Glucocorticoid-induced RPE injury in ARPE-19 cells: association with excessive autophagy and AMPK/mTOR signaling.
AIM To investigate whether excessive glucocorticoid (GC) exposure disrupts retinal pigment epithelial (RPE) homeostasis through glucocorticoid receptor (GR)-dependent autophagy hyperactivation. METHODS Human retinal pigment epithelial cell line (ARPE-19 cells) were exposed to GC (0-100 µmol/L, 48h). GR subcellular localization was analyzed via mitochondrial fractionation and immunofluorescence. Functional assessments included barrier integrity [zonula occludens-1 (ZO-1) immunostaining], phagocytic capacity (FluoSpheres™-labeled photoreceptor outer segment uptake), and ultrastructural analysis (transmission electron microscopy, TEM). Autophagic flux was quantified using mCherry-green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (LC3) B reporters. Molecular mechanisms were probed through AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway activity and autophagic flux assessed by LC3-II levels. GR dependency was confirmed using the antagonist RU486, and cell-type specificity was assessed using mouse photoreceptor (661W) cells. RESULTS GC induced GR upregulation and mitochondrial translocation in a dose- and time-dependent manner, correlating with pathological autophagy activation. Key findings included: 1) tight junction disruption, evidenced by ZO-1 fragmentation, 2) reduction in phagocytic uptake efficiency, 3) mitochondrial shrinkage and autolysosome accumulation (TEM), 4) altered AMPK/mTOR signaling (p-AMPK up, p-mTOR down) associated with autophagosome formation, 5) elevated LC3-II levels confirming excessive autophagic flux. Critically, all GC-induced effects, including GR upregulation, signaling changes, and increased autophagy, were abolished by GR antagonism with RU486. CONCLUSION This study shows that GR-dependent GC exposure induces autophagic flux with ultrastructural evidence suggestive of mitochondria-targeted autophagy and alters AMPK/mTOR signaling, correlating with RPE barrier collapse and phagocytic failure, a possible link to steroid-associated central serous chorioretinopathy (CSC).