Roles of GalNAc-disialyl Lactotetraosyl Antigens in Renal Cancer Cells
Akiko TsuchidaMotohiro SendaAkihiro ItoSeiichi SaitoMakoto KisoTakayuki AndoAnne Harduin‐LepersAkio MatsudaKeiko FurukawaKoichi Furukawa
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Abstract:
GalNAc-disialyl Lc4 (GalNAc-DSLc4) was reported as a novel antigen that associated with malignant features of renal cell cancers (RCCs). To clarify roles of GalNAc-DSLc4 in malignant properties of RCCs, we identified B4GalNAc-T2 as a responsible gene for the synthesis of GalNAc-DSLc4, and prepared stable transfectants of GalNAc-T2 cDNA using VMRC-RCW cells, resulting in the establishment of high expressants of GalNAc-DSLc4. They showed increased proliferation and invasion, and specific adhesion to laminin. In the transfectants, PI3K/Akt signals were highly activated by serum stimulation or adhesion to laminin. GalNAc-DSLc4 was co-localized in lipid rafts with integrin β1 and caveolin-1 in both immunoblotting of fractionated detergent extracts and immunocytostaining, particularly when stimulated with serum. Masking of GalNAc-DSLc4 with antibodies as well as PI3K inhibitor suppressed malignant properties of the transfectants. These results suggested that GalNAc-DSLc4 is involved in malignant properties of RCCs by forming a molecular complex with integrins in lipid rafts.Keywords:
Lipid raft
To analyse the interactions between glioma cells and extracellular matrix (ECM) proteins, the adhesive and migratory capacity of five human glioma cell lines (D37MG, D54MG, GaMG, U118MG and U251MG) were studied. The expression of integrins was analysed and correlated to the adhesive and migratory abilities of the cells. All cell lines were able to adhere to and migrate on the extracellular matrix proteins collagen IV, fibronectin, laminin and vitronectin. Laminin was superior in propagating adhesion and migration in all five cell lines. As analysed by flow cytometry, the expression of the integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, beta 3, beta 4, alpha v, and integrin alpha v beta 5 proved to be uniform between the cell lines. All integrins except alpha 4, alpha 6, beta 3 and beta 4 were expressed on more than 85% of the cells. Inhibition of adhesion with synthetic peptides and antibodies directed against integrins demonstrated that adhesion on laminin was independent of integrins and the 67 kD laminin receptor. On the other hand, migration was shown to be integrin-dependent on all substrates. The results indicate that the mechanism responsible for cell adhesion in human gliomas differ from those present during migration, and that integrins play an important role in regulating these two mechanisms.
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Abstract The basement membrane (BM) protein laminin‐332 (Lm332) (laminin‐5) has unique activity and structure as compared with other laminins: it strongly promotes cellular adhesion and migration, and its α3, β3, and γ2 chains are all truncated in their N‐terminal regions (short arms). In the present study, we investigated the biological function of the laminin β3 chain. When the β3 chain short arm (β3SA) was overexpressed in HEK293 cells (β3SA‐HEK), they deposited a large amount of β3SA and a small amount of laminin‐511 (Lm511) (laminin‐10) on culture plates. Control HEK293 cells secreted Lm511 but failed to deposit it. The extracellular matrix (ECM) deposited by β3SA‐HEK cells strongly promoted cell attachment and spreading. The β3SA‐HEK ECM did not directly bind Lm511, but it stimulated control HEK293 cells to deposit Lm511 on the culture plates. Although purified β3SA did not support cell adhesion by itself, it enhanced the cell adhesion activity of Lm511. Experiments with anti‐integrin antibodies also suggested that the strong cell adhesion activity of the β3SA‐HEK ECM was derived from the synergistic action of β3SA and Lm511. It has previously been found that β3SA binds an unknown cell surface receptor. Taken together, the present study suggests that the short arm of the laminin β3 chain enhances the matrix assembly of Lm511 and its cell adhesion activity by interacting with its receptor. J. Cell. Biochem. 100: 545–556, 2007. © 2006 Wiley‐Liss, Inc.
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Integrins and syndecans mediate cell adhesion to extracellular matrix and their synergistic cooperation is implicated in cell adhesion processes. We previously identified two active peptides, AG73 and EF1, from the laminin α1 chain LG4 module, that promote cell attachment through syndecan‐ and α2β1 integrin‐binding, respectively. Here, we examined time‐dependent cell attachment on the mixed peptides AG73/EF1. The AG73/EF1 promoted stronger and more rapid cell attachment, spreading, FAK phosphorylation that reached a maximum at 20 min than that on AG73 (40 min) or EF1 (90 min) supplied singly. Thus, the syndecan‐ and α2β1 integrin‐binding peptides synergistically affect cells and accelerate cell adhesion.
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Abstract The basement membrane protein laminin‐5 promotes cell adhesion and migration. The carboxyl‐terminal G3 domain in the α3 chain is essential for the unique activity of laminin‐5. To investigate the function of the G3 domain, we prepared various recombinant laminin‐5 forms with a partially deleted or mutated G3 domain. The deletion of the carboxyl‐terminal 28 amino acids (region III) markedly decreased the cell adhesion activity with a slight loss of the cell motility activity toward BRL and EJ‐1 cells. This change was attributed to the loss of Lys‐Arg‐Asp sequence. Further deletion of 83 amino acids (region II) led to almost complete loss of the cell motility activity. All charged amino acid residues tested in this region were not responsible for the activity loss. These results suggest that the G3 domain contains two distinct regions that differently regulate cell adhesion and migration. Analysis of laminin‐5 receptors showed that integrins α3β1, α6β1, and α6β4 had different but synergistic effects on cell adhesion and migration on laminin‐5. However, the structural change of the G3 domain appeared not to change integrin specificity. The present study demonstrates that the G3 domain in laminin‐5 plays a central role to produce different biological effects on cells. © 2002 Wiley‐Liss, Inc.
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Laminin 5 is a basement membrane component that actively promotes adhesion and migration of epithelial cells. Laminin 5 undergoes extracellular proteolysis of the γ2 chain that removes the NH2-terminal short arm of the polypeptide and reduces the size of laminin 5 from 440 to 400 kD. The functional consequence of this event remains obscure, although lines of evidence indicate that cleavage of the γ2 chain potently stimulated scattering and migration of keratinocytes and cancer cells. To define the biological role of the γ2 chain short arm, we expressed mutated γ2 cDNAs into immortalized γ2-null keratinocytes. By immunofluorescence and immunohistochemical studies, cell detachment, and adhesion assays, we found that the γ2 short arm drives deposition of laminin 5 into the extracellular matrix (ECM) and sustains cell adhesion. Our results demonstrate that the unprocessed 440-kD form of laminin 5 is a biologically active adhesion ligand, and that the γ2 globular domain IV is involved in intermolecular interactions that mediate integration of laminin 5 in the ECM and cell attachment.
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