Impaired selective turnover of p62 by autophagy causes severe liver injury accompanied by the formation of p62-positive inclusions and upregulation of detoxifying enzymes. These phenotypes correspond closely to the pathological conditions seen in human liver diseases, including alcoholic hepatitis and hepatocellular carcinoma. However, the molecular mechanisms and pathophysiological processes in these events are still unknown. Here we report the identification of a novel regulatory mechanism by p62 of the transcription factor Nrf2, whose target genes include antioxidant proteins and detoxification enzymes. p62 interacts with the Nrf2-binding site on Keap1, a component of Cullin-3-type ubiquitin ligase for Nrf2. Thus, an overproduction of p62 or a deficiency in autophagy competes with the interaction between Nrf2 and Keap1, resulting in stabilization of Nrf2 and transcriptional activation of Nrf2 target genes. Our findings indicate that the pathological process associated with p62 accumulation results in hyperactivation of Nrf2 and delineates unexpected roles of selective autophagy in controlling the transcription of cellular defence enzyme genes.

Cancer cells consume large quantities of nutrients and maintain high levels of anabolism. Recent studies revealed that various oncogenic pathways are involved in modulation of metabolism. Nrf2, a key regulator for the maintenance of redox homeostasis, has been shown to contribute to malignant phenotypes of cancers including aggressive proliferation. However, the mechanisms with which Nrf2 accelerates proliferation are not fully understood. Here, we show that Nrf2 redirects glucose and glutamine into anabolic pathways, especially under the sustained activation of PI3K-Akt signaling. The active PI3K-Akt pathway augments the nuclear accumulation of Nrf2 and enables Nrf2 to promote metabolic activities that support cell proliferation in addition to enhancing cytoprotection. The functional expansion of Nrf2 reinforces the metabolic reprogramming triggered by proliferative signals.

The Keap1-Nrf2 regulatory pathway plays a central role in the protection of cells against oxidative and xenobiotic damage. Under unstressed conditions, Nrf2 is constantly ubiquitinated by the Cul3-Keap1 ubiquitin E3 ligase complex and rapidly degraded in proteasomes. Upon exposure to electrophilic and oxidative stresses, reactive cysteine residues of Keap1 become modified, leading to a decline in the E3 ligase activity, stabilization of Nrf2 and robust induction of a battery of cytoprotective genes. Biochemical and structural analyses have revealed that the intact Keap1 homodimer forms a cherry-bob structure in which one molecule of Nrf2 associates with two molecules of Keap1 by using two binding sites within the Neh2 domain of Nrf2. This two-site binding appears critical for Nrf2 ubiquitination. In many human cancers, missense mutations in KEAP1 and NRF2 genes have been identified. These mutations disrupt the Keap1-Nrf2 complex activity involved in ubiquitination and degradation of Nrf2 and result in constitutive activation of Nrf2. Elevated expression of Nrf2 target genes confers advantages in terms of stress resistance and cell proliferation in normal and cancer cells. Discovery and development of selective Nrf2 inhibitors should make a critical contribution to improved cancer therapy.

The Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system is essential for cytoprotection against oxidative and electrophilic insults. Under unstressed conditions, Keap1 serves as an adaptor for ubiquitin E3 ligase and promotes proteasomal degradation of Nrf2, but Nrf2 is stabilized when Keap1 is inactivated under oxidative/electrophilic stress conditions. Autophagy-deficient mice show aberrant accumulation of p62, a multifunctional scaffold protein, and develop severe liver damage. The p62 accumulation disrupts the Keap1-Nrf2 association and provokes Nrf2 stabilization and accumulation. However, individual contributions of p62 and Nrf2 to the autophagy-deficiency-driven liver pathogenesis have not been clarified. To examine whether Nrf2 caused the liver injury independent of p62, we crossed liver-specific Atg7::Keap1-Alb double-mutant mice into p62-and Nrf2-null backgrounds. Although Atg7::Keap1-Alb:: p62 −/− triple-mutant mice displayed defective autophagy accompanied by the robust accumulation of Nrf2 and severe liver injury, Atg7:: Keap1-Alb::Nrf2 −/− triple-mutant mice did not show any signs of such hepatocellular damage. Importantly, in this study we noticed that Keap1 accumulated in the Atg7-or p62-deficient mouse livers and the Keap1 level did not change by a proteasome inhibitor, indicating that the Keap1 protein is constitutively degraded through the autophagy pathway. This finding is in clear contrast to the Nrf2 degradation through the proteasome pathway. We also found that treatment of cells with tert-butylhydroquinone accelerated the Keap1 degradation. These results thus indicate that Nrf2 accumulation is the dominant cause to provoke the liver damage in the autophagy-deficient mice. The autophagy pathway maintains the integrity of the Keap1-Nrf2 system for the normal liver function by governing the Keap1 turnover.electrophile | polyubiquitination

Diesel exhaust particles (DEP) contain quinones that are capable of catalyzing the generation of reactive oxygen species in biological systems, resulting in induction of oxidative stress. In the present study, we explored sulfhydryl oxidation by phenanthraquinone, a component of DEP, using thiol compounds and protein preparations. Phenanthraquinone reacted readily with dithiol compounds such as dithiothreitol (DTT), 2,3-dimercapto-1-propanol (BAL), and 2,3-dimercapto-1-propanesulfonic acid (DMPS), resulting in modification of the thiol groups, whereas minimal reactivities of this quinone with monothiol compounds such as GSH, 2-mercaptoethanol, and N-acetyl-L-cysteine were seen. The modification of DTT dithiol caused by phenanthraquinone proceeded under anaerobic conditions but was accelerated by molecular oxygen. Phenanthraquinone was also capable of modifying thiol groups in pulmonary microsomes from rats and total membrane preparation isolated from bovine aortic endothelial cells (BAEC), but not bovine serum albumin (BSA), which has a Cys34 as a reactive monothiol group. A comparison of the thiol alkylating agent N-ethylmaleimide (NEM) with that of phenanthraquinone indicates that the two mechanisms of thiol modification are distinct. Studies revealed that thiyl radical intermediates and reactive oxygen species were generated during interaction of phenanthraquinone with DTT. From these findings, it is suggested that phenanthraquinone-mediated destruction of protein sulfhydryls appears to involve the oxidation of presumably proximal thiols and the reduction of molecular oxygen.

p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

Cancer cells first adapt to the microenvironment and then propagate. Mutations in tumor suppressor genes or oncogenes are frequently found in cancer cells. Comprehensive genomic analyses have identified somatic mutations and other alterations in the KEAP1 or NRF2 genes and in well-known tumor suppressor genes or oncogenes, such as TP53, CDKN2A, PTEN, and PIK3CA, in various types of cancer. Aberrant NRF2 activation in cancer cells occurs through somatic mutations in the KEAP1 or NRF2 gene as well as through other mechanisms that disrupt the binding of KEAP1 to NRF2. Unregulated NRF2 confers on cancer cells high-level resistance to anticancer drugs and reactive oxygen species (ROS) and directs cancer cells toward metabolic reprogramming. Therefore, NRF2 has been studied as a therapeutic target molecule in cancer. Two strategies have been used to target NRF2 via therapeutic drugs: inhibition of NRF2 and induction of NRF2. NRF2 inhibitors may be effective against NRF2-addicted cancer cells in which NRF2 is aberrantly activated. These inhibitors have not yet been established as NRF2-targeted anticancer drugs for the treatment of human cancers. Diagnosis of NRF2 activation could facilitate the use of NRF2 inhibitors for the treatment of patients with NRF2-addicted cancers. Conversely, NRF2 inducers have been used or are being developed for non-cancer diseases. In addition, NRF2 inducers may be useful for cancer chemotherapy in combination with conventional anticancer agents or even NRF2 inhibitors.

The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-boxmediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock ''gated'' pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic Clock D19 mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.