Miyano Takashi, Suzuki Atsushi, Konta Hisaaki, Sakamoto Naoya
Department of Medical and Robotic Engineering Design, Tokyo University of Science, Tokyo, Japan.
Department of Mechanical Systems Engineering, Graduate School of Systems Design, Tokyo Metropolitan University, Tokyo, Japan.
Cell Mol Bioeng. 2025 Jan 21;18(1):39-52. doi: 10.1007/s12195-024-00839-6. eCollection 2025 Feb.
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 signaling pathways, contributing to understanding cellular responses to mechanical stress.
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 chelator, BAPTA-AM, and a calcineurin inhibitor were used to dissect the Ca 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 channel, was assessed using pharmacological inhibition to determine its impact on TFEB translocation and autophagy marker LC3-II levels.
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 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.
Hyperosmotic stress promotes TFEB nuclear localization, and TRPML1-induced activation of calcineurin is involved in the mechanism of hyperosmolarity-induced autophagy.
The online version contains supplementary material available at 10.1007/s12195-024-00839-6.
我们之前证明，作为机械应力的高渗应激可诱导肾小管上皮细胞自噬。本研究旨在阐明高渗诱导自噬的分子机制。该研究问题探讨了高渗应激如何通过转录因子EB（TFEB）和钙信号通路激活自噬，有助于理解细胞对机械应力的反应。
使用含甘露醇的培养基对NRK-52E正常大鼠肾细胞施加高渗应激。利用荧光显微镜观察TFEB核转位，这是自噬调节中的一个关键事件。使用细胞内钙螯合剂BAPTA-AM和钙调神经磷酸酶抑制剂来剖析参与TFEB转位的钙信号通路。分析雷帕霉素复合物1激酶的哺乳动物靶点的底物p70S6K的磷酸化，以探讨其在TFEB定位中的作用。此外，使用药理学抑制来评估细胞内钙通道瞬时受体电位黏脂蛋白1（TRPML1）的功能，以确定其对TFEB转位和自噬标志物LC3-II水平的影响。
甘露醇诱导的高渗应激促进了TFEB的核转位，而用BAPTA-AM处理可完全消除这种转位。钙调神经磷酸酶的抑制在高渗条件下抑制了TFEB核转位，表明由细胞内钙控制的信号通路参与了TFEB的核转位。相反，高渗应激并未显著改变p70S6K的磷酸化。TRPML1的药理学抑制减弱了高渗应激诱导的TFEB核转位和LC3-II上调。
高渗应激促进TFEB核定位，TRPML1诱导的钙调神经磷酸酶激活参与高渗诱导自噬的机制。
在线版本包含可在10.1007/s12195-024-0https://doi.org/10.1007/s12195-024-00839-6获取的补充材料。
