Persistent Activation of Nrf2 through p62 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: H2O2 treatment resulted in time-dependent accumulation of LC3-II and the expression of GFP-LC3 in HEI-OC1 cells. Autophagic vacuoles were confirmed under transmission electron microscope in H2O2-treated HEI-OC1. Necrosis and autophagy were coincidentally identified in H2O2-treated HEI-OC1 cells. There were significant differences of cell viability between H2O2 only and H2O2 and Rapamycin-treated cells. 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 p62 could be a molecular hub of the Nrf2/Keap1 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.