研究者詳細 - 大塚　基之

Differential cellular gene expression induced by hepatitis B and C viruses 査読

M Otsuka, H Aizaki, N Kato, T Suzuki, T Miyamura, M Omata, N Seki

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 300 ( 2 ) 443 - 447 2003年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Hepatitis B virus (HBV) is a hepatotropic virus that causes acute and chronic hepatocellular injury and hepatocellular carcinoma. To clarify how HBV proteins regulate host cellular gene expression, we used our in-house cDNA microarray and HepG2.2.15 cells, which are derived from HepG2 cells and produce all HBV proteins. Of 2304 genes investigated, several genes were differentially expressed in HepG2.2.15 cells compared with HepG2 cells. These genes included insulin-like growth factor II and alpha-fetoprotein, consistent with previous reports. Furthermore. we previously performed similar microarray analyses to clarify the effects of hepatitis C virus (HCV) proteins on host cells, using a HepG2-derivative cell line, which produces all HCV proteins. Using these two microarray results, we compared the differences in cellular gene expression induced by HBV and HCV proteins. The expression of the majority of genes investigated differed only slightly between HBV and HCV protein-producing cells. However, HBV and HCV proteins clearly regulated several genes in a reciprocal manner. Combined, these microarray results shed new light on the effects of HBV proteins on cellular gene expression and on the differences in the pathogenic activities of these two hepatitis viruses. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)02861-9

Signals induced by HCV proteins 査読

M Otsuka, N Kato, H Taniguchi, H Yoshida, Y Shiratori, M Omata

HCV/OXIDATIVE STRESS AND LIVER DISEASE 32 - 47 2003年

記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）出版者・発行元：SPRINGER-VERLAG TOKYO

Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. To explore the influence of HCV infection on hepatocytes, the effects of HCV proteins on intracellular signal transduction pathways, especially those related to apoptosis, fibrosis, and cell growth, were investigated. The effects of nine HCV proteins (core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) on the CRE-, SRE-, NFB-, AP-1-, SRF-, ISRE-, HSE-, GRE-, and p53-associated pathways were investigated by use of a reporter assay. The effects of core protein on apoptosis were examined by DNA laddering and Western blotting after induction of apoptosis by Fas stimulation. The possible mechanisms of anti-apoptotic effects of core protein were investigated by RNase protection assay and a reporter assay. The effects of HCV proteins on TGF beta production were determined by a reporter assay. Among seven HCV proteins investigated, core protein had the strongest influence on intracellular signaling, especially for the SRE-, AP-1-, NFkappaB-, and p53-associated pathways. Core protein promoted Bcl-x(L) expression through the mitogen-activated protein kinase (MAPK) pathway and inhibited apoptosis. Among HCV proteins, only core protein caused TGF beta promoter activity through the MAPK pathway. From these results, core protein was considered to regulate cell growth exquisitely in hepatocytes, inhibit apoptosis, and promote liver fibrosis directly without any inflammation or the mediation of any other cells. These functions may reflect the direct action of HCV proteins on the intracellular signal transduction pathways related to chronic hepatitis pathogenesis.

Otsuka M, Kato N, Yoshida H, Yoshida H, Shiratori Y, Omata M

Uirusu 52 ( 2 ) 295 - 300 2002年12月

Expression profiling of liver cell lines expressing entire or parts of hepatitis C virus open reading frame 査読

H Aizaki, T Harada, M Otsuka, N Seki, M Matsuda, YW Li, H Kawakami, Y Matsuura, T Miyamura, T Suzuki

HEPATOLOGY 36 ( 6 ) 1431 - 1438 2002年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：W B SAUNDERS CO

Although hepatitis C virus (HCV) is a causative agent of liver diseases, its mechanism of pathogenesis is still unclear, mainly because of the lack of adequate cell culture systems to support HCV infection and replication. In this report, we describe development and characterization of human hepatoma cell lines constitutively expressing entire (Hep394) or parts (Hep352, Hep3294) of the HCV open reading frame (ORF). The viral and cellular proteolytic machinery involved in the viral precursor processing was consistently functional, and processed HCV proteins were synthesized in these established cell lines. By using a cDNA microarray analysis coupled with semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), we identified 12 genes up-regulated and 4 genes down-regulated in Hep394 cells. With regard to genes related to cell growth regulation, we found up-regulation of forkhead transcription factor FREAC-1, poly (A) binding protein PABP2,, and Ras suppressor Rsu-1. Another category of changes in gene expression includes MHC antigens, which play an important role in the T-cell-mediated immune reaction in the liver. In conclusion, functional genomic approaches comparing expression among the different cell lines expressing parts of the HCV genome may promote our understanding of the molecular basis of pathogenicity of HCV infection.

DOI： 10.1053/jhep.2002.36937

Identification of candidate genes for Sjögren's syndrome using MRL/lpr mouse model of Sjögren's syndrome and cDNA microarray analysis. 査読

Azuma T, Takei M, Yoshikawa T, Nagasugi Y, Kato M, Otsuka M, Shiraiwa H, Sugano S, Mitamura K, Sawada S, Masuho Y, Seki N

Immunology letters 81 ( 3 ) 171 - 176 2002年5月

Hepatitis C virus core protein inhibits apoptosis via enhanced Bcl-x(L) expression 査読

M Otsuka, N Kato, H Taniguchi, H Yoshida, T Goto, Y Shiratori, M Omata

VIROLOGY 296 ( 1 ) 84 - 93 2002年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Previous studies indicated that hepatitis C virus core protein influences cellular apoptosis. However, the precise mechanisms of the effects are not fully understood. Therefore, in this study, we examined the mechanisms of the effects on cell apoptosis by core protein, using transiently transfected and magnetically collected core-producing HepG2 cells. First, to elucidate the target site of core protein in the apoptotic pathway, we examined the activation of caspases after anti-Fas antibody stimulation. Core protein inhibited the apoptotic cascade downstream from caspase 8 and upstream from caspase 3. Next, to clarify more direct mechanisms of this effect, mRNA levels of several bcl-2-related genes were examined. An RNase protection assay showed that the mRNA of bcl-xl increased in the core-producing cells. We showed that this increase was mediated by the enhancement of bcl-x promoter activity by core protein through an extracellular-regulated kinase pathway. These results suggest that core protein inhibits apoptosis at the mitochondria level through augmentation of Bcl-x expression, resulting in an inhibition of caspase 3 activation. (C) 2002 Elsevier Science (USA).

DOI： 10.1006/viro.2002.1371

The evaluation of putative virulence factors of Helicobacter pylori for gastroduodenal disease by use of a short-term Mongolian gerbil infection model 査読

M Akanuma, S Maeda, K Ogura, Y Mitsuno, Y Hirata, T Ikenoue, M Otsuka, T Watanabe, Y Yamaji, H Yoshida, T Kawabe, Y Shiratori, M Omata

JOURNAL OF INFECTIOUS DISEASES 185 ( 3 ) 341 - 347 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：UNIV CHICAGO PRESS

Few virulence determinants of Helicobacter pylori have been tested in vivo. We conducted this study to establish an animal model for their screening. Six-week-old male Mongolian gerbils were inoculated with wild-type H. pylori (TN2) or its isogenic mutant with deletion of cagE (TN2DeltacagE), total cag pathogenicity island (TN2Deltacag PAI), HP0499 (TN2DeltaHP499), or HP0638 (TN2DeltaHP638) (n = 5 each). The animals were killed 3 weeks later, and the density of bacteria and the degree of inflammation in the stomach were compared. Infection was established in all animals except those inoculated with TN2DeltaHP638. TN2 and TN2DeltaHP499, but not TN2DcagE and TN2Deltacag PAI, induced intense inflammation, although the densities of bacteria were similar. The Mongolian gerbil model was useful for the screening of virulence determinants in vivo, which confirmed the importance of cag PAI while questioning that of HP0499.

DOI： 10.1086/338772

Identification of genes associated with sensitivity to 5-fluorouracil and cisplatin in hepatoma cells 査読

Y Hoshida, M Moriyama, M Otsuka, N Kato, T Goto, H Taniguchi, Y Shiratori, N Seki, M Omata

JOURNAL OF GASTROENTEROLOGY 37 92 - 95 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER-VERLAG TOKYO

Background. The prognosis of patients with advanced hepatoma is grim. Although chemotherapy is adapted to such patients, the efficacy is low and the outcome cannot be predicted before therapy. In this study, we aimed to identify genes associated with sensitivity to 5-fluorouracil and cisplatin, drugs widely used in treatment, using gene expression profiles. Methods: Gene expression was evaluated in eight human hepatoma cell lines using an in-house cDNA microarray including 2300 known genes. The 50% growth inhibitory concentrations (Gl(50)) of 5-fluorouracil and cisplatin were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and designated as chemosensitivity. Genes with expression ratios associated with Gl(50) were selected using the permutation test. Results: For 5-fluorouracil and cisplatin, 21 and 40 genes, respectively, were selected. From among the genes associated with 5-fluorouracil and cisplatin, several encoding metabolic enzymes were selected. In addition, several genes involved in the cell cycle and transcription were identified. Conclusions: We identified genes that may be associated with sensitivity to 5-fluorouracil and cisplatin. A list of these genes may be useful to elucidate how these drugs work on human hepatoma.

Induction of proliferation-related signals by hepatitis C virus 査読

N Kato, H Yoshida, M Otsuka, Y Shiratori, M Omata

GROWTH, PROLIFERATION, AND APOPTOSIS IN HEPATOCYTES 81 - 98 2002年

記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）出版者・発行元：SPRINGER-VERLAG TOKYO

Hepatitis C virus (HCV) causes persistent infection, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. To explore the influence of HCV infection on hepatocytes, the effects of HCV proteins on intracellular signal transduction pathways were investigated. The effects of seven HCV proteins (core, nonstructural [NS]2, NS3, NS4A, NS4B, NS5A, and NS5B) on cyclic AMP response element (CRE)-, serum response element (SRE)-, nuclear factor (NF)-kappaB-, activator protein (AP)-1-, serum response factor (SRF)-, and p53-associated pathways were investigated by use of a reporter assay. The activation of signals by HCV proteins was examined using a reporter plasmid with an interleukin (IL)-8 or p21(waf1) promoter. The possible mechanisms by which HCV proteins activate these pathways were investigated. Among the seven HCV proteins investigated, core protein had the strongest influence on intracellular signaling, especially SRE-, AP-1-, NF-kappaB-, and p53-associated pathways. Core protein activated IL-8 promoter through NF-kappaB and AP-1 and activated p21 promoter having a p53-binding site. Core protein activated the NF-kappaB pathway mainly through IKKbeta and tumor necrosis factor receptor-associated factor 2/6 and augmented p53 function by increasing both p53-DNA binding affinity and transcriptional ability itself. Direct interaction between core protein and the C-terminus of p53 was detected. In addition, the interaction between core protein and human TBP-associated factor 1128, a component of the transcriptional factor complex, was also demonstrated. Core protein may directly promote cell proliferation and induce an inflammatory reaction by activating SRE, AP-1-, and NF-kappaB-associated pathways. On the other hand, core protein could enhance p53 function. These opposing functions may result in exquisitely balancing the proliferation of hepatocytes infected with HCV.

Differential expression of the L-plastin gene in human colorectal cancer progression and metastasis 査読

M Otsuka, M Kato, T Yoshikawa, H Chen, EJ Brown, Y Masuho, M Omata, N Seki

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 289 ( 4 ) 876 - 881 2001年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

To identify molecular alterations in the progression of colorectal carcinoma, we analyzed gene expression profiles of colon cancer cell lines derived from primary and metastatic tumors from a single patient. Of 2280 cDNAs investigated using our in-house microarray, the expression of 6 genes (tumor-associated antigen L6, L-plastin, the human homologue of yeast ribosomal protein S28, the B-cell translocation gene, mitochondrial aspartate-aminotransferase, and HLA-A) increased, while that of 2 genes (keratin 5 and phosphoglucomutase) decreased in metastatic-tumor-derived cells compared with primary-tumor-derived cells. Of these genes, we assessed the L-plastin gene, an actin-bundling protein, at the protein level using a tissue microarray consisting of 58 clinically stratified colorectal cancer specimens. Consistent with our microarray results, the expression of L-plastin was significantly correlated with the progression of cancer staging. Therefore, our results suggest that the L-plastin gene is a potential metastatic marker. In addition, combining cDNA microarrays and tissue arrays, as shown here, is thought to facilitate the rapid characterization of candidate biomarkers. (C) 2001 Elsevier Science.

DOI： 10.1006/bbrc.2001.6047

cDNA microarray analysis of Helicobacter pylori-mediated alteration of gene expression in gastric cancer cells 査読

S Maeda, M Otsuka, Y Hirata, Y Mitsuno, H Yoshida, Y Shiratori, Y Masuho, M Muramatsu, N Seki, M Omata

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 284 ( 2 ) 443 - 449 2001年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC

Helicobacter pylori infection stimulates several intracellular signaling pathways and is accompanied by increased gene expression in gastric epithelial cells. High-density cDNA microarray was used to characterize the mRNA expression profile of genes in human gastric cancer cells (MKN45, AGS) cocultured with H. pylori. Coculture with cag pathogenicity island (PAI)-positive H. pylori (wild-type) significantly upregulated mRNA expression in 8 of 2304 genes tested. In 6 (interleukin-8, I kappaB alpha, A20, ERF-1, keratin K7, glutathione peroxidase) of the 8 genes, up-regulation was confirmed by RT-PCR. In coculture with isogenic cagE-negative mutant (Delta cagE), which encodes a type TV secretion system with other genes in the cag PAI, no significant up-regulation was found. We further analyzed the role of A20. Transfection of expression vector encoding A20 resulted in an inhibition of H. pylori-mediated NF-KB activation, indicating that H, pylori-mediated A20 expression could be a negative regulator of NF-KB activation. Taken together, these results indicate the importance of microarray technology as a tool for analyzing the complex interplay between H. pylori and the host. (C) 2001 Academic Press.

DOI： 10.1006/bbrc.2001.5006

Hepatitis C virus core protein activates nuclear factor kappa B-dependent signaling through tumor necrosis factor receptor-associated factor 査読

H Yoshida, N Kato, Y Shiratori, M Otsuka, S Maeda, J Kato, M Omata

JOURNAL OF BIOLOGICAL CHEMISTRY 276 ( 19 ) 16399 - 16405 2001年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Hepatitis C virus (HCV) core protein, a viral nucleocapsid, has been shown to affect various intracellular events including the nuclear factor kappaB (NF-kappaB) signaling supposedly associated with inflammatory response, cell proliferation, and apoptosis. In order to elucidate the effect of HCV core protein on the NF-kappaB signaling in HeLa and HepG2 cells, a reporter assay was utilized. HCV core protein significantly activated NF-kappaB signaling in a dose-dependent manner not only in HeLa and HepG2 cells transiently transfected with core protein expression plasmid, but also in HeLa cells induced to express core protein under the control of doxycycline. HCV core protein increased the DNA binding affinity of NF-kappaB in the electrophoretic mobility shift assay. Acetyl salicylic acid, an IKK beta -specific inhibitor, and dominant negative form of IKK beta significantly blocked NF-kappaB activation by HCV core protein, suggesting HCV core protein activates the NF-kappaB pathway mainly through IKK beta. Moreover, the dominant negative forms of TRAF2/6 significantly blocked activation of the pathway by HCV core protein, suggesting HCV core protein mimics proinflammatory cytokine activation of the NF-kappaB pathway through TRAF2/6. In fact, HCV core protein activated interleukin-lp promoter mainly through NF-kappaB pathway. Therefore, this function of HCV core protein may play an important role in the inflammatory reaction induced by this hepatotropic virus.

Improvement of Chemosensitivity Prediction by Transcriptional Profiling in Hepatoma Cells

Hoshida Yujin, Moriyama Masaru, Otsuka Motoyuki, Kato Naoya, Shiratori Yasushi, Seki Naohiko, Omata Masao

Genome Informatics 12 257 - 258 2001年

Large isoform of hepatitis delta antigen activates serum response factor-associated transcription 査読

T Goto, N Kato, SK Ono-Nita, H Yoshida, M Otsuka, Y Shiratori, M Omata

JOURNAL OF BIOLOGICAL CHEMISTRY 275 ( 48 ) 37311 - 37316 2000年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Hepatitis delta virus infection sometimes causes severe and fulminant hepatitis as a coinfection or superinfection along with the hepatitis B virus. To elucidate the underlying mechanism of injury caused by hepatitis delta virus, we examined whether two isoforms of the hepatitis delta antigen (HDAg) had any effect on five well defined intracellular signal transduction pathways: serum response factor (SRF)-, serum response element (SRE)-, nuclear factor kappaB-, activator protein 1-, and cyclic AMP response element-dependent pathways. Reporter assays revealed that large HDAg (LIDDAg) activated the SRF- and SRE-dependent pathways. In contrast, small HDAg (LHDAg) did not activate any of five pathways. LHDAg enhanced the transcriptional ability of SRF without changing its DNA binding affinity in an electrophoretic mobility shift assay. In addition, LHDAg activated a rat SM22 alpha promoter containing SRF binding site and a human c-fos promoter containing SRE. In conclusion, LHDAg, but not SHDAg, enhances SRF-associated transcriptions. Despite structural similarities be tween the two HDAgs, there are significant differences in their effects on intracellular signal transduction pathways. These results may provide clues that will aid in the clarification of functional differences between LHDAg and SHDAg and the pathogenesis of delta hepatitis.

Hepatitis C virus core protein enhances p53 function through augmentation of DNA binding affinity and transcriptional ability 査読

Motoyuki Otsuka, Naoya Kato, Keng-Hsin Lan, Hideo Yoshida, Jun Kato, Tadashi Goto, Yasushi Shiratori, Masao Omata

Journal of Biological Chemistry 275 ( 44 ) 34122 - 34130 2000年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Biochemistry and Molecular Biology Inc.

Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis, and hepatocellular carcinoma. Since there are several reports indicating that some viruses influence the tumor suppressor p53 function, we determined the effects of HCV proteins on p53 function and its mechanism determined by use of a reporter assay. Among seven HCV proteins investigated (core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), only core protein augmented the transcriptional activity of p53 and increased the expression of p21(waf1) protein, which is a major target of p53. Core protein increased both DNA-binding affinity of p53 in electrophoretic morbidity shift assay and transcriptional ability of p53 itself in a reporter assay. The direct interaction between core protein and C terminus of p53 was also shown by glutathione S-transferase fusion protein binding assay. In addition, core protein interacted with hTAF(II)28, a component of the transcriptional factor complex in vivo and in vitro. These results suggest that HCV core protein interacts with p53 and modulates p53-dependent promoter activities during HCV infection.

DOI： 10.1074/jbc.M000578200

Scopus

Activation of intracellular signaling by hepatitis B and C viruses: C-viral core is the most potent signal inducer 査読

N Kato, H Yoshida, SK Ono-Nita, J Kato, T Goto, M Otsuka, KH Lan, K Matsushima, Y Shiratori, M Omata

HEPATOLOGY 32 ( 2 ) 405 - 412 2000年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：W B SAUNDERS CO

To clarify the effects of hepatitis C virus (HCV) infection on hepatocytes, we analyzed and compared the induction of intracellular signals by HCV and hepatitis B virus (HBV) proteins. We examined the influence of 7 HCV (core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and 4 HBV (precore, core, polymerase, and X) proteins on 5 well-defined intracellular signaling pathways associated with cell proliferation, differentiation, and apoptosis by use of a reporter assay. Viral protein-expression vectors were cotransfected into mammalian cells with reporter vectors having a luciferase gene driven by the following inducible cis-enhancer elements: the cyclic adenosine monophosphate response element, the serum response element (SRE), and the binding sites for nuclear factor kappa B (NF-kappa B), activator protein 1 (AP-1), and serum response factor (SRF). In addition, the activation of signals by HCV proteins was examined in a reporter plasmid having a natural interleukin-8 (IL-8) promoter upstream of a luciferase gene. Of 11 HCV and HBV proteins, HCV core had the strongest influence on intracellular signals, especially NF-kappa B-, AP-1-, and SRE-associated pathways. HCV core's activation level exceeded that of HBV X protein, a well-characterized transactivator of these signals. Moreover, HCV core activated the IL-8 promoter through NF-kappa B and AP-1. For the other proteins, HCV NS4B showed signal activation, but signals were activated at a lesser extent. The luciferase reporter assay, a recently introduced technique, helped in the elucidation of molecular events underlying the inflammatory and proliferation process in the liver induced by HCV.