Biomedical subjects

Yoshiaki Fujii-Kuriyama

Publications and source records attributed to Yoshiaki Fujii-Kuriyama.

At least 19 recordsLinked to original sources

PubMed2007

Identification of amino acid residues in the Ah receptor involved in ligand binding.

The Ah receptor (AhR) is a ligand-activated transcription factor. Five amino acids as candidate amino acids necessary for ligand binding within or near the ligand-binding domain were selected based on their evolutional conservation and their aromatic nature that could interact with xenobiotic ligands. These amino acids were changed to Ala, and the mutated AhRs were subjected to a test of their transactivation activity in HeLa cells. Mutation of Phe318 completely lost its activity whereas other mutations only weakly impaired activity. The Leu-substituted mutant, AhR(Phe318Leu), activated the luciferase activity to the level comparable to wild type in the cells treated with 3-methylcholanthrene (MC) but not at all with beta-naphthoflavone (beta-NF). Ligand-binding activity of mutants was examined with [3H]MC in vitro. AhR(Phe318Ala) could not bind to [3H]MC. [3H]MC bound by AhR(Phe318Leu) was competed with unlabeled MC but not with beta-NF. A structural model of the ligand-binding domain was constructed.

Amino Acid Sequence↗

PubMed2006

Quantitative analysis of benzo[a]pyrene biotransformation and adduct formation in Ahr knockout mice.

Benzo[a]pyrene (BP) is an ubiquitous environmental pollutant with potent mutagenic and carcinogenic properties. The Ah receptor (Ahr) is involved in the metabolic activation of BP and is therefore important in the induction of chemical carcinogenesis. In this study, the relationship between Ahr genotype and biotransformation of BP in internal organs was investigated in Ahr (+/+), Ahr (+/-) and Ahr (-/-) mice. The mice were treated with BP (100mg/kg) by gavage. Gene expression was measured after 24h by real-time RT-PCR and showed induction of Cyp1a1 in liver and lung, and Cyp1b1 in lung in both Ahr (+/+) and Ahr (+/-). No induction of the Cyp genes was observed in the Ahr (-/-). There was a significant basal expression of Cyp1b1 in the liver of all genotypes, and this expression was independent of the BP exposure. Analyzed by HPLC-fluorescence, there were increased levels of protein and DNA adducts, metabolites, conjugates and unmetabolized BP in the internal organs of Ahr (-/-) as compared to Ahr (+/+) and Ahr (+/-) mice. This may be partly explained by a delayed bioactivation of BP in the Ahr deficient mice. The BP metabolism observed in the Ahr (-/-) mice is also evidence of an Ahr independent biotransformation of BP.

Animals↗

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

Constitutively active aryl hydrocarbon receptor expressed specifically in T-lineage cells causes thymus involution and suppresses the immunization-induced increase in splenocytes.

The aryl hydrocarbon receptor (AhR) is a transcription factor belonging to the basic helix-loop-helix-PER-ARNT-SIM superfamily. Xenobiotics, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, bind the receptor and trigger diverse biological reactions. Thymocyte development and T cell-dependent immune reactions are sensitive targets of AhR-dependent 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity. However, the exact role of the AhR in T cells in animals exposed to exogenous ligands has not been clarified because indirect effects of activated AhR in other cell types cannot be excluded. In this study, we generated transgenic (Tg) mice expressing a constitutively active mutant of AhR under the regulation of a T cell-specific CD2 promoter to examine AhR function in T cells. The mRNAs of the constitutively active mutant of AhR and an AhR-induced gene, CYP1A1, were expressed in the thymus and spleen of the Tg mice. The transgene expression was clearly detected in the thymocytes, CD4, and CD8 T cells, but not in the B cells or thymus stromal cells. These Tg mice had a decreased number of thymocytes and an increased percentage of CD8 single-positive thymocytes, but their splenocytes were much less affected. By contrast, the increase in number of T cells and B cells taking place in the spleen after immunization was significantly suppressed in the Tg mice. These results clearly show that AhR activation in the T-lineage cells is directly involved in thymocyte loss and skewed differentiation. They also indicate that AhR activation in T cells and not in B cells suppresses the immunization-induced increase in both T cells and B cells.

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

Altered thyroxin and retinoid metabolic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin in aryl hydrocarbon receptor-null mice.

To determine whether the disruption of thyroid hormone and retinoid homeostasis that occurs after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can be mediated by the arylhydrocarbon receptor (AhR), pregnant AhR-heterozygous (AhR+/-) mice were administered a single oral dose of 10 microg kg(-1) TCDD at gestation day 12.5. Serum and liver were collected on postnatal day 21 from vehicle-treated control or TCDD-treated AhR+/- and AhR-null (AhR-/-) mouse pups. Whereas TCDD exposure resulted in a marked reduction of total thyroxin (TT4) and free T4 (FT4) levels in the serum of AhR+/- mice, TCDD had no effects on AhR-/- mice. Gene expression of UDP-glucuronosyltransferase (UGT)1A6, cytochrome P450 (CYP)1A1, and CYP1A2 in the liver was induced markedly by TCDD in AhR+/- but not AhR-/- mice. Induction of CYP1A1 in response to TCDD was confirmed by immunohistochemical evidence in that CYP1A1 protein was conspicuously localized in the cytoplasm of hepatocytes in the centrilobular region. Levels of retinyl palmitate were greatly reduced in the liver of TCDD-exposed AhR+/- mice, but not in vehicle-treated AhR+/- mice. No effects of TCDD on retinoid levels in the liver were found in AhR-/- mice. We conclude that disruption of thyroid hormone and retinoid homeostasis is mediated entirely via AhR. Induction of UGT1A6 is thought to be responsible at least partly for reduced serum thyroid hormone levels in TCDD-exposed mice.

Administration, Oral↗

PubMed2004

A point mutation in a silencer module reduces the promoter activity for the human mercaptopyruvate sulfurtransferase.

A promoter region of human mercaptopyruvate sulfurtransferase (MST) [EC 2.8.1.2] is G+C-rich and TATA-less, showing features of a house-keeping gene. In the core promoter, a GC box (-284:GGGGCGTGGC:-275) and an initiator (-219:TTATATG:-225) are found. A cap site hunting analysis for human liver cDNA revealed four possible transcriptional start sites, nucleotides -223, -159, -35 and -25. Point mutagenesis and deletion studies suggest that a module of the silencer element is -394:GCTG:-391. A replacement of -391G to C lost the silencer function; on the other hand, a replacement of -394G to T or C, -393C to T or -392T to G markedly reduced the promoter activity.

Base Sequence↗

PubMed2004

Dibenzo[A,L]pyrene-induced genotoxic and carcinogenic responses are dramatically suppressed in aryl hydrocarbon receptor-deficient mice.

Dibenzo[a,l]pyrene (DB[a,l]P), a notorious air pollutant, is the most powerful carcinogenic polycyclic aromatic hydrocarbon (PAH) ever tested. Although the carcinogenicity of PAH may be primarily mediated by the aryl hydrocarbon receptor (AhR), the in vivo role of AhR in skin carcinogenesis remains to be defined. In this context, we investigated the genotoxic and carcinogenic responses of the AhR-deficient mouse skin to DB[a,l]P. A single painting resulted in a striking epidermal hyperplasia in AhR+/+ mice but not in AhR-/- mice. Bromodeoxyuridine-labeling index and accumulation of p53 protein in epidermal cells of AhR+/+ mice were 8- and 33-fold higher than those of AhR-/- mice, respectively. 32P-Postlabeling assay for DB[a,l]P-DNA adducts displayed a 2-fold increase in the AhR+/+ mouse skin. After DB[a,l]P exposure, AhR-/- mice arranged a nearly 60% reduction in the induction of epidermal cytochrome P450 (CYP)1A1, but CYP1B1 was constitutively expressed in both genotypes of mice, irrespective of DB[a,l]P treatment. As compared with AhR+/+ mice, AhR-/- mice had both significantly lower incidence (100% vs. 33%) and multiplicity (2.7 vs. 0.46) of skin tumors by the complete carcinogenesis study. These observations indicate that a reduced tumor yield in AhR-/- mice may be secondary to reduction of inducible CYP1A1 activation and subsequent DNA adduction. It is evident from our continuous work that although AhR is likely to play a central role in epidermal proliferation and possibly neoplastic transformation, the relative importance of AhR for carcinogenesis may be different among PAH examined.

Administration, Cutaneous↗

PubMed2004

Testicular cytochrome P450scc and LHR as possible targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the mouse.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in adult animals has been reported to perturb the regulation of steroidogenesis in the testis, possibly by arylhydrocarbon receptor (AhR). To clarify how AhR is involved in the testicular steroidogenesis, we carried out comparative experiments using wild-type and AhR-null male mice that were intraperitoneally administered TCDD. The TCDD administration to wild-type mice showed significant reduction of P450scc and LHR in the testis, whereas the levels in the AhR-null mouse testis were unchanged. To compare anti-androgenic properties on hypothalamo-pituitary-gonadal (HPG) axis, estradiol-3-benzoate (EB), a synthetic estrogen agonist, was administered to mice, the expression of the LHalpha/FSHalpha, LHbeta, FSHbeta and GnRHR genes was severely impaired in the pituitary gland, in contrast to no observed effects in the TCDD-treated mice. In addition, the expression of the LHR gene was increased in the testis of the EB-treated mice. These observations suggest that the target of TCDD is different from that of EB on HPG axis and that TCDD treatment suppresses the P450scc and LHR genes in the testis in an AhR-dependent manner.

Adrenal Glands↗

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

Altered DNA binding specificity of Arnt by selection of partner bHLH-PAS proteins.

The Ah receptor (AhR) and HLF are transcription factors involved in xenobiotic metabolism and hypoxic response, respectively. AhR and HLF heterodimerize with Arnt as the common partner, and bind to asymmetric E-boxes termed XRE and HRE, respectively. In order to investigate nucleotide preference of the heterodimers, reporter plasmids with oligonucleotides for XREs or HREs with systematic mutations were constructed and their activity was determined. Comparison of the activity revealed that DNA length and nucleotide preference recognized by Arnt subunit in the two heterodimers were largely different between XRE and HRE. We expressed AhR-Arnt and HLF-Arnt in Escherichia coli and used them for DNA binding. The dissociation constant of HLF-Arnt-HRE was 10.4 +/- 1.6 nM. Competition activity of mutated XREs or HREs with wild type was consistent with their transcription activity. Bending of XRE and HRE induced by binding of the relevant heterodimers was observed with stronger bending of XRE than of HRE. By deletional and mutational analyses, an alanine and three arginine (Ala 8, Arg 9, Arg 11 and Arg 12) residues in the basic sequence of HLF were found to be indispensable for the transcriptional activity.

Amino Acid Sequence↗

PubMed2004

A novel induction mechanism of the rat CYP1A2 gene mediated by Ah receptor-Arnt heterodimer.

We have identified an enhancer responsible for induction by 3-methylcholanthrene in the upstream region of the CYP1A2 gene. The enhancer does not contain the invariant core sequence of XREs that are binding sites for the Ah receptor (AhR) and Arnt heterodimer. The enhancer did not show any inducible expression in Hepa-1-derived cell lines, C4 and C12, deficient of Arnt and AhR, respectively. On the other hand, bacterially expressed AhR-Arnt heterodimer could not bind to the enhancer. Mutational analysis of the enhancer revealed that a repeated sequence separated by six nucleotides is important for expression. A factor binding specifically to the enhancer was found by using gel shift assays. Bacterially expressed AhR-Arnt heterodimer interacted with the factor. A dominant negative mutant of the AhR to XRE activated the enhancer. Collectively, these results demonstrate that a novel induction mechanism is present in which the AhR-Arnt heterodimer functions as a coactivator.

Animals↗

PubMed2004

Aryl hydrocarbon receptor-mediated induction of microsomal drug-metabolizing enzyme activity by indirubin and indigo.

Indirubin and indigo, which are thought to be natural ligands for aryl hydrocarbon receptor (AhR), showed marked AhR ligand activities in a reporter gene assay using recombinant yeast. Their activities were comparable with or more potent than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. When indirubin and indigo were administered to mice, ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in the liver were increased, but subsequently decreased within 2 days. Indirubin was more potent than indigo. Levels of cytochrome P450 1A1/2 proteins and mRNAs in the liver of mice dosed with indirubin were also enhanced. These enhancing effects of indirubin and indigo were not observed in AhR knock-out mice. Ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in rat hepatocytes and HepG2 cells were enhanced by the addition of indirubin or indigo, but less potently than by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Indigocarmine, a sulfate derivative of indigo, which is used as food additive, did not show these inducing effects on drug-metabolizing enzymes. Our results suggest that indirubin and indigo act as inducers for cytochrome P450 1A1/2 mediated by AhR in mammals in vivo.

Animals↗