EPB-53

Cobalt is a transition element that abundantly exists in the environment. Besides direct hypoxia stress, cobalt ions indirectly induce hypoxia-reoxygenation injury (HRI), the main cause of acute kidney injury (AKI), a life-threatening clinical syndrome characterized by the necrosis of the proximal tubular epithelial cells (PTECs) and inflammation. Pyroptosis, a type of inflammatory programmed cell death, might play an essential role in HRI-AKI. However, whether pyroptosis is involved in cobalt chloride (CoCl₂)-induced HRI-AKI remains unknown. Autophagy is a cellular biological process maintaining cell homeostasis that is involved in cell damage in AKI, yet the underlying regulatory mechanism of autophagy on pyroptosis has not been fully understood. In this study, the in vitro and in vivo models of CoCl₂-induced HRI-AKI were established with HK-2 cell line and C57BL/6J mouse. Pyroptosis-related markers were detected with western blotting and immunofluorescence assays, and results showed that gasdermin E (GSDME)-mediated pyroptosis was involved in the cell damage in HRI-AKI. Specific chemical inhibitors of caspase 3, caspase 8, and caspase 9 significantly inhibited GSDME-mediated pyroptosis, verifying that GSDME-mediated pyroptosis was induced via the activation of caspase 3/8/9. The western blotting and immunofluorescence assays were adopted to detect the accumulation of the autophagosomes, and results suggested that HRI increased the autophagic level. The effects of autophagy on apoptosis and pyroptosis were evaluated using lentivirus transfection assays to knock down autophagy-specific genes atg5 and fip200, and results demonstrated that autophagy induced GSDME-mediated pyroptosis via apoptotic pathways in HRI-AKI. Our results revealed the involvement of GSDME-mediated pyroptosis in CoCl₂-induced HRI-AKI and promoted the understanding of the regulatory mechanism of GSDME cleavage. Our study might provide a potential therapeutic target for HRI-AKI, and will be helpful for the risk evaluation of cobalt exposure.

Heme-regulated inhibitor (HRI) kinase is a serine-threonine kinase, controlling the initiation of protein synthesis via phosphorylating α subunit of eIF2 on serine 51 residue, mainly in response to heme deprivation in erythroid cells. However, recent studies showed that HRI is also activated by several diverse signals, causing dysregulations in intracellular homeostatic mechanisms in non-erythroid cells. For instance, it was reported that the decrease in protein synthesis upon the 26S proteasomal inhibition by MG132 or bortezomib is mediated by increased eIF2α phosphorylation in an HRI-dependent manner in mouse embryonic fibroblast cells. The increase in eIF2α phosphorylation level through the activation of HRI upon 26S proteasomal inhibition is believed to protect cells against the buildup of misfolded and ubiquitinated proteins, having the potential to trigger the apoptotic response. In contrast, prolonged and sustained HRI-mediated eIF2α phosphorylation can induce cell death, which may involve ATF4 and CHOP expression. Altogether, these studies suggest that HRI-mediated eIF2α phosphorylation may be cytoprotective or cytotoxic depending on the cells, type, and duration of pharmacological agents used. It is thus hypothesized that both HRI activators, inducing eIF2α phosphorylation or HRI inhibitors causing disturbances in eIF2α phosphorylation, may be effective as novel strategies in cancer treatment if the balance in eIF2α phosphorylation is shifted in favor of autophagic or apoptotic response in cancer cells. It is here aimed to review the role of HRI in various biological mechanisms as well as the therapeutic potentials of recently developed HRI activators and inhibitors, targeting eIF2α phosphorylation in cancer cells.