Suppression of AMPK/aak-2 by NRF2/SKN-1 downregulates autophagy during prolonged oxidative stress

Abstract

NF-E2-related factor 2 (NRF2) transcription factor has a fundamental role in cell homeostasis maintenance as one of the master regulators of oxidative and electrophilic stress responses. Previous studies have shown that a regulatory connection exists between NRF2 and autophagy during reactive oxygen species-generated oxidative stress. The aim of the present study was to investigate how autophagy is turned off during prolonged oxidative stress, to avoid overeating and destruction of essential cellular components.AMPKis a key cellular energy sensorhighlyconservedineukaryoticorganisms, andithas anessential role inautophagy activationat various stress events. Here the role of human AMPK and its Caenorhabditis elegans counterpart AAK-2 was explored upon oxidative stress. We investigated the regulatory connection between NRF2 and AMPK during oxidative stress induced by tert-butyl hydroperoxide (TBHP) in HEK293T cells and C. elegans. Putative conserved NRF2/protein skinhead-1 binding sites were found in AMPK/aak-2 genes by in silico analysis and were later confirmed experimentally by using EMSA. After addition of TBHP, NRF2 and AMPK showed a quick activation; AMPK was later down-regulated,however,whileNRF2 level remainedhigh.Autophagosome formationandUnc-51-likeautophagy activating kinase 1 phosphorylation were initially stimulated, but they returned to basal values after 4 h of TBHP treatment.The silencing ofNRF2 resulted in a constant activation ofAMPKleading to hyperactivation of autophagy during oxidative stress. We observed the same effects in C. elegans demonstrating the conservation of this selfdefensemechanism to save cells fromhyperactivated autophagy upon prolonged oxidative stress.Weconclude that NRF2 negatively regulates autophagy through delayed down-regulation of the expression of AMPK upon prolonged oxidative stress. This regulatory connection between NRF2 and AMPK may have an important role in understanding how autophagy is regulated in chronic humanmorbidities characterized by oxidative stress, such as neurodegenerative diseases, certain cancer types, and in metabolic diseases.