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Biomedical subjects

Makoto Hijikata

Publications and source records attributed to Makoto Hijikata.

At least 19 recordsLinked to original sources
PubMed2006

The expression of phosphatidic acid phosphatase 2a, which hydrolyzes lipids to generate diacylglycerol, is regulated by p73, a member of the p53 family.

p73, a p53-related gene, is essential for a development of animals, while p53 is important for tumor formation. And little is known about the target genes specifically regulated by p73. Identifying the specific targets of p73 is important to understand the physiological roles of p73. To identify the genes specifically regulated by p73, we conducted serial analysis of gene expression to quantitatively evaluate messenger RNA populations. We found that the gene for phosphatidic acid phosphatase 2a (PAP2a), an enzyme that hydrolyzes lipids to generate diacylglycerol, was specifically upregulated by ectopic production of p73beta. The promoter region of this gene contains an element that is functionally responsive to p73beta. And the quantity of PAP2a protein was upregulated by ectopic production of p73beta. These results suggest that the expression of PAP2a is directly regulated by p73.

Cell Line↗
PubMed2006

Serum-derived hepatitis C virus infectivity in interferon regulatory factor-7-suppressed human primary hepatocytes.

BACKGROUND/AIMS: The development of an efficient in vitro infection system for HCV is important in order to develop new anti-HCV strategy. Only Huh7 hepatocyte cell lines were shown to be infected with JFH-1 fulminant HCV-2a strain and its chimeras. Here we aimed to establish a primary hepatocyte cell line that could be infected by HCV particles from patients' sera. METHODS: We transduced primary human hepatocytes with human telomerase reverse transcriptase together with human papilloma virus 18/E6E7 (HPV18/E6E7) genes or simian virus large T gene (SV40 T) to immortalize cells. We also established the HPV18/E6E7-immortalized hepatocytes in which interferon regulatory factor-7 was inactivated. Finally we analyzed HCV infectivity in these cells. RESULTS: Even after prolonged culture HPV18/E6E7-immortalized hepatocytes exhibited hepatocyte functions and marker expression and were more prone to HCV infection than SV40 T-immortalized hepatocytes. The susceptibility of HPV18/E6E7-immortalized hepatocytes to HCV infection was further improved, in particular, by impairing signaling through interferon regulatory factor-7. CONCLUSIONS: HPV18/E6E7-immortalized hepatocytes are useful for the analysis of HCV infection, anti-HCV innate immune response, and screening of antiviral agents with a variety of HCV strains.

Antigens, CD↗
PubMed2006

Evaluation of the anti-hepatitis C virus effects of cyclophilin inhibitors, cyclosporin A, and NIM811.

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. We recently discovered that the immunosuppressant cyclosporin A (CsA) and its analogue lacking immunosuppressive function, NIM811, strongly suppress the replication of HCV in cell culture. Inhibition of a cellular replication cofactor, cyclophilin (CyP) B, is critical for its anti-HCV effects. Here, we explored the potential use of CyP inhibitors for HCV treatment by analyzing the HCV replicon system. Treatment with CsA and NIM811 for 7 days reduced HCV RNA levels by 2-3 logs, and treatment for 3 weeks reduced HCV RNA to undetectable levels. NIM811 exerted higher anti-HCV activity than CsA at lower concentrations. Both CyP inhibitors rapidly reduced HCV RNA levels even further in combination with IFNalpha without modifying the IFNalpha signal transduction pathway. In conclusion, CyP inhibitors may provide a novel strategy for anti-HCV treatment.

Antiviral Agents↗
PubMed2006

Diverse effects of cyclosporine on hepatitis C virus strain replication.

Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents.

Amino Acid Sequence↗
PubMed2005

Enhancement of internal ribosome entry site-mediated translation and replication of hepatitis C virus by PD98059.

Translation initiation of hepatitis C virus (HCV) occurs in an internal ribosome entry site (IRES)-dependent manner. We found that HCV IRES-dependent protein synthesis is enhanced by PD98059, an inhibitor of the extracellular signal-regulated kinase (ERK) signaling pathway, while cellular cap-dependent translation was relatively unaffected by the compound. Treatment of cells with PD98059 allowed for robust HCV replication following cellular incubation with HCV-positive serum. Though the molecular mechanism underlying IRES enhancement remains elusive, PD98059 is a potent accelerator of HCV RNA replication.

Base Sequence↗
PubMed2005

Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase.

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

Carcinoma, Hepatocellular↗
PubMed2005

Centrosomal P4.1-associated protein is a new member of transcriptional coactivators for nuclear factor-kappaB.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor important for various cellular events such as inflammation, immune response, proliferation, and apoptosis. In this study, we performed a yeast two-hybrid screening using the N-terminal domain of the p65 subunit (RelA) of NF-kappaB as bait and isolated centrosomal P4.1-associated protein (CPAP) as a candidate for a RelA-associating partner. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments followed by Western blotting also showed association of CPAP with RelA. When overexpressed, CPAP enhanced NF-kappaB-dependent transcription induced by tumor necrosis factor-alpha (TNFalpha). Reduction of the protein level of endogenous CPAP by RNA interference resulted in decreased activation of NF-kappaB by TNFalpha. After treatment with TNFalpha, a portion of CPAP was observed to accumulate in the nucleus, although CPAP was found primarily in the cytoplasm without any stimulation. Moreover, CPAP was observed in a complex recruited to the transcriptional promoter region containing the NF-kappaB-binding motif. One hybrid assay showed that CPAP has the potential to activate gene expression when tethered to the transcriptional promoter. These data suggest that CPAP functions as a coactivator of NF-kappaB-mediated transcription. Since a physiological interaction between CPAP and the coactivator p300/CREB-binding protein was also observed and synergistic activation of NF-kappaB-mediated transcription was achieved by these proteins, CPAP-dependent transcriptional activation is likely to include p300/CREB-binding protein.

Amino Acid Motifs↗
PubMed2005

Suppression of hepatitis C virus replicon by TGF-beta.

Hepatitis C virus (HCV) is one of the major causative agents of liver diseases, such as liver inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Using an efficient HCV subgenomic replicon system, we demonstrate that transforming growth factor-beta (TGF-beta) suppresses viral RNA replication and protein expression from the HCV replicon. We further show that the anti-viral effect of this cytokine is associated with cellular growth arrest in a manner dependent on Smad signaling, not mitogen-activated protein kinase (MAPK) signaling. These results suggest a novel insight into the mechanisms of liver diseases caused by HCV.

Cell Line, Tumor↗
PubMed2005

Analysis of the 5' end structure of HCV subgenomic RNA replicated in a Huh7 cell line.

OBJECTIVE: Recently, HCV subgenomic RNA that replicates in vitro in a certain cell line have been elucidated. Since the 5' end of the genome of positive strand RNA viruses is often modified with a cap structure or a covalently linked protein, we have assessed structural feature of the HCV genome obtained from Huh7 cells in which HCV subgenomic RNA has been shown to efficiently self-replicate. METHODS: HCV subgenomic RNA was obtained from the Huh7 and was analyzed for its 5' end. RESULTS: Phosphorylation of the genomic RNA by polynucleotide kinase was observed only after treatment with phosphatase. The labeling efficiency of the genome with polynucleotide kinase was not enhanced by treatment with pyrophosphatase. CONCLUSION: It is suggested that the 5' end of HCV genomic RNA obtained from HCV replicon cells is not modified except phosphorylation. Furthermore, analysis of the 5' end of the HCV RNA obtained from the HCV subgenome self-replicating cells revealed the presence of two types of subgenomic RNA that contained either guanylate or adenylate at the 5' end. This result indicates that the 5' end of the subgenome in Huh7 cells is redundant and there is no significant evolutionary advantage between the two genomes.

Adenosine Monophosphate↗
PubMed2004

Siah-1L, a novel transcript variant belonging to the human Siah family of proteins, regulates beta-catenin activity in a p53-dependent manner.

Beta-catenin is a potent oncogenic protein whose cytoplasmic accumulation is a frequent event in cancer cells. The level of beta-catenin is regulated by two mechanisms: the adenomatous polyposis coli/Axin/glycogen synthase kinase 3beta-dependent degradation pathway and the Siah-1/Siah interacting protein/Ebi-mediated degradation pathway. In this study, we have investigated the functional significance of p53-inducible human Siah-family protein expression in the regulation of beta-catenin activity. We show here by reverse-transcriptase polymerase chain reaction that two mRNA transcripts, designated human Siah-1 and Siah-1L, are generated from the human Siah-1 locus. Interestingly, the expression of Siah-1L was upregulated by p53, whereas human Siah-1 expression was constant. Furthermore, introduction of exogenous Siah-1L protein downregulated beta-catenin protein and promoted apoptosis induced by anticancer drugs in cancer cells that lack endogenous p53. Thus, Siah-1L represents a new member of the human Siah family that is induced in response to p53 and plays an important role in the regulation of beta-catenin activity in tumor cells. These findings also suggest new strategies for restoring tumor suppressive pathways lost in cancer cells that have suffered p53 inactivation.

Base Sequence↗
PubMed2004

The role of notch signaling in the development of intrahepatic bile ducts.

BACKGROUND & AIMS: Mutations in Jagged1 , a Notch ligand, cause Alagille syndrome (AGS), a disorder characterized by a paucity of intrahepatic bile ducts (IHBD). The mechanism underlying the contribution of the Notch signaling pathway to IHBD formation, however, remains unknown. Here we investigated the role of Notch signaling in IHBD development. METHODS: The expression patterns of Jagged1, Notch2, and Hes1 during mouse liver development were analyzed by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), immunoblot, and immunohistochemistry. The hepatocyte maturation level and IHBD development were studied in Hes1 null mice in comparison with wild-type mice. The effect of Jagged1 on biliary differentiation was assessed by using an in vitro 2-cell coculture system with WB-F344 cells, a cell line derived from normal adult rat liver. RESULTS: Jagged1 was expressed in the portal mesenchyme during the neonatal period. During the same period, Notch2 and Hes1 expression was observed in the biliary epithelial cells adjacent to the Jagged1-positive cells. During ductal plate remodeling, Notch2 and Hes1 were up-regulated exclusively in the biliary epithelial cells that form tubular structures. In contrast, the tubular formation of IHBD was completely absent in Hes1 null mice. Coculture with Balb3T3 cells stably overexpressing Jagged1 induced transactivation of the Hes1 promoter and increased expression of biliary lineage markers, such as cytokeratin-19 and gamma-glutamyl transpeptidase, in WB-F344 cells. CONCLUSIONS: Our results suggest that Notch signaling has an important role in the differentiation of biliary epithelial cells and is essential for their tubular formation during IHBD development.

Alagille Syndrome↗
PubMed2004

Effect of interaction between hepatitis C virus NS5A and NS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon.

Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S232I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by alpha interferon. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.

Amino Acid Sequence↗
PubMed2003

Hepatitis C virus non-structural proteins in the probable membranous compartment function in viral genome replication.

The molecular mechanism of hepatitis C virus(HCV) RNA replication is still unknown. Recently, a cell culture system in which the HCV subgenomic replicon is efficiently replicated and maintained for a long period in Huh-7 cells has been established. Taking advantage of this replicon system, we detected the activity to synthesize the subgenomic RNA in the digitonin-permeabilized replicon cells. To elucidate how and where this viral RNA replicates in the cells, we monitored the activity for HCV RNA synthesis in the permeabilized replicon cells under several conditions. We obtained results suggesting that HCV replication complexes functioning to synthesize the replicon RNA are protected from access of nuclease and proteinase by possible cellular lipid membranes. We also found that a large part of the replicon RNA, including newly synthesized RNA, was present in such a membranous structure but a large part of each NS protein was not. A small part of each NS protein that was resistant to the proteinase action was shown to contribute sufficiently to the synthesis of HCV subgenomic RNA in the permeabilized replicon cells. These results suggested that a major subcellular site of HCV genome replication is probably compartmentalized by lipid membranes and that only a part of each NS protein forms the active replication complex in the replicon cells.

Blotting, Northern↗
PubMed2003

Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes.

Persistent infection of hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Searching for a substance with anti-HCV potential, we examined the effects of a variety of compounds on HCV replication using a HCV subgenomic replicon cell culture system. Consequently, the immunosuppressant cyclosporin A (CsA) was found to have a suppressive effect on the HCV replicon RNA level and HCV protein expression in these cells. CsA also inhibited multiplication of the HCV genome in a cultured human hepatocyte cell line infected with HCV using HCV-positive plasma. This anti-HCV activity of CsA appeared to be independent of its immunosuppressive function. In conclusion, our results suggest that CsA may represent a new approach for the development of anti-HCV therapy.

Antiviral Agents↗
PubMed2003

Modulation of retinoid signaling by a cytoplasmic viral protein via sequestration of Sp110b, a potent transcriptional corepressor of retinoic acid receptor, from the nucleus.

Hepatitis C virus (HCV) core protein (core) plays a significant role in the development of chronic liver diseases caused by HCV infection. We have discovered that the core sensitized all-trans-retinoic acid (ATRA)-induced cell death in MCF-7 cells. Activation of retinoic acid receptor alpha (RARalpha)-mediated transcription by the core was also seen in all the cell lines tested. By use of a yeast two-hybrid system, we identified Sp110b as a candidate for a core-interacting cellular factor. Although the function of Sp110b has remained unknown, we observed that Sp110b interacts with RARalpha and suppresses RARalpha-mediated transcription. These data suggest that Sp110b is a transcriptional cofactor negatively regulating RARalpha-mediated transcription. RNA interference-mediated reduction of endogenous Sp110b levels depressed the ability of the core to activate RARalpha-mediated transcription, suggesting an essential role for Sp110b in this pathway. The normal nuclear subcellular localization of Sp110b was altered by molecular interaction with the core to the cytoplasmic surface of the endoplasmic reticulum. This evidence suggests a model in which the core sequesters Sp110b from the nucleus and inactivates its corepressor function to activate RARalpha-mediated transcription. These findings likely describe a novel system in which a cytoplasmic viral protein regulates host cell transcription.

Active Transport, Cell Nucleus↗
PubMed2002

Wnt/beta-catenin signaling suppresses apoptosis in low serum medium and induces morphologic change in rodent fibroblasts.

Wnt/beta-catenin signaling plays important roles in tumorigenesis in certain tumors as well as during development. However, the mechanism of tumorigenesis mediated by this signaling remains to be elucidated. We investigated the response of rodent fibroblasts to activation of Wnt/beta-catenin signaling by treatment with conditioned medium containing soluble Wnt-3a protein (W3a-CM) and by expression of a constitutive active beta-catenin gene harbored by an adenovirus vector. W3a-CM induced transcriptional activation of a beta-catenin/T-cell factor (Tcf)-responsive promoter in rodent fibroblasts such as NIH3T3, Rat-1, Swiss3T3 and Balb3T3 cells. In these cells, an increase in saturation density and an inhibition of apoptosis and/or promotion of growth in low-serum medium were induced by treatment with W3a-CM. In Rat-1 cells, morphologic changes were also induced. All these alterations were reversible. Moreover, the inhibition of apoptosis of NIH3T3 cells in low-serum medium and the morphologic changes in Rat-1 cells, but not the increase in saturation density, were also induced by ectopic expression of a constitutive active beta-catenin gene. These results suggested that activation of Wnt/beta-catenin signaling induces inhibition of apoptosis and morphologic changes in these cells.

3T3 Cells↗
PubMed2002

Subgenomic replicon derived from a cell line infected with the hepatitis C virus.

Recently, cell culture systems have been established, where a hepatitis C virus (HCV) subgenomic replicon was efficiently replicated and maintained for a long period. To see whether a HCV sequence derived from HCV-infected cultured cell sequence can be used for the construction of a functional replicon, a HCV subgenomic RNA carrying a neomycin-resistant gene was constructed using the HCV genome RNA obtained from cultured cells infected with HCV. After transfection, G418-resistant Huh-7 cells were selected and subcloned. Finally, the production of HCV proteins and de novo synthesis of subgenomic RNA were confirmed in the selected cell clone, indicating that this subgenomic RNA replicated in cultured cells and functioned as a replicon. These results suggest that the HCV genome obtained from an in vitro HCV infection system with cultured cells can be used to develop a subgenomic replicon system with diverse HCV sequences.

Cell Line↗