Matrix stiffness disrupts tight junction integrity in retinal pigment epithelial cells via YAP1-mediated autophagy...

The extracellular matrix (ECM) of retinal pigment epithelial (RPE) cells undergoes progressive stiffening during aging and under pathological conditions. However, how such mechanical effects affect RPE cell function remains incompletely understood. This study aims to investigate the effect of stiffness of ECM on autophagy in RPE cells and its regulatory role on tight junctions.

ARPE-19 cells were cultured on polyacrylamide hydrogel substrates with low (5 kPa) and high (40 kPa) stiffness. The expression and subcellular localization of tight junction proteins and autophagy-related markers were assessed by Western blotting, quantitative PCR, and immunofluorescence staining. Autophagy was pharmacologically modulated using the autophagy activator rapamycin and the inhibitor chloroquine to evaluate the effects of autophagy on tight junction integrity. In addition, Yes-associated protein 1(YAP1) was knocked down using shRNA to investigate its role in ECM stiffness-mediated regulation of autophagy and tight junction alterations.

Polyacrylamide hydrogel substrates with low stiffness (5 kPa) and high stiffness (40 kPa) had no significant effect on the growth or proliferation of ARPE-19 cells. High-stiffness substrates markedly suppressed autophagic activity in ARPE-19 cells and downregulated the expression of tight junction proteins zonula occludens-1 (ZO-1), occludin, and claudin-19, resulting in disruption of tight junction integrity. Pharmacological inhibition of autophagy further exacerbated tight junction damage, whereas autophagy activation partially reversed the tight junction impairment induced by high matrix stiffness. Moreover, knockdown of YAP1 substantially attenuated the inhibitory effects of high stiffness on autophagy and tight junction protein expression.

Increased ECM stiffness impairs tight junction integrity in ARPE-19 cells through a YAP1-mediated suppression of autophagy.