Molecular Crosstalk between Nrf2/Keap1 Signaling Pathway, Autophagy and Necrosis in Auditory Cells

Objective: Oxidative stress affects auditory cellular functions. The Nrf2/Keap1 signaling pathway is the main pathway responsible for cell defense against oxidative stress. The purposes of this study were to elucidate how Keap1 and Nrf2 provide molecular foundation for a possible crosstalk between autophagy and necrosis pathways in auditory cells. Method: We used auditory cell line (HEI-OC1) in this study. The viability of HEI-OC1 was determined by cell viability assays. The samples after treatment of HEI-OC1 were analyzed by a flow cytometer. Immunofluorescent confocal laser microscopy was used. Western blot was performed. HEI-OC1 was transfected with Nrf2, Keap1, and p62 siRNA. Results: After H2O2 treatment, not apoptotic cells but necrotic cells were detected by FAC scan analysis. The pretreatment of HEI-OC1 with rapamycin protects against H2O2-induced necrotic cell death. After treatment with rapamycin, the accumulation of LC3-I/II ratio and the expression of GFP-LC3 were observed in HEI-OC1. The knockdown of p62 by siRNA, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 up regulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in auditory cells. Conclusion: We demonstrated a promotion of autophagy through the mTOR signaling pathway to enhance cell survival in auditory cells under oxidative stress. A crosstalk between Nrf2/Keap1 pathway in the autophagy related p62 are also presented. The selective autophagy substrate p62 activates the oxidative stress responsive factor Nrf2 through inactivation of keap1.