Tomonori Hosoya

Transcription Factor Nrf2 Plays a Pivotal Role in Protection against Elastase-Induced Pulmonary Inflammation and Emphysema

Emphysema is one of the major pathological abnormalities associated with chronic obstructive pulmonary disease. The protease/antiprotease imbalance and inflammation resulting from oxidative stress have been attributed to the pathogenesis of emphysema. Nrf2 is believed to protect against oxidative tissue damage through the transcriptional activation of a battery of antioxidant enzymes. In this study, we investigated the protective role of Nrf2 in the development of emphysema using elastase-induced emphysema as our model system. We found that elastase-provoked emphysema was markedly exacerbated in Nrf2-knockout (KO) mice compared with wild-type mice. The severity of emphysema in Nrf2-KO mice correlated intimately with the degree of lung inflammation in the initial stage of elastase treatment. The highly inducible expression of antioxidant and antiprotease genes observed in wild-type alveolar macrophages was significantly attenuated in the lungs of Nrf2-KO mice. Interestingly, transplantation of wild-type bone marrow cells into Nrf2-KO mice retarded the development of initial lung inflammation and subsequent emphysema, and this improvement correlated well with the appearance of macrophages expressing Nrf2-regulated antiprotease and antioxidant genes. Thus, Nrf2 appears to exert its protective effects through the transcriptional activation of antiprotease and antioxidant genes in alveolar macrophages.

Distinct response to dioxin in an arylhydrocarbon receptor (AHR)-humanized mouse

There are large inter- and intraspecies differences in susceptibility to dioxin-induced toxicities. A critical question in risk assessment of dioxin and related compounds is whether humans are sensitive or resistant to their toxicities. The diverse responses of mammals to dioxin are strongly influenced by functional polymorphisms of the arylhydrocarbon receptor (AHR). To characterize responses mediated by the human AHR (hAHR), we generated a mouse possessing hAHR instead of mouse AHR. Responses of these mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene were compared with the responses of naturally sensitive (C57BL/6J) and resistant (DBA/2) mice. Mice homozygous for hAHR exhibited weaker induction of AHR target genes such as cyp1a1 and cyp1a2 than did C57BL/6J (Ahrb-1/b-1) mice. DBA/2 (Ahrd/d) mice were less responsive to induction of cyp genes than C57BL/6J mice. hAHR and DBA/2 AHR exhibit similar ligand-binding affinities and homozygous hAHR and Ahrd/d mice displayed comparable induction of AHR target genes by 3-methylcholanthrene. However, when TCDD was administered, a greatly diminished response was observed in homozygous hAHR mice compared with Ahrd/d mice, indicating that hAHR expressed in mice is functionally less responsive to TCDD than DBA/2 AHR. After maternal exposure to TCDD, homozygous hAHR fetuses developed embryonic hydronephrosis, but not cleft palate, whereas fetuses possessing Ahrb-1 or Ahrd developed both anomalies. These results suggest that hAHR may define the specificity of the responses to various AHR ligands. Thus, the hAHR knock-in mouse is a humanized model mouse that may better predict the biological effects of bioaccumulative environmental toxicants like TCDD in humans.

BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress

ABSTRACT NF-E2-related factor 2 (Nrf2) regulates antioxidant-responsive element-mediated induction of cytoprotective genes in response to oxidative stress. The purpose of this study was to determine the role of BRG1, a catalytic subunit of SWI2/SNF2-like chromatin-remodeling complexes, in Nrf2-mediated gene expression. Small interfering RNA knockdown of BRG1 in SW480 cells selectively decreased inducible expression of the heme oxygenase 1 (HO-1) gene after diethylmaleate treatment but did not affect other Nrf2 target genes, such as the gene encoding NADPH:quinone oxidoreductase 1 (NQO1). Chromatin immunoprecipitation analysis revealed that Nrf2 recruits BRG1 to both HO-1 and NQO1 regulatory regions. However, BRG1 knockdown selectively decreased the recruitment of RNA polymerase II to the HO-1 promoter but not to the NQO1 promoter. HO-1, but not other Nrf2-regulated genes, harbors a sequence of TG repeats capable of forming Z-DNA with BRG1 assistance. Similarly, replacement of the TG repeats with an alternative Z-DNA-forming sequence led to BRG1-mediated activation of HO-1. These results thus demonstrate that BRG1, through the facilitation of Z-DNA formation and subsequent recruitment of RNA polymerase II, is critical in Nrf2-mediated inducible expression of HO-1.

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.

Conditional Gata2 inactivation results in HSC loss and lymphatic mispatterning.

The transcription factor GATA-2 plays vital roles in quite diverse developmental programs, including hematopoietic stem cell (HSC) survival and proliferation. We previously identified a vascular endothelial (VE) enhancer that regulates GATA-2 activity in pan-endothelial cells. To more thoroughly define the in vivo regulatory properties of this enhancer, we generated a tamoxifen-inducible Cre transgenic mouse line using the Gata2 VE enhancer (Gata2 VECre) and utilized it to temporally direct tissue-specific conditional loss of Gata2. Here, we report that Gata2 VECre-mediated loss of GATA-2 led to anemia, hemorrhage, and eventual death in edematous embryos. We further determined that the etiology of anemia in conditional Gata2 mutant embryos involved HSC loss in the fetal liver, as demonstrated by in vitro colony-forming and immunophenotypic as well as in vivo long-term competitive repopulation experiments. We further documented that the edema and hemorrhage in conditional Gata2 mutant embryos were due to defective lymphatic development. Thus, we unexpectedly discovered that in addition to its contribution to endothelial cell development, the VE enhancer also regulates GATA-2 expression in definitive fetal liver and adult BM HSCs, and that GATA-2 function is required for proper lymphatic vascular development during embryogenesis.

Nrf2 regulates ferroportin 1-mediated iron efflux and counteracts lipopolysaccharide-induced ferroportin 1 mRNA suppression in macrophages

Iron is an essential element of hemoglobin, and efficient iron recycling from senescent erythrocytes by splenic macrophages is required for erythrocyte hemoglobin synthesis during erythropoiesis. Ferroportin 1 (Fpn1) is the sole iron exporter in mammals, and it also regulates iron reutilization. In this study, we demonstrated genetically that a redox-sensitive transcription factor, Nrf2, regulates Fpn1 mRNA expression in macrophages. Nrf2 activation by several electrophilic compounds commonly resulted in the upregulation of Fpn1 mRNA in bone marrow-derived and peritoneal macrophages obtained from wild-type mice but not from Nrf2 knockout mice. Further, Nrf2 activation enhanced iron release from the J774.1 murine macrophage cell line. Previous studies showed that inflammatory stimuli, such as LPS, downregulates macrophage Fpn1 by transcriptional and hepcidin-mediated post-translational mechanisms leading to iron sequestration by macrophages. We showed that two Nrf2 activators, diethyl maleate and sulforaphane (SFN; a natural Nrf2 activator found in broccoli), restored the LPS-induced suppression of Fpn1 mRNA in human and mouse macrophages, respectively. Furthermore, SFN counteracted the LPS-induced increase of Hepcidin mRNA by an Nrf2-independent mechanism in mouse peritoneal macrophages. These results demonstrate that Nrf2 regulates iron efflux from macrophages through Fpn1 gene transcription and suggest that Nrf2 may control iron metabolism during inflammation.

Nrf2 Neh5 domain is differentially utilized in the transactivation of cytoprotective genes

The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) contains two transcription activation domains, Neh4 (Nrf2 ECH homology 4) and Neh5, which co-ordinately regulate transactivation of cytoprotective genes. In the present study we aimed to clarify the role of the Neh5 domain in Nrf2-mediated gene regulation. Deletion of the complete Neh5 domain reduces expression of endogenous Nrf2 target genes, such as HO-1 (haem oxygenase 1), NQO1 [NAD(P)H:quinone oxidoreductase 1] and GCLM (glutamate cysteine ligase modulatory subunit), in human kidney epithelial cells. Furthermore, the deletion of Neh5 markedly repressed CBP [CREB (cAMP-response-element-binding protein)-binding protein] and BRG1 (Brahma-related gene 1) from associating with Nrf2, diminishing their co-operative enhancement of HO-1 promoter activity. Mutational analysis of the Neh5 domain revealed a motif that shares significant homology with beta-actin and ARP1 (actin-related protein 1). Mutagenesis of this motif selectively decreased HO-1, but not NQO1 and GCLM, expression. Taken together, these results indicate that the Neh5 domain has the ability to regulate Nrf2 target gene transcription, yet the role of the Neh5 domain in transcription varies from gene to gene.

GATA-3 regulates hematopoietic stem cell maintenance and cell-cycle entry

Maintaining hematopoietic stem cell (HSC) quiescence is a critical property for the life-long generation of blood cells. Approximately 75% of cells in a highly enriched long-term repopulating HSC (LT-HSC) pool (Lin(-)Sca1(+)c-Kit(hi)CD150(+)CD48(-)) are quiescent, with only a small percentage of the LT-HSCs in cycle. Transcription factor GATA-3 is known to be vital for the development of T cells at multiple stages in the thymus and for Th2 differentiation in the peripheral organs. Although it is well documented that GATA-3 is expressed in HSCs, a role for GATA-3 in any prethymic progenitor cell has not been established. In the present study, we show that Gata3-null mutant mice generate fewer LT-HSCs and that fewer Gata3-null LT-HSCs are in cycle. Furthermore, Gata3 mutant hematopoietic progenitor cells fail to be recruited into an increased cycling state after 5-fluorouracil-induced myelosuppression. Therefore, GATA-3 is required for the maintenance of a normal number of LT-HSCs and for their entry into the cell cycle.

Keap1/Nrf2 system regulates neuronal survival as revealed through study of keap1 gene-knockout mice

Keap1 is proposed to be a sensor protein of electrophilic compounds and a transducer of the signal from electrophilic compounds for transcriptional activation. Thus, the use of keap1 gene-knockout (KO) mice is a straightforward approach in order to clarify the molecular background for the use of electrophilies as neuroprotective compounds. In the present report, we investigated the question as to how the deletion of the keap1 gene affects the activities of Nrf2 and survival of immature cortical neurons. In cortical cultures prepared from wild-type (WT) mice, Keap1 was expressed in the neurons, and Nrf2 protein was retained in their cytoplasm; whereas Nrf2 was translocated into the nuclei of neurons and phase 2 enzymes were constitutively activated in the cortical cultures from KO mice. Consistent with these results, cortical neurons from KO mice showed increased resistance to oxidative stress induced by high concentrations of glutamate and rotenone. These results suggest that the absence of Keap1 constitutively activates Nrf2, which then induces the phase 2 enzymes in neurons and induces increased resistance of cortical neurons to oxidative stress. This report is the first report to show that Keap1 is a key regulator of cell defense mechanisms of CNS neurons against oxidative stress.

Transgenic rescue of erythroid 5-aminolevulinate synthase-deficient mice results in the formation of ring sideroblasts and siderocytes.

Molecular defects in erythroid 5‐aminolevulinate synthase (ALAS‐E), the first enzyme in the heme biosynthetic pathway, cause X‐linked sideroblastic anemia (XLSA). However, ring sideroblasts, the hallmark of XLSA, were not found in ALAS‐E‐deficient mouse embryos, indicating that simple ALAS‐E‐deficiency is not sufficient for ring sideroblast formation. To investigate the developmental stage‐specific pathogenesis caused by heme‐depletion, we attempted a complementation rescue of ALAS‐E‐deficiency. We exploited transgenic mouse lines expressing human ALAS‐E at approximately half that of wild‐type levels. In these hypomorphic embryos, most of the primitive erythroid cells were transformed into ring sideroblasts. The majority of the circulating definitive erythroid cells became siderocytes, enucleated erythrocytes containing iron deposits, and definitive ring sideroblasts were also observed. These iron‐overloaded cells suffered from an α/β globin chain imbalance. Despite the iron overload, transferrin receptors were highly expressed in the erythroid cells, suggesting they contribute to the formation of ring sideroblasts and siderocytes. These results indicate that a partially depleted heme supply provokes ring sideroblast formation. The experimental generation of ring sideroblasts in animals would contribute to our understanding of the iron metabolism and its disorder in erythroid cells.