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Junji Takeda

Publications and source records attributed to Junji Takeda.

At least 37 records · Page 2Linked to original sources
PubMed2004

Ahnak/Desmoyokin is dispensable for proliferation, differentiation, and maintenance of integrity in mouse epidermis.

Desmoyokin was first isolated from bovine muzzle epidermis and thought to be an epidermal desmosome-related protein. We previously demonstrated that the Desmoyokin gene is identical to the Ahnak gene, which is expressed ubiquitously and downregulated in neuroblastomas. It was assumed Ahnak/Desmoyokin was associated with epidermal cell adhesion, tumorigenesis, cell proliferation and differentiation, and embryonic development. To determine the precise biological function of Ahnak/Desmoyokin, we generated a null mutation in ES cells and mice. The resultant Ahnak/Desmoyokin-deficient ES cells normally differentiated into embryoid bodies and neural cells. The mutant mice were viable and fertile and showed no gross developmental defects. Electron microscopic examination of skin sections demonstrated that the ultrastructure of epidermal intercellular junctions, including desmosomes, of the mutant mice was indistinguishable from that of wild-type mice. Two-stage chemical skin carcinogenesis experiments showed no difference in frequency or onset of cutaneous tumor formation between wild-type and mutant mice. Moreover, no tumorigenesis was observed in other tissues and organs of mutant mice up to 2 y of age. These results lead us to conclude that Ahnak/Desmoyokin deficiency has only a minimal effect on epidermal cell adhesion, tumorigenesis, cell proliferation and differentiation, and overall mouse development.

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

p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload.

The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38alpha is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38alpha in hearts. First, we generated mice with floxed p38alpha alleles and crossbred them with mice expressing the Cre recombinase under the control of the alpha-myosin heavy-chain promoter to obtain cardiac-specific p38alpha knockout mice. These cardiac-specific p38alpha knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38alpha plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.

Animals↗
PubMed2004

Enhancement of Sleeping Beauty transposition by CpG methylation: possible role of heterochromatin formation.

The Sleeping Beauty (SB) transposase is the most active transposase in vertebrate cells, and the SB transposon system has been used as a tool for insertional mutagenesis and gene delivery. Previous studies have indicated that the frequency of chromosomal transposition is considerably higher in mouse germ cells than in mouse embryonic stem cells, suggesting the existence of unknown mechanisms that regulate SB transposition. Here, we demonstrated that CpG methylation of the transposon region enhances SB transposition. The transposition efficiencies of a methylated transposon and an unmethylated transposon which had been targeted in the same genomic loci by recombination-mediated cassette exchange in mouse erythroleukemia cells were compared, and at least a 100-fold increase was observed in the methylated transposon. CpG methylation also enhanced transposition from plasmids into the genome. Chromatin immunoprecipitation assays revealed that histone H3 methylated at lysine-9, a hallmark of condensed heterochromatin, was enriched at the methylated transposon, whereas the unmethylated transposon formed a relaxed euchromatin structure, as evidenced by enrichment of acetylated histone H3 and reporter gene expression. Possible roles of heterochromatin formation in the transposition reaction are discussed. Our findings indicate a novel relationship between CpG methylation and transposon mobilization.

Animals↗
PubMed2004

Disruption of Stat3 reveals a critical role in both the initiation and the promotion stages of epithelial carcinogenesis.

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a wide spectrum of human malignancies. Here, we have assessed the effect of Stat3 deficiency on skin tumor development using the 2-stage chemical carcinogenesis model. The epidermis of Stat3-deficient mice showed a significantly reduced proliferative response following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) because of a defect in G1-to-S-phase cell cycle progression. Treatment with the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) resulted in a significant increase in the number of keratinocyte stem cells undergoing apoptosis in the bulge region of hair follicles of Stat3-deficient mice compared with nontransgenic littermates. Notably, Stat3-deficient mice were completely resistant to skin tumor development when DMBA was used as the initiator and TPA as the promoter. Abrogation of Stat3 function using a decoy oligonucleotide inhibited the growth of initiated keratinocytes possessing an activated Ha-ras gene, both in vitro and in vivo. In addition, injection of Stat3 decoy into skin tumors inhibited their growth. To our knowledge, these data provide the first evidence that Stat3 is required for de novo epithelial carcinogenesis, through maintaining the survival of DNA-damaged stem cells and through mediating and maintaining the proliferation necessary for clonal expansion of initiated cells during tumor promotion. Collectively, these data suggest that, in addition to its emerging role as a target for cancer therapy, Stat3 may also be a target for cancer prevention strategies.

Animals↗
PubMed2003

T cell-specific disruption of arylhydrocarbon receptor nuclear translocator (Arnt) gene causes resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced thymic involution.

The arylhydrocarbon receptor nuclear translocator (ARNT) is a member of the basic helix-loop-helix, PER-ARNT-SIM family of heterodimeric transcription factors, and serves as a dimerization partner for arylhydrocarbon receptor (AHR) and hypoxia-inducible factor-1alpha. To assess the function of ARNT in T cells, we disrupted the Arnt gene specifically in T cells of mice by conditional gene targeting using T cell-specific p56(lck)-Cre (Lck-Cre) transgenic Arnt-floxed mice. Thus generated, T cell-specific Arnt-disrupted mice (Lck-Cre;Arnt(flox/Delta) transgenic mice) exhibited complete loss of the expression of ARNT protein only in T cells, and were viable and appeared normal. The Arnt-disrupted T cells in the thymus were phenotypically and histologically normal. The Arnt-deficient T cells in the spleen were capable of responding to TCR stimulation in vitro. However, unlike normal mice in which exposure to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an AHR ligand, resulted in thymic involution, the thymus of Lck-Cre;Arnt(flox/Delta) mice were resistant to TCDD treatment in vivo. In contrast, benzo(a)pyrene, another AHR ligand, still caused thymic involution in Lck-Cre;Arnt(flox/Delta) mice. Finally, fetal thymus organ culture using Lck-Cre;Arnt(flox/Delta) and K5-Cre;Arnt(flox/Delta) (epithelial cell-specific Arnt-disrupted mice) showed that thymocytes rather than thymic epithelial cells are predominantly responsible for TCDD-induced thymic atrophy. Our results indicate that ARNT in T lineage cells is essential for TCDD-mediated thymic involution.

Animals↗
PubMed2003

Infertility in female mice with an oocyte-specific knockout of GPI-anchored proteins.

Glycosylphosphatidylinositol-anchored proteins on the egg surface have been proposed to play a role in gamete fusion on the basis of in vitro experiments. We tested this hypothesis by asking if oocyte GPI-anchored proteins are required for fertilization in vivo. Oocyte-specific knockout mice were created using the Cre/loxP system to delete a portion of the Pig-a gene, which encodes an enzyme involved in GPI anchor biosynthesis. Conditional Pig-a-knockout females are infertile, and eggs recovered from the females after mating are unfertilized. In in vitro assays, the knockout eggs are severely deficient in their ability to fuse with sperm. These results demonstrate that GPI-anchored proteins are required for gamete fusion. Loss of the GPI-anchored complement of plasma membrane proteins could prevent fusion by altering the organization and function of GPI-anchored protein-containing lipid domains. Alternatively, a single GPI-anchored protein may be required in the fusion process. To distinguish between these possibilities, we have begun to identify the GPI-anchored proteins on the egg surface. We have identified one egg GPI-anchored protein as CD55, an approximately 70 kDa complement regulatory protein. It has previously been found that CD55-knockout mice are fertile, demonstrating that CD55 is not essential for fertilization. This finding also means that the presence of the full complement of egg GPI-anchored proteins is not necessary for gamete fusion. Other egg GPI-anchored proteins acting in the fusion process can now be investigated, with the goal of understanding the mechanism of their function in sperm-egg fusion.

Animals↗
PubMed2003

Keratinocyte-specific Pten deficiency results in epidermal hyperplasia, accelerated hair follicle morphogenesis and tumor formation.

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate a keratinocyte-specific null mutation of Pten in mice (k5Pten(flox/flox) mice). k5Pten(flox/flox) mice exhibit wrinkled skin because of epidermal hyperplasia and hyperkeratosis and ruffled, shaggy, and curly hair. Histological examination revealed that skin morphogenesis is accelerated in k5Pten(flox/flox) mice. Within 3 weeks of birth, 90% of k5Pten(flox/flox) mice die of malnutrition possibly caused by hyperkeratosis of the esophagus. All k5Pten(flox/flox) mice develop spontaneous tumors within 8.5 months of birth, and chemical treatment accelerates the onset of tumors. k5Pten(flox/flox) keratinocytes are hyperproliferative and resistant to apoptosis and show increased activation of the Pten downstream signaling mediators Akt/protein kinase B (PKB) and extracellular signal-regulated kinase. Pten is thus an important regulator of normal development and oncogenesis in the skin.

Animals↗
PubMed2003

T cell-specific gene targeting reveals that alpha4 is required for early T cell development.

Alpha4-mediated signaling is involved in a variety of functions in mammalian cells. To determine whether this is true for immunocompetent cells, we generated mutant (Lck-alpha4-) mice in which the alpha4 gene was deleted in a T cell-specific manner using the Cre/loxP system. These mice showed impaired early T cell development. Thymi at most ages were small and their architecture was disorganized. This defect was not due to increased thymocyte apoptosis but to decreased cell proliferation. T cell development was found to be severely arrested at the CD4/CD8 double-negative 3 stage and the thymus contained very few double-positive or single-positive (SP) mature thymocytes. The mutant thymocytes showed impaired proliferative responses to anti-CD3 monoclonal antibody (mAb) stimulation or to the cytokines IL-2, IL-1 or TNF. In the spleen, the numbers of mature SP T cells were decreased and their proliferative responses to anti-CD3 plus IL-2 or to anti-CD28 mAb were impaired. A severe impairment of CD3-induced expression of CD25 was also observed. These data suggest that alpha4 plays a critical role in the proliferation of thymocytes, which is necessary for early T cell development.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Dissecting the role of lymphotoxin in lymphoid organs by conditional targeting.

Mice with inactivation of lymphotoxin beta receptor (LTbetaR) system have profound defects in the development and maintenance of peripheral lymphoid organs. As surface LT is expressed by lymphocytes, natural killer cells, and lymphoid tissue-initiating cells as well as by some other cell types, we dissected cell type-specific LT contribution into the complex LT-deficient phenotype by conditional gene targeting. B-LTbeta knockout (KO) mice displayed an intermediate phenotype in spleen as compared with mice with complete LTbeta deficiency. In contrast, T-LTbeta KO mice displayed normal structure of the spleen. However, inactivation of LTbeta in both T and B cells resulted in additional defects in the structure of the marginal zone and in the development of follicular dendritic cells in spleen. Structure of lymph nodes (LN) and Peyer's patches (PP) was normal in both B-LTbeta KO and T- and B-LTbeta KO mice, except that PPs were of reduced size. When compared across the panel of lymphocyte-specific LT KOs, the defects in antibody responses to T-cell-dependent antigens correlated with the severity of defects in spleen structure. Expression of CCL21 and CCL19 chemokines was not affected in spleen, LN and PP of B-LTbeta KO and T- and B-LTbeta KO mice, while CXCL13 was slightly reduced only in spleen. Collectively, our data suggest the following: (i). requirements for LT signaling to support architecture of spleen, LN and PP are different; (ii). LT complex expressed by B cells plays a major role in the maintenance of spleen structure, while surface LT expressed by T cells provides a complementary but distinct signal; and (iii). in a non-transgenic model, expression of lymphoid tissue chemokines is only minimally dependent on the expression of surface LT complex on B and T lymphocytes.

Animals↗
PubMed2003

Exacerbated and prolonged allergic and non-allergic inflammatory cutaneous reaction in mice with targeted interleukin-18 expression in the skin.

Interleukin 18 induces both T helper 1 and T helper 2 cytokines, proinflammatory cytokines, chemokines, and IgE and IgG1 production. A role of interleukin 18 in inflammatory cutaneous reactions is still unclear, however. Here we generated keratin 5/interleukin 18 transgenic mice overexpressing mature murine interleukin 18 in the skin using a human keratin 5 promoter. In the contact hypersensitivity model, trinitrochlorobenzene elicited a stronger ear swelling in keratin 5/interleukin 18 transgenic mice compared with control littermate wild-type or immunoglobulin/interleukin 18 transgenic mice in which mature interleukin 18 was expressed by B and T cells under the control of the immunoglobulin promoter. Application of an irritant, croton oil, induced stronger and more sustained ear swelling in keratin 5/interleukin 18 transgenic mice than in immunoglobulin/interleukin 18 transgenic or wild-type mice. Repetitive topical application (weekly for six consecutive weeks) of trinitrochlorobenzene to their ears also elicited a stronger cutaneous inflammation in keratin 5/interleukin 18 transgenic mice than seen in immunoglobulin/interleukin 18 transgenic or wild-type mice. After these six trinitrochlorobenzene applications, the expression of interferon-gamma, interleukin-4, and CCL20 mRNA in the ear tissue was increased and dermal changes, such as acanthosis and eosinophilic, neutrophilic, and mast cell infiltration, were greater in keratin 5/interleukin 18 transgenic mice than in wild-type mice. Furthermore, the repetitive application elicited a significant increase in serum IgE levels and the number of B cells in the draining lymph node in keratin 5/interleukin 18 transgenic mice. These results suggest that overexpression of interleukin 18 in the skin aggravates allergic and nonallergic cutaneous inflammation, which is accompanied by high expression of T helper 1 and T helper 2 cytokines and chemokines in the skin.

Animals↗
PubMed2003

In vivo cooperation between Bcl-xL and the phosphoinositide 3-kinase-Akt signaling pathway for the protection of epidermal keratinocytes from apoptosis.

To investigate the function of Bcl-xL in the skin, we established keratinocyte-specific Bcl-x gene-targeted mice under the keratin 5 promoter (K5). K5.Bcl-xL-/- mice were viable, devoid of alteration in the development of skin or appendages. However, they harbored spontaneous apoptotic keratinocytes in the epidermis. Bcl-xL-deficient keratinocytes cultured in vitro readily underwent apoptosis in the absence of growth factors, but the addition of HGF or EGF resulted in restoration of cell survival, which was reversed by treatment with wortmannin, an inhibitor of phosphoinositide-3 kinase (PI3K). Topical treatment of K5.Bcl-xL-/- mice with wortmannin sensitized the skin for apoptosis induced by UV (UV) B, although wild-type epidermis was only marginally affected by this treatment, suggesting that the resistance to UVB largely depended on PI3K-Akt signaling in Bcl-xL-deficient mice but not in wild-type mice. Furthermore, UVB irradiation resulted in redistribution of phosphorylated Akt from the basal layer to the suprabasal layer, indicating that Akt could spatially cooperate with Bcl-xL upon UVB exposure in the upper epidermis where Bcl-xL is normally localized. These results suggest that Bcl-xL and the PI3K-Akt pathway form a cooperative, intercompensatory axis for the protection of epidermal keratinocytes from apoptosis in vivo.

Androstadienes↗
PubMed2003

Characterization of Sleeping Beauty transposition and its application to genetic screening in mice.

The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.

Animals↗
PubMed2003

Alteration of the 4-sphingenine scaffolds of ceramides in keratinocyte-specific Arnt-deficient mice affects skin barrier function.

Aryl hydrocarbon receptor nuclear translocator (ARNT), a transcription factor of the Per/AHR/ARNT/Sim family, regulates gene expression in response to environmental stimuli including xenobiotics and hypoxia. To examine its role in the epidermis, the Cre-loxP system was used to disrupt the Arnt gene in a keratinocyte-specific manner. Gene-targeted, newborn mice with almost normal appearance died neonatally of severe dehydration caused by water loss. Histology showed small changes in the architecture of cornified layers, with apparently preserved intercorneocyte lamellar structures responsible for the skin barrier function. In contrast, HPLC/ion-trap mass spectrometry revealed significant alterations in the compositions of ceramides, the major components of the lamellae. The murine epidermal ceramides normally contain 4-sphingenine and 4-hydroxysphinganine. In Arnt-null epidermis, 4-sphingenine was largely replaced by sphinganine and the amounts of ceramides with 4-hydroxysphinganine were greatly decreased, suggesting deficiency of dihydroceramide desaturases that catalyze the formation of both 4-sphingenyl and 4-hydroxysphinganyl moieties. A desaturase isoenzyme, DES-1, prefers desaturation, but DES-2 catalyzes both reactions to a similar extent. Transcript levels of Des-2, but not Des-1, were considerably decreased in cultured keratinocytes from Arnt-null epidermis. These results indicate that proper ceramide compositions through 4-desaturation regulated by ARNT are crucial for maintaining the epidermal barrier function.

Animals↗
PubMed2002

Prostacyclin-deficient mice develop ischemic renal disorders, including nephrosclerosis and renal infarction.

BACKGROUND: Prostacyclin (PGI2) is a short-lived endogenous inhibitor of platelet aggregation and a potent vasodilator and regulator of the growth of vascular smooth muscle cells. To study the role of PGI2 in the vascular system in vivo, PGI2-deficient (PGID) mice were established by genetic disruption of the PGI2 synthase gene. METHODS AND RESULTS: PGI2 synthase-null mice were generated by replacing the exons of PGI2 synthase gene that encodes for the catalytic site of the enzyme with a neomycin resistance gene. In these mice, PGI2 levels in the plasma, kidneys, and lungs were reduced, whereas thromboxane and prostaglandin E2 levels became elevated. Blood pressure and the amounts of urea nitrogen and creatinine in plasma of the PGID mice were significantly higher than those of wild-type mice (P<0.05). They developed progressive morphological abnormalities in the kidneys, accompanied by atrophy, surface irregularity, fibrosis, cyst, arterial sclerosis, and hypertrophy of vessel walls. Thickening of the thoracic aortic media and adventitia were observed in aged PGID mice. Importantly, these phenotypes have not been reported in PGI2 receptor-deficient mice. CONCLUSIONS: PGI2 deficiency resulted in the development of vascular disorders with the thickening of vascular walls and interstitial fibrosis, especially in mouse kidneys. The findings demonstrated in vivo that PGI2 is important in the homeostasis of blood vessels. Our established PGID mice are useful for studies on the initiation and development of vascular diseases, such as ischemic renal disorders with arterial sclerosis and infarction, and also for studies on the novel signaling pathway of PGI2.

Age Factors↗