Autophagy through 4EBP1 and AMPK regulates oxidative stress-induced premature senescence in auditory cells

The aim of this study was to determine whether autophagy and AMPK contribute to premature senescence in auditory cells. Incubating HEI-OC1 auditory cells with 5 mM H(2)O(2) for 1 h induced senescence, as demonstrated by senescence-associated β-galactosidase (SA-β-gal) staining. H(2)O(2) treatment significantly delayed population-doubling time, leaving cell viability unchanged. Furthermore, the proportion of SA-β-gal-positive cells significantly increased. Autophagy-related protein expression increased, with Atg7 and LC3-II peaking 6 h and Lamp2 peaking 24 h after H(2)O(2) treatment. The expression of these proteins decreased 48 h after treatment. Transmission electron microscopy revealed lipofuscin and aggregates within autolysosomes, which accumulated markedly in the cytoplasm of HEI-OC1 cells 48 h after treatment. Akt and P70S6 phosphorylation markedly decreased after H(2)O(2) treatment, but 4EBP1 phosphorylation significantly increased 48 h after treatment. After RNAi-mediated knockdown (KD) of Atg7 and AMPK, H(2)O(2)-treated cells displayed dense SA-β-gal staining. Also, premature senescence was significantly induced. These suggest that a negative feedback loop may exist between autophagy and AMPK signaling pathways in HEI-OC1 cells. In our model, oxidative stress-induced premature senescence occurred due to impaired autophagy function through 4EBP1 phosphorylation. Our results also indicate that AMPK may regulate premature senescence in auditory cells in an autophagy-dependent and independent manner.