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Molecular and Cellular Biology, November 2004, p. 9763-9770, Vol. 24, No. 22
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.22.9763-9770.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Calcium Binding of ARC Mediates Regulation of Caspase 8 and Cell Death

Dong-Gyu Jo, Joon-Il Jun, Jae-Woong Chang, Yeon-Mi Hong, Sungmin Song, Dong-Hyung Cho, Sang Mi Shim, Ho-June Lee, Chunghee Cho, Do Han Kim, and Yong-Keun Jung^*

Department of Life Science, Gwangju Institute of Science and Technology, Gwangju, South Korea

Received 23 April 2004/ Returned for modification 26 May 2004/ Accepted 20 August 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Apoptosis repressor with CARD (ARC) possesses the ability not only to block activation of caspase 8 but to modulate caspase-independent mitochondrial events associated with cell death. However, it is not known how ARC modulates both caspase-dependent and caspase-independent cell death. Here, we report that ARC is a Ca²⁺-dependent regulator of caspase 8 and cell death. We found that in Ca²⁺ overlay and Stains-all assays, ARC protein bound to Ca²⁺ through the C-terminal proline/glutamate-rich (P/E-rich) domain. ARC expression reduced not only cytosolic Ca²⁺ transients but also cytotoxic effects of thapsigargin, A23187, and ionomycin, for which the Ca²⁺-binding domain of ARC was indispensable. Conversely, direct interference of endogenous ARC synthesis by targeting ARC enhanced such Ca²⁺-mediated cell death. In addition, binding and immunoprecipitation analyses revealed that the protein-protein interaction between ARC and caspase 8 was decreased by the increase of Ca²⁺ concentration in vitro and by the treatment of HEK293 cells with thapsigargin in vivo. Caspase 8 activation was also required for the thapsigargin-induced cell death and suppressed by the ectopic expression of ARC. These results suggest that calcium binding mediates regulation of caspase 8 and cell death by ARC.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Apoptosis or programmed cell death is genetically controlled and plays a central role in normal development and tissue homeostasis, including development of the nervous system and regulation of the immune system (8, 25, 35). Dysregulated apoptosis has been implicated in the pathogenesis of cancer and autoimmune, neurodegenerative, and cardiovascular diseases (28). The cell death machinery that is conserved throughout evolution is composed of activators, inhibitors, and effectors (14). The effector arm of the cell death pathway consists of a family of cysteinyl aspartate-specific proteases called caspases (2). Data suggest that apoptotic cell death can be brought about by the loss of Ca²⁺ homeostatic control, but it can also be finely tuned positively or negatively by more subtle changes in Ca²⁺ distribution within intracellular compartments (29). While protein kinases such as AKT and ERK have been reported to modulate caspase activity through phosphorylation (1, 7), there have been few regulatory molecules directly linking cytotoxic Ca²⁺ signaling to caspase activity.

Based on sequence similarities, three prominent interaction motifs involved in apoptosis are recognized. The death domain superfamily consists of death domain (DD), death effector domain (DED), and caspase recruitment domain (CARD) families (16, 26). In recent years, a number of CARD-containing proteins have been identified and participate in various signaling pathways during apoptosis and NF-B activation. ARC is a CARD protein that selectively interacts with the initiator caspase 8 and significantly attenuates death receptor-induced apoptosis (22). Recently, ARC was also found capable of blocking caspase-independent events such as hypoxia-induced cytochrome c release and hydrogen peroxide (H₂O₂)-induced necrotic cell death (10, 27). We also described the protective role of ARC during hypoxia of hippocampal neurons (17). In addition, ARC is known to be phosphorylated by protein kinase CK2, modulating the subcellular localization of ARC (23). While increasing evidence suggests an inhibitory role for ARC in the diverse cell death processes, the precise mechanism by which ARC interferes with caspase-dependent and caspase-independent cell death has not been defined yet. In the present study, we postulate that ARC is a Ca²⁺-binding CARD protein that modulates activation of caspase 8.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmid construction. To construct mammalian expression plasmids, ARC, NARC_(1-98), and CARC_(99-208) were amplified by PCR and subcloned into EcoRI/XhoI sites of pcDNA3.1 (pcDNA3.1-ARC, pcDNA3.1-NARC, and pcDNA3.1-CARC; Invitrogen) and EcoRI/BamHI sites of pEGFP (pARC-GFP, pNARC-GFP, and pCARC-GFP) and pDsRed (pARC-RFP, pNARC-RFP, and pCARC-RFP) (Clontech). Caspase 8 cDNA was subcloned into pEGFP (pCaspase-8-GFP). For glutathione S-transferase (GST) fusion proteins, ARC, NARC_(1-98), and CARC_(99-208) were inserted into the EcoRI/XhoI sites of pGEX4T-3 (Amersham Pharmacia Biotech).

Cell culture and transfection. SK-N-BE(2)C, HeLa, B103, HEK293, and COS-7 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Jurkat cells were maintained with RPMI 1640 supplemented with 10% fetal bovine serum. Cells were transfected with Lipofectamine reagent (GIBCO BRL) and selected with G418 for 2 weeks to generate stable cell clones (B103/vector and B103/ARC) or mixed populations (B103/ARC-mix).

Antibody production and Western blot analysis. GST-ARC fusion protein was purified from Escherichia coli BL21(DE 3) with glutathione-Sepharose 4B (Amersham Pharmacia Biotech) and injected into a rabbit following standard immunization procedures. Anti-ARC antibody was purified by ARC affinity chromatography.

⁴⁵Ca²⁺ overlay assays. ⁴⁵Ca²⁺ overlay assays were performed as previously described (24). Briefly, GST-ARC, GST-NARC, GST-CARC, and calmodulin proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, incubated with ⁴⁵Ca²⁺, and exposed to X-ray film.

Staining of Ca²⁺-binding fusion proteins. Detection of the Ca²⁺-binding proteins following SDS-PAGE was performed using the cationic carbocyanine dye Stains-all {1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazoline-2-ylidene)-2-methylpropenyl]-naphtho-[1,2-d]thiazolium bromide} as previously described (5, 6).

Quantification of [Ca²⁺]_i by ratiometric fluorescence imaging. Cells were resuspended in Krebs-Ringer solution containing 0.1% bovine serum albumin and incubated with 2 µM fura-2/AM (Molecular Probes) for 45 min at 30°C. The fluorescence ratio was recorded at excitation wavelengths of 340 and 380 nm and at an emission wavelength of 510 nm with a PTI Deltascan (Photon Technology International). Values for R_max (the fluorescence ratio of the Ca²⁺-saturated sample) and R_min (the fluorescence ratio of the Ca²⁺-free sample) were determined with the addition of 10% (vol/vol) Triton X-100 and 400 mM EGTA, respectively. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) levels were calculated from the following formula: [Ca²⁺]_i = K_d[(R – R_min)/(R_max – R)](Sf2/Sb2), where K_d is the effective dissociation constant for the Ca²⁺-fura 2/AM complex and Sf2 and Sb2 are the fluorescence proportionality coefficients obtained at 380 nm under R_min and R_max conditions, respectively.

Measurement of Ca²⁺ transients by confocal microscopy. Briefly, cells were loaded with 10 µM fluo-3/AM (Molecular Probes) at 30°C in Krebs-Ringer solution containing 140 mM NaCl, 5 mM KCl, 0.5 mM MgCl₂, 5.5 mM HEPES (pH 7.4), 10 mM glucose, and 2 mM CaCl₂ for 60 min. Cells were stimulated with ATP and observed under a laser scanning confocal microscope (Leica). Fluo-3 was excited with light at a wavelength of 488 nm, and the emitted fluorescence was measured at 515 nm. Fluo-3 fluorescence was expressed as the normalized increase in fluorescence compared with the resting level (F/F₀). The fluorescence ratio F/F₀ was produced by dividing the fluorescence intensity (F) of each pixel in the original fluorescence image by its intensity at the beginning of the image (defined as F₀).

Antisense oligonucleotide treatment. Antisense oligonucleotide complementary to human ARC mRNA was generated: 5'-TCAGGCAGTGCATCCAAT-3'. It is located 240 bp downstream of the translation initiation site within the coding region. Comparison of the oligonucleotide sequence with the database detected only homology to the ARC sequence. Scrambled sequences were used as a control: C-1 (5'-ATGCTGCAGCATGATCTA-3') and C-2 (5'-GCTACTAGTAGCAGCTAC-3').

Fusion protein-binding assay. GST fusion proteins were immobilized by incubating with glutathione-Sepharose 4B and incubated with in vitro-translated caspase 8 labeled with [³⁵S]methionine (Amersham Pharmacia Biotech). Beads were then washed with 20 mM Tris-Cl (pH 7.4), 0.15 M NaCl, and 0.2% Triton X-100, and the bound proteins were subjected to SDS-PAGE.

Coimmunoprecipitation. HEK293 cells were lysed for 40 min in ice-cold lysis buffer (50 mM HEPES [pH 7.5], 250 mM NaCl, 0.2% NP-40, and protease inhibitors) supplemented with 1 mM CaCl₂ or 1 mM EGTA. Antibodies were incubated with lysate for 2 h at 4°C, and complexes were immunoprecipitated with protein A-Sepharose.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ca²⁺-binding ability of ARC. ARC protein contains an N-terminal CARD motif (amino acids 1 to 98) and an acidic C-terminal domain containing proline-glutamate dipeptide tandem repeats (amino acids 99 to 208) (Fig. 1A). The fact that ARC contains a highly acidic C terminus with an isoelectric point of 3.4 prompted us to assess its capability to bind to Ca²⁺. To this end, full-length ARC, the N-terminal CARD motif (NARC), and the C-terminal acidic region (CARC) were constructed as GST fusion proteins. Affinity-purified GST fusion proteins were stained with Coomassie blue (Fig. 1B) and examined for the Ca²⁺-binding ability in a ⁴⁵Ca²⁺ overlay assay (Fig. 1C). Calmodulin, a well-known Ca²⁺-binding protein, and GST served as positive and negative controls, respectively. ⁴⁵Ca²⁺ ligand blotting showed that GST-ARC and calmodulin bound to ⁴⁵Ca²⁺. Similar ⁴⁵Ca²⁺ binding was also detected with GST-CARC but not with GST-NARC. A number of well-documented Ca²⁺-binding proteins, such as calsequestrin and calmodulin, show a typical interaction with the metachromatic carbocyanine dye Stains-all and stain blue (5, 6). Stains-all staining revealed that GST-ARC, GST-CARC, and calmodulin stained blue, while other proteins, including GST and GST-NARC, stained pink (Fig. 1D). These observations indicate that ARC may bind to Ca²⁺ through the C terminus.

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FIG. 1. In vitro Ca2+ binding and domain mapping of ARC. (A) Schematic representation of full-length ARC, CARC, and NARC. CARD and the P/E-rich domain are illustrated. (B) GST, calmodulin (CMD), GST-ARC (ARC), GST-NARC (NARC), and GST-CARC (CARC) fusion proteins were purified from bacteria, resolved by SDS-PAGE, and stained with Coomassie brilliant blue. (C) Resolved proteins were transferred to nitrocellulose membrane, incubated with 45Ca2+, and exposed to X-ray film. (D) The SDS-polyacrylamide gel was stained with Stains-all.

ARC expression suppresses intracellular free Ca²⁺ transient. Extracellular ATP, via the plasma membrane P₂ purinergic receptor, generates inositol (1,4,5) triphosphate (InsP₃) and increases [Ca²⁺]_i by enhancing Ca²⁺ release from the endoplasmic reticulum (ER) (13). To examine the effects of ARC expression on [Ca²⁺]_i, an intracellular Ca²⁺ transient triggered by treatment with ATP was examined with confocal microscopic analysis using fluo-3. Compared to the nontransfected control cells (Fig. 2A), the cytosolic profiles of ATP-induced Ca²⁺ transients exhibited a decrease in ARC-expressing cells (Fig. 2B).

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FIG. 2. Suppression of Ca2+ transient by the C-terminal P/E-rich region of ARC. (A and B) COS-7 cells plated on coverslips were transfected with ARC-RFP, loaded with the Ca2+-sensitive dye fluo-3 in a chamber containing Ca2+-free medium, and stimulated with 20 µM ATP (arrows). ATP-induced Ca2+ transient was quantified by ratiometric fluorescence monitoring. One channel was used to detect the fluorescence intensity of Ca2+ indicator (large graphs) and, simultaneously, another channel was used to monitor the red fluorescence intensity of ARC-RFP protein (small graphs). In the same field of the confocal microscope, five cells were observed. Three of them were untransfected control cells (A), and two cells showed red fluorescence (B). (C and D) COS-7 cells were transfected with the indicated plasmids for 24 h and then loaded with 2 µM fura-2 for 45 min at 30°C. A dose of 20 µM ATP was then added to the cells (arrow), and the fluorescence ratio was recorded with a PTI Deltascan. Representative traces of Ca2+ transients are shown in the cells transfected with pcDNA3.1 (vector) or pcDNA3.1-ARC (ARC) (C) and with pcDNA3.1 (vector), pcDNA3.1-NARC (NARC), or pcDNA3.1-CARC (CARC) (D).

In addition, PTI Deltascan analysis using fura-2 also showed a decreased amplitude of cytosolic Ca²⁺ transient in the cells transfected with ARC (Fig. 2C). ATP-evoked increase of [Ca²⁺]_i (364 ± 20 nM) in control cells was reduced (195 ± 12 nM) in the cells expressing ARC. Because ATP causes capacitative Ca²⁺ entry in the presence of extracellular Ca²⁺, we performed similar experiments in Ca²⁺-free medium to monitor the effects of NARC and CARC on [Ca²⁺]_i, mainly contributed by the Ca²⁺ released from intracellular stores. ATP treatment evoked a rise of [Ca²⁺]_i to 89 ± 3 nM in the cells expressing NARC but less increase in the cells expressing CARC (55 ± 8 nM) (Fig. 2D). The inhibitory effects of ARC on ATP-induced Ca²⁺ transients were almost the same as those of CARC (data not shown). These results indicate that ARC suppresses the ATP-stimulated increase of [Ca²⁺]_i through the C terminus.

Antiapoptotic function of ARC in Ca²⁺-mediated cell death. We next examined the contribution of ARC to Ca²⁺-mediated cell death by directly targeting ARC expression using antisense oligonucleotides. Transfection with ARC antisense oligonucleotides effectively sensitized SK-N-BE(2)C cells to death (19 to 60% at 24 h) triggered by A23187, a Ca²⁺ ionophore that is known to increase cytosolic free Ca²⁺ concentration (Fig. 3A). Endogenous expression of ARC was diminished in the cells treated with ARC antisense oligonucleotides but not with scrambled oligonucleotides as examined by Western blot analysis. The protective role of ARC was further explored in the cells exposed to several Ca²⁺-dependent or Ca²⁺-independent cell death triggers. Ectopic expression of ARC effectively suppressed cell death triggered by A23187 and thapsigargin, an ER Ca²⁺-ATPase inhibitor, but not by etoposide, a DNA damaging agent (Fig. 3B). Similar antiapoptotic function of ARC was observed in the B103 cell line stably expressing ARC (B103/ARC). B103/ARC cells showed a higher expression level of ARC than the mixed populations of B103 cells (B103/ARC-mix) (Fig. 3C). Compared to B103 and B103/ARC-mix cells, B103/ARC cells were more resistant to cell death triggered by thapsigargin and A23187, but not by staurosporin, a kinase inhibitor (Fig. 3D to F). Consistent with previous reports, cell death induced by tumor necrosis factor alpha (TNF-) was also attenuated by the stable expression of ARC (Fig. 3G).

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FIG. 3. Down-regulation of ARC expression increases Ca2+-mediated cell death. (A) SK-N-BE(2)C cells were transfected with 5 µM antisense (AS-ARC) or scrambled (control) oligonucleotides for 48 h and then treated with 2 µM A23187 for the indicated times. The expression level of ARC was examined by Western blot analysis, and the percentage of dead cells was assessed by trypan blue exclusion. Values are means ± standard deviations (SD; n = 3). (B) SK-N-BE(2)C cells were transiently transfected with pEGFP (vector) or pARC-GFP (ARC) for 24 h and then treated with 5 µM thapsigargin (TG), 2 µM A23187, or 40 µM etoposide for 48 h. Cell viability was determined based on the morphology of GFP-positive cells under a fluorescence microscope. Values are means ± SD (n = 4). (C) Western blot analysis showing the expression level of ARC in stable B103 cells. B103 cells were transfected with pcDNA3.1 (B103/vector) or pcDNA3.1-ARC and selected for a single clone (B103/ARC) or mixed together (B103/ARC-Mix) in the presence of G418. (D to G) Dose-dependent kinetics of cell death. B103/vector, B103/ARC-mix, and B103/ARC cells were treated with increasing concentrations of thapsigargin, A23187, staurosporin, or TNF- with cycloheximide (10 µg/ml) for 24 h. Values are means ± SD (n = 3).

Next we mapped the domain responsible for the protective function of ARC. While ectopic expression of NARC or Nop30, an alternative splicing variant of ARC containing the common N-terminal CARD and the divergent C terminus (34), did not affect cell viability, CARC reduced cell death triggered by thapsigargin and ionomycin that increased the cytosolic free Ca²⁺ concentration (Fig. 4B and C). ARC and CARC were not effective in suppressing cell death triggered by broad doses (50 to 150 µg/ml) of etoposide (Fig. 4D; see also Fig. S1 in the supplemental material). These results indicate that the antiapoptotic activity of ARC in Ca²⁺-mediated cell death resides in the C-terminal Ca²⁺-binding region. Interestingly, TNF--induced cell death was significantly suppressed by only ARC but not by CARC (Fig. 4E). Ectopic expression of these GFP fusion proteins was confirmed by Western blotting, with lower expression of CARC (Fig. 4F).

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FIG. 4. Protective role of the C-terminal Ca2+-binding domain of ARC in Ca2+-mediated cell death. HeLa cells were transiently transfected with pEGFP (vector), pEGFP-ARC (ARC), pEGFP-NARC (NARC), pEGFP-CARC (CARC), or pEGFP-Nop30 (Nop30) and left untreated (A) or exposed to either thapsigargin (B), ionomycine (C), etoposide (D), or TNF- with cycloheximide (10 µg/ml) (E). Cell viability of the GFP-positive cells was then assessed. Values are means ± standard deviations (n = 3). (F) Expression levels of the exogenous GFP fusion proteins (arrowheads) were examined by Western blot analysis using anti-GFP antibody.

Ca²⁺-dependent dissociation of caspase 8 from ARC in vitro and in vivo. Our observations that ARC bound to Ca²⁺ led us to address whether the protein-protein interaction between ARC and caspase 8 could be affected by Ca²⁺. In vitro GST-pull-down assays using the purified GST-ARC protein showed that the interaction of ARC with caspase 8 began to be weakened by the presence of 0.1 mM Ca²⁺ and was markedly reduced by the presence of 1 mM Ca²⁺ in the binding buffer (Fig. 5A, lower panel), indicating that ARC interacts with caspase 8 in a Ca²⁺-sensitive and dose-dependent manner. In vitro binding assays further revealed that GST-ARC, but not GST-NARC or GST-CARC, bound to caspase 8 in the absence of Ca²⁺ (Fig. 5B, lower panel), indicating that, while both ARC and CARC are able to bind to Ca²⁺, full-length ARC only interacts with caspase 8, at least in vitro. These observations are in line with the results that ARC, but not NARC or CARC, can reduce TNF--induced and caspase 8-dependent cell death (Fig. 4E).

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FIG. 5. Ca2+-dependent dissociation of caspase 8 from ARC in vitro. (A) In vitro binding assays. GST or GST-ARC fusion protein was incubated with in vitro-translated [35S]methionine-labeled caspase 8 in the presence of increasing concentrations of Ca2+. Samples were pulled down with glutathione-Sepharose beads, separated by SDS-PAGE, and then stained with Coomassie blue (upper panel) or exposed to X-ray film (lower panel). (B) The purified GST fusion proteins, including GST, GST-ARC (ARC), GST-NARC (NARC), and GST-CARC (CARC), were incubated with [35S]methionine-labeled caspase 8 in the presence of 1 mM EGTA or 1 mM Ca2+ for in vitro binding assays as described above.

The Ca²⁺-dependent intracellular interaction of endogenous ARC with caspase 8 was also evident from coimmunoprecipitation analysis. As reported earlier, ARC was detected by Western blotting in the caspase 8-containing immunocomplexes isolated from HEK293 cell extracts in the absence of Ca²⁺ (Fig. 6A, lane 8). However, the presence of 1 mM CaCl₂ in the cell extracts abolished the formation of a caspase 8-ARC complex (Fig. 6A, lane 9). We then investigated whether or not the protein-protein interaction between ARC and caspase 8 within cells was affected by the increase of intracellular Ca²⁺. Compared to the control (Fig. 6A, lane 2), treatment of HEK293 cells with thapsigargin for 2 h decreased the amounts of ARC protein detected in the immunocomplexes isolated with anti-caspase 8 antibody (Fig. 6A, lane 5). In contrast, while FADD was detected in the immunocomplex, its dissociation was not affected by the increase of Ca²⁺ (Fig. 6A, middle panel). Reciprocally, caspase 8 was also detected in the ARC-containing immunocomplex isolated with anti-ARC antibody (Fig. 6B, lane 8). Consistently, its interaction with ARC was decreased in the presence of Ca²⁺ (Fig. 6B, lanes 5 and 9), whereas caspase 2 remained in the complex independent of Ca²⁺ (Fig. 6B, middle panel).

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FIG. 6. Thapsigargin treatment dissociates ARC from caspase 8. Total extracts prepared from HEK293 cells (Total) were either used as a control for Western blotting (lane 1) or treated with 1 mM EGTA (lanes 6 and 8) or 1 mM CaCl2 (lanes 7 and 9). HEK293 cells were left untreated (lane 2) or treated with 3 µM thapsigargin (TG) for the indicated times (lanes 3 to 5). Cell extracts were prepared and immunoprecipitated (IP) with preimmune immunoglobulin G (IgG), anti-caspase 8 (A), or anti-ARC (B) antibody. The immunoprecipitates were subjected to immunoblotting with anti-ARC, anti-caspase 8, anti-FADD, and anti-caspase 2 antibodies. Preimmune IgG and anti-FADD or anti-caspase 2 antibodies were used as negative and positive controls for the interaction, respectively. Heavy chains (HC) and light chains (LC) of the immunoglubulins are indicated.

The effects of calcium on the interaction of ARC and caspase 8 were further examined on a single-cell level. Ectopic expression showed that ARC-red fluorescent protein (RFP) and caspase 8-green fluorescent protein (GFP) were colocalized mainly in perinuclear and cytosolic regions (Fig. 7, upper panel). After treatment with thapsigargin for 3 h, subcellular localization of ARC-GFP was apparently changed and detected in the inclusions of the cytosol and nucleus, which did not completely overlap with that of caspase 8-GFP (Fig. 7, lower panel). These results strongly suggest that intracellular Ca²⁺ levels may regulate the protein-protein interaction of ARC and caspase 8.

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FIG. 7. Colocalization of ARC and caspase 8 is disrupted by treatment with thapsigargin. HEK293 cells were cotransfected with pARC-RFP and pCaspase-8-GFP for 24 h and then left untreated (control) or treated with 3 µM thapsigargin for 3 h in the presence of 20 µM zVAD-fmk. After staining nuclei with 4',6'-diamidino-2-phenylindole (DAPI), specimens were imaged under a fluorescence microscope.

Inhibition of thapsigargin-induced activation of caspase 8 by ARC. We found that B103 cells exposed to submicromolar concentrations of thapsigargin underwent apoptosis, which was inhibited by zVAD, a pan-caspase inhibitor (data not shown). Interestingly, Western blotting revealed that caspase 8 was proteolytically activated in the thapsigargin-treated B103 cells (Fig. 8A, left panel), while overexpression of ARC significantly suppressed the activation of caspase 8 triggered by thapsigargin (Fig. 8A, right panel). ARC expression remained constant during thapsigargin-induced cell death (Fig. 8A, middle panel). In addition, immunocytochemical analysis using an antibody specific for the active form (p20) of caspase 8 showed the appearance of anti-active caspase 8 antibody-reactive cells with condensed or fragmented nuclei in B103/vector cells (Fig. 8B, upper panel) but few in B103/ARC cells after thapsigargin treatment (Fig. 8B, lower panel). These results indicate that the thapsigargin-induced activation of caspase 8 is suppressed by the increased expression of ARC.

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FIG. 8. ARC-dependent requirement of caspase 8 in thapsigargin-induced cell death. (A and B) Suppression of caspase 8 activation by ARC. B103/vector and B103/ARC cells were treated with thapsigargin (TG) for 24 h. Western blot analyses were performed using anti-caspase 8 and anti-ARC antibodies. Pro-caspase 8 (Casp8) and its processed large subunit (p20) are indicated (A). Cells treated with thapsigargin were stained with anti-active caspase 8 antibody and Hoechst dye. Arrowheads indicate dying cells showing condensed and fragmented nuclei. The results shown are representative of three independent experiments (B). (C to F) Lack of thapsigargin-mediated cell death in caspase 8–/– Jurkat cells. Wild-type or caspase 8-deficient Jurkat cells were treated with different concentrations of thapsigargin for the indicated times (C and D) or transiently transfected with pARC-GFP for 36 h and then treated with 5 µM thapsigargin (TG), 0.5 µM staurosporin (Staur), or 100 ng of TNF--related apoptosis-inducing ligand (TRAIL)/ml for an additional 24 h (E and F). The results shown are from four independent experiments.

The finding that caspase 8 is activated in dying cells after treatment with thapsigargin does not necessarily mean that caspase 8 plays an essential role in the thapsigargin signaling leading to cell death. We therefore analyzed the thapsigargin-induced cell death using Jurkat cells lacking caspase 8 (4, 20, 33). Compared to parental cells (A3), caspase 8-deficient Jurkat cells (I9-2) were viable during prolonged exposures to high concentrations of thapsigargin (Fig. 8C and D). Similarly, thapsigargin-induced cell death was decreased in HEK293 cells transfected with antisense cDNA of caspase 8 (data not shown). We also confirmed that the ectopic expression of ARC in Jurkat cells significantly reduced cell death triggered by thapsigargin but not by staurosporin (Fig. 8E and F), while partial reduction of cell death was also observed in caspase 8-deficient Jurkat cells (Fig. 8E). As previously reported (4, 20, 33), caspase 8-deficient Jurkat cells were resistant to TNF--related apoptosis-inducing ligand (Fig. 8F). These results indicate that ARC regulates caspase 8 activation that is required for thapsigargin-induced cell death, providing more evidence for the role of ARC in the regulation of caspase 8 by intracellular Ca²⁺ levels.

Top Abstract Introduction Materials and Methods Results Discussion References

 
ARC seems to be a calcium-binding protein and functions as a cytosolic Ca²⁺ buffer in cells. Indeed, ectopic expression of ARC reduced the ATP-induced increase of [Ca²⁺]_i in the cells. The C-terminal region of ARC was sufficient to reduce the ATP-induced Ca²⁺ transient, consistent with the in vitro Ca²⁺-binding ability of ARC as evidenced by ⁴⁵Ca²⁺ overlay assay and blue staining with Stains-all, which have widely been used to identify Ca²⁺-binding proteins. The C-terminal domain was crucial for the Ca²⁺-binding ability of ARC; presumably, Ca²⁺ binds to a Ca²⁺ coordination site in the cluster of negatively charged amino acids. While ARC may alternatively down-regulate Ca²⁺ release from the ER, these observations led us to propose that ARC is effective in buffering intracellular free Ca²⁺. We also suggest that this Ca²⁺ buffering function is important for the antiapoptotic role of ARC. Expressional regulation of ARC interfered with various types of Ca²⁺-mediated cell death, including caspase 8-dependent (e.g., TNF-) and caspase 8-independent (e.g., ionomycin and A23187) cell death. Especially, the C-terminal Ca²⁺-binding region, but not the N-terminal CARD of ARC and Nop30, was sufficient for the attenuation of caspase 8-independent and Ca²⁺-mediated cell death, which might mainly be attributed to its ability to chelate cytosolic free Ca²⁺.

It was previously suggested that ARC also suppressed hypoxia and H₂O₂-induced cell death by interfering with a noncaspase factor, upstream of cytochrome c release (10, 27). Hypoxia itself causes an increase in [Ca²⁺]_i (9, 18). Also, oxidative stress including H₂O₂ induces rapid increases in [Ca²⁺]_i by stimulating Ca²⁺ influx from the extracellular environment and efflux from intracellular stores (11, 31). Considering ARC as a calcium-binding protein, the noncaspase factor would be Ca²⁺. An elevated [Ca²⁺]_i may act directly on mitochondria and cause cytochrome c to be released (3, 12, 32). These observations suggest that the mechanism by which ARC interferes with cytochrome c release is the modulation of [Ca²⁺]_i through a C-terminal P/E-rich domain. Recently, it was reported that ARC associates with Bax to inhibit Bax function in cells (15). We observed that ARC did not directly bind to BAX in vitro but suppressed the release of cytochrome c through its C terminus during thapsigargin-induced cell death (data not shown), probably by modulating Bax function. Thus, an additional factor may be associated with the ARC-BAX complexes which remains to be further clarified.

There is increasing evidence showing that caspase 8 plays a crucial role in Ca²⁺ signaling. We also found that thapsigargin treatment induced activation of proximal caspase 8 as a necessary step leading to cell death, which was effectively blocked by ARC. Thapsigargin seems to induce caspase 8-dependent cell death as well as a caspase 8-independent signal. Based on our observations that the elevated levels of Ca²⁺ caused dissociation of ARC and caspase 8 in vitro and in vivo, we hypothesize that in caspase 8-mediated apoptosis an increase of [Ca²⁺]_i dissociates ARC from pro-caspase 8 and leaves pro-caspase 8 prone to be activated. Therefore, full-length ARC is required for the interaction with caspase 8, which may explain why ARC, but not CARC, suppressed TNF--induced cell death (Fig. 4E).

ARC is known to localize in mitochondria, where ARC is phosphorylated and shows an inhibitory effect on caspase 8-mediated cell death (23). In overexpression analysis, ARC tends to localize in perinuclear or mitochondrial regions. After treatment with thapsigargin, subcellular localization of ARC apparently changed. However, Western blot analysis following two-dimensional electrophoresis showed that treatment with thapsigargin did not affect the phosphorylation status of ARC (see Fig. S2 in the supplemental material). The mechanism by which calcium modulates subcellular localization of ARC independent of phosphorylation status is yet unclear.

In many studies, a sustained elevation of [Ca²⁺]_i is observed during apoptosis. Whether there is an early Ca²⁺ signal existing in the commitment phase is still controversial; there are numerous observations, however, suggesting that Ca²⁺ signal may play an important role in the regulation of cell death. For example, Fas activation causes a marked [Ca²⁺]_i rise (19), and T lymphocytes with a deficiency of IP3R, which are incapable of mobilizing intracellular Ca²⁺, are resistant to Fas-mediated apoptosis. TNF- treatment also induces the release of Ca²⁺ from the ER Ca²⁺ pool (21, 30). These death ligand-induced [Ca²⁺]_i responses may allow caspase 8 to be dissociated from ARC and recruited into the death receptor-induced signaling complex. Further, though local concentrations of free Ca²⁺ vary in the cells, another calcium-activated proteins(s) may be required to facilitate dissociation of ARC-caspase 8 protein complexes responding to physiological changes in [Ca²⁺]_i. Thus, ARC function during TNF--induced cell death should be evaluated, because ARC not only exhibits Ca²⁺-buffering activity but also binds to caspase 8. While the molecular architecture of ARC and the caspase 8 complex remains to be further clarified, the present study sheds new light on the role of ARC in the regulation of caspase 8 and in Ca²⁺-mediated cell death.

 
We thank J. Blenis (Harvard University) for Jurkat cells (A3, I9-2), S. Stamm (Max-Planck Institute) for Nop30 cDNA, and Mark P. Mattson and S. L. Chan (National Institute on Aging Intramural Research Program, National Institutes of Health) for a critical reading.

This work was supported in part by the National Research Laboratory program (to Y. K. Jung), Systems Biology, Brain Research Center, and Functional Analysis of Human Genome of the 21st Century Frontier Research Program of the Korean Ministry of Science and Technology.

 
* Corresponding author. Mailing address: Department of Life Science, Gwangju Institute of Science and Technology, 1 Oryongdong, Bukgu, Gwangju 500-712, South Korea. Phone: 82-62-970-2492. Fax: 82-62-970-2484. E-mail: ykjung{at}gist.ac.kr.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, NIH, Baltimore, MD 21224.

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Molecular and Cellular Biology, November 2004, p. 9763-9770, Vol. 24, No. 22
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.22.9763-9770.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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