Phosphorylation of eIF2α suppresses the impairment of GSH/NADPH homeostasis and mitigates the activation of cell death pathways, including ferroptosis, during ER stress.

eIF2α Phosphorylation helps maintain cellular homeostasis and overcome endoplasmic reticulum (ER) stress through transcriptional and translational reprogramming. This study aims to elucidate the transcriptional regulation of glutathione (GSH) and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) homeostasis through eIF2α phosphorylation and its impact on cell death during ER stress. eIF2α phosphorylation-deficient (A/A) cells exhibited decreased expression of multiple genes involved in GSH synthesis and NADPH production, leading to an exacerbated depletion of both cellular and mitochondrial GSH, as well as mitochondrial NADPH, during ER stress. Impaired GSH homeostasis resulted from deficient expression of ATF4 and/or its dependent factor, Nrf2, which are key transcription factors in the antioxidant response during ER stress. In contrast, the exacerbation of NADPH depletion may primarily be attributed to the dysregulated expression of mitochondrial serine-driven 1-carbon metabolism pathway genes, which are regulated by an unidentified eIF2α phosphorylation-dependent mechanism during ER stress. Moreover, the eIF2α phosphorylation-ATF4 axis was responsible for upregulation of ferroptosis-inhibiting genes and downregulation of ferroptosis-activating genes upon ER stress. Therefore, ER stress strongly induced ferroptosis of A/A cells, which was significantly inhibited by treatments with cell-permeable GSH and the ferroptosis inhibitor ferrostatin-1. ATF4 overexpression suppressed impairment of GSH homeostasis in A/A cells during ER stress by promoting expression of downstream target genes. Consequently, ATF4 overexpression mitigated ferroptosis as well as apoptosis of A/A cells during ER stress. Our findings underscore the importance of eIF2α phosphorylation in maintaining GSH/NADPH homeostasis and inhibiting ferroptosis through ATF4 and unidentified eIF2α phosphorylation-dependent target(s)-mediated transcriptional reprogramming during ER stress.