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J. Biol. Chem., Vol. 276, Issue 34, 32257-32263, August 24, 2001

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Synergistic Movements of Ca²⁺ and Bax in Cells Undergoing Apoptosis^*

Zui Pan^§, Manjunatha B. Bhat^§, Anna-Liisa Nieminen^¶, and Jianjie Ma

From the Departments of  Physiology and Biophysics and ^¶ Anatomy, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106

Received for publication, January 9, 2001, and in revised form, June 13, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Apoptosis is a physiological counterbalance to mitosis and plays important roles in tissue development and homeostasis. Cytosolic Ca²⁺ has been implicated as a proapoptotic second messenger involved in both triggering apoptosis and regulating cell death-specific enzymes. A critical early event in apoptosis is associated with the redistribution of Bax from cytosol to mitochondria and endoplasmic reticulum (ER) membranes; however, the molecular mechanism of Bax translocation and its relationship to Ca²⁺ is largely unknown. Here we provide functional evidence for a synergistic interaction between the movements of intracellular Ca²⁺ and cytosolic Bax in the induction of apoptosis. Overexpression of Bax in cultured cells causes a loss of ER Ca²⁺ content. Depletion of ER Ca²⁺ through activation of the ryanodine receptor enhances the participation of Bax into the mitochondrial membrane. Neither Bax translocation nor Bax-induced apoptosis is affected by buffering of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, suggesting that depletion of ER Ca²⁺ rather than elevation of cytosolic Ca²⁺ is the signal for cell apoptosis. This dynamic interplay of Ca²⁺ and Bax movements may serve as an amplifying factor in the initial signaling steps of apoptosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Apoptosis or programmed cell death is a highly regulated process of selective cell deletion involved in development, normal cell turnover, cell-mediated immunity, pathological disorders, and tumor regression (1, 2). The Bcl-2 family members are active mediators of apoptosis that either inhibit (e.g. Bcl-2, Bcl-xL, and Mcl-1) or facilitate (e.g. Bax, Bad, Bak, Bid, Bik/Nbk, and Bim) apoptotic cell death (3, 4). The proapoptotic Bax proteins are largely cytosolic or loosely associate with intracellular membranes (5). After an appropriate death signal, Bax targets and integrates into intracellular membranes, especially the mitochondrial outer membrane, which induces opening of the permeability transition pore, release of cytochrome c, and activation of downstream caspase pathways (6-8). Recent studies have shown that this process may involve Bax dimerization or oligomerization and conformational changes (9-11), but the precise mechanism by which Bax is activated to translocate from cytosol to the intracellular membrane has not been elucidated.

Several studies have suggested that changes in intracellular Ca²⁺ homeostasis play an important role in the modulation of apoptosis. Many cell death stimuli including growth factor withdrawal, hormonal stimulation, and drug treatment are known to alter the concentration of Ca²⁺ in the cytosol and the storage of Ca²⁺ in the intracellular organelles (12-14). Compounds that directly affect the intracellular Ca²⁺ homeostasis and capacitative Ca²⁺ entry such as Ca²⁺ ionophores and thapsigargin (TG)¹ have been shown to induce apoptosis in a variety of cells (15, 16). Ca²⁺-release channels in the ER, i.e. inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR), seem to participate in the signal transduction pathway of apoptosis (17-19). Furthermore, recent evidence shows that Bcl-2 may suppress apoptosis via a mechanism that is linked to intracellular Ca²⁺ compartmentalization (20-22).

Our previous study demonstrated that depletion of ER Ca²⁺ by caffeine and ryanodine through the activation of RyR/Ca²⁺-release channels induces apoptosis in Chinese hamster ovary (CHO) cells transfected with RyR, and Bcl-xL prevents cell death at a stage downstream of ER Ca²⁺ release and capacitative Ca²⁺ entry (23). However, the functional interplay between Ca²⁺ and proapoptotic Bcl-2 members in apoptosis, e.g. Bax, has not been studied. Here we report that overexpression of Bax affected the ER Ca²⁺ homeostasis, and perturbation of intracellular Ca²⁺ could feed back to the translocation process of Bax. Our data suggest a synergistic action between the movements of Bax and Ca²⁺ in the initiation of apoptosis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture and Gene Transfection-- Stable clones of Chinese hamster ovary (CHO) cells permanently expressing RyR and Bcl-xL proteins were maintained at 37 °C and 5% CO₂ in Ham's F-12 medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (23). To eliminate possible bias caused by antibiotic selection, the control cells (CHO-WT) and cells stably expressing RyR (CHO-RyR) were transfected with mock pCEP4 vector (Invitrogen) and subjected to double selection with G418 (0.5 mg/ml) and hygromycin (0.26 mg/ml) in the same way as cells co-expressing RyR and Bcl-xL (RyR-xL). The cDNA of the mouse Bax gene was cloned into the pCMS-EGFP expression vector (CLONTECH) with transcription driven by the CMV promoter. The pCMS-EGFP plasmid also contains a separate SV40-driven green fluorescent protein (GFP) sequence, providing a convenient way of selecting cells transiently transfected with Bax and GFP genes. The GFP-Bax fusion construct was cloned into the pcDNA3 expression vector. Plasmids carrying Bax or GFP-Bax cDNAs were introduced into CHO cells using the LipofectAMINE Plus reagents following manufacturer instructions (Life Technologies, Inc.). The plasmid containing GFP alone was used as control. Transient expressions of Bax and GFP-Bax proteins were assayed 10-36 h after gene transfection by confocal microscopy and Western blot.

Confocal Microscopy Imaging and Immunocytochemistry-- For subcellular localization of transiently expressed GFP-Bax, CHO cells were grown on glass-bottomed TC3 dishes (Bioptechs, Inc., Butler, PA) and visualized with a Zeiss LSM-510 laser scanning microscope using a Plan-Apochromat ×63 oil or C-Apochromat ×40 water immersion objective (24). Anti-cytochrome oxidase subunit IV (COX IV) monoclonal antibody (Molecular Probes) was used to label the mitochondria. 18 h after transfection with GFP-Bax, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 10 min. The cells were incubated for 1 h with PBS containing 2% normal goat serum, 2 h with the anti-COX IV monoclonal antibody (diluted 1:100 in PBS + 5% normal goat serum), and developed with tetramethylrhodamine-conjugated goat anti-mouse IgG antibody (Molecular Probes, 1:1000 dilution). Relative green and red channels of the LSM-510 were used to observe the fluorescence of GFP-Bax and mitochondrial marker protein. The cell death process was monitored by Hoechst 33442 staining of the cell nucleus as described (23).

Intracellular Ca²⁺ Measurement-- CHO cells were grown in TC3 dishes and loaded with 2 µM Fura-2 AM fluorescent Ca²⁺ indicator. The changes in intracellular Ca²⁺ were measured with a dual wavelength excitation (340 and 380 nm, emission at 510 nm) spectrofluorometer (Photon Technology International, Inc.). The release of intracellular Ca²⁺ in individual cells was measured after exposure to caffeine, ryanodine, ATP, or thapsigargin in a Ca²⁺-free balanced salt solution (140 mM NaCl, 2.8 mM KCl, 2 mM MgCl₂, 0.5 mM EGTA, and 10 mM HEPES, pH 7.2) by rapid solution exchange. A separate set of fluorescein isothiocyanate filters was used to select CHO cells transiently transfected with the pcDNA3.1 (GFP-Bax) or pCMS-EGFP (Bax) plasmids to test the effect of GFP-Bax and Bax on intracellular Ca²⁺ release.

Quantitative Assay of Bax Integration into Intracellular Membranes-- CHO cells transfected with GFP-Bax were visualized under a confocal microscope. The cells were grouped according to the pattern of GFP-Bax distribution: healthy cells with a diffuse pattern of green fluorescence, indicating cytosolic distribution of GFP-Bax, and preapoptotic cells with a punctate pattern of green fluorescence, indicating integration of GFP-Bax into intracellular membranes. At different time points, cells with punctate and diffused patterns of GFP-Bax were counted from at least 20 fields, which were randomly selected. The percentage of transfected cells with membrane-bound Bax was defined as the number of cells with a punctated pattern of GFP-Bax divided by the total number of green fluorescent cells (in both punctated and diffused patterns). The data are from 4-10 independent experiments.

Apoptosis Assay-- The detailed procedure of quantitative assay of cell apoptosis upon treatment of cells with caffeine/ryanodine or after transient transfection with Bax has been described elsewhere (23, 25). Briefly, the differential uptake of fluorescent DNA binding dyes acridine orange (4 µg/ml) and ethidium bromide (8 µg/ml) was used to measure viable and nonviable cells in a given population. A viable cell will have a red cytoplasm with bright green nucleus because of the intercalation of acridine orange into the DNA. Ethidium bromide is only taken up in nonviable cells after disruption of the plasma membrane, and this dye also intercalates into DNA, making it appear orange. Thus, a dying cell will have a bright nucleus (the ethidium overwhelms the acridine), and its cytoplasm will appear dark red.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transient Expression of Bax in CHO Cells Stably Transfected with RyR and Bcl-xL-- To study the effect of Bcl-2-related proteins on the Ca²⁺ signaling of apoptosis, we derived several clones of CHO cells stably transfected with RyR alone (CHO-RyR) or together with Bcl-xL (RyR-xL), an antiapoptotic protein (Fig. 1a, lanes 2 and 4) (23). The ryanodine receptors are located in the ER membrane and provide the conduction pore of Ca²⁺-release channels (26). Activation of these Ca²⁺-release channels by caffeine and ryanodine leads to sustained depletion of the intracellular Ca²⁺ stores (27, 28) (Fig. 1b), indicated by the lack of thapsigargin-induced Ca²⁺ release even after prolonged incubation with 2 mM Ca²⁺ in the bath solution.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Expression of RyR, Bcl-xL, Bax, and GFP-Bax in CHO cells. a, Western blot of RyR, Bcl-xL, Bax, or GFP-Bax expressed in CHO cells. CHO-RyR and RyR-xL are stable clones of CHO cells that were permanently transfected with RyR or co-transfected with RyR and Bcl-xL, respectively. The Bax and GFP-Bax cDNAs were introduced into the cells using the LipofectAMINE reagent. Cells were harvested 10 h after transfection, and proteins (60 µg/lane) from the cell lysate were separated on a 3-12% SDS-polyacrylamide gel electrophoresis gradient gel and probed with monoclonal antibodies against RyR (molecular mass, 560 kDa, upper portion), against Bcl-xL (molecular mass, 26 kDa), or Bax (molecular mass, 21 kDa) (lower portions). Lane 1, CHO-WT cells; lane 2, CHO-RyR cells; lane 3, Bax expressed in CHO-RyR cells; lane 4, RyR-xL cells; lane 5, GFP-Bax (molecular mass, 46 kDa) expressed in CHO-RyR cells. b, caffeine and ryanodine-induced Ca2+ release from CHO-RyR cells. Application of 10 µM ryanodine (rya) to the cell at resting state did not affect intracellular Ca2+ release, because ryanodine only binds to the open state of the Ca2+-release channel (26). Caffeine (caff, 10 mM) activated the RyR channel and caused rapid Ca2+ release from the ER. Subsequent binding of ryanodine to RyR induced permanent opening of the Ca2+-release channel (28), which emptied the ER Ca2+ content revealed by the lack of response to TG.

The endogenous Bax proteins in CHO cells were not detectable with Western blot (data not shown). To study the interaction of Bax with Bcl-xL and Ca²⁺ in the apoptosis of CHO cells, we have used a transient expression system. 10 h after transfection with either Bax or GFP-Bax gene into CHO-RyR cells, an ample amount of Bax proteins could be detected, as revealed by the distinct band of the 21-kDa protein recognized by the anti-Bax antibody (Fig. 1a, lane 3) or a band of the 46-kDa protein recognized by the anti-GFP antibody (lane 5). Under identical transfection conditions, the expression levels of Bax or GFP-Bax were similar in CHO-RyR and RyR-xL cells (data not shown), suggesting that the presence of Bcl-xL does not affect the transient expression of Bax in CHO cells.

Apoptosis of CHO Cells Induced by Overexpression of Bax-- In Fig. 2, we show that overexpression of Bax in CHO-RyR and RyR-xL cells can induce apoptotic cell death based on the following features of apoptosis: genomic DNA laddering, nuclear chromatin condensation, and mitochondrial membrane clustering. As shown in Fig. 2a, DNA laddering was clearly visible in CHO-RyR cells 24 h after transfection with either Bax or GFP-Bax (lanes 4 and 5) but not in control cells transfected with GFP only (lane 1). As described in our previous study (Ref. 23), depletion of ER Ca²⁺ stores via activation of the RyR/Ca²⁺-release channel induced apoptosis in CHO-RyR cells but not in RyR-xL cells (Fig. 2a, lanes 2 and 3), whereas genomic DNA isolated from RyR-xL cells overexpressing GFP-Bax showed a typical laddering pattern, although the nucleus fragmentation process was delayed compared with that in CHO-RyR cells. This result indicates that Bxl-xL cannot prevent apoptosis completely when Bax is active.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Overexpression of Bax induced apoptosis in CHO cells. a, DNA laddering assay. Genomic DNA isolated from various CHO cells was separated on a 1.5% agarose gel. Lane 1, control CHO-RyR cells transfected with GFP only; lane 2, CHO-RyR cells treated with caffeine and ryanodine; lane 3, RyR-xL cells treated with caffeine and ryanodine; lane 4, CHO-RyR cells 24 h after transfection with GFP-Bax; lane 5, CHO-RyR cells 24 h after transfection with Bax; lane 6, RyR-xL cells 36 h after transfection with GFP-Bax. b, representative confocal images of CHO cell with Hoechst 33342 staining and GFP fluorescence. The pictures were taken 12 h after transfection with GFP-Bax. Hoechst dye staining (blue color) revealed characteristic nuclear condensation and chromatin fragmentation in cells undergoing apoptosis (indicated by arrows) with punctated GFP-Bax but not in cells with a diffuse pattern of GFP-Bax distribution. c, changes in mitochondria morphology in cells undergoing apoptosis. Immunostaining of COX IV (red fluorescence) revealed organized mitochondrial structure in RyR-xL cells with a diffuse pattern of GFB-Bax (upper panel). The clustering of mitochondria became evident in cells with a punctated distribution of GFP-Bax (lower panel). In overlay images, a yellow color shows the localization of GFP-Bax in mitochondria (Mito), and a green color shows thatGFP-Bax traffics to other intracellular membranes.

More obvious condensation of the nucleus in individual transfected cells is revealed in Fig. 2b by using Hoechst 33342 dye staining. Clearly, most cells with a punctate pattern of GFP-Bax distribution showed condensed nuclei with fragmented chromatin structures, a characteristic feature of cells undergoing apoptosis (Fig. 2b, indicated by arrows). And the translocation of Bax from cytosol to intracellular membranes seems essential for Bax-induced apoptosis, because only cells with a diffuse pattern of GFP-Bax distribution remained healthy and morphologically intact and with normal nuclear structure, consistent with previous studies (5, 7).

Apoptosis is also accompanied with rearrangement of mitochondria from an organized membrane network in cells with a diffuse distribution of GFB-Bax into a clustered vesicular pattern in cells with a punctate pattern of GFP-Bax. Fig. 2c shows the immunohistochemical structure of mitochondria labeled with the monoclonal antibody against COX IV (a mitochondria marker protein). A similar phenomenon was also reported by Margineantu et al. (29). The punctated GFP-Bax was mostly in the peri-nuclear region and co-localized with mitochondria (yellow spots in Fig. 2c overlay) but did not exclude ER or other intracellular membranes.

Depletion of ER Ca²⁺ Enhances Translocation of Bax from Cytosol into Intracellular Membranes-- In the transient expression assays, we observed that GFP-Bax was predominantly distributed in the cytosol with a diffuse pattern of green fluorescence 4-10 h after gene transfection. By 24-36 h, most of the transfected cells showed a punctate distribution of GFP-Bax. Interestingly, close to 60% of the cells expressing RyR (CHO-RyR) exhibited a punctate distribution of GFP-Bax as early as 6 h after gene transfection, a number that is significantly higher than in control CHO cells (CHO-WT) (Fig. 3b, left panel). Thus, it seems that the presence of RyR in the ER membrane may serve as an intrinsic factor for the translocation of Bax from cytosol to intracellular membranes. We have exercised separate assays with CHO cells permanently transfected with a nonconducting mutant of RyR, the E4032A-RyR1 mutant (Ref. 23) and found no difference between CHO-WT and CHO-E4032A cells in the initial distribution of GFP-Bax. Thus, it is likely that the intrinsic Ca²⁺-release function of RyR may play a role in the movement of GFP-Bax. Consistent with the antiapoptotic effect of Bcl-xL, the RyR-xL cells transfected with GFP-Bax maintained a cytosolic distribution of GFP-Bax for as long as 24-36 h with delayed induction of apoptosis (see Fig. 3b, right panel).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Depletion of ER Ca2+ enhances translocation of Bax from cytosol to intracellular membranes in cells undergoing apoptosis. a, Ca2+ release-mediated redistribution of GFP-Bax from cytosol to intracellular membranes. Representative images were taken from CHO-RyR (upper panels) and RyR-xL cells (lower panels) 12 h after transfection with GFP-Bax (Control). Most of the RyR-xL cells exhibited a diffused pattern of GFP-Bax distribution, whereas a portion of the CHO-RyR cells exhibited a punctated pattern of GFP-Bax distribution. Caffeine (10 mM) and ryanodine (10 µM) were added to the medium 6 h after transfection of cells with GFP-Bax (+caff/rya). As a result, both CHO-RyR and RyR-xL cells exhibited a more punctated distribution of GFP-Bax. b, time dependence of Bax translocation. The time-dependent changes in Bax redistribution under control conditions (filled symbols) or after treatment with caffeine and ryanodine (arrow, open symbols) were followed in a period of 60 h after transfection of GFB-Bax into CHO-RyR (upper panel) or RyR-xL cells (lower panel). For comparison, CHO-WT cells are presented as filled diamonds. The membrane-bound Bax is defined as the percentage of cells with a punctated pattern of GFB-Bax distribution in total green cells. 6 h was the earliest time at which measurable expression of GFP-Bax could be detected after transfection.

The application of caffeine and ryanodine to CHO-RyR and RyR-xL cells, which led to the depletion of ER Ca²⁺ (see Fig. 1b), caused significant redistribution of GFP-Bax (Fig. 3a). The percentage of cells with punctate distribution of GFP-Bax, a measure of the membrane-bound Bax, was increased significantly within 6 h after depletion of ER Ca²⁺. This enhanced movement of Bax from cytosol to mitochondria and ER could be seen clearly with the RyR-xL cells, which appeared to maintain the cytosolic distribution of GFP-Bax under control conditions (Fig. 3b). 24 h after transfection of GFP-Bax into RyR-xL cells, the addition of caffeine and ryanodine caused a redistribution of GFP-Bax from mostly cytosolic (22 ± 3%) to mostly membrane-bound (63% ± 9%). Thus, the combined activation of ER Ca²⁺ release and Bax overexpression overcame the antiapoptotic effect of Bcl-xL and led to eventual cell death.

Overexpression of Bax Causes Depletion of Intracellular Ca²⁺ Stores-- The integration of Bax into intracellular membranes might have a reciprocal effect on the intracellular Ca²⁺ homeostasis and contribute to the overall apoptotic cell death process. Therefore, we measured the ability of ATP, caffeine, or thapsigargin to release Ca²⁺ from intracellular membranes in individual CHO-RyR cells after transient transfection with either GFP-Bax or Bax; in the latter case, the Bax cDNA was cloned into the pCMS-EGFP vector, allowing selection of transfected cells with GFP fluorescence. ATP is an agonist of the purinergic receptor on the plasma membrane, which activates IP₃Rs on the ER membrane via generation of IP₃ (30). Thapsigargin is a specific inhibitor of the sarcoplasmic reticulum Ca²⁺ pump, which permits the direct assessment of the ER Ca²⁺ pool (15, 16, 31).

As shown in Fig. 4a, CHO-RyR cells transfected with GFP alone respond to the rapid release of Ca²⁺ from ER upon application of caffeine and ATP, indicating that functional RyR and IP₃R are present in the ER membrane and share a common pool of releasable Ca²⁺. A striking difference was observed with CHO-RyR cells transfected with Bax, in which the amount of ATP- and caffeine-induced Ca²⁺ release was significantly smaller as a result of Bax overexpression (Fig. 4b). To further correlate the subcellular localization of Bax with its effect on ER Ca²⁺ homeostasis, we selected the CHO-RyR cells on the basis of their pattern of GFP-Bax distribution. CHO-RyR cells with GFP-Bax present in the cytosol contain an ER Ca²⁺ pool that is significantly smaller than in control cells (compare Fig. 4c with 4a); those cells with GFP-Bax localized to the intracellular membranes appeared to have an empty ER Ca²⁺ pool, because they lost response to caffeine, ATP, and thapsigargin (Fig. 4d). Based on transmission images, these cells could have normal morphology with intact plasma membrane even with a punctate distribution of GFP-Bax and thus are likely to be in the preapoptotic stages. Similar results, i.e. reduced or even empty ER Ca²⁺ stores, were observed in CHO-WT and CHO-E4032A (mutant RyR protein lacking Ca²⁺ channel activation) cells transfected with GFP-Bax by using ATP and thapsigargin stimulation (data not shown). Interestingly, RyR-xL cells with diffuse distribution pattern of GFP-Bax are capable of releasing intracellular Ca²⁺ upon stimulation with caffeine in a manner similar to the CHO-RyR cells (compare Fig. 4e with 4a); but the extent of ER Ca²⁺ release is significantly reduced in response to a second application of ATP.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Bax-mediated changes in intracellular Ca2+ in cells undergoing apoptosis. The changes in cytosolic [Ca2+] were monitored by Fura-2, a Ca2+ indicator, and are represented as the ratio of fluorescence at 340/380 nm excitation wavelength. The release of intracellular Ca2+ was triggered by caffeine (caff, 10 mM), ATP (0.2 mM), or TG (1 µM) applied to a Ca2+-free balanced salt solution. a, CHO-RyR cells transfected with GFP alone (for control purpose) contain active RyR and IP3R Ca2+-release channels, indicated by the rapid response to caffeine and ATP. TG induced a slower rise in cytosolic Ca2+ via inhibition of the sarcoplasmic reticulum Ca2+ pump. b, transient transfection of pCMS-EGFP(Bax) into CHO-RyR cells led to a significant reduction in the ER Ca2+ content, because the response of the cells to caffeine, ATP, and TG was smaller compared with the controls (a). c, CHO-RyR cells with cytosolic distribution of GFP-Bax also exhibited reduced ER Ca2+ content. d, CHO-RyR cells with intracellular membrane-bound GFP-Bax contained empty ER Ca2+ content, because the cell lost its response to caffeine, ATP, and TG. The cell had an intact plasma membrane structure and was likely in the preapoptotic stage. e, RyR-xL cells with a diffuse pattern of GFP-Bax distribution exhibited a close to normal response to caffeine stimulation compared with the controls (a), but the second application of ATP resulted in significantly less intracellular Ca2+ release.

The results with separate cells in multiple transfections are summarized in Fig. 5 and Table I. Overexpression of Bax led to a significant reduction in the ER Ca²⁺ content, because ~70% of the CHO-RyR cells transfected with GFP-Bax contained an empty ER Ca²⁺ pool, whereas only a small population of cells transfected with GFP alone (~18%) appeared to have lost their ER Ca²⁺ content likely because of the toxic effect of the LipofectAMINE reagent. Interestingly, the resting [Ca²⁺] in CHO-RyR and RyR-xL cells did not appear to be affected by the transfection with either Bax or GFP-Bax genes (Table I), suggesting that the overexpressed Bax, in addition to affecting the intracellular Ca²⁺-release process, may also interfere with the extracellular Ca²⁺ entry pathway.²

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Reduction of ER Ca2+ storage induced by Bax overexpression in CHO cells. The bar chart represents data from multiple cells in multiple transfections. The percentage of cells with full ER Ca2+ content (ratio of 340/380 nm Fura-2 fluorescence > 1.3), reduced ER Ca2+ content (Fura-2 fluorescence range 0.3-1.2), or empty ER Ca2+ (Fura-2 fluorescence < 0.2) was calculated from a total of 33 cells (n = 11 transfections) with CHO-RyR cells transfected with GFP alone, a total of 168 cells (n = 17) with CHO-RyR cells transfected with GFP-Bax, and a total of 126 cells (n = 16) with RyR-xL cells transfected with GFP-Bax.

View this table: [in this window] [in a new window]   Table I Bax-mediated changes in intracellular Ca2+ homeostasis Entry points are average values of F340/F380 (mean ± S.E., n = 5  90) in individual cells loaded with 2 µM Fura-2 AM. The resting [Ca]i was measured with 2 mM Ca2+ present in balanced salt solution. Caffeine- (10 mM) and ATP- (0.2 mM) induced Ca2+ release from ER was performed in Ca2+-free balanced salt solution (+0.5 mM EGTA).

Effects of BAPTA on Bax Translocation and Bax-mediated Apoptosis-- The depletion of intracellular Ca²⁺ results in transient elevation of cytosolic Ca²⁺, which may directly activate caspase-like enzymes or indirectly influence the release of cytochrome c from mitochondria and trigger the apoptotic cascade (32, 33). It is not clear whether the elevation of cytosolic Ca²⁺ per se or the depletion of intracellular Ca²⁺ store serves as the primary trigger for apoptosis (20-22, 34, 35). To further test the effect of cytosolic Ca²⁺ on Bax translocation and Bax-mediated apoptosis, we pretreated the cells with BAPTA-AM, a membrane-permeable Ca²⁺ chelator. As shown in Fig. 6a, in cells pretreated with BAPTA-AM, the caffeine/ryanodine-induced rapid cytosolic Ca²⁺ elevation was reduced dramatically (compare trace ii with trace i). The buffering of cytosolic Ca²⁺ by BAPTA had no effect on the movement of GFP-Bax in both CHO-RyR and RyR-xL cells, because neither the pattern of GFB-Bax distribution (Fig. 6b) nor the time course of spontaneous GFP-Bax translocation changed with or without the addition of BAPTA-AM (Fig. 7a). In addition, Hoechst dye staining of the cell nucleus did not reveal a visible difference between control and BAPTA-treated cells (Fig. 6b). Notice that the untransfected CHO-RyR and RyR-xL cells are not affected by the pretreatment with BAPTA-AM; in other words, buffering of cytosolic Ca²⁺ does not appear to interfere with the apoptotic process of CHO cells. Furthermore, quantitative assays of cell apoptosis using differential uptake of fluorescent DNA binding dyes acridine orange and ethidium bromide give similar cell death indices irrespective of treatment with BAPTA, either 6 or 24 h after transfection with GFP-Bax (Fig. 7b). Together, these data suggest that depletion of intracellular Ca²⁺ store rather than elevation of cytosolic Ca²⁺ serves as the signal in Bax-mediated apoptosis.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   BAPTA-AM reduced elevation of cytosolic Ca2+ and did not affect apoptosis in CHO cells. Cells were incubated with 10 µM BAPTA-AM in extracellular medium at 37 °C for 90 min. a, representative traces of caffeine/ryanodine-induced Ca2+ release were plotted in control (i, black line) and 2 h after pretreatment with BAPTA-AM (ii, gray line) in CHO-RyR cells. b, cells were incubated with BAPTA-AM 3 h after transfection with GFP-Bax. The pretreatment of BAPTA did not alter the subcellular distribution of GFB-Bax. The blue color of Hoechst dye staining revealed similar characteristic nuclear condensation and chromatin fragmentation in cells undergoing apoptosis (indicated by arrows) regardless of the treatment with BAPTA (compared with Fig. 2c).

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Effects of BAPTA on Bax translocation and Bax-mediated apoptosis. a, the time course of GFP-Bax translocation from cytosol to intracellular membranes (defined as the membrane-bound Bax) was not affected by BAPTA treatment (open symbols) in either CHO-RyR (filled squares) or RyR-xL (filled circles) cells. b, quantitative assays of Bax-induced apoptosis. The chart was plotted as the percentages of apoptosis in CHO-RyR cells as a result of the overexpression of Bax or GFP-Bax or the depletion of ER Ca2+ (with the addition of caffeine and ryanodine). Pretreatment with BAPTA did not affect the degree of apoptosis either 6 or 24 h after transfection with GFB-Bax.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we demonstrate a functional interaction between the movements of intracellular Ca²⁺ and cytosolic Bax in the apoptotic pathway. We show that the overexpression of Bax can affect the storage of Ca²⁺ in the intracellular membranes, and changes in intracellular Ca²⁺ storage can feed back to the translocation of Bax from cytosol to intracellular membranes. The spontaneous Bax redistribution between cytosol and membrane association appears to be accelerated by the release of Ca²⁺ from the ER, whereas it is not affected by the resting Ca²⁺ level, because buffering of cytosolic Ca²⁺ by BAPTA does not influence the movement of Bax. These data add further insights into the cellular and molecular mechanism of Ca²⁺ signaling in apoptosis.

ER as a multifaceted organelle participates in protein synthesis and trafficking, cellular responses to stress, and intracellular Ca²⁺ signaling. Accumulating evidence shows that the ER plays important roles not only in cell proliferation but also in apoptosis (35, 36). It has been shown that changes in ER luminal environment by overexpression of calreticulin affect cell sensitivity to apoptosis (37). The activation of caspase-12 by ER stress induces apoptosis in a mitochondria-independent pathway (32). In the initial stage of apoptosis, although a majority of Bax distributes to the mitochondrial outer membrane, a portion of Bax also participates in other intracellular membranes such as the ER (38). Presumably, that ER-bound Bax may affect apoptosis by interfering with the ionic homeostasis of the ER and introducing cellular stress to the intracellular organelles. The Bax-induced depletion of ER Ca²⁺ storage may represent one of the functional effects of Bax in apoptosis. Alternatively, the Bax-mediated reduction of ER Ca²⁺ may represent a secondary effect of cells entering the apoptotic cycle. In any event, the combined effects of Ca²⁺ and Bax movements add an amplifying factor in triggering the fast execution of apoptotic cell death.

Exactly how Bax affects the intracellular Ca²⁺ homeostasis is presently unknown, and understanding its molecular and cellular mechanisms will require extensive studies. Several possibilities can be proposed here. First, the membrane-bound Bax may oligomerize and create a nonselective ion pore across the ER membrane and may therefore cause leakage of Ca²⁺ from the intracellular stores (39). Second, Bax may interact directly or indirectly with the sarcoplasmic reticulum Ca²⁺ pump similar to Bcl-2 (40), the IP₃R, or the RyR Ca²⁺-release channels and thus may affect the overall Ca²⁺ transport across the ER membrane. Another intriguing possibility could be that Bax can affect the communication between ER and mitochondria in the movement of Ca²⁺. With the presence of Bax in both mitochondria and ER membranes, a toxic loading of Ca²⁺ into the mitochondria matrix may cause morphological changes in the mitochondria network, thus leading to apoptotic cell death. The antiapoptotic effects of Bcl-2 have been implicated in changes of intracellular Ca²⁺ homeostasis. It is not surprising, therefore, that the balancing effect of Bax and Bcl-2 on ER Ca²⁺ homeostasis could play a role in determining the fate of the cells to either undergo proliferation or apoptosis (41, 42).

Calcium can function either as a promitotic or proapoptotic messenger, depending on its localization, cytosolic concentration, or oscillating pattern (23, 35, 36). One of the interesting results from this study is that Ca²⁺ movement across the ER membrane accelerated Bax movement from cytosol to intracellular membrane and therefore accelerated apoptosis. The balance between mitosis and apoptosis is essential for tissue development and homeostasis. Abnormal cell growth or cell death has been implicated as the primary cause for cancer or degenerative diseases, respectively (1-3). Proper control of apoptosis requires rigorous signaling communication and ordered function of regulatory molecules, which provide a rapid and effective response to diverse extracellular and intracellular apoptotic triggers. Ca²⁺- and Bcl-2-related proteins affect the morphology and function of the ER and mitochondria, two of the key intracellular organelles participating in apoptosis. Our data establish the concept that the dynamic interplay between the movements of Ca²⁺ and Bax may provide an important feedback mechanism for the rapid execution of apoptosis.

	ACKNOWLEDGEMENTS

We thank Drs. D. Danielpour and G. Dubyak for helpful discussions, D. Damron for help with the intracellular Ca²⁺ measurement, and C. Distelhorst for providing the Bax cDNA. We also appreciate the generous support from the Charlotte Geyer Foundation.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants RO1-AG15556 and RO1-CA85834 (to J. M.) and RO1-NS39469 and S10-RR14690 (to A. L. N.).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.

^§ Both authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854.Tel.: 732-235-4552; Fax: 732-235-5038; E-mail: jxm63@po.cwru.edu.

Published, JBC Papers in Press, June 18, 2001, DOI 10.1074/jbc.M100178200

² Z. Pan and J. Ma, unpublished data.

	ABBREVIATIONS

The abbreviations used are: TG, thapsigargin; ER, endoplasmic reticulum; IP₃, inositol 1,4,5-trisphosphate; IP₃R, IP₃ receptor; RyR, ryanodine receptor; CHO, Chinese hamster ovary; GFP, green fluorescent protein; WT, wild type; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; AM, acetoxymethyl ester.

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