Kiyoshi Nagata

Tohoku Pharmaceutical University, Japan

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Publications (186)455.84 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: There is a large discrepancy between the inter-individual difference in the hepatic expression level of cytochrome P450 (CYP) 3A4 and that of drug clearance mediated by this enzyme. However, the reason for this discrepancy remains largely unknown. As CYP3A4 interacts with UDP-glucuronosyltransferase 2B7 (UGT2B7) to alter its function, the reverse regulation is expected to modulate CYP3A4-catalyzed activity. To address this issue, we investigated whether protein-protein interaction between CYP3A4 and UGT2B7 modulates CYP3A4 function. For this purpose, we co-expressed CYP3A4, NADPH-cytochrome P450 reductase and UGT2B7 using a baculovirus-insect cell system. The activity of CYP3A4 was significantly suppressed by co-expressing UGT2B7, and this suppressive effect was lost when UGT2B7 was replaced with calnexin (CNX). These results strongly suggest that UGT2B7 negatively regulates CYP3A4 activity through a protein-protein interaction. In order to identify the UGT2B7 domain associated with CYP3A4 suppression we generated 12 mutants including chimeras with CNX. Mutations introduced into the UGT2B7 carboxyl terminal trans-membrane helix caused a loss of the suppressive effect on CYP3A4. Thus, this hydrophobic region is necessary for the suppression of CYP3A4 activity. Replacement of the hydrophilic end of UGT2B7 with that of CNX produced a similar suppressive effect as the native enzyme. The data using chimeric protein demonstrated that the internal membrane-anchoring region of UGT2B7 is also needed for the association with CYP3A4. These data suggest that 1) UGT2B7 suppresses CYP3A4 function, and 2) both hydrophobic domains located near the C-terminus and within UGT2B7 are needed for interaction with CYP3A4. The American Society for Pharmacology and Experimental Therapeutics.
    Molecular pharmacology 08/2015; 88(4). DOI:10.1124/mol.115.098582 · 4.13 Impact Factor
  • Keita Inami · Takamitsu Sasaki · Takeshi Kumagai · Kiyoshi Nagata ·
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    ABSTRACT: The bioavailability of orally administered therapies are often significantly limited in the human intestine by the metabolic activities of Cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp). Predicting whether candidate compounds induce CYP3A4 and P-gp is a crucial stage in the drug development process, as drug-drug interactions may result in the induction of intestinal CYP3A4 and P-gp. However, the assay systems needed to evaluate both CYP3A4 and P-gp induction in the intestine are yet to be established. To address this urgent requirement, we used LS174T cells to create two stable cell lines expressing the CYP3A4 or ATP-binding cassette subfamily B member 1 (ABCB1, encoding P-gp) reporter genes. First, we tested these stable cells by treatment with 1α, 25-dihydroxyvitamin D3 (1,25(OH)2D3), all-trans-retinoic acid (ATRA), and 9-cis-retinoic acid (9-cis RA) that induce CYP3A4 and P-gp in the intestines. All these compounds significantly increased both CYP3A4 and ABCB1 reporter activities in the stable cell lines. To simultaneously assess induction of CYP3A4 and ABCB1, both stable cells were co-cultivated to measure their reporter activities. The mixed cells showed a significant increase in the CYP3A4 and ABCB1 reporter activities following treatment with 1,25(OH) 2D3, ATRA, and 9-cis RA. These activity levels were maintained after passaging for more than 20 times and following multiple freeze-thaw cycles. These results demonstrate that our established cell lines can be used to simultaneously evaluate CYP3A4 and ABCB1 induction in the intestines, providing a valuable in vitro model for the evaluation of future drug candidates. This article is protected by copyright. All rights reserved.
    Biopharmaceutics & Drug Disposition 11/2014; 36(3). DOI:10.1002/bdd.1927 · 2.34 Impact Factor
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    ABSTRACT: In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.
    PLoS ONE 08/2014; 9(8):e104010. DOI:10.1371/journal.pone.0104010 · 3.23 Impact Factor
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    ABSTRACT: The use of human induced pluripotent stem (iPS) cells would be of great value for a variety of applications involving drug development studies. Several reports have been published on the differentiation of human iPS cells into hepatocyte-like cells; however, the cells were insufficient for application in drug metabolism studies. In this study, we aimed to establish effective methods for differentiation of human iPS cells into hepatocytes. Two human iPS cell lines were differentiated by addition of activin A, dimethyl sulfoxide, hepatocyte growth factor, oncostatin M, and dexamethasone. The differentiated cells expressed hepatocyte markers and drug-metabolizing enzymes, revealing that the human iPS cells were differentiated into hepatocyte-like cells. Expression of CYP3A4 and UGT1A1 mRNAs increased by treatment with typical inducers of the enzymes, and the response of the cells against the inducers was similar to that of human hepatocytes. Furthermore, the drug-metabolizing activity of CYP3A4, as monitored by testosterone 6β-hydroxylase activity, was elevated by these inducers. In conclusion, we established methods for differentiation of hepatocyte-like cells expressing drug metabolizing activity from human iPS cells. The hepatocyte-like cells derived from human iPS cells will be useful for drug metabolism studies.
    Drug Metabolism and Pharmacokinetics 12/2013; 29(3). DOI:10.2133/dmpk.DMPK-13-RG-104 · 2.57 Impact Factor
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    ABSTRACT: Functional protein-protein interactions between UDP-glucuronosyltransferase (UGT)1A isoforms and cytochrome P450 (CYP)3A4 were studied. To this end, UGT1A-catalyzed glucuronidation was assayed in Sf-9 cells which simultaneously expressed UGT and CYP3A4. In the kinetics of UGT1A6-catalyzed glucuronidation of 5-HT, both Km and Vmax were increased by CYP3A4. When CYP3A4 was co-expressed either with UGT1A1 or 1A7, the Vmax for the glucuronidation of the irinotecan metabolite (SN-38) was significantly increased. The S50 or Km was little affected by simultaneous expression of CYP3A4. This study also examined the catalytic properties of the allelic variants of UGT1A1 and 1A7, and their effects on the interaction with CYP3A4. While the UGT1A1-catalyzing activity of 4-methylumbelliferone glucuronidation was reduced in its variant, UGT1A1*6, the co-expression of CYP3A4 restored the impaired function to a level comparable with the wild-type. Similarly, simultaneous expression of CYP3A4 increased the Vmax of UGT1A7*1 (wild-type) and *2 (R129K and R131K), whereas the same was not observed in UGT1A7*3 (R129K, R131K and W208A). In the kinetics involving different concentrations of UDP-glucuronic acid (UDP-GlcUA), the Km for UDP-GlcUA was significantly higher for UGT1A7*2 and *3 than *1. The Km of UGT1A7*1 and *3 was increased by CYP3A4, whereas *2 did not exhibit any such change. These results suggest that 1) CYP3A4 changes the catalytic function of the UGT1A subfamily in a UGT isoform-specific manner and 2) non-synonymous mutations in UGT1A7*3 reduce not only the ability of UGT to utilize UDP-GlcUA, but also CYP3A4-mediated enhancement of catalytic activity while CYP3A4 is able to restore the UGT1A1*6 function.
    Drug metabolism and disposition: the biological fate of chemicals 11/2013; 42(2). DOI:10.1124/dmd.113.054833 · 3.25 Impact Factor
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    ABSTRACT: Human induced pluripotent stem (iPS) cells were differentiated into the endoderm using activin A and were then treated with fibroblast growth factor 2 (FGF2) for differentiation into intestinal stem cell-like cells. These immature cells were then differentiated into enterocyte-like cells using epidermal growth factor (EGF) in 2% fetal bovine serum (FBS). At the early stage of differentiation, mRNA expression of caudal type homeobox 2 (CDX2), a major transcription factor related to intestinal development and differentiation, and leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), an intestinal stem cell marker, was markedly increased by treatment with FGF2. When cells were cultured in medium containing EGF and a low concentration of FBS, mRNAs of specific markers of intestinal epithelial cells, including sucrase-isomaltase, the intestinal oligopeptide transporter SLC15A1/peptide transporter 1 (PEPT1), and the major metabolizing enzyme CYP3A4, were expressed. In addition, sucrase-isomaltase protein expression and uptake of β-Ala-(L)-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA), a fluorescence-labeled substrate of the oligopeptide transporter, were detected. These results demonstrate a simple and direct method for differentiating human iPS cells into functional enterocyte-like cells.
    Drug Metabolism and Pharmacokinetics 07/2013; 29(1). DOI:10.2133/dmpk.DMPK-13-RG-005 · 2.57 Impact Factor
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    ABSTRACT: Human induced pluripotent stem cells (iPSCs) are a valuable source of hepatocytes for applications in drug metabolism studies. However, the current protocols for generating iPSC-derived hepatocyte-like cells (iPSHCs) are still very inefficient, and iPSHCs do not have sufficient hepatocyte-specific features, which include expression of a series of hepatocyte-specific genes, such as those encoding cytochrome P450 (CYP). In this study, we investigated whether introduction of human hepatocyte nuclear factor 6 (HNF6) could modulate the expression of CYP3A4 and other CYP genes in iPSHCs as well as in HepG2 cells, a fetal liver cell line (HFL cells), and in hepatocytes. CYP3A4 mRNA could be detected in iPSHCs, but the expression level was very low compared with those in HepG2 cells and hepatocytes. However, the CYP3A4 mRNA levels markedly increased on introduction of HNF6 and reached one-tenth of those in hepatocytes. We also found that HNF6 introduction increased CYP3A4 gene transcription in HFL cells and HepG2 cells, which have features similar to those of fetal hepatocyte-like cells; however, it did not affect CYP3A4 mRNA expression in hepatocytes. These results suggest that HNF6 plays an important role in the gene regulation of CYP3A4 during development from the fetal period to the postnatal period.
    Drug Metabolism and Pharmacokinetics 06/2013; 28(3). DOI:10.2133/dmpk.DMPK-12-RG-132 · 2.57 Impact Factor
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    ABSTRACT: Distinctive response patterns of CYP3A4 and CYP3A7 to cobalt chloride (CoCl2) in human fetal liver (HFL) cells were observed and compared with those under hypoxic conditions. The expression levels of CYP3A4 and CYP3A7 mRNAs were decreased by CoCl2 and hypoxia, although significance could not be determined in HFL cells cultured under 3% O-2. The hypoxia-inducible factor-1 alpha (HIF-1 alpha) protein content in HFL cells was significantly increased by CoCl2 and 3% O-2. Transcriptional activities of CYP3A4 and CYP3A7 were not altered by 3% 02 when reporter plasmids containing the promoter region ranging up to about 10 kb and 12 kb upstream, respectively, were transfected into HFL cells, although the activity was significantly suppressed by CoCl2. These results suggested that the mechanisms controlling CYP3A gene expression of HIF-1 alpha chemical stabilizer in fetal hepatocytes might be different from those in adult hepatocytes, and that HIF-1 alpha is not directly involved in regulation of CYP3A4 or CYP3A7 expression.
    Drug Metabolism and Pharmacokinetics 08/2012; 27(4):398-404. DOI:10.2133/dmpk.DMPK-11-RG-074 · 2.57 Impact Factor
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    ABSTRACT: Human fetal liver (HFL) cells express major drug metabolic enzymes CYP3A4, CYP3A5 and CYP3A7. In the fetal hepatocytes, betamethasone and dexamethasone (DEX) markedly enhanced the expression levels of CYP3A4 and CYP3A7 mRNAs and slightly increased expression level of CYP3A5 mRNA. Interestingly, a high correlation between the CYP3A induction ability and the intensity of anti-inflammatory effect was observed. Human glucocorticoid receptor (GR)-small interfering RNA clearly attenuated the expression level of GR mRNA, and diminished the DEX-stimulated CYP3A4, CYP3A5 and CYP3A7 expression in HFL cells. These findings indicate that GR mediates the induction of CYP3A4 and CYP3A7 expression in human fetal hepatocytes as well as the CYP3A5.
    Drug Metabolism and Pharmacokinetics 05/2012; 27(6). DOI:10.2133/dmpk.DMPK-12-NT-018 · 2.57 Impact Factor
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    ABSTRACT: Distinctive response patterns of CYP3A4 and CYP3A7 to cobalt chloride (CoCl(2)) in human fetal liver (HFL) cells were observed and compared with those under hypoxic conditions. The expression levels of CYP3A4 and CYP3A7 mRNAs were decreased by CoCl(2) and hypoxia, although significance could not be determined in HFL cells cultured under 3% O(2). The hypoxia-inducible factor-1α (HIF-1α) protein content in HFL cells was significantly increased by CoCl(2) and 3% O(2). Transcriptional activities of CYP3A4 and CYP3A7 were not altered by 3% O(2) when reporter plasmids containing the promoter region ranging up to about -10 kb and -12 kb upstream, respectively, were transfected into HFL cells, although the activity was significantly suppressed by CoCl(2). These results suggested that the mechanisms controlling CYP3A gene expression of HIF-1α chemical stabilizer in fetal hepatocytes might be different from those in adult hepatocytes, and that HIF-1α is not directly involved in regulation of CYP3A4 and CYP3A7 expression.
    Drug Metabolism and Pharmacokinetics 01/2012; · 2.57 Impact Factor
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    ABSTRACT: Aryl hydrocarbon receptor (AhR) activators have been shown to induce members of the cytochrome P450 (P450) 1 family. Here we demonstrate that the AhR activators induce CYP3A4 through human pregnane X receptor (PXR). AhR activators, polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased CYP3A4 reporter activity and CYP3A4 mRNA expression in HepG2 cells. The CYP3A4 reporter activity was also increased by treatment with cigarette tar. The increased CYP3A4 reporter activity was clearly knocked down by the introduction of human PXR-small interfering RNA, but not by that of human AhR-small interfering RNA. The CYP3A4 reporter activity enhanced by overexpression of human PXR was further increased by treatment with PAHs and TCDD as well as by treatment with rifampicin. These results suggest that PAHs contained in cigarette smoke induce CYP3A4 in human liver.
    Drug Metabolism and Pharmacokinetics 11/2011; 27(2):200-6. DOI:10.2133/dmpk.DMPK-11-RG-094 · 2.57 Impact Factor
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    ABSTRACT: The mouse cholesterol sulfotransferase St2b2 contributes to epidermal differentiation by biosynthesizing cholesterol sulfate (CS) from cholesterol in the epidermis. 12-O-Tetradecanoylphorbol-13-acetate (TPA) causes epidermal hyperplasia, an abnormal increase in epidermal cell numbers resulting from aberrant cell differentiation and an increase in St2b2 protein levels. The mechanisms underlying enhanced St2b2 expression and the pathophysiologic significance of the increased expression are unclear, however. To verify whether increased St2b2 levels are necessary for TPA-induced epidermal hyperplasia, the effects of St2b2-specific small hairpin RNA (St2b2-shRNA) on hyperplasia were examined in mice. St2b2-shRNA clearly suppressed TPA-induced epidermal hyperplasia and the expression of a marker of epidermal differentiation, involucrin (INV). Interestingly, treating mouse epidermal cells with tumor necrosis factor-alpha (TNFα) increased St2b2 expression. Furthermore, treatment with TNFα-siRNA or anti-TNF receptor antibodies reduced the TPA-induced enhancement of St2b2 expression. Treatment with BAY 11-7082, a specific inhibitor of nuclear factor-kappa B (NF-κB), diminished TPA-induced St2b2 expression. These results suggested that enhancement of St2b2 expression by TPA treatment occurs mainly through the TNFα-NF-κB inflammatory signaling pathway, which in turn leads to increased CS concentrations in epidermal cells and hyperplasia.
    Biological & Pharmaceutical Bulletin 02/2011; 34(2):183-90. DOI:10.1248/bpb.34.183 · 1.83 Impact Factor
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    ABSTRACT: Growing evidence indicates that the innate immune system and oxidative stress caused by gut-derived endotoxins play a key role in alcoholic liver disease (ALD). Intracellular mechanisms associated with endotoxin-induced signaling play a crucial role in the initiation and progression of ALD. It is now widely accepted that activation of the innate immune system and increased release of pro-inflammatory cytokines and other mediators play an important role in the development of ALD. Accumulating evidence suggests that alcohol-mediated upregulation of CYP2E1 expression may initiate lipid peroxidation via reactive oxygen species. Non-alcoholic steatohepatitis (NASH) is a liver disease characterized by histopathological features similar to those observed in ALD, but in the absence of significant alcohol consumption. Initial efforts to clarify the mechanisms that promote the progression from steatosis to steatohepatitis somewhat artificially divided disease mechanisms into "first and second hits." This model considered the development of steatosis to be the "first hit," increasing the sensitivity of the liver to the putative "second hit," leading to hepatocyte injury, inflammation, and oxidative stress. We have emphasized the important role of gut-derived bacterial toxins, the innate immune system, and oxidative stress in the common pathogenic mechanism in ALD and NASH progression.
    Drug Metabolism and Pharmacokinetics 12/2010; 26(1):30-46. DOI:10.2133/dmpk.DMPK-10-RV-087 · 2.57 Impact Factor
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    ABSTRACT: Malondialdehyde-modified low-density lipoprotein (MDA-LDL) and oxidized LDL (Ox-LDL), which accelerate the pathogenesis of arteriosclerosis, are thought to be involved in parthenogenesis caused by smooth muscle cell proliferation. In this study, we investigated the suppression mechanism of polycyclic aromatic hydrocarbons (PAHs) on the growth of an MDA-LDL-induced human acute monocyte leukemia suspension cell line (THP-1 cells). We found that PAHs suppressed MDA-LDL-induced THP-1 cell growth. Cotreatment with benzo[a]pyrene (BaP) or 3-methylchoranthrene (3-MC) decreased MDA-LDL-induced THP-1 cell growth, whereas treatment with benzo[e]pyrene (BeP) or pyrene, which is not a ligand for the arylhydrocarbon receptor (AhR), did not decrease THP-1 cell growth. Our findings clearly demonstrated that THP-1 cell growth, which was suppressed by PAHs, was restored by the addition of alpha-naphtoflavone, which is a partial antagonist to AhR. Moreover, it was shown that cotreatment with MDA-LDL and BaP markedly induced the expression of human cytochrome P4501A1 (hCYP1A1) messenger ribonucleic acid (mRNA) and significantly induced the expressions of p53 and p21 mRNAs. In support of these findings, AhR small interfering RNA suppressed the induced level of p21 mRNA and by BaP and the overexpression of hCYP1A1 significantly induced levels of p21 mRNA. On the other hand, the uptake rate of [(14)C]BaP into cells was increased more significantly by cotreatment with MDA-LDL than by treatment with [(14)C]BaP alone. These results strongly suggest that the suppression of MDA-LDL-induced THP-1 cell growth is caused by the increased uptake of PAHs, which strongly activate the AhR signal pathway accompanying DNA damage.
    The Journal of Toxicological Sciences 01/2010; 35(2):137-47. DOI:10.2131/jts.35.137 · 1.29 Impact Factor
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    ABSTRACT: Human CYP1A1 and CYP1A2 genes are in a head-to-head orientation on chromosome 15 and are separated by a 23-kb intergenic space. To our knowledge, this is the first report on a stable cell line that contains the 23-kb full-length regulatory region and is able to simultaneously assess the transcriptional activation of CYP1A1 and CYP1A2 genes. The stable cell line that constitutively expresses the reporter activities was constructed by inserting the dual reporter plasmid containing the 23-kb region between the CYP1A1 and CYP1A2 genes into the chromosome. Transcriptional activation of the CYP1A1 and CYP1A2 genes was measured simultaneously using luciferase (Luc) and secreted alkaline phosphatase (SEAP) activities, respectively. To demonstrate the utility of the stable cell line, CYP1A1/1A2 induction by the majority of compounds previously identified as CYP1A1/1A2 inducers was measured. The results clearly show that all compounds caused induction of reporter activities. In addition to assessing transcriptional activation of the CYP1A1 and CYP1A2 genes by measuring reporter activities, we determined the intrinsic CYP1A1 and CYP1A2 mRNA levels by treating them with the same compounds. The results suggest that this stable cell line may be used to rapidly and accurately predict CYP1A1/1A2 induction.
    Drug Metabolism and Pharmacokinetics 01/2010; 25(2):180-9. DOI:10.2133/dmpk.25.180 · 2.57 Impact Factor
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    ABSTRACT: ABCB1 (P-glycoprotein) is an efflux transporter that limits the cellular uptake levels of various drugs in intestine, brain, and other tissues. The expression of human ABCB1 has recently been reported to be under the control of nuclear receptor NR1I subfamily members, pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3). Here, we have investigated the involvement of another NR1I member, vitamin D receptor (VDR, NR1I1), in ABCB1 expression. In the human colorectal adenocarcinoma cell line LS174T, which abundantly expresses VDR, both 1alpha,25-dihydroxyvitamin D(3) (1,25-VD3) and lithocholic acid (LCA) increased ABCB1 mRNA levels. Reporter gene assays in LS174T cells with constructs containing various lengths of the ABCB1 regulatory region revealed that the region containing multiple nuclear receptor binding motifs located at -7.8 kilobases [termed nuclear receptor-responsive module (NURREM)], to which PXR and CAR also bind, is essential for the VDR-mediated ABCB1 transactivation. Further reporter assays with constructs containing truncated NURREM and gel shift assays suggested simultaneous binding of multiple VDR/retinoid X receptor alpha heterodimers to NURREM. Furthermore, knockdown of VDR expression in LS174T cells blocked the LCA- and the 1,25-VD3-induced transcription of ABCB1 reporter genes. In human hepatoma HepG2 cells, in contrast with LS174T cells, 1,25-VD3 activated the ABCB1 transcription only in the presence of ectopically expressed VDR. These results suggest that the NR1I subfamily members regulate the ABCB1 expression sharing the binding sites within NURREM and that the physiologically produced LCA and 1,25-VD3 may modulate the ABCB1 expression in human intestines, possibly associated with interindividual variations of ABCB1 expression.
    Drug metabolism and disposition: the biological fate of chemicals 06/2009; 37(8):1604-10. DOI:10.1124/dmd.109.027219 · 3.25 Impact Factor
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    ABSTRACT: Pregnane X receptor (PXR) has been shown to form a heterodimer with retinoid X receptor alpha (RXRalpha) and to bind to the distal nuclear receptor-binding element 1 and an everted repeat separated by six nucleotides in the proximal promoter of the CYP3A4 gene. In the present study, a new rifampicin-responsive region, located at -7.6 kilobases upstream from the transcription initiation site, has been identified using reporter assays in HepG2 cells. This region contains a cluster of possible nuclear receptor-binding half-sites, AG(G/T)TCA-like sequence. Of these putative half-sites, we focused six half-sites and termed them alpha-eta half-sites. Introduction of a mutation into either an alpha or beta half-site of CYP3A4 reporter genes almost completely diminished the rifampicin-induced transcription. In electrophoretic mobility shift assays, PXR/RXRalpha heterodimer bound to the direct repeat separated by four nucleotides (DR4) formed with alpha and beta half-sites. HepG2-based transactivation assays with the reporter gene constructs with or without mutations in the PXR binding element(s) demonstrated that this DR4 motif is essential for the transcriptional activation not only by rifampicin but also by various human PXR activators. In addition, reporter assays performed in human hepatocytes and mice with adenoviruses expressing luciferase derived from various CYP3A4 reporter genes and that expressing human PXR supported the results of experiments in HepG2 cells. These results suggest the obligatory role of the newly identified direct repeat separated by four nucleotides-type PXR binding element of the CYP3A4 gene for xenobiotic induction of CYP3A4.
    Molecular pharmacology 03/2009; 75(3):677-84. DOI:10.1124/mol.108.050575 · 4.13 Impact Factor
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    ABSTRACT: We have reported that the protein-protein interaction between UDP-glucuronosyltransferase (UGT) 2B7 and cytochrome P450 3A4 (CYP3A4) alters UGT2B7 function. However, the domain(s) involved in the interaction are largely unknown. To address this issue, we examined in more detail the CYP3A4-UGT2B7 association by means of immunoprecipitation, overlay assay, and cross-linking involving 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Purified CYP3A4 or glutathione transferase (GST)-tagged CYP3A4 was cross-linked to UGT2B7 in solubilized baculosomes. The formation of the cross-linked complex was detected by immunoblotting using both antibodies against CYP3A4 and UGTs. Although the GST-tagged CYP3A4 containing the region ranging from Tyr25 to Ala503 was cross-linked to UGT2B7, the same did not occur when another construct containing Met145 to His267 was used. This observation was consistent with the result of the overlay assay indicating that CYP3A4 lacking the N-terminal hydrophobic segment retains the ability to associate with UGT2B7, whereas the Met145-to-His267 region loses this capacity. Although the Met145-to-His267 peptide was recognized by one anti-CYP3A4 antibody that has the ability to coimmunoprecipitate UGT2B7, it was not recognized by another antibody incapable of coimmunoprecipitating UGT2B7. The epitope of the latter antibody was mapped to the Leu331-to-Lys342 region, which is located on the J-helix of CYP3A4. Taken together, the results obtained suggest that 1) CYP3A4 and UGT2B7 are a pair of enzymes in proximity to each other and 2) either the Leu331-to-Lys342 domain or the surrounding region plays a role in the interaction with UGT2B7, whereas the hydrophobic Met145-to-His267 region does not contribute to this interaction.
    Molecular pharmacology 02/2009; 75(4):956-64. DOI:10.1124/mol.108.052001 · 4.13 Impact Factor
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    ABSTRACT: Human hepatocyte culture is widely used to predict human drug metabolism for new drug development. The limited supply and lot-to-lot (donor-to-donor) variations in enzymatic activity, however, hamper its applicability. In the present study, we explore a new cell system with adenovirus-mediated expression of cytochrome P450s (P450s) as an alternative for hepatocytes. In this system, P450 apoprotein levels and catalytic activity increased depending on the amounts of adenoviruses infected for the individual expression of CYP3A4 or CYP2C19 in HepG2 cells. Similar results were observed in the system with co-expression of CYP3A4 and CYP2C19. When HepG2 cells were infected with adednovirus for CYP3A4 and that for CYP2C19 simultaneously at a ratio of 10:1, the ratio of their apoprotein levels was similar to that observed in human hepatocytes and the metabolic profile of diazepam in the system was almost identical to that observed in hepatocyets. These results indicate that this adenovirus-mediated system makes it possible to reproducibly prepare cells expressing multiple P450s at a desired ratio, suggesting a possible use of this system in preclinical metabolic tests for a drug candidate(s), particularly to assess the influence of inter-individual variation in P450 activity.
    Drug Metabolism and Pharmacokinetics 02/2009; 24(3):209-17. DOI:10.2133/dmpk.24.209 · 2.57 Impact Factor
  • M Shimada · T Matsuda · A Sato · T Akase · T Matsubara · K Nagata · Y Yamazoe ·
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    ABSTRACT: 1. St2b2, a mouse cytosolic sulfotransferase, predominantly catalyses epidermal cholesterol sulfation. St2b2 was found in the basement layer by immunohistochemical analysis of normal mouse skin. The highest expression level was detected in epidermis from 3-day-old mice and then decreased before maturation. There was a good correlation between expression levels of skin St2b2 and a differentiation marker, involucrin. 2. To understand the role of St2b2 in epidermal cell differentiation, recombinant St2b2 was expressed in primary epidermal cells. The expression of St2b2 enhanced the involucrin expression with an increase of cholesterol sulfate. Furthermore, by down-regulation of the St2b2 gene expression, involucrin was decreased in dorsal skin of 1-3-day-old mice by 67% of the control. 3. These results strongly suggest a possibility that St2b2 expression plays a trigger of epidermal cell differentiation by controlling cholesterol sulfate level in the cells.
    Xenobiotica 01/2009; 38(12):1487-99. DOI:10.1080/00498250802488593 · 2.20 Impact Factor

Publication Stats

4k Citations
455.84 Total Impact Points


  • 2006-2014
    • Tohoku Pharmaceutical University
  • 1996-2011
    • Tohoku University
      • • Graduate School of Pharmaceutical Sciences
      • • Department of Life and Pharmaceutical Science
      Miyagi, Japan
  • 1981-2007
    • Kyushu University
      • • Graduate School of Pharmaceutical Sciences
      • • Faculty of Pharmaceutical Sciences
      Hukuoka, Fukuoka, Japan
  • 2003
    • Taisho Pharmaceutical
      Edo, Tōkyō, Japan
  • 2001
    • Sendai University
      Sendai, Kagoshima, Japan
  • 1998
    • National Institute of Public Health
      Saitama, Saitama, Japan
  • 1987-1996
    • Keio University
      • School of Medicine
      Edo, Tōkyō, Japan
  • 1986-1994
    • National Heart, Lung, and Blood Institute
      베서스다, Maryland, United States
  • 1987-1990
    • National Institutes of Health
      • Laboratory of Human Carcinogenesis
      베서스다, Maryland, United States