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

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.