Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA)

Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system.

The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.

Although inflammation and protease/antiprotease imbalance have been postulated to be critical in cigarette smoke-induced (CS-induced) emphysema, oxidative stress has been suspected to play an important role in chronic obstructive pulmonary diseases. Susceptibility of the lung to oxidative injury, such as that originating from inhalation of CS, depends largely on its upregulation of antioxidant systems. Nuclear factor, erythroid-derived 2, like 2 (Nrf2) is a redox-sensitive basic leucine zipper protein transcription factor that is involved in the regulation of many detoxification and antioxidant genes. Disruption of the Nrf2 gene in mice led to earlier-onset and more extensive CS-induced emphysema than was found in wild-type littermates. Emphysema in Nrf2-deficient mice exposed to CS for 6 months was associated with more pronounced bronchoalveolar inflammation; with enhanced alveolar expression of 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of oxidative stress; and with an increased number of apoptotic alveolar septal cells--predominantly endothelial and type II epithelial cells--as compared with wild-type mice. Microarray analysis identified the expression of nearly 50 Nrf2-dependent antioxidant and cytoprotective genes in the lung that may work in concert to counteract CS-induced oxidative stress and inflammation. The responsiveness of the Nrf2 pathway may act as a major determinant of susceptibility to tobacco smoke-induced emphysema by upregulating antioxidant defenses and decreasing lung inflammation and alveolar cell apoptosis.

Although inflammation and protease/antiprotease imbalance have been postulated to be critical in cigarette smoke–induced (CS-induced) emphysema, oxidative stress has been suspected to play an important role in chronic obstructive pulmonary diseases. Susceptibility of the lung to oxidative injury, such as that originating from inhalation of CS, depends largely on its upregulation of antioxidant systems. Nuclear factor, erythroid-derived 2, like 2 (Nrf2) is a redox-sensitive basic leucine zipper protein transcription factor that is involved in the regulation of many detoxification and antioxidant genes. Disruption of the Nrf2 gene in mice led to earlier-onset and more extensive CS-induced emphysema than was found in wild-type littermates. Emphysema in Nrf2-deficient mice exposed to CS for 6 months was associated with more pronounced bronchoalveolar inflammation; with enhanced alveolar expression of 8-oxo-7,8-dihydro-2′-deoxyguanosine, a marker of oxidative stress; and with an increased number of apoptotic alveolar septal cells — predominantly endothelial and type II epithelial cells — as compared with wild-type mice. Microarray analysis identified the expression of nearly 50 Nrf2-dependent antioxidant and cytoprotective genes in the lung that may work in concert to counteract CS-induced oxidative stress and inflammation. The responsiveness of the Nrf2 pathway may act as a major determinant of susceptibility to tobacco smoke–induced emphysema by upregulating antioxidant defenses and decreasing lung inflammation and alveolar cell apoptosis.

The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central alpha-helix followed by a mini antiparallel beta-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the beta-propeller structure of Keap1. In contrast, the central alpha-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two beta-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central alpha-helix in a correct position to benefit ubiquitin signaling.

BACKGROUND: Nrf2 belongs to the Cap-N-Collar (CNC) transcription factor family and is essential for the antioxidant responsive element (ARE)-mediated expression of a group of detoxifying and antioxidant genes. The forced expression of Nrf2 in mammalian cells activates the expression of target genes through the ARE, with Nrf2 showing the highest transactivation activity among the CNC family of transcription factors. To elucidate the molecular mechanisms generating this potent transactivation activity, we examined the functions of the domains within Nrf2. RESULT: We found that Nrf2 contains two transcription activation domains, Neh4 and Neh5, which act synergistically to attain maximum a activation of reporter gene expression. Neh4 and Neh5 both individually and cooperatively bind to CBP (CREB (cAMP Responsive Element Binding protein) Binding Protein). In fact, the specific inhibitor of CBP, adenovirus E1A protein, significantly reduced Nrf2 activity. Importantly, the CBP-binding activity of Nrf2 deletion mutants positively correlated with their transactivation activity. Neh5 contains a motif which is commonly conserved among the CNC factors, whereas Neh4 contains the novel CBP-interacting motif recently identified in p53 and E2F. CONCLUSIONS: Our results indicate that Nrf2 exploits the cooperative binding of two independent transactivation domains to CBP in the acquisition of a potent transactivation activity.

Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice, tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody against 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), it was observed that macrophages from pleural lavage accumulate 15d-PGJ(2). We show that in mouse peritoneal macrophages 15d-PGJ(2) can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy caused persistence of neutrophil recruitment and, in macrophages, attenuated the 15d-PGJ(2) accumulation and PrxI expression. Administration of 15d-PGJ(2) into the pleural space of NS-398-treated wild-type mice largely counteracted both the decrease in PrxI and persistence of neutrophil recruitment. In contrast, these changes did not occur in the Nrf2-deficient mice. These results demonstrate that Nrf2 regulates the inflammation process downstream of 15d-PGJ(2) by orchestrating the recruitment of inflammatory cells and regulating the gene expression within those cells.

Menthol and other counterstimuli relieve itch, resulting in an antipruritic state that persists for minutes to hours. However, the neural basis for this effect is unclear, and the underlying neuromodulatory mechanisms are unknown. Previous studies revealed that Bhlhb5(-/-) mice, which lack a specific population of spinal inhibitory interneurons (B5-I neurons), develop pathological itch. Here we characterize B5-I neurons and show that they belong to a neurochemically distinct subset. We provide cause-and-effect evidence that B5-I neurons inhibit itch and show that dynorphin, which is released from B5-I neurons, is a key neuromodulator of pruritus. Finally, we show that B5-I neurons are innervated by menthol-, capsaicin-, and mustard oil-responsive sensory neurons and are required for the inhibition of itch by menthol. These findings provide a cellular basis for the inhibition of itch by chemical counterstimuli and suggest that kappa opioids may be a broadly effective therapy for pathological itch.

Nrf1 is a member of the vertebrate Cap'n'Collar (CNC) transcription factor family that commonly contains a unique basic-leucine zipper domain. Among CNC family members, Nrf2 is known to regulate a battery of antioxidant and xenobiotic-metabolizing enzyme genes through the antioxidant response element (ARE). Although Nrf1 has also been shown to bind the ARE, it is unclear whether it plays a distinct role from Nrf2 in regulating genes with this element. To address this issue in vivo, we generated mice bearing a hepatocyte-specific disruption of the Nrf1 gene. AlthoughNrf2 knock-out mice did not exhibit liver damage when they were maintained in an unstressed condition, hepatocyte-specific deletion of Nrf1 caused liver damage resembling the human disease non-alcoholic steatohepatitis. Gene expression analysis revealed that the disruption of Nrf1 causes stress that activates a number of ARE-driven genes in an Nrf2-dependent manner, indicating that Nrf2 cannot compensate completely for loss of Nrf1 function in the liver. In contrast, expression of metallothionein-1 and -2 (MT1 and MT2) genes, each of which harbors at least one ARE in its regulatory region, was decreased in the Nrf1-null mutant mice. Whereas Nrf1 and Nrf2 bound the MT1 ARE with comparable affinity, Nrf1 preferentially activated the reporter gene expression through the MT1 ARE. This study has, thus, identified the first ARE-dependent gene that relies exclusively on Nrf1, suggesting that it plays a distinct functional role in regulating ARE-driven genes.

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.

The isothiocyanate sulforaphane [SF; 1-isothiocyanato-4(R)-methylsulfinylbutane] is abundant in broccoli sprouts in the form of its glucosinolate precursor (glucoraphanin). SF is powerfully bactericidal against Helicobacter pylori infections, which are strongly associated with the worldwide pandemic of gastric cancer. Oral treatment with SF-rich broccoli sprouts of C57BL/6 female mice infected with H. pylori Sydney strain 1 and maintained on a high-salt (7.5% NaCl) diet reduced gastric bacterial colonization, attenuated mucosal expression of tumor necrosis factor-alpha and interleukin-1beta, mitigated corpus inflammation, and prevented expression of high salt-induced gastric corpus atrophy. This therapeutic effect was not observed in mice in which the nrf2 gene was deleted, strongly implicating the important role of Nrf2-dependent antioxidant and anti-inflammatory proteins in SF-dependent protection. Forty-eight H. pylori-infected patients were randomly assigned to feeding of broccoli sprouts (70 g/d; containing 420 micromol of SF precursor) for 8 weeks or to consumption of an equal weight of alfalfa sprouts (not containing SF) as placebo. Intervention with broccoli sprouts, but not with placebo, decreased the levels of urease measured by the urea breath test and H. pylori stool antigen (both biomarkers of H. pylori colonization) and serum pepsinogens I and II (biomarkers of gastric inflammation). Values recovered to their original levels 2 months after treatment was discontinued. Daily intake of sulforaphane-rich broccoli sprouts for 2 months reduces H. pylori colonization in mice and improves the sequelae of infection in infected mice and in humans. This treatment seems to enhance chemoprotection of the gastric mucosa against H. pylori-induced oxidative stress.

A subtractive hybridization strategy was used to isolate putative genes involved in the development of mouse primordial germ cells (PGC). Complimentary DNA was amplified on RNA isolated from the base of the allantois where PGC are located in the 7.5 days post coitum (dpc) mouse embryo. It was then subtracted by hybridization with cDNA amplified on RNA of the anterior region where PGC are absent. A novel gene thus isolated is designated as Mesp1 and encodes a possible transcription factor MesP1 containing a basic helix-loop-helix motif. Its earliest expression was observed at the onset of gastrulation, as early as 6.5 dpc, in the nascent mesodermal cells that first ingressed at the end of the primitive streak. These expressing cells in the lateral and extraembryonic mesoderm showed a wing-shaped distribution. Its initial expression was soon down-regulated at 7.5 dpc before the completion of gastrulation, except at the proximal end of the primitive streak which included the extraembryonic mesoderm and the base of allantois. At 8 dpc, the expression at the base of the allantois moved laterally. This distribution between 7.0 and 8.0 dpc was similar to that of PGC detected by the alkaline phosphatase activity. However, the expression of Mesp1 was down-regulated thereafter, when PGC entered in the migration stage. After birth, Mesp1 expression was detected only in mature testes, but in a different isoform from that expressed in the embryo. Mesp1 was mapped to the mid region of chromosome 7, near the mesodermal deficiency gene (mesd). However, a Southern hybridization study clearly showed that Mesp1 was distinctly different from mesd. The amino acid sequence and its expression pattern suggest that MesP1 plays an important role in the development of the nascent mesoderm including PGC.

GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1-deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation.

In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type-specific expression of the Epo gene.

The LacZ gene, which encodes Escherichia coli β-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.

The poliovirus receptor CD155 and its family member CD112 (nectin-2) are the ligands for the activating cell-surface receptor DNAM-1 on CD8+ T cells and natural killer (NK) cells. Here, we demonstrate that, whereas the RMA tumor grew in syngeneic mice, DNAM-1 ligand-transduced RMA was rejected, in which CD8+ T cells and NK cells played an essential role. Importantly, CD8+ memory cytotoxic T cells to parental RMA were generated in these mice. We found that DNAM-1 was also expressed on CD8alpha+, rather than CD8alpha-, dendritic cells (DCs). Cross-linking DNAM-1 induced maturation of CD8alpha+ DCs. Antigen presentation by these stimulated DCs drove Th1 cells. Moreover, the rejection of DNAM-1 ligand-transduced RMA was canceled in CD4+ T-cell-depleted and major histocompatibility complex class II-deficient mice. Taken together, these results suggest that DNAM-1 ligands stimulate innate immunity by CD8alpha+ DCs as well as NK cells, which efficiently prime cell-mediated tumor-specific immunity.

The EWS gene when fused to transcription factors such as the ETS family ATF-1, Wilms' tumor-1, and nuclear orphan receptors upon chromosomal translocation is thought to contribute the development of Ewing sarcoma and several malignant tumors. Although EWS is predicted to be an RNA-binding protein, an inherent EWS nuclear function has not yet been elucidated. In this study, we found that EWS associates with a transcriptional co-activator CREB-binding protein (CBP) and the hypophosphorylated RNA polymerase II, which are included preferentially in the transcription preinitiation complex. These interactions suggest the potential involvement of EWS in gene transcription, leading to the hypothesis that EWS may function as a co-activator of CBP-dependent transcription factors. Based on this hypothesis, we investigated the effect of EWS on the activation of nuclear receptors that are activated by CBP. Of nuclear receptors examined, hepatocyte nuclear factor 4-dependent transcription was selectively enhanced by EWS but not by an EWS mutant defective for CBP binding. These results suggest that EWS as a co-activator requires CBP for hepatocyte nuclear factor 4-mediated transcriptional activation.

Water-dispersible gold nanoparticles with narrow size distributions were prepared by irradiation of nonequilibrium atmospheric-pressure dielectric-barrier-discharge (DBD) helium plasma jets to tetrachloroauric acid solutions containing poly(ethylene glycol) (PEG) that had a pentaethylenehexamine at one end of each polymer chain (N6-PEG). N6-PEG is known to interact with aurate cations to facilitate nucleation and growth of gold nanoparticles as a surface-modification agent. The helium-based DBD plasma jets contain energetic electrons and free radicals, which can induce chemical reactions on the surface of and/or in a solution at room temperature. For gold nanoparticles formed by DBD plasma-jet irradiation with the molar ratio of tetrachloroauric acid/N6-PEG being 1:20 at pH 10, the average particle size was in the range of 5−10 nm in diameter from transmission electron microscopy observation. Furthermore, gold nanoparticles prepared in this manner are found to have remarkable stability under physiological conditions because of the immobilization of PEG-derived materials on the gold surfaces. Such gold nanoparticles may therefore be considered excellent candidates for high-performance bionanoparticles.

T cell line-tropic (T-tropic) HIV type 1 strains enter cells by interacting with the cell-surface molecules CD4 and CXCR4. We have generated transgenic mice predominantly expressing human CD4 and CXCR4 on their CD4-positive T lymphocytes (CD4+ T cells). Their primary thymocytes are susceptible to T-tropic but not to macrophage-tropic HIV-1 infection in vitro, albeit with a viral antigen production less efficient than human peripheral blood mononuclear cells. Interestingly, even without HIV infection, transgenic mice display a CD4+ T cell depletion profile of peripheral blood reminiscent of that seen in AIDS patients. We demonstrate that CD4+ T cell trafficking in transgenic mice is biased toward bone marrow essentially due to CXCR4 overexpression, resulting in the severe loss of CD4+ T cells from circulating blood. Our data suggest that CXCR4 plays an important role in lymphocyte trafficking through tissues, especially between peripheral blood and bone marrow, participating in the regulation of lymphocyte homeostasis in these compartments. Based on these findings, we propose a hypothetical model in which the dual function of CXCR4 in HIV-1 infection and in lymphocyte trafficking may cooperatively induce progressive HIV-1 infection and CD4+ T cell decline in patients.

The selenocysteine tRNA (tRNA(Sec)) molecule is the sight of synthesis for the amino acid selenocysteine and the adaptor for its translational insertion into selenoprotein enzymes, the majority of which contribute to cellular redox homeostasis. To examine the consequences of selenoprotein depletion on the oxidative environment of the cell, we generated a conditional knock-out mouse for the tRNA(Sec) gene (Trsp). Deletion of Trsp in either macrophages or liver elevated oxidative stress and activated the transcriptional induction of cytoprotective antioxidant and detoxification enzyme genes, including glutathione S-transferase P1 and NAD(P)H:quinone oxidoreductase 1, and other well known target genes of the transcription factor Nrf2 (NF-E2-related factor 2). Simultaneous disruption of Trsp and Nrf2 severely compromised the cytoprotective response. Double knock-out macrophages displayed reduced viability, elevated oxidative stress, and increased susceptible to hydrogen peroxide treatment compared with deletion of either gene alone. Mice carrying a liver-specific deletion of Trsp on an Nrf2-null background experienced hepatocellular apoptosis and displayed a severely reduced survival rate compared with loss of Trsp alone. Our results thus demonstrate that reduced selenoprotein activity is counterbalanced by an Nrf2-mediated cytoprotective response, which is essential for maintaining cellular redox homeostasis and viability.