Hyperosmotic Stress Promotes the Nuclear Translocation of TFEB in Tubular Epithelial Cells Depending on Intracellular Ca2+ Signals via TRPML Channels

Abstract Purpose We previously demonstrated that hyperosmotic stress, which acts as mechanical stress, induces autophagy of tubular epithelial cells. This study aims to elucidate the molecular mechanisms of hyperosmolarity-induced autophagy. The research question addresses how hyperosmotic stress activates autophagy through transcription factor EB (TFEB) and Ca 2+ signaling pathways, contributing to understanding cellular responses to mechanical stress. Methods NRK-52E normal rat kidney cells were subjected to hyperosmotic stress using mannitol-containing medium. Fluorescence microscopy was utilized to observe TFEB nuclear translocation, a crucial event in autophagy regulation. An intracellular Ca 2+ chelator, BAPTA-AM, and a calcineurin inhibitor were used to dissect the Ca 2+ signaling pathway involved in TFEB translocation. The phosphorylation of p70S6K, a substrate of the mammalian target of rapamycin complex 1 kinase, was analyzed to explore its role in TFEB localization. Additionally, the function of transient receptor potential mucolipin 1 (TRPML1), an intracellular Ca 2+ channel, was assessed using pharmacological inhibition to determine its impact on TFEB translocation and autophagy marker LC3-II levels. Results Mannitol-induced hyperosmotic stress promoted the nuclear translocation of TFEB, which was completely abolished by treatment with BAPTA-AM. Inhibition of calcineurin suppressed TFEB nuclear translocation under hyperosmolarity, indicating that a signaling pathway governed by intracellular Ca 2+ is involved in TFEB’s nuclear translocation. In contrast, hyperosmotic stress did not significantly alter p70S6K phosphorylation. Pharmacological inhibition of TRPML1 attenuated both TFEB nuclear translocation and LC3-II upregulation in response to hyperosmotic stress. Conclusions Hyperosmotic stress promotes TFEB nuclear localization, and TRPML1-induced activation of calcineurin is involved in the mechanism of hyperosmolarity-induced autophagy.