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Toru Nakano

Publications and source records attributed to Toru Nakano.

At least 19 recordsLinked to original sources
PubMed2007

Growth arrest-specific gene 6 and Axl signaling enhances gastric cancer cell survival via Akt pathway.

Activation of tyrosine kinases is an important factor during cancer development. Axl, one of the receptor tyrosine kinases, binds to the specific ligand growth arrest-specific gene 6 (Gas6), which encodes a vitamin K-dependent gamma-carboxyglutamyl protein. Although many receptor tyrosine kinases and their ligands are involved in gastric carcinogenesis, whether Gas6-Axl signaling is involved in gastric carcinogenesis has not been elucidated. The aim of this study was to investigate the expression of Gas6 and Axl in gastric cancer and also their roles during gastric carcinogenesis. mRNA and protein of Gas6 and Axl were highly expressed in a substantial proportion of human gastric cancer tissue and cell lines, and Gas6 expression was significantly associated with lymph node metastasis. With recombinant Gas6 and a decoy-receptor of Axl in vitro, we demonstrated that Gas6-Axl signaling pathway enhanced cellular survival and invasion and suppressed apoptosis via Akt pathway. Our results suggests that Gas6-Axl signaling plays a role during gastric carcinogenesis, and that targeting Gas6-Axl signaling could be a novel therapeutic for gastric cancer.

Base Sequence↗
PubMed2006

The Pten/PI3K pathway governs the homeostasis of Valpha14iNKT cells.

The tumor suppressor PTEN is mutated in many human cancers. We previously used the Cre-loxP system to generate mice (LckCrePten mice) with a Pten mutation in T-lineage cells. Here we describe the phenotype of Pten-deficient Valpha14iNKT cells. A failure in the development of Valpha14iNKT cells occurs in the LckCrePten thymus between stage 2 (CD44(high)NK1.1(-)) and stage 3 (CD44(high)NK1.1(+)), resulting in decreased numbers of peripheral Valpha14iNKT cells. In vitro, Pten-deficient Valpha14iNKT cells show reduced proliferation and cytokine secretion in response to alphaGalCer stimulation but enhanced inhibitory Ly49 receptor expression. Following interaction with dendritic cells (DCs) loaded with alphaGalCer, Pten-deficient Valpha14iNKT cells demonstrate activation of PI3K. Indeed, the effects of the Pten mutation require intact function of the PI3K subunits p110gamma and p110delta. In vivo, LckCrePten mice display reduced serum IFNgamma after alphaGalCer administration. Importantly, Valpha14iNKT cell-mediated protection against the metastasis of melanoma cells to the lung was impaired in the absence of Pten. Thus, the Pten/PI3K pathway is indispensable for the homeostasis and antitumor surveillance function of Valpha14iNKT cells.

Animals↗
PubMed2006

PGC7/Stella protects against DNA demethylation in early embryogenesis.

DNA methylation is an important means of epigenetic gene regulation and must be carefully controlled as a prerequisite for normal early embryogenesis. Although global demethylation occurs soon after fertilization, it is not evenly distributed throughout the genome. Genomic imprinting and epigenetic asymmetry between parental genomes, that is, delayed demethylation of the maternal genome after fertilization, are clear examples of the functional importance of DNA methylation. Here, we show that PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. After determining that PGC7/Stella binds to Ran binding protein 5 (RanBP5; a nuclear transport shuttle protein), mutant versions of the two proteins were used to examine exactly when and where PGC7/Stella functions within the cell. It is likely that PGC7/Stella protects the maternal genome from demethylation only after localizing to the nucleus, where it maintains the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogramming after fertilization.

Animals↗
PubMed2006

Gas6/Axl-PI3K/Akt pathway plays a central role in the effect of statins on inorganic phosphate-induced calcification of vascular smooth muscle cells.

Apoptosis is essential for the initiation and progression of vascular calcification. Recently, we showed that 3-hydroxy-3-methylglutaryl (HMG) CoA reductase inhibitors (statins) have a protective effect against vascular smooth muscle cell calcification by inhibiting apoptosis, where growth arrest-specific gene 6 (Gas6) plays a pivotal role. In the present study, we clarified the downstream targets of Gas6-mediated survival signaling in inorganic phosphate (Pi)-induced apoptosis and examined the effect of statins. We found that fluvastatin and pravastatin significantly inhibited Pi-induced apoptosis and calcification in a concentration-dependent manner in human aortic smooth muscle cells (HASMC), as was found with atorvastatin previously. Gas6 and its receptor, Axl, expression were downregulated in the presence of Pi, and recombinant human Gas6 (rhGas6) significantly inhibited apoptosis and calcification in a concentration-dependent manner. During apoptosis, Pi suppressed Akt phosphorylation, which was reversed by rhGas6. Wortmannin, a specific phosphatidylinositol 3-OH kinase (PI3K) inhibitor, abolished the increase in Akt phosphorylation by rhGas6 and eliminated the inhibitory effect of rhGas6 on both Pi-induced apoptosis and calcification, suggesting that PI3K-Akt is a downstream signal of the Gas6-mediated survival pathway. Pi reduced phosphorylation of Bcl2 and Bad, and activated caspase 3, all of which were reversed by rhGas6. The inhibitory effect of statins on Pi-induced apoptosis was accompanied by restoration of the Gas6-mediated survival signal pathway: upregulation of Gas6 and Axl expression, increased phosphorylation of Akt and Bcl2, and inhibition of Bad and caspase 3 activation. These findings indicate that the Gas6-mediated survival pathway is the target of statins' effect to prevent vascular calcification.

Androstadienes↗
PubMed2006

Hyperplasia and carcinomas in Pten-deficient mice and reduced PTEN protein in human bladder cancer patients.

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate an urothelium-specific null mutation of Pten in mice [FabpCrePten(flox/flox) (FPten(flox/flox)) mice]. Histologic examination revealed that all FPten(flox/flox) mice exhibited urothelial hyperplasia in which component cells showed enlarged nuclei and increased cell size. With time, 10% of FPten(flox/flox) mice spontaneously developed pedicellate papillary transitional cell carcinomas (TCC). This type of tumor also arose in FPten(flox/flox) mice treated with the chemical carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine. FPten(flox/flox) urothelial cells were hyperproliferative and showed increased activation of the survival signaling molecules Akt and extracellular signal-regulated kinase. In humans, 53% of primary bladder cancer patients exhibited decreased or absent expression of PTEN protein in either the cytoplasm or nucleus of tumor cells. In early bladder cancers, PTEN expression was repressed in 42% of superficial papillary TCC but in only 8% of cases of carcinoma in situ (CIS). In advanced bladder cancers, PTEN protein was significantly reduced (particularly in the nucleus) in 94% of cases, and this decrease in PTEN correlated with disease stage and grade. Thus, PTEN deficiency may contribute to bladder cancer both by initiating superficial papillary TCC and by promoting the progression of CIS to advanced invasive and metastatic forms.

Animals↗
PubMed2006

The Grb2/Mek pathway represses Nanog in murine embryonic stem cells.

The homeobox gene Nanog is a key intrinsic determinant of self renewal in embryonic stem (ES) cells, and its repression leads ES cells to selectively differentiate into primitive endoderm. Although Nanog repression occurs at the outermost layer of ES cell aggregates independent of the leukemia inhibitory factor (LIF)/STAT3 pathway, it is largely undetermined what external cues and intracellular signals cause the event. Of interest, addition of the tyrosine phosphatase inhibitor, sodium vanadate, selectively repressed Nanog transcription without any detectable changes in upstream transcriptional regulators Oct3/4 and Sox2. Furthermore, sodium vanadate induced primitive endoderm differentiation, even in the inner cells of ES cell aggregates. Expression of Gata6 and Zfp42, two putative downstream Nanog effectors, was also increased and decreased by the addition of sodium vanadate, respectively, but these changes were eliminated by exogenous Nanog expression. The effects of sodium vanadate were abrogated by Grb2 deficiency or by the addition of the Mek inhibitor, PD98059. Indeed, PD98059 prevented Nanog repression induced by ES cell aggregation as well. Furthermore, transfection of a constitutive active Mek mutant into ES cells induced Nanog repression and primitive endoderm differentiation. These data indicate that the Grb2/Mek pathway primarily mediates Nanog gene repression upon ES cell differentiation into primitive endoderm.

Animals↗
PubMed2006

The stabilization of beta-catenin leads to impaired primordial germ cell development via aberrant cell cycle progression.

Primordial germ cells (PGCs) are germ cell precursors that are committed to sperm or oocytes. Dramatic proliferation during PGC development determines the number of founder spermatogonia and oocytes. Although specified to a germ lineage, PGCs produce pluripotent embryonic germ (EG) cells in vitro and testicular teratomas in vivo. Wnt/beta-catenin signaling regulates pluripotency and differentiation in various stem cell systems, and dysregulation of this signaling causes various human cancers. Here, we examined the role of Wnt/beta-catenin signaling in PGC development. In normal PGC development, Wnt/beta-catenin signaling is suppressed by the GSK3beta-mediated active degradation of beta-catenin and the low expression of canonical Wnt molecules. The effects of aberrant activation of Wnt/beta-catenin signaling in PGCs were analyzed using mice carrying a deletion of the exon that encodes the GSK3beta phosphorylation sites in the beta-catenin locus. Despite the potential activity of Wnt/beta-catenin signaling in stem cell maintenance and carcinogenesis in various cell lineages, teratomas were not induced in the mice expressing the nuclear-localized beta-catenin in PGCs. Instead, the mutant mice showed germ cell deficiency caused by the delayed cell cycle progression of the proliferative phase PGCs. Our results show that the suppression of Wnt/beta-catenin signaling is a prerequisite for the normal development of PGCs.

Animals↗
PubMed2006

Characterization of the piRNA complex from rat testes.

Small noncoding RNAs regulate processes essential for cell growth and development, including mRNA degradation, translational repression, and transcriptional gene silencing (TGS). During a search for candidate mammalian factors for TGS, we purified a complex that contains small RNAs and Riwi, the rat homolog to human Piwi. The RNAs, frequently 29 to 30 nucleotides in length, are called Piwi-interacting RNAs (piRNAs), 94% of which map to 100 defined (< or = 101 kb) genomic regions. Within these regions, the piRNAs generally distribute across only one genomic strand or distribute on two strands but in a divergent, nonoverlapping manner. Preparations of piRNA complex (piRC) contain rRecQ1, which is homologous to qde-3 from Neurospora, a gene implicated in silencing pathways. Piwi has been genetically linked to TGS in flies, and slicer activity cofractionates with the purified complex. These results are consistent with a gene-silencing role for piRC in mammals.

Adenosine Triphosphatases↗
PubMed2006

A novel class of small RNAs bind to MILI protein in mouse testes.

Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes. A subgroup of the Argonaute proteins--known as the 'Piwi family'--is required for germ- and stem-cell development in invertebrates, and two Piwi members--MILI and MIWI--are essential for spermatogenesis in mouse. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5' uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26-31 nucleotides (nt) in length--clearly distinct from the 21-23 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)--and we refer to them as 'Piwi-interacting RNAs' or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis.

Animals↗
PubMed2006

Statins protect human aortic smooth muscle cells from inorganic phosphate-induced calcification by restoring Gas6-Axl survival pathway.

Vascular calcification is clinically important in the development of cardiovascular disease. It is reported that hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) inhibited vascular calcification in several clinical trials. However, the mechanism is poorly understood. Recently, it has been suggested that apoptosis is one of the important processes regulating vascular smooth muscle cell (VSMC) calcification. In this study, we investigated the effect of statins on VSMC calcification by testing their effect on apoptosis, focusing in particular on regulation of the survival pathway mediated by growth arrest-specific gene 6 (Gas6), a member of the vitamin K-dependent protein family, and its receptor, Axl. In human aortic smooth muscle cells (HASMC), statins significantly inhibited inorganic phosphate (Pi)-induced calcification in a concentration-dependent manner (reduced by 49% at 0.1 micromol/L atorvastatin). The inhibitory effect of statins was mediated by preventing apoptosis, which was increased by Pi in a concentration-dependent manner, and not by inhibiting sodium-dependent phosphate cotransporter (NPC) activity, another mechanism regulating HASMC calcification. Furthermore, the antiapoptotic effect of statins was dependent on restoration of Gas6, whose expression was downregulated by Pi. Restoration of Gas6 mRNA by statins was mediated by mRNA stabilization, and not by an increase in transcriptional activity. Suppression of Gas6 using small interfering RNA and the Axl-extracellular domain abolished the preventive effect of statins on Pi-induced apoptosis and calcification. These data demonstrate that statins protected HASMC from Pi-induced calcification by inhibiting apoptosis via restoration of the Gas6-Axl pathway.

Aorta↗
PubMed2006

Multipotential differentiation ability of GATA-1-null erythroid-committed cells.

GATA-1, a zinc finger transcription factor, has been believed to be indispensable for the survival of proerythroblasts. However, we found that GATA-1-null proerythroblasts could survive and proliferate on OP9 stroma cells in the presence of erythropoietin. Furthermore, myeloid and mast cells were induced from the GATA-1-null proerythroblasts by the stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), respectively, but lymphoid differentiation was not achieved by in vivo transfer. Thus, without activity of the transcription factor required for terminal differentiation, even relatively mature and committed cells proliferate continuously with the differentiation capacity to other lineages. Our data suggest that GATA-1 is a critical transcription factor to fix erythroid progenitors to the erythroid lineage.

Animals↗
PubMed2005

Redirecting differentiation of hematopoietic progenitors by a transcription factor, GATA-2.

GATA-2 is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells. We analyzed the function of GATA-2 by a combined method of tetracycline-dependent conditional gene expression and in vitro hematopoietic differentiation from mouse embryonic stem (ES) cells using OP9 stroma cells (OP9 system). In the presence of macrophage colony-stimulating factor (M-CSF), the OP9 system induced macrophage differentiation. GATA-2 expression in this system inhibited macrophage differentiation and redirected the fate of hematopoietic differentiation to other hematopoietic lineages. GATA-2 expression commencing at day 5 or day 6 induced megakaryocytic or erythroid differentiation, respectively. Expression levels of PU.1, a hematopoietic transcription factor that interferes with GATA-2, appeared to play a critical role in differentiation to megakaryocytic or erythroid lineages. Transcription of PU.1 was affected by histone acetylation induced by binding of GATA-2 to the PU.1 promoter region. This study demonstrates that the function of GATA-2 is modified in a context-dependent manner by expression of PU.1, which in turn is regulated by GATA-2.

Animals↗
PubMed2005

Efficient and repetitive production of hematopoietic and endothelial cells from feeder-free monolayer culture system of primate embryonic stem cells.

We have established an innovative culture system for the efficient differentiation of hematopoietic and endothelial cells from primate embryonic stem (ES) cells without feeder cells, embryoid bodies, or cell-sorting processes. After several days' culture in murine stromal OP9-conditioned medium supplemented with a cytokine cocktail on collagen-coated dishes, ES cells differentiated into a very unique population of cells with a finger-like appearance. These finger-like cells were positive for mesodermal and/or hemangioblastic markers of kinase insert domain receptor (KDR) and T-cell acute lymphocytic leukemia 1 (TAL1), and produced large amounts of protein tyrosine phosphatase, receptor type, C-positive hematopoietic cells. These hematopoietic cells showed the morphology of immature hematopoietic cells, formed blast cell colonies with high efficiency, and were positive for CD34 antigen, KDR, TAL1, and GATA binding protein 1, suggesting that these blast cells are equivalent to the multipotent hematopoietic progenitor cells. Moreover, they produced functional macrophages in murine stromal MS-5-conditioned medium and primitive erythroblasts in the presence of erythropoietin. The finger-like cells, putative mesodermal progenitors and/or hemangioblasts, actively proliferated and repetitively produced hematopoietic cells as long as they were maintained on the original dish. By contrast, the majority of the finger-like cells differentiated into endothelial cells with specific markers and specific functions after transfer to fresh dishes, indicating that conditions established in the original dish supported the proliferation and hematopoietic differentiation of the finger-like cells. Our method provides a highly controllable culture protocol for repetitive production of hematopoietic and endothelial cells from feeder-free monolayer cultivation of primate ES cells.

Animals↗
PubMed2005

Differential effects of GATA-1 on proliferation and differentiation of erythroid lineage cells.

The zinc finger transcription factor GATA-1 is essential for both primitive (embryonic) and definitive (adult) erythropoiesis. To define the roles of GATA-1 in the production and differentiation of primitive and definitive erythrocytes, we established GATA-1-null embryonic stem cell lines in which GATA-1 was able to be conditionally expressed by using the tetracycline conditional gene expression system. The cells were subjected to hematopoietic differentiation by coculturing on OP9 stroma cells. We expressed GATA-1 in the course of primitive and definitive erythropoiesis and analyzed the ability of GATA-1 to rescue the defective erythropoiesis caused by the GATA-1 null mutation. Our results show that GATA-1 functions in the proliferation and maturation of erythrocytes in a distinctive manner. The early-stage expression of GATA-1 during both primitive and definitive erythropoiesis was sufficient to promote the proliferation of red blood cells. In contrast, the late-stage expression of GATA-1 was indispensable to the terminal differentiation of primitive and definitive erythrocytes. Thus, GATA-1 affects the proliferation and differentiation of erythrocytes by different mechanisms.

Anti-Bacterial Agents↗
PubMed2005

The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis.

PTEN is an important tumor suppressor gene. Hereditary mutation of PTEN causes tumor-susceptibility diseases such as Cowden disease. We used the Cre-loxP system to generate an endothelial cell-specific mutation of Pten (Tie2CrePten) in mice. Tie2CrePten(flox/+) mice displayed enhanced tumorigenesis due to an increase in angiogenesis driven by vascular growth factors. This effect was partially dependent on the PI3K subunits p85alpha and p110gamma. In vitro, Tie2CrePten(flox/+) endothelial cells showed enhanced proliferation/migration. Tie2CrePten(flox/flox) mice died before embryonic day 11.5 (E11.5) due to bleeding and cardiac failure caused by impaired recruitment of pericytes and vascular smooth muscle cells to blood vessels, and of cardiomyocytes to the endocardium. These phenotypes depend strongly on p110gamma rather than on p85alpha and were associated with decreased expression of Ang-1, VCAM-1, connexin 40, and ephrinB2 but increased expression of Ang-2, VEGF-A, VEGFR1, and VEGFR2. Pten is thus indispensable for normal cardiovascular morphogenesis and post-natal angiogenesis, including tumor angiogenesis.

Animals↗
PubMed2005

Incadronate inhibits aminopeptidase N expression in prostatic PC-3 cells.

Bone metastasis is an important cause of morbidity in advanced prostate cancer. Bisphosphonates are widely used for the treatment and prevention of osteoporosis, but recently have been observed to be effective in controlling prostate cancer metastasis. Since aminopeptidase N (AP-N) is known to be involved in the metastasis of prostate cancer, we investigated the effect of bisphosphonate on AP-N expression. Incadronate induced inhibition of AP-N mRNA and protein expression in PC-3 cells. The inhibitory effect of AP-N mRNA expression was also observed in the cells treated with pravastatin and other nitrogen-containing bisphosphonates, which inhibit the key enzyme in the isoprenoid biosynthesis pathway. The decrease of AP-N mRNA expression induced by incadronate was inhibited by co-incubation with geranylgeranyl diphosphate (GGPP). Moreover, GGTI-286 treatment also resulted in reduced AP-N mRNA expression. The translocation of small G protein Rap1 from the cytosol to the membrane was inhibited by incadronate and pravastatin, respectively. These above results indicate that the decrease in AP-N expression elicited by bisphosphonate is related to the inhibition of the mevalonate pathway.

Antineoplastic Agents↗
PubMed2005

Pluripotential competence of cells associated with Nanog activity.

Nanog is a novel pluripotential cell-specific gene that plays a crucial role in maintaining the undifferentiated state of early postimplantation embryos and embryonic stem (ES) cells. We have explored the expression pattern and function of Nanog and a Nanog-homologue, Nanog-ps1.Nanog-ps1 was mapped on Chromosome 7 and shown to be a pseudogene. Immunocytochemical analysis in vivo showed that the NANOG protein was absent in unfertilized oocytes, and was detected in cells of morula-stage embryos, the inner cell mass of blastocysts and the epiblast of E6.5 and E7.5 embryos, but not in primordial germ cells of early postimplantation embryos. In monkey and human ES cells, NANOG expression was restricted to undifferentiated cells. Furthermore, reactivation of the somatic cell-derived Nanog was tightly linked with nuclear reprogramming induced by cell hybridization with ES cells and by nuclear transplantation into enucleated oocytes. Notably, mouse Nanog (+/-) ES cells, which produced approximately half the amount of NANOG produced by wild-type ES cells, readily differentiated to multi-lineage cells in culture medium including LIF. The labile undifferentiated state was fully rescued by constitutive expression of exogenous Nanog. Thus, the activity of Nanog is tightly correlated with an undifferentiated state of cells even in nuclear reprogrammed somatic cells. Nanog may function as a key regulator for sustaining pluripotency in a dose-dependent manner.

Amino Acid Sequence↗