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Junsei Mimura

Publications and source records attributed to Junsei Mimura.

15 recordsLinked to original sources
PubMed2006

Unique and overlapping transcriptional roles of arylhydrocarbon receptor nuclear translocator (Arnt) and Arnt2 in xenobiotic and hypoxic responses.

Arnt and the homologous Arnt2 share a high degree of sequence similarity and are believed to function as obligate common partners for a number of basic helix-loop-helix (bHLH)-PAS transcription factors including arylhydrocarbon receptor (AhR) and HIFalpha. Genetic disruption of both Arnt and Arnt2 demonstrated both unique and overlapping functions in response to environmental stimuli and during mouse development. Either stably or transiently expressed Arnt/Arnt2 wild type and various mutants or chimeric constructs in Hepa1-c4 cells exhibit similar levels of hypoxic response element-driven reporter gene expression and the induction of endogenous Glut-1 through binding with HIFalpha in response to hypoxia. In contrast, we observed clear functional differences in the ability of Arnt and Arnt2 to induce xenobiotic response element-driven reporter and endogenous CYP1A1 gene expression. In contrast with Arnt, Arnt2 was practically incapable of interacting with ligand-activated AhR to induce the expression of target genes for xenobiotic-metabolizing enzymes in response to xenobiotics. The differential binding of AhR by Arnt and Arnt2 can be ascribed to a single His/Pro amino acid difference in the PASB region of Arnt and Arnt2, suggesting that the PASB/PASB interaction between bHLH-PAS transcription factors plays a selective role for their specific partner molecule.

Amino Acid Sequence↗
PubMed2005

Effects of aryl hydrocarbon receptor signaling on the modulation of TH1/TH2 balance.

An orally active antiallergic agent, M50367, skews the Th1/Th2 balance toward Th1 dominance by suppressing naive Th cell differentiation into Th2 cells in vitro. Administration results in the suppression of IgE synthesis and peritoneal eosinophilia in vivo. In this report, we determined that M50354 (an active metabolite of M50367) was a ligand for the aryl hydrocarbon receptor (AhR); the immunological effects of this compound on in vitro Th1/Th2 differentiation from naive Th cells and Th1/Th2 balance in vivo were manifested through binding to AhR. These effects were completely abolished in AhR-deficient mice. AhR expression in the naive Th cell was significantly up-regulated by costimulation of TCR and CD28. Suppression of naive Th cell differentiation into Th2 cells via binding of M50354 to AhR was associated with inhibition of GATA-3 expression in Th cells. In addition, forced expression of a constitutively active form of AhR or activation of AhR by the addition of representative ligands suppressed naive Th cell differentiation into Th2 cells. Based on these results, we conclude that AhR functions as a modulator of the in vivo Th1/Th2 balance through activation in naive Th cells.

Animals↗
PubMed2005

Identification of the promoter region of the parathyroid hormone receptor gene responsible for transcriptional suppression by insulin-like growth factor-I.

We investigated parathyroid hormone (PTH)/PTH-related protein receptor (PTH1R) gene suppression induced by insulin-like growth factor (IGF)-I using a rat osteoblast-like cell line (UMR-106). Observations were made with PD98059, a specific ERK signaling pathway inhibitor, and UMR-106 cells transfected with dominant negative or constitutively active forms of MAP kinase kinase. IGF-I inhibited PTH1R gene expression via an ERK1/2 MAP kinase pathway. We cloned the 8-kb promoter region of the rat PTH1R gene and characterized the U3 promoter, a major IGF-I-responsive promoter among the two present in rat osteoblasts. The IGF-I-suppressive region was between +1 and +25, identical to the previously described PTH-suppressive region (PTHSR). Gel mobility-shift detected a specific DNA-protein complex decreased by IGF-I. Mutation involving a three base sequence (+1 to +3) among more than 3.5 kb constituting the PTH1R promoter region completely abolished IGF-I action. Thus, IGF-I signaling may act at the osteoblast exon U3 transcription initiation site to repress the transcriptional activity.

Animals↗
PubMed2005

A dioxin sensitive gene, mammalian WAPL, is implicated in spermatogenesis.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disruptor that produces a variety of toxic effects. We have isolated a mouse homolog of the hWAPL gene, termed mouse WAPL (mWAPL), as a target of TCDD by cDNA representational difference analysis from mouse embryonic stem cells. A statistically significant increase in mWAPL expression was observed at 0.1 microM TCDD in AhR-/- mouse embryonic fibroblast cells. Interestingly, at 1 microM TCDD, mWAPL mRNA levels decreased in AhR+/+ cells, but further increased in AhR-/- cells. hWAPL and mWAPL were highly expressed only in testes among normal tissue samples, and we observed mWAPL localization in the synaptonemal complex of testicular chromosomes. In addition, mouse testes decreased the expression of mWAPL mRNA after a single intraperitoneal injection of TCDD. Thus, mammalian WAPL such as hWAPL and mWAPL may be involved in spermatogenesis and be target genes mediating the reproductive toxicity induced by TCDD.

Amino Acid Sequence↗
PubMed2005

Constitutive expression of aryl hydrocarbon receptor in keratinocytes causes inflammatory skin lesions.

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.

Animals↗
PubMed2005

Intrinsic function of the aryl hydrocarbon (dioxin) receptor as a key factor in female reproduction.

Dioxins exert a variety of adverse effects on organisms, including teratogenesis, immunosuppression, tumor promotion, and estrogenic action. Studies using aryl hydrocarbon receptor (AhR)-deficient mice suggest that the majority of these toxic effects are mediated by the AhR. In spite of the adverse effects mediated by this receptor, the AhR gene is conserved among a number of animal species, ranging from invertebrates to vertebrates. This high degree of conservation strongly suggests that AhR possesses an important physiologic function, and a critical function is also supported by the reduced fertility observed with AhR-null female mice. We demonstrate that AhR plays a crucial role in female reproduction by regulating the expression of ovarian P450 aromatase (Cyp19), a key enzyme in estrogen synthesis. As revealed by in vitro reporter gene assay and in vivo chromatin immunoprecipitation assay, AhR cooperates with an orphan nuclear receptor, Ad4BP/SF-1, to activate Cyp19 gene transcription in ovarian granulosa cells. Administration to female mice of an AhR ligand, DMBA (9,10-dimethyl-1,2-benzanthracene), induced ovarian Cyp19 gene expression, irrespective of the intrinsic phase of the estrus cycle. In addition to elucidating a physiological function for AhR, our studies also suggest a possible mechanism for the toxic effects of exogenous AhR ligands as endocrine disruptors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

Phosphorylation analysis of 90 kDa heat shock protein within the cytosolic arylhydrocarbon receptor complex.

The arylhydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that regulates the transcription of genes encoding xenobiotic metabolizing enzymes and also mediates most of the toxic effects caused by dioxins and polycyclic aromatic hydrocarbons. The cytosolic AhR complex exists as a transcriptionally cryptic complex, consisting of the 90 kDa heat shock protein (HSP90) and the hepatitis B virus X-associated protein 2 (XAP2). The posttranslational modifications, especially phosphorylation, of the cytosolic AhR-HSP90-XAP2 complex are poorly understood, although the phosphorylation of a transcriptionally active heterodimer of AhR and an AhR nuclear translocator is critically involved in AhR function. To reveal the phosphorylation status involved in AhR function, we used mass spectrometry to determine the site-specific phosphorylation of the steady-state cytosolic AhR complex, prepared from Chinese hamster ovary cells stably expressing mouse AhR. We identified phosphorylations of the HSP90 subunits within the AhR complex at Ser225 and Ser254 of HSP90beta and Ser230 of HSP90alpha. By site-directed mutagenesis, these serine residues were substituted with alanine and glutamic acid to elucidate the role of the HSP90beta serine phosphorylations in the AhR function. Immunoprecipitation assays using COS7 transfectants showed that the replacement of Ser225 and Ser254 by Ala, S225/254A, increased the binding affinity for AhR, as compared with the Glu replacement. In a ligand-induced AhR transcription activity assay using Hepa1 transfectants, the S255/254A mutant exhibited more potent transcription activity than the S225/254E mutant, which had activity similar to that of wild-type HSP90beta. These results suggest that the phosphorylations in the charged linker region of the HSP90 molecule modulate the formation of the functional cytosolic AhR complex.

Amino Acid Sequence↗
PubMed2004

Expression of hypothalamic neuropeptides after acute TCDD treatment and distribution of Ah receptor repressor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant originating from industrial waste. At sublethal concentrations it induces anorexia and weight loss as part of the so-called wasting syndrome. To gain insight into its possible underlying mechanisms, mRNA expression of some key hypothalamic neuropeptides involved in the regulation of body weight was studied using in situ hybridization histochemistry in adult male Sprague-Dawley rats 6 days after single oral administration of TCDD (15 microg/kg) and in age-paired control rats. In TCDD-treated rats which displayed a decrease in body weight gain vs. controls, arcuate nucleus expression of neuropeptide Y (NPY), proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) mRNA was increased. In the lateral hypothalamic area, melanin-concentrating hormone (MCH) mRNA expression was also increased, while levels of CART and orexin/hypocretin mRNA were not significantly changed. Since TCDD is known to bind to the aryl hydrocarbon receptor (AhR), the distribution of the AhR repressor (AhRR), which is co-expressed with AhR in the same cells, was studied by immunohistochemistry in the mouse hypothalamus using mouse AhRR specific antiserum. AhRR immunoreactivity was present in the nuclei of neurons found in all main hypothalamic groups including NPY, CART, MCH and orexin/hypocretin neurons. Xenobiotic response elements were found in these neuropeptide genes with the exception of MCH. Thus changes in expression of orexigenic and anorexigenic neuropeptides after TCDD treatment may help to explain the occurrence of the TCDD-induced weight loss, which may be either directly or indirectly related to the effects of TCDD on neuropeptide expression.

Administration, Oral↗
PubMed2004

A constitutively active arylhydrocarbon receptor induces growth inhibition of jurkat T cells through changes in the expression of genes related to apoptosis and cell cycle arrest.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress T cell-dependent immune reactions through the activation of the arylhydrocarbon receptor (AhR). Our previous findings suggest that TCDD inhibits the activation and subsequent expansion of T cells following antigen stimulation in mice, leading to a decreased level of T cell-derived cytokines involved in antibody production. In the present study, we investigated the effects of activated AhR on T cells by transiently expressing a constitutively active AhR (CA-AhR) mutant in AhR-null Jurkat T cells. In agreement with our previous findings, CA-AhR markedly inhibited the growth of Jurkat T cells. The inhibited cell growth was found to be concomitant with both an increase in the annexin V-positive apoptotic cells and the accumulation of cells in the G(1) phase. The growth inhibition was also shown to be mediated by both xenobiotic response element (XRE)-dependent and -independent mechanisms, because an A78D mutant of the CA-AhR, which lacks the ability of XRE-dependent transcription, partially inhibited the growth of Jurkat T cells. Furthermore, we demonstrated that CA-AhR induces expression changes in genes related to apoptosis and cell cycle arrest. These expression changes were shown to be solely mediated in an XRE-dependent manner, because the A78D mutant of the CA-AhR did not induce them. To summarize, these results suggest that AhR activation causes apoptosis and cell cycle arrest, especially through expression changes in genes related to apoptosis and cell cycle arrest by the XRE-dependent mechanism, leading to the inhibition of T cell growth.

Apoptosis↗
PubMed2003

Modulation of oestrogen receptor signalling by association with the activated dioxin receptor.

Environmental contaminants affect a wide variety of biological events in many species. Dioxins are typical environmental contaminants that exert adverse oestrogen-related effects. Although their anti-oestrogenic actions are well described, dioxins can also induce endometriosis and oestrogen-dependent tumours, implying possible oestrogenic effects. However, the molecular mechanism underlying oestrogen-related actions of dioxins remains largely unknown. A heterodimer of the dioxin receptor (AhR) and Arnt, which are basic helix-loop-helix/PAS-family transcription factors, mediates most of the toxic effects of dioxins. Here we show that the agonist-activated AhR/Arnt heterodimer directly associates with oestrogen receptors ER-alpha and ER-beta. This association results in the recruitment of unliganded ER and the co-activator p300 to oestrogen-responsive gene promoters, leading to activation of transcription and oestrogenic effects. The function of liganded ER is attenuated. Oestrogenic actions of AhR agonists were detected in wild-type ovariectomized mouse uteri, but were absent in AhR-/- or ER-alpha-/- ovariectomized mice. Our findings suggest a novel mechanism by which ER-mediated oestrogen signalling is modulated by a co-regulatory-like function of activated AhR/Arnt, giving rise to adverse oestrogen-related actions of dioxin-type environmental contaminants.

Animals↗
PubMed2003

Functional role of AhR in the expression of toxic effects by TCDD.

Cytochrome P450 1A1 (CYP1A1) is one of the xenobiotic metabolizing enzymes (XMEs), which is induced by polycyclic aromatic hydrocarbons (PAHs). The most potent inducer of CYP1A1 is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, TCDD induces a broad spectrum of biochemical and toxic effects, such as teratogenesis, immunosuppression and tumor promotion. Most, if not all, of the effects caused by TCDD and other PAHs are known to be mediated by AhR (aryl hydrocarbon receptor or dioxin receptor) which has a high binding affinity to TCDD. The liganded AhR translocates from cytoplasm to nuclei where it switches its partner molecule from Hsp90 to Arnt. Thus formed AhR/Arnt heterodimer binds a specific DNA sequence designated XRE in the promoter region of the target genes including CYP1A1, UDP-glucuronosyl transferase and others to enhance their expression. Although it remains to be studied how AhR is involved in the other TCDD-induced biological effects such as teratogenesis and immunosuppression than induction of XMEs, it is believed that these adverse TCDD effects are caused by untimely activation of gene expression by ligand-activated AhR in the biological process. We summarize the present knowledge about functional role of AhR in TCDD-induced biological effects.

Animals↗
PubMed2003

Heterodimers of bHLH-PAS protein fragments derived from AhR, AhRR, and Arnt prepared by co-expression in Escherichia coli: characterization of their DNA binding activity and preparation of a DNA complex.

AhR (aryl hydrocarbon receptor), AhRR (AhR repressor), and Arnt (AhR nuclear translocator) are members of the bHLH (basic-helix-loop-helix)-PAS (Per-AhR/Arnt-Sim homology sequence) transcription factor superfamily. They associate with each other to form heterodimers, AhR/Arnt or AhRR/Arnt, and bind the XRE (xenobiotic responsive element) sequences in the promoter regions of the target genes to regulate their expression. Their basic regions and HLH motifs mediate DNA binding activity and protein dimerization, respectively. The PAS domain includes two incomplete repeats, PAS-A and PAS-B, and is considered to determine the specificity on protein dimerization. However, the three-dimensional structures of PAS folds reported so far are all monomeric, therefore, little is known about the structural basis of protein interaction through PAS domains. In the present study, we prepared heterodimeric bHLH-PAS domains derived from AhR and Arnt, and AhRR and Arnt by co-expressing each pair in E. coli, and showed that the heterodimers formed exhibited full DNA-binding activity, which was not apparently affected by deletion of the highly basic amino acid cluster most N-terminal as to the HLH region of AhR or AhRR. Methylation of the two CpG sites in the XRE core sequence reduced the binding affinity to heterodimeric proteins, with 5-methylcytosine in the AhR recognition site exhibiting a greater inhibitory effect than that in the Arnt recognition site.

Amino Acid Sequence↗
PubMed2002

Dioxin stimulates synthesis and secretion of IgE-dependent histamine-releasing factor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is the most toxic man-made member of the class of environmental pollutants represented by polychlorinated dibenzo-p-dioxins and dibenzofurans. TCDD produces a wide variety of toxic effects. However, the downstream genes targeted by TCDD and their relation to the diversity of dioxin toxicity symptoms are poorly understood. To identify the target genes of TCDD, we used a cDNA representational difference analysis (RDA) to compare the mRNA patterns of mouse embryonic stem (ES) cells that had and had not been exposed to TCDD. Here we show that TCDD stimulated the expression of IgE-dependent histamine-releasing factor (HRF) mRNA via an aryl hydrocarbon receptor (AhR)-dependent pathway. TCDD also induced the synthesis and secretion of HRF. To our knowledge, this is the first example of HRF being a direct transcriptional target of a toxic agent. HRF has previously been shown to induce histamine release in a dose-dependent manner, at least in vitro. Thus, our data suggest that "endocrine-disrupting" agents may have the potential to influence allergic disorders in the human body.

Animals↗