Activation of the Keap1/Nrf2 stress response pathway in autophagic vacuolar myopathies

Abstract

Nrf2 (nuclear factor [erythroid-derived 2]-like 2; the transcriptional master regulator of the antioxidant stress response) is regulated through interaction with its cytoplasmic inhibitor Keap1 (Kelch-like ECH-associated protein 1), which under basal conditions targets Nrf2 for proteasomal degradation. Sequestosome 1 (SQSTM1)/p62–a multifunctional adapter protein that accumulates following autophagy inhibition and can serve as a diagnostic marker for human autophagic vacuolar myopathies (AVMs)–was recently shown to compete with Nrf2 for Keap1 binding, resulting in activation of the Nrf2 pathway. In this study, we used 55 human muscle biopsies divided into five groups [normal control, hydroxychloroquine- or colchicine-treated non-AVM control, hydroxychloroquine- or colchicine-induced toxic AVM, polymyositis, and inclusion body myositis (IBM)] to evaluate whether Keap1-SQSTM1 interaction led to increased Nrf2 signaling in human AVMs. In toxic AVMs and IBM, but not in control muscle groups or polymyositis, Keap1 antibody labeled sarcoplasmic protein aggregates that can be used as an alternate diagnostic marker for both AVM types; these Keap1-positive aggregates were co-labeled with the antibody against SQSTM1 but not with the antibody against autophagosome marker LC3 (microtubule-associated protein 1 light chain 3). In human AVM muscle, sequestration of Keap1 into the SQSTM1-positive protein aggregates was accompanied by an increase in mRNA and protein levels of Nrf2 target genes; similarly, treatment of differentiated C2C12 myotubes with autophagy inhibitor chloroquine led to an increase in the nuclear Nrf2 protein level and an increase in expression of the Nrf2-regulated genes. Taken together, our findings demonstrate that Nrf2 signaling is upregulated in autophagic muscle disorders and raise the possibility that autophagy disruption in skeletal muscle leads to dysregulation of cellular redox homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0384-6) contains supplementary material, which is available to authorized users.

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“…This accumulation may enhance the isolation of Keap1 by p62. In addition, there is reported that CQ can activate Nrf2/HO-1 pathway ( 14 ). Therefore, we speculated that the accumulation of p62 and degradation of Keap1 induced by DC32 were controlled by PI3K-III.…”

“…In addition to competing with Nrf2 for the Keap1 binding site and sequestering Keap1 into aggregates, p62 also brings Keap1 to autolysosomes for degradation in selective autophagy. The Nrf2-Keap1 interaction, which is blocked by the accumulation of p62 and Nrf2, is increased in the cytoplasm, resulting in activation of the Nrf2/HO-1 pathway ( 12 , 14 – 16 ). In addition, p62/SQSTM1 is also a target gene of Nrf2, and its expression can be induced by Nrf2 under oxidative stress conditions, further facilitating the activation of Nrf2 by blocking its degradation ( 17 ).…”

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“…Intriguingly, several studies have indicated that accumulation of p62 in addition to ubiquitylated protein aggregates results in various disorders, which are caused by disruption of Nrf2 degradation and its persistent activation under autophagy-deficient conditions (Duleh et al, 2016). Notably, to date, the biological functions of p62 under diverse pathophysiological processes remain elusive and modulating functions of accumulated p62 in liver disease have seldom been reported in autophagy sufficiency conditions.…”

A comparative analysis of in vitro toxicity of diesel exhaust particles from combustion of 1st- and 2nd-generation biodiesel fuels in relation to their physicochemical properties—the FuelHealth project

“…Further signs of antioxidant responses include overexpression of HMOX1, which is considered one of the most sensitive and reliable indicators of cellular oxidative stress (Maamoun et al 2016 ), TXNRD1, a pivotal intracellular redox sensor and antioxidant enzyme (Schmidt 2015 ), NQO1, which is essential for cell defense against ROS (Di Francesco et al 2016 ), and FTH1, a major iron storage protein that can protect cells from the toxic effects of iron as well as other free radical producing stresses (Funauchi et al 2015 ). In addition, downregulation of the pro-apoptotic BNIP3L, which plays an important role in hypoxia-dependent cell death (Bellot et al 2009 ) and upregulation of SQSTM1, a multifunctional adapter protein that accumulates following autophagy inhibition (Duleh et al 2016 ), was also observed. This could explain the relatively moderate impact of the tested DEPs on cell cytotoxicity.…”