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Biomedical subjects

Toru Nakano

Publications and source records attributed to Toru Nakano.

At least 37 records · Page 2Linked to original sources
PubMed2005

Reduced expression of IL-12 receptor beta2 and IL-18 receptor alpha genes in natural killer cells and macrophages derived from B6-mi/mi mice.

The mi transcriptional factor (MITF) is a basic helix-loop-helix leucine zipper-type transcriptional factor. The mi mutant allele encodes an abnormal MITF, in which one out of four consecutive arginines is deleted in the basic domain. The VGA-9-tg (tg) allele is another mutant allele and considered to be a null mutant allele. C57BL/6 (B6)-mi/mi mice showed abnormal phenotypes of natural killer (NK) cells and macrophages, whereas B6-tg/tg mice did not. The expression levels of the genes for the interleukin-12 receptor (IL-12R) beta2 and IL-18Ralpha were reduced in both the NK cells and macrophages of B6-mi/mi mice, while the expression levels of the corresponding genes in B6-tg/tg mice were unaffected. The B6-mi/mi NK cells and B6-mi/mi macrophages showed impaired responses to stimulation with IL-12, IL-18, and IL-12 plus IL-18 stimulation. The abnormal NK cell and macrophage of B6-mi/mi mice appear to be due to decreased expression of the IL-12Rbeta2 and IL-18Ralpha genes.

Animals↗
PubMed2005

Gas6 induces Akt/mTOR-mediated mesangial hypertrophy in diabetic nephropathy.

BACKGROUND: We have already reported Gas6 is involved in glomerular hypertrophy observed in diabetic nephropathy. However, the molecular mechanisms involved in glomerular hypertrophy are still unknown, especially in vivo. METHODS: In vivo, diabetes was induced in rats and mice by streptozotocin (STZ) and the activation of the Akt/mTOR pathway in glomeruli was examined. In vitro, mesangial hypertrophy was assessed by [(3)H]leucine incorporation and measuring cell areas. RESULTS: Akt, p70 S6 kinase, and 4E-BP-1 were induced and phosphorylated in rat glomerular lysates after 12 weeks of STZ injection when mesangial and glomerular hypertrophy was observed. We then examined the role of Gas6 by treating STZ-rats with warfarin, and found that warfarin treatment inhibited the phosphorylation of these molecules as well as the hypertrophy. We next examined whether high glucose stimulation can induce the expression of Gas6/Axl in mesangial cells. Stimulation of the cells with 25 mmol/L of glucose increased the expression of Gas6/Axl and mesangial cell size compared with that with 5.6 mmol/L of glucose. This hypertrophic effect was abolished in mesangial cells derived from Gas6 knockout mice. We also found that LY294002 and rapamycin blocked Gas6-induced activation of the Akt/mTOR pathway and mesangial hypertrophy. Furthermore, less phosphorylated Akt-positive or 4E-BP-1-positive areas were found in STZ-treated Gas6 knockout mice than in STZ-treated wild-type mice. CONCLUSION: Our study indicates that the Akt/mTOR pathway is a key signaling cascade in Gas6-mediated mesangial and glomerular hypertrophy and revealed a crucial role of Gas6/Axl and the Akt/mTOR pathway in the development of diabetic nephropathy.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Enhanced insulin sensitivity, energy expenditure and thermogenesis in adipose-specific Pten suppression in mice.

Pten is an important phosphatase, suppressing the phosphatidylinositol-3 kinase/Akt pathway. Here, we generated adipose-specific Pten-deficient (AdipoPten-KO) mice, using newly generated Acdc promoter-driven Cre transgenic mice. AdipoPten-KO mice showed lower body and adipose tissue weights despite hyperphagia and enhanced insulin sensitivity with induced phosphorylation of Akt in adipose tissue. AdipoPten-KO mice also showed marked hyperthermia and increased energy expenditure with induced mitochondriagenesis in adipose tissue, associated with marked reduction of p53, inactivation of Rb, phosphorylation of cyclic AMP response element binding protein (CREB) and increased expression of Ppargc1a, the gene that encodes peroxisome proliferative activated receptor gamma coactivator 1 alpha. Physiologically, adipose Pten mRNA decreased with exposure to cold and increased with obesity, which were linked to the mRNA alterations of mitochondriagenesis. Our results suggest that altered expression of adipose Pten could regulate insulin sensitivity and energy expenditure. Suppression of adipose Pten may become a beneficial strategy to treat type 2 diabetes and obesity.

Adiponectin↗
PubMed2004

Involvement of connective tissue-type mast cells in Th1 immune responses via Stat4 expression.

Mast cells are the sentinels of immune systems and, like other immuno-competent cells, they are produced by hematopoietic stem cells. We analyzed the expression of signal transducer and activator of transcription 4 (Stat4), and investigated its role in mast cells. Murine mast cells are usually divided into 2 distinct populations by their distribution and contents of their granules: mucosal mast cells (MMCs) and connective tissue-type mast cells (CTMCs). Stat4 protein was detected in CTMCs but not in MMCs. The absence of Stat4 expression in cultured mast cells was due to the presence of Stat6. In T-helper (Th) cells, Stat4 plays an important role in Th1 shift by inducing a set of genes, such as interferon gamma (IFN-gamma) and interleukin-18 receptor alpha subunit (IL-18Ralpha). As in Th1 shift, we found that Stat4 trans-activated these genes in the Stat4-expressing cultured mast cells, namely, microphthalmia transcription factor (MITF)-deficient cultured MMCs, Stat6-deficient cultured MMCs, and cultured CTMCs. Stat4 also enhanced expression of nitric oxide synthase 2 (NOS2) in CTMCs, which brought about increased levels of NO-dependent cytotoxic activity. These data indicate that expression of Stat4 in CTMCs plays an important role on Th1 immune responses.

Animals↗
PubMed2004

Upstream stimulatory factors stimulate transcription through E-box motifs in the PF4 gene in megakaryocytes.

Platelet factor 4 (PF4) is expressed during megakaryocytic differentiation. We previously demonstrated that the homeodomain proteins (myeloid ecotropic integration site 1 [MEIS1], Pbx-regulating protein 1 [PREP1], and pre-B-cell leukemia transcription factors [PBXs]) bind to the novel regulatory element tandem repeat of MEIS1 binding element [TME] and transactivate the rat PF4 promoter. In the present study, we investigated and identified other TME binding proteins in megakaryocytic HEL cells using mass spectrometry. Among identified proteins, we focused on upstream stimulatory factor (USF1) and USF2 and investigated their effects on the PF4 promoter. USF1 and 2 bound to the E-box motif in the TME and strongly transactivated the PF4 promoter. Furthermore, physiologic bindings of USF1 and 2 to the TME in rat megakaryocytes were demonstrated by the chromatin immunoprecipitation (ChIP) assay. Interestingly, the E-box motif in the TME was conserved in TME-like sequences of both the human and mouse PF4 promoters. USF1 and 2 also bound to the human TME-like sequence and transactivated the human PF4 promoter. Expressions of USF1 and 2 were detected by reverse-transcriptase-polymerase chain reaction (RT-PCR) in the human megakaryocytes derived from CD34+ cells. Thus, these studies demonstrate that the novel TME binding transcription factors, USF1 and 2, transactivate rat and human PF4 promoters and may play an important role in megakaryocytic gene expression.

Amino Acid Motifs↗
PubMed2004

Mili, a mammalian member of piwi family gene, is essential for spermatogenesis.

The piwi family genes, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. To reveal the function of the mammalian homolog of piwi, we produced and analyzed mice with targeted mutations in the Mili gene, which is one of three mouse homologs of piwi. Spermatogenesis in the MILI-null mice was blocked completely at the early prophase of the first meiosis, from the zygotene to early pachytene, and the mice were sterile. However, primordial germ cell development and female germ cell production were not disturbed. Furthermore, MILI bound to MVH, which is an essential factor during the early spermatocyte stage. The similarities in the phenotypes of the MILI- and MVH-deficient mice and in the physical binding properties of MILI and MVH indicate a functional association of these proteins in post-transcriptional regulation. These data indicate that MILI is essential for the differentiation of spermatocytes.

Animals↗
PubMed2004

Tumorigenesis facilitated by Pten deficiency in the skin: evidence of p53-Pten complex formation on the initiation phase.

Pten, a tumor suppressor gene, is mutated in various human cancers and in hereditary cancer syndromes, such as Cowden disease. We have previously developed a knockout mouse in which Pten is specifically disrupted in the skin, resulting in hyperproliferation and spontaneous tumorigenesis of the skin keratinocytes. In this study, we further clarified the effects of Pten deficiency in tumorigenesis, by using a two-step model in intact skin of Pten knockout mouse. Although the conventional protocol requires serial exposures to DMBA and TPA, mice deficient for Pten developed skin papilloma within 6 weeks after a single exposure to DMBA, indicating that loss of Pten has a tumor-promoting effect. Serial exposure to DMBA-TPA ointments produced 10-fold more papillomas in the skin of knockout mice than in the wild-type counterpart, suggesting an increased rate of initiation. Therefore, we precisely examined the effect of DMBA. This treatment was highly apoptotic in wild-type mice, whereas the number of apoptotic cells was diminished in Pten-deficient skin. Moreover, primary keratinocytes isolated from Pten-deficient mice were also resistant to the apoptotic effect of DMBA. The status of p53, Pten proteins and downstream targets of p53, such as p21, 14-3-3, and Reprimo, were also examined, and we found that accumulation of p53 protein and up-regulation of p53 targets were delayed in Pten-knockout skin. These observations suggest that Pten is involved in rapid recruitment of p53 in the tumor initiation phase.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

Differentiation status dependent function of FOG-1.

The molecular interactions between transcription factors and cofactors play crucial roles in various biological processes, including haematopoiesis. FOG-1 is a cognate cofactor of GATA-1, and the FOG-1/GATA-1 complex is essential for the haematopoietic differentiation of erythroid cells and megakaryocytes. In order to elucidate the biological functions of FOG-1 in the different contexts of cell differentiation, we analysed the effects of FOG-1 expression on haematopoietic cell differentiation, using a combination of in vitro differentiation of mouse embryonic stem (ES) cells and conditional gene expression. FOG-1 suppressed the proliferation of primitive and definitive erythroid cells in all stages of differentiation. However, FOG-1 inhibited and enhanced megakaryopoiesis in the early and late differentiation stages, respectively, through different molecular mechanisms. In addition, FOG-1 inhibited the proliferation of ES cells, the molecular mechanism of which differs from those of erythroid and megakaryocytic cells. These results suggest that FOG-1 functions in a cell differentiation context-dependent manner.

Animals↗
PubMed2004

Cross talk between retinoic acid signaling and transcription factor GATA-2.

All-trans-retinoic acid (RA) stimulates differentiation of normal hematopoietic progenitors and acute myeloid leukemia cells. GATA-2 is a transcription factor expressed in early progenitor cells and implicated in the control of the fate of hematopoietic stem cells and progenitor cells. We have investigated the possibility that the GATA and nuclear hormone receptor pathways are functionally linked through direct protein-protein interaction. Here we demonstrate that in human myeloid KG1 cells, RA receptor alpha (RARalpha), the major RAR expressed in hematopoietic cells, associates with GATA-2. This association is mediated by the zinc fingers of GATA-2 and the DNA-binding domain of RARalpha. As a consequence of this interaction, RARalpha is tethered to the DNA sites that are recognized and bound by GATA-2, and the transcriptional activity of GATA-2 becomes RA responsive. The RA responsiveness of GATA-dependent transcription is eliminated by expression of either a dominant negative form of RARalpha or a GATA-2 mutant that fails to interact with RARalpha. Overexpression of RXRalpha inhibits RARalpha binding to the GATA-2-DNA complex, thus resulting in attenuation of the effects of RARalpha on GATA-2 activity. In addition, inhibition by RA of GATA-2-dependent hematopoietic colony formation in an embryonic stem cell model of hematopoietic differentiation provided biological evidence for functional cross talk between RA and GATA-2-dependent pathways.

Amino Acid Motifs↗
PubMed2004

Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas.

PTEN is a tumor suppressor gene mutated in many human cancers, and its expression is reduced or absent in almost half of hepatoma patients. We used the Cre-loxP system to generate a hepatocyte-specific null mutation of Pten in mice (AlbCrePten(flox/flox) mice). AlbCrePten(flox/flox) mice showed massive hepatomegaly and steatohepatitis with triglyceride accumulation, a phenotype similar to human nonalcoholic steatohepatitis. Adipocyte-specific genes were induced in mutant hepatocytes, implying adipogenic-like transformation of these cells. Genes involved in lipogenesis and beta-oxidation were also induced, possibly as a result of elevated levels of the transactivating factors PPARgamma and SREBP1c. Importantly, the loss of Pten function in the liver led to tumorigenesis, with 47% of AlbCrePten(flox/flox) livers developing liver cell adenomas by 44 weeks of age. By 74-78 weeks of age, 100% of AlbCrePten(flox/flox) livers showed adenomas and 66% had hepatocellular carcinomas. AlbCrePten(flox/flox) mice also showed insulin hypersensitivity. In vitro, AlbCrePten(flox/flox) hepatocytes were hyperproliferative and showed increased hyperoxidation with abnormal activation of protein kinase B and MAPK. Pten is thus an important regulator of lipogenesis, glucose metabolism, hepatocyte homeostasis, and tumorigenesis in the liver.

Adipocytes↗
PubMed2004

Stage-specific expression of mouse germ cell-less-1 (mGCL-1), and multiple deformations during mgcl-1 deficient spermatogenesis leading to reduced fertility.

A mouse homologue of Drosophila germ cell-less, mouse germ cell-less-1 (mgcl-1), is highly expressed in the testis. Previous report revealed that the fertility of the mgcl-1(-/-) male mice is reduced significantly as a result of various morphological abnormalities in the sperm (Kimura et al., 2003). To elucidate the function of mgcl-1 in spermatogenesis, the expression of mGCL-1 in the wild-type testis was examined. Immunohistochemical studies demonstrated that mGCL-1 first appeared in the nuclei of the pachytene spermatocytes at stage VI of the seminiferous epithelium, and existed in those of spermatids until step 8 during spermatogenesis. mGCL-1 was not detectable after step 9 spermatids. The testicular cells and epididymal sperm were further analyzed morphologically using mgcl-1(-/-) mice. In the testis, deformed nuclei first occurred in the pachytene spermatocytes at stage VI, which is consistent with the time of the first appearance of the mGCL-1 protein in the wild-type testis. Abnormal nuclei and acrosomes were found in spermatids after step 5, and nuclei of the spermatids and epididymal sperm were frequently invaginated. In addition, variously deformed sperm such as bent-neck, multi-headed or multi-nucleated sperm were observed in the mgcl-1(-/-) cauda epididymidis. However, several key structures such as the acroplaxome marginal ring (Kierszenbaum et al., 2003), postacrosomal sheath, and posterior ring apparently formed. In addition, MN7 and MN13, essential substances for fertilization that are located in sperm heads, were detectable in the mgcl-1 null sperm. These observations provide important insights into the mechanisms regulating the nuclear architecture and causes of human infertility.

Animals↗
PubMed2003

Stella is a maternal effect gene required for normal early development in mice.

stella is a novel gene specifically expressed in primordial germ cells, oocytes, preimplantation embryos, and pluripotent cells. It encodes a protein with a SAP-like domain and a splicing factor motif-like structure, suggesting possible roles in chromosomal organization or RNA processing. Here, we have investigated the effects of a targeted mutation of stella in mice. We show that while matings between heterozygous animals resulted in the birth of apparently normal stella null offspring, stella-deficient females displayed severely reduced fertility due to a lack of maternally inherited Stella-protein in their oocytes. Indeed, we demonstrate that embryos without Stella are compromised in preimplantation development and rarely reach the blastocyst stage. stella is thus one of few known mammalian maternal effect genes, as the phenotypic effect on embryonic development is mainly a consequence of the maternal stella mutant genotype. Furthermore, we show that STELLA that is expressed in human oocytes is also expressed in human pluripotent cells and in germ cell tumors. Interestingly, human chromosome 12p, which harbours STELLA, is consistently overrepresented in these tumors. These findings suggest a similar role for STELLA during early human development as in mice and a potential involvement in germ cell tumors.

Amino Acid Sequence↗
PubMed2003

Cadherin-mediated cell interaction regulates germ cell determination in mice.

The germ cell lineage segregates from the somatic cell lineages in early embryos. Germ cell determination in mice is not regulated by maternally inherited germplasm, but is initiated within the embryo during gastrulation. However, the mechanisms of germ cell specification in mice remain unknown. We located precursors to primordial germ cells (PGCs) within early embryos, and show here that cell-cell interaction among these precursors is required for germ cell specification. We found that the expression of a calcium-dependent cell adhesion molecule, E-cadherin, is restricted to the proximal region of extra-embryonic mesoderm that contains PGC precursors, and that blocking the functions of E-cadherin with an antibody inhibits PGC formation in vitro. These results showed that E-cadherin-mediated cell-cell interaction among cells containing PGC precursors is essential to directing such cells to the germ cell fate.

Animals↗
PubMed2003

Effect of Gas6 on secretory phospholipase A(2)-IIA-induced apoptosis in cortical neurons.

Gas6, a product of the growth-arrest-specific gene 6, protects cortical neurons from amyloid beta protein (Abeta)-induced apoptosis. Neuronal apoptosis is also caused by human group IIA secretory phospholipase A(2) (sPLA(2)-IIA), which is expressed in the cerebral cortex after brain ischemia. sPLA(2)-IIA induces Ca(2+) influx via L-type voltage-sensitive calcium channels (L-VSCCs), leading to its neurotoxicity. In the present study, we investigated effects of Gas6 on sPLA(2)-IIA-induced cell death in primary cultures of rat cortical neurons. sPLA(2)-IIA caused neuronal cell death in a concentration- and time-dependent manner. Gas6 significantly prevented neurons from sPLA(2)-IIA-induced cell death. Gas6 suppressed sPLA(2)-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Prior to cell death, sPLA(2)-IIA increased the influx of Ca(2+) into neurons through L-VSCCs. Gas6 significantly inhibited the sPLA(2)-IIA-induced Ca(2+) influx. The blocker of L-VSCCs also suppressed sPLA(2)-IIA-induced neuronal cell death. The cortical cultures contained few non-neuronal cells, indicating that Gas6 affected the survival of neurons directly, but not indirectly via non-neuronal cells. In conclusion, we demonstrate that Gas6 rescues cortical neurons from sPLA(2)-IIA-induced apoptosis. Furthermore, the present study indicates that inhibition of L-VSCC contributes to the neuroprotective effect of Gas6.

Animals↗
PubMed2003

Enhanced degradation of MDM2 by a nuclear envelope component, mouse germ cell-less.

A mouse homologue of Drosophila germ cell less, mouse germ cell less-1 (mgcl-1), encodes a nuclear envelope component essential for nuclear integrity. To analyze the molecular function of mGCL-1, we carried out two hybrid screening and found that mGCL-1 bound to the gene product of tumor susceptibility gene 101 (tsg101). Effects of mGCL-1 on the expression of MDM2-p53 axis were examined, since TSG101 has been shown to elevate the amount of MDM2 by inhibiting the ubiquitination. mGCL-1 significantly reduced the amount of MDM2 probably by changing the sub-cellular localization of the MDM2 and facilitating the ubiquitination of MDM2. In addition, the amount of p53 was increased and transactivation by p53 was enhanced by mGCL-1. Thus, mGCL-1 turned out to be a factor modulating MDM2-p53 axis by enhanced degradation of MDM2.

Animals↗
PubMed2003

A stromal cell-derived membrane protein that supports hematopoietic stem cells.

Hematopoietic stem cells cannot be maintained in vitro without stromal cells, even if they are provided with growth factors, and it is likely that supportive cells in the bone marrow express membrane or secreted proteins that maintain hematopoiesis. Here we show that mKirre, a mammalian homolog of the gene kirre of Drosophila melanogaster, encodes a type Ia membrane protein that is involved in the hematopoietic supportive capacity of OP9 mouse stromal cells. Repressing mKirre expression with a short interfering RNA significantly reduced this supportive capacity. Our data suggest that mKirre is cleaved by metalloproteinases and that the extracellular domain of mKirre is responsible for supporting hematopoietic stem cells. These results contribute to our understanding of the mechanisms by which the hematopoietic microenvironment regulates hematopoiesis.

Amino Acid Sequence↗