Tatsuhiko Kodama

The University of Tokyo, Tōkyō, Japan

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Publications (549)2776.59 Total impact

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    ABSTRACT: Selective PPARα modulators (SPPARMα) are under development for use as next-generation lipid lowering drugs. In the current study, to predict the pharmacological and toxicological effects of a novel SPPARMα K-877, comprehensive transcriptome analyses of K-877-treated primary human hepatocytes and mouse liver tissue were carried out. Total RNA was extracted from the K-877 treated primary human hepatocytes and mouse liver and adopted to the transcriptome analysis. Using a cluster analysis, commonly and species specifically regulated genes were identified. Also, the profile of genes regulated by K-877 and fenofibrate were compared to examine the influence of different SPPARMα on the liver gene expression. Consequently, a cell-based transactivation assay showed that K-877 activates PPARα with much greater potency and selectivity than fenofibric acid, the active metabolite of clinically used fenofibrate. K-877 upregulates the expression of several fatty acid β-oxidative genes in human hepatocytes and the mouse liver. Almost all genes up- or downregulated by K-877 treatment in the mouse liver were also regulated by fenofibrate treatment. In contrast, the K-877-regulated genes in the mouse liver were not affected by K-877 treatment in the Ppara-null mouse liver. Depending on the species, the peroxisomal biogenesis-related gene expression was robustly induced in the K-877-treated mouse liver, but not human hepatocytes, thus suggesting that the clinical dose of K-877 may not induce peroxisome proliferation or liver toxicity in humans. Notably, K-877 significantly induces the expression of clinically beneficial target genes (VLDLR, FGF21, ABCA1, MBL2, ENPEP) in human hepatocytes. These results indicate that changes in the gene expression induced by K-877 treatment are mainly mediated through PPARα activation. K-877 regulates the hepatic gene expression as a SPPARMα and thus may improve dyslipidemia as well as metabolic disorders, such as metabolic syndrome and type 2 diabetes, without untoward side effects.
    Journal of atherosclerosis and thrombosis 06/2015; DOI:10.5551/jat.28720 · 2.77 Impact Factor
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    ABSTRACT: Highly protonated histone-derived peptides impede a sufficient mass spectrometry (MS) -based epigenetic analysis since their relatively low m/z due to a high degree of proton addition to peptides would difficult to analyze the resulting complex MS/MS spectra. In order to reduce the degree of protonations, we have developed a new interface, the IVU, in which peptides are ionized under a vaporized organic solvent, and it has demonstrated that the doubly-charged histone-tail H2B peptide, PEPAKSAPAPKKGSKKAVTKAQKK (m/z 1238.243 , +2), has been detected by using the IVU interface and sequenced, which was not detectable before. Copyright © 2015. Published by Elsevier Inc.
    Analytical Biochemistry 06/2015; DOI:10.1016/j.ab.2015.06.002 · 2.31 Impact Factor
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    ABSTRACT: ROBO1 is a membrane protein that contributes to tumor metastasis and angiogenesis. We previously reported that 90Y-labeled anti-ROBO1 monoclonal antibody (90Y-anti-ROBO1 IgG) showed an antitumor effect against ROBO1-positive tumors. In this study, we performed a biodistribution study and radioimmunotherapy (RIT) against ROBO1-positive small cell lung cancer (SCLC) models. For the biodistribution study, 111In-labeled anti-ROBO1 monoclonal antibody (111In-anti-ROBO1 IgG) was injected into ROBO1-positive SCLC xenograft mice via the tail vein. To evaluate antitumor effects, an RIT study was performed, and SCLC xenograft mice were treated with 90Y-anti-ROBO1 IgG. Tumor volume and body weight were periodically measured throughout the experiments. The tumors and organs of mice were then collected, and a pathological analysis was carried out. As a result of the biodistribution study, we observed tumor uptake of 111In-anti-ROBO1 IgG. The liver, kidney, spleen, and lung showed comparably high accumulation of 111In-labeled anti-ROBO1. In the RIT study, 90Y-anti-ROBO1 IgG significantly reduced tumor volume compared with baseline. Pathological analyses of tumors revealed coagulation necrosis and fatal degeneration of tumor cells, significant reduction in the number of Ki-67-positive cells, and an increase in the number of apoptotic cells. A transient reduction of hematopoietic cells was observed in the spleen, sternum, and femur. These results suggest that RIT with 90Y-anti-ROBO1 IgG is a promising treatment for ROBO1-positive SCLC.
    PLoS ONE 05/2015; 10(5):e0125468. DOI:10.1371/journal.pone.0125468 · 3.53 Impact Factor
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    ABSTRACT: Lysophosphatidylcholine (LPC) and oxysterols which are major components in oxidized low-density lipoprotein (oxLDL) have been shown to possess an opposite effect on the expression of sterol regulatory element-binding protein-2 (SREBP-2) target genes in endothelial cells. In the present study, we aimed at elucidating the mechanisms of activation of SREBP-2 by LPC and evaluating the effects of LPC and 25-hydroxycholesterol (25-HC) on the release of inflammatory cytokines. Human umbilical vein endothelial cells (HUVEC) were treated with LPC or oxysterols including 25-HC. LPC activated SREBP-2 within 15 min, resulting in induction of expression of SREBP-2 target genes which were involved in intracellular cholesterol homeostasis. The rapid activation of SREBP-2 was caused by enhanced efflux of intracellular cholesterol, which was evaluated using (14)C-acetate. The LPC-induced activation of SREBP-2 was inhibited by addition of 25-HC. In contrast, both LPC and 25-HC increased release of interleukin-6 (IL-6) and IL-8, respectively and additively. In conclusion, LPC activated SREBP-2 via enhancement of cholesterol efflux, which was suppressed by 25-HC. The release of inflammatory cytokines such as IL-6 and IL-8 in endothelial cells was SREBP-2-independent. LPC and 25-HC may act competitively in cholesterol homeostasis but additively in inflammatory cytokine release. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
    Journal of Biochemistry 05/2015; DOI:10.1093/jb/mvv044 · 3.07 Impact Factor
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    ABSTRACT: Histone 3 lysine 9 (H3K9) demethylase JMJD1A regulates b-adrenergic-induced systemic metabolism and body weight control. Here we show that JMJD1A is phosphorylated at S265 by protein kinase A (PKA), and this is pivotal to activate the b1-adrenergic receptor gene (Adrb1) and downstream targets including Ucp1 in brown adipocytes (BATs). Phosphorylation of JMJD1A by PKA increases its interaction with the SWI/SNF nucleosome remodelling complex and DNA-bound PPARg. This complex confers b-adrenergic-induced rapid JMJD1A recruitment to target sites and facilitates long-range chromatin interactions and target gene activation. This rapid gene induction is dependent on S265 phosphorylation but not on demethylation activity. Our results show that JMJD1A has two important roles in regulating hormone-stimulated chromatin dynamics that modulate thermogenesis in BATs. In one role, JMJD1A is recruited to target sites and functions as a cAMP-responsive scaffold that facilitates long-range chromatin interactions, and in the second role, JMJD1A demethylates H3K9 di-methylation.
    Nature Communications 05/2015; 6. DOI:10.1038/ncomms8052 · 10.74 Impact Factor
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    ABSTRACT: Most gnathostomata craniofacial structures derive from pharyngeal arches (PAs), which are colonized by cranial neural crest cells (CNCCs). The anteroposterior and dorsoventral identities of CNCCs are defined by the combinatorial expression of Hox and Dlx genes. The mechanisms associating characteristic Hox/Dlx expression patterns with the topology and morphology of PAs derivatives are only partially known; a better knowledge of these processes might lead to new concepts on the origin of taxon-specific craniofacial morphologies and of certain craniofacial malformations. Here we show that ectopic expression of Hoxa2 in Hox-negative CNCCs results in distinct phenotypes in different CNCC subpopulations. Namely, while ectopic Hoxa2 expression is sufficient for the morphological and molecular transformation of the first PA (PA1) CNCC derivatives into the second PA (PA2)-like structures, this same genetic alteration does not provoke the transformation of derivatives of other CNCC subpopulations, but severely impairs their development. Ectopic Hoxa2 expression results in the transformation of the proximal Meckel's cartilage and of the malleus, two ventral PA1 CNCCs derivatives, into a supernumerary styloid process (SP), a PA2-derived mammalian-specific skeletal structure. These results, together with experiments to inactivate and ectopically activate the Edn1-Dlx5/6 pathway, indicate a dorsoventral PA2 (hyomandibular/ceratohyal) boundary passing through the middle of the SP. The present findings suggest context-dependent function of Hoxa2 in CNCC regional specification and morphogenesis, and provide novel insights into the evolution of taxa-specific patterning of PA-derived structures. Copyright © 2015. Published by Elsevier Inc.
    Developmental Biology 04/2015; 121(2). DOI:10.1016/j.ydbio.2015.04.007 · 3.64 Impact Factor
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    ABSTRACT: ROBO1, fibronectin type-III domain (Fn)-containing protein, is a novel immunotherapeutic target for hepatocellular carcinoma in humans. The crystal structure of the antigen-binding fragment (Fab) of B2212A, the monoclonal antibody against the third Fn domain (Fn3) of ROBO1, was determined in pursuit of antibody drug for hepatocellular carcinoma. This effort was conducted in the presence or absence of the antigen, with the chemical features being investigated by determining the affinity of the antibody using molecular dynamics and thermodynamics. The structural comparison of B2212A Fab between the complex and the free form revealed that the interfacial Tyr(L) 50 (superscripts L, H, and F stand for the residues in the light chain, heavy chain, and Fn3, respectively) played important roles in Fn3 recognition. That is, the aromatic ring of Tyr(L) 50 pivoted toward Phe(F) 68, forming a CH/π interaction and a new hydrogen bond with the carbonyl O atom of Phe(F) 68. Molecular dynamics simulations predicted that the Tyr(L) 50-Phe(F) 68 interaction almost entirely dominated Fab-Fn3 binding, and Ala-substitution of Tyr(L) 50 led to a reduced binding of the resultant complex. On the contrary, isothermal titration calorimetry experiments underscored that Ala-substitution of Tyr(L) 50 caused an increase of the binding enthalpy between B2212A and Fn3, but importantly, it induced an increase of the binding entropy, resulting in a suppression of loss in the Gibbs free energy in total. These results suggest that mutation analysis considering the binding entropy as well as the binding enthalpy will aid in the development of novel antibody drugs for hepatocellular carcinoma. This article is protected by copyright. All rights reserved. Copyright © 2014 The Protein Society.
    Protein Science 03/2015; 24(3). DOI:10.1002/pro.2619 · 2.86 Impact Factor
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    ABSTRACT: In this study, we propose a supercomputer-assisted drug design approach involving all-atom molecular dynamics (MD)-based binding free energy prediction after the traditional design/selection step. Because this prediction is more accurate than the empirical binding affinity scoring of the traditional approach, the compounds selected by the MD-based prediction should be better drug candidates. In this study, we discuss the applicability of the new approach using two examples. Although the MD-based binding free energy prediction has a huge computational cost, it is feasible with the latest 10 petaflop-scale computer. The supercomputer-assisted drug design approach also involves two important feedback procedures: The first feedback is generated from the MD-based binding free energy prediction step to the drug design step. While the experimental feedback usually provides binding affinities of tens of compounds at one time, the supercomputer allows us to simultaneously obtain the binding free energies of hundreds of compounds. Because the number of calculated binding free energies is sufficiently large, the compounds can be classified into different categories whose properties will aid in the design of the next generation of drug candidates. The second feedback, which occurs from the experiments to the MD simulations, is important to validate the simulation parameters. To demonstrate this, we compare the binding free energies calculated with various force fields to the experimental ones. The results indicate that the prediction will not be very successful, if we use an inaccurate force field. By improving/validating such simulation parameters, the next prediction can be made more accurate.
    CHEMICAL & PHARMACEUTICAL BULLETIN 03/2015; 63(3):147-55. DOI:10.1248/cpb.c14-00596 · 1.38 Impact Factor
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    ABSTRACT: We synthesized two new biotin analogues, biotin carbonate 5 and biotin carbamate 6. These molecules were designed to reversibly bind with streptavidin by replacing the hydrogen bond donor NH group(s) of biotin's cyclic urea moiety to oxygen. Biotin carbonate 5 was synthesized from L-arabinose (7), which furnishes the desired stereochemistry at the 3,4-cis-dihydroxy groups, in 11% overall yield (10 steps). Synthesis of biotin carbamate 6 was accomplished from L-cysteine-derived chiral aldehyde 33 in 11% overall yield (7 steps). Surface plasmon resonance analysis of water-soluble biotin carbonate analogue 46 and biotin carbamate analogue 47 revealed that KD values of these compounds for binding to streptavidin were 6.7x10-6 M (46) and 1.7x10-10 M (47), respectively. These values were remarkably greater than that of biotin (KD = 10-15 M), and thus indicate the importance of the nitrogen atoms of biotin's cyclic urea motif for the strong binding between biotin and streptavidin. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    Chemistry - An Asian Journal 02/2015; 10(4). DOI:10.1002/asia.201500120 · 3.94 Impact Factor
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    ABSTRACT: For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenicity, termed LISA-314, was produced previously as a drug delivery tool. However, endogenous biotins with high affinity (Kd < 10(-10) M) for the binding pocket of LISA-314 prevents access of exogenous biotin-labeled anticancer drugs. In the present study, we improve the binding pocket of LISA-314 to abolish its affinity for endogenous biotin species, therefore ensuring that the newly designed LISA-314 binds only artificial biotin analogue. The replacement of three amino acid residues was performed in two steps to develop a mutant termed V212, which selectively binds to 6-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoic acid (IMNtail). Surface plasmon resonance results showed that V212 has a Kd value of 5.9 × 10(-7) M towards IMNtail, but no binding affinity for endogenous biotin species. This V212/IMNtail system will be useful as a novel delivery tool for anticancer therapy. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
    Journal of Biochemistry 02/2015; 157(6). DOI:10.1093/jb/mvv004 · 3.07 Impact Factor
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    ABSTRACT: The streptavidin/biotin interaction has been widely used as a useful tool in research fields. For application to a pre-targeting system, we previously developed a streptavidin mutant that binds to an iminobiotin analog while abolishing affinity for natural biocytin. Here, we design a bivalent iminobiotin analog that shows 1000-fold higher affinity than before, and determine its crystal structure complexed with the mutant protein.
    Bioscience Biotechnology and Biochemistry 01/2015; 79(4):1-3. DOI:10.1080/09168451.2014.991692 · 1.21 Impact Factor
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    ABSTRACT: Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms including ubiquitination of H2A and chromatin compaction. However, whether it regulates the step-wise progression of adipogenesis is unknown. Here we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10 but it does require the F-box and leucine rich repeat (LRRs) domains which we show recruit a non-canonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1 and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10 mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell-cycle and adipogenic genes Cdk1, Uhrf1, Pparg1 and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis through targeting a non-canonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 12/2014; 290(7). DOI:10.1074/jbc.M114.626929 · 4.60 Impact Factor
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    ABSTRACT: The Tokyo Medical University Hospital in Japan and the Lund University hospital in Sweden have recently initiated a research program with the objective to impact on patient treatment by clinical disease stage characterization (phenotyping), utilizing proteomics sequencing platforms. By sharing clinical experiences, patient treatment principles, and biobank strategies, our respective clinical teams in Japan and Sweden will aid in the development of predictive and drug related protein biomarkers. Data from joint lung cancer studies are presented where protein expression from Neuro- Endocrine lung cancer (LCNEC) phenotype patients can be separated from Small cell- (SCLC) and Large Cell lung cancer (LCC) patients by deep sequencing and spectral counting analysis. LCNEC, a subtype of large cell carcinoma (LCC), is characterized by neuroendocrine differentiation that small cell lung carcinoma (SCLC) shares. Pre-therapeutic histological distinction between LCNEC and SCLC has so far been problematic, leading to adverse clinical outcome. An establishment of protein targets characteristic of LCNEC is quite helpful for decision of optimal therapeutic strategy by diagnosing individual patients. Proteoform annotation and clinical biobanking is part of the HUPO initiative (http://www.hupo.org) within chromosome 10 and chromosome 19 consortia.
    12/2014; 3(1):61. DOI:10.1186/s40169-014-0038-x
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    ABSTRACT: We previously created a low-immunogenic core streptavidin mutant No. 314 (LISA-314) by replacing six amino-acid residues for use as a delivery tool for an antibody multistep pre-targeting process (Yumura et al., Protein science, 22, 213-221, 2013). Here, we performed high-resolution X-ray structural analyses of LISA-314 and wild-type streptavidin to investigate the effect of substitutions on the protein function and the three-dimensional structure. LISA-314 forms a tetramer in the same manner as wild-type streptavidin. The binding mode of d-biotin in LISA-314 is also completely identical to that in wild-type streptavidin, and conformational changes were observed mostly at the side chains of substituted sites. Any large conformational changes corresponding to the reduction of B factors around the substituted sites were not observed. These results demonstrated the LISA-314 acquired low immunogenicity without losing structural properties of original wild-type streptavidin. Copyright © 2014. Published by Elsevier B.V.
    Journal of Bioscience and Bioengineering 11/2014; 119(6). DOI:10.1016/j.jbiosc.2014.10.025 · 1.79 Impact Factor
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    ABSTRACT: Pentraxins belong to the superfamily of conserved proteins that are characterized by a cyclic multimeric structure. Pentraxin 3 (PTX3) is a long pentraxin which can be produced by different cell types upon exposure to various inflammatory signals. Inside the neutrophil PTX3 is stored in form of granules localized in the cytoplasm. Neutrophilic granules are divided into three types: azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. PTX3 has been considered to be localized in specific (secondary) granules. Immunofluorescent analyses using confocal laser microscopic examination were performed to clarify the localization of all three groups of granules within the cytoplasm of the mature neutrophils and neutrophils stimulated with IL-8. Furthermore, PTX3 was localized in primary granules of promyelocyte cell line HL-60. As a result, we suggest that PTX3 is localized not only in specific granules, but is also partly expressed in primary and tertiary granules. After the stimulation with IL-8, irregular reticular structures called neutrophil extracellular traps (NETs) were formed, three types of granules were trapped by NETs and PTX3 showed partial colocalization with these granular components. PTX3 localized in all three types of granules in neutrophils may play important roles in host defense. Copyright © 2014. Published by Elsevier Inc.
    Experimental and Molecular Pathology 11/2014; 98(1). DOI:10.1016/j.yexmp.2014.11.009 · 2.88 Impact Factor
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    ABSTRACT: VEGF is a key regulator of endothelial cell migration, proliferation and inflammation, which leads to activation of several signaling cascades including the calcineurin-NFAT pathway. NFAT is important for not only immune responses but also cardiovascular development and pathogenesis of Down syndrome. We recently showed that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis by using Down syndrome model mice and clinical patient samples. However, the connection between genome-wide views of the NFAT mediated gene regulation, and the downstream gene function in endothelium has not been extensively studied. Here, we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells, and elucidate functional consequences of the VEGF-NFATc1 mediated phenotypic changes. Comparison of NFATc1 ChIP-sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. SiRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyper-activation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1-binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target-genes with multiple functions including regulation of cell migration, tube formation, and barrier formation in endothelial cells.
    Journal of Biological Chemistry 08/2014; 289(42). DOI:10.1074/jbc.M114.555235 · 4.60 Impact Factor
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    ABSTRACT: Macrophages are important for maintaining intestinal immune homeostasis. Here, we show that PPARβ/δ (peroxisome proliferator-activated receptor β/δ) directly regulates CD300a in macrophages that express the immunoreceptor tyrosine based-inhibitory motif (ITIM)-containing receptor. In mice lacking CD300a, high-fat diet (HFD) causes chronic intestinal inflammation with low numbers of intestinal lymph capillaries and dramatically expanded mesenteric lymph nodes. As a result, these mice exhibit triglyceride malabsorption and reduced body weight gain on HFD. Peritoneal macrophages from Cd300a-/- mice on HFD are classically M1 activated. Activation of toll-like receptor 4 (TLR4)/MyD88 signaling by lipopolysaccharide (LPS) results in prolonged IL-6 secretion in Cd300a-/- macrophages. Bone marrow transplantation confirmed that the phenotype originates from CD300a deficiency in leucocytes. These results identify CD300a-mediated inhibitory signaling in macrophages as a critical regulator of intestinal immune homeostasis.
    Scientific Reports 06/2014; 4:5412. DOI:10.1038/srep05412 · 5.58 Impact Factor
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    ABSTRACT: BackgroundROBO1 is a membrane protein that functions in axon guidance. ROBO1 contributes to tumour metastasis and angiogenesis and may have potential as a target protein of immunotherapy because ROBO1 is specifically expressed at high levels in hepatocellular carcinoma. In this study, we examined biodistribution and radioimmunotherapy (RIT) using a radioisotope-labelled anti-ROBO1 monoclonal antibody (MAb) against hepatocellular carcinoma models.MethodsROBO1-positive HepG2 human hepatocellular carcinoma xenograft nude mice were used in this study. We conjugated anti-ROBO1 MAb with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and the conjugates were labelled with 111In and 90Y. To study biodistribution, the 111In-DOTA-anti-ROBO1 MAb was injected into HepG2 xenograft mice via the tail vein. To evaluate any antitumour effect, a RIT study was performed, and the 90Y-DOTA-anti-ROBO1 MAb was injected via the tail vein. Tumour volume, mouse weight, and blood cell count were periodically measured throughout the experiments. The tumours and organs of mice were collected, and a histopathological analysis was carried out.ResultsThe tumour uptake of 111In-anti-ROBO1 MAb in HepG2 xenograft mice was 15.0% ± 0.69% injected dose per gram at 48 h after injection.Immunotherapy with cold-anti-ROBO1 MAb (70 μg) did not cause a significant antitumour effect. RIT with 6.7 MBq of 90Y-anti-ROBO1 MAb caused significant tumour growth suppression. Transient body weight loss and bone-marrow suppression were observed. Histopathological analyses of tumours revealed the fatal degeneration of tumour cells, significant reduction of the Ki-67 index, and an increase of the apoptosis index. Normal organs showed no significant injury, but a transient reduction of hematopoietic cells was observed in the spleen and in the sternal bone marrow.ConclusionsThese results suggest that RIT with 90Y-anti-ROBO1 MAb is a promising treatment for ROBO1-positive hepatocellular carcinoma.
    06/2014; 4:29. DOI:10.1186/s13550-014-0029-3
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    ABSTRACT: Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.
    PLoS ONE 05/2014; 9(5):e96005. DOI:10.1371/journal.pone.0096005 · 3.53 Impact Factor
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    ABSTRACT: Synergistic transcriptional activation by different stimuli has been reported along with a diverse array of mechanisms, but the full scope of these mechanisms has yet to be elucidated. We present a detailed investigation of hypoxia-inducible factor (HIF) 1 dependent gene expression in endothelial cells which suggests the importance of crosstalk between the peroxisome proliferator-activated receptor (PPAR) beta/delta and HIF signaling axes. A migration assay shows a synergistic interaction between these two stimuli, and we identify angiopoietin-like 4 (ANGPTL4) as a common target gene by using a combination of microarray and ChIP-seq analysis. We profile changes of histone marks at enhancers under hypoxia, PPARbeta/delta agonist and dual stimulations and these suggest that the spatial proximity of two response elements is the principal cause of the synergistic transcription induction. A newly developed quantitative chromosome conformation capture assay shows the quantitative change of the frequency of proximity of the two response elements. To the best of our knowledge, this is the first report that two different transcription factors cooperate in transcriptional regulation in a synergistic fashion through conformational change of their common target genes.
    Genome biology 04/2014; 15(4):R63. DOI:10.1186/gb-2014-15-4-r63 · 10.47 Impact Factor

Publication Stats

22k Citations
2,776.59 Total Impact Points


  • 1982–2015
    • The University of Tokyo
      • • Research Center for Advanced Science and Technology
      • • Division of Internal Medicine
      Tōkyō, Japan
  • 2005–2011
    • Beth Israel Deaconess Medical Center
      • • Division of Molecular and Vascular Medicine
      • • Center for Vascular Biology Research
      Boston, Massachusetts, United States
  • 2005–2010
    • Tokyo Medical and Dental University
      • Department of Cell Signaling
      Edo, Tōkyō, Japan
  • 2009
    • National Institute of Advanced Industrial Science and Technology
      Tsukuba, Ibaraki, Japan
    • Showa General Hospital
      Edo, Tōkyō, Japan
  • 2003–2007
    • Juntendo University
      Edo, Tōkyō, Japan
    • Harvard University
      Cambridge, Massachusetts, United States
  • 2001–2007
    • Niigata University
      • Division of Cellular and Molecular Pathology
      Niahi-niigata, Niigata, Japan
  • 2006
    • Yokohama City University
      • Department of Gastroenterology
      Yokohama, Kanagawa, Japan
  • 2003–2005
    • University of Tsukuba
      • Institute of Clinical Medicine
      Tsukuba, Ibaraki-ken, Japan
  • 2004
    • Kyorin University
      Edo, Tōkyō, Japan
    • Saitama University
      Saitama, Saitama, Japan
  • 2000–2004
    • National Hospital Organization Kyushu Cancer Center
      Hukuoka, Fukuoka, Japan
  • 1996–2002
    • University of Oxford
      • Sir William Dunn School of Pathology
      Oxford, ENG, United Kingdom
    • University of Kuopio
      • Department of Medicine
      Kuopio, Northern Savo, Finland
  • 1981–2002
    • National Cancer Center, Japan
      • • Center for Cancer Control and Information Services
      • • Endoscopy Division
      Edo, Tōkyō, Japan
  • 1999
    • The University of Tokushima
      Tokusima, Tokushima, Japan
  • 1995–1999
    • Chiba-East National Hospital
      Tiba, Chiba, Japan
    • Jichi Medical University
      Totigi, Tochigi, Japan
  • 1998
    • Leiden University
      • Leiden Amsterdam Center for Drug Research
      Leiden, South Holland, Netherlands
    • Dartmouth College
      • Department of Biochemistry
      Hanover, New Hampshire, United States
  • 1992–1998
    • Kumamoto University
      • • Department of Medical Biochemistry
      • • Department of Metabolic Medicine
      Kumamoto, Kumamoto, Japan
  • 1997
    • National Cancer Research Institute
      Londinium, England, United Kingdom
  • 1994–1995
    • National Institute of Health and Nutrition
      Edo, Tōkyō, Japan
  • 1990
    • Higashi-Matsudo Municipal Hospital
      Matsudo, Chiba, Japan