1 Department of Radiation Oncology, University of Tübingen, Hoppe-Seyler Str. 3, D-72076 Tübingen, Germany
2 Department of Physiology I, University of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany
3 Department of Immunology and Cell Biology, Institute of Experimental Dermatology, University of Münster, Röntgenstr. 21, D-48149 Münster, Germany
4 Department of Internal Medicine I, University of Tübingen, Ottfried-Müller Str. 10, D-72076 Tübingen, Germany
*Author for correspondence (e-mail: claus.belka{at}uni-tuebingen.de)
Accepted August 16, 2001
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SUMMARY |
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Bcl-2/WT and Bcl-2/MT strongly inhibited radiation-induced apoptosis and caspase activation, whereas Bcl-2/TM had completely lost its anti-apoptotic effect. Interestingly, Bcl-2/ER conferred protection against radiation-induced mitochondrial damage and apoptosis similarly to Bcl-2/MT. The finding that ER-targeted Bcl-2 interfered with mitochondrial
m breakdown and caspase-9 activation indicates the presence of a crosstalk between both organelles in radiation-induced apoptosis. By contrast, Bcl-2 in either subcellular position did not influence CD95- or TRAIL-mediated apoptosis.
Key words: Bcl-2, Mitochondria, Endoplasmic reticulum, Apoptosis, Radiation, CD95
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INTRODUCTION |
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The search for human homologues revealed that CED-3 is a cysteine protease resembling the caspase protease family in higher eukaryotes (Miura et al., 1993), whereas CED-4 is both structurally and functionally related to APAF-1 (Zou et al., 1997). However, the linear model of apoptosis induction in C. elegans is opposed by more complex apoptosis systems in vertebrates. Up to now two major signaling cascades have been analyzed in greater detail.
A widely accepted mammalian pathway with significant similarities to the apoptotic cascade in C. elegans is involved in cell death in response to DNA damage and cellular stress. The transcriptional activation of Noxa or similar pro-apoptotic molecules are key events of apoptosis mediated by p53 (Lowe et al., 1993; Oda et al., 2000). Other pro-apoptotic members of the Bcl-2 protein family such as Bim may be involved in apoptosis since deletion of Bim was shown to confer partial resistance against apoptosis induced by gamma-irradiation (Bouillet et al., 1999).
Noxa and Bax induce mitochondrial damage with the breakdown of the mitochondrial membrane potential and release of cytochrome c (Jurgensmeier et al., 1998; Oda et al., 2000; Pastorino et al., 1998). The translocation of cytochrome c triggers the mitochondrial apoptosis pathway in a strictly Bcl-2/BclxL-controlled manner (Hu et al., 1998; Yang et al., 1997). Cytoplasmic cytochrome c forms a complex with APAF-1 which oligomerizes and recruits the initiator caspase-9 for autoactivation (Li et al., 1997). Activated caspase-9 in turn activates caspase-3 and other effector caspases leading to the apoptotic phenotype through the cleavage of several vital proteins (Li et al., 1997).
The second well-characterized cascade is activated by binding of ligands of the tumor necrosis factor family (e.g. CD95/Fas/Apo-1 ligand (CD95L) or TRAIL/Apo-2L) to their cognate surface receptors. The CD95 signaling pathway is paradigmatic for the whole family of death receptors. Binding of CD95L to its receptor mediates the recruitment of the adapter molecule FADD, which in turn attracts procaspase-8, which undergoes autoproteolytic activation (Muzio et al., 1996). Subsequently, caspase-8 directly activates downstream effectors including caspase-3 (Stennicke et al., 1998). This simple model of CD95-mediated apoptosis is complicated by the existence of an amplification loop that is required for the rapid execution of cell death in cases of low initial caspase-8 activation (Scaffidi et al., 1998). In this scenario, caspase-8 activates the pro-apoptotic Bcl-2 member BID by removing its N-terminal domain. Truncated BID then translocates to mitochondria where it induces cytochrome c release (Li et al., 1998; Luo et al., 1998). Cytoplasmic cytochrome c in turn triggers the APAF1/caspase-9 cascade, thereby amplifying the initial death receptor signal. This amplification mechanism is particularly observed in so-called type-II cells, which are efficiently protected by Bcl-2, whereas in many other cell types (type-I cells) Bcl-2 does not strongly interfere with death receptor-mediated apoptosis (Strasser et al., 1995; Scaffidi et al., 1998; Huang et al., 1999; Belka et al., 2000; Engels et al., 2000).
Although it is generally accepted that Bcl-2 and Bcl-xL inhibit mitochondrial damage, a precise understanding of their function is hampered by the complexity of mammalian apoptotic cascades. The localization of Bcl-2/Bcl-xL in different subcellular compartments and a multitude of described interactions of Bcl-2/Bcl-xL with various components of the apoptotic machinery may contribute to the anti-apoptotic effects. Attempts have been made to predict functions from the molecular structure of Bcl-2/Bcl-xL. Through interaction of the hydrophobic cleft that is formed by critical residues within the BH1, BH2 and BH3 domains, Bcl-xL interacts with the BH3 domain of pro-apoptotic molecules including Bax, Bak and Bid (Muchmore et al., 1996; Oltvai et al., 1993; Sattler et al., 1997; Wang et al., 1996). The ability to form heterodimers was shown to be crucial for the anti-apoptotic function of Bcl-xL in some models (Sedlak et al., 1995). By contrast, other studies (Cheng et al., 1996; Minn et al., 1999) provided evidence that those Bcl-xL mutants that do not interact with Bax, retain significant anti-apoptotic potency in yeast and higher eukaryotes. Thus, the anti-apoptotic capacity of Bcl-2 proteins may be only partially mediated by the functional sequestration of pro-apoptotic family members. Independently of the capability to heterodimerize with pro-apoptotic proteins, Bcl-2/Bcl-xL were shown to form ion channels in artificial lipid bilayers. Indeed, the 3D structures of Bcl-2 and Bcl-xL are related to pore-forming bacterial toxins. The ion channel activity of Bcl-2/Bcl-xL may maintain ion homeostasis across mitochondrial and other biological membranes. Thus, evidence exists that, in addition to the interaction with pro-apoptotic family members, the capability to form ion channels may be a key element of Bcl-2 function.
Although the analysis of the Bcl-2/Bcl-xL structure helps to clarify functional aspects of the anti-apoptotic action, several aspects remain unclear. For example, Bcl-2 and Bcl-xL interfere with many stages of the mitochondrial apoptosis pathway. Most importantly, it was shown that Bcl-2 blocks the release of cytochrome c from mitochondria (Kluck et al., 1997). However, other mitochondrial functions are influenced by Bcl-2/Bcl-xL as well. It has been shown that Bcl-xL abolished growth factor deprivation-induced cell death by facilitating mitochondrial ATP/ADP exchange (Vander Heiden et al., 1999). In addition, Bcl-xL prevents mitochondrial swelling and outer membrane rupture in response to CD95 stimulation or staurosporine treatment (Vander Heiden et al., 1997).
Apart from direct effects on mitochondria, Bcl-2 was shown to block apoptosis induction in response to microinjection of cytochrome c into cells, suggesting that Bcl-2 may also act downstream of cytochrome c (Brustugun et al., 1998). Since CED-9 directly interferes with CED-4 in C. elegans (Yang et al., 1998), Bcl-2/BclxL were proposed to directly bind to and antagonize mammalian APAF-1. A specific mutation of CED-9, namely the G169E substitution in the CED-9(n1950) mutant, leads to a loss of CED-9 interaction with the BH3-only protein EGL-1. As a result of this impaired interaction, EGL-1-induced release of CED-4 from CED-4/CED-9 complexes is also impaired in the gain-of-function mutant (Parrish et al., 2000). In contrast to CED-4, it was shown recently that Bcl-2/Bcl-xL do not physically interact with APAF-1 and the proteins are not found in similar cellular locations (Hausmann et al., 2000; Moriishi et al., 1999). However, this does not exclude the possibility that additional CED-4-like adapter molecules might be inactivated by Bcl-2. Alternatively, the recently described protein Aven, which binds to both APAF-1 and Bcl-xL, may act as bridging protein and thus potentially mediate Bcl-2/Bcl-xL effects downstream of cytochrome c (Chau et al., 2000).
In addition to mitochondria, Bcl-2 localizes to other biological membranes, in particular the endoplasmic reticulum and nuclear membrane (Lithgow et al., 1994), raising the possibility that Bcl-2 acts at extramitochondrial sites. However, the role of Bcl-2 in the ER is poorly understood. It was suggested that Bcl-2 and related proteins interfere with intracellular calcium stores and calcium release, which may be relevant for apoptosis induction and related to its ion channel activity. Bcl-2 decreases the ER calcium content by increasing the calcium permeability of the ER membrane (Foyouzi-Youssefi et al., 2000; Pinton et al., 2000). Other studies, by contrast, demonstrated that Bcl-2 causes an interference with ER calcium release and shift from the ER to mitochondria (Baffy et al., 1993; He et al., 1997).
Although a multitude of data on potential functions of Bcl-2 and other family members is available, the precise mechanisms still remain elusive. In particular, the contribution of the subcellular localization of Bcl-2 to its anti-apoptotic function is unclear. In order to gain further insight into the role of the Bcl-2 subcellular localization for its anti-apoptotic effects, we expressed organelle-specific mutants of Bcl-2 in Jurkat cells and investigated their effect on apoptosis induced by either ionizing irradiation or death receptors. We assessed the effect of wild-type Bcl-2, a cytoplasmic version lacking the transmembrane domain and Bcl-2 mutants targeted to either mitochondria or the endoplasmic reticulum. Our data indicate the existence of a molecular crosstalk between the endoplasmic reticulum and mitochondria in radiation-induced apoptosis, which can be interrupted by Bcl-2 at the level of the endoplasmic reticulum.
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MATERIALS AND METHODS |
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Cell culture and transfections
Jurkat E6 T-lymphoma cells were from ATCC (Bethesda, MD). For all experiments cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Gibco Life Technologies, Eggenstein, Germany) and maintained in a humidified incubator at 37°C and 5% CO2. Expression vectors encoding wild-type Bcl-2 (Bcl-2/WT), cytoplasmic Bcl-2 lacking the transmembrane domain (Bcl-2/TM) and Bcl-2 mutants restricted to the outer mitochondrial membrane (Bcl-2/MT) or endoplasmic reticulum (Bcl-2/ER) were kindly provided by B. Leber (Ontario, Canada). Mitochondrial and ER-specific targeting of Bcl-2 was achieved by exchanging the C-terminal insertion sequence of Bcl-2 with equivalent sequences of the listerial protein ActA and rat cytochrome b5, respectively (Zhu et al., 1996). Jurkat cells stably expressing the different versions of Bcl-2 and the respective vector control were prepared by electroporation using a Gene pulser II (BioRad, Munich, Germany) and were primarily used as pool transfectants. Parallel experiments were performed with individual clones derived from the bulk transfected culture by limiting dilution.
Radiation, CD95 and TRAIL stimulation
Cells were irradiated with 6 MV photons from a linear accelerator (LINAC SL25 Phillips) with a dose rate of 4 Gy/min at room temperature (RT). CD95 stimulation was performed using the agonistic IgM antibody CH11 (100 ng/ml; UBI Biomol, Hamburg, Germany). TRAIL receptors were stimulated using recombinant human flag-tagged TRAIL and a crosslinking antibody (Alexis Biochemicals, Grünberg, Germany)
Confocal microscopy
The subcellular localization of Bcl-2 was verified by confocal microcopy using a Leica TCS NT microscope. In brief, cells were immobilized on coverslips with 0.1% poly-L-lysine, fixed with 2.5% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 10% fetal calf serum cells were stained with primary antibodies for 1 hour at RT. Cells were washed several times and incubated with secondary antibodies for 30 minutes. Finally, the coverslips were mounted with Mowiol (Sigma). Bcl-2 staining was performed with a specific rabbit antibody (Santa-Cruz-Biotech, Heidelberg, Germany). Localization of Bcl-2 in the ER or mitochondria was confirmed by costaining with a murine antibody against the calcium pump SERCA (UBI Biomol) and a mouse anti-cytochrome c antibody (PharMingen, Hamburg, Germany). As secondary antibodies Alexa FluorTM-conjugated anti-mouse (Molecular Probes, Mobitech, Göttingen, Germany) and CyTM5-conjugated anti-rabbit antibodies (Dianova, Hamburg, Germany) were employed.
Determination of apoptosis
Apoptosis was quantified using scatter characteristics in a FACS Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany). In addition, apoptosis induction was measured by DNA fluorescence microscopy. To this end, cells were incubated with Hoechst 33342 at a final concentration of 1.5 µM for 15 minutes. Cell morphology was then determined by fluorescence microscopy (Zeiss Axiovert 135, Carl Zeiss, Jena, Germany) using an excitation wavelength filter of 380 nm. At least five independent experiments were performed. The given error bars represent the standard deviation from independent measurements of the same cell batch.
Immunoblotting
Cells (1x105) were lysed in a buffer containing 25 mM Hepes, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, 10 mM EDTA, 10 mM NaF and 125 mM NaCl. After removing insoluble material by centrifugation for 10 minutes at 12,000 g, 20 µg of lysate was separated by SDS-PAGE. Blotting was performed in a tank blotting apparatus (BioRad) onto Hybond ECL membranes (Amersham, Braunschweig, Germany). Equal protein loading was confirmed by Ponceau S staining. Blots were blocked in TBS buffer containing 0.05% Tween-20 and 5% fetal calf serum at RT for 15 minutes. Then, the primary antibodies were applied for 1 hour. After repeated washings with TBS/Tween-20 (0.05%) the membrane was incubated with the secondary antibody (anti IgG-AP 1:20.000, Santa-Cruz-Biotech) in TBS/Tween for 30 minutes at RT and washed three times with TBS/Tween. Antibody binding was detected by enhanced chemoluminescence staining. The anti-caspase-3, anti-caspase-9 and anti-PARP antibodies were from cell signaling (NEB, Frankfurt, Germany). Caspase-8 activation was detected as described previously (Belka et al., 2000) employing a mouse monoclonal antibody directed against the p18 subunit at a 1:50 dilution of the hybridoma supernatant. Bcl-2 expression was tested using a Bcl-2 antibody (Santa-Cruz-Biotech). Caspase-12 was detected using a rat polyclonal serum against caspase-12 (Ab-2, Oncogene Research Products, San Diego, CA)
Determination of the mitochondrial potential
The mitochondrial transmembrane potential (m) was analyzed using the
m-specific stain TMRE (Molecular Probes, Mobitech). In brief, 1x105 cells were stained at the indicated time points in a solution containing 25 nM TMRE for 30 minutes. Staining was quantified by flow cytometry using the FL2 channel and scatter characteristics.
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RESULTS |
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Confocal analysis of cells overexpressing Bcl-2/WT revealed a localization at the ER, the perinuclear region and mitochondria (Fig. 1D). A discrete background staining detectable in all cell lines was due to a low-level expression of endogenous Bcl-2 (Fig. 2A).
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We therefore asked whether an apoptotic pathway that is essentially controlled by mitochondria is affected by the subcellular distribution of Bcl-2. As shown in Fig. 2B, ionizing radiation induced time-dependent apoptosis in Jurkat cells, which was strongly inhibited by wild-type Bcl-2. The Bcl-2 mutant lacking the transmembrane domain was unable to interfere with radiation-induced apoptosis indicating a critical need for a membrane anchor. Surprisingly, however, mitochondrial and ER-targeted Bcl-2 inhibited radiation-induced cell death to a very similar extent, suggesting that both the ER and mitochondria are key elements in radiation-induced apoptosis.
We next analyzed whether the activation of caspases was influenced by overexpression of Bcl-2 in different subcellular compartments (Fig. 3). In contrast to the death receptor pathway, caspase-9 is the most apical caspase activated by ionizing radiation downstream of mitochondrial release of cytochrome c. Thus, we expected that overexpression of Bcl-2/WT and Bcl-2/MT would interfere with radiation-induced activation of caspase-9. Irradiation of Jurkat cells induced a time-dependent processing of caspase-9 as demonstrated by the appearance of the p35 cleavage fragment that is generated through autocatalytic activation of caspase-9 at D315. (Fig. 3). As expected, capase-9 activation was inhibited by Bcl-2/WT but not by Bcl-2/TM. Interestingly, Bcl-2/ER and Bcl-2/MT efficiently prevented caspase-9 processing also.
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In addition, we analyzed the effect of Bcl-2 on radiation-induced caspase-8 activation which, in contrast to CD95, is not mediated by FADD-induced proximity but by a postmitochondrial event (Strasser et al., 1995; Huang et al., 1999; Belka et al., 2000; Huang et al., 1999; Newton and Strasser, 2000). Caspase-8 is synthesized as two isoforms of 54 and 52 kDa (caspase-8a and -8b) which, following formation of intermediate cleavage products of 43 and 41 kDa, are processed to a p18 and p10 heterodimer. Radiation induced a pronounced processing of caspase-8 that was strongly prevented by both mitochondrial and ER-targeted Bcl-2. The slight activation of caspases corresponded to the background level of apoptosis and was presumably due to a lower expression level of Bcl-2 and the mutants in a few cells of the pooled transfectants.
Since mitochondrial damage is considered as a key step for DNA damage-induced apoptosis, it was speculated that Bcl-2-like proteins generally act by protecting mitochondrial integrity. Our data suggest that at least some aspects of Bcl-2 action may be also regulated at the level of the ER. To determine how far mitochondrial damage is influenced by Bcl-2 in different subcellular positions, the integrity of mitochondrial function was analyzed by determining the mitochondrial potential (m).
m was measured using the potential-sensitive stain TMRE and subsequent flow cytometry. In a first set of experiments we analyzed how far radiation-induced breakdown of
m in Jurkat cells is a primary event and does not occur secondarily to caspase activation. Therefore, Jurkat cells were pretreated with the broad-spectrum caspase inhibitor zVAD (20 µM), leading to a complete inhibition of effector caspases during radiation-induced apoptosis (not shown). In parallel, PARP cleavage was fully abrogated (Fig. 4). However, no influence on the breakdown of
m could be detected (Fig. 4A). Thus, the breakdown of
m is an initial event during radiation-induced apoptosis and therefore suitable to determine the maintenance of mitochondrial integrity by Bcl-2.
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In addition, caspase-9 activation was analyzed using an antibody directed against its p35 subunit. Again, Bcl-2/WT and Bcl-2/MT delayed and weakened the activation of caspase-9 (Fig. 7). In parallel, cleavage of the caspase-3 substrate PARP to its characteristic 85 kDa fragment was determined (Fig. 7). In accordance with the previous data, Bcl-2 targeted to mitochondria and the wild-type form weakly interfered with the time course of caspase activation, whereas the soluble, cytosolic and ER-specific Bcl-2 mutants had almost no effect.
An additional feature of CD95-induced apoptosis is the breakdown of the mitochondrial membrane potential (m). Therefore, we tested how far Bcl-2 and the differentially expressed mutants interfered with
m breakdown. As shown in Fig. 8, 24 hours after treatment of cells with anti-CD95 or TRAIL a complete loss of the mitochondrial potential was observed in almost all cells regardless of Bcl-2 expression. However, slight differences in the proportion of cells with low and high
m were detectable 6 hours and still 12 hours after stimulation in those cells, in which either Bcl-2/WT or Bcl-2/MT were expressed.
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Involvement of caspase-12
Up to this point our results suggested that ER-controlled steps are involved in radiation-induced apoptosis. Recent data suggested that the activation of caspase-12 is involved in a specific form of apoptosis in the ER unfolded protein response that can be induced by brefeldin A or tunicamycin (Nakagawa et al., 2000). In knockout mice it was shown that apoptosis induction by other stimuli including CD95 and staurosporine did not require the presence of caspase-12. Using a polyclonal serum directed against caspase-12 a single band corresponding to the p53 proform of caspase-12 was detected (Fig. 9). After irradiation with 10 Gy a smaller fragment of approximately 17 kDa became visible. Overexpression of Bcl-2 in either position abrogated the processing of caspase-12 (Fig. 9A). Since caspase-9 is considered as the key caspase for radiation-induced apoptosis, we tested whether the observed weak cleavage of caspase-12 might be secondary to the activation of caspase-9. As shown in Fig. 9B, pretreatment with 50 µM of the caspase-9 LEHD-fmk abrogated caspase-9, caspase-3 as well as caspase-12 processing, suggesting that the observed cleavage of caspase-12 was downstream of caspase-9.
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DISCUSSION |
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There are only a few data available regarding the importance of Bcl-2 localization for its anti-apoptotic effects. In our experiments, we overexpressed Bcl-2 mutants that had the hydrophobic C-terminus exchanged for that of cytochrome b5 or the mitochondrial protein ActA. Such Bcl-2 constructs have been previously shown to block apoptosis in certain but not all cell types. Whereas MDCK epithelial cells were protected from serum deprivation-induced apoptosis by wild-type and mitochondrial Bcl-2, no survival was conferred by Bcl-2 targeted to the ER (Zhu et al., 1996). By contrast, in Rat-1/Myc cells Bcl-2 located at the ER was more effective than Bcl-2 targeted to mitochondria (Zhu et al., 1996). A recent study provided evidence that ER stress triggered by brefeldin A, a compound that blocks the retrograde transport of Golgi vesicles, or tunicamycin, which induces aberrant protein folding, was inhibited by Bcl-2 in the ER (Hacki et al., 2000). It was also shown that ER-targeted Bcl-2 could inhibit c-Myc- but not etoposide-induced apoptosis in Rat-1 fibroblasts (Lee et al., 1999).
The role of membrane anchoring for the function of Bcl-2 has not been determined in detail. Our data provide evidence that the transmembrane domain is required for the anti-apoptotic function of Bcl-2, since neither CD95- nor radiation-induced apoptosis was affected by the Bcl-2/TM construct in Jurkat cells. This finding may be important as the apoptosis pathway in C. elegans suggested that Bcl-2 might eventually act via direct interference with cytosolic APAF-1. Indeed, coprecipitation experiments indicated that Bcl-2 interacts with APAF-1 (Pan et al., 1998), whereas later, more detailed studies excluded a physical interaction between molecules (Hausmann et al., 2000; Moriishi et al., 1999). Our data show that the presence of free cytoplasmic Bcl-2 is not sufficient for the anti-apoptotic function, suggesting that functional interactions with cytoplasmic APAF-1 are very unlikely.
The influence of Bcl-2 on death receptor-mediated apoptosis is still a matter of debate (Huang et al., 2000; Schmitz et al., 1999). Based on the lack of efficient DISC formation after CD95 stimulation Jurkat cells have been categorized as type-II cells (Scaffidi et al., 1998). According to this model, apoptosis in Jurkat cells at least partially relies on a Bcl-2-inhibitable mitochondrial pathway. We and others, however, have shown that stimulation with either the anti-CD95 antibody CH11 (Belka et al., 2000) or CD95L (Huang et al., 1999) clearly induced apoptosis in Bcl-2-overexpressing cells. Furthermore, Huang et al. suggested that differences between type-I and type-II cells might be solely attributed to the anti-CD95 antibody used (Huang et al., 1999; Huang et al., 2000). The data presented here suggest that Bcl-2 only weakly interferes with the time course of CD95-induced caspase activation. The effect of Bcl-2 was observed only in very early phases resulting in a reduced activation of caspase-8, whereas the final outcome of cell death was not affected by Bcl-2 expression. This finding is compatible with the notion that caspase-8 initially triggers both a direct pathway leading to activation of caspase-3 and an indirect pathway leading to activation of caspase-3 via BID and caspase-9. Bcl-2 overexpression only interferes with the activation of amplifying mitochondrial pathways, thereby reducing the initial activation of caspase-8. Therefore, our data do not favor a general role of Bcl-2 for the regulation of CD95-mediated apoptosis. Since Bcl-2 also did not interfere with TRAIL-induced apoptosis in Jurkat cells, a similar argumentation applies to the TRAIL receptor system.
Up to now most data suggest that the anti-apoptotic action of Bcl-2 relies on its interference with mitochondrial pathways, in particular cytochrome c release. Our data show that mitochondrial and ER-targeted Bcl-2 confer survival against radiation to a similar extent. Furthermore, we found that apoptosis induced by other stimuli, such as ER stress-inducing agents such as brefeldin A, was influenced by the two versions of Bcl-2 (data not shown). A protective effect by ER-targeted Bcl-2 and inhibition of mitochondrial cytochrome c release by staurosporine, brefeldin A and tunicamycin has been recently reported (Hacki et al., 2000), although in this study the impact of mitochondrial Bcl-2 was not assessed. Thus, our data reveal that, unlike the death receptor pathway, during irradiation-induced apoptosis Bcl-2 can also function in the ER to inhibit mitochondrial events. These findings therefore imply a molecular crosstalk between both organelles in radiation-induced apoptosis. It will be interesting to investigate whether Bcl-2 targeted to other membranes, such as lysosomes or the plasma membrane, retains its anti-apoptotic activity.
The role of the ER in apoptosis has not yet been analyzed extensively. It is not known how irradiation perturbs the ER and how these perturbations impinge on mitochondria and the caspase cascade. Several possible explanations can be considered. Caspase-8 could be a candidate since it can reside at the ER and mediate mitochondrial damage via BID activation. Bcl-2 has been shown to bind to and neutralize the endoplasmic pro-apoptotic caspase-8 substrate Bap31 (Ng et al., 1997; Ng and Shore, 1998). However, several data argue against a contribution of caspase-8. It has been demonstrated that neither a deficiency of caspase-8 nor of Bid abolishes radiation-induced apoptosis (Belka et al., 2000; Yin et al., 1999). Thus, an initial radiation-induced activation of caspase-8 at the ER with the propagation of this signal to mitochondria is unlikely. Furthermore, Nakagawa et al.s results and our results do not favor an important role of caspase-12 for radiation-induced apoptosis. Only a weak processing of caspase-12 could be observed that was apparently downstream of caspase-9 activation.
An alternative way of crosstalk may involve calcium. Both the ER and mitochondria act as calcium stores controlling the capacitive calcium influx and cytoplasmic calcium homeostasis (Pozzan and Rizzuto, 2000). Indeed, Bcl-2 was shown to control calcium storage at mitochondria and the ER (Baffy et al., 1993; Foyouzi-Youssefi et al., 2000; He et al., 1997; Zhu et al., 1996). Bcl-2 may modulate ER permeability by forming an ion channel or regulating ER-based channels, such as the IP3 receptor or the calcium ATPase SERCA. In addition, other members of the Bcl-2 family were shown to interfere with calcium flux-mediated apoptosis. For instance, lymphocytes from mice lacking Bim did not undergo apoptosis when the calcium homeostasis was disrupted by calcium ionophores (Bouillet et al., 1999).
Only a few data are currently available on a potential calcium-mediated crosstalk between the ER and mitochondria during apoptosis. Apoptosis in response to thapsigargin, a specific inhibitor of the ER calcium pump, was shown to involve Bcl-2 inhibitable cytochrome c release (Srivastava et al., 1999). However, the contribution of Bcl-2 in either localization was not tested. In contrast to thapsigargin, the influence of ionizing radiation on cellular calcium homeostasis is unknown. Nevertheless, some molecules critically involved in calcium homeostasis appear to be mediators in radiation-induced apoptosis. IP3 receptor-1, which is involved in TCR receptor-mediated calcium response in T cells, also critically controls radiation-induced cell death (Jayaraman and Marks, 1997). In addition, the tyrosine kinase Lck, which is also required for the regulation of calcium release from the ER (Keizer et al., 1995; Straus and Weiss, 1992) is involved in the control of radiation and ceramide-induced apoptosis (Belka et al., 1999; Manna et al., 2000). By contrast, studies using the intracellular calcium chelator AM-BAPTA revealed contradictory results. Whereas in Ly cells calcium chelation interfered with apoptotic DNA degradation in response to ionizing radiation (Voehringer et al., 1997), only minor effects were observed in MOLT-4 cells (Takahashi et al., 1999). The use of calcium chelators is problematic, since calcium levels in all cellular compartments are affected. Thus, more precise techniques to measure and manipulate calcium within cellular compartments will have to be employed to assess the role of calcium in the crosstalk between ER and mitochondria during radiation-induced apoptosis.
Taken together, the following model system of radiation- and receptor-induced apoptosis can be derived from the effects of Bcl-2 in cellular compartments (Fig. 10). In the death receptor pathway, triggering of CD95 leads to initial activation of caspase-8, which subsequently activates downstream molecules including BID and caspase-3. BID triggers an amplifying mitochondrial pathway leading to activation of caspase-9 and downstream caspases. The entire network is complicated by the existence of several feedback loops. For example, caspase-3 may also activate caspase-9, caspase-8 and BID, and caspase-8 may directly activate other caspases. Hence, inhibition of cytochrome c release will only affect the time course of caspase activation but not the final outcome of apoptosis in cases of optimal CD95/TRAIL stimulation. By contrast, radiation presumably initially triggers perturbations in the ER and mitochondria, which may involve changes of calcium homeostasis or activation of pro-apoptotic mediators that may crosstalk between both organelles. Such an ER/mitochondrial loop may lead to profound mitochondrial damage, cytochrome c release and activation of caspase-9 and downstream caspases. It is interesting to note that mitochondria have been observed to exist as a continuous interconnected reticulum closely associated with the ER. Changes in calcium homeostasis in the ER might thus be preferentially transferred into the mitochondrial matrix. Future experiments will therefore have to investigate whether the close proximity of mitochondria and the ER and the tight coupling of calcium signaling plays an essential role in the cell sensitivity to radiation-induced apoptosis.
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ACKNOWLEDGMENTS |
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