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. 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. One of the unique features of transcription factors is that they can be categorized into structurally related groups (or families) based on their DNA binding motifs. It appears that in addition to their common basic functions each member within a specific transcription factor family is likely to have acquired novel properties during its molecular evolution. Consequently, it is probable that a family of transcription factors collectively possesses abilities that enable individual members to fine-tune the expression of target genes during disparate biological processes through a common cis-acting element. The Cap'n'Collar (CNC) 3The abbreviations used are: CNC, Cap'n'Collar; NASH, non-alcoholic steatohepatitis; ARE, antioxidant response element; ER, endoplasmic reticulum; MT, metallothionein; RT, reverse transcription; NQO1, NAD(P)H dehydrogenase quinone 1; GCLC, glutamate-cysteine ligase catalytic subunit; HO-1, heme oxygenase-1; GSTP1, glutathione S-transferase π1. 3The abbreviations used are: CNC, Cap'n'Collar; NASH, non-alcoholic steatohepatitis; ARE, antioxidant response element; ER, endoplasmic reticulum; MT, metallothionein; RT, reverse transcription; NQO1, NAD(P)H dehydrogenase quinone 1; GCLC, glutamate-cysteine ligase catalytic subunit; HO-1, heme oxygenase-1; GSTP1, glutathione S-transferase π1. family of transcription factors has been well described. It comprises four closely related factors, Nrf1, Nrf2, Nrf3, and p45 NF-E2 (1Motohashi H. O'Connor T. Katsuoka F. Engel J.D. Yamamoto M. Gene (Amst.). 2002; 294: 1-12Crossref PubMed Scopus (377) Google Scholar, 2Blank V. J. Mol. Biol. 2008; 376: 913-925Crossref PubMed Scopus (114) Google Scholar). These CNC members contain a CNC domain juxtaposed with a conserved basic region-leucine zipper (bZip) domain. The CNC domain is represented in an evolutionally distant homologue, SKN1 in the nematode Caenorhabditis elegans (3An J.H. Blackwell T.K. Genes Dev. 2003; 17: 1882-1893Crossref PubMed Scopus (497) Google Scholar). Although SKN1 binds to DNA as a monomer, all vertebrate CNC members each heterodimerize with one of three small Maf proteins, and the resulting heterodimers bind to the Maf recognition element (TGCTGA(G/C)TCAGCA) or related sequences. Because the vertebrate CNC members cannot bind to Maf recognition element sequences as monomers, small Maf proteins are indispensable partners of CNC bZip transcription factors (1Motohashi H. O'Connor T. Katsuoka F. Engel J.D. Yamamoto M. Gene (Amst.). 2002; 294: 1-12Crossref PubMed Scopus (377) Google Scholar, 2Blank V. J. Mol. Biol. 2008; 376: 913-925Crossref PubMed Scopus (114) Google Scholar). The Maf recognition element-related sequence that is referred to as an antioxidant or electrophile response element (ARE/EpRE; hereafter designated ARE for simplicity) has been identified in the transcriptional regulatory regions of antioxidant and xenobiotic-metabolizing enzyme genes (1Motohashi H. O'Connor T. Katsuoka F. Engel J.D. Yamamoto M. Gene (Amst.). 2002; 294: 1-12Crossref PubMed Scopus (377) Google Scholar, 2Blank V. J. Mol. Biol. 2008; 376: 913-925Crossref PubMed Scopus (114) Google Scholar) and is thought to account for the coordinated up-regulation of these genes during oxidative stress. The ARE has been shown to recruit Nrf2 and small Maf proteins (1Motohashi H. O'Connor T. Katsuoka F. Engel J.D. Yamamoto M. Gene (Amst.). 2002; 294: 1-12Crossref PubMed Scopus (377) Google Scholar, 2Blank V. J. Mol. Biol. 2008; 376: 913-925Crossref PubMed Scopus (114) Google Scholar). In Nrf2-null mutant mice the up-regulation of many antioxidant and detoxification enzyme genes has been found to be severely impaired (4Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3088) Google Scholar). Nrf1 has also been shown to contribute to the regulation of ARE-dependent genes (5Chen L. Kwong M. Lu R. Ginzinger D. Lee C. Leung L. Chan J.Y. Mol. Cell. Biol. 2003; 23: 4673-4686Crossref PubMed Scopus (108) Google Scholar, 6Kwong M. Kan Y.W. Chan J.Y. J. Biol. Chem. 1999; 274: 37491-37498Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Unlike Nrf2-null mice, Nrf1-null mice die by embryonic day 13.5 (E13.5) due to anemia (7Chan J.Y. Kwong M. Lu R. Chang J. Wang B. Yen T.S.B. Kan Y.W. EMBO J. 1998; 17: 1779-1787Crossref PubMed Scopus (214) Google Scholar). Furthermore, Nrf1:Nrf2 compound mutant mice die by E11.5, suggesting that Nrf2 can compensate, albeit partially, for the absence of Nrf1 in embryonic mice (8Leung L. Kwong M. Hou S. Lee C. Chan J.Y. J. Biol. Chem. 2003; 278: 48021-48029Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Based on the observation that an evolutionally distant CNC homolog SKN1 also contributes to the induction of antioxidant genes (9Inoue H. Hisamoto N. An J.H. Oliveira R.P. Nishida E. Blackwell T.K. Matsumoto K. Genes Dev. 2005; 19: 2278-2283Crossref PubMed Scopus (282) Google Scholar), it can be hypothesized that the regulation of antioxidant genes is one of the original functions descended from a common ancestral CNC protein. It is clear that Nrf1 and Nrf2 have acquired specific functions during their molecular evolution. It has been reported that hepatocyte-specific conditional targeting of the Nrf1 gene (Nrf1 cKO) causes a liver pathology resembling human non-alcoholic steatohepatitis (NASH) (10Xu Z. Chen L. Leung L. Yen T.S.B. Lee C. Chan J.Y. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4120-4125Crossref PubMed Scopus (225) Google Scholar); this previous study also reported that the expression of several ARE-dependent genes was decreased in the livers of Nrf1 cKO mice, suggesting that Nrf1 has an overlapping function with Nrf2 in the regulation of antioxidant and detoxifying genes. In stark contrast, however, Nrf2-null mutant mice do not show any liver damage when housed in an unstressed environment (11Chanas S.A. Jiang Q. McMahon M. McWalter G.K. McLellan L.I. Elcombe C.R. Henderson C.J. Wolf C.R. Moffat G.J. Itoh K. Yamamoto M. Hayes J.D. Biochem. J. 2002; 365: 405-416Crossref PubMed Scopus (360) Google Scholar). Thus, perturbations in the expression of antioxidant genes in Nrf1 cKO mice appear unlikely to account for the NASH-like disorder. Collectively, these observations suggest that Nrf1 possesses unique functions that are not exhibited by Nrf2. To execute its unique activity, Nrf1 protein possesses several distinct domains that are conserved among cross-species Nrf1 homologues (12Zhang Y. Crouch D.H. Yamamoto M. Hayes J.D. Biochem. J. 2006; 399: 373-385Crossref PubMed Scopus (96) Google Scholar). For instance, Nrf1 has an endoplasmic reticulum (ER) targeting sequence in its N-terminal domain that is responsible for it being anchored to the ER membrane and an Asn/Ser/Thr-rich (NST) domain through which it is glycosylated within the ER (13Zhang Y. Lucocq J.M. Yamamoto M. Hayes J.D. Biochem. J. 2007; 408: 161-172Crossref PubMed Scopus (30) Google Scholar). It has also been reported that Nrf1 is cleaved and translocated from the ER to the nucleus in response to ER stress (14Wang W. Chan J.Y. J. Biol. Chem. 2006; 281: 19676-19687Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), although the functional contribution of Nrf1 to the ER stress response has not been well described. Because the distinct phenotypes of Nrf1 and Nrf2 knock-out mice are highly likely to be caused by differences in the expression of their relevant target genes, it is necessary to examine gene expression profiles in mutant mice to the molecular for the disruption of In this study we generated hepatocyte-specific Nrf1 cKO mice and gene expression To expression of Nrf2 target genes in the liver was by the loss of Nrf1 in Nrf2-dependent manner, indicating that of Nrf1 target genes causes an of the response also found that several ARE-driven genes were in the Nrf1 cKO mice with mice. Among we found that expression of metallothionein-1 and -2 genes is on Nrf1 not on Nrf2. reporter that Nrf1 preferentially activates the MT1 ARE. in livers from mutant mice Nrf2 activates its target genes any H. H. A. H. Yamamoto M. Biochem. Biophys. Res. Commun. 2006; PubMed Scopus Google Scholar), induction of MT1 expression was These thus, that Nrf1 has acquired specific distinct from of the CNC family of the Nrf1 Nrf1 DNA was from a the Nrf1 gene from the at a Nrf1 targeting to a of a into the Nrf1 and a of protein was also into the at a of the was of the Nrf1 gene to we identified one that a The of with the was by analysis of and DNA a the targeting The and The were into and mice were with to mice. deletion of the Nrf1 was through with the mice from The The mice were with mice to mice, which were with each to hepatocyte-specific mice. of mice with Nrf2 mutant mice (4Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3088) Google Scholar) or mutant mice H. H. A. H. Yamamoto M. Biochem. Biophys. Res. Commun. 2006; PubMed Scopus Google Scholar), mice or mice were These were to the compound mutant and and was from livers of three mice and mice and was in that were to by the for Nrf1 were as and specific to have been K. H. Katsuoka F. Engel J.D. Yamamoto M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). was an or as reported N. Takahashi S. T. Yamamoto M. 1998; PubMed Google Scholar). The and sequences used to NAD(P)H dehydrogenase quinone the glutamate-cysteine ligase catalytic heme and glutathione S-transferase have been F. H. Ishii T. H. Engel J.D. Yamamoto M. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar, T. H. Takahashi S. S. Yamamoto M. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). Nrf1, and were the and MT1 MT1 MT1 The and were from were being and to the for the Gene were to the were with and in were and with and To livers were with and in The were with and with was by at and or at and were with an T. M. T. T. Engel J.D. H. Yamamoto M. Genes 2006; PubMed Scopus Google Scholar), and M. Yamamoto T. H. T. T. T. Engel J.D. B. Yamamoto M. Genes PubMed Scopus Google Scholar) were with at their and by The was with and with the To a of and and their mutant and were a of was to individual of the and proteins with or was as K. M. M. Mol. Cell. Biol. PubMed Google Scholar). the and were by on a in MT1 gene with its from K. of D. G.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) was into the reporter the ARE was generated by sequences to to the transcriptional from the reporter Nrf1 was into Nrf1 its ER (14Wang W. Chan J.Y. J. Biol. Chem. 2006; 281: 19676-19687Abstract Full Text Full Text PDF PubMed Scopus (131) Google were with or with or or of each and the to the of the were for the expression of and was a reporter was to for of J.D. Res. PubMed Scopus Google Scholar) from that been with or were to analysis as were with To were and with was were by of Nrf1 the in functions of Nrf1, we generated conditional Nrf1 mutant mice the targeting was in which the of was with sequences Because this the bZip DNA binding we that the a of Nrf1 that was of binding DNA and gene also an protein in the of Nrf1 that its expression be by protein. the targeting and the Nrf1 in by DNA was to a to the of the Nrf1 gene. was identified by the of an DNA by the of a in the of the element. mice were by into and the resulting were with mice to of the Nrf1 In mice were with mice, which and embryonic by not with a previous Nrf1 gene knock-out analysis (7Chan J.Y. Kwong M. Lu R. Chang J. Wang B. Yen T.S.B. Kan Y.W. EMBO J. 1998; 17: 1779-1787Crossref PubMed Scopus (214) Google Scholar). also that of into the did not the expression of Nrf1 in livers of mice Nrf1 a hepatocyte-specific deletion of Nrf1 in mice, we mice with mice, mice. of the and with liver DNA are shown in The of the to be and analysis revealed that Nrf1 were severely in livers of mutant mice of liver from mutant mice with and revealed that they many To the we also with The that the of mice a of when with mice. In of mice at of and in mice were in mice These the previous that knock-out of Nrf1 gene causes in the liver that human (10Xu Z. Chen L. Leung L. Yen T.S.B. Lee C. Chan J.Y. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4120-4125Crossref PubMed Scopus (225) Google Scholar). of Genes in in a Nrf2-dependent of from livers of mice livers from as Because it has been reported that Nrf1 contributes to the expression of enzyme genes that are known to be by Nrf2, we whether the expression of Nrf2 target genes was in The revealed that the expression of a of genes is decreased in the absence of Nrf1 of Nrf1 on expression of Nrf2-dependent of quinone catalytic in a To these we four ARE-dependent genes GSTP1, GCLC, and and found that the expression of these genes was in livers when with livers Thus, in up-regulation of at least genes. To examine whether the expression of these genes on Nrf2, we generated a compound mutant bearing a deletion of Nrf2 with hepatocyte-specific deletion of The in expression of these four Nrf2 target genes was completely in the Nrf1:Nrf2 compound knock-out liver. This that the expression of antioxidant and xenobiotic-metabolizing enzyme genes is in an Nrf2-dependent in response to stress caused by the of An observation is that Nrf1 not contribute to the expression of these four genes in the absence of Nrf2, suggesting at least Nrf1 not contribute to the regulation of Nrf2 target genes of Genes in the we genes that were in the found that genes were decreased in the livers when with the categorized these genes into groups to their function To three of the genes be categorized as to the and family member the we found that many of the genes that were in Nrf1-null livers to the of This of genes proteins, a and These thus, that Nrf1 is in the regulation of a number of biological functions in the which are distinct from of expression were decreased in mice with and family member dehydrogenase protein protein member domain and and gene protein factor protein family protein and protein protein domain domain family member domain protein domain domain factor in a Genes in the genes identified in the MT1 and have been reported to contain at least one ARE in their regulatory regions D. G.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To these we and that the expression of MT1 and genes was in livers when with their expression in mice It is well known that the expression of is in response to and that this is by the transcription factor through the response element in gene F. R. M. M. Z. W. EMBO J. PubMed Scopus Google Scholar). that expression did not in the livers not indicating that the of MT1 in the livers was not caused by of Because the ARE-dependent genes were in mice in an Nrf2-dependent these the that in liver the MT1 and genes are preferentially and by Nrf1, not by Nrf2, through the ARE in their regulatory MT1 ARE by Nrf1 by of the MT1 gene has been with to transcription factor and its binding to the response element sequence F. R. M. M. Z. W. EMBO J. PubMed Scopus Google Scholar). In addition to this response it has been reported that the MT1 gene has a conserved ARE in its regulatory G.K. Biochem. PubMed Scopus Google Scholar). also that are in the regions of vertebrate genes, as from and contribution of the CNC family factors to the regulation of MT1 expression has not been well To examine whether Nrf1 and Nrf2 bind to the MT1 ARE, we For this Nrf1, Nrf2, and proteins their DNA binding domains and were in a expression found that and heterodimers bind to the MT1 ARE with The binding was specific it was by of an ARE not by a mutant ARE that Nrf1 and Nrf2 exhibit binding for the MT1 ARE. the that Nrf1 and Nrf2 transcription through the MT1 ARE. a reporter The MT1 gene regulatory the ARE was to a and the reporter was into with or expression Although the of and was found to be comparable the MT1 reporter gene was activated by was activated by and were severely when the MT1 ARE sequence was from the reporter that is on the ARE. In stark contrast, activated a reporter gene by ARE did These thus, that the MT1 gene is an ARE-dependent gene by Nrf1 not by Nrf2. MT1 by of Nrf2 in that Nrf2 not regulate we whether or not MT1 is in the livers of mutant mice Nrf2 can its target genes. Nrf2 target genes were in mice, as has been reported H. H. A. H. Yamamoto M. Biochem. Biophys. Res. Commun. 2006; PubMed Scopus Google Scholar). was in the MT1 gene expression and mice, that the MT1 gene is not by Nrf2 in Because Nrf1 and Nrf2 show binding and expression these factors have been to overlapping target genes and overlapping (1Motohashi H. O'Connor T. Katsuoka F. Engel J.D. Yamamoto M. Gene (Amst.). 2002; 294: 1-12Crossref PubMed Scopus (377) Google Scholar, 2Blank V. J. Mol. Biol. 2008; 376: 913-925Crossref PubMed Scopus (114) Google Scholar). in mice revealed that Nrf1-null mice (7Chan J.Y. Kwong M. Lu R. Chang J. Wang B. Yen T.S.B. Kan Y.W. EMBO J. 1998; 17: 1779-1787Crossref PubMed Scopus (214) Google Scholar) exhibit a from that of the Nrf2-null mice (4Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3088) Google Scholar), suggesting that these transcription factors distinct the analysis of Nrf1 has been knock-out of the Nrf1 gene in mice embryonic to specific that Nrf1 we have a of its function in this we generated hepatocyte-specific Nrf1 gene knock-out mice and the and in the livers of the mutant mice. revealed that loss of Nrf1 did not in the of well known genes, genes were in Nrf1 cKO mice. Furthermore, we found that Nrf1 stress response genes, as and DNA genes as well as genes in and the of These thus, that Nrf1 has acquired unique functions that cannot be by the CNC family transcription factors during its molecular evolution. An during the study was that expression of genes were in mice when with their expression in mice. This induction is on Nrf2 as the induction of these genes in mice was deletion of Nrf2. that in the absence of Nrf1, Nrf2 can be activated by It is well known that Nrf2 is activated by and ER C. K. M. Yamamoto M. S. Biochem. Biophys. Res. Commun. 2006; PubMed Scopus Google Scholar, B. Yamamoto M. J. 2007; PubMed Scopus Google Scholar, D. M. E. Mol. Cell. Biol. 2003; 23: PubMed Scopus Google Scholar). a is that the disruption of Nrf1 ER which to the of Nrf2 (13Zhang Y. Lucocq J.M. Yamamoto M. Hayes J.D. Biochem. J. 2007; 408: 161-172Crossref PubMed Scopus (30) Google Scholar, W. Chan J.Y. J. Biol. Chem. 2006; 281: 19676-19687Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). with the that Nrf2, we that Nrf2 target genes are in liver specific knock-out of T. H. Takahashi S. S. Yamamoto M. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). In this during into a number of antioxidant glutathione and T. H. Takahashi S. S. Yamamoto M. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). These show that the Nrf2 gene battery a of that to with stress that is of an Thus, Nrf2 is for stress In contrast, Nrf1 is indispensable for stress and the of Nrf1 activates Nrf2 as a the that are by the of CNC members has on the and they In the livers of Nrf1 knock-out mice, of the genes to the are MT1 and are MT1 and genes have been reported to at least one ARE in their These have also been reported to the regulation by bZip proteins and G.K. Res. 2003; PubMed Scopus Google Scholar) and Nrf2 Yen 2005; PubMed Scopus Google Scholar). In contrast, of reported that MT1 expression was not decreased in the livers of Nrf2 knock-out mice N. Itoh K. H. Yamamoto M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). In with the we found that in the livers of mice, MT1 gene expression is not in response to the of Nrf2. The analysis revealed that in the absence of Nrf1, MT1 and are Nrf2 target genes are in an Nrf2-dependent These that MT1 and genes are activated by The reporter that Nrf1 and Nrf2 regulate members of the ARE gene Nrf2 not Nrf1 activates the reporter gene Nrf1 not Nrf2 activates reporter gene expression through the MT1 ARE. In contrast, however, that Maf and Maf heterodimers bind to the MT1 ARE with DNA binding not to be to account for their target gene that Nrf1 and Nrf2 bind the MT1 ARE, Nrf1 can a that activates transcription of the MT1 gene. in to the of the DNA binding and the domain is not conserved Nrf1 and Nrf2. Thus, analysis of the of Nrf1 and Nrf2 as a to this and and G.K. Biochem. PubMed Scopus Google Scholar). For instance, knock-out of the MT1 gene mice to liver damage by Z. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, MT1 and mutant mice have a to from and although they show was in these mutant mice J.H. I. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). a and regulation of is Nrf1 target genes responsible for the of to be for ARE sequences within the of genes in as many functional have been found in the found genes that have ARE sequences in the have not been reported to be among these genes, dehydrogenase is by E. R. I. 2003; PubMed Scopus (377) Google Scholar). is a regulatory enzyme in the of from to in response to K. Biochem. J. 2002; PubMed Scopus Google Scholar, J. 2003; PubMed Scopus Google Scholar). The expression of was comparable in mice with the of expression in mice not also found that genes, as protein and and stress response gene one or in their Although it to be whether is a the of these genes and the NASH-like liver damage in mice, this to an to of we have generated hepatocyte-specific Nrf1 knock-out mice from the of Chan and (10Xu Z. Chen L. Leung L. Yen T.S.B. Lee C. Chan J.Y. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4120-4125Crossref PubMed Scopus (225) Google Scholar). have found that the NASH-like liver pathology and in the mutant mice are in with the previous gene expression has not been reported in Nrf1 knock-out mice. Furthermore, among the genes we found a differences in the hepatocyte-specific Nrf1 knock-out mice. that this be due to a or it be due to In we have that Nrf1 contributes to the expression of a of ARE-dependent genes that is distinct from by Nrf2, indicating that each CNC family member has acquired a specific target gene of the individual function of CNC members into the regulation of the ARE gene the of Y. for the H. for and and members for with

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 (tRNASec) 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 tRNASec gene (Trsp). Deletion of Trsp in either macrophages or liver elevated oxidative stress and the of and and and of the of Trsp and the knock-out macrophages elevated oxidative and to of either gene a of Trsp on and a of Trsp selenoprotein is which is for cellular redox and The selenocysteine tRNA (tRNASec) 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. 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