Biomedical subjects

Noriko Sanzen

Publications and source records attributed to Noriko Sanzen.

10 recordsLinked to original sources

PubMed2006

Extracellular matrix of the human cyclic corpus luteum.

Extracellular matrix regulates many cellular processes likely to be important for development and regression of corpora lutea. Therefore, we identified the types and components of the extracellular matrix of the human corpus luteum at different stages of the menstrual cycle. Two different types of extracellular matrix were identified by electron microscopy; subendothelial basal laminas and an interstitial matrix located as aggregates at irregular intervals between the non-vascular cells. No basal laminas were associated with luteal cells. At all stages, collagen type IV alpha1 and laminins alpha5, beta2 and gamma1 were localized by immunohistochemistry to subendothelial basal laminas, and collagen type IV alpha1 and laminins alpha2, alpha5, beta1 and beta2 localized in the interstitial matrix. Laminin alpha4 and beta1 chains occurred in the subendothelial basal lamina from mid-luteal stage to regression; at earlier stages, a punctate pattern of staining was observed. Therefore, human luteal subendothelial basal laminas potentially contain laminin 11 during early luteal development and, additionally, laminins 8, 9 and 10 at the mid-luteal phase. Laminin alpha1 and alpha3 chains were not detected in corpora lutea. Versican localized to the connective tissue extremities of the corpus luteum. Thus, during the formation of the human corpus luteum, remodelling of extracellular matrix does not result in basal laminas as present in the adrenal cortex or ovarian follicle. Instead, novel aggregates of interstitial matrix of collagen and laminin are deposited within the luteal parenchyma, and it remains to be seen whether this matrix is important for maintaining the luteal cell phenotype.

Collagen Type IV↗

PubMed2006

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.

Amino Acid Sequence↗

PubMed2005

A novel large-scale production system for modified basement membrane matrices using gene-swapped parietal endoderm cells.

Parietal endoderm-like cells, including Engelbreth-Holm-Swarm tumor and differentiated F9 embryonal carcinoma cells, produce huge amounts of basement membrane components, including laminin-1 (alpha1beta1gamma1). We employed a double-lox system-based gene-swapping strategy in F9 cells to replace the laminin alpha1 gene with a laminin alpha5 minigene. The gene-swapped F9 cells secreted laminin-10 (alpha5beta1gamma1) consisting of the exogenous alpha5 subunit and endogenous beta1 and gamma1 subunits on differentiation. The laminin-10 concentration in the conditioned medium exceeded 10 mg/l, which is 10-fold higher than the concentrations achieved by conventional recombinant expression systems. The gene-swapped F9 cells deposited basement membrane-like matrices containing laminin-10 on culture dishes, offering a novel microenvironment for in vitro cell manipulation.

Animals↗

PubMed2005

Potentiation of the ligand-binding activity of integrin alpha3beta1 via association with tetraspanin CD151.

CD151, one of the tetraspanins, forms a stable complex with integrin alpha3beta1, the major laminin receptor on the cell surface. We found that 8C3, an anti-CD151 mAb obtained by screening for reactivity with integrin alpha3beta1-CD151 complexes, was capable of dissociating CD151 from integrin alpha3beta1, thereby allowing us to deplete CD151 from purified integrin alpha3beta1-CD151 complexes. The CD151-free integrin alpha3beta1 thus obtained showed a significant reduction in its ability to bind to laminin-10/11, a high-affinity ligand for integrin alpha3beta1, with a concomitant reduction in its reactivity with mAb AG89, which recognizes activated beta1-containing integrins. These results raised the possibility that the association of integrin alpha3beta1 with CD151 potentiates the ligand-binding activity of the integrin through sustaining its activated conformation. In support of this possibility, the ligand-binding activity was restored when CD151-free integrin alpha3beta1 was reassociated with purified CD151. 8C3-induced dissociation of CD151 from integrin alpha3beta1 was also demonstrated on the surface of living cells by fluorescent resonance energy transfer imaging, accompanied by a concomitant reduction in the cell adhesion to laminin-10/11-coated substrates. CD151 knock-down by RNA interference also resulted in a reduction in the adhesive activity of the cells. Taken together, these results indicate that CD151 association modulates the ligand-binding activity of integrin alpha3beta1 through stabilizing its activated conformation not only with purified proteins but also in a physiological context.

Animals↗

PubMed2005

Laminin gamma2-chain fragment circulating level increases in patients with metastatic pancreatic ductal cell adenocarcinomas.

Laminin-5 (LN5) is expressed solely by epithelial cells and considered to enhance cell migration after being cleaved by proteases, leading to the shedding of the N-terminal fragment of the LN5 gamma2-chain (G2F). We estimated circulating G2F level in 181 patients with various digestive diseases and 15 healthy subjects. The G2F level in pancreatic ductal cell adenocarcinoma patients with liver metastases was markedly elevated, but that in patients without liver metastases was not significantly elevated. The G2F levels in patients with benign pancreatic tumours (pancreatic cysts and intraductal papillary mucinous tumours) were similar to that in healthy volunteers. On the other hand, the level of the gamma1-chain is a common constituent of several laminin heterotrimers. The N-terminal fragment of gamma1-chain (G1F) in the circulation of these patients was also determined, and a slight increase in G1F levels was observed only in hepatocellular carcinoma patients. Interestingly, a significant increase in circulating G2F/G1F ratio was observed in patients with bile duct and gallbladder carcinoma, as well as in those with metastatic pancreatic ductal cell adenocarcinoma. The increase in the circulating G2F level, particularly compared with circulating G1F level, should correlated with LN5 overexpression in invasive carcinomas, independently of basement membrane metabolism in the entire body.

Adenocarcinoma↗

PubMed2004

Laminin isoforms differentially regulate adhesion, spreading, proliferation, and ERK activation of beta1 integrin-null cells.

The presence of many laminin receptors of the beta1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin alpha6beta4 and dystroglycan. We therefore tested the binding of a beta1 integrin-null cell line GD25 to four different laminin variants. The cells were shown to produce dystroglycan, which based on affinity chromatography bound to laminin-1, -2/4, and -10/11, but not to laminin-5. The cells also expressed the integrin alpha6Abeta4A variant. GD25 beta1 integrin-null cells are known to bind poorly to laminin-1, but we demonstrate here that these cells bind avidly to laminin-2/4, -5, and -10/11. The initial binding at 20 min to each of these laminins could be inhibited by an integrin alpha6 antibody, but not by a dystroglycan antibody. Hence, integrin alpha6Abeta4A of GD25 cells was identified as a major receptor for initial GD25 cell adhesion to three out of four tested laminin isoforms. Remarkably, cell adhesion to laminin-5 failed to promote cell spreading, proliferation, and extracellular signal-regulated kinase (ERK) activation, whereas all these responses occurred in response to adhesion to laminin-2/4 or -10/11. The data establish GD25 cells as useful tools to define the role integrin alpha6Abeta4A and suggest that laminin isoforms have distinctly different capacities to promote cell adhesion and signaling via integrin alpha6Abeta4A.

Animals↗

PubMed2003

CD151 regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actin cytoskeletal reorganization.

CD151, a member of the tetraspanin family proteins, tightly associates with integrin alpha3beta1 and localizes at basolateral surfaces of epithelial cells. We found that overexpression of CD151 in A431 cells accelerated intercellular adhesion, whereas treatment of cells with anti-CD151 mAb perturbed the integrity of cortical actin filaments and cell polarity. E-Cadherin puncta formation, indicative of filopodia-based adhesion zipper formation, as well as E-cadherin anchorage to detergent-insoluble cytoskeletal matrix, was enhanced in CD151-overexpressing cells. Levels of GTP-bound Cdc42 and Rac were also elevated in CD151-overexpressing cells, suggesting the role of CD151 in E-cadherin-mediated cell-cell adhesion as a modulator of actin cytoskeletal reorganization. Consistent with this possibility, engagement of CD151 by the substrate-adsorbed anti-CD151 mAb induced prominent Cdc42-dependent filopodial extension, which along with E-cadherin puncta formation, was strongly inhibited by calphostin C, a protein kinase C (PKC) inhibitor. Together, these results indicate that CD151 is involved in epithelial cell-cell adhesion as a modulator of PKC- and Cdc42-dependent actin cytoskeletal reorganization.

Actins↗

PubMed2003

Laminin gamma2-chain fragment in the circulation: a prognostic indicator of epithelial tumor invasion.

Laminin (LN) 5, the major component of epithelial-derived extracellular matrix (ECM), plays a major role in cell adhesion and motility. Previous reports stated that proteolytic processing of the NH(2)-terminal region of the gamma2 chain enhances cell motility on LN5, indicating that the degraded gamma2 chain NH(2)-terminal region would be shed from the ECM. However, soluble LN gamma2 NH(2)-terminal fragment (G2F) have not been detected in biological fluids. Here, we developed a double-monoclonal electrochemiluminescence immunoassay for human G2F and detected its presence in the normal human circulation (mean +/- SD: 39.2 +/- 10.3 ng/ml; n = 10). We also measured serum levels of G2F in nude mice orthotopically transplanted with three different human pancreatic carcinoma cell lines: MIApaca-II (secreting no LN5), HPAC (secreting the alpha3beta3gamma2 heterotrimer of LN5), or KP-1 (secreting the monomeric gamma2 chain of LN5). Serum levels of G2F drastically increased in the nude mice transplanted with HPAC (mean +/- SD: 351 +/- 33 ng/ml, 5 weeks after transplantation), the most invasive tumor cells to generate extensive peritoneal dissemination in vivo. A moderate increase in serum levels of G2F was also observed in mice transplanted with KP-1 (87.9 +/- 82 ng/ml, 5 weeks after transplantation), but no antigen was detected in the sera of MIApaca-II-transplanted mice. Therefore, circulating G2F was demonstrated to be a sensitive marker for LN5 production of primary pancreatic carcinomas, even if it was produced only as a monomeric gamma2 chain. In 11 established human pancreatic tumor cell lines (6 of LN5-producing cells and 5 of nonproducing cells), LN5-secreting cells have significantly higher levels of cell surface expression of beta4 integrin than nonsecreting cells. Thus, LN5 secretion is accompanied by cell surface expression of alpha6beta4 integrin, participating in hemidesmosome reorganization to form invading edges of malignant epithelial carcinomas. These data reveal that the level of circulating G2F is a new, prognostic, tumor-characterizing marker for estimating the invasiveness and malignancy of epithelial carcinomas in cancer patients.

Adenocarcinoma↗

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↗

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↗