Fumiki Katsuoka

Two-site substrate recognition model for the Keap1-Nrf2 system: a hinge and latch mechanism.

Abstract Cells are equipped with a number of transcriptional factors that safeguard against various environmental insults. Proteasomal protein degradation plays an important role in the Keap1-Nrf2 cytoprotection system, with molecular machinery similar to that for other environmental defense systems such as inflammatory and hypoxic responses. While Nrf2 protein stabilization is known to be redox-sensitive, the transcription factors NF-κB and HIF-1α for inflammatory and hypoxic responses, respectively, are also influenced by the cellular redox conditions. In this review we present the recently proposed two-site substrate recognition model of the Keap1-Nrf2 system, which regulates the cellular responses against oxidative and xenobiotic stresses. The implications of two destructive motifs in Nrf2, the ETGE and DLG motifs, which appear to function as a hinge and latch attenuating Keap1 activity in different redox states, are discussed.

Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes

Synthetic triterpenoids have been developed, which are potent inducers of cytoprotective enzymes and inhibitors of inflammation, greatly improving on the weak activity of naturally occurring triterpenoids. An imidazolide triterpenoid derivative, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im or TP235), has been previously shown to potently protect against hepatic tumorigenesis, acting in part by inducing cytoprotective genes through Keap1-Nrf2-antioxidant response element (ARE) signaling. In these studies, the pharmacodynamic activity of CDDO-Im is characterized in two distinct lines of ARE reporter mice and by measuring increases in Nqo1 transcript levels as a marker of cytoprotective gene induction. Oral administration of CDDO-Im induces ARE-regulated cytoprotective genes in many tissues in the mouse, including liver, lung, kidney, intestines, brain, heart, thymus, and salivary gland. CDDO-Im induces Nqo1 RNA transcripts in some organs at doses as low as 0.3 μmol/kg body weight (orally). A structure activity evaluation of 15 additional triterpenoids (a) confirmed the importance of Michael acceptor groups on both the A and C rings, (b) showed the requirement for a nitrile group at C-2 of the A ring, and (c) indicated that substituents at C-17 dramatically affected pharmacodynamic action in vivo. In addition to CDDO-Im, other triterpenoids, particularly the methyl ester CDDO-Me (TP155) and the dinitrile TP225, are extremely potent inducers of cytoprotective genes in mouse liver, lung, small intestine mucosa, and cerebral cortex. This pharmacodynamic characterization highlights the chemopreventive promise of several synthetic triterpenoids in multiple target organs. [Mol Cancer Ther 2007;6(1):154–62]

Genetic Evidence that Small Maf Proteins Are Essential for the Activation of Antioxidant Response Element-Dependent Genes

ABSTRACT While small Maf proteins have been suggested to be essential for the Nrf2-mediated activation of antioxidant response element (ARE)-dependent genes, the extent of their requirement remains to be fully documented. To address this issue, we generated mafG::mafF double-mutant mice possessing MafK as the single available small Maf. Induction of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene was significantly impaired in double-mutant mice treated with butylated hydroxyanisole, while other ARE-dependent genes were less affected. Similarly, in a keap1-null background, where many of the ARE-dependent genes are constitutively activated in an Nrf2-dependent manner, only a subset of ARE-dependent genes, including NQO1, were sensitive to a simultaneous deficiency in MafG and MafF. Examination of single and double small maf mutant cells revealed that MafK also contributes to the induction of ARE-dependent genes. To obtain decisive evidence, we established mafG::mafK::mafF triple-mutant fibroblasts that completely lack small Mafs and turned out to be highly susceptible to oxidative stress. We found that induction in response to diethyl maleate was abolished in a wider range of ARE-dependent genes in the triple-mutant cells. These data explicitly demonstrate that small Mafs play critical roles in the inducible expression of a significant portion of ARE-dependent genes.

Nrf2–MafG heterodimers contribute globally to antioxidant and metabolic networks

NF-E2-related factor 2 (Nrf2) is a key transcription factor that is critical for cellular defense against oxidative and xenobiotic insults. Nrf2 heterodimerizes with small Maf (sMaf) proteins and binds to antioxidant response elements (AREs) to activate a battery of cytoprotective genes. However, it remains unclear to what extent the Nrf2–sMaf heterodimers contribute to ARE-dependent gene regulation on a genome-wide scale. We performed chromatin immunoprecipitation coupled with high-throughput sequencing and identified the binding sites of Nrf2 and MafG throughout the genome. Compared to sites occupied by Nrf2 alone, many sites co-occupied by Nrf2 and MafG exhibit high enrichment and are located in species-conserved genomic regions. The ARE motifs were significantly enriched among the recovered Nrf2–MafG-binding sites but not among the Nrf2-binding sites that did not display MafG binding. The majority of the Nrf2-regulated cytoprotective genes were found in the vicinity of Nrf2–MafG-binding sites. Additionally, sequences that regulate glucose metabolism and several amino acid transporters were identified as Nrf2–MafG target genes, suggesting diverse roles for the Nrf2–MafG heterodimer in stress response. These data clearly support the notion that Nrf2–sMaf heterodimers are complexes that regulate batteries of genes involved in various aspects of cytoprotective and metabolic functions through associated AREs.

Positive or Negative MARE-Dependent Transcriptional Regulation Is Determined by the Abundance of Small Maf Proteins

The small Maf transcription factor proteins bind to Maf Recognition Elements (MAREs) by dimerizing with CNC proteins or themselves. We undertook experiments to clarify the functional relationship between the small Mafs and their partners in vivo. Embryos expressing abundant transgene-derived MafK died of severe anemia, while lines expressing lower levels of small Maf lived to adulthood. Megakaryocytes from the latter overexpressing lines exhibited reduced proplatelet formation and MARE-dependent transcription, phenocopying mafG null mutant mice. When the mafG null mutants were bred to small Maf-overexpressing transgenic animals, both loss- and gain-of-function phenotypes were reversed. These results provide direct in vivo evidence that transcriptional regulation through MARE elements hinges on an exquisitely sensitive balance of activating CNC molecules and their small Maf partners.

Constitutive Expression of Aryl Hydrocarbon Receptor in Keratinocytes Causes Inflammatory Skin Lesions

ABSTRACT Occupational and environmental exposure to polycyclic aromatic hydrocarbons (PAHs) has been suggested to provoke inflammatory and/or allergic disorders, including asthma, rhinitis, and dermatitis. The molecular mechanisms of this PAH-mediated inflammation remain to be clarified. Previous studies implied the involvement of PAHs as irritants and allergens, with the reactive oxygen species generated from the oxygenated PAHs believed to be an exacerbating factor. It is also possible that PAHs contribute to the pathogenesis through activation of aryl-hydrocarbon receptor (AhR)-mediated transcription, since PAHs are potent inducers of the AhR. To address this point, we generated transgenic mouse lines expressing the constitutive active form of the AhR in keratinocytes. In these lines of mice, the AhR activity was constitutively enhanced in the absence of ligands, so that any other direct effects of PAHs and their metabolites could be ignored. At birth, these transgenic mice were normal, but severe skin lesions with itching developed postnatally. The skin lesions were accompanied by inflammation and immunological imbalance and resembled typical atopic dermatitis. We demonstrate that constitutive activation of the AhR pathway causes inflammatory skin lesions and suggests a new mechanism for the exacerbation of inflammatory diseases after exposure to occupational and environmental xenobiotics.

Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals

The Tohoku Medical Megabank Organization reports the whole-genome sequences of 1,070 healthy Japanese individuals and construction of a Japanese population reference panel (1KJPN). Here we identify through this high-coverage sequencing (32.4 × on average), 21.2 million, including 12 million novel, single-nucleotide variants (SNVs) at an estimated false discovery rate of <1.0%. This detailed analysis detected signatures for purifying selection on regulatory elements as well as coding regions. We also catalogue structural variants, including 3.4 million insertions and deletions, and 25,923 genic copy-number variants. The 1KJPN was effective for imputing genotypes of the Japanese population genome wide. These data demonstrate the value of high-coverage sequencing for constructing population-specific variant panels, which covers 99.0% SNVs of minor allele frequency ≥0.1%, and its value for identifying causal rare variants of complex human disease phenotypes in genetic association studies.

Solution structure of the DNA-binding domain of MafG.

The Maf family proteins, which constitute a subgroup of basic region-leucine zipper (bZIP) proteins, function as transcriptional regulators of cellular differentiation. Together with the basic region, the Maf extended homology region (EHR), conserved only within the Maf family, defines the DNA binding specific to Mafs. Here we present the first NMR-derived structure of the DNA-binding domain (residues 1–76) of MafG, which contains the EHR and the basic region. The structure consists of three α-helices and resembles the fold of the DNA-binding domain of Skn-1, a developmental transcription factor of Caenorhabditis elegans. The structural similarity between MafG and Skn-1 enables us to propose a possible mechanism by which Maf family proteins recognize their consensus DNA sequences.

DNA methyltransferase 3a regulates osteoclast differentiation by coupling to an S-adenosylmethionine–producing metabolic pathway

Metabolic reprogramming occurs in response to the cellular environment to mediate differentiation, but the fundamental mechanisms linking metabolic processes to differentiation programs remain to be elucidated. During osteoclast differentiation, a shift toward more oxidative metabolic processes occurs. In this study we identified the de novo DNA methyltransferase 3a (Dnmt3a) as a transcription factor that couples these metabolic changes to osteoclast differentiation. We also found that receptor activator of nuclear factor-κB ligand (RANKL), an essential cytokine for osteoclastogenesis, induces this metabolic shift towards oxidative metabolism, which is accompanied by an increase in S-adenosylmethionine (SAM) production. We found that SAM-mediated DNA methylation by Dnmt3a regulates osteoclastogenesis via epigenetic repression of anti-osteoclastogenic genes. The importance of Dnmt3a in bone homeostasis was underscored by the observations that Dnmt3a-deficient osteoclast precursor cells do not differentiate efficiently into osteoclasts and that mice with an osteoclast-specific deficiency in Dnmt3a have elevated bone mass due to a smaller number of osteoclasts. Furthermore, inhibition of DNA methylation by theaflavin-3,3′-digallate abrogated bone loss in models of osteoporosis. Thus, this study reveals the role of epigenetic processes in the regulation of cellular metabolism and differentiation, which may provide the molecular basis for a new therapeutic strategy for a variety of bone disorders.

NF-E2 domination over Nrf2 promotes ROS accumulation and megakaryocytic maturation

In megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, whereas Nrf2 is a key activator of stress-responsive genes. Because p45 and Nrf2 have similar DNA-binding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently used as signaling molecules. We conducted comprehensive gene expression profiling with wild-type and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that 2 characteristic gene clusters are defined within p45 target genes: platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation.