b Nrf2 (nuclear factor erythroid 2-related factor 2) is an antioxidant transcription factor. AMP-activated protein kinase (AMPK) functions as a central regulator of cell survival in response to stressful stimuli. Nrf2 should be coordinated with the cell survival pathway controlled by AMPK, but so far the mechanistic connections remain undefined. This study investigated the role of AMPK in Nrf2 trafficking and its activity regulation. A subnetwork integrating neighbor molecules suggested direct interaction between AMPK and Nrf2. In cells, AMPK activation caused nuclear accumulation of Nrf2. In the in vitro kinase and peptide competition assays, AMPK phosphorylated Nrf2 at the Ser558 residue (Ser550 in mouse) located in the canonical nuclear export signal. Nrf2 with an S550A mutation failed to be accumulated in the nucleus after AMPK activation. Leptomycin B, a nuclear export inhibitor, did not enhance nuclear accumulation of wild-type Nrf2 (WT-Nrf2) activated by AMPK or a phospho-Ser550-mimetic Nrf2 mutant, corroborating the finding that AMPK facilitated nuclear accumulation of Nrf2, probably by inhibiting nuclear export. Activated glycogen synthase kinase 3␤ (GSK3␤) diminished the basal nuclear level of Myc-S550A-Nrf2. Taking the data collectively, AMPK phosphorylates Nrf2 at the Ser550 residue, which, in conjunction with AMPK-mediated GSK3␤ inhibition, promotes nuclear accumulation of Nrf2 for antioxidant response element (ARE)-driven gene transactivation. Redox homeostasis in the cell may be disturbed by a deficiency in energy production as well as by reactive oxygen species (ROS) generated from xenobiotic biotransformation or the process of fuel oxidation (1-3). Nrf2 (nuclear factor erythroid 2-related factor 2), a key antioxidant transcription factor, participates in maintaining redox homeostasis in the cell (4). Various physiological or pathological circumstances accompanying free radical generation activate Nrf2 as a consequence of an adaptive response to oxidative stress (5). However, the regulation of Nrf2 has been minimally studied in the context of cellular energy deficiency (e.g., starvation, hypoxia, and exercise).The response of Nrf2 to oxidative challenge stress rapidly occurs mainly through phosphorylation at Ser40; protein kinase C␦ (PKC␦) induces activation of phosphorylation of Nrf2 at the residue, and the phosphorylated form is then released from Keap-1 and is stabilized for antioxidant response element (ARE)-driven gene expression (6, 7). An active form of Nrf2 has been shown to localize in the nucleus and binds to the ARE(s) present in the promoter regions of target genes (8). In this process, phosphoinositide 3-kinase controls nuclear translocation of Nrf2 (9). In contrast, glycogen synthase kinase 3␤ (GSK3␤) catalyzes the inhibitory phosphorylation of Nrf2 for the tight activity control (10, 11). Other kinases, including mitogen-activated protein kinases and Fyn, may also affect Nrf2 activity (12, 13). Despite the identification of oxidative stress-associated Nrf2 kinases, the characterist...

MicroRNAs (miRNAs) have a role in the cellular defense mechanism. Nuclear factor erythroid-2-related factor 2 (Nrf2) increases antioxidant enzyme capacity. However, miRNA transcriptionally controlled by Nrf2 had been uncharacterized. Here we report that miR-125b is transactivated by Nrf2 and inhibits aryl hydrocarbon receptor (AhR) repressor (AhRR). Bioinformatic approaches enabled us to extract six candidate miRNAs. Of them, only miR-125b was increased in the kidney of mice treated with oltipraz. Nrf2 overexpression enhanced primary, precursor and mature miR-125b levels. Functional assays revealed MIR125B1 is a bona fide target gene of Nrf2. Oltipraz treatment protected the kidney from cisplatin toxicity with increase of miR-125b. Consistently, Nrf2 knockout abrogated an adaptive increase of miR-125b elicited by cisplatin, augmenting kidney injury. An integrative network of miRNA and messenger RNA changes enabled us to predict miR-125b as an inhibitor of AhRR for the control of AhR activity and cell survival. In our molecular study, miR-125b inhibited AhRR and thereby activated AhR, leading to the induction of mdm2. Consistently, p53 activation by cisplatin was diminished by either miR-125b or oltipraz treatment. The results of experiments using miR-125b mimic or small interfering RNA of AhRR verified the role of miR-125b in AhRR regulation for kidney protection. In conclusion, miR-125b is transcriptionally activated by Nrf2 and serves as an inhibitor of AhRR, which contributes to protecting kidney from acute injury.

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Background and Aims Fat accumulation results from increased fat absorption and/or defective fat metabolism. Currently, the lipid‐sensing nuclear receptor that controls fat utilization in hepatocytes is elusive. Liver X receptor alpha (LXRα) promotes accumulation of lipids through the induction of several lipogenic genes. However, its effect on lipid degradation is open for study. Here, we investigated the inhibitory role of LXRα in autophagy/lipophagy in hepatocytes and the underlying basis. Approach and Results In LXRα knockout mice fed a high‐fat diet, or cell models, LXRα activation suppressed the function of mitochondria by inhibiting autophagy/lipophagy and induced hepatic steatosis. Gene sets associated with “autophagy” were enriched in hepatic transcriptome data. Autophagy flux was markedly augmented in the LXRα knockout mouse liver and primary hepatocytes. Mechanistically, LXRα suppressed autophagy‐related 4B cysteine peptidase (ATG4B) and Rab‐8B, responsible for autophagosome and ‐lysosome formation, by inducing let‐7a and microRNA (miR)‐34a. Chromatin immunoprecipitation assay enabled us to find LXRα as a transcription factor of let‐7a and miR‐34a. Moreover, 3’ untranslated region luciferase assay substantiated the direct inhibitory effects of let‐7a and miR‐34a on ATG4B and Rab‐8B. Consistently, either LXRα activation or the let‐7a/miR‐34a transfection lowered mitochondrial oxygen consumption rate and mitochondrial transmembrane potential and increased fat levels. In obese animals or nonalcoholic fatty liver disease (NAFLD) patients, let‐7a and miR‐34a levels were elevated with simultaneous decreases in ATG4B and Rab‐8B levels. Conclusions LXRα inhibits autophagy in hepatocytes through down‐regulating ATG4B and Rab‐8B by transcriptionally activating microRNA let‐7a‐2 and microRNA 34a genes and suppresses mitochondrial biogenesis and fuel consumption. This highlights a function of LXRα that culminates in the progression of liver steatosis and steatohepatitis, and the identified targets may be applied for a therapeutic strategy in the treatment of NAFLD.

BackgroundThe injured liver loses normal function, with concomitant decrease of key identity genes. Super-enhancers contribute to mammalian cell identity. Here, we identified core transcription factors (TFs) that are active in hepatocytes, using genome-wide analysis and hierarchical ordering of super-enhancer distribution.MethodsExpression of core TFs was assessed in a cohort of patients with hepatitis or cirrhosis and animal models. Quantitative PCR, chromatin immunoprecipitation assays, and hydrodynamic gene delivery methods were used to assess gene regulation and hepatocyte viability. RNA-sequencing data were generated to investigate the role of LRH1 in hepatocyte protection from injury.ResultsNetwork analysis of super-enhancer-associated gene interactions and expression arrays for cohorts of patients with hepatitis and cirrhosis enabled us to identify a super-enhancer-associated network, and LRH1, HNF4α, PPARα, and RXRα as core TFs. In mouse models, expression of core TFs was robustly inhibited by single and multiple challenge(s) with liver toxicant. RNA-seq analysis revealed changes in expression in the super-enhancer-associated genes sensitively biased toward repression by intoxication. LRH1 gene delivery prevented the loss of hepatic super-enhancer-associated signaling circuitry in toxicant-challenged mice, and protected the liver from injury, indicating the role of LRH1 in hepatocyte identity and viability. In hepatocytes, overexpression of each core TF promoted induction of other TFs.ConclusionOverall, this study identified LRH1-driven pathway as a circuitry responsible for hepatocyte identity by using cistromic analysis, improving our understanding of liver pathophysiology and identifying novel therapeutic targets.