ATF3 inhibits retinal pigment epithelial cell injury by activating HSPA5:A mechanistic insight for diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of vision loss in working-age adults. Heat shock protein family A (Hsp70) member 5 (HSPA5) has been reported to be a core ferroptosis-related gene in DR, while its regulatory mechanism remains unclear. DEGs were screened from the GSE233164 dataset with strict thresholds (P < 0.01, |log2FC| > 3). Bioinformatics enrichment and PPI network analyses were performed to screen genes. Clinical samples from 49 DR patients and 49 healthy controls were used for external validation. HG-induced ARPE-19 cell models were established to evaluate cell viability, proliferation, apoptosis, oxidative stress, ferroptosis, and inflammation. The ATF3-HSPA5 transcriptional regulatory relationship was predicted by JASPAR and verified via ChIP and dual-luciferase assays. 171 DEGs were screened, and 39 core genes were retained for network analysis. HSPA5 was consistently downregulated in HG-stimulated ARPE-19 cells and DR clinical samples, with a high diagnostic AUC of 0.93. HG exposure triggered RPE cell dysfunction, oxidative damage, ferroptosis, and inflammation, all of which were significantly rescued by HSPA5 overexpression. ATF3 was also downregulated in DR models and clinical samples and positively correlated with HSPA5. Mechanistically, ATF3 directly bound and transcriptionally activated the HSPA5 promoter. Rescue experiments confirmed that HSPA5 knockdown completely abolished the protective effects of ATF3 against HG-induced cell injury and ferroptosis. ATF3 alleviates HG-induced retinal pigment epithelial cell injury and ferroptosis by transcriptionally activating HSPA5. The ATF3-HSPA axis represents a potential therapeutic target for DR.