30 nM concentration of the drug, suggesting only partial emptying of Ca²⁺ in the ER. In the presence of SLF, however, Tg (30 nM)-induced [Ca²⁺]_c returned to basal levels in under 10 min, supporting the interpretation that SLF enhances Tg-induced emptying of ER Ca²⁺ content but does not affect cytoplasmic Ca²⁺ levels.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   SLF enhances thapsigargin-induced mobilization of Ca2+. A, exposure of BMMCs to thapsigargin (Tg; 30 nM) results in slow leakage of Ca2+ into the cytosol which is enhanced by the addition of SLF (1 µg/ml). B, in the presence of Ni2+ (3 mM), Tg (30 nM) induced Ca2+ release is sustained in the absence of SLF (1 µg/ml) only.

Tg and Ec Effects on EIF2 Phosphorylation and Protein Synthesis-- Tg has previously been shown to stimulate EIF-2 phosphorylation resulting in decreases in the rate of protein synthesis. Although some controversy exists regarding the identity of the protein kinase which initiates the unfolded protein response (PERK versus PKR), the target protein for both molecules is EIF-2. We therefore measured the effect of Tg and Ec on EIF-2 phosphorylation and protein synthesis. As shown in Fig. 7 (panels A and B), 30 nM Tg plus SLF stimulated EIF-2 phosphorylation in BMMCs, correlating with the ER depleting effects of this combination (Fig. 6B). In the presence of SLF, Ec alone also stimulated EIF2 phosphorylation, however, only at the highest concentration tested. In contrast, in the presence of IL-3, 30 nM Tg was insufficient to stimulate significant levels of EIF2 phosphorylation. In fact, in the presence of IL-3, significant EIF2 phosphorylation was only observed following treatment with both 30 nM Tg plus 12 µM Ec. When protein synthesis rates were measured, we observed that both Tg and Ec were able to suppress protein synthesis both alone and in combination (Fig. 7C). However, additive effects of the two compounds were only observed at the higher Ec concentrations.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Econazole and thapsigargin synergistically block protein synthesis via EIF-2 phosphorylation. A, BMMCs were incubated in the presence or absence of Ec (4, 8, and 12 µM) ± Tg (30 nM) for 30 min in the presence of SLF (500 ng/ml; left) or IL-3 (10% WEHI-cm; right). To detect the phosphorylation of EIF-2 (upper blots), treated cells (106/lane) were lysed, separated by SDS-PAGE, electroblotted to nitrocellulose, and hybridized to an antibody specific to EIF-2 phosphorylated at serine 51 (BIOSOURCE). To determine protein loading, hybridized antibody was removed by acid hydrolysis and the membranes were reprobed with an antibody specific to actin (middle blots). B, EIF-2 phosphorylation and actin levels were measured by densitometry and normalized to control. Reported data is an average of three independent experiments. C, to measure the rate of protein synthesis, treated cells (7.5 × 105/sample) were incubated with [3H]leucine for the final 10 min of incubation. Proteins were separated using trichloroacetic acid precipitation and counts were determined in a liquid scintillation counter. The data are representative of three separate experiments.

Previous studies have reported that Tg-induced loss of protein synthesis is reversible and that its toxicity can be attenuated by blocking protein synthesis through other means (13, 28). To determine whether Ec-induced EIF-2 phosphorylation and protein synthesis inhibition is similarly reversible, EIF-2 phosphorylation and protein synthesis rates were evaluated following a 6-h incubation. As shown in Fig. 8 (panels A and B), both Ec- and Tg-induced EIF2 phosphorylation levels were reduced in all treated cells compared with the 30-min end point. When protein synthesis was measured, we observed that all treated cells showed some degree of recovery, however, significant suppression of protein synthesis was maintained for all cells exposed to toxic levels of Ec alone or Ec plus Tg. These results therefore indicate that sustained suppression of protein synthesis correlates with Ec-mediated toxicity. However, the ability of Ec to protect Tg-treated cells does not correlate with either enhancement or suppression of protein synthesis.

View larger version (38K): [in this window] [in a new window]   Fig. 8.   Partial recovery of EIF-2 phosphorylation and protein synthesis following 6 h incubation with econazole and thapsigargin. BMMCs were incubated in the presence or absence of Ec (4, 8, and 12 µM) ± Tg (30 nM) for 6 h in the presence of SLF (500 ng/ml; left) or IL-3 (10% WEHI-cm; right). To detect the phosphorylation of EIF-2 (upper blots), treated cells (106/lane) were lysed, separated by SDS-PAGE, electroblotted to nitrocellulose, and hybridized to an antibody specific to EIF-2 phosphorylated at serine 51 (BIOSOURCE). To determine protein loading, hybridized antibody was removed by acid hydrolysis and the membranes were reprobed with an antibody specific to actin (middle blots). B, EIF-2 phosphorylation and actin levels were measured by densitometry and normalized to control. Reported data is an average of three independent experiments. C, to measure the rate of protein synthesis, treated cells (7.5 × 105/sample) were incubated with [3H]leucine for the final 10 min of incubation. Proteins were separated using trichloroacetic acid precipitation and counts were determined in a liquid scintillation counter. The data are representative of three separate experiments.

Cycloheximide Protects from Tg Toxicity but Enhances Ec-induced Cell Death-- To further investigate the role of protein synthesis in mediating ER-associated toxicity, we determined the effect of cycloheximide on both Tg and Ec-induced cell death. As shown in Fig. 9, cycloheximide partially protected BMMCs from 30 nM Tg between 150 ng/ml and 1.5 µg/ml irrespective of the presence of SLF (Fig. 9A) or IL-3 (Fig. 9B). However, at higher concentrations of cycloheximide, this protection was reversed. In contrast, we observed that cycloheximide profoundly enhanced BMMC cell death induced by Ec beginning at 500 ng/ml. Moreover, SLF-induced enhancement of this interaction was observed at 500 ng/ml and 1.5 µg/ml. Since these concentrations of cycloheximide are considerably lower than those typically used in previous studies, we confirmed that protein synthesis inhibition occurs at these concentrations. We observed a 74.2 ± 8.2% decrease in the rate of protein synthesis following a 30-min incubation with 500 ng/ml cycloheximide in the presence of SLF and a comparable 72.5 ± 4.9% decrease in the presence of IL-3. These observations therefore confirm that modest suppression of protein synthesis can partially protect cells from Tg. In contrast, Ec-induced cell death is strongly enhanced by protein synthesis suppression.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Cycloheximide enhances econazole-induced loss of clonogenicity but protects from Tg-induced loss of clonogenicity. BMMCs (5 × 105 cells/ml) were incubated in the presence or absence of cycloheximide (0.05, 0.15, 0.5, 1.5, 5, or 15 µg/ml) ± thapsigargin (30 nM) or econazole (12 µM) in the presence of either SLF (1 µg/ml; panel A) or IL-3 (10% WEHI; panel B). After a 24-h incubation period, 104 cells were transferred into methylcellulose (0.9%) containing IL-3 (10% WEHI), SLF (1 µg/ml), and IL-4 (1.5 ng/ml) incubated for 7 to 9 days. Data are expressed as the number of colony forming units per plate (CFU/104 cells).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we have investigated the relationship between ER Ca²⁺ depletion, inhibition of protein synthesis, and the induction of cell death in bone marrow-derived mast cells. We found that mast cell Ca²⁺ stores consist of a Tg-sensitive ER compartment, the Tg-insensitive SIC store and FCCP-sensitive mitochondrial stores. We observed that low levels of Ec interfered with Tg-stimulated mitochondrial Ca²⁺ loading while promoting progressive leakage of Ca²⁺ from the ER. Low levels of Ec completely reversed Tg toxicity while higher levels blocked store-operated influx and induced cell death. Both Ec and Tg inhibited protein synthesis, however, only SLF plus Tg or very high levels of Ec were able to significantly stimulate EIF-2 phosphorylation. In addition, we observed that cycloheximide provided only partial protection against Tg toxicity yet profoundly synergized with Ec to induce cell death. These results therefore indicate that although both Tg and Ec deplete ER Ca²⁺ levels, their mechanism of toxicity in BMMCs is different. Tg's mechanism of toxicity involves primarily mitochondrial loading with Ca²⁺ and secondarily from ER stress associated with Ca²⁺ depletion while Ec induces mast cell death through profound, sustained suppression of protein synthesis.

Tg induces ER Ca²⁺ depletion, sustained increases in [Ca²⁺]_c and loading of mitochondria with Ca²⁺. Several previous studies have attempted to determine which of these events is key to Tg toxicity. Decreased ER Ca²⁺ content has been shown to prevent normal protein synthesis and trafficking, which can contribute to cell death while sustained increases in cytosolic Ca²⁺ concentrations can lead to Ca²⁺ overload-induced apoptosis (7, 29). In distinguishing among these mechanisms, the current study provides some insight. First, we observed no difference in steady-state [Ca²⁺]_c (~500 nM) following the addition of either 30 nM or 2 µM Tg, despite the fact that exposure to these two concentrations leads to a several order of magnitude difference in BMMC clonogenicity. Second, we found that 4 µM Ec could completely protect against 30 nM Tg-induced loss of clonogenicity, although this concentration of Ec had no effect on steady-state Tg-induced [Ca²⁺]_c and in fact increased peak cytoplasmic Ca²⁺ levels. These two observations both suggest that a large increase in [Ca²⁺]_c does not inevitably lead to toxicity. However, Ec concentrations observed to be completely protective for Tg were found to block loading of mitochondria with Ca²⁺ with little effect on mitochondrial membrane polarization (data not shown). In addition, under the conditions of the current study, low concentrations of cycloheximide provided only partial protection from the toxic effects of Tg. Taken together, these results suggest that the ER stress associated with Ca²⁺ depletion plays only a partial role, while mitochondrial events play a greater role in mediating Tg toxicity.

As shown in Figs. 7 and 8, both Tg and Ec stimulated EIF-2 phosphorylation and suppressed protein synthesis. However, the relationship between these end points, Ca²⁺ depletion, and mast cell viability is complex. Conditions resulting in enhanced Ca²⁺ depletion from the ER in general led to increased EIF-2 phosphorylation and enhanced suppression of protein synthesis. However, by 6 h, EIF-2 phosphorylation was lost, even in conditions where significant suppression of protein synthesis was maintained. Furthermore, by the 6-h time point, significant Ec-induced suppression of protein synthesis was only observed at concentrations where it is lethal to mast cells. These observations provide additional support for the interpretation that Ec does not protect Tg-exposed BMMCs through protein synthesis suppression. However, sustained inhibition of protein synthesis does correlate with loss of clonogenicity. The fact that cycloheximide strongly synergized with Ec to promote mast cell death further suggests that profound, sustained protein synthesis inhibition is the major mechanism by which Ec kills mast cells. Protein synthesis inhibition was also identified as a likely cause of growth arrest observed with the related compound clotrimazole (28).

The fact that both Ec and Tg were found to inhibit protein synthesis at concentrations where EIF-2 phosphorylation was not observed may indicate that small, undetectable changes in EIF-2 phosphorylation are sufficient to effect substantial changes in the rate of protein synthesis. Alternatively, ER Ca²⁺ depletion caused by these compounds may simply block protein synthesis through other pathways (8, 30) or in a nonspecific manner. Future studies may help to clarify the mechanism by which Ec and Tg inhibit protein synthesis. Nevertheless, our observations indicate that ER depletion through the combination of Ca²⁺ influx blockade with mobilizing agents and biological agonists such as SLF, represents a potent approach for inducing ER-based cell death.

A role for mitochondria as Ca²⁺ buffers has been recognized for many years. It has been reported that mitochondria can accumulate in the immediate vicinities of the ER (5) or SOCs (31). Since it is well established that local [Ca²⁺]_c at the site of Ca²⁺ entry far exceeds global [Ca²⁺]_c and mitochondria cannot fill with Ca²⁺ at concentrations less than 300-500 nM, it is likely that the cytosolic locations of mitochondria play a key role in their ability to buffer [Ca²⁺]_c. Given our observation that Ec could disrupt mitochondrial Ca²⁺ filling without affecting mitochondrial membrane polarization, it is possible that the imidazole disupts ER-mitochondrial junctions, perhaps by initiating cytoplasmic remodeling systems. Alternatively, Ec may have direct effects on mitochondrial Ca²⁺ loading via the electrophoretic uniporter. Ec's unique ability to deplete ER Ca²⁺ stores while preventing mitochondrial loading may prove to be a useful tool in clarifying the role of mitochondria as buffers of Ca²⁺ transients.

Several recent studies have provided evidence that Tg can activate caspases. In particular, the ER-resident cysteine protease caspase 12 is thought to play a pivotal role as an inducer of apoptosis in response to ER stress (12, 32). Caspase 12 can be activated by Ca²⁺-regulated calpains (33) and by caspases 7 and 9 (32). Since caspase 9 is activated upon stimulation of the mitochondrial permeability transition, which can occur in response to mitochondrial loading with Ca²⁺ (34), it is possible that the caspase 9-caspase 12 pathway is a major effector of Tg-induced apoptosis. In this context, it is interesting to note that following treatment with Ec that result in greater than 99.9% decreases in BMMC clonogenicity, we observed that only a relatively small percentage of cells display apoptotic phenotypes detectable by Hoechst assays.² These results therefore suggest that loss of clonogenicity induced by Ec may occur independent of full caspase activation.

In conclusion, we have found that inhibition of protein synthesis associated with the depletion of the Tg-sensitive ER compartment can be partially protective or toxic depending upon the extent and duration of the Ca²⁺ depletion. Moreover, our observations support a role for both ER Ca²⁺ depletion and mitochondrial filling rather than cytosolic Ca²⁺ overload as key inducers of cell death. Since biological agonists such as SLF can enhance the effects of ER Ca²⁺-depleting agents, our results highlight the therapeutic potential of targeting ER Ca²⁺ stores, particularly in highly stimulated or activated cells.

	ACKNOWLEDGEMENT

We thank C. Cantin for assistance with the Ca²⁺ measurements.

	FOOTNOTES

^* This work was supported by a grant from the National Cancer Institute of Canada (to S. A. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom all correspondence and reprint requests should be addressed: Arthritis and Immune Disorder Research Center, University Health Network, 620 University, Suite 700, Toronto, Ontario M5G 2M9, Canada. Tel.: 416-946-6541; Fax: 416-946-6589; E-mail: Berger@uhnres.utoronto.ca.

Published, JBC Papers in Press, February 8, 2002, DOI 10.1074/jbc.M112129200

² J. Soboloff and S. A. Berger, unpublished data.

	ABBREVIATIONS

The abbreviations used are: [Ca²⁺]_c, cytoplasmic Ca²⁺ concentration; SLF, Steel Factor; BMMC, bone marrow-derived mast cell; SOC, store-operated Ca²⁺ channel; ER, endoplasmic reticulum; Tg, thapsigargin; Ec, econazole; SIC, stimulus-induced calcium store; IL, interleukin; BSA, bovine serum albumin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; EIF-2, eukaroytic initiation factor 2.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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