Peroxiredoxin 6 suppresses erastin‐induced ferroptosis in lung endothelial cells

Abstract

Ferroptosis is a newly described form of regulated cell death driven by an iron‐dependent accumulation of phospholipid hydroperoxides. Glutathione peroxidase 4 (GPx4) suppresses ferroptosis through its ability to reduce phospholipid hydroperoxides. Peroxiredoxin 6 (Prdx6) expresses phospholipid hydroperoxide glutathione peroxidase activity, but its role in the regulation of ferroptosis remains unexplored. We compared Prdx6 and GPx4 mRNA levels in murine lungs. Prdx6 expression was five times higher than GPx4 expression. We then visualized the spatial localization of lung Prdx6. Prdx6 was expressed in both pulmonary epithelial (epCAM+) and endothelial (Ve‐cadherin+) cells. We studied the role of Prdx6 on lung ferroptosis induced by erastin, an inhibitor of the cystine‐glutamate antiporter, which is critical to glutathione production. Erastin (1‐5μM, 24h) increased lipid peroxidation and cell death in mouse pulmonary microvascular endothelial cells (MPMVECs) in primary culture; this effect was higher in Prdx6‐KO MPMVECs than in WT‐MPMVECs. Co‐treatment with the ferroptosis suppressor ferrostatin‐1 prevented erastin‐induced lipid peroxidation and cell death in both WT and Prdx6‐KO MPMVECs. Similarly, knockdown (KD) of Prdx6 in human pulmonary microvascular endothelial cells (HPMVECs) increased erastin‐induced cell death, suggesting that Prdx6 limits ferroptosis in the pulmonary endothelium. We then conducted extracellular flux assays and RNA‐seq analyses in Prdx6‐deficient cells to further explore how the loss of Prdx6 sensitizes cells to ferroptosis. Cellular respiration and mitochondrial function were blunted in Prdx6‐deficient cells compared to corresponding controls. Top differentially expressed genes in Prdx6‐KD HPMVECs include known regulators of iron metabolism and ferroptosis such as hmox1, cybrd1, and sfxn1. Upregulated Reactome pathways enriched in Prdx6‐KD HPMVECs include mitochondrial and amino acid metabolism and p53‐mediated DNA damage response, while downregulated pathways include biological oxidations, transport of bile salts and organic acids, metal ions and amine compounds, cytochrome P450, and phase 1 metabolism. These results show that loss of Prdx6 sensitizes lung endothelial cells to ferroptosis by promoting lipid peroxidation and altering transcriptional signatures associated with cell cycle progression and mitochondrial metabolism.