Biomedical subjects

Junji Takeda

Publications and source records attributed to Junji Takeda.

At least 19 recordsLinked to original sources

PubMed2007

Transposon-tagged mutagenesis in the rat.

Although the laboratory rat (Rattus norvegicus) is an indispensable experimental animal for biomedical research and drug development, the lack of embryonic stem cell lines hampers gene-knockout studies. Here we report the successful generation of insertional mutant rats using the Sleeping Beauty (SB) transposon system. This would benefit a variety of biomedical research fields for which the rat model is better suited than the mouse model.

Adenosine Triphosphatases↗

PubMed2006

Sleeping beauty transposase has an affinity for heterochromatin conformation.

The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influenced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the transposition event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with intense DAPI (4',6'-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for heterochromatin conformation leads to enhancement of SB transposition efficiency.

Animals↗

PubMed2006

Bloom's syndrome gene-deficient phenotype in mouse primary cells induced by a modified tetracycline-controlled trans-silencer.

We recently reported genome-wide bi-allelic mutagenesis and phenotype-based genetic screening by tetracycline-regulated disruption of the Bloom's syndrome gene (Blm) in mouse embryonic stem (ES) cells. However, the same approach was hampered in mouse tissues owing to leaky expression of the Blm gene, which is the major obstacle in the tetracycline regulatory system. Here we describe a single-chain reverse tetracycline-controlled trans-silencer (sc rtTS) which reduces leaky expression in the tet-off system. The sc rtTS consists of two silencer moieties linked by a 36 amino acid linker. Although the silencer moiety contained a dimerization domain compatible with the tetracycline-controlled transactivator (tTA), heterodimerization with tTA was prevented because intramolecular self-assembly between linked silencer moieties was preferred. The system was applied to mouse splenic lymphocytes and elevation of sister chromatid exchange, the hallmark of Blm dysfunction, was observed in the presence of doxycycline. A cassette containing both sc rtTS and tTA was introduced into the Blm allele in ES cells and reduction of basal activity was observed upon doxycycline treatment. Our data demonstrate effectiveness of sc rtTS in the tet-off system. Application of sc rtTS in mice may allow us to implement bi-allelic mutagenesis in vivo.

Adenosine Triphosphatases↗

PubMed2006

In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice.

The visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice. Here we report the generation of two strains of mice, each expressing a spectrally distinct lipid-modified GFP-variant fluorescent protein fusion. The CAG::GFP-GPI strain exhibited widespread expression of a glycosylphosphatidylinositol-tagged green fluorescent protein (GFP) fusion, while the CAG::myr-Venus strain exhibited widespread expression of a myristoyl-Venus yellow fluorescent protein fusion. Imaging of live transgenic embryonic stem (ES) cells, either live or fixed embryos and postnatal tissues demonstrated that glycosylphosphatidyl inositol- and myristoyl-tagged GFP-variant fusion proteins are targeted to and serve as markers of the plasma membrane. Moreover, our data suggest that these two lipid-modified protein fusions are dynamically targeted both to overlapping as well as distinct lipid-enriched compartments within cells. These transgenic strains not only represent high-contrast reporters of cell morphology and plasma membrane dynamics, but also may be used as in vivo sensors of lipid localization. Furthermore, combining these reporters with the study of mouse mutants will be a step forward in understanding the inter- and intracellular behaviors underlying morphogenesis in both normal and mutant contexts.

Animals↗

PubMed2006

Rad54 is dispensable for the ALT pathway.

Some immortal cells use the alternative lengthening of telomeres (ALT) pathway to maintain their telomeres instead of telomerase. Previous studies revealed that homologous recombination (HR) contributes to the ALT pathway. To further elucidate molecular mechanisms, we inactivated Rad54 involved in HR, in mouse ALT embryonic stem (ES) cells. Although Rad54-deficient ALT ES cells showed radiosensitivity in line with expectation, cell growth and telomeres were maintained for more than 200 cell divisions. Furthermore, although MMC-stimulated sister chromatid exchange (SCE) was suppressed in the Rad54-deficient ALT ES cells, ALT-associated telomere SCE was not affected. This is the first genetic evidence that mouse Rad54 is dispensable for the ALT pathway.

Animals↗

PubMed2006

Sleeping beauty transposon-based phenotypic analysis of mice: lack of Arpc3 results in defective trophoblast outgrowth.

The Sleeping Beauty (SB) transposon system has generated many transposon-insertional mutant mouse lines, some of which have resulted in embryonic lethality when bred to homozygosity. Here we report one such insertion mapped to the mouse actin-related protein complex subunit 3 gene (Arpc3). Arpc3 is a component of the Arp2/3 complex, which plays a major role in actin nucleation with Y-shaped branching from the mother actin filament in response to migration signaling. Arpc3 transposon-inserted mutants developed only to the blastocyst stage. In vitro blastocyst culture of Arpc3 mutants exhibited severe spreading impairment of trophoblasts. This phenotype was also observed in compound heterozygotes generated using conventional gene-targeted and transposon-inserted alleles. Arpc3-deficient mutants were shown to lack actin-rich structures in the spreading trophoblast. Electron microscopic analysis demonstrated the lack of mesh-like structures at the cell periphery, suggesting a role of Arpc3 in Y-shaped branching formation. These data indicate the importance of Arpc3 in the Arp2/3 complex for trophoblast outgrowth and suggest that Arpc3 may be indispensable for implantation.

Actin-Related Protein 2-3 Complex↗

PubMed2005

Signal transducer and activator of transcription 3 is a key regulator of keratinocyte survival and proliferation following UV irradiation.

UVB irradiation of signal transducer and activator of transcription 3 (Stat3)-deficient keratinocytes resulted in a high incidence of apoptosis compared with controls. Conversely, forced expression of Stat3 desensitized keratinocytes to UVB-induced apoptosis. Upon UVB exposure, keratinocyte Stat3 was rapidly dephosphorylated, followed by decreases of both Stat3 mRNA and protein levels in a p53-independent manner. Vanadate treatment reversed the UVB-induced down-regulation of Stat3 and generation of apoptotic keratinocytes, suggesting the involvement of a tyrosine phosphatase. Furthermore, Stat3 was required for UVB-induced proliferation of follicular keratinocytes, leading to epidermal thickening. Finally, constitutive activation of Stat3 was observed in UVB-induced squamous cell carcinomas of either mice or human origin. These data suggest that Stat3 is required for survival and proliferation of keratinocytes following UVB exposure and that Stat3 is tightly regulated as part of a novel protective mechanism against UVB-induced skin cancer.

Animals↗

PubMed2005

Raf-1 regulates Rho signaling and cell migration.

Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

Animals↗

PubMed2005

Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization.

The angiotensin-converting enzyme (ACE) is a key regulator of blood pressure. It is known to cleave small peptides, such as angiotensin I and bradykinin and changes their biological activities, leading to upregulation of blood pressure. Here we describe a new activity for ACE: a glycosylphosphatidylinositol (GPI)-anchored protein releasing activity (GPIase activity). Unlike its peptidase activity, GPIase activity is weakly inhibited by the tightly binding ACE inhibitor and not inactivated by substitutions of core amino acid residues for the peptidase activity, suggesting that the active site elements for GPIase differ from those for peptidase activity. ACE shed various GPI-anchored proteins from the cell surface, and the process was accelerated by the lipid raft disruptor filipin. The released products carried portions of the GPI anchor, indicating cleavage within the GPI moiety. Further analysis by high-performance liquid chromatography-mass spectrometry predicted the cleavage site at the mannose-mannose linkage. GPI-anchored proteins such as TESP5 and PH-20 were released from the sperm membrane of wild-type mice but not in Ace knockout sperm in vivo. Moreover, peptidase-inactivated E414D mutant ACE and also PI-PLC rescued the egg-binding deficiency of Ace knockout sperms, implying that ACE plays a crucial role in fertilization through this activity.

Animals↗

PubMed2005

Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system.

Recent consolidation of the whole-genome sequence with genome-wide transcriptome profiling revealed the existence of functional units within the genome in specific chromosomal regions, as seen in the coordinated expression of gene clusters and colocalization of functionally related genes. An efficient region-specific mutagenesis screen would greatly facilitate research in addressing the importance of these clusters. Here we use the 'local hopping' phenomenon of a DNA-type transposon, Sleeping Beauty (SB), for region-specific saturation mutagenesis. A transgenic mouse containing both transposon (acts as a mutagen) and transposase (recognizes and mobilizes the transposon) was bred for germ-cell transposition events, allowing us to generate many mutant mice. All genes within a 4-Mb region of the original donor site were mutated by SB, indicating the potential of this system for functional genomic studies within a specific chromosomal region.

Animals↗

PubMed2004

Target-site preferences of Sleeping Beauty transposons.

The Sleeping Beauty (SB) transposon is a Tc1/mariner family transposon that has applications in vertebrate animals for gene transfer, gene-tagging, and human gene therapy. In this study, we analyzed the target-site preferences of the SB transposon. At the genomic level, integration of SB transposons with respect to genes (exons and introns) and intergenic regions appears fairly random but not on a micro-scale. Although there appears to be a consensus sequence around the vicinity of the target sites, the primary sequence is not the determining factor for target selection. When integrations were examined over a limited topography, the sites used most often for integration did not match the consensus sequence. Rather, a unique deformation inherent in the sequence may be a recognition signal for target selection. The deformation is characterized by an angling of the target site such that the axis around the insertion site is off-center, the rotation of the helix (twisting) is non-uniform and there is an increase in the distance between the central base-pairs. Our observations offer several hypothetical insights into the transposition process. Our observations suggest that particular deformations of the double helix predicted by the V(step) algorithm can distinguish TA sites that vary by about 16-fold in their preferences for accommodating insertions of SB transposons.

AT Rich Sequence↗

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

Enhanced responses of glycosylphosphatidylinositol anchor-deficient T lymphocytes.

The functions of GPI-anchored proteins in T lymphocyte activation have been controversial. This issue was addressed by studying the responses of T lymphocytes from T lymphocyte-specific GPI anchor-deficient mice to different stimuli that normally allow coligation of TCR and GPI-anchored proteins. Stimulation of GPI anchor-deficient T lymphocytes with ConA induced 2-fold higher proliferative responses than did normal cells. In response to allogeneic stimulation, proliferation of GPI anchor-deficient T lymphocytes was enhanced 2- to 3-fold. The response to ConA of a GPI anchor-deficient anti-OVA T lymphocyte clone generated from these mice was approximately 3-fold higher than that of cells from the same clone in which GPI anchor expression was restored by retroviral transduction. The response of the GPI anchor-deficient cloned anti-OVA T lymphocytes to antigenic stimulation was similar to that of the retrovirally restored cells. These results indicate that coligation with GPI-anchored proteins counteracts the response to TCR stimulation by ConA or alloantigen but not protein Ag.

Animals↗

PubMed2004

GPI-anchor deficiency in myeloid cells causes impaired FcgammaR effector functions.

Signaling by transmembrane immunoglobulin G (IgG)-Fc receptors (FcgammaRs) in response to ligand involves association with membrane microdomains that contain glycosyl phosphatidylinositol (GPI)-anchored proteins. Recent in vitro studies showed enhancement of FcgammaR signaling by forced monoclonal antibody-mediated cocrosslinking with various GPI-anchored proteins. Here, the possibility that GPI-anchored proteins are involved in normal physiologic FcgammaR effector functions in response to a model ligand was studied using myeloid-specific GPI-anchor-deficient mice, generated by Cre-loxP conditional targeting. GPI-anchor-deficient primary myeloid cells exhibited normal FcgammaR expression and binding or endocytosis of IgG-immune complexes (IgG-ICs). Strikingly, after stimulation with IgG-ICs, tumor necrosis factor-alpha release, dendritic cell maturation, and antigen presentation were strongly reduced by GPI-anchor deficiency. Tyrosine phosphorylation of the FcR gamma-chain in response to IgG-IC was impaired in GPI-anchor-deficient cells. Myeloid GPI-anchor deficiency resulted in attenuated in vivo inflammatory processes during IgG-IC-mediated alveolitis. This study provides the first genetic evidence for an essential role of GPI-anchored proteins in physiologic FcgammaR effector functions in vitro and in vivo.

Animals↗

PubMed2004

Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene.

The chief limitation of phenotype-based genetic screening in mammalian systems is the diploid nature of the genome. Cells deficient in the Bloom's syndrome gene (Blm) show an increased rate of loss of heterozygosity. Here we have used a tetracycline-regulated Blm allele (Blm(tet)) to introduce bi-allelic mutations across the genome in mouse embryonic stem (ES) cells. Transient loss of Blm expression induces homologous recombination not only between sister chromatids but also between homologous chromosomes. We considered that the phenotype of ES cells bearing bi-allelic mutations would be maintained after withdrawal of the tetracycline analogue doxycycline. Indeed, a combination of N-ethyl-N-nitrosourea mutagenesis and transient loss of Blm expression enabled us to generate an ES cell library with genome-wide bi-allelic mutations. The library was evaluated by screening for mutants of glycosylphosphatidylinositol-anchor biosynthesis, which involves at least 23 genes distributed throughout the genome. Mutants derived from 12 different genes were obtained and two unknown mutants were simultaneously isolated. Our results indicate that phenotype-based genetic screening with Blm(tet) is very efficient and raises possibilities for identifying gene functions in ES cells.

Adenosine Triphosphatases↗

PubMed2004

Identification of a cytokine-induced antiapoptotic molecule anamorsin essential for definitive hematopoiesis.

Many growth factors and cytokines prevent apoptosis. Using an expression cloning method, we identified a novel antiapoptotic molecule named Anamorsin, which does not show any homology to known apoptosis regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. The expression of Anamorsin was completely dependent on stimulation with growth factors such as interleukin 3, stem cell factor, and thrombopoietin in factor-dependent hematopoietic cell lines, and forced expression of Anamorsin conferred resistance to apoptosis caused by growth factor deprivation in vitro. Furthermore, Anamorsin was found to act as an antiapoptotic molecule in vivo because Anamorsin-/- mice die in late gestation due to defective definitive hematopoiesis in the fetal liver (FL). Although the number of hematopoietic stem/progenitor cells in the FL did not decrease in these mice, myeloid, and particularly erythroid colony formation in response to cytokines, was severely disrupted. Also, Anamorsin-/- erythroid cells initiated apoptosis during terminal maturation. As for the mechanism of Anamorsin-mediated cell survival, a microarray analysis revealed that the expression of Bcl-xL and Jak2 was severely impaired in the FL of Anamorsin-/- mice. Thus, Anamorsin is considered to be a necessary molecule for hematopoiesis that mediates antiapoptotic effects of various cytokines.

Amino Acid Sequence↗

PubMed2004

Downregulation of STAT3 activation is required for presumptive rod photoreceptor cells to differentiate in the postnatal retina.

Ciliary neurotrophic factor (CNTF) has been known to inhibit the differentiation of presumptive rod photoreceptor cells; however, the underlying mechanisms have remained to be elucidated. We demonstrated that STAT3 activation, but not SHP2 activation, is responsible for the CNTF/gp130 signaling that inhibits expression of Rhodopsin and its upstream activator, crx, in the retinal explants derived from P0 mice (P0 retinal explants), utilizing STAT3-deficient retina and electroporation of dominant-negative form of STAT3 (STAT3F). We also demonstrated that STAT3 activation in presumptive rod photoreceptor cells at E18.5 is rapidly downregulated at P0, when Rhodopsin expression starts during retinal development. Persistent STAT3 activation in the P0 retinal explants prevented Rhodopsin expression and rapid upregulation of crx expression. STAT3-deficient retinas did not exhibit precocious rod photoreceptor cell differentiation as a whole, although they occasionally exhibited precocious upregulation of crx mRNA. Thus, we conclude that downregulation of STAT3 activation is required, but insufficient, for rod photoreceptor cell differentiation in the postnatal retina.

Animals↗

PubMed2004

Epidermal-specific defect of GPI anchor in Pig-a null mice results in Harlequin ichthyosis-like features.

We previously demonstrated that the epidermal-specific glycosylphosphatidylinositol (GPI)-anchor-deficient mice, generated by Pig-a gene disruption (Pig-a null mice), exhibited wrinkled and dry skin with hyperkeratosis and abnormal differentiation, and they died within a few days after birth. Here, we investigated the basis for the early demise of these animals, and the potential role of epidermal structural and biochemical abnormalities. The rapid demise of these animals was associated with both diminished epidermal permeability barrier function and decreased stratum corneum (SC) water content. The barrier abnormality could be attributed abnormal internal contents of lamellar bodies, with a downstream failure to generate normal extracellular lamellar bilayers in the SC. Moreover, processing profilaggrin to its monomeric form was impaired in Pig-a null mouse epidermis, while levels of the differentiation-specific proteins, involucrin, loricrin and profilaggrin were normal. Failure of filaggrin processing was accompanied by decreased activity of protein phosphatase 2A, an enzyme involved in profilaggrin to filaggrin processing. Thus, these studies demonstrate a critical role for GPI anchor and GPI-anchored proteins in divergent arms of epidermal terminal differentiation. While the permeability barrier abnormality can be attributed to defects in the lamellar body secretory system, the hydration abnormality is, in part, due to lack of availability of filaggrin-derived proteolytic products. Finally, since the dual abnormalities in the lamellar body secretory system and filaggrin processing resemble two key features of human Harlequin ichthyosis, Pig-a null mice could provide an appropriate analog for further studies of this disease.

Animals↗