It has been known that apoptotic morphological changes are observed in cell death caused by ER stress (Imaizumi et al., 2001
). Caspases are activated to transmit apoptotic signals transcending the difference in species (Alnemri et al., 1996
). In rodents, caspase-12 mediates apoptosis specifically in response to ER stress (Nakagawa et al., 2000
). Although human caspase-12 gene is transcribed into mRNA, mature caspase-12 protein would not be produced, because the gene is interrupted by frame shift and premature stop codon (Fischer et al., 2002
). Furthermore, it contains amino acid substitution in the critical site, which leads to loss of function in several caspases (Fischer et al., 2002
). Thus, human caspase-12 does not seem to function in ER stress-induced apoptosis, and some other caspases with similar structure might substitute functionally for caspase-12 in humans. The caspase-12 gene is located within a region where caspase-1/ICE subfamily genes cluster (caspases 1, 4, 5, 12 in human and caspases 1, 11, 12 in mouse). No locus with a comparably high homology to rodent caspase-12 could be found in the human genome. Caspases 4 and 5 are located between caspases 1 and 12 in human genome, whereas only caspase-11 is located between caspases 1 and 12 in mouse. Although it is not known why the region in human genome contains gene duplication, caspases 4 and 5 have been thought to function similarly to caspases 11 and 12. Mouse caspase-11 is essential for the activation of caspase-1/ICE to promote pro–IL-1β (interleukin-1β) processing (Wang et al., 1996
). On the other hand, caspase-5 is likely involved in processing of pro–IL-1β together with caspase-1/ICE (Martinon et al., 2002
) and the caspase-5 gene resembles the mouse caspase-11 in its lipopolysaccharide inducibility (Lin et al., 2000
). Therefore, caspase-5 should be the orthologue of caspase-11. Here, the screening process yielded the caspase-4 gene as the homologous gene to mouse caspase-12. Thus, caspase-4 is the best candidate that would function similarly to mouse caspase-12 in ER stress-induced cell death in humans.
Here, we examined the localization of human caspase-4 using several methods. The immunostaining analysis using anti–caspase-4 antibody and fluorescent analysis for caspase-4/GFP fusion protein in showed the predominant localization of caspase-4 on the ER. On the other hand, the immuno-EM showed the nearly equal distribution of caspase-4 on the ER and mitochondria, and subcellular fractionation showed that caspase-4 was recovered in the microsome-enriched and mitochondria-enriched fractions, and also in cytosolic fraction. Although we could not eliminate contamination of ER marker proteins in the mitochondria-enriched fraction in subcellular fractionation using SK-N-SH cells, microsome-enriched fraction does not seem to contain mitochondria and cytosol. Under these conditions, caspase-4 was recovered in both mitochondria-enriched fraction and microsome-enriched fraction, and amounts of caspase-4 recovered in the microsome-enriched and mitochondria-enriched fractions were comparable to those of ER marker, presenilin-1. Therefore, considering all the results shown in , we concluded that caspase-4 was localized to the ER membrane, and probably to the mitochondria in addition.
Caspase-4 on the ER is supposed to function in ER stress-induced apoptosis similarly to caspase-12. In supporting this hypothesis, caspase-4 was cleaved specifically by ER stress and Aβ-treatment, but not by other apoptotic stimuli including etoposide, staurosporine, and UV. Additionally because Bcl-2 that can completely inhibit the signaling pathway at least downstream from mitochondria, did not prevent the cleavage of caspase-4 by ER stress, it should be most probable that caspase-4 on the ER but not on mitochondria is primarily cleaved. We also showed that reduction of the level of caspase-4 by RNA interference (RNAi) resulted in decrease in ER stress-induced cell death and Aβ-induced cell death, but did not affect cell death induced by etoposide. The characteristics of human caspase-4 shown here are very similar to those of mouse caspase-12 reported previously (Nakagawa et al., 2000
), and therefore, caspase-4 is able to substitute the caspase-12 functions in ER stress-induced apoptosis and Aβ-induced cell death. Because caspase-4 was also localized to the mitochondria in addition to the ER membrane, whereas caspase-12 was shown to localize predominantly to the ER, but not to the mitochondria (Nakagawa et al., 2000
), caspase-4 might have additional function compared with caspase-12, although the function of caspase-4 on mitochondria is not clear.
Several mechanisms that activate caspase-12 have been proposed in mouse system. For example, calpain, a protease that can be activated by calcium released from ER upon ER stress, starts cleavage of caspase-12 (Nakagawa and Yuan, 2000
), caspase-7 activates caspase-12 upon prolonged ER stress (Rao et al., 2001
), or TRAF2 mediates caspase-12 activation, which is regulated by IRE1α (Yoneda et al., 2001
). It is not clear which mechanism is involved in activation of caspase-4, but because final cleavage products were not observed in cells with activated caspase-7 ( a), activation of caspase-7 does not seem to be enough for full activation of caspase-4. To know the precise mechanism that activate caspase-4, it should be important to find proteins that interact with pro–caspase-4 during ER stress-induced apoptosis.
The inhibition of apoptosis induced by ER stress exposure by RNAi to caspase-4 was incomplete. It is possible that the residual activity of caspase-4 after RNAi would be responsible for the cell death. Alternatively, other apoptotic mechanisms might also operate simultaneously. Several possible pathways have been postulated for ER stress-induced apoptosis. ER stress is reported to activate ASK–c-Jun NH2
-terminal kinase pathway through the IRE1–TRAF2–ASK1 complex formation (Nishitoh et al., 2002
). Other signaling pathway is mediated by transcriptional activation of genes encoding proapoptotic function. Activation of stress transducer IRE1, PERK, or ATF6 leads to transcriptional activation of CHOP/GADD153, a bZIP transcription factor that potentiates apoptosis (Oyadomari et al., 2002
). Operation of these mechanisms might account for incomplete inhibition of ER stress-induced apoptosis by knockout of caspase-12 and knockdown of caspase-4 in mouse and humans, respectively. It is possible that caspase-dependent mechanism and other mechanisms function in parallel in initiating ER stress-induced apoptosis, and the mechanism that mainly operates could differ depending on cell types. We have shown that the extent to decrease in cell death by decreasing caspase-4 level of SK-N-SH cells was comparable to that reported for caspase-12 knockout mouse (Nakagawa et al., 2000
), whereas that of HeLa cells was relatively less. We also find some cells, like HUVEC, in which decrease in caspase-4 did not affect the ER stress-induced apoptosis (unpublished data). Thus, caspase-4 has been shown to function in ER stress-induced apoptosis at least in several cell lines, including SK-N-SH and HeLa cells, but not all cells.
Cell death caused by Aβ treatment was also partially inhibited by RNAi to caspase-4. Although it is controversial whether Aβ-induced cell death involves ER stress-induced apoptosis, the results are consistent that these two types of cell death are mediated by common mechanism at least in part. Recent report described that the cell death induced by Aβ was inhibited by the broad-spectrum caspase inhibitor z-VAD and more specifically by the down-regulation of caspase-2 with antisense oligonucleotides (Haviv et al., 1998
). Neuronal culture derived from caspase-2 null mice was also shown to be partially resistant to Aβ1-42
toxicity (Troy et al., 2000
). Thus Aβ1-42
-induced cell death might be mediated by caspase-2 as well as caspase-12. It is possible that both caspases 2 and 4 are involved in Aβ-induced cell death also in the human system.
Because caspase-4 seems to be responsible for cell death after Aβ treatment, caspase-4 might be involved in pathogenesis of AD. Consistently to this hypothesis, our preliminary analysis showed an increase in cytoplasmic staining for caspase-4 in the pyramidal cell layer of the hippocampal CA1-2 region in AD patients, but not in control brains (unpublished data). All of the AD brains tested had stronger caspase-4 immunoreactivity than disease control brains from patients with other neurodegenerative disorders (n = 4 for AD and n = 3 for disease control), and the strong staining was remarkable in the pyramidal neurons around deposits of β-amyloid. Increased caspase-4 might elevate the vulnerability of neurons to apoptosis, and therefore may be involved in the pathogenesis of AD.
Bcl-2 family proteins play essential roles in regulating apoptosis. Although antiapoptotic family members (Bcl-2, Bcl-xL
) and multidomain proapoptotic members (Bak, Bax) are thought to function mainly on mitochondria, recent studies suggest that they may also function on the ER where they reside as well. Overexpression of Bcl-2 (Foyouzi-Youssefi et al., 2000
; Pinton et al., 2000
) or knockout of both Bak and Bax (Scorrano et al., 2003
) are reported to reduce ER Ca2+
concentration, but other reports showed that Bcl-2 enhanced the retention of Ca2+
in the ER lumen (Distelhorst et al., 1996
; He et al., 1997
). Thus, although it is still controversial, Bcl-2 family members may contribute to regulating ER stress-induced apoptosis on the ER (Ferri and Kroemer, 2001
; Scorrano et al., 2003
; Zong et al., 2003
) in addition to their main function on mitochondria. Our results demonstrate that overexpressed Bcl-2 and Bcl-xL
, which can completely inhibit the signaling pathway at least downstream from mitochondria, did not prevent, but slightly affected, the cleavage of caspase-4 by ER stress. The slight decrease might be due to the absence of its feed back cleavage by downstream effector caspases, such as caspase-3. Therefore, without regard to the mitochondria or the ER where Bcl-2 family functions, activation of caspase-4 should be the primary reaction in ER stress-induced activation of caspases.
In this work, we discovered that caspase-4 plays a key role in ER stress-induced apoptosis in humans. Caspase-4 also seems to act in the β-amyloid–induced cell death, suggesting that human caspase-4 corresponds to rodent caspase-12 to initiate cell death signaling pathway, and that the activation of caspase-4 would mediate neuronal cell death in neurodegenerative disorder. Caspase-4 could be the potential target to develop treatments for such diseases including AD.