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

In recent years, autophagy, as a crucial intracellular degradation and recycling mechanism, has played a key role in maintaining cellular homeostasis and responding to various stress conditions. Particularly in renal proximal tubular epithelial cells, autophagy activity is essential for coping with common renal injuries such as ischemia, toxic damage, and inflammation. However, although the role of autophagy in cellular stress adaptation has been extensively studied, the molecular mechanisms by which hyperosmotic stress induces autophagy remain unclear. As a form of mechanical stress, hyperosmotic stress can affect cellular function by altering osmotic pressure differences between the intracellular and extracellular environments, but how it specifically regulates the autophagy pathway remains an unresolved mystery.

Transcription factor EB (TFEB) is a major transcriptional regulator of the autophagy-lysosome pathway. It promotes autophagy by regulating the expression of autophagy and lysosome-related genes. The activity of TFEB is controlled by phosphorylation events. When dephosphorylated, TFEB translocates from the cytoplasm to the nucleus, thereby activating the transcription of autophagy-related genes. However, how hyperosmotic stress regulates TFEB nuclear translocation through calcium ion (Ca²⁺) signaling pathways and TRPML1 channels remains a question that requires further exploration.

Source of the Paper

The paper, titled “Hyperosmotic Stress Promotes the Nuclear Translocation of TFEB in Tubular Epithelial Cells Depending on Intracellular Ca²⁺ Signals via TRPML Channels,” was co-authored by Takashi Miyano, Atsushi Suzuki, Hisaaki Konta, and Naoya Sakamoto. They are affiliated with the Graduate School of Mechanical Systems Engineering at Tokyo University of Science and Tokyo Metropolitan University. The paper was published online on January 21, 2025, in the journal Cellular and Molecular Bioengineering, with the DOI 10.1007/s12195-024-00839-6.

Research Process

Research Objectives

This study aims to elucidate how hyperosmotic stress activates autophagy through TFEB and Ca²⁺ signaling pathways, thereby revealing the mechanisms of cellular responses to mechanical stress. Specifically, the research team induced hyperosmotic stress using mannitol, observed the nuclear translocation of TFEB, and explored the roles of TRPML1 channels and calcineurin in this process.

Experimental Methods

1. Cell Culture and Hyperosmotic Stimulation
   NRK-52E rat renal tubular epithelial cells were used as the experimental model. The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum. When the cells reached 80% confluency, they were treated with hyperosmotic medium containing mannitol. To study the role of the Ca²⁺ signaling pathway, the intracellular Ca²⁺ chelator BAPTA-AM and the calcineurin inhibitor FK-506 were used. Additionally, the TRPML1 antagonist ML-SI3 was employed to assess the function of TRPML1 channels.

2. Immunofluorescence Staining and Nuclear Translocation Evaluation
   The nuclear translocation of TFEB was observed through immunofluorescence staining. Cells were fixed with 4% paraformaldehyde and stained with anti-TFEB antibody and Alexa Fluor 488-labeled secondary antibody. Nuclei were stained with Hoechst 33342. The fluorescence intensities in the nucleus and cytoplasm were quantified using ImageJ software to evaluate the extent of TFEB nuclear translocation.

3. RNA Extraction and Quantitative Real-Time PCR
   Total RNA was extracted from NRK-52E cells and reverse-transcribed into cDNA. The expression levels of TFEB target genes (e.g., LC3, VPS18, LAMP1, and LAMP2) were detected by quantitative real-time PCR (RT-qPCR). Data were normalized using the 2−ΔΔCt method.

4. Western Blot Analysis
   The phosphorylation level of p70S6K was analyzed by Western blot to assess mTORC1 activity. Additionally, the protein level of LC3-II was analyzed as an autophagy marker. Nuclear and cytoplasmic fractions were separated using a nuclear/cytosolic fractionation kit, with GAPDH and Lamin A/C serving as internal controls.

Key Findings

1. Mannitol-Induced Hyperosmotic Stress Promotes TFEB Nuclear Translocation
   The experimental results showed that mannitol treatment significantly promoted the translocation of TFEB from the cytoplasm to the nucleus, with this phenomenon peaking at 1 hour. In contrast, urea treatment did not significantly alter the nuclear localization of TFEB, indicating that TFEB nuclear translocation is closely related to mechanical stress induced by cell shrinkage.

2. Ca²⁺ Signaling Pathway Regulates TFEB Nuclear Translocation
   Chelation of intracellular Ca²⁺ with BAPTA-AM completely inhibited TFEB nuclear translocation, while chelation of extracellular Ca²⁺ with EGTA had no significant effect. This suggests that intracellular Ca²⁺ plays a critical role in hyperosmotic stress-induced TFEB activation.

3. Role of Calcineurin in TFEB Nuclear Translocation
   Hyperosmotic stress significantly enhanced calcineurin activity, as verified by the nuclear translocation of its downstream transcription factor NFAT. Inhibition of calcineurin with FK-506 significantly suppressed TFEB nuclear translocation, indicating that calcineurin plays an important role in hyperosmotic stress-induced TFEB activation.

4. Role of TRPML1 Channels in TFEB Nuclear Translocation
   The TRPML1 antagonist ML-SI3 significantly inhibited hyperosmotic stress-induced TFEB nuclear translocation and LC3-II upregulation, suggesting that TRPML1 channels activate calcineurin by releasing Ca²⁺, thereby promoting TFEB nuclear translocation and autophagy.

Conclusion

This study demonstrates that mannitol-induced hyperosmotic stress activates TFEB through TRPML1 channels and calcineurin, promoting its nuclear translocation and the expression of autophagy-related genes. This finding not only reveals the molecular mechanisms of hyperosmotic stress-induced autophagy but also provides new insights into how cells translate mechanical stress into biological responses.

Research Highlights

1. Reveals a New Mechanism of Hyperosmotic Stress-Induced Autophagy
   This study is the first to elucidate the critical roles of TRPML1 channels and calcineurin in hyperosmotic stress-induced TFEB nuclear translocation, filling a knowledge gap in this field.

2. Provides Potential Therapeutic Targets for Kidney Diseases
   By regulating the TFEB and TRPML1 pathways, this research may offer new therapeutic strategies for treating kidney diseases associated with autophagy dysfunction.

3. Innovative Experimental Design
   The research team systematically revealed the molecular mechanisms of hyperosmotic stress-induced autophagy by combining immunofluorescence staining, Western blot, and RT-qPCR, providing important experimental references for related fields.

Research Significance

This study not only deepens our understanding of the mechanisms of hyperosmotic stress-induced autophagy but also provides a theoretical basis for developing novel treatments for kidney diseases. Additionally, the findings offer new research directions for exploring other mechanical stress-related cellular responses.