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    • Pharmacological inhibition of PKR seems to be

    2021-09-14

    Pharmacological inhibition of PKR seems to be an interesting strategy for revealing the role of PKR in oxytosis and ferroptosis. The oxindole/imidazole derivative C16 was identified by screening a library of 26 different ATP-binding site-directed inhibitors of varying structure that is more selective than previously available inhibitors (Jammi et al., 2003). C16 was further used in different studies as an inhibitor of PKR in vitro and in vivo (Ingrand et al., 2007; Joshi et al., 2013; Shimazawa and Hara, 2006). In this study, we used the specific inhibitor of PKR, C16, to investigate the role of PKR and its downstream components in the intracellular signaling of glutamate- and erastin-induced cell death in HT22 cells. The selectivity of C16 was confirmed using PKR knockout (KO) HT22 cells. We also evaluated real time changes of cellular metabolic rates to investigate the effect of C16 on impaired mitochondrial function.
    Materials and methods
    Results
    Discussion We reported here the role of PKR in glutamate-induced oxytosis and erastin-induced ferroptosis of HT22 cells. C16, a PKR inhibitor, prevented oxidative stress and affected various signaling molecules in the ER stress pathway and the MAPK pathways. Previous studies demonstrated that IRE1, which activates chaperone genes in response to ER stress, also activated JNK (Nishitoh et al., 2002; Urano et al., 2000). Our results suggest that IRE1 activates JNK in response to not only ER stress but also oxidative stress, and PKR regulates the IRE1-JNK pathway. This study also showed that glutamate and erastin affected limited components of the ER stress pathway. The treatment of HT22 YM-155 hydrochloride with glutamate or erastin increased the expression of GADD153 and Chac1, downstream components in the PERK-eIF2α pathway (Mungrue et al., 2009), and the phosphorylation of IRE1; however, it did not increase the phosphorylation of PERK and eIF2α, enhance splicing of XBP1 mRNA downstream components of the IRE1 pathway, or cause activation of the ATF6 pathway. The observation of the increased expression of GADD153 and Chac1 and the lack of evidence for enhanced splicing of the XBP1 mRNA were consistent with a previous study (Dixon et al., 2014). These results suggest that pharmacological inhibition of system Xc does not cause typical ER stress but activates the IRE1-JNK pathway (Fig. 6). The role of PKR in the activation of JNK and p38 MAPK, representatives of the stress-activated MAPKs, has been reported in double-stranded RNA-treated HeLa cells (Iordanov et al., 2000). Many studies have shown that JNK phosphorylation is increased in response to apoptotic cell death; however, the contribution of this pathway to oxytosis or ferroptosis is not well known. It has been reported that binding of activated JNK to the outer mitochondrial membrane protein Sab, which leads to increased mitochondrial ROS, mediates ER stress-induced apoptosis in cultured cells and ischemic necrosis in the brain (Chambers et al., 2013; Win et al., 2014). Thus, non-transcriptional targets of phospho-JNK such as Sab could play a significant role not only in ER stress but also in oxytosis and ferroptosis, in addition to traditional transcriptional targets of activated JNK such as c-Jun, c-Myc, ATF2 and Elk1 (Win et al., 2018). This hypothesis is supported by the observation that a pharmacological inhibitor of JNK SP600125 did not have a prominent protective effect on glutamate- and erastin-induced cell death (Fig. S4). Another pathway that could modify oxidative stress-induced cell death is the ERK pathway. It has been reported that the ERK pathway serve as an endogenous neuroprotective signaling pathway against various neurotoxic insults including oxidative stress (Maher, 2001; Shibata et al., 2017). Interestingly, PKR inhibitor C16 increased the phosphorylation of p44/42 MAPK and MEK1/2 both in control and glutamate or erastin-treated HT22 cells, suggesting that PKR inhibition enhances a cell survival pathway. Furthermore, genetic deletion of PKR potentiates the basal level of the phospho-p44/42 signal (Takada et al., 2007). Taken together, these results suggest that PKR negatively regulates the ERK pathway and cell survival. Further studies of the role of PKR on the activation of the ERK pathway will shed light on the neuroprotective mechanism of C16.