Mitochondrial apoptosis induced by BH3-only molecules in the exclusive presence of endoplasmic reticular Bak
2009; Springer Nature; Volume: 28; Issue: 12 Linguagem: Inglês
10.1038/emboj.2009.90
ISSN1460-2075
AutoresMartina Klee, Kathrin Pallauf, Sonia Alcalá, Aarne Fleischer, Felipe X. Pimentel‐Muiños,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle2 April 2009free access Mitochondrial apoptosis induced by BH3-only molecules in the exclusive presence of endoplasmic reticular Bak Martina Klee Martina Klee Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Kathrin Pallauf Kathrin Pallauf Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Sonia Alcalá Sonia Alcalá Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Aarne Fleischer Aarne Fleischer Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Felipe X Pimentel-Muiños Corresponding Author Felipe X Pimentel-Muiños Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Martina Klee Martina Klee Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Kathrin Pallauf Kathrin Pallauf Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Sonia Alcalá Sonia Alcalá Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Aarne Fleischer Aarne Fleischer Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Felipe X Pimentel-Muiños Corresponding Author Felipe X Pimentel-Muiños Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Author Information Martina Klee1, Kathrin Pallauf1, Sonia Alcalá1, Aarne Fleischer1 and Felipe X Pimentel-Muiños 1 1Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain *Corresponding author. Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca 37007, Spain. Tel.: +34 923 294 818; Fax: +34 923 294 795; E-mail: [email protected] The EMBO Journal (2009)28:1757-1768https://doi.org/10.1038/emboj.2009.90 There is a Have you seen ...? (June 2009) associated with this Article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Bak and Bax are critical apoptotic mediators that naturally localize to both mitochondria and the endoplasmic reticulum (ER). Although it is generally accepted that mitochondrial expression of Bak or Bax suffices for apoptosis initiated by BH3-only homologues, it is currently unclear whether their reticular counterparts may have a similar potential. In this study, we show that cells exclusively expressing Bak in endoplasmic membranes undergo cytochrome c mobilization and mitochondrial apoptosis in response to BimEL and Puma, even when these BH3-only molecules are also targeted to the ER. Surprisingly, calcium was necessary but not sufficient to drive the pathway, despite normal ER calcium levels. We provide evidence that calcium functions coordinately with the ER-stress surveillance machinery IRE1α/TRAF2 to transmit apoptotic signals from the reticulum to mitochondria. These results indicate that BH3-only mediators can rely on reticular Bak to activate an ER-to-mitochondria signalling route able to induce cytochrome c release and apoptosis independently of the canonical Bak,Bax-dependent mitochondrial gateway, thus revealing a new layer of complexity in apoptotic regulation. Introduction Bcl-2 family members have important functions in the integration of multiple apoptotic pathways (Danial and Korsmeyer, 2004). These proteins are structurally related by the presence of at least one of the four characteristic family domains (Bcl-2-homology domains 1–4 (BH1–4)) and can be further categorized according to functional and structural criteria (Adams and Cory, 2007). Thus, the BH3-only subgroup includes initiators of apoptosis only sharing the BH3 domain (Puthalakath and Strasser, 2002). In addition, Bak and Bax are multidomain agonists containing BH1–3, and the apoptotic activity of both subfamilies is counteracted by protective homologues presenting a BH4 domain (Adams and Cory, 2007). The current view regarding how apoptotic signalling pathways work is that specific BH3-only molecules are induced by a wide variety of stimuli, and convey the death signal to a common downstream machinery constituted by Bak and Bax (Danial and Korsmeyer, 2004; Adams and Cory, 2007). These are critical mediators, as cells lacking both proteins are resistant to apoptosis induced by BH3-only effectors (Wei et al, 2001; Zong et al, 2001). Bak and Bax are known to have in this context a prominent mitochondrial function by promoting the release of cytochrome c. In fact, their exclusive expression in this organelle is sufficient to trigger BH3-only-induced death (Scorrano et al, 2003), thus supporting the notion that Bak and Bax constitute a mitochondrial gateway for apoptotic routes channelled through BH3-only molecules (Wei et al, 2001; Zong et al, 2001). However, Bak and Bax also localize to the endoplasmic reticulum (ER) (Scorrano et al, 2003; Zong et al, 2003), and accumulated evidence shows that their presence in this organelle is linked to the regulation of calcium metabolism (Breckenridge et al, 2003; Rong and Distelhorst, 2008). Thus, enforced expression of Bak and Bax provokes ER calcium release (Nutt et al, 2002; Zong et al, 2003). In addition, double knockout mouse embryonic fibroblasts (MEFs) lacking Bak and Bax show decreased calcium levels in the reticular lumen that result in reduced apoptotic responses to stimuli that use ER calcium to activate the death program, like C2-ceramide or oxidative stress (Scorrano et al, 2003). Recovery of endoplasmic calcium by alternative means other than reexpressing Bak or Bax restores death induced by these agents, thus underscoring that the main apoptotic function of both proteins in the reticulum is the regulation of calcium levels (Scorrano et al, 2003). Therefore, ER-localized Bak or Bax seem to act as an apoptotic gateway for stimuli that rely on reticular calcium to activate programmed cell death. Certain BH3-only mediators are known to function at the ER to induce apoptosis and, although calcium fluxes have been involved in these pathways, other mechanisms have also been proposed. For example, Puma is a key participant in p53-induced cell death (Villunger et al, 2003), and activates apoptosis by inducing ER calcium liberation (Shibue et al, 2006). Spike is a less-studied homolog that localizes to the ER, where it regulates the cleavage of Bap31 to trigger mitochondrial apoptosis (Mund et al, 2003). Bim mediates death induced by ER stress (Puthalakath et al, 2007), a process that involves Bim translocation to reticular membranes (Morishima et al, 2004). In addition, Bik is known to induce mitochondrial apoptosis by promoting both the oligomerization of reticular Bak and ER calcium release (Mathai et al, 2005). However, to what extent these death signals initiated at the ER by BH3-only molecules are autonomous inducers of apoptosis or simple facilitators of the conventional Bak,Bax-dependent mitochondrial gateway is not entirely clear. Here, we address this issue by generating cells expressing reticular Bak in a cellular background devoid of both Bak and Bax. We found that the BH3-only molecules Bim and Puma are able to fully induce cytochrome c release and apoptosis in the sole presence of reticular Bak. We also show that this activity involves an ER-to-mitochondria communication pathway mediated by calcium and a novel configuration of the IRE1α/TRAF2 stress signalling machinery. Results and discussion Generation of a cellular system exclusively expressing Bak at the ER Earlier studies show that a fusion protein between Bak and the reticular targeting signal of cytochrome b5 (Bak–cb5) localizes to endoplasmic membranes (Zong et al, 2003). Consistently, transfected Bak–cb5 showed in our hands tight reticular localization in various cell types, including MEFs deficient for both Bak and Bax (bak,bax−/−) (Figure 1A). These cells were then retrovirally engineered to stably express an AU-tagged form of Bak–cb5 (AU-Bak–cb5; Supplementary Figure 1A). The resulting polyclonal cultures were tested for cell death in response to staurosporine (STS) or serum starvation, two conventional apoptotic inducers. Interestingly, STS treatment, but not starvation, killed the Bak–cb5-expressing cells (Figure 1B), whereas control bak,bax−/− MEFs were insensitive (Figure 1B). Double-deficient MEFs expressing a version of Bak targeted to mitochondria (Bak–ActA; Zong et al, 2003) were susceptible to both stimuli (Figure 1B; Supplementary Figure 1A and B), thus confirming previous indications that mitochondrial Bak or Bax suffice to transmit a wide range of apoptotic signals (Scorrano et al, 2003). Figure 1.Reticular expression of Bak–cb5 in bak,bax−/− MEFs. (A) Transfected Bak–cb5 localizes to the ER. 293T cells (left panels) were co-transfected with AU-Bak–cb5 and erGFP or mGFP, and stained for AU 24 h later. Bak,bax−/− MEFs (right panels) were transfected with GFP-Bak–cb5 in the absence or presence of mRFP, and stained for calnexin (top) or directly mounted (bottom), 24 h later. Pictures show colocalization of Bak–cb5 with reticular (erGFP, left; calnexin, right), but not mitochondrial (mGFP, left; mRFP, right) markers. (B) Polyclonal Bak–cb5-expressing MEFs die in response to STS treatment, but not serum starvation. The indicated cells were treated with STS (1 μM) or serum starvation for 24 or 36 h, respectively, and death was evaluated by propidium iodide (PI) staining. (C) Polyclonal AU-Bak–cb5-expressing cells undergo Bim- and Puma-induced apoptosis. Cells were transduced with the indicated BH3-only molecules and analysed for cell death by PI staining 24 h later. The inset shows expression of HA-Bim, HA-tBid and HA-Puma in bak,bax−/− MEFs, 24 h after transduction (anti-HA western blot). Bak,bax-deficient cells were used to avoid an influence of the induced death in expression levels. (D) Expression of AU-Bak–cb5, Bcl2 and Bcl-XL in clones #2 and #6. Total lysates were subjected to western blotting. (E) AU-Bak–cb5 is excluded from mitochondria in clones #2 and #6. Equal protein amounts were subjected to western blotting for AU, calnexin and VDAC. HM, heavy membranes; S3, post-mitochondria supernatant. (F) AU-Bak–cb5 co-purifies with calnexin in the microsomal fraction. Equal amounts of protein from the post-mitochondria supernatant (S3), light membranes (LM) or the post-microsome supernatant (S100) were subjected to western blotting for AU, calnexin or actin, as shown. (G) Recovered levels of reticular calcium in Bak–cb5-expressing clones. The amount of TG-releasable calcium (left bars) and the FRET signals provided by transduced erYC4.3 (right bars) are represented as a fraction of the values obtained for wild-type MEFs. Shown are averages and standard deviations (error bars) of the values obtained from three independent experiments. Asterisks indicate significant differences (Student's t-test) with respect to bak,bax−/− MEFs (P<0.05). Download figure Download PowerPoint These results suggest that certain stimuli can fully activate the death program in the sole presence of reticular Bak, revealing the existence of signalling cascades capable of transmitting these signals. To explore these routes, we excited molecularly-defined apoptotic pathways by overexpressing the BH3-only molecules BimEL (Bim hereafter), tBid and Puma, three family members known to be particularly potent activators of apoptosis (Adams and Cory, 2007). Expression of Bim and Puma, but not tBid, provoked cell death in MEFs expressing Bak–cb5 (Figure 1C), whereas Bak–ActA-expressing MEFs were susceptible to all three inducers (Figure 1C). These data point to the notion that reticular Bak might suffice to relay death signals in response to certain BH3-only activators. To prevent drifting of the polyclonal population, we cloned the Bak–cb5-expressing cells and chose two clones for further studies (#2, #6). Both strains showed expression of the transgene (Figure 1D) and presented unaltered levels of Bcl-2 and Bcl-XL (Figure 1D), suggesting that the cloning process did not involve a parallel selection for these protective molecules. Such bias was conceivable, given that excessive expression of Bak–cb5 induces cell death (Zong et al, 2003). Biochemical fractionation studies indicated that Bak–cb5 was excluded from the mitochondrial (Figure 1E) and cytoplasmic fractions (Figure 1F), and localized to light membranes (Figure 1F). Expression levels of the subcellular markers VDAC (mitochondria) and calnexin (ER) were comparable in all strains (Supplementary Figure 2). Bak and Bax are necessary to maintain high calcium concentrations in reticular cisternae (Scorrano et al, 2003). Consistently, the clones showed increased calcium mobilization induced by thapsigargin (TG, an ER calcium-releasing agent; Breckenridge et al, 2003) as well as elevated FRET signals provided by the calcium reporter erYC4.3 (Palmer et al, 2004), when compared with bak,bax−/− MEFs (Figure 1G), indicating recovered calcium levels in the ER lumen. ErYC4.3 was in fact able to measure reticular calcium, as it showed responsiveness to forced ER calcium depletion (Supplementary Figure 3). Both clones remained susceptible to cell death induced by STS, but not serum starvation (Supplementary Figure 4). Bim and Puma induce cytochrome c release and apoptosis in Bak–cb5-expressing cells We used the clones expressing Bak–cb5 as a model system to dissect the apoptotic role of reticular Bak in response to BH3-only mediators. Expression of Bim and Puma in these cells induced significant levels of cell death, similar to those observed in bax−/− MEFs (Figure 2A). However, although tBid efficiently killed wild-type and bax−/− cells, both clones were strikingly resistant (Figure 2A). This result further suggests that no functional Bak is expressed at the mitochondria in these cells, given that mitochondrial expression of multidomain pro-apoptotic effectors is sufficient for tBid-induced apoptosis (see Figure 1C; Scorrano et al, 2003). The death process activated by Bim and Puma in Bak–cb5-expressing MEFs had apoptotic features, because the dying cells presented Annexin-V reactivity (Figure 2B), cleavage of caspases 9 and 3 (Figure 2C) and cytochrome c release (Figure 2D). However, no signs of caspase-12 cleavage were detected (Supplementary Figure 5), thus arguing against a role of this protease in the pathway. An involvement of the general caspase machinery was revealed by the capacity of the pan-caspase inhibitor zVAD.fmk to block death (Figure 2E). Treatment with the caspase-9 inhibitor zLEHD.fmk also diminished cell death (Figure 2F), a result that, together with the data showing concomitant cytochrome c mobilization (see Figure 2D), points to mitochondria as the main executioners of the apoptotic program. Taken together, these results indicate that certain BH3-only activators, like Bim and Puma, can induce cytochrome c mobilization and mitochondrial apoptosis in the sole presence of reticular Bak. Figure 2.Expression of Bim and Puma in MEFs expressing Bak–cb5 induces mitochondrial apoptosis. (A) Retroviral transduction of Bim and Puma leads to a progressive accumulation of PI-permeable cells in clones #2 and #6. Cells were transduced with the shown BH3-only molecules, and cell death was evaluated by PI-staining at the indicated times. (B) Bim and Puma induce Annexin-V reactivity before membrane disruption. PI-negative cells were analysed for Annexin-V binding 24 h after transduction. (C) Processing of caspases-3 and -9. Transduced cells were lysed at the shown times for western blotting. (D) Induction of cytochrome c release. Cells were transduced in the presence of zVAD.fmk (100 μM), and the percentage of cells showing diffuse cytochrome c staining was evaluated 17 h after transduction. (E) Cell death provoked by Bim and Puma is blocked by the pan-caspase inhibitor zVAD.fmk. Cells were transduced in the presence of zVAD.fmk (80 μM) and analysed for death by PI-staining 24 h later. (F) Inhibition of cell death by the caspase-9 inhibitor zLEHD.fmk. Cells were transduced in the presence of zLEHD.fmk (80 μM). Death was evaluated 17 h after transduction. Download figure Download PowerPoint Bim and Puma targeted to the ER retain the capacity to activate mitochondrial apoptosis in Bak–cb5-expressing cells Bim and Puma are thought to act at the level of mitochondria as well as the ER (Yamaguchi and Wang, 2002; Morishima et al, 2004; Shibue et al, 2006). To explore the relative contribution of both subcellular localizations to their apoptotic effect in the Bak–cb5 clones, we generated versions of these BH3-only effectors directed to the ER (cb5 fusions) or the mitochondrial outer membrane (ActA fusions). Bim chimeras lacking the native C-terminal hydrophobic region (BimΔTM–cb5 or BimΔTM–ActA) showed superior targeting specificity than constructs based on full-length Bim (data not shown) and, therefore, were used in these studies. All chimeric proteins presented proper subcellular localization (Figure 3A). We found that mitochondria-localized BimΔTM–ActA had a diminished capacity to activate cytochrome c exit and cleavage of caspases 9 and 3 compared with wild-type Bim (Figure 3C and D), despite similar expression levels (Figure 3B). On the other hand, BimΔTM–cb5 retained both activities (Figure 3C and D) and induced a death process blocked by caspase-9 and pan-caspase inhibitors (Figure 3E). These results argue that the apoptotic effect of unaltered Bim in Bak–cb5-expressing cells (see Figure 2) is likely to be mainly reticular. Figure 3.Reticular Bim and Puma stimulate mitochondrial apoptosis in MEFs expressing Bak–cb5. (A) The cb5 and ActA derivatives of BimΔTM and Puma are targeted to the ER and mitochondria, respectively. GFP-tagged versions of Bim and Puma were transfected into clone #2, sometimes in combination with a plasmid expressing mRFP (as shown), in the presence of zVAD.fmk (100 μM). After 48 h, cells were stained for calnexin or directly mounted (mRFP samples). Pictures show co-localization of the cb5 and ActA chimeras with reticular (Calnexin) and mitochondrial (mRFP) markers, respectively. Shown are representative examples. (B) Expression of the different constructs in bak,bax−/− MEFs. Cells were lysed for anti-HA western blotting 24 h after transduction. Bak,bax-deficient cells were used to prevent cell death. (C) Cytochrome c release induced by BimΔTM and Puma–cb5 and ActA fusions. Cells were transduced in the presence of zVAD.fmk (100 μM) and stained for cytochrome c 17 h later. (D) Caspase cleavage provoked by the chimeras. Cells were transduced and lysed 17 h later for western blotting. (E) BimΔTM–cb5 and Puma–cb5-induced death is inhibited by zVAD.fmk and the capase-9 inhibitor zLEHD.fmk. Cells were transduced and treated with zVAD.fmk (80 μM) or zLEHD.fmk (80 μM). Cell death was evaluated 24 h (zVAD.fmk) or 17 h (zLEHD.fmk) later by PI staining and is represented as a fraction of the value obtained in the absence of inhibitor. Download figure Download PowerPoint Unexpectedly, however, Puma–ActA retained the potential of the wild-type molecule to excite the pathway in both Bak–cb5-expressing clones (Figure 3C and D). Given that bak,bax−/− MEFs remained unresponsive (Figure 3C and D), this result suggests that reticular Bak can somehow restore susceptibility of double-deficient mitochondria to certain apoptotic insults. The effect of Puma–ActA was inhibited by 2-aminoethyl-diphenyl-borinate (2-APB; Supplementary Figure 6A), an agent that reduces the overall calcium content of the cell (Peppiatt et al, 2003), thus pointing to a role of calcium in this phenomenon. How a global calcium decrease can inhibit apoptosis caused by direct mitochondrial engagement is unclear but, as expected, 2-APB reduced the basal calcium levels present in mitochondria (Supplementary Figure 6B), suggesting that mitochondrial calcium could play a relevant role. Importantly, 2-APB did not have a significant impact on ATP levels (Supplementary Figure 7). These data suggest that Bak–cb5 might be able to influence mitochondrial susceptibility to apoptosis by regulating their calcium metabolism, perhaps through its effect on reticular stores. This is a plausible notion, given that both organelles have a privileged interaction in terms of calcium metabolism (Walter and Hajnoczky, 2005). But more interesting in the context of this work, Puma–cb5 provoked cytochrome c release (Figure 3C) and caspase cleavage (Figure 3D) as efficiently as wild-type Puma, and induced a death process mediated by caspases 9 and 3 (Figure 3E). Therefore, unlike Bim, the apoptotic effect of native Puma in the Bak–cb5 clones (see Figure 2) is probably a mixture of events initiated at both reticular and mitochondrial membranes. Taken together, these results show that Bim and Puma targeted to the ER retain the ability to provoke cytochrome c release and mitochondrial apoptosis in Bak–cb5-expressing MEFs. This scenario implies the activation of ER-to-mitochondria apoptotic communication mechanisms able to induce cytochrome c mobilization in the absence of Bak and Bax expressed in mitochondrial membranes, although a minor fraction of Bak–cb5 mislocalized to mitochondria might also contribute to cytochrome c release. We sought to study the signalling events that participate in this pathway. ER calcium mobilization is insufficient to activate mitochondrial apoptosis in cells expressing Bak–cb5 Calcium transits have been involved in the apoptotic communication between the reticulum and mitochondria (Walter and Hajnoczky, 2005). To evaluate whether a discharge of reticular calcium is sufficient to activate mitochondrial apoptosis in our system, we tested the death response of both Bak–cb5-expressing clones to stimuli that induce ER calcium liberation. TG treatment induced a higher degree of death in these cells when compared with bak,bax−/− MEFs (Figure 4A), a result consistent with earlier observations in bak,bax−/− cells engineered to have recovered ER calcium levels by overexpression of the SERCA pump (Scorrano et al, 2003). However, TG-induced death was not accompanied by cytochrome c release (Figure 4B) or cleavage of caspases 9 and 3 (Figure 4C), thus excluding an involvement of the mitochondrial apoptotic machinery. Similarly, other stimuli that rely on ER calcium to trigger apoptosis (C2-ceramide, H2O2; Scorrano et al, 2003) provoked a higher death rate in Bak–cb5-expressing MEFs compared with bak,bax−/− cells (Supplementary Figure 8), and were also unable to induce caspase processing (Figure 4D). These data indicate that calcium released from the reticulum, although capable of promoting death, is inefficient in inducing cytochrome c exit and apoptosis in cells lacking mitochondrial Bak and Bax. Consequently, it seems unlikely that ER calcium mobilization, if involved at all, is the only mediator of apoptosis induced by reticular Bim and Puma in our Bak–cb5-expressing cells. How calcium-dependent agents can activate death without mitochondrial involvement is unclear, but calcium mobilization can also trigger alternative death pathways like necrosis (Leung and Halestrap, 2008) or calpain-dependent apoptosis (Mathiasen et al, 2002). Figure 4.ER calcium agonists are unable to stimulate mitochondrial apoptosis in MEFs expressing Bak–cb5. (A) TG induces increased death in clones #2 and #6. Cells were treated with TG (5 μM) and cell death was measured by PI-staining 24 h later. (B) TG does not induce cytochrome c release. Cells were treated as in (A) in the presence of 100 μM zVAD.fmk, and stained for cytochrome c at the shown times. (C) Absence of caspase-3 and -9 processing induced by TG. Cells were treated with TG (5 μM) for the indicated times and lysed for western blotting. (D) C2-ceramide and H2O2 fail to activate caspases in Bak–cb5-expressing cells. Cells were treated for the indicated times with C2-ceramide (100 μM) or H2O2 (1 mM). Download figure Download PowerPoint ER-to-mitochondria apoptotic communication mediated by a cooperative action of calcium and the IRE1/TRAF2 stress signalling system To explore a potential necessary role of calcium in this system, we used calcium antagonists like 2-APB or the calcium chelator Bapta-AM. Treatment with these agents inhibited cleavage of caspases 9 and 3 caused by BimΔTM–cb5 and Puma–cb5 in both clones (Figure 5A). Bapta-AM also inhibited cytochrome c release (Figure 5B) and, similar to 2-APB, did not cause reduction in ATP levels (see Supplementary Figure 7). Therefore, calcium mobilization is involved in this signalling system. However, since calcium cannot autonomously activate the pathway (see Figure 4), these data suggest that additional mechanisms mediate the apoptotic crosstalk between the ER and mitochondria in the Bak–cb5-expressing cells. Figure 5.ER-to-mitochondria apoptotic communication in MEFs-Bak–cb5 is mediated by calcium and IRE1/TRAF2. (A) 2-APB and Bapta-AM reduce cleavage of caspases 9 and 3 induced by BimΔTM–cb5 and Puma–cb5. Cells were transduced, treated with 2-APB (A; 75 μM) or Bapta-AM (B; 15 μM) 8 h later, and lysed 17 h after transduction for western blotting. (B) Treatment with Bapta-AM inhibits cytochrome c release. Cells were transduced in the presence of zVAD.fmk (100 μM), treated with Bapta-AM and stained for cytochrome c 17 h after transduction. (C) Effect of TRAF2-DN and Bapta-AM in cytochrome c release. Cells were pretransduced twice with retroviruses expressing HA-TRAF2-DN (HA-TF2-DN) and, 36 h later, treated as in (B). The insets show TRAF2-DN expression (anti-HA western blot). (D) Effect of TRAF2-DN and Bapta-AM in caspase processing. Cells were treated as in (C) without zVAD.fmk, and lysed 17 h later for western blotting. (E) Effect of TRAF2-DN plus Bapta-AM in cell death. Death was evaluated by PI staining, 24 h after transduction. Data are represented as percentages of the values obtained in the absence of TRAF2-DN and Bapta-AM. (F) Effect of IRE1α depletion and Bapta-AM in cytochrome c release. Cells were transfected with the siRNAs, transduced 48 h later in the presence of 100 μM zVAD.fmk, treated with Bapta-AM as in (C) and stained for cytochrome c 17 h after transduction. The insets show IRE1α depletion (anti-IRE1α western blot). Download figure Download PowerPoint Reticular Bak is known to participate in the cellular response to ER stress by binding and activating the stress sensor IRE1α (Hetz et al, 2006). Stimulated IRE1α directly generates a spliced, active form of the transcription factor XBP1 (sXBP1) that in turn promotes transcription of stress-relieving molecules. A second signalling branch emanates from active IRE1α, and sequentially engages the adaptor TRAF2 and the kinases ASK1 and JNK (Ron and Walter, 2007). Although a recent study argues that the main function of IRE1α is to promote survival (Lin et al, 2007), other reports show that the IRE1α/TRAF2/ASK1 route contributes to apoptosis if the reticular damage is improperly managed (Nishitoh et al, 2002; Szegezdi et al, 2006). To evaluate whether the IRE1α signalling machinery might be involved in promoting apoptosis in the Bak–cb5-expressing clones, we tested a possible dominant-negative effect of a deleted version of TRAF2 (TRAF2-DN) known to interfere with this ER stress route (Ron and Walter, 2007). Expression of TRAF2-DN reduced cytochrome c release induced by BimΔTM–cb5 or Puma–cb5 in both clones expressing reticular Bak (Figure 5C). Interestingly, such diminution was turned into a nearly complete blockade by the additional treatment with Bapta-AM (Figure 5C). This cooperative reduction in cytochrome c release had a parallel impact both in caspase processing (Figure 5D) and cell death (Figure 5E). In addition, knock-down of IRE1α inhibited cytochrome c liberation induced by reticular Bim and Puma (Figure 5F) and, again, Bapta-AM further reduced the activity (Figure 5F). These results reveal that IRE1α and TRAF2 mediate the apoptotic consequence of Bak activation by Bim and Puma at the ER, working in coordination with calcium to transmit death signals to Bak,Bax-devoid mitochondria. Since the simultaneous impairment of both pathways routinely reduced cytochrome c mobilization, caspase processing and cell death to marginal levels (see Figure 5), the IRE1α/TRAF2 and calcium routes are likely to be the main players involved. Unconventional IRE1 signalling in response to reticular Bim and Puma To further explore whether reticular Bim and Puma can activate IRE1α, we studied the induction of downstream signalling events. Treatment of Bak–cb5-expressing cells with tunicamycin (an inducer of ER st
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