ER stress can be induced pharmacologically in cells with agents that perturb protein modification or trafficking at the ER, including BFA (an inhibitor of ER-Golgi protein transport), tunicamycin (specific N glycosylation inhibitor), and TG (inhibitor of the ER Ca2+
pump). Our laboratory and others have previously shown that Bax−/− Bak−/−
(DKO) fibroblasts are strikingly resistant to apoptosis in response to these ER stress-inducing agents (15
). Moreover, targeting BAX stably and exclusively to the outer mitochondrial membrane in DKO cells restores the cell death response to BFA and other ER stress-inducing agents (26
), which argues that an accumulation of misfolded proteins in the ER lumen triggers an apoptotic signal that is transmitted to mitochondria, where it induces activation of BAX and/or BAK. Once active, BAX and/or BAK permeabilize the outer mitochondrial membrane, which results in the release of prodeath mitochondrial proteins into the cytosol (e.g., cytochrome c
and Smac/DIABLO) and activation of the executioner caspases (e.g., caspase-3) (6
). Therefore, we hypothesized that the resistance of the Bax−/− Bak−/−
cells to ER stress would allow us to capture and identify the early (premitochondrial) apoptotic signals induced by this form of injury while the cells remained alive and free of prodeath activities resulting from mitochondrial permeabilization.
To this end, we induced ER stress in SV40-transformed DKO MEFs via treatment with BFA for 24 h. Following treatment, a cytosolic extract (the S100 fraction) was isolated by mechanical disruption of the cells in an isotonic buffer (to keep organelles intact) followed by high-speed (100,000 × g) centrifugation. To test for the presence of ER stress-induced BAX/BAK activating signals, we incubated the S100 fractions from untreated and BFA-treated DKO MEFs with mitochondria isolated from human Jurkat T cells and then assayed them for cytochrome c release. As shown in Fig. , the S100 fraction from untreated DKO MEFs did not induce significant mitochondrial cytochrome c release above the level seen with buffer alone. In contrast, the S100 of BFA-treated DKO cells released greater than 90% of the mitochondrial cytochrome c into the supernatant (as measured by an enzyme-linked immunosorbent assay against human cytochrome c). The addition of the Ca2+ chelator EGTA or the pancaspase inhibitor zVAD-fmk had no effect on the cytochrome c-releasing activity of the S100 of BFA-treated DKO cells (data not shown). Moreover, to confirm that the S100 fraction from the BFA-treated MEFs was causing cytochrome c release by specifically activating BAX/BAK and not by generally disrupting mitochondrial integrity, we incubated the S100 fractions from BFA-treated and untreated DKO MEFs with isolated liver mitochondria from age-matched wild-type (WT) or conditionally targeted (Baxf/f Bak−/−) mice with a liver-specific Cre deletion (albumin-Cre) (Fig. ). The S100 fraction from treated DKO MEFs induced robust cytochrome c release from the WT mitochondria but not from BAX/BAK-deficient mitochondria (Fig. ). Together, these data strongly indicate that ER stress induces a cytosolic activity capable of triggering BAX/BAK-dependent cytochrome c release from isolated mitochondria.
FIG. 1. BID is proteolytically activated following ER stress and induces BAX/BAK-dependent mitochondrial cytochrome c release. (A) Cytosolic (S100) extracts from untreated (UNT) and BFA-treated DKO MEFs, as well as recombinant tBID (positive control) and reaction (more ...)
In response to various forms of cell injury, mitochondrial BAX and BAK can be activated either directly or indirectly by a subclass of proapoptotic BCL-2 family members known as the BH3-only proteins, so named because of their sequence homology solely in the amphipathic α-helical BH3 domain (4
). Therefore, we assayed S100 fractions from untreated and BFA-treated DKO MEFs for differences in expression or posttranslational modifications of known BH3-only proteins. The cytosolic levels of most of the known BH3-only proteins, including BAD, PUMA, and BIK, remain unchanged with ER stress (Fig. ). The BH3-protein BIM has recently been implicated in ER stress-induced apoptosis (23
). While the levels of mitochondrion-associated BIM clearly increased upon BFA treatment, there was no detectable BIM in our cytosolic extracts either before or after ER stress (Fig. ), ruling out the possibility that BIM significantly contributes to the BFA-induced cytosolic cytochrome c
-releasing activity. Surprisingly, we found that while the vast majority of the BH3-only protein BID in the untreated S100 is full-length (~22 kDa), nearly all the BID present in the S100 from the BFA-treated cells is of a smaller size (~14 kDa) (Fig. ). In response to certain death signals, BID is activated by proteolytic cleavage into a smaller 14 -kDa truncated form, termed tBID, which then translocates to mitochondria, where it potently activates BAX and/or BAK (12
). The presence of tBID in the BFA S100 fraction from DKO cells suggested the possibility that this activated protein might be an apoptotic signal that links ER stress to the mitochondrial death machinery. To test this hypothesis, we immunodepleted BID/tBID from the S100 fraction of BFA-treated cells and subsequently assayed the ability of this fraction to induce cytochrome c
release (Fig. ). Interestingly, BFA-treated S100 extract that had been immunodepleted for BID contained approximately a third less cytochrome c
releasing-activity than the nonimmunodepleted sample (Fig. ). These data indicate that tBID represented one of the major ER stress-induced apoptotic activities in our cytosolic extract.
If BID is essential for ER stress-induced death in vivo, then the loss of BID should confer resistance to this form of cell injury. To test this, we challenged SV40-transformed WT, DKO, and Bid−/−
MEFs with various concentrations of BFA and TG. The Bid−/−
MEFs displayed significant resistance to treatment with both BFA (Fig. ) and TG (Fig. ), albeit less than did the DKO cells. To rule out the possibility that the apoptotic resistance of Bid−/−
and DKO cells was due to changes in UPR signaling, we measured the major downstream targets of both IRE1α and PERK in response to the same doses of BFA and TG used in the apoptotic studies. Unfolded proteins in the ER activate the RNase domain of IRE1α to initiate splicing of the XBP1 mRNA (2
). As a consequence of IRE1α-mediated intron removal, XBP1 mRNA is frame shifted and translated to produce XBP1 protein, a transcription factor whose downstream effects increase protein-folding capacity (25
). As assessed through an reverse transcription-PCR-based assay, the levels of spliced XBP1 mRNA were essentially identical among the three cell types at baseline and in response to the same doses of BFA and TG used in our apoptosis studies (Fig. ). PERK is an ER-localized transmembrane kinase that, when activated by misfolded proteins, phosphorylates eIF2α at Ser51 (7
), leading to a global attenuation in cap-dependent protein translation. As shown in Fig. , ER stress-induced phosphorylation of eIF2α at Ser51 occurs equally among the three cell types. These results suggest that the apoptotic resistance of the Bid−/−
and DKO cells to ER stress is not attributable to gross differences in UPR signaling at the ER membrane. Moreover, the Bid−/−
MEFs did not display resistance to STS (a pankinase inhibitor) treatment, a death stimulus known to induce mitochondrial BAX/BAK-dependent apoptosis (Fig. ). These results indicate that Bid−/−
cells have a specific resistance to apoptosis induced by ER stress but not to all intrinsic apoptotic stimuli. To ensure that the transformed nature of the MEFs was not affecting our results, we also challenged primary WT and Bid−/−
thymocytes with various concentrations of BFA and TG. Again, the Bid−/−
thymocytes displayed significantly greater resistance to both BFA (Fig. ) and TG (Fig. ) than their WT counterparts. Together, these data demonstrate that loss of BID confers cytoprotection against ER stress, confirming that tBID is one of the major apoptotic signals that link the ER and the mitochondrion.
FIG. 2. Bid−/− cells are resistant to treatment with ER stress agents. Wild-type, Bax−/− Bak−/− (DKO), and Bid−/− simian virus 40-transformed MEFs were treated with the indicated concentrations of (more ...)
To investigate where BID is cleaved in response to ER stress, we transiently expressed V5-tagged versions of WT BID or a D59E BID mutant in DKO MEFs. The D59E mutation replaces the aspartate residue in BID that is targeted by caspase-8 and other caspases, rendering BID uncleavable by these proteases (34
). We monitored changes in the levels of full-length V5-tagged WT or D59E BID as indications of BID cleavage, since the anti-V5 antibody does not recognize V5-tagged tBID. Upon treatment of the transfected cells with TNF-α-cycloheximide (which is known to induce BID cleavage via caspase-8 [12
]), the level of full-length V5-tagged WT BID was greatly reduced, indicative of its being cleaved (Fig. ). In contrast, the level of full-length D59E BID remained constant in the TNF-α-cycloheximide-treated cells, as it is resistant to caspase-8 activity. Moreover, through activation of initiator caspase-8, TNF-α-cycloheximide led to the processing of caspase-3 and the cleavage of its target, PARP, independently of mitochondrial permeabilization. When the transfected cells were treated with BFA, the level of full-length V5 WT BID was substantially reduced, consistent with our finding that BID is cleaved during ER stress. Yet the D59E mutant of BID remained at a constant level after BFA treatment (Fig. ). As expected, BFA was unable to activate caspase-3 in the DKO cells where mitochondrial permeabilization was blocked, which suggests that the ER stress-induced protease is an initiator (premitochondrial) caspase and cleaves BID at this site. As mentioned previously, the classical BID protease is caspase-8, but we found no evidence to suggest that it is cleaved or enzymatically activated in MEFs following BFA treatment (Fig. ).
FIG. 3. ER stress induces BID cleavage at position D59 independently of caspase-8 activation. (A) Nontransfected (NT) DKO MEFS or DKO MEFS transfected with either BID-V5 (WT) or D59E BID-V5 (D59E) were left untreated or treated with 2.5 μg/ml of BFA or (more ...)
To investigate the mechanism of BID cleavage following ER stress, we asked whether we could detect its proteolytic cleavage in our cell extracts. Following depletion of endogenous BID/tBID by preclearing (pc) the S100 extracts with agarose beads coated with an antiapoptotic binding partner (BCL-xL), we incubated full-length recombinant BID (recBID) with the pcS100 extracts from untreated and BFA-treated DKO MEFs. While the extract from untreated cells had no effect on the size of recBID, the pcS100 from the BFA-treated cells contained a definite BID cleavage activity (Fig. ). Interestingly, this BID proteolytic activity was not substantially reduced by the broad-spectrum caspase inhibitor z-VAD-fmk (Fig. ), which is known to block some but not all caspases. Moreover, pretreatment with the specific caspase-8 inhibitor Ac-IETD-CHO did not block this in vitro BID cleavage activity (Fig. ), consistent with our previously mentioned results showing that caspase-8 is not activated by ER stress (Fig. ). These results suggest that another caspase might be responsible for cleaving BID under conditions of ER stress.
As caspase-2 is resistant to z-VAD-fmk and has been reported to cleave BID in response to heat shock (1
), we decided to further investigate whether this protease plays a role in ER stress. The caspase-2-specific inhibitor z-VDVAD-fmk completely blocked the in vitro cleavage of recBID by the pcS100 fraction from BFA-treated DKO MEFs (Fig. ). Moreover, we found that caspase-2 is cleaved in the S100 extract from BFA-treated DKO MEFs (Fig. ), indicating that it is activated by ER stress upstream of mitochondrial permeabilization. To determine if caspase-2 is responsible for BID activation within intact cells, DKO MEFs were left untreated or were pretreated with z-VDVAD-fmk and then challenged with BFA. In whole-cell extracts, we were not able to consistently detect tBID with our antibody; however, a reduction in the level of full-length BID was obvious. Similar to the in vitro results, caspase-2 inhibition blocked ER stress-induced cleavage of BID (Fig. ). To confirm that the observed effects on BID cleavage were a direct result of caspase-2 inhibition and not an off-target effect of the inhibitor, we transfected DKO MEFs with control siRNA or siRNA directed against caspase-2 (Fig. ) and then induced ER stress with BFA. In cells with reduced caspase-2 expression, the level of full-length BID did not decrease after BFA treatment (Fig. ). Together, these results strongly indicate that caspase-2 is the primary protease that activates BID following ER stress.
FIG. 4. Caspase-2 cleaves BID in response to ER stress. (A) S100 extracts from untreated (UNT) and BFA-treated DKO MEFs were precleared (pc) of endogenous BID/tBID and then incubated with recBID with or without pretreatment with the specific caspase-2 inhibitor (more ...)
If caspase-2 is critical for linking ER stress to the mitochondrion via BID cleavage, then reducing caspase-2 activity should confer resistance to ER stress-induced apoptosis in a manner similar to genetic loss of BID. To test this, WT MEFs were left untreated or were pretreated with z-VDVAD-fmk and then challenged with BFA, TG, or STS. The cells pretreated with the caspase-2 inhibitor displayed significant resistance to ER stress-induced apoptosis but not to STS, compared to the cells that were not pretreated with z-VDVAD-fmk (Fig. ). To rule out nonspecific effects of the inhibitor, we again transiently transfected WT MEFs with either control siRNA or siRNA directed against caspase-2. The transfected cells were then treated with BFA or TG. The cells transfected with control siRNA were significantly more sensitive to ER stress than those treated with caspase-2 siRNA (Fig. ). Moreover, expression of V5-tagged WT BID in the Bid−/− MEFs significantly increased their apoptotic response to BFA and TG (Fig. ). Importantly, pharmacological inhibition or siRNA knockdown of caspase-2 did not further protect Bid−/− cells against these ER stress agents, suggesting that BID is the major apoptotic target of caspase-2 in response to this form of cell injury (Fig. ). Together, these data demonstrate that caspase-2 activity is required for BID processing following ER stress and that activated BID is one of the major signals that link the ER to the mitochondrion.
FIG. 5. Absence of caspase-2 (Casp2) activity confers resistance to ER stress-induced apoptosis. (A) WT MEFs were pretreated with dimethyl sulfoxide (DMSO) or 50 μM z-VDVAD-fmk (a caspase-2 inhibitor) and challenged with 0.25 μg/ml BFA (24 h), (more ...)
Although BID is known to be important in transducing apoptotic signals for several other death stimuli, we are the first to show that it is a critical mediator of ER stress. Loss of BID increases resistance to ER stress, confirming the importance of BID in apoptotic signaling downstream of this organelle. Nonetheless, Bid−/−
cells are not completely resistant to ER stress-induced apoptosis, indicating that BID is not the only signal that can link the ER to the mitochondrial death machinery. Consistent with this finding, depletion of BID from the cytosolic extract of BFA-treated DKO cells only partially reduced the ability of the extract to release cytochrome c
from isolated mitochondria in vitro. Given that ER stress conditions are encountered by the most ancient of eukaryotic organisms, it is not surprising that mammalian cells have evolved several pathways to induce apoptosis once the ER is terminally damaged by misfolded proteins. Indeed, it has recently been reported that another BH3-only protein, BIM, can contribute to apoptotic signaling downstream of ER stress (23
). However, despite finding increased BIM at mitochondria following BFA treatment, we tested our cytosolic extracts from untreated and BFA-treated DKO cells and found that they contained no detectable BIM. Therefore, it is plausible that other BH3-only proteins (in addition to BID and BIM) can signal from the ER to the mitochondria under certain conditions (10
Compared to caspase-8, little is known about the activation and function of caspase-2. A role for caspase-2 is best documented in response to genotoxic stress, where it seems to act upstream of mitochondrial permeabilization (11
). While there have been hints that caspase-2 may be involved in ER stress (3
), we are the first to show that it plays an essential role in this apoptotic pathway and to define its critical target. However, it remains unclear how caspase-2 is activated by ER stress and whether it is downstream of one or more of the stress sensors (IRE1α, ATF6, and PERK) that monitor ER homeostasis. This is a topic we are actively investigating.
Our data offer novel insights into the biochemical links between the ER and the mitochondria during times of ER stress. Given the role of ER stress in the pathogenesis of many prevalent human degenerative disorders, our findings may promote the development of rational antiapoptotic therapeutic strategies to preserve cell viability and function in such diseases.