A Peptide Derived from Tenascin-C Induces β1 Integrin Activation through Syndecan-4

Abstract

Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated ␤1 integrinmediated cell adhesion of nonadherent and adherent cell types, by inducing activation of ␤1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate (HS) proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced ␤1 integrin activation: 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photoaffinity labeling; 2) heparitinase I treatment of cells abrogated ␤1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) small interfering RNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced ␤1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of ␤1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with protein kinase C␣ inhibitor Gö6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of the TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains and then induced conformational change in ␤1 integrin necessary for its functional activation. A lateral interaction of ␤1 integrin with the extracellular region of the syndecan-4 molecule may be involved in this conformation change. Tenascin (TN)-C2 is one of the most intriguing extracellular matrix (ECM) proteins (1-3). TN-C is expressed predominantly during embryogenesis, wound healing, and neoplastic processes, in which alternative mRNA splicing within the fibronectin (FN) type III-like (FN-III) repeats can generate different TN-C isoforms (4). Multifunctional properties have been identified for TN-C, including effects on cell adhesion, migration, proliferation, survival, and differentiation. The effects of TN-C on cell adhesion are particularly complex; the TN-C substrate supports attachment of some cell types, but is nonadhesive or even repulsive for other cell types (5-7). Based on these antipodal effects on cell adhesion, TN-C is multifunctional and is therefore classified as an adhesion modulatory ECM protein, a so-called "matricellular" protein (8). Various TN-C molecule domains, especially FN-III repeats, including the alternative splicing domains, have been implicated in its function as a matricellular protein. However, their contributions to the adhesion modulatory effects of TN-C are not completely understood.Interactions of cells with the ECM are largely mediated by members of the integrin superfamily of adhesive receptors. The most unique feature of integrins is their ability to alter ligand binding and signaling activities. Because ...

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“…Anoikis-NIH3T3 cells adhere to the fibronectin substratum mainly via integrin ␣5␤1 (28). When NIH3T3 cells adhering to the fibronectin were cultured under serum-starved conditions, the number of viable cells decreased in a time-dependent manner, which was in parallel with an increase in the detached cell number (Fig.…”

“…For cell adhesion, cells suspended with the serum-free medium with or without the sample to be tested were seeded onto a 96-well plate coated with fibronectin, incubated for 30 -60 min, and then fixed with formalin. After washing off unadhered cells, cells adhered to the fibronectin substratum were counted as described previously (28). For ␤1-integrin activation, activation of ␤1 integrins on the cells was evaluated by flow cytometric analysis (FACS Aria, BD Biosciences) using 9EG7, a monoclonal antibody recognizing the active ␤1-integrin conformation-specific epitope (29), as described previously (18).…”

“…Cell Survival-Cell survival during serum starvation culture was determined as described previously (28). Briefly, cells suspended with serum-free medium were seeded onto a 96-well plate coated with fibronectin and cultured in the presence or absence of the sample to be tested for the indicated periods.…”

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“…Here, a 22mer fusion peptide containing an adhesive YTITIRG sequence bound to the heparin sulfated side chains of the b1 integrin co-receptor synecan-4, hence inducing cell adhesion and spreading on FN. 132 This study found that mutation of the cytosolic region of syndecan-4 had no effect on promoting the inhibition of b1 integrin action, suggesting the extracellular portion of syndecan-4 binds FNIII-A2 and hence promotes b1 integrin clustering and activation, rather than mediating this effect via downstream targets of syndecan-4. Elevated levels of both activated MMP-2 and degraded tenascin-C were identified in NSCLC, with proteolytically degraded tenascin-C serving as a good indicator of stage-1 NSCLC recurrence.…”

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“…[8][9][10][11][12][13] However, the complete molecule is considered anti-adhesive. This function has been mapped primarily to the EGFL component of TNC.…”