MOAP‐1‐mediated dissociation of p62/SQSTM1 bodies releases Keap1 and suppresses Nrf2 signaling

Nrf2 signaling is vital for protecting cells against oxidative stress. However, its hyperactivation is frequently found in liver cancer through excessive build‐up of p62/SQSTM1 bodies that sequester Keap1, an adaptor of the E3‐ubiquitin ligase complex for Nrf2. Here, we report that the Bax‐binding protein MOAP‐1 regulates p62‐Keap1‐Nrf2 signaling through disruption of p62 bodies. Upon induction of cellular stresses that stimulate formation of p62 bodies, MOAP‐1 is recruited to p62 bodies and reduces their levels independent of the autophagy pathway. MOAP‐1 interacts with the PB1‐ZZ domains of p62 and interferes with its self‐oligomerization and liquid–liquid phase separation, thereby disassembling the p62 bodies. Loss of MOAP‐1 can lead to marked upregulation of p62 bodies, enhanced sequestration of Keap1 by p62 and hyperactivation of Nrf2 antioxidant target genes. MOAP‐1‐deficient mice exhibit an elevated tumor burden with excessive levels of p62 bodies and Nrf2 signaling in a diethylnitrosamine (DEN)‐induced hepatocarcinogenesis model. Together, our data define MOAP‐1 as a negative regulator of Nrf2 signaling via dissociation of p62 bodies.     

The involvement of p62–Keap1–Nrf2 antioxidative signaling pathway and JNK in the protection of natural flavonoid quercetin against hepatotoxicity

Quercetin, one of the most abundant dietary flavonoids, is reported to have protective function against various hepatotoxicant-induced hepatotoxicity. The present study aims to investigate the critical role of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidative signaling pathway in the protection of quercetin against hepatotoxicity. Quercetin prevented the cytotoxicity induced by a variety of hepatotoxicants including clivorine (Cliv), acetaminophen (APAP), ethanol, carbon tetrachloride (CCl₄), and toosendanin (TSN) in human normal liver L-02 cells. Quercetin induced the nuclear translocation of Nrf2, along with the increased expression of the antioxidant responsive element (ARE)-dependent genes like catalytic or modify subunit of glutamate-cysteine ligase (GCLC/GCLM), and heme oxygenase-1 (HO-1). In addition, the HO-1 inhibitor zinc protoporphyrin (ZnPP) and the GCL inhibitor L-buthionine-(S,R)-sulfoximine (BSO) both reduced the hepatoprotection induced by quercetin. Quercetin had no effect on kelch-like ECH-associated protein-1(Keap1) expression, but molecular docking results indicated the potential interaction of quercetin with the Nrf2-binding site in Keap1 protein. Quercetin increased the expression of p62, and p62 siRNA decreased quercetin-induced hepatoprotection. Quercetin induced the activation of c-Jun N-terminal kinase (JNK) in hepatocytes. JNK inhibitor SP600125 and JNK siRNA both reduced quercetin-induced hepatoprotection. SP600125 and JNK siRNA decreased the increased p62 expression induced by quercetin. In addition, SP600125 also decreased the increased mRNA and protein expression of GCLC, GCLM, and HO-1 induced by quercetin. Taken together, our present study demonstrates that quercetin prevents hepatotoxicity by inducing p62 expression, inhibiting the binding of Keap1 to Nrf2, and thus leading to the increased expression of antioxidative genes dependent on Nrf2. Meanwhile, our study indicates that JNK plays some regulation in this process.     

Arsenic Inhibits Autophagic Flux,Activating the Nrf2-Keap1 Pathway in a p62-Dependent Manner

The Nrf2-Keap1 signaling pathway is a protective mechanism promoting cell survival. Activation of the Nrf2 pathway by natural compounds has been proven to be an effective strategy for chemoprevention. Interestingly, a cancer-promoting function of Nrf2 has recently been observed in many types of tumors due to deregulation of the Nrf2-Keap1 axis, which leads to constitutive activation of Nrf2. Here, we report a novel mechanism of Nrf2 activation by arsenic that is distinct from that of chemopreventive compounds. Arsenic deregulates the autophagic pathway through blockage of autophagic flux, resulting in accumulation of autophagosomes and sequestration of p62, Keap1, and LC3. Thus, arsenic activates Nrf2 through a noncanonical mechanism (p62 dependent), leading to a chronic, sustained activation of Nrf2. In contrast, activation of Nrf2 by sulforaphane (SF) and tert-butylhydroquinone (tBHQ) depends upon Keap1-C151 and not p62 (the canonical mechanism). More importantly, SF and tBHQ do not have any effect on autophagy. In fact, SF and tBHQ alleviate arsenic-mediated deregulation of autophagy. Collectively, these findings provide evidence that arsenic causes prolonged activation of Nrf2 through autophagy dysfunction, possibly providing a scenario similar to that of constitutive activation of Nrf2 found in certain human cancers. This may represent a previously unrecognized mechanism underlying arsenic toxicity and carcinogenicity in humans.     

Antioxidant activities of ginsenoside Rg1 against cisplatin-induced hepatic injury through Nrf2 signaling pathway in mice

Oxidative stress is mainly caused by reactive oxygen species (ROS). The damage causes a net stress on normal organs, leading to a gradual loss of vital physiological function. ROS, such as free radicals, represent a class of molecules which are derived from the metabolism of oxygen and exist inherently. However, excessive produced ROS can damage all aerobic organisms. Ginseng is one of the most commonly used alternative herbal medicines, also as a traditional Chinese medicine. The aim of this study is to investigate the antioxidant potential function of ginsenoside Rg1 against cisplatin-caused hepatic damage. Male mice were treated with cisplatin to induce oxidative stress to mimic the side effect of anti-cancer drug cisplatin. Ginsenoside Rg1 effectively prevented against cisplatin-induced hepatotoxicity, alleviating histological lesions. Antioxidant functions of Rg1 were restrained by the activation of p62–Keap1–Nrf2 signaling pathway, simultaneously accompanied with expression of protein products. Accumulative p62 and increased activation of JNK in hepatocytes promoted the activation of Nrf2. For the other, degradation of Nrf2 was guided by tyrosine phosphorylation, ubiquitin, and Keap1. In summary, Rg1 prevents hepatotoxicity mainly by inhibiting the binding of Keap1 and Nrf2, partly by p62 accumulation, and more importantly by increasing the production of antioxidative proteins associated to Nrf2. Pharmacological activation of Nrf2 is an effective way in combating against liver injury.     

Keap1-Knockdown Decreases Fasting-Induced Fatty Liver via Altered Lipid Metabolism and Decreased Fatty Acid Mobilization from Adipose Tissue

Aims

The purpose of this study was to determine whether Nrf2 activation, via Keap1-knockdown (Keap1-KD), regulates lipid metabolism and mobilization induced by food deprivation (e.g. fasting).

Methods and Results

Male C57BL/6 (WT) and Keap1-KD mice were either fed ad libitum or food deprived for 24 hours. After fasting, WT mice exhibited a marked increase in hepatic lipid accumulation, but Keap1-KD mice had an attenuated increase of lipid accumulation, along with reduced expression of lipogenic genes (acetyl-coA carboxylase, stearoyl-CoA desaturase-1, and fatty acid synthase) and reduced expression of genes related to fatty acid transport, such as fatty acid translocase/CD36 (CD36) and Fatty acid transport protein (FATP) 2, which may attribute to the reduced induction of Peroxisome proliferator-activated receptor (Ppar) α signaling in the liver. Additionally, enhanced Nrf2 activity by Keap1-KD increased AMP-activated protein kinase (AMPK) phosphorylation in liver. In white adipose tissue, enhanced Nrf2 activity did not change the lipolysis rate by fasting, but reduced expression of fatty acid transporters — CD36 and FATP1, via a PPARα-dependent mechanism, which impaired fatty acid transport from white adipose tissue to periphery circulation system, and resulted in increased white adipose tissue fatty acid content. Moreover, enhanced Nrf2 activity increased glucose tolerance and Akt phosphorylation levels upon insulin administration, suggesting Nrf2 signaling pathway plays a key role in regulating insulin signaling and enhanced insulin sensitivity in skeletal muscle.

Conclusion

Enhanced Nrf2 activity via Keap1-KD decreased fasting-induced steatosis, pointing to an important function of Nrf2 on lipid metabolism under the condition of nutrient deprivation.     

Nrf2 Enhances Cholangiocyte Expansion in Pten-Deficient Livers

Keap1-Nrf2 system plays a central role in the stress response. While Keap1 ubiquitinates Nrf2 for degradation under unstressed conditions, this Keap1 activity is abrogated in response to oxidative or electrophilic stresses, leading to Nrf2 stabilization and coordinated activation of cytoprotective genes. We recently found that nuclear accumulation of Nrf2 is significantly increased by simultaneous deletion of Pten and Keap1, resulting in the stronger activation of Nrf2 target genes. To clarify the impact of the cross talk between the Keap1-Nrf2 and Pten–phosphatidylinositide 3-kinase–Akt pathways on the liver pathophysiology, in this study we have conducted closer analysis of liver-specific Pten::Keap1 double-mutant mice (Pten::Keap1-Alb mice). The Pten::Keap1-Alb mice were lethal by 1 month after birth and displayed severe hepatomegaly with abnormal expansion of ductal structures comprising cholangiocytes in a Nrf2-dependent manner. Long-term observation of Pten::Keap1-Alb::Nrf2^+/− mice revealed that the Nrf2-heterozygous mice survived beyond 1 month but developed polycystic liver fibrosis by 6 months. Gsk3 directing the Keap1-independent degradation of Nrf2 was heavily phosphorylated and consequently inactivated by the double deletion of Pten and Keap1 genes. Thus, liver-specific disruption of Keap1 and Pten augments Nrf2 activity through inactivation of Keap1-dependent and -independent degradation of Nrf2 and establishes the Nrf2-dependent molecular network promoting the hepatomegaly and cholangiocyte expansion.     

Assurance of mitochondrial integrity and mammalian longevity by the p62-Keap1-Nrf2-Nqo1 cascade

Sqstm1/p62 functions in the non-canonical activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, its physiological relevance is not certain. Here, we show that p62(-/-) mice exhibited an accelerated presentation of ageing phenotypes, and tissues from these mice created a pro-oxidative environment owing to compromised mitochondrial electron transport. Accordingly, mitochondrial function rapidly declined with age in p62(-/-) mice. In addition, p62 enhanced basal Nrf2 activity, conferring a higher steady-state expression of NAD(P)H dehydrogenase, quinone 1 (Nqo1) to maintain mitochondrial membrane potential and, thereby, restrict excess oxidant generation. Together, the p62-Nrf2-Nqo1 cascade functions to assure mammalian longevity by stabilizing mitochondrial integrity.