Biomedical subjects

Hiroshi Nishina

Publications and source records attributed to Hiroshi Nishina.

At least 19 recordsLinked to original sources

PubMed2026

YAP1 induces hepatocellular carcinoma via DNA demethylation rather than by canonical driver gene mutations.

Large-scale genome sequencing analyses have identified driver gene mutations (DGMs) in most cancers as well as their associated tumorigenic mechanisms. However, a small fraction of cancers are not positive for these canonical DGMs, leaving the mechanisms underpinning their formation a mystery. We hypothesized that canonical DGM-negative cancers might be driven by activation of the transcriptional coactivator YAP1 that led to the induction of epigenetic changes. To test this theory, we established a mouse mosaic model of hepatocellular carcinoma (HCC) in which we induced YAP1-TEAD activation in a few hepatocytes. Whole-exome sequencing did not identify canonical DGMs in HCCs, but bisulfite sequencing revealed widespread DNA demethylation leading to the transcriptional activation of multiple oncogenes. Knockdown of the DNA demethylation-promoting gene, Tet1, attenuated HCC formation in these mice. Single-cell spatial transcriptomics identified a Tet1-high subpopulation of HCC cells that interacted with other hepatic cell types. Our mechanistic mouse data align with the observation that YAP1-TEAD-TET1-associated signatures were also elevated in hepatocytes from patients with Fontan-associated liver disease (FALD), a condition associated with the development of HCCs with lower frequencies of canonical DGMs. Our study suggests that the YAP1-TEAD-TET1 axis promotes canonical DGM-negative HCC development, and provides new insights into the molecular processes involved.

Animals↗

PubMed2006

Administration of fibroblast growth factor 2 in combination with bone marrow transplantation synergistically improves carbon-tetrachloride-induced liver fibrosis in mice.

We previously reported that fibroblast growth factor 2 (FGF2) facilitated the differentiation of transplanted bone marrow cells (BMCs) into hepatocytes. Our earlier study also demonstrated that administration of FGF2 in combination with bone marrow transplantation (BMT) synergistically activated tumor necrosis factor-alpha signaling and significantly improved liver function and prognosis more than BMT alone. However, the way that it affected the extracellular matrix remained unclear. Here, we investigated the effect of FGF2 treatment together with BMT on liver fibrosis in mice treated with carbon tetrachloride (CCl(4)). Transplantation of BMCs and concurrent treatment with FGF2 caused a statistically significant reduction in CCl(4)-induced liver fibrosis that was accompanied by strong expression of matrix metalloproteinase 9 as compared with FGF2-only treatment or BMT alone. Moreover, in this process, the proliferation of bone-marrow-derived cells was accelerated without causing apoptosis. Thus, the administration of FGF2 in combination with BMT synergistically improves CCl(4)-induced liver fibrosis in mice. This treatment has the potential of being an effective therapy for patients with liver cirrhosis.

Animals↗

PubMed2006

Release of RASSF1C from the nucleus by Daxx degradation links DNA damage and SAPK/JNK activation.

Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) responds to a variety of stress stimuli and controls cell fates such as cell cycle entrance, apoptosis and senescence. Stimuli such as ultraviolet irradiation and chemical reagents that damage genomic DNA induce the activation of the SAPK/JNK signaling pathway. However, it is unclear how the signal arising in the nucleus owing to DNA damage is transmitted to SAPK/JNK in the cytoplasm. Here, we report that the nuclear components Daxx and Ras-association domain family 1C (RASSF1C) link DNA damage to SAPK/JNK activation in HeLa cells. In response to DNA damage, Daxx localized in promyelocytic leukaemia-nuclear bodies (PML-NBs) undergoes ubiquitination and degradation. RASSF1C, a tumor suppressor and newly identified binding partner of Daxx, is constitutively anchored by Daxx in PML-NBs but is released from the nucleus when Daxx is degraded. This released RASSF1C translocates to cytoplasmic microtubules and participates in the activation of SAPK/JNK. Our data define a novel mechanism by which the Daxx-RASSF1C complex in PML-NBs couples nuclear DNA damage to the cytoplasmic SAPK/JNK signaling pathway.

Active Transport, Cell Nucleus↗

PubMed2006

Proteomic analysis of serum marker proteins in recipient mice with liver cirrhosis after bone marrow cell transplantation.

We previously found that transplantation with bone marrow cells (BMCs) improves liver function and liver fibrosis in cirrhotic mice. In the presence of liver damage induced by carbon tetrachloride (CCl4), transplanted BMC migrated into the peri-portal region and trans-differentiated into hepatocytes that produce albumin. Thus under these conditions, BMC transplantation induces liver regeneration. Detecting serum marker proteins is important to monitor the recovery of liver function of cirrhotic mice after BMC transplantation. We therefore initially resolved proteins extracted from serum samples at 48 h after BMC transplantation by 2-DE and compared spot intensity between control and BMC groups of mice. Six protein spots increased in the BMC group compared with the control group. MS revealed that these spots comprised apolipoprotein A1 (apoA1), apolipoprotein C3 (apoC3), vitamin D-binding protein, alpha-1-antitrypsin and proteasome subunit alpha type 1. We subsequently confirmed the levels of apoA1 in serum and liver samples by immunoblotting. ApoA1 increased at early stage (48 h and 1 wk) after BMC transplantation in this mouse model of liver cirrhosis. The early elevation of apoA1 might be useful to predict liver regeneration in cirrhotic mice after BMC transplantation.

Animals↗

PubMed2005

Autoamplification of NFATc1 expression determines its essential role in bone homeostasis.

NFATc1 and NFATc2 are functionally redundant in the immune system, but it was suggested that NFATc1 is required exclusively for differentiation of osteoclasts in the skeletal system. Here we provide genetic evidence that NFATc1 is essential for osteoclast differentiation in vivo by adoptive transfer of NFATc1(-/-) hematopoietic stem cells to osteoclast-deficient Fos(-/-) mice, and by Fos(-/-) blastocyst complementation, thus avoiding the embryonic lethality of NFATc1(-/-) mice. However, in vitro osteoclastogenesis in NFATc1-deficient cells was rescued by ectopic expression of NFATc2. The discrepancy between the in vivo essential role of NFATc1 and the in vitro effect of NFATc2 was attributed to selective autoregulation of the NFATc1 gene by NFAT through its promoter region. This suggested that an epigenetic mechanism contributes to the essential function of NFATc1 in cell lineage commitment. Thus, this study establishes that NFATc1 represents a potential therapeutic target for bone disease and reveals a mechanism that underlies the essential role of NFATc1 in bone homeostasis.

Animals↗

PubMed2005

Fibroblast growth factor 2 facilitates the differentiation of transplanted bone marrow cells into hepatocytes.

We have developed an in vivo mouse model, the green fluorescent protein (GFP)/carbon tetrachloride (CCl(4)) model, and have previously reported that transplanted GFP-positive bone marrow cells (BMCs) differentiate into hepatocytes via hepatoblast intermediates. Here, we have investigated the growth factors that are closely related to the differentiation of transplanted BMCs into hepatocytes, and the way that a specific growth factor affects the differentiation process in the GFP/CCl(4) model. We performed immunohistochemical analysis to identify an important growth factor in our model, viz., fibroblast growth factor (FGF). In liver samples, the expression of FGF1 and FGF2 and of FGF receptors (FGFRs; FGFR1, FGFR2) was significantly elevated with time after bone marrow transplantation (BMT) compared with other factors, and co-expression of GFP and FGFs or FGFRs could be detected. We then analyzed the effect and molecular mechanism of FGF signaling on the enhancement of BMC differentiation into hepatocytes by immunohistochemistry, immunoblotting, and microarray analysis. Treatment with recombinant FGF (rFGF), especially rFGF2, elevated the repopulation rate of GFP-positive cells in the liver and significantly increased the expression of both Liv2 (hepatoblast marker) and albumin (hepatocyte marker). Administration of rFGF2 at BMT also raised serum albumin levels and improved the survival rate. Transplantation of BMCs with rFGF2 specifically activated tumor necrosis factor-alpha (TNF-alpha) signaling. Thus, FGF2 facilitates the differentiation of transplanted BMCs into albumin-producing hepatocytes via Liv2-positive hepatoblast intermediates through the activation of TNF-alpha signaling. Administration of FGF2 in combination with BMT improves the liver function and prognosis of mice with CCl(4)-induced liver damage.

Animals↗

PubMed2005

Lesson from the GFP/CCl4 model--translational research project: the development of cell therapy using autologous bone marrow cells in patients with liver cirrhosis.

The plasticity of bone marrow has been confirmed by the analysis of autopsy findings in female recipients of bone marrow cells transplanted from male donors. To establish new clinical cell therapies using autologous bone marrow cells for patients with liver failure, we developed a new in vivo model, the "green fluorescent protein (GFP)/carbon tetrachloride (CCl(4)) model". Using the GFP/CCl(4) model, we found that transplanted Liv8-negative cells efficiently repopulated into cirrhotic liver tissue and trans-differentiated into albumin-producing hepatocytes under conditions of persistent liver damage induced by CCl(4). Moreover, one marrow cell transplantation into liver cirrhosis mice improved their liver function, ameliorated liver fibrosis, and improved their survival rate. Results from the GFP/CCl(4) model showed that cell therapy using autologous bone marrow cells has the potential to become an effective treatment for patients with liver failure. Here we describe the findings from the GFP/CCl(4) model and the scope of the translational research project.

Animals↗

PubMed2005

Development of cell therapy using autologous bone marrow cells for liver cirrhosis.

The plasticity of bone marrow has been confirmed by the autopsy of a female recipient of bone marrow cell transplantation from a male donor. To establish new clinical cell therapies using autologous bone marrow cells for patients with liver failure, we developed a new in vivo model named the green fluorescent protein (GFP)/carbon tetrachloride (CCl4) model. Using the GFP/CCl4 model, we found that transplanted Liv8-negative cells efficiently repopulated into cirrhotic liver tissue and differentiated into albumin-producing hepatocytes under persistent liver damage induced by carbon tetrachloride. Moreover, bone marrow cell transplantation into mice with liver cirrhosis improved liver function and liver fibrosis with the strong expression of matrix metalloproteinases (MMPs), especially MMP-9 activity, resulting in an improved survival rate. Results from the GFP/CCl4 model showed that cell therapy using autologous bone marrow cells has the potential to become an effective treatment for patients with liver failure. A summary of findings from the GFP/CCl4 model is described.

Animals↗

PubMed2005

Purification and analysis of RIN family-novel Rab5 GEFs.

The small GTPase Rab5 plays important roles in membrane budding and trafficking in the early endocytic pathways, and the activation of this GTPase is mediated by several guanine nucleotide exchange factors (GEFs) at each of the transport steps. The RIN family has been identified as GEFs for Rab5 and shown to possess unique biochemical properties. The RIN family preferentially interacts with an activated form of Rab5, although it enhances guanine nucleotide exchange reaction. Moreover, biochemical analysis indicates that the RIN family functions as a tetramer. In this chapter, we describe the isolation of the recombinant RIN family via expression in Spodoptera frugiperda (Sf9) insect cells and in mammalian cells. In addition, functional analysis is also provided to assess the physiological properties of the RIN family.

Animals↗

PubMed2005

Human homologue of maid is a useful marker protein in hepatocarcinogenesis.

BACKGROUND & AIMS: Human homologue of maid (HHM) is a helix-loop-helix (HLH) transcriptional regulatory protein that is involved in the hepatic stem cell development and differentiation. We analyzed the potential involvement of HHM in hepatocarcinogenesis. METHODS: We analyzed HHM expression in the choline-deficient L-amino acid defined (CDAA) diet model of rat hepatocarcinogenesis and in human adenomatous hyperplasia (AH) and hepatocellular carcinoma (HCC) biopsy samples. We assessed the effects of HHM on cell proliferation. We screened proteins that bind to HHM protein using a yeast 2-hybrid screen. RESULTS: High HHM expression was seen in foci and HCC induced in the rat CDAA diet model. HHM protein was expressed in 23 of 32 AH samples (72%), 19 of 28 well-differentiated HCC samples (68%), and 9 of 18 poorly-moderately differentiated HCC samples (50%). Over-expressed HHM enhanced the S phase. HHM interference RNA significantly inhibited cell proliferation. A yeast 2-hybrid screen identified Jun activation domain-binding protein 1 (Jab1) as a binding partner for HHM. We confirmed HHM and Jab1 binding by immunoprecipitation and immunofluorescent histochemistry. The expression of Jab1 was found in human AH and HCC samples. We found an association between levels of expression of HHM and those of Jab1 in AH and HCC tissues examined (P = .027 by chi2 test). CONCLUSIONS: High-level HHM expression was found from the very early stages of hepatocarcinogenesis, suggesting that HHM may be a useful marker protein to detect.

Adenoma↗

PubMed2005

Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin.

The Ras-homologous GTPase Rheb that is conserved from yeast to human appears to be involved not only in cell growth but also in nutrient uptake. Recent biochemical analysis revealed that tuberous sclerosis complex (TSC), a GTPase-activating protein (GAP), deactivates Rheb and that phosphatidylinositol 3'-kinase (PI3k)-Akt/PKB kinase pathway activates Rheb through inhibition of the GAP-mediated deactivation. Although mammalian target of rapamycin (mTOR) kinase is implicated in the downstream target of Rheb, the direct effector(s) and exact functions of Rheb have not been fully elucidated. Here we identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles, which are characterized as late endocytic (late endosome- and lysosome-like) components. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of the previously identified mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression.

Animals↗

PubMed2004

Molecular signature associated with plasticity of bone marrow cell under persistent liver damage by self-organizing-map-based gene expression.

The mechanism that regulates the plasticity of bone marrow cells (BMCs) into hepatocytes is poorly understood. We developed a green fluorescent protein/carbon tetrachloride model to find that BMC transplantation recovered liver damage. Serum albumin level and liver fibrosis were recovered by BMC transplantation. To understand the mechanism, we used DNA-chip technology to profile the change of transient gene expression before and after BMC transplantation. On the basis of gene expression with self-organizing map using specific equation, genes were classified into 153 clusters. The information is useful to understand the dramatic gene activation during the process of the plasticity of BMC.

Animals↗

PubMed2004

Requirement of MKK4 and MKK7 for CdCl2- or HgCl2-induced activation of c-Jun NH2-terminal kinase in mouse embryonic stem cells.

c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase (SAPK), is activated primarily by inflammatory cytokines and environmental stresses including toxic metal exposure. To reveal the upstream kinase responsible for JNK activation by toxic metals, the phosphorylation status and the activity of JNK were examined in mouse embryonic stem (ES) cells lacking MKK4 or MKK7 following exposure to CdCl(2) or HgCl(2). Treatment with CdCl(2) or HgCl(2) induced the phosphorylation of JNK in a dose- and time-dependent manner in wild-type ES cells. In both mkk4(-/-) and mkk7(-/-) ES cells, CdCl(2)- or HgCl(2)-induced phosphorylation and activation of JNK were suppressed significantly. However, in mkk7(-/-) ES cells treated with CdCl(2) and HgCl(2), JNK activation was not abolished (suppressed by 56% and 78%, respectively). These findings suggest that the full activation of JNK by toxic metal exposure requires both MKK4 and MKK7, and these upstream kinases might contribute differentially in JNK activation between mouse ES cells exposed to CdCl(2) and HgCl(2).

Animals↗

PubMed2004

MKK7 couples stress signalling to G2/M cell-cycle progression and cellular senescence.

During the development of multicellular organisms, concerted actions of molecular signalling networks determine whether cells undergo proliferation, differentiation, death or ageing. Here we show that genetic inactivation of the stress signalling kinase, MKK7, a direct activator of JNKs in mice, results in embryonic lethality and impaired proliferation of hepatocytes. Beginning at passage 4-5, mkk7(-/-) mouse embryonic fibroblasts (MEFs) display impaired proliferation, premature senescence and G2/M cell cycle arrest. Similarly, loss of c-Jun or expression of a c-JunAA mutant in which the JNK phosphorylation sites were replaced with alanine results in a G2/M cell-cycle block. The G2/M cell-cycle kinase CDC2 was identified as a target for the MKK7-JNK-c-Jun pathway. These data show that the MKK7-JNK-c-Jun signalling pathway couples developmental and environmental cues to CDC2 expression, G2/M cell cycle progression and cellular senescence in fibroblasts.

Animals↗

PubMed2004

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver.

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.

Albumins↗

PubMed2004

Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice.

We investigated the effect of bone marrow cell (BMC) transplantation on established liver fibrosis. BMCs of green fluorescent protein (GFP) mice were transplanted into 4-week carbon tetrachloride (CCl4)-treated C57BL6 mice through the tail vein, and the mice were treated for 4 more weeks with CCl4 (total, 8 weeks). Sirius red and GFP staining clearly indicated migrated BMCs existing along with fibers, with strong expression of matrix metalloproteinase (MMP)-9 shown by anti-MMP-9 antibodies and in situ hybridization. Double fluorescent immunohistochemistry showed the expression of MMP-9 on the GFP-positive cell surface. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area coinciding with the location of MMP-9-positive BMCs. Four weeks after BMC transplantation, mice had significantly reduced liver fibrosis, as assessed by hydroxyproline content of the livers, compared to that of mice treated with CCl4 alone. Subpopulation of Liv8-negative BMCs was responsible for this fibrolytic effect. In conclusion, mice with BMC transplants with continuous CCl4 injection had reduced liver fibrosis and a significantly improved survival rate after BMC transplantation compared with mice treated with CCl4 alone. This finding introduces a new concept for the therapy of liver fibrosis.

Animals↗

PubMed2004

Genetic dissection of the formation of the forebrain in Medaka, Oryzias latipes.

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.

Animals↗

PubMed2004

Mutations affecting somite formation in the Medaka (Oryzias latipes).

The metameric structure of the vertebrate trunk is generated by repeated formation of somites from the unsegmented presomitic mesoderm (PSM). We report the initial characterization of nine different mutants affecting segmentation that were isolated in a large-scale mutagenesis screen in Medaka (Oryzias latipes). Four mutants were identified that show a complete or partial absence of somites or somite boundaries. In addition, five mutations were found that cause fused somites or somites with irregular sizes and shapes. In situ hybridization analysis using specific markers involved in the segmentation clock and antero-posterior (A-P) polarity of somites revealed that the nine mutants can be compiled into two groups. In group 1, mutants exhibit defects in tailbud formation and PSM prepatterning, whereas A-P identity in the somites is defective in group 2 mutants. Three mutants (planlos, pll; schnelles ende, sne; samidare, sam) have characteristic phenotypes that are similar to those in zebrafish mutants affected in the Delta/Notch signaling pathway. The majority of mutants, however, exhibit somitic phenotypes distinct from those found in zebrafish, such as individually fused somites and irregular somite sizes. Thus, these Medaka mutants can be expected to provide clues to uncovering novel components essential for somitogenesis.

Animals↗