When unfolded proteins become excessive, ER stress-induced apoptosis is mediated by three primary signal transducers, i.e., PERK, Ire1, and ATF6 (41
). These proteins act by several mechanisms, including direct activation of caspases, kinases, transcription factors, and Bcl-2-family proteins (50
). Other studies showed that in ER stress, JNK activation, coupled with Ire1α-TRAF2-ASK1 signaling, induced apoptotic cell death (23
). We have demonstrated that SubAB-induced apoptosis did not follow a pathway involving ER stress sensor Ire1α activation in HeLa cells (52
). A recent study showed that in NRK-52E rat renal tubular epithelial cells, dominant negative inhibition of PERK enhanced SubAB-induced apoptosis and reduced phosphorylation of ERK and Akt (44
). We show here that in HeLa cells, PERK knockdown by siRNA triggers a significant attenuation of SubAB-induced cytotoxicity with decreased Bax/Bak conformational changes and oligomerization, reduced cytochrome c
release, and thus decreased caspase activation. We also observed that, in PERK knockdown G401 cells and mPERK knockdown MEF cells, similar to the findings in HeLa cells, SubAB-induced caspase-7 activation was inhibited. There are cases in which PERK activation promotes or protects against ER stress-induced apoptosis (11
). These studies imply that PERK plays different roles in regulation of cell proliferation or apoptosis, depending on the cell type or species.
Here we show that proteasome inhibitor, e.g., lactacystin, MG132, pretreatment of HeLa cells inhibited toxin-induced proteosomal degradation of ubiquitinated proteins and apoptotic signaling at an early stage. The ubiquitin-proteasome pathway plays an essential role in protein homeostasis, which is fundamental to the control of cell survival (14
). In addition, we demonstrated in PERK knockdown HeLa cells that SubAB-induced loss of ubiquitinated proteins, including c-Myc and cyclin D1, was inhibited, resulting in suppression of apoptosis. Consistent with our results, previous studies found a functional cross talk between PERK-mediated eIF2α phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation was dependent upon eIF2α phosphorylation (4
). They observed that PERK serves as a critical effector of ER stress-induced growth arrest, linking stress in the ER to control of cell cycle progression rather than cell death. Thus, our study demonstrates that in HeLa cells, SubAB-induced activation of PERK leads to protein degradation by the ubiquitin-proteasome pathway, resulting in activation of apoptosis.
ER stress-induced eIF2α phosphorylation represses translation initiation of most transcripts, with loss of short-lived proteins (12
). Our previous data showed that after treatment of cells with SubAB, protein synthesis was transiently suppressed for 2 h and then moderately recovered; these events were probably regulated by a pathway downstream of eIF2α phosphorylation (30
). A recent study showed that eIF2α kinases may mediate UV light-induced apoptosis via eIF2α-dependent or -independent signaling pathways (36
). In contrast, studies of β-amyloid (Aβ)-mediated cell death in SK-N-SH human neuroblastoma cells demonstrated that maintaining levels of phosphorylation of eIF2α inhibited cell death (22
). Thus, eIF2α kinase plays different roles in regulation of apoptosis due to various stimuli or in different cell types.
In HeLa cells, SubAB-induced eIF2α phosphorylation was decreased in PERK knockdown cells (). Further, pretreatment with Sal003, an eIF2α phosphatase inhibitor, with or without SubAB enhanced eIF2α phosphorylation and slightly promoted SubAB-induced caspase activation. eIF2α phosphatase inhibitor is known not only to enhance cell survival even when apoptosis is induced by ER stressors (3
) but also to restrict cell growth (42
). In HeLa cells, protein synthesis inhibition by toxin-induced eIF2α phosphorylation may trigger caspase activation.
Although phosphorylation of eIF2α by ER stress leads to inhibition of general protein translation, transcription factor ATF4 content is selectively enhanced. ATF4 regulates the expression of downstream target genes of both antiapoptotic and apoptotic pathways (43
). Our results show that transient suppression of ATF4 by siRNA did not inhibit SubAB-induced caspase activation; however, prolonged ATF4 suppression enhanced SubAB-induced cytochrome c
release via a Bax/Bak-independent pathway and promoted caspase activation, leading to apoptosis (). These data suggest that ATF4 targets, e.g., CHOP (43
), do not contribute to SubAB-induced apoptosis; rather, ATF4 probably plays an important role in HeLa cell survival. Consistent with our data, Ye and collaborators demonstrated that ATF4 knockdown inhibited HT1080 cell proliferation and enhanced apoptosis (55
Our previous study showed that signaling pathways associated with functional SubAB receptors may be required for activation of SubAB-dependent apoptotic pathways (54
). A recent study showed that in chondrocytes, the presence of extracellular matrix alters the response to ER stressors through delayed Grp78 expression and inhibition of ER stress-induced apoptosis, suggesting that signaling by the extracellular matrix affects the chondrocyte ER stress response (34
). Since SubAB receptors, e.g., α2β1 integrin and NG2, are known to associate with extracellular matrix (24
), we propose that knockdown of SubAB receptors affects the ER stress response, resulting in inhibition of SubAB-induced apoptosis. Further studies are necessary to determine how SubAB-induced PERK-mediated apoptotic signals are linked to signals from SubAB receptors.
In conclusion, we identified a new role for PERK signaling in the regulation of SubAB-induced apoptosis (). BiP cleavage by SubAB in the ER causes PERK activation, followed by eIF2α phosphorylation, leading to transient inhibition of protein synthesis. During transient shutdown of protein synthesis, certain antiapoptotic or proapoptotic proteins, e.g., Mcl-1, p53, and c-Myc, are rapidly turned over; they are ubiquitinated and then degraded in the ubiquitin-proteasome system, leading to Bak/Bax conformational changes and their oligomerization, followed by cytochrome c release, and caspase activation. PERK knockdown by siRNA blocked SubAB-induced apoptosis, with inhibition of eIF2α phosphorylation, ATF4 expression, Bak/Bax conformational changes, cytochrome c release, caspase activation, and ubiquitinated protein degradation. In addition, proteasome inhibitors (MG132 and lactacystin) could suppress SubAB-induced cytochrome c release, followed by caspase activation. Thus, SubAB-induced PERK-mediated Bax/Bak conformational changes are probably regulated by certain ubiquitinated factors, leading to activation of apoptosis. Knockdown of eIF2α induces Bax/Bak conformational changes and oligomerization, followed by cytochrome c release, resulting in caspase activation, a pathway that is enhanced by SubAB-induced BiP cleavage. In addition, increased ATF4 expression is likely to contribute to resistance to cell death. Further studies are necessary to determine the ubiquitinated factors involved in SubAB-induced PERK-mediated Bax/Bak conformational changes, followed by activation of apoptosis.
Proposed model of SubAB-induced PERK-mediated apoptosis signaling pathway in HeLa cells. See the text for additional details.