The role of syndecan-2 in regulation of actin-cytoskeletal organization of Lewis lung carcinoma-derived metastatic clones

Abstract

Syndecans, a family of transmembrane heparan sulphate proteoglycans, contribute to various biological processes, including adhesion, motility, proliferation, differentiation and morphogenesis. We document here the involvement of syndecan-2 acting alone or co-operatively with integrin alpha5beta1, for regulation of actin-cytoskeletal organization on cell adhesion to fibronectin, using fibronectin-recombinant polypeptides containing the ligands for either or both of these receptors as substrata. Lewis lung carcinoma-derived low-metastatic P29 cells binding to the substrata by both receptors formed actin stress fibres, whereas those binding by syndecan-2 or integrin alpha5beta1 alone formed filopodia or cortex actin. In contrast, higher metastatic LM66-H11 cells formed cortex actin even on substrata containing both ligands. Northern-blot and flow-cytometric analyses revealed that syndecan-2 expression in LM66-H11 cells was significantly lower (1/4.5 in mRNA and 1/8 in cell-surface expression) than in P29 cells, whereas expression levels of integrin alpha5beta1 and other syndecans were similar in both cell types. These results suggest that the failure of LM66-H11 to form stress fibres is due to a lower expression of syndecan-2 than that due to a threshold for its function. This was confirmed by the finding that overexpression of syndecan-2 by transfection of its cDNA into LM66-H11 cells caused the formation of stress fibres on the fibronectin substratum. These in vitro cellular responses of the two clones might reflect their in vivo situation in primary tumours in which P29 cells with a stroma-inducing capacity were immediately surrounded by fibronectin-rich matrix formed by the induced stromal cells, whereas LM66-H11 cells without such capacity were not surrounded by a similar matrix.

A Novel Function of Syndecan-2, Suppression of Matrix Metalloproteinase-2 Activation, Which Causes Suppression of Metastasis

“…For tumor cells responding to and migrating through a host microenvironment, it is reasonable that cell adhesion to ECM through ECM component-specific receptors, such as integrins, not only reorganizes the cytoskeleton of the cells but also triggers the formation of a (or the) molecular cluster on the cell surface involving MMPs. It is noteworthy that syndecan-2 of the tumor cells used in this study cooperated with integrin ␣5␤1 on the fibronectin substratum to regulate organization of the cytoskeleton (14,15). In cell surface molecular clusters formed in response to ECM, the regulation of degradation enzymes such as MMP-2 in addition to MT1-MMP might occur through the heparan sulfate side chains of syndecan.…”

“…Construction of Syndecan-2 Core Protein and a Mutant of It-cDNA of mouse syndecan-2 core protein containing the entire coding region was generated as described previously (15). The mutant core protein of syndecan-2, of which the glycosaminoglycan-attachable serine residues were changed to alanine residues (S41A, S53A, S55A, and S57A), was generated using a TaKaRa Mutan-Super Express Km Kit (TaKaRa BIO Inc.).…”

“…Northern blot analysis was performed as described previously (15). In brief, 2 g of each poly(A) ϩ RNA from cells was electrophoresed on a 1.0% agarose gel containing 1.1 M formaldehyde, transferred to a Hybond Nϩ nylon membrane (GE Healthcare Bio-Sciences Corp.), and then hybridized to a cDNA fragment of mouse syndecan-1 (nucleotides 409 -967), syndecan-2 (nucleotides 487-1175), syndecan-3 (nucleotides 370 -931), syndecan-4 (nucleotides 92-534), MMP-2 (nucleotides 683-1484), MT1-MMP (nucleotides 1115-1696), TIMP-2 (nucleotides 227-691), ␤-actin (nucleotides 183-722), or glyceraldehyde-3-phosphate dehydrogenase (nucleotides 319 -1047), which had been labeled with [␣-32 P]dCTP by the random labeling method.…”

“…syndecan-1, -2, and -4). Among them the expression level of only syndecan-2, a key molecule for regulation of actin cytoskeletal organization in cooperation with integrin ␣5␤1, was different between the clones, although the expression levels of the other two syndecans were very similar in the two clones (14,15). In this study, to verify the correlation between the metastatic potential and the expression level of syndecan-2, another clone, LM12-3, with intermediate metastatic potential (28), was used.…”

“…Moreover, it is well known that syndecans cooperate with integrins as co-receptors for fibronectin, which constitutes various types of ECM, and participate in cell adhesion and/or migration through regulation of focal contact and stress fiber formation (12)(13)(14)(15)(16). Furthermore, besides the function regarding this signal transduction, cell surface proteoglycans provide initial docking sites for the binding to various viruses (17,18).…”