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Junji Takeda

Publications and source records attributed to Junji Takeda.

41 records · Page 3Linked to original sources
PubMed2002

Inefficient response of T lymphocytes to glycosylphosphatidylinositol anchor-negative cells: implications for paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder in which clonal cells defective in glycosylphosphatidylinositol (GPI) biosynthesis are expanded, leading to complement-mediated hemolysis. PNH is often associated with bone marrow suppressive conditions, such as aplastic anemia. One hypothetical mechanism for the clonal expansion of GPI(-) cells in PNH is that the mutant cells escape attack by autoreactive cytotoxic cells that are thought to be responsible for aplastic anemia. Here we studied 2 model systems. First, we made pairs of GPI(+) and GPI(-) EL4 cells that expressed major histocompatibility complex (MHC) class II molecules and various types of ovalbumin. When the GPI-anchored form of ovalbumin was expressed on GPI(+) and GPI(-) cells, only the GPI(+) cells presented ovalbumin to ovalbumin-specific CD4(+) T cells, indicating that if a putative autoantigen recognized by cytotoxic cells is a GPI-anchored protein, GPI(-) cells are less sensitive to cytotoxic cells. Second, antigen-specific as well as alloreactive CD4(+) T cells responded less efficiently to GPI(-) than GPI(+) cells in proliferation assays. In vivo, when GPI(-) and GPI(+) fetal liver cells, and CD4(+) T cells alloreactive to them, were cotransplanted into irradiated hosts, the contribution of GPI(-) cells in peripheral blood cells was significantly higher than that of GPI(+) cells. The results obtained with the second model suggest that certain GPI-anchored protein on target cells is important for recognition by T cells. These results provide the first experimental evidence for the hypothesis that GPI(-) cells escape from immunologic attack.

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PubMed2002

Efficient biallelic mutagenesis with Cre/loxP-mediated inter-chromosomal recombination.

The isolation of mutant cells with phenotypes caused by random mutagenesis has been hampered in mammalian cells because there are two alleles per gene and the disruption of both alleles is extremely rare. We describe a method for the efficient biallelic mutagenesis in embryonic stem cells. loxP sites were introduced near the centromeric regions of a pair of chromosome 1s. A mutant neo gene was inserted at the distal part of one of the loxP sites so that biallelic mutants would be selected by high-dose G418. Expression of Cre induced the recombination between homologous chromosomes and led to an elevation in the number of biallelic mutants. This system will facilitate phenotype-driven gene function study in the mammalian system.

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PubMed2002

STAT3 deficiency in keratinocytes leads to compromised cell migration through hyperphosphorylation of p130(cas).

We previously reported that STAT3 plays a crucial role in transducing a signal for migration of keratinocytes (Sano, S., Itami, S., Takeda, K., Tarutani, M., Yamaguchi, Y., Miura, H., Yoshikawa, K., Akira, S., and Takeda, J. (1999) EMBO J. 18, 4657-4668). To clarify the role of STAT3 in signaling the migration, we studied the intracellular signaling pathway through an integrin receptor in STAT3-deficient keratinocytes. STAT3-deficient keratinocytes demonstrated increased adhesiveness and fast spreading on a collagen matrix. Staining with anti-phosphotyrosine antibody revealed that STAT3-deficient keratinocytes had an increased number of tyrosyl-hyperphosphorylated focal adhesions. Analyses with immunoprecipitation revealed that p130(cas) was constitutively hyperphosphorylated on tyrosine residues, while other focal adhesion molecules such as focal adhesion kinase and paxillin were not. Transfection of STAT3-deficient keratinocytes with an adenoviral vector encoding the wild-type Stat3 gene reversed not only impaired migration but also the increased tyrosine phosphorylation of p130(cas). These results strongly suggest that STAT3 in keratinocytes plays a critical role in turnover of tyrosine phosphorylation of p130(cas), modulating cell adhesiveness to the substratum leading to growth factor-dependent cell migration.

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PubMed2002

Rapid compensation for glycosylphosphatidylinositol anchor deficient keratinocytes after birth: visualization of glycosylphosphatidylinositol-anchored proteins in situ.

Pig-a, an X-linked gene, is a key component of glycosylphosphatidylinositol (GPI) anchor biosynthesis based on the fact that lack of this gene causes deficiencies of hundreds of GPI-anchored proteins. We previously demonstrated an essential role for the GPI-anchor in keratinocyte differentiation using male Pig-a knockout mice (K5-Cre:Pig-a flox). Here we analyzed keratinocytes of the female K5-Cre: Pig-a flox/+ mice with heterozygous knockout of Pig-a. These cells exhibited the mosaic pattern of GPI-anchor positive and negative expression typical of random inactivation of the X chromosome. The female K5-Cre:Pig-a flox/+ mice appeared slightly wrinkled with dry skin at birth and white scales starting from 4 d after birth without any histologic abnormality. This phenotype was temporary and milder than that seen in the male knockout mice. To characterize the fate of GPI-anchor-positive cells more clearly, we introduced a transgenic mouse line that expresses enhanced green fluorescent protein in GPI-anchored form into female K5-Cre:Pig-a flox/+ mice and monitored GPI-anchor-positive keratinocytes in situ. Within 36 h after birth, the upper layer of the GPI-anchor-negative zone in epidermis was replaced by the GPI-anchor-positive counterpart. This tissue replacement was accompanied by recovery in trans-epidermal water loss over a similar time course. These observations suggest that the GPI-anchoring is associated with the barrier function as well as with organized differentiation of the epidermis after birth.

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