Biomedical subjects

Kiyotoshi Sekiguchi

Publications and source records attributed to Kiyotoshi Sekiguchi.

43 records · Page 3Linked to original sources

PubMed2003

Laminin expression patterns in human ureteral tissue.

PURPOSE: Laminins are extracellular matrix proteins that are involved in various cellular functions, including adhesion, proliferation and differentiation. In this study we examined the expression patterns of the laminin chains in human ureteral tissue in vitro and in vivo. MATERIALS AND METHODS: We screened the expression of laminin chains at the mRNA level and determined the major laminins expressed in epithelial and stromal cells of human ureteral tissue by reverse transcriptase-polymerase chain reaction and Western blot analysis. We also examined their expression in vivo by immunofluorescence study. RESULTS: Reverse transcriptase-polymerase chain reaction analysis revealed that urothelial cells in vitro expressed laminins alpha1, alpha3, alpha5, beta1, beta2, beta3, gamma1 and gamma2, while stromal cells expressed alpha1, alpha2, alpha4, alpha5, beta1, beta2 and gamma1. Western blot analysis under reduced conditions confirmed that urothelial cells expressed laminin alpha3 and alpha5, while stromal cells strongly expressed alpha4 and alpha2. Immunofluorescence labeling with antilaminin alpha chain antibodies in human ureteral tissues confirmed that laminin alpha2, alpha3 and alpha5 chains were strongly expressed in the basement membrane of the urothelium, while alpha4 was expressed predominantly in smooth muscle cells. CONCLUSIONS: The most prominent laminin of urothelial cells was laminin 5 (alpha3beta3gamma2), whereas in stromal cells we noted laminins 8/9 (alpha4beta1/2gamma1). Laminin alpha5 showed the widest distribution pattern in vivo. Our data may be useful for understanding the mechanism involved not only in cell growth and differentiation, but also in cancer invasion in the urinary tract.

Basement Membrane↗

PubMed2003

Antisense inhibition of laminin-8 expression reduces invasion of human gliomas in vitro.

Using gene array technology, we recently observed for the first time an up-regulation of laminin alpha4 chain in human gliomas. The data were validated by semiquantitative reverse transcription-PCR for RNA expression and immunohistochemistry for protein expression. Moreover, increase of the alpha4 chain-containing laminin-8 correlated with poor prognosis for patients with brain gliomas. Therefore, we hypothesized that inhibition of laminin-8 expression by a new generation of highly specific and stable antisense oligonucleotides (Morpholino) against chains of laminin-8 could slow or stop the spread of glioma and its recurrence and thus might be a promising approach for glioma therapy. We next sought to establish an in vitro model to test the feasibility of this approach and to optimize conditions for Morpholino treatment. To develop a model, we used human glioblastoma multiforme cell lines M059K and U-87MG cocultured with normal human brain microvascular endothelial cells (HBMVEC). Using Western blot analysis and immunohistochemistry, we confirmed that antisense treatment effectively blocked laminin-8 protein synthesis. Antisense oligonucleotides against both alpha4 and beta1 chains of laminin-8 were able to block significantly the invasion of cocultures through Matrigel. On average, the invasion was blocked by 62% in cocultures of U-87MG with HBMVEC and by 53% in cocultures of M059K with HBMVEC. The results show that laminin-8 may contribute to glioma progression and recurrence not only as part of the neovascularization process but also by directly increasing the invasive potential of tumor cells.

Blotting, Western↗

PubMed2002

Identification and recombinant production of human laminin alpha4 subunit splice variants.

Laminins, the major basement membrane glycoproteins, are composed of three subunits. We identified a splice variant of the human laminin alpha4 subunit transcript containing 21 extra nucleotides. A heptapeptide sequence, MDCPTIS, was inserted close to the two cysteine residues possibly involved in the intersubunit disulfide bonds. Both the authentic alpha4 subunit (alpha4A) and the variant with the heptapeptide insertion (alpha4B) were readily secreted as laminin-8 trimers (alpha4Abeta1gamma1 or alpha4Bbeta1gamma1) upon cotransfection with expression vectors for the beta1 and gamma1 subunits. The purified recombinant laminin-8 containing the alpha4B subunit was more potent in promoting cell spreading than that containing alpha4A, raising the possibility that the alternative splicing of the alpha4 subunit transcript regulates the cell-adhesive activity of laminin-8. Since both alpha4A and alpha4B transcripts were detected by RT-PCR in several human cell lines, these two isoforms of laminin-8 with differing cell-adhesive activities are present in the basement membranes of human tissues.

Alternative Splicing↗

PubMed2002

Mice lacking the EDB segment of fibronectin develop normally but exhibit reduced cell growth and fibronectin matrix assembly in vitro.

Fibronectins (FNs) are major cell-adhesive proteins in the extracellular matrix and are essential for embryonic development. FNs are encoded by a single gene, but heterogeneity is introduced by alternative pre-mRNA splicing. One of the alternatively spliced segments, extra domain B (EDB), is prominently expressed during embryonic development and in tumor tissues, although it is mostly eliminated from FN in normal adult tissues. To examine the function of the EDB segment in vivo, we generated mice lacking the EDB exon using the Cre-loxP system. Although EDB-containing FNs are highly expressed throughout early embryogenesis, EDB-deficient mice developed normally and were fertile. Despite the absence of any significant phenotypes observed in vivo, however, fibroblasts obtained from EDB-deficient mice grew slowly in vitro and deposited less FN in the pericellular matrix than fibroblasts from wild-type mice. These results indicate that expression of EDB-containing isoforms is dispensable during embryonic development, yet may play a modulating role in the growth of connective tissue cells via the FN matrix.

Amino Acid Sequence↗

PubMed2002

Laminin-10/11 and fibronectin differentially prevent apoptosis induced by serum removal via phosphatidylinositol 3-kinase/Akt- and MEK1/ERK-dependent pathways.

Cell adhesion to the extracellular matrix inhibits apoptosis, but the molecular mechanisms underlying the signals transduced by different matrix components are not well understood. Here, we examined integrin-mediated antiapoptotic signals from laminin-10/11 in comparison with those from fibronectin, the best characterized extracellular adhesive ligand. We found that the activation of protein kinase B/Akt in cells adhering to laminin-10/11 can rescue cell apoptosis induced by serum removal. Consistent with this, wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, or ectopic expression of a dominant-negative mutant of Akt selectively accelerated cell death upon serum removal. In contrast to laminin-10/11, fibronectin rescued cells from serum depletion-induced apoptosis mainly through the extracellular signal-regulated kinase pathway. Cell survival on fibronectin but not laminin was significantly reduced by treatment with PD98059, a specific inhibitor of mitogen- or extracellular signal-regulated kinase kinase-1 (MEK1) and by expression of a dominant-negative mutant of MEK1. Laminin-10/11 was more potent than fibronectin in preventing apoptosis induced by serum depletion. These results, taken together, demonstrate laminin-10/11 potency as a survival factor and demonstrate that different extracellular matrix components can transduce distinct survival signals through preferential activation of subsets of multiple integrin-mediated signaling pathways.

Apoptosis↗

PubMed2002

Localization of laminin alpha4-chain in developing and adult human tissues.

Recent studies suggest important functions for laminin-8 (Ln-8; alpha4beta1gamma1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln alpha4-chain. Immunoreactivity for the Ln alpha4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln alpha4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln alpha4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln alpha4- and Ln alpha2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced Ln alpha4-chain as M(r) 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum. In cell-free extracellular matrices of human kidney and lung, Ln alpha4-chain was found as M(r) 180,000 protein.

Antibodies, Monoclonal↗

PubMedDate not supplied

Differential regulation of focal adhesion kinase and paxillin phosphorylation by the small GTP-binding protein Rho in human corneal epithelial cells.

PURPOSE: Lysophosphatidic acid (LPA), which activates the small guanosine triphosphate (GTP)-binding protein Rho, was previously shown to promote migration of the rabbit corneal epithelium in culture. The signaling pathway responsible for this effect of LPA was examined in this study with a human corneal epithelial (HCE) cell line. METHODS: The activation of Rho was detected with a pull-down assay. Tyrosine phosphorylation of paxillin and of focal adhesion kinase (FAK) were examined both by immunofluorescence staining and by immunoprecipitation and immunoblot analyses. Expression of integrins alpha5 and beta1 was evaluated by immunoblot analysis. RESULTS: Incubation of cells with LPA (10 micro M) for 2 min resulted in marked activation of Rho, and this effect was blocked by pretreatment with the Rho inhibitor exoenzyme C3 (2 micro g/ml) for 24 h. Tyrosine phosphorylation of both paxillin and FAK was detected in HCE cells under basal conditions by immunofluorescence staining, immunoprecipitation, and immunoblot analyses. LPA induced a concentration- and time-dependent increase in the tyrosine phosphorylation of paxillin, which was maximal at a concentration of 10 micro M and a time of 2 min. Exoenzyme C3 inhibited LPA-induced paxillin phosphorylation. Neither LPA nor exoenzyme C3 affected tyrosine phosphorylation of FAK or expression of integrins alpha5 and beta1. CONCLUSIONS: LPA induces Rho activation and the consequent tyrosine phosphorylation of paxillin in HCE cells, and these effects likely contribute to the promotion of corneal epithelial migration by this agent.

Cell Line↗