Biomedical subjects

Hitoshi Niwa

Publications and source records attributed to Hitoshi Niwa.

36 records · Page 2Linked to original sources

PubMed2004

Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells.

Oct-3/4 is a key transcriptional factor whose expression level governs the fate of primitive inner cell mass and embryonic stem (ES) cells. Previously, an upstream 3.3-kb distal enhancer (DE) fragment was identified to be responsible for the specific expression of mouse Oct-3/4 in the inner cell mass and ES cells. However, little is known about the cis-elements and trans-factors required for DE activity. In this study, we identified a novel cis-element, called Site 2B here, located approximately 30 bp downstream from Site 2A, which was previously revealed in DE by an in vivo chemical modification experiment. Using the luciferase reporter assay, we demonstrated that both Site 2A and Site 2B are necessary and sufficient for activating DE in the contexts of both the native Oct-3/4 promoter and the heterologous thymidine kinase minimal promoter. In an electrophoretic mobility shift assay we showed that Site 2B specifically binds to Oct-3/4 and Sox2 when ES-derived cell extracts were used, whereas Site 2A binds to a factor(s) present in both ES and NIH 3T3 cells. Furthermore, we showed that the physiological level of Oct-3/4 in ES cells is required for Site 2B-mediated DE activity using the inducible knock-out system of Oct-3/4 in ES cells. These results indicate that Oct-3/4 is a member of the gene family regulated by Oct-3/4 and Sox2, as reported before for the FGF-4, UTF1, Sox2, and Fbx15 genes. Thus, Oct-3/4 and Sox2 comprise a regulatory complex that controls the expression of genes important for the maintenance of the primitive state, including themselves. This autoregulatory circuit of the Sox2.Oct-3/4 complex may contribute to maintaining robustly the precise expression level of Oct-3/4 in primitive cells.

Animals↗

PubMed2004

Nonylphenol induces the death of neural stem cells due to activation of the caspase cascade and regulation of the cell cycle.

Endocrine disruptors (EDs) are a great concern throughout the world, because they have adverse effects on human health and wildlife. In the present study, we investigated the effects of EDs on the proliferation and survival of murine neural stem cells (NSCs). In contrast to bisphenol A, phthalic acid benzyl n-butyl ester, phthalic acid di-n-butyl ester and phthalic acid di(2-ethylhexyl) ester, the treatment of NSCs with 4-nonylphenol for 24 h inhibited cell growth in a concentration-dependent manner. In addition, treatment with 4-nonylphenol resulted in nuclear condensation and DNA fragmentation (morphological changes due to apoptosis) in NSCs after 12 h of exposure, and activated caspase-3 after 6 h and 9 h of exposure. Furthermore, an exposure to 4-nonylphenol led to the accumulation of cells at the G2/M phase interface and down-regulated the protein levels of cyclin A and B1, which are the major regulatory proteins at the G2 to M transition of the cell cycle. Together, these results indicate that, in contrast to other EDs, 4-nonylphenol may exhibit a potent cytotoxicity through apoptosis via the caspase cascade and cell cycle arrest at the G2/M phase, and suggest that 4-nonylphenol may affect neurogenesis in the CNS.

Animals↗

PubMed2004

A novel mechanism for regulating clonal propagation of mouse ES cells.

Self-renewal and differentiation of embryonic stem (ES) cells are controlled by the combinatorial action of extracellular signals and regulation of gene expression. For characterizing the entire molecular mechanism governing these events, we first established a feeder- and serum-free culture system in which mouse ES cells could propagate in clonal density in keeping with proper pluripotency. Supplementation of peptide hormones such as adrenocorticotropic hormone (ACTH) is required to remove serum, and the key event in this phenomenon may be the inhibition of the adenylyl cyclase (AC) activity, as it replaces the effect of these peptides. Because ES cells themselves produce the same activity, the finding suggests a novel mechanism in which activation of AC restricts clonal propagation of pluripotent stem cells.

Adenylyl Cyclase Inhibitors↗

PubMed2004

The Sox-2 regulatory regions display their activities in two distinct types of multipotent stem cells.

The Sox-2 gene is expressed in embryonic stem (ES) cells and neural stem cells. Two transcription enhancer regions, Sox-2 regulatory region 1 (SRR1) and SRR2, were described previously based on their activities in ES cells. Here, we demonstrate that these regulatory regions also exert their activities in neural stem cells. Moreover, our data reveal that, as in ES cells, both SRR1 and SRR2 show their activities rather specifically in multipotent neural stem or progenitor cells but cease to function in differentiated cells, such as postmitotic neurons. Systematic deletion and mutation analyses showed that the same or at least overlapping DNA elements of SRR2 are involved in its activity in both ES and neural stem or progenitor cells. Thus, SRR2 is the first example of an enhancer in which a single regulatory core sequence is involved in multipotent-state-specific expression in two different stem cells, i.e., ES and neural stem cells.

Animals↗

PubMed2003

Diclofenac inhibits proliferation and differentiation of neural stem cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in clinical situations as anti-inflammatory, analgesic and antipyretic drugs. However, it is still unknown whether NSAIDs have effects on the development of the central nervous system. In the present study, we investigated the effects of NSAIDs on neural stem cell (NSC) proliferation and differentiation into neurons. In contrast to aspirin, naproxen, indomethacin and ibuprofen, treatment with diclofenac (10 microM) for 2 days induced the death of NSCs in a concentration-dependent manner. Diclofenac also inhibited the proliferation of NSCs and their differentiation into neurons. Treatment with diclofenac resulted in nuclear condensation (a morphological change due to apoptosis of NSCs) 24hr after the treatment and activated caspase-3 after 6 hr, indicating that diclofenac may cause apoptosis of neuronal cells via activation of the caspase cascade. These results suggest that diclofenac may affect the development of the central nervous system.

Animals↗

PubMed2003

Genomic organization and promoter analysis of the Dnmt3b gene.

The Dnmt3b gene encodes a de novo DNA methyltransferase that is essential for normal mouse development. It is highly expressed in early embryos and embryonic stem (ES) cells but downregulated in most adult somatic tissues. To gain insight into the regulation of Dnmt3b, we have isolated a mouse genomic bacterial artificial chromosome clone that contains the Dnmt3b gene. Complete sequence analysis of the clone demonstrated that Dnmt3b consists of at least 24 exons and spans 38 kilobases. S1 nuclease analysis identified two adjacent transcriptional start sites located downstream of a unique TATA-like element in a CpG island. There was an unknown gene which we named mU(3) 17 kb upstream of the Dnmt3b locus, and it was transcribed ubiquitously and in the opposite direction of Dnmt3b. Transfection analysis revealed that the minimal promoter region containing an Sp1 site was active even in somatic cells, and that there were several repressor elements within 7.9 kb upstream of Dnmt3b downregulated this gene specifically in somatic cells but not in ES cells. These findings provide a basis for future detailed studies of the mechanisms controlling Dnmt3b expression.

3T3 Cells↗

PubMed2003

HEX acts as a negative regulator of angiogenesis by modulating the expression of angiogenesis-related gene in endothelial cells in vitro.

OBJECTIVE: The hematopoietically expressed homeobox (HEX) is transiently expressed in endothelial cells (ECs) during vascular formation in embryo. Here, we investigated whether HEX played any role in angiogenesis-related properties of ECs in vitro. METHODS AND RESULTS: We transiently overexpressed HEX in human umbilical vein ECs (HUVECs). To our surprise, HEX completely abrogated the response of HUVECs to vascular endothelial growth factor (VEGF) with regard to proliferation, migration, and invasion and abolished network formation by HUVECs on Matrigel. cDNA microarray analysis and quantitative real-time reverse transcription-polymerase chain reaction combined with Western blotting revealed that HEX significantly repressed the expression of VEGF receptor-1, VEGF receptor-2, neuropilin-1, tyrosine kinase with Ig and EGF homology domains (TIE)-1, TIE-2, and the integrin alpha(v) subunit, whereas it augmented the expression of endoglin in HUVECs. We established murine embryonic stem cells that were stably transfected with HEX sense cDNA or antisense cDNA, and we examined the in vitro differentiation to ECs. Although the expression of VEGF receptor-2 was decreased in sense transfectants, the number of cells expressing VE-cadherin, a specific marker of ECs, was not altered. CONCLUSIONS: Our present results suggest that HEX may not affect the differentiation of ECs but acts as a negative regulator of angiogenesis.

Animals↗

PubMed2003

Essential role for ERK2 mitogen-activated protein kinase in placental development.

BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) has been implicated in cell proliferation, differentiation, and survival. However, its role in vivo remains to be determined. RESULTS: Here we show that the targeted disruption of the mouse ERK2 gene results in embryonic lethality by E11.5 and severe abnormality of the placenta. In these animals, the labyrinthine layer of the placenta is very thin and few foetal blood vessels are observed. ERK2 mutants can be rescued by the transgenic expression of ERK2, demonstrating that these abnormalities are caused by ERK2-deficiency. Although ERK2-deficient fetuses are much smaller than wild-type littermates, this seems to be secondary to malfunction of the placenta. When the placental defect is rescued by tetraploid-aggregation, ERK2-deficient foetuses grow as well as littermate controls. CONCLUSION: These observations indicate that ERK2 is essential for placental development and suggest that ERK2 in the trophoblast compartment may be indispensable for the vascularization of the labyrinth.

Animals↗

PubMed2003

Enhanced genomic instability and defective postreplication repair in RAD18 knockout mouse embryonic stem cells.

In lower eukaryotes, Rad18 plays a crucial role in postreplication repair. Previously, we isolated a human homologue of RAD18 (hRAD18) and showed that human cells overexpressing hRad18 protein with a mutation in the RING finger motif are defective in postreplication repair. Here, we report the construction of RAD18-knockout mouse embryonic stem cells by gene targeting. These cells had almost the same growth rate as wild-type cells and manifested phenotypes similar to those of human cells expressing mutant Rad18 protein: hypersensitivity to multiple DNA damaging agents and a defect in postreplication repair. Mutation was not induced in the knockout cells with any higher frequencies than in wild-type cells, as shown by ouabain resistance. In the knockout cells, spontaneous sister chromatid exchange (SCE) occurred with twice the frequency observed in normal cells. After mild DNA damage, SCE was threefold higher in the knockout cells, while no increase was observed in normal cells. Stable transformation efficiencies were approximately 20-fold higher in knockout cells, and gene targeting occurred with approximately 40-fold-higher frequency than in wild-type cells at the Oct3/4 locus. These results indicate that dysfunction of Rad18 greatly increases both the frequency of homologous as well as illegitimate recombination, and that RAD18 contributes to maintenance of genomic stability through postreplication repair.

Amino Acid Motifs↗

PubMed2003

Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development.

Embryonic stem (ES) cells are immortal and pluripotent cells derived from early mammalian embryos. Transcription factor Oct3/4 is essential for self-renewal of ES cells and early mouse development. However, only a few Oct3/4 target genes have been identified. In this study, we found that F-box-containing protein Fbx15 was expressed predominantly in mouse undifferentiated ES cells. Inactivation of Oct3/4 in ES cells led to rapid extinction of Fbx15 expression. Reporter gene analyses demonstrated that this ES cell-specific expression required an 18-bp enhancer element located approximately 500 nucleotides upstream from the transcription initiation site. The enhancer contained an octamer-like motif and an adjacent Sox-binding motif. Deletion or point mutation of either motif abolished the enhancer activity. The 18-bp fragment became active in NIH 3T3 cells when Oct3/4 and Sox2 were coexpressed. A gel mobility shift assay demonstrated cooperative binding of Oct3/4 and Sox2 to the enhancer sequence. In mice having a beta-galactosidase gene knocked into the Fbx15 locus, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining was detected in ES cells, early embryos (two-cell to blastocyst stages), and testis tissue. Despite such specific expression of Fbx15, homozygous mutant mice showed no gross developmental defects and were fertile. Fbx15-null ES cells were normal in morphology, proliferation, and differentiation. These data demonstrate that Fbx15 is a novel target of Oct3/4 but is dispensable for ES cell self-renewal, development, and fertility.

3T3 Cells↗

PubMed2003

Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures.

Oct3/4 plays a critical role in maintaining embryonic stem cell pluripotency. Regulatable transgene-mediated sustained Oct3/4 expression in ES cells cultured in serum-free LIF-deficient medium caused accelerated differentiation to neuroectoderm-like cells that expressed Sox2, Otx1 and Emx2 and subsequently differentiated into neurons. Neurogenesis of ES cells is promoted by SDIA (stromal cell-derived inducing activity), which accumulates on the PA6 stromal cell surface. Oct3/4 expression in ES cells was maintained by SDIA whereas without it expression was promptly downregulated. Suppression of Oct3/4 abolished neuronal differentiation even after stimulation by SDIA. In contrast, sustained upregulated Oct3/4 expression enhanced SDIA-mediated neurogenesis of ES cells. Therefore, Oct3/4 appears to promote neuroectoderm formation and subsequent neuronal differentiation from ES cells.

Animals↗

PubMed2002

Nitration of PPARgamma inhibits ligand-dependent translocation into the nucleus in a macrophage-like cell line, RAW 264.

Nitration of tyrosine residues in proteins has been observed in many inflammatory tissues of arthritis, ulcerative colitis, septic shock and ischemia-reperfusion injury. Although several studies have been carried out, it is still unclear what type of protein is nitrated and whether tyrosine nitration interferes with protein function. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor whose activation is linked to several physiological pathways including regulation of insulin sensitivity and control of inflammation. PPARgamma possesses several tyrosine residues, which might be potential targets for nitration by peroxynitrite during inflammatory responses. Here we have investigated whether PPARgamma is nitrated in macrophage-like RAW 264 cells and the effect of nitration on the translocation of PPARgamma into the nucleus. Western blot analysis showed that tumor necrosis factor-alpha, lipopolysaccharide or peroxynitrite treatment significantly increases the nitration of PPARgamma. Cell fractionation analysis and immunofluorescence coupled with confocal laser microscopy revealed that nitration of PPARgamma inhibits its ligand-dependent translocation from the cytosol into the nucleus. Together, these results indicate that nitration of PPARgamma during inflammation may be involved in a reduction in the control of inflammatory responses and also in the development of resistance to PPARgamma ligand-based therapies against inflammation.

Active Transport, Cell Nucleus↗

PubMed2002

Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex.

Sox-2 is a transcriptional cofactor expressed in embryonic stem (ES) cells as well as in neuronal cells. It has been demonstrated that Sox-2 plays an important role in supporting gene expression in ES cells, especially by forming a complex with embryonic Octamer factor, Oct-3/4. Here, we have analyzed the regulatory regions of the Sox-2 gene and identified two enhancers which stimulate transcription in ES cells as well as in embryonal carcinoma cells. These regulatory regions, which we termed Sox regulatory regions (SRR) 1 and 2, exert their function specifically when cells are in an undifferentiated state. Interestingly, like the regulatory elements of FGF-4 and UTF1 genes, combinatorial action of Octamer and Sox-2 binding sites support the SRR2 activity. However, biochemical analyses reveal that, due to the unique sequence and/or its organization, the SRR2 bears distinct characteristics from those of FGF-4 and UTF1 regulatory elements. That is, unlike the FGF-4 gene enhancer, the SRR2 precludes the binding of the Oct-1-Sox-2 complex. The difference between the SRR2 and UTF1 regulatory element is in the ability of SRR2 to recruit the Oct-6-Sox-2 complex as well as the Oct-3/4-Sox-2 complex. Co-transfection analyses confirm that both complexes are able to stimulate transcription through the SRR2 element.

Animals↗

PubMed2002

Differentiation of embryonic stem cells is induced by GATA factors.

Extraembryonic endoderm (ExE) is differentiated from the inner cell mass of the late blastocyst-stage embryo to form visceral and parietal endoderm, both of which have an important role in early embryogenesis. The essential roles of Gata-6 and Gata-4 on differentiation of visceral endoderm have been identified by analyses of knockout mice. Here we report that forced expression of either Gata-6 or Gata-4 in embryonic stem (ES) cells is sufficient to induce the proper differentiation program towards ExE. We believe that this is the first report of a physiological differentiation event induced by the ectopic expression of a transcription factor in ES cells.

Animals↗

PubMed2002

Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis.

Neuropilins (NP1 and NP2) are vascular endothelial growth factor (VEGF) receptors that mediate developmental and tumor angiogenesis. Transgenic mice, in which both NP1 and NP2 were targeted (NP1(-/-)NP2(-/-)) died in utero at E8.5. Their yolk sacs were totally avascular. Mice deficient for NP2 but heterozygous for NP1 (NP1(+/-)NP2(-/-)) or deficient for NP1 but heterozygous for NP2 (NP1(-/-)NP2(+/-)) were also embryonic lethal and survived to E10-E10.5. The E10 yolk sacs and embryos were easier to analyze for vascular phenotype than the fragile poorly formed 8.5 embryos. The vascular phenotypes of these E10 mice were very abnormal. The yolk sacs, although of normal size, lacked the larger collecting vessels and had less dense capillary networks. PECAM staining of yolk sac endothelial cells showed the absence of branching arteries and veins, the absence of a capillary bed, and the presence of large avascular spaces between the blood vessels. The embryos displayed blood vessels heterogeneous in size, large avascular regions in the head and trunk, and blood vessel sprouts that were unconnected. The embryos were about 50% the length of wild-type mice and had multiple hemorrhages. These double NP1/NP2 knockout mice had a more severe abnormal vascular phenotype than either NP1 or NP2 single knockouts. Their abnormal vascular phenotype resembled those of VEGF and VEGFR-2 knockouts. These results suggest that NRPs are early genes in embryonic vessel development and that both NP1 and NP2 are required.

Animals↗

PubMed2002

Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity.

Large-scale gene expression profiling was performed on embryo-derived stem cell lines to identify molecular signatures of pluripotency and lineage specificity. Analysis of pluripotent embryonic stem (ES) cells, extraembryonic-restricted trophoblast stem (TS) cells, and terminally-differentiated mouse embryo fibroblast (MEF) cells identified expression profiles unique to each cell type, as well as genes common only to ES and TS cells. Whereas most of the MEF-specific genes had been characterized previously, the majority (67%) of the ES-specific genes were novel and did not include known differentiated cell markers. Comparison with microarray data from embryonic material demonstrated that ES-specific genes were underrepresented in all stages sampled, whereas TS-specific genes included known placental markers. Investigation of four novel TS-specific genes showed trophoblast-restricted expression in cell lines and in vivo, whereas one uncharacterized ES-specific gene, Esg-1, was found to be exclusively associated with pluripotency. We suggest that pluripotency requires a set of genes not expressed in other cell types, whereas lineage-restricted stem cells, like TS cells, express genes predictive of their differentiated lineage.

Animals↗

PubMed2002

Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells.

Transcription factors of the POU family govern cell fate through combinatorial interactions with coactivators and corepressors. The POU factor Oct-3/4 can define differentiation, dedifferentation, or self-renewal of pluripotent embryonic stem (ES) cells in a sensitive, dose-dependent manner (H. Niwa, J.-I. Miyazali, and A. G. Smith, Nat. Genet. 24:372-376, 2000). Here we have developed a complementation assay based on the ability of Oct-3/4 transgenes to rescue self-renewal in conditionally null ES cells and used this to define which domains of Oct-3/4 are required to sustain the undifferentiated stem cell phenotype. Surprisingly, we found that molecules lacking either the N-terminal or C-terminal transactivation domain, though not both, can effectively replace full-length Oct-3/4. Furthermore, a fusion of the heterologous transactivation domain of Oct-2 to the Oct-3/4 POU domain can also sustain self-renewal. Thus, the unique function of Oct-3/4 in ES cell propagation resides in combination of the specific POU domain with a generic proline-rich transactivation domain. Interestingly, however, Oct-3/4 target gene expression elicited by the N- and C-terminal transactivation domains is not identical, indicating that at least one class of genes activated by Oct-3/4 is not required for ES cell propagation.

Animals↗

PubMedDate not supplied

Dental management prior to hematopoietic stem cell transplantation.

The authors investigated oral health treatment in patients scheduled for hematopoietic stem cell transplantation. Fifteen patients were scheduled for bone marrow transplantation: 12 had leukemia, two had aplastic anemia, and one had myelodysplastic syndrome. Twenty-three patients were scheduled to undergo peripheral blood stem cell transplantation: 20 had malignant lymphoma and three had leukemia. Patients with platelet counts of < or = 50,000/mm3 who required tooth extraction and those with platelet counts of < or = 30,000 /mm3 who required scaling received platelet transfusions before the dental treatment. We saw no bleeding problems in these patients. Dental care for patients who had neutrophil counts of > or = 500/mm3 included tooth extractions or scaling, with no postoperative infection. Even those with severe immunosuppression (neutrophil count: 100-150/mm3) tolerated treatment when prophylactic measures, such as intravenous administration of antibiotics, were used. One patient with a neutrophil count of < or = 50/mm3 developed an infection after even conservative therapy. Thus, caution should be exercised when treating patients scheduled for HSCT as even conservative therapy can initiate and infection.

Adult↗