SP1-activated CFL2 promotes high glucose-induced retinal pigment epithelial cell injury and involves the AMPK/mTOR pathway.

Abstract

Background: Cofilin-2 (CFL2) belongs to the cofilin family of actin-binding proteins and plays an important role in the actin homeostasis of muscle cells. CFL2 has been confirmed to regulate diabetic retinopathy (DR) progression. However, the current research is limited and more evidence is needed to reveal its role and mechanism in the DR process.

Methods: Retinal pigment epithelial (RPE) cells (ARPE-19) were cultured in high-glucose (HG; 30 mM) conditions to mimic DR cell models. Cell proliferation and apoptosis were examined by CCK8 assay, EdU assay, flow cytometry, and caspase 3 activity detection. Cell oxidative stress, ferroptosis, and inflammation were evaluated by detecting ROS, MDA, SOD, GSH, Fe²⁺, TNF-α, and IL-1β levels. The mRNA and protein levels of CFL2 and special protein 1 (SP1) were tested by qRT-PCR and western blot. CFL2 and SP1 interaction was assessed by ChIP assay and dual-luciferase reporter assay.

Results: HG suppressed ARPE-19 cell proliferation, while inducing apoptosis, oxidative stress, ferroptosis, and inflammation. Silencing of CFL2 alleviated HG-induced ARPE-19 cell injury by inhibiting cell apoptosis, oxidative stress, ferroptosis, and inflammation. SP1 could bind to CFL2 promoter regions to increase its expression. SP1 knockdown relieved HG-induced ARPE-19 cell injury via decreasing CFL2 expression. Besides, SP1 knockdown inhibited the activity of the AMPK/mTOR pathway, and CFL2 overexpression could reverse this effect.

Conclusions: CFL2, activated by SP1, promoted HG-induced RPE cell injury through regulating the AMPK/mTOR pathway, which might provide a potential target for DR.