FOXO6 inhibits SMURF2‐mediated ubiquitination and degradation of NR4A1 to promote retinal pigment epithelial cell ferroptosis in diabetic retinopathy

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Diabetic retinopathy (DR) is the main cause of vision loss, with retinal pigment epithelial (RPE) cell dysfunction as a key contributor. Ferroptosis is implicated in RPE injury under hyperglycemia; however, its upstream regulatory mechanisms remain unclear. High glucose (HG)-induced human RPE ARPE-19 cells and a diabetic mouse model were used. Ferroptosis was assessed through biochemical assays, Western blot, fluorescence probes, and histological staining. Protein interactions and transcriptional regulation were examined using Co-IP, glutathione S-transferase pull-down, ChIP, and luciferase reporter assays. Immunofluorescence staining was used as key indicator expression in retinal tissues. Nuclear receptor 4A1 (NR4A1) expression was upregulated under HG-induced ARPE-19 cells. NR4A1 knockdown alleviated HG-induced iron accumulation, lipid peroxidation, and ferroptosis marker changes. Mechanistically, SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2) interacted with NR4A1 and promoted its ubiquitination and degradation. Forkhead box O6 (FOXO6) was upregulated by HG and repressed SMURF2 transcription. SMURF2 overexpression mitigated HG-induced ARPE-19 ferroptosis and retinal injury in DR mice, an effect reversed by NR4A1 co-overexpression. SMURF2 knockdown reversed the effect of FOXO6 knockdown on HG-induced ferroptosis in ARPE-19 cells and retinal injury in DR mice. The FOXO6-SMURF2-NR4A1 axis critically regulates ferroptosis in RPE cells during DR.