Yoshiko Hoshikawa

Detection of Epstein-Barr virus in salivas and throat washings in healthy adults and children.

It is well known that Epstein-Barr virus (EBV) is excreted from oral regions in the patients with infectious mononucleosis. We analyzed the prevalence of EBV in saliva and throat washings from healthy people in Japan by the polymerase chain reaction assay. EBV DNA was detected in 43 (90%) of the 48 throat washings from healthy adults (21 to 57 years old) and in 35 (38%) of the 93 salivas from healthy children (0 to 6 years old). The percentages of the EBV DNA-positive ratio in salivas increased in proportion relative to the increase of the childrens ages. EBV type 1 was predominant and was detected in 86 and 94% of adults and children, respectively. Umbilical cord lymphocytes were transformed by some throat washings from EBV seropositive donors. EBV DNA was detected in throat washings from two healthy adults whose EBV antibody was not detected. In both cases, higher amounts of EBV DNA were detected in their peripheral blood mononuclear cells than in those of other, EBV antibody-positive donors. These results demonstrated the incidence of EBV excretion in oral regions of healthy individuals in Japan and defined a novel type of EBV infection in healthy adults.

Hepatic differentiation of human bone marrow–derived mesenchymal stem cells by tetracycline-regulated hepatocyte nuclear factor 3β†

Human bone marrow–derived mesenchymal stem cells (BM‐MSCs) are expected to be a potential source of cells for transplantation. Although recent reports have shown that isolated MSCs can differentiate into hepatocytes, the efficiency of differentiation is insufficient for therapeutic application. To circumvent this problem, it is necessary to understand the mechanisms of hepatic differentiation of human BM‐MSCs. Hepatocyte nuclear factor 3β (HNF3β), a forkhead/winged helix transcription factor, is essential for liver development. In the present study, we established a tetracycline (Tet)‐regulated expression system for HNF3β in UE7T‐13 BM‐MSCs. HNF3β expression significantly enhanced expression of albumin, α‐fetoprotein (AFP), tyrosine amino transferase (TAT) and epithelial cell adhesion molecule (EpCAM) genes. The differentiated cells showed hepatocyte‐specific functions including glycogen production and urea secretion. During treatment with the Tet‐on system for 8 days, over 80% of UE7T‐13 cells turned out to express albumin. Furthermore, the combination of Tet with basic fibroblast growth factor (bFGF) efficiently induced the genes such as albumin and TAT, which are associated with maturity of hepatocytes; however, it suppressed genes such as AFP and EpCAM, which are associated with immaturity of hepatocytes, suggesting that Tet‐induced HNF3β expression sensitizes BM‐MSCs to bFGF signals. Finally, the results of the present study suggest that down‐regulation of Wnt/β‐catenin signals caused by translocation of β‐catenin to cytoplasmic membrane is associated with hepatic differentiation of human BM‐MSCs. Conclusion: HNF3β expression induced efficient differentiation of UE7T‐13 human BM‐MSCs. (HEPATOLOGY 2008;48:597–606.)

Synthetic retinoid CD437 induces mitochondria-mediated apoptosis in hepatocellular carcinoma cells

Retinoids play an important role in the regulation of cell growth and death. Synthetic retinoid CD437 reportedly induces apoptosis in various cancer cell lines. However, the mechanism of inducing apoptosis in hepatocellular carcinoma (HCC) cells by this agent remains to be clarified. In this study, we investigated the signaling pathway by which CD437 induces apoptosis in HCC cell lines. Apoptosis of six human HCC cell lines was induced by treatment with CD437. Caspase-3 and -9 were activated by CD437, suggesting that the apoptosis is mediated by mitochondrial pathways. Consistent with these findings, the treatment with CD437 upregulated Bax protein, downregulated Bcl-2 protein and released cytochrome c into the cytoplasm. Moreover, rhodamine123 staining revealed mitochondrial depolarization in the cells treated with CD437. These data of the present study suggest that CD437 induces apoptosis in HCC cells via mitochondrial pathways.

Involvement of microRNAs in regulation of osteoblastic differentiation in mouse induced pluripotent stem cells.

Backgoround MicroRNAs (miRNAs), which regulate biological processes by annealing to the 3′-untranslated region (3′-UTR) of mRNAs to reduce protein synthesis, have been the subject of recent attention as a key regulatory factor in cell differentiation. The effects of some miRNAs during osteoblastic differentiation have been investigated in mesenchymal stem cells, however they still remains to be determined in pluripotent stem cells. Methodology/Principal Findings Bone morphogenic proteins (BMPs) are potent activators of osteoblastic differentiation. In the present study, we profiled miRNAs during osteoblastic differentiation of mouse induced pluripotent stem (iPS) cells by BMP-4, in which expression of important osteoblastic markers such as Rux2, osterix, osteopontin, osteocalcin, PTHR1 and RANKL were significantly increased. A miRNA array analysis revealed that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a were significantly downregulated. Interestingly, miR-124a and miR-181a directly target the transcription factors Dlx5 and Msx2, both of which were increased by about 80-and 30-fold, respectively. In addition, transfection of miR-124a and miR-181a into mouse osteo-progenitor MC3T3-E1 cells significantly reduced expression of Dlx5, Runx2, osteocalcin and ALP, and Msx2 and osteocalcin, respectively. Finally, transfection of the anti-miRNAs of these six miRNAs, which are predicted to target Dlx5 and Msx2, into mouse iPS cells resulted in a significant increase in several osteoblastic differentiation markers such as Rux2, Msx2 and osteopontin. Conclusions/Significance In the present study, we demonstrate that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a miRNAs, especially miR-124a and miR-181a, are important regulatory factors in osteoblastic differentiation of mouse iPS cells.

Genetic analysis of expression profile involved in retinoid metabolism in non-alcoholic fatty liver disease

Aim:  The patients with non‐alcoholic fatty liver disease (NAFLD) have been reported to be at greater risk for progression to chronic liver disease including liver cirrhosis (LC). To examine the mechanisms for the progression of NAFLD, a genetic analysis of hepatic expression profile in retinoid metabolism in NAFLD was performed since the loss of retinoid signaling is associated with the progression of liver disease via reactive oxygen species (ROS) generation.

Suppressive Effects of Retinoids on Iron-Induced Oxidative Stress in the Liver

BACKGROUND & AIMS We previously reported that impaired retinoid signaling in the liver causes steatohepatitis and hepatocellular carcinoma. Recently, oxidative stress induced by hepatic iron overload has emerged as an important factor for the progression of liver disease in patients with chronic hepatitis C, alcoholic liver disease, and nonalcoholic steatohepatitis. In this study, the relationship between retinoid signaling and iron metabolism in the liver was investigated. METHODS The effect of retinoids on the iron metabolism was examined in HuH7 cells treated with all-trans retinoic acid and acyclic retinoid NIK-333. In in vivo experiments, we used the mice expressing the dominant negative form of retinoic acid receptor alpha gene under the control of albumin enhancer/promoter (RAR-E Tg) and iron-overloaded wild mice fed with retinoid-deficient and retinoid-excess diets. RESULTS Hepatic iron accumulation and increased expression of hemojuvelin were observed in RAR-E Tg mouse liver. Retinoid treatment significantly suppressed expression of hemojuvelin and mildly suppressed expression of transferrin receptor type 2 and hepcidin, accompanied by decreased hepatic iron content and iron-induced oxidative stress in vitro and in vivo. Overexpression of hemojuvelin in HuH7 hepatoma cells led to a significant increase in cellular iron content. CONCLUSIONS Our results suggest that retinoids are involved in hepatic iron metabolism through transcriptional regulation of hemojuvelin. This study demonstrated a novel functional role of retinoids in preventing iron-induced oxidative stress in the liver.

Hepatic differentiation of human bone marrow-derived UE7T-13 cells: Effects of cytokines and CCN family gene expression.

Aim:  Bone marrow‐derived mesenchymal stem cells (MSC) are expected to be an excellent source of cells for transplantation. We aimed to study the culture conditions and involved genes to differentiate MSC into hepatocytes.

The Interaction of Mitogen-Activated Protein Kinases to Epstein-Barr Virus Activation in Akata Cells

To understand the mechanism by which Epstein-Barr virus (EBV) is activated in Akata cells by cross-linking of surface immunoglobulin, the interaction between mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and EBV activation was investigated. Immunoblotting using an anti-phosphoMAPK antibody (Ab) revealed that anti-IgG Ab induced rapid phosphorylation of MAPK in the cells. The phosphorylation was inhibited by MAPK/ERK kinase specific inhibitor, PD98059. The expressions of the EBV immediate early BZLF1 mRNA and its protein product ZEBRA, and early antigen were also inhibited by the inhibitor. These results indicate that MAPK is involved in the pathways of EBV activation.

The vIL-10 gene of the Epstein-Barr virus (EBV) is conserved in a stable manner except for a few point mutations in various EBV isolates.

A gene of the Epstein-Barr virus (EBV), BamHI-C fragment rightward reading frame 1 (BCRF1), codes viral interleukin-10 (vIL-10), which is a close homolog to human IL-10. EBV strain variations are known at EBV latent membrane protein 1 (LMP1), and the distinct forms of LMP1 have been identified. In order to further elucidate the variations of EBV strains, the BCRF1 (vIL-10) gene was analyzed using PCR-direct sequencing in African Burkitt’s lymphoma (BL) cell lines Raji, P3HR-1, EB1 and Daudi, Japanese BL cell line Akata, lymphoblastoid cell line OB and 22 wild EBV isolates from eight gastric carcinoma tissues and 14 throat washes. We found only five variations of the vIL-10 gene in them with one silent mutation and three non-silent mutations. Raji had no mutation to the prototype gene of B95-8. EB1 and P3HR-1 had non-silent mutations in the sequences leading to the arginine/serine and threonine/proline interchanges at residues 4 and 166, respectively. The silent mutation was detected at valine 102 in Daudi and also in the Japanese cell lines Akata, OB and 20 (90.9%) of the wild EBV isolates. The type of variations in the vIL-10 gene had a common relationship with those in the LMP1 gene. All of the variants of valine 102 had China1-type LMP1 sequences except for Daudi with Med-type LMP1 and other minorities with B95-8 type LMP1. The conservativeness of vIL-10 with a few variations suggests the indispensability of the vIL-10 gene in EBV and that the variations of the vIL-10 gene may depend upon the geographical prevalence of the EBV strains. This is the first report regarding the variations of the vIL-10 gene in cell lines and other wild isolates.

Reactive oxygen species and NADPH oxidase 4 induced by transforming growth factor β1 are the therapeutic targets of polyenylphosphatidylcholine in the suppression of human hepatic stellate cell activation

Objective and designTo clarify the molecular mechanism of polyenylphosphatidylcholine (PPC), we examined the involvement of reactive oxygen species (ROS) and NADPH oxidase 4 (Nox4) in human hepatic stellate cells (HSCs).MaterialUsing human LX-2 HSC cells, we examined the effects of PPC on expression of α-smooth muscle actin (α-SMA) and collagen 1, generation of ROS, Nox4 expression, p38 activation and cell proliferation, induced by transforming growth factor β1 (TGFβ1).ResultsPPC suppressed ROS which are induced by TGFβ1, phosphorylation of p38MAPK, and expression levels of α-SMA and collagen 1 in a dose-dependent manner. Higher concentrations of PPC also suppressed Nox4 levels.ConclusionThese results suggest that ROS and Nox4 induced by TGFβ1 are the therapeutic targets of PPC in the suppression of human hepatic stellate cell activation.