Kiyotoshi Sekiguchi

Integrin α3β1‐mediated interaction with laminin‐5 stimulates adhesion, migration and invasion of malignant glioma cells

Gliomas, characterized by their progressively invasive phenotype, express integrin α3β1 as a major receptor for the extracellular matrix both in vivo and in vitro. Since the integrin α3β1 has been shown to be a specific receptor for laminin‐5 (α3β3γ2), we examined the effects of purified human laminin‐5 on adhesion, migration and invasion of human glioma cells. Among different types of laminin variants and other matrix proteins including fibronectin and vitronectin, laminin‐5 was most potent in promoting adhesion and migration of different kinds of glioma cells. Laminin‐5‐mediated adhesion and migration were specifically inhibited by monoclonal antibodies against integrin α3 and β1 chains, confirming the role of integrin α3β1 as the major laminin‐5 receptor. Invasion of the reconstituted basement membrane (i.e., Matrigel) by glioma cells was also selectively stimulated by laminin‐5. Out results show that laminin‐5 is the major extracellular stimulant for glioma cell adhesion, migration and invasion. The immunohistochemical distribution of laminin γ2 chain, a laminin subunit unique to laminin‐5, showed that it was expressed in the tumor parenchyma of human glioma tissues. Expression of laminin α3, β3 and γ2 chains in glioma tissues and in glioma cell lines was also demonstrated at the messenger RNA level by reverse transcription polymerase chain reaction. Our results, taken together, show that laminin‐5 may be involved in the invasive phenotype of malignant gliomas both in vitro and in vivo. Int. J. Cancer76:63–72, 1998.© 1998 Wiley‐Liss, Inc.

Alternatively spliced EDA segment regulates fibronectin-dependent cell cycle progression and mitogenic signal transduction.

Fibronectin (FN) is comprised of multiple isoforms arising from alternative splicing of a single gene transcript. One of the alternatively spliced segments, EDA, is expressed prominently in embryonic development, malignant transformation, and wound healing. We showed previously that EDA+ FN was more potent than EDA− FN in promoting cell spreading and cell migration because of its enhanced binding affinity to integrin α5β1 (Manabe, R., Oh-e, N., Maeda, T., Fukuda, T., and Sekiguchi, K. (1997) J. Cell Biol. 139, 295–307). In this study, we compared the cell cycle progression and its associated signal transduction events induced by FN isoforms with or without the EDA segment to examine whether the EDA segment modulates the cell proliferative potential of FN. We found that EDA+ FN was more potent than EDA− FN in inducing G1-S phase transition. Inclusion of the EDA segment potentiated the ability of FN to induce expression of cyclin D1, hyperphosphorylation of pRb, and activation of mitogen-activated protein kinase extracellular signal regulated kinase 2 (ERK2). EDA+ FN was also more potent than EDA− FN in promoting FN-mediated tyrosine phosphorylation of p130Cas, but not focal adhesion kinase, which occurred in parallel with the activation of ERK2, suggesting that p130Cas may be involved in activation of ERK2. These results indicated that alternative splicing at the EDA region is a novel mechanism that promotes FN-induced cell cycle progression through up-regulation of integrin-mediated mitogenic signal transduction.

Identification and recombinant production of human laminin α4 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.

The domains of human fibronectin mediating the binding of alpha antigen, the most immunopotent antigen of mycobacteria that induces protective immunity against mycobacterial infection.

We have recently shown that alpha antigen (alpha-Ag), the immunodominant antigen of mycobacteria, has a novel fibronectin (FN)-binding motif that is unique among mycobacteria [Naito, Ohara, Matsumoto and Yamada (1998) J. Biol. Chem. 273, 2905-2909]. In this study, we examined the domains of human FN that interacted with alpha-Ag. Fragments of FN generated by either proteolysis or recombinant DNA techniques were compared for their ability to bind to alpha-Ag. Fragments containing either the C-terminal heparin-binding domain or the central cell-binding domain consistently bound to alpha-Ag. The fragment of the C-terminal heparin-binding domain, upon mutation that resulted in the loss of its heparin-binding activity, could not bind with alpha-Ag. These findings suggested that the mutated site, i.e. the main heparin-binding site of FN, was also the principal site for binding to alpha-Ag. The alpha-Ag-binding domains of FN could bind whole mycobacterial bacilli, suggesting that these two domains are important contributors to mycobacterial infection.

Decreased fibronectin expression in Met/HGF-mediated tumorigenesis

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor (HGF)/scatter factor are involved in the etiology and progression of a number of human cancers. Coexpression of Met and HGF in mesenchymal cells increases the tumorigenic and metastatic potential of the cells. In the studies described here, we used differential display screening to identify changes in gene expression that are initiated by Met/HGF, and that may lead to these phenotypes. We learned that Met/HGF signaling resulted in greatly decreased fibronectin mRNA production in three different human and mouse tumor cell lines; these decreases in fibronectin mRNA were paralleled by decreases in fibronectin protein. We also found a progressive decrease in fibronectin in tumor explants and metastases derived from the Met/HGF transformed cells. The absence of fibronectin expression is a frequent cancer phenotype; our results indicate that decreases in fibronectin correlate with, but are not essential for, MetHGF/SF-mediated tumorigenesis.

Csk regulates integrin-mediated signals: involvement of differential activation of ERK and Akt

Csk phosphorylates Src family tyrosine kinases and down-regulates their activities in vitro and in vivo. To gain insight into the integrin-mediated cellular functions of this negative regulator of the Src family, we examined integrin-mediated signals in Csk-deficient fibroblasts (Csk(-) cells) and their stable transfectants expressing re-introduced Csk (Csk(-)/Csk cells). Integrin-mediated activation of extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase in Csk(-)/Csk cells upon adhesion to fibronectin or laminin-10/11 was down-regulated, whereas Akt activation increased. Interestingly, the suppression of ERK-MAP kinase activation in Csk(-)/Csk cells was restored by overexpression of a dominant-negative Akt. In agreement with these results, Csk(-)/Csk cells were more resistant to apoptosis induced by serum depletion, but were less proliferative, compared with Csk(-) cells. These results, taken together, demonstrate that Csk is an important regulator of integrin-mediated signaling and cellular behavior.

Matrix metalloproteinase-2 is involved in A549 cell migration on laminin-10/11

We have reported that laminin-10/11 strongly promotes migration of A549 human lung carcinoma cells by activating the alpha3beta1 integrin-dependent signaling pathway. To elucidate the mechanism involved, we investigated whether matrix metalloproteinases (MMPs) are involved in cell migration on laminin-10/11. Here, we demonstrate that laminin-10/11, but not fibronectin which does not greatly promote A549 cell movement, stimulated MMP-2 secretion approximately 3-fold. The cell migration-promoting activity of laminin-10/11 was down-regulated by an MMP inhibitor. In addition, cell motility was significantly increased when cells adhered to a mixture of fibronectin and laminin-10/11 with a concomitant decrease of focal contacts, compared with those adhering to fibronectin alone. The enhanced cell migration was partially suppressed by the MMP inhibitor. Furthermore, an anti-alpha3 integrin, but not an anti-alpha5 integrin, antibody induced the activated form of MMP-2. These data suggest that MMP-2 may play an important role in A549 cell migration on laminin-10/11 through an alpha3beta1 integrin-dependent pathway.