There are two well-described caspase-dependent pathways that induce apoptotic cell death. One is the extrinsic pathway, in which binding of death receptors by death ligands is followed by recruitment of adaptor molecules and activation of caspase-8. The other is the intrinsic pathway, in which cytochrome c
release from mitochondria triggers the formation of the apoptosome composed of Apaf-1, procaspase-9, and cytochrome c
, which results in the activation of caspase-3. SubAB is bound to cells, internalized by endocytosis, and transported from Golgi apparatus to ER in a retrograde manner. In the ER lumen, it cleaved BiP, leading to ER stress, which was demonstrated by activation of PERK and eIF2α, leading to transient protein synthesis inhibition and stress-inducible CHOP induction (17
). A catalytically inactive mutant, SubAB(S272A), did not cleave BiP and did not induce apoptosis. Therefore, SubAB-induced apoptosis is believed to be initiated not by cell receptor recognition but by ER stress resulting from BiP cleavage. The precise mechanisms of how ER stress results in the activation of caspases have not been fully elucidated (25
). Murine caspase-12 and human caspase-4, the counterpart of murine caspase-12, are candidates for involvement in the initial events of ER stress-induced apoptosis (12
); however, recent reports questioned their participation. Further ER stress-induced apoptosis required mitochondrion-dependent apoptosome formation by a caspase-12-independent mechanism (8
). SubAB induced changes in mitochondrial permeability and released cytochrome c
in a caspase-independent manner, suggesting that caspase activation by SubAB might be downstream of the changes in mitochondrial membrane permeability. Similar to the case for the intrinsic pathway, in HeLa cells, a decrease in Apaf-1 expression clearly suppressed procaspase-9 and -3 cleavage with no change in cytochrome c
release, suggesting that apoptosome formation was necessary to induce caspase-3 activation. Treatment with SubAB might induce a similar pathway for caspase-3 activation in Vero cells. Caspase inhibitors did not suppress cytochrome c
release by SubAB, also suggesting that there might be caspase-independent apoptosis, i.e., mitochondrial release of apoptosis-inducing factor (2
). Further studies are necessary to define the potential function of apoptosis-inducing factor and other factors.
We found that activation of procaspase-8, known as an initiator caspase, was downstream of caspase-3. We reached this conclusion because caspase-8 appeared later than caspase-3 and caspase-8-specific inhibitors did not suppress procaspase-3 cleavage, while a caspase-3-specific inhibitor suppressed procaspase-8 cleavage. However, this pharmacological approach may be compromised by possible off-target effects of the inhibitors. The use of a specific caspase siRNA may yield more conclusive results. Also, there is still a possibility that caspase-8 activation may partially occur independently of caspase-3. With regard to caspase-8, however, a recent report showed that unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 functions as an amplifying executioner caspase (9
). Similar to the results in that report, cytochrome c
release by SubAB was not suppressed by caspase inhibitors, and caspase-8 activation was a postmitochondrial event initiated by caspase-3. Therefore, caspase-8 might enhance the apoptotic signal initiated by mitochondria.
The mechanisms of how ER stress by SubAB induces mitochondrial damage have not been identified. Bcl-2 family proteins regulate apoptosis by controlling mitochondrial permeability. CHOP, a transcription factor, involved in ER stress-induced apoptosis that reduces expression of Bcl-2 (16
), was activated in SubAB-treated cells. Further, ER stress also leads to increased cytosolic calcium levels, which activated m-calpain and resulted in cleavage of Bcl-XL
). The study of Bcl-2 family proteins and their modulators may clarify signal transduction following ER stress (1
Finally, we show a proposed activation pathway of SubAB in Fig. . SubAB first binds to cell membranes (step 1), is delivered to ER by endocytosis (step 2), and cleaves BiP (step 3), which is a necessary step in apoptosis. BiP cleavage triggers a mitochondrial membrane permeability change and induces cytochrome c release (step 4). Cytochrome c may form an apoptosome with Apaf-1 and procaspase-9 (step 5) and produce caspase-9 (step 6), caspase-3 (step 7), and caspase-8 (step 8) consecutively.
Proposed model of the SubAB-induced apoptosis signaling pathway. See text for additional details.