Article |
Address correspondence to Craig B. Thompson, Department of Cancer Biology, Abramson Cancer Center, 421 Curie Blvd., BRB II/III, 445, Philadelphia, PA 19104-6160. Tel.: (215) 746-5515. Fax: (215) 746-5511. E-mail: craig{at}mail.med.upenn.edu
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Abstract |
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Key Words: Bak; Bax; Ca2+; caspase 12; ER
* Abbreviations used in this paper: BMH, bismaleimidohexane; cb5, cytochrome b5; HM, heavy membrane; IRES, internal ribosomal entry site; LM, light membrane; MEF, murine embryonic fibroblast; t-caspase, truncated caspase; UPR, unfolded protein response.
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Introduction |
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The ER is the cytoplasmic compartment where proteins and lipids are synthesized and modified. Due to the presence of high concentrations of protein, numerous protein chaperones exist in the ER. These chaperones facilitate the proper folding of proteins, or maintain proteins in a folding-competent state and prevent protein folding intermediates from aggregating. In response to a number of stimuli that disrupt ER function, such as Ca2+ depletion from the ER lumen, inhibition of asparagine (N)-linked glycosylation, reduction of disulfide bonds, or overexpression of some proteins, protein misfolding occurs and unfolded proteins accumulate and aggregate in the ER. The misfolded and aggregated proteins then trigger the unfolded protein response (UPR).* Under less severe situations, UPR results in the reduction of general protein synthesis and selectively activates the expression of proteins facilitating the chaperone function (Kaufman, 1999). Under conditions of severe ER stress that the cells are not able to adapt to, UPR triggers apoptosis. UPR-induced apoptosis is associated with the up-regulation of the transcriptional factor CHOP/GADD153 and with eIF-2 phosphorylation (Kaufman, 1999), which may lead to the activation of a caspase cascade that involves the ER-specific caspase 12 (Nakagawa et al., 2000).
Bcl-2 family proteins play essential roles in regulating apoptosis. Whereas some members of the Bcl-2 family antagonize cell death, others exhibit proapoptotic activities. Bax and Bak are the proapoptotic members of the Bcl-2 family that are ubiquitously expressed in different tissues. They reside in the cytosol or on mitochondria and are kept in an inactive form in healthy cells. Overexpression of the multidomain death-promoting Bax-like proteins results in cell death in both mammalian and yeast cells (Oltvai et al., 1993; Chittenden et al., 1995; Hanada et al., 1995; Greenhalf et al., 1996; Jurgensmeier et al., 1997). When a cell is committed to apoptosis, Bax and Bak undergo conformational change, leading to their oligomerization and insertion in the mitochondria outer membrane (Hsu et al., 1997; Gross et al., 1998; Desagher et al., 1999; Griffiths et al., 1999; Wei et al., 2000; Makin et al., 2001). This process is followed by the release of cytochrome c from mitochondria to the cytosol. Cytochrome c binds to Apaf-1 and caspase 9, resulting in the activation of caspase 9, the subsequent activation of caspases 3 and 7, and ultimately cell death. Studies of the Bax/Bak doubly deficient mice indicate that the multidomain proapoptotic Bax and Bak proteins are required for mitochondria-mediated cell death (Lindsten et al., 2000; Wei et al., 2001; Zong et al., 2001).
Although intensive studies have been focusing on how Bcl-2 family proteins regulate apoptosis at the mitochondria level, evidence exists that Bcl-2 proteins may also function at the ER level. In addition to mitochondria, Bcl-2 is also found in the ER membranes (Cleary and Sklar, 1985; Krajewski et al., 1993; Akao et al., 1994; Lithgow et al., 1994; Nguyen et al., 1994). Overexpression of Bcl-2 and Bcl-xL blocks thapsigargin-induced apoptosis (Srivastava et al., 1999). A Bcl-2cytochrome b5 (cb5) mutant selectively targeted to the ER blocks apoptosis induced by ER stress resulting from tunicamycin and brefeldin A treatment (Hacki et al., 2000; Annis et al., 2001). This ER-targeted Bcl-2 mutant also protects cells from Bax-induced cell death (Wang et al., 2001). Fibroblasts lacking both Bax and Bak are resistant to ER stressinduced apoptosis, and ectopic expression of Bax in these doubly deficient cells restores their sensitivity (Wei et al., 2001; Zong et al., 2001). These findings suggest that Bax and Bak can either sense the ER damage and function on mitochondria to induce mitochondrial membrane permeabilizationmediated apoptosis, or that there exists an unknown function of Bax and Bak at the ER level. In the present study, we report that the multidomain proapoptotic Bax and Bak proteins also reside at the ER in addition to their mitochondrial localization. ER stress can induce conformational changes and oligomerization of Bax and Bak on the ER membrane. ER stressinduced cleavage of the ER-specific caspase 12 is dependent on Bax and Bak. Targeted expression of Bak in the ER results in ER Ca2+ depletion, caspase 12 processing, and ultimately cell death. These findings indicate that in addition to their roles on mitochondria, Bax/Bak proteins may also be involved in initiating apoptosis from the ER.
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Results |
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Caspase 12 activation is Bax/Bak dependent
Caspase 12 is specifically localized to the ER and has been reported to be cleaved during ER stressinduced apoptosis (Nakagawa et al., 2000). To investigate whether ER stressinduced caspase 12 cleavage is dependent on Bax and Bak, wild-type, bax-/-bak-/-, and NIH3T3 cells were treated with brefeldin A, thapsigargin, and tunicamycin. No consistent differences in total caspase 12 expression were observed between wild-type MEFs, NIH3T3 cells, and bax-/-bak-/- MEFs. However, in both wild-type MEFs and NIH3T3 cells, an 47-kD fragment was revealed in untreated cells, indicating that there exists a basal processing of caspase 12 in these cells, which does not lead to apoptosis (Fig. 3 A). The ER stress treatment resulted in further caspase 12 cleavage, shown as the appearance of an
42-kD fragment in both wild-type MEFs and NIH3T3 cells. In contrast, the caspase 12 processing was not observed in bax-/-bak-/- cells (Fig. 3 A). Interestingly, the UPR-responsive transcriptional factor CHOP was induced by ER stress treatment in all three cell lines, including bax-/-bak-/- cells, indicating that cells deficient in Bax and Bak are still responding to ER stressinduced UPR (Fig. 3 A). If caspase 12 activation is dependent on Bax and Bak, it can be postulated that the killing activity of caspase 12 lies downstream in the apoptotic pathway initiated by these multidomain proapoptotic Bcl-2 proteins. We examined whether enforced activation of caspase 12 can kill cells deficient in Bax and Bak. As expected, ectopically expressed caspase 3 and caspase 9 resulted in apoptosis in both wild-type and bax-/-bak-/- MEFs (Fig. 3 B). Similarly, overexpression of full-length and truncated caspase 12 (t-caspase 12) induced cell death in both wild-type and bax-/-bak-/- cells, and t-caspase 12 had a higher killing activity than the full-length caspase 12, as previously described (Fig. 3 B; Nakagawa and Yuan, 2000).
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The ER-targeted Bak can induce apoptosis
Expression of wild-type Bak, Bak-ActA, and Bak-cb5 induced apoptosis in wild-type MEFs (Fig. 6), as well as in HeLa, MCF7, and 293T cells (unpublished data), whereas the carboxy terminusdeleted Bak-C mutant did not cause cell death. It is possible that the mitochondria- and ER-targeted Bak mutants kill cells by simply breaking the balance between the endogenous anti- and proapoptotic Bcl-2 family proteins. Previous studies have demonstrated that cells deficient in both Bax and Bak are defective in apoptosis. Therefore, we expressed the mitochondria- and ER-targeted Bak mutants in bax-/-bak-/- cells to examine whether the multidomain proapoptotic Bak-like proteins can directly initiate apoptosis at the ER. As shown in Fig. 6 A, wild type and mitochondria- and ER-targeted Bak mutants all killed the bax-/-bak-/- cells (Fig. 6 A). Quantitation of cell death by DAPI exclusion showed that the ER-targeted Bak has more potent killing activity than the wild type and the mitochondria-targeted Bak in wild-type and bax-/-bak-/- cells (Fig. 6 B). This higher ability of Bak-cb5 to induce apoptosis when compared with wild-type Bax could not be accounted for by a difference in the level of expression, as wild-type Bak was consistently expressed at higher levels than Bak-cb5 after retroviral infection (unpublished data). These data indicate that in the absence of endogenous proapoptotic proteins Bax and Bak, the multidomain proapoptotic Bcl-2 proteins can initiate apoptosis directly from the ER. Overexpression of Bcl-xL blocked the proapoptotic activities of all Bak mutants (Fig. 6 B), indicating that these mutants, including Bak-cb5, induce apoptosis through a physiological manner that is blockable by the antiapoptotic molecule Bcl-xL. Interestingly, the killing activities of the Bak mutants were also blocked by the pan-caspase inhibitor z-VAD-fmk, suggesting that caspase activation may be involved in the death-inducing activities of these mutants (Fig. 6 B).
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The ER-targeted Bak mutant specifically induces caspase 12, but not caspase 7, cleavage
As caspase 12 cleavage is dependent on Bax and Bak, and the ER-targeted Bak can lead to apoptosis, we determined whether expression of the ER-targeted Bak in bax-/-bak-/- cells results in caspase 12 processing. Wild-type Bak and the ER- and mitochondria-targeted Bak mutants were retrovirally expressed in bax-/-bak-/- MEFs. Expression of Bak-cb5 resulted in cleavage of caspase 12, whereas little caspase 12 cleavage was observed in wild-type Bakexpressing cells, and virtually no caspase 12 cleavage was observed in Bak-ActAexpressing cells (Fig. 7). In contrast, expression of either wild-type Bak or Bak-ActA mutant resulted in cleavage of caspase 7 and PARP, whereas little to no caspase 7 and PARP cleavage was observed in Bak-cb5infected cells (Fig. 7). Thus, despite the fact that Bak-cb5 induces apoptosis, it does not appear to activate the mitochondrial apoptosis machinery in the bax-/-bak-/-cells that lack the mitochondrial Bax and Bak. Moreover, these findings indicate that increased cleavage of caspase 12 in Bak-cb5expressing cells is not merely due to higher levels of cell death, but rather that distinct caspases are selectively activated depending on whether Bak is localized primarily to the ER or mitochondria.
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Discussion |
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The ER is the main intracellular storage compartment for Ca2+, which is an important secondary messenger that is required for numerous cellular functions. Apoptosis occurs upon the perturbation of cellular Ca2+ homeostasis, such as cytosolic Ca2+ overload, ER Ca2+ depletion, and mitochondrial Ca2+ increase. The close physical contact of mitochondria and the ER results in a higher exposure to Ca2+ of mitochondria than the rest of the cytosol, when Ca2+ is released from the ER (Rizzuto et al., 1998). Bcl-2 family proteins have been previously implicated in controlling apoptosis by affecting cellular Ca2+ homeostasis. Overexpression of Bcl-2 leads to reduced mitochondrial Ca2+ stores and enhanced ER sequestration, whereas overexpression of either wild-type Bax or Bak has the opposite effect (Pinton et al., 2000; Pan et al., 2001; Abeele et al., 2002; Li et al., 2002; Nutt et al., 2002a, b). This has been interpreted to mean that Bax and Bak interfere with mitochondrial Ca2+ homeostasis or initiate apoptosis by affecting the function of the mitochondrial permeability transition pore. The present data suggest that Bax and Bak localized to the ER can directly affect ER Ca2+ stores. In bax-/-bak-/- cells, targeting the Bak-cb5 mutant to the ER resulted in acute ER Ca2+ depletion, indicating a direct effect of the multidomain proapoptotic proteins on ER Ca2+ stores (Fig. 5). The effect on ER Ca2+ appears to result from the fact that the ER-targeted Bak-cb5 protein undergoes activation directly upon insertion in the ER membrane, as demonstrated by its adopting an active Bak configuration when introduced into cells by retroviral infection. In contrast, secondary factors, such as endogenous BH3-only proteins, must activate wild-type Bak, as it retains an inactivated configuration and does not induce significant Ca2+ release. When apoptosis is initiated in wild-type Bakexpressing cells, it induces a mixture of events associated with apoptosis from the two targeted Bak constructs.
In mitochondria-initiated apoptosis, the activation of Bax and Bak involves BH3-only proteins. The presence of several BH3-only proteins, such as tBid, Bim, Noxa, and Puma, at mitochondria when apoptosis is triggered has suggested that the activation of Bax/Bak by BH3-only proteins may take place at this site (Li et al., 1998; Luo et al., 1998; Puthalakath et al., 1999; Oda et al., 2000; Nakano and Vousden, 2001; Yu et al., 2001). It is possible that some of the BH3-only proteins may also localize to the ER and trigger Bax and Bak activation at the ER, as suggested by recent findings that the ER localization of the BH3-only protein BIK/NBK was induced by apoptotic stimuli, and that a BIK/NBK mutant specifically targeted to the ER retained the capability of inducing cytochrome c release and apoptosis (Germain et al., 2002; Mathai et al., 2002). It is of interest to determine whether other BH3-only proteins can also function at the ER and whether novel ER-specific BH3-only proteins may exist.
Caspase 12 has been demonstrated to be specifically localized to the ER (Nakagawa et al., 2000). Although a required role for caspase 12 in the induction of apoptosis has not been established, the finding that caspase 12 cleavage is dependent on Bax and Bak suggests that caspase 12 processing lies downstream of Bax and Bak. Interestingly, expression of the ER-targeted Bak-cb5 resulted in a significant increase in caspase 12 processing, whereas expression of wild-type or the mitochondrial Bak-ActA resulted in preferential cleavage of caspase 7 and PARP. These data suggest that in addition to their role in mitochondrial apoptosis, Bax and Bak localized to the ER can initiate an alternative pathway of caspase activation that results in selective caspase 12 activation (Fig. 8).
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Materials and methods |
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DNA constructs
Murine Bak cDNA was cloned into pBabeMN-IRES-GFP to generate plasmid pBabe-Bak-GFP (Bak-IRES-GFP). A stop codon was introduced after amino acid 183 by PCR to generate plasmid pBabe-Bak-C-IRES-GFP (Bak-
C-IRES-GFP). Cb5 and ActA DNA fragments (Zhu et al., 1996) were generated by PCR and subsequently subcloned after amino acid 183 to make pBabe-Bak-cb5-IRES-GFP (Bak-cb5-IRES-GFP) and pBabe-Bak-ActA-IRES-GFP (Bak-ActA-IRES-GFP), respectively. Plasmids used for localization experiments were made by subcloning appropriate DNA fragments from pBabeMN-IRES-GFP into pEGFP-C1 (CLONTECH Laboratories, Inc.). Expression constructs for caspase 3 (pBK5-caspase 3) and caspase 9 (pBK5-caspase 9) are a gift from X. Yang (University of Pennsylvania, Philadelphia, PA). Expression constructs for caspase 12 (pcDNA3.1-caspase 12) and t-caspase 12 (pcDNA3.1-t-caspase 12) were previously described (Nakagawa and Yuan, 2000).
Retroviral infection
Phoenix packaging cells were transfected with retroviral constructs. Viral supernatants were collected during the 4896-h period after transfection and centrifuged at 2,000 rpm for 10 min to remove contaminating packaging cells. MEFs were infected with the viral supernatant in the presence of 10 µg/ml of polybrene (Sigma-Aldrich).
Cell death assay
Cells were retrovirally infected with GFP or Bak mutantGFP. 48 h after infection, a total of 1 µg/ml DAPI was added to cell culture. Cells were observed and photographed with the Nikon Eclipse TE300 microscope and subjected to flow cytometry to determine the number of DAPI-positive cells over that of GFP-positive cells. In transient transfection assays, cells were transfected with 0.3 µg pEGFP together with 1.8 µg pBK5-caspase 3, pBK5-caspase 9, pcDNA3.1-caspase 12, or pcDNA3.1-t-caspase 12 using Geneporter2 transfection reagent (Gene Therapy Systems). 24 h later, cells were stained with 1 µg/ml DAPI, and the percentage of cell death was determined by the ratio of DAPI-positive cells to GFP-positive cells.
Immunoelectronic microscopy
Cells were rinsed briefly with prewarmed serum-free medium and then fixed with 2% paraformaldehyde and 0.05% glutaraldehyde for 30 min. Cells were collected to make a pellet by centrifugation, washed with PBS, and dehydrated at -25°C with ethanol. Cells were infiltrated with a mixture of ethanol/Lowicryl K4M medium, embedded in pure Lowicryl K4M, and polymerized at -25°C for 5 d with UV light at 365 nm. Ultrathin sections (80 nm) were cut with a diamond knife, mounted on Formvar-coated thin-bar nickel grids, and labeled with established protocols (Allen et al., 1996) using the antibodies against Bax (6A7; BD Biosciences) and Bak (NT; Upstate Biotechnology).
Subcellular fractionation
Cells were resuspended in hypotonic buffer A (250 mM sucrose, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1x protease inhibitor complex [Roche]) on ice for 30 min. Cells were disrupted by passing through 26-gauge needles 30 times, and then 30-gauge needles 20 times. Cell lysates were centrifuged at 750 g for 10 min at 4°C to get rid of unlysed cells and nuclei. The supernatant was centrifuged at 10,000 g for 20 min at 4°C. The pellet was saved as the HM (mitochondria) fraction. The supernatant was centrifuged at 100,000 g for 1 h at 4°C. The supernatant was saved as the S-100 (cytosol) fraction and the pellet as the LM (ER/microsome) fraction. The HM and LM fractions were lysed in RIPA buffer (1% sodium deoxycholine, 0.1% SDS, 1% Triton X-100, 10 mM Tris, pH 8.0, 0.14 M NaCl) for Western blotting. For Fig. 1 B, a mouse liver was homogenized in buffer A with four strokes using Dounce homogenizer. The lysate was centrifuged at 750 g for 10 min at 4°C to get rid of unlysed cells and nuclei. The postnuclei supernatant was centrifuged at 6,000 g for 30 min to obtain the pellet as crude mitochondria. Crude mitochondria were resuspended in 0.5 ml buffer A, top loaded to a 12 M continuous sucrose gradient, and centrifuged at 25,000 rpm in an SW28 rotor for 90 min. Fractions were collected using a 16G needle into collection tubes at a rate of 0.75 ml/tube. The postmitochondrial supernatant was adjusted with 2.5 M sucrose to the final concentration of 1.8 M and bottom loaded into an SW41 centrifuge tube. 1.5 ml each of 1.5, 1.3, 1.0, 0.86, and 0.25 M sucrose was added to fill the centrifuge tube. The sample was centrifuged at 41,000 rpm for 6 h at 4°C. The material formed at the interface between the 1.5 and 1.8 M sucrose layers was collected. All fractions were diluted with threefold buffer A and centrifuged at 41,000 rpm for 90 min in an SW55 rotor. Pellets were dissolved in 100 µl RIPA buffer. 10 µl of each fraction was loaded into a 412% NuPAGE gradient gel for immunoblotting analysis.
Measurement of [Ca2+]i
Cells were resuspended in RPMI medium 1640 with 1% FBS and loaded with 3 µM Indo-1 (Molecular Probes) in the presence of 4 mM probenecid at 37°C for 30 min. Loaded cells were washed twice and resuspended in HBSS without calcium (Invitrogen). The measurement of cytosolic free Ca2+ concentration ([Ca2+]i) was performed on an LSR flow cytometer (Becton Dickinson). In brief, 2 min before analysis, EGTA was added to each sample to a final concentration of 1.5 mM. After stable basal [Ca2+]i was achieved, cells were treated with thapsigargin (5 µM) to release the ER Ca2+ pool. Data analysis was performed by using FlowJo software (Tree Star), and [Ca2+]i was calculated from the equation [Ca2+]i = Kd · (R - Rmin) · F( = 475)min/(Rmax - R) · F(
= 475)max, where the values of Rmin, Rmax, Fmin, and Fmax were determined by in situ calibration. Indo-1loaded cells were resuspended in RPMI.
Detection of Bax and Bak conformational changes
Analysis of the conformational changes of Bax and Bak was performed as described previously (Griffiths et al., 1999; Mandic et al., 2001). Cells were treated with thapsigargin (2 µM) or tunicamycin (5 µg/ml) for 48 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were washed three times with PBS and incubated with 1:50 antimouse IgG1 (BD Biosciences), anti-Bax (clone 6A7; BD Biosciences), anti-Bak (AM03; Oncogene Research Products), or anti-Bak (NT; Upstate Biotechnology) in 100 µg/ml digitonin in PBS for 30 min. After being washed with PBS three times, cells were incubated with 1:100 FITC-conjugated antimouse Ig and 1 µg/ml DAPI for 30 min. Cells were subjected to analysis using an LSR cytometer.
Chemical cross-linking
Wild-type MEFs or NIH3T3 cells were treated with brefeldin A (10 µg/ml), thapsigargin (2 µM), or tunicamycin (1 µg/ml) for 24 h. Cells were harvested and disrupted with 26-gauge needles. The cell lysates were incubated with 5 mM BMH (Pierce Chemical Co.) for 30 min at room temperature. Subcellular fractionation was performed to obtain HM and LM pellets. The pellets were lysed in RIPA buffer for immunoblotting.
Immunoblotting and immunofluorescence
20 µg of total protein was resolved in precast 412% NuPAGE gradient gels. Immunoblotting was performed by use of antibodies against Bax (N-20; Santa Cruz Biotechnology, Inc.), Bak (Upstate Biotechnology), COX IV (Molecular Probes), Tom40 (Santa Cruz Biotechnology, Inc.), calnexin (C-20; Santa Cruz Biotechnology, Inc.), caspase 12, and CHOP (B-3; Santa Cruz Biotechnology, Inc.). For immunofluorescence, cells were fixed in 4% paraformaldehyde and permeabilized for 10 min in PBS containing 0.2% Triton X-100. Cells were washed with PBS containing 0.02% Triton X-100 and 1.5% FBS, followed by incubation with antibodies against COX IV or calnexin (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Cells were incubated with rhodamine red or FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Nuclei were visualized by staining with 1 µg/ml DAPI. Images were captured on a 510 LSM confocal microscope (Carl Zeiss MicroImaging, Inc.).
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Acknowledgments |
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W.X. Zong is a recipient of the postdoctoral fellowship from the Cancer Research Institute. C. Li is supported by National Institutes of Health training grant T32 (CA09140). G. Hatzivassiliou is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG1714-02).
Submitted: 14 February 2003
Revised: 13 May 2003
Accepted: 23 May 2003
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