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

// Nana Akagi Tsuchihashi 1, 2 , Ken Hayashi 1, 3 , Katsuaki Dan 4 , Fumiyuki Goto 1 , Yasuyuki Nomura 5 , Masato Fujioka 1 , Sho Kanzaki 1 , Shizuo Komune 2 , Kaoru Ogawa 1 1 Department of Otorhinolaryngology, Head and Neck Surgery, Keio University, School of Medicine, Tokyo 160–8582, Japan 2 Department of Otorhinolaryngology, Head and Neck Surgery, Kyushu University, School of Medicine, Fukuoka 812–0054, Japan 3 Department of Otorhinolaryngology, Kamio Memorial Hospital, Tokyo 101–0063, Japan 4 Collaborative Research Resources, Core Instrumentation Facility, Keio University, Tokyo 160–8582, Japan 5 Department of Otorhinolaryngology-Head and Neck Surgery, Nihon University, School of Medicine, Tokyo 173–8610, Japan Correspondence to: Kaoru Ogawa, e-mail: [email protected] Keywords: premature senescence(definition), autophagy(definition), AMPK, oxidative stress, auditory cell Received: November 17, 2014      Accepted: December 08, 2014      Published: December 30, 2014 ABSTRACT 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.