Physiological Significance of p62-mediated Autophagy in Auditory Cells

Abstract

Objectives:

Elucidate how Keap1, Nrf2 and p62 provide molecular foundation for a possible crosstalk between autophagy and necrosis pathways in auditory cells.

Methods:

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. Morphological observation was performed by transmission electron microscope (TEM). Transient GFP-LC3 and p62 siRNA were transfected into HEI-OC1 with electroporation.

Results:

HEI-OC1 treated with H2O2 exhibited dose- and time-dependent cell death. After treatment of H2O2, not apoptotic cell but necrotic cell was detected by a flow cytometer. H2O2 treatment resulted in time-dependent accumulation of LC3-II and the expression of GFP-LC3 in HEI-OC1 cells. Autophagosomes were confirmed under TEM in H2O2 treated-HEI-OC1. Immunostaining analysis demonstrated that the majority proportion of Nrf2 translocated into the nucleus in p62 KD HEI-OC1 cells, while the majority proportion of Nrf2 disappeared from nucleus after treatment of H2O2 in p62 KD HEI-OC1. The suppression of autophagy by p62 knockdown accelerated cell death in H2O2-treated HEI-OC1.

Conclusions:

Our results demonstrated that the intracellular clearance is decreased under oxidative stress due to the impairment of crosstalk of p62, Nrf2 and Keap1. This leads to the progression of auditory cell death. Taken together, a crosstalk between Keap1/Nrf2 pathway and the p62-mediated autophagy play an important role in cell survival of auditory cells under oxidative stress.