Search PubMed⌕ Search

Biomedical subjects

Isao Kii

Publications and source records attributed to Isao Kii.

9 recordsLinked to original sources
PubMed2026

PUF60 is a Critical Regulator of PKM Splicing During Myogenesis.

Pyruvate kinase M (PKM) catalyzes the conversion of phosphoenolpyruvate to pyruvate in glycolysis and exists as two splice isoforms, PKM1 and PKM2, generated from alternative splicing of mutually exclusive exons 9 or 10, respectively. The expression balance between PKM1 and PKM2 is tightly regulated in a cell-type-specific manner. PKM1 is predominantly expressed in tissues such as skeletal muscle, heart, and brain, whereas PKM2 is prevalent in most other tissues and various cancer cells. Despite its importance, the trans-acting factors promoting exon 9 selection in a tissue-specific context remain largely unknown. Here, using a multi-color splicing reporter system for cell-based cDNA screening, we identified PUF60 as a novel trans-acting factor that promotes PKM1-type splicing. We also demonstrated that PUF60 induction and the resulting splicing switch are essential for myotube formation during C2C12 differentiation. This study establishes PUF60 as a critical regulator of muscle-specific splicing and provides new insights into the fundamental mechanisms governing skeletal muscle differentiation.

Animals↗
PubMed2006

Periostin is an extracellular matrix protein required for eruption of incisors in mice.

A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin-/- mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin-/- mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone.

Animals↗
PubMed2006

Identification of cysteine residues critically involved in homodimer formation and protein expression of human ATP-binding cassette transporter ABCG2: a new approach using the flp recombinase system.

Since ABCG2 is a half-transporter in the ATP-binding cassette (ABC) transporter family, it has been suspected that ABCG2 functions as a homodimer. In the present study, we have investigated the molecular mechanism underlying homodimer formation of ABCG2. Based on the amino acid sequence of ABCG2, three cysteine residues (Cys592, Cys603, and Cys608) are expected to exist in the extracellular loop. To identify a cysteine residue(s) required for homodimer formation, we have substituted those cysteine residues to glycine by site-directed mutagenesis and stably expressed the resulting variants in Flp-In-293 cells. Substitution of the amino acid at position 603 from cysteine to glycine (C603G) completely diminished homodimer formation, whereas substitution of both Cys592 and Cys608 to glycine residues (C592G/C608G) had no effect on homodimer formation. These results strongly suggest that Cys603 is prerequisite for homodimer formation of ABCG2 via a disulfide bond. On the other hand, immunohistochemistry experiments revealed that the C592G/C608G variant is mainly located in intracellular compartments. The C592G/C608G variant exhibited lower activity of ATP-dependent methotrexate (MTX) transport, and its expression did not confer Flp-In-293 cells resistance to SN-38 or mitoxantrone. Cys592 and Cys608 appear to be important for intracellular sorting of the de novo synthesized ABCG2 protein to the plasma membrane. Taken together, cysteine residues in the extra-cellular loop are considered to play pivotal roles in homodimer formation and plasma membrane localization of ABCG2.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2004

Cell-cell interaction mediated by cadherin-11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage.

UNLABELLED: We studied cadherin-11 function in the differentiation of mesenchymal cells. Teratomas harboring the cadherin-11 gene generated bone and cartilage preferentially. Cadherin-11 transfectants of C2C12 cells and cadherin-11 and/or N-cadherin transfectants of L cells showed that cadherin-11 together with N-cadherin-induced expression of ALP and FGF receptor 2. These results suggest that cadherin-11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage in a different manner from N-cadherin. INTRODUCTION: Cell-cell interaction is an essential event for tissue formation; however, the role of cell-cell adhesion in mesenchymal tissue formation as well as in cell differentiation in this tissue remains unclear. cadherins, which are calcium-dependent cell adhesion receptors, form adherence junctions after adherence and aggregation of cells. Because cadherin-11 as well as N-cadherin has been reported to be a mesenchyme-related cadherin, we examined the cadherin-11 action in teratomas and in the cell lines C2C12 and L cell. Herein, we show that cell-cell interaction mediated by cadherin-11 is responsible for bone and cartilage formation. MATERIALS AND METHODS: It has been previously reported that N-cadherin-expressing E-cadherin-/- ES transfectants formed neuroepithelium and cartilage in teratomas. Thus, we transfected the E-cadherin-/- ES cell line with the cadherin-11 gene. Moreover, we also transfected C2C12 cells and L cells with the cadherin-11 gene for morphological analysis and study of the induced differentiation at the molecular level. RESULTS AND CONCLUSION: Teratomas derived from embryonic stem cells in which the cadherin-11 gene had been expressed exogenously contained bone and cartilage preferentially, showing that cadherin-11 is involved in mesenchymal tissue formation, specifically in controlling the differentiation of these cells into osteoblasts and chondrocytes. Therefore, we further examined the functional difference between cadherin-11 and N-cadherin. The expression patterns of cadherin-11 and N-cadherin in cells of the mouse osteoblastic cell line MC3T3-E1 showed that each cadherin was located independently of the cell-cell adhesion site and acted individually. In hanging drop cultures, cadherin-11 L cell transfectants aggregated in a sheet-like structure, whereas N-cadherin transfectants aggregated in a spherical form, indicating that each cadherin confers a different 3D architecture because of its individual adhesive property. To investigate the molecular mechanism of cadherin-11 action in cell differentiation, we analyzed cadherin-11 transfectants of C2C12 cells and cadherin-11 and/or N-cadherin transfectants of L cells and showed that cadherin-11, together with N-cadherin, induced expression of alkaline phosphatase (ALP) and fibroblast growth factor receptor 2. These results suggest that cadherin-11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage in a different manner from N-cadherin.

3T3 Cells↗
PubMed2004

Inactivation of Rho/ROCK signaling is crucial for the nuclear accumulation of FKHR and myoblast fusion.

Myoblast fusion is a critical process for the terminal differentiation of skeletal muscle. To elucidate the intracellular mechanisms regulating myoblast fusion, we studied the roles of signaling through the small GTPase Rho and its effector, the Rho-associated kinase ROCK, in myoblast fusion of mouse C2C12 cells. We found that Rho activity, which was high in proliferating myoblasts, decreased during myogenesis. Expression of a constitutively active form of Rho blocked myoblast fusion, but not the earlier steps of differentiation. Consistently, ROCK activity was also decreased in differentiating C2C12 cells, and an active ROCK mutant prevented their fusion. Furthermore, inactivation of ROCK by the specific inhibitor Y-27632 enhanced myoblast fusion, even in cells expressing the active Rho mutant. Thus, the down-regulation of Rho/ROCK signaling is required for myoblast fusion. We also found that Rho/ROCK signaling was required for retaining FKHR, a transcription factor implicated in myoblast fusion, in the cytoplasm and that inactivation of ROCK was essential for the nuclear accumulation of FKHR that took place just before the onset of myoblast fusion. Moreover, ROCK directly phosphorylated FKHR in vitro. We conclude that the inactivation of Rho/ROCK signaling is a prerequisite for FKHR nuclear translocation and myoblast fusion in C2C12 cells, providing evidence for a novel regulatory role of Rho/ROCK signaling in myogenic differentiation.

Active Transport, Cell Nucleus↗
PubMed2004

Immunohistochemical localization of periostin in tooth and its surrounding tissues in mouse mandibles during development.

Previous reports have shown expression of immunoreactivity for periostin, originally identified as osteoblast-specific factor-2, in the periosteum and periodontal ligament. However, the developmental changes in its expression and the detailed immunolocalization have remained veiled. The present study was undertaken to examine the spatiotemporal expression of this protein in teeth and their associated tissues of mice during development at light and electron microscopic levels. In tooth germs at cap stage, periostin immunoreactivity was recognizable in the interface between inner enamel epithelium and preodontoblasts as well as in the mesenchymal tissues around cervical loop. Dental follicles around tooth germs at bell stage localized periostin immunopositivity in addition to the immunopositive areas observed in cap-staged tooth germs, although the functional significance of periostin has remained unclear in tooth development. Furthermore, periostin immunoreactivity was also found in the alveolar bone surface. In the incisors of both 7- and 21-day-old mice, immunoreaction for periostin was discernible in the lingual periodontal ligament and labial fibrous tissue adjacent to the papillary layer. After postnatal day 7, immunoreaction for periostin came to be restricted to the fibrous bundles in the periodontal ligament in accordance with the organization of the periodontal fibers, indicating its localization matched the morphogenesis of the periodontal ligament. Immunoelectron microscopic observation of the mature periodontal ligament verified the localization of periostin between the cytoplasmic processes of periodontal fibroblasts and cementoblasts and the adjacent collagen fibrils. Our findings suggest that periostin is involved at the sites of the cell-to-matrix interaction, serving as adhesive equipment for bearing mechanical forces, including occlusal force and tooth eruption.

Animals↗
PubMed2003

A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport.

Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg(482), Gly(482), and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg(482)]ABCG2 transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)). Transport of methotrexate by [Arg(482)]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S -octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg(482)]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg(482) is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

ATP Binding Cassette Transporter, Subfamily G, Mem↗