Syndecan-4 Deficiency Impairs Focal Adhesion Formation Only under Restricted Conditions

Abstract

Two domains of fibronectin deliver two different but cooperative signals required for focal adhesion formation. The signal from the cell-binding domain is mediated by integrins, whereas the signal from the heparinbinding domain is recognized by heparan sulfate proteoglycans, of which syndecan-4 has been hypothesized to be involved in focal adhesion formation. We generated mice deficient in syndecan-4 to study its role directly. Even in fibroblasts from syndecan-4-deficient mice, focal adhesions were formed, and actin fibers terminated normally at focal adhesions when they were cultured on coverslips coated with fibronectin or with a mixture of its cell-binding and heparin-binding fragments. However, when the cells were cultured on the cell-binding fragment and the heparin-binding fragment was added to the medium, focal adhesion formation was impaired in the syndecan-4 null fibroblasts as compared with that in wild-type cells. Therefore, syndecan-4 is essential for promoting focal adhesion formation only when the signal of the heparin-binding domain of fibronectin is delivered as a soluble form, most probably from the apical surface. When the signal is delivered as a substratum-bound form, other molecule(s) also participate(s) in the signal reception.Interactions between cells and extracellular matrices are highly important in the regulation of various biological processes such as development, growth, and repair. Focal adhesions are macromolecular complexes found at the sites of cell adhesion to extracellular matrices (1, 2). Focal adhesions are linked to actin stress fibers and also serve as signaling complexes involved in triggering intracellular signaling cascades.When cells are plated on fibronectin, focal adhesion formation requires two independent signals delivered from the cellbinding domain and the heparin-binding domain of the molecule (2-4). The signal from the RGD-containing cell-binding domain is mediated through integrins (5, 6), whereas the heparin-binding domain is recognized by heparan sulfate proteoglycan(s) (2-4). Among these, syndecan-4 (also called ryudocan or amphiglycan; see Refs. 7-9) is believed to be important, because this transmembrane protein is a component of focal adhesions (10, 11) and antibodies directed against the ectodomain of syndecan-4 cooperate with the cell-binding fragment of fibronectin to form focal adhesions (12). To evaluate the precise role of syndecan-4 in focal adhesion formation, we generated mice deficient in the syndecan-4 gene (Synd4) 1 and examined fibroblasts from these animals. EXPERIMENTAL PROCEDURESMaterials-Intact human fibronectin and its cell-binding fragment (CBF) were purchased from Wako Chemical Co. Heparin-binding fragment of fibronectin (HBF), tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin, anti-vinculin monoclonal antibody, fluorescein isothiocyanate-conjugated anti-mouse IgG antibody, and TRITC-conjugated anti-rabbit IgG antibody were obtained from SigmaAldrich. Anti-syndecan-4 ectodomain antibody (@-Synd4) was prepared as descri...

Syndecan-4 signaling via NFAT regulates extracellular matrix production and cardiac myofibroblast differentiation in response to mechanical stress

“…In line with previous work [21,[27][28][29], vinculin confocal immunofluorescence revealed fewer and smaller focal adhesions in fibroblasts from syndecan-4 -/-mice when compared to those from WT mice (Supplemental Figure S4) which may imply impaired focal adhesion signaling in cardiac fibroblasts from syndecan-4 -/-mice.…”

“…Fibroblasts will spontaneously differentiate into myofibroblasts when cultured on a fibronectin-coated rigid substrate due to effects of fibronectin on focal adhesion assembly [21] and increased mechanical tension compared to the in vivo situation [22]. We took advantage of this property and compared the expression of SMA and the percentage of cells exhibiting organized SMA fibers in WT and syndecan-4 -/-cardiac fibroblasts after 24 and 48 hrs of culture on fibronectin-coated glass cover slips (Figure 2A and B).…”

Coregulation of Fibronectin Signaling and Matrix Contraction by Tenascin-C and Syndecan-4

“…Analysis of actin cytoskeleton organization over time shows that filopodia and lamellae are formed as early as 15 min and continue to extend up to 60 min (our unpublished results). On planar FN-coated substrates, both wild-type and syndecan-4 -deficient fibroblasts became well spread and formed stress fibers and focal adhesions (our unpublished results; Ishiguro et al, 2000;Echtermeyer et al, 2001). In contrast, syndecan-4 -null cells attached poorly and did not spread on 3D matrix composed of fibrin alone, similar to the behavior of NIH3T3 fibroblasts (Wenk et al, 2000), which indicates that the contribution of fibrin to the response is limited.…”

“…On a surface coated with a mixture of cell-and heparin-binding (HepII) domains from FN, stress fibers terminating at focal adhesions were formed by these fibroblasts at levels comparable to intact FN (Ishiguro et al, 2000). However, addition of HepII fragment in soluble form to cells on the cell-binding domain induced very few focal adhesions.…”

Differential Regulation of Extracellular Tissue Inhibitor of Metalloproteinases-3 Levels by Cell Membrane-bound and Shed Low Density Lipoprotein Receptor-related Protein 1

“…MEF-1 and PEA-13 (homozygous LRP-1-deficient) (29) cells were generated as described previously. Syndecan-4-deficient and wild-type MEF (30) were kindly provided by John R. Couchman (Department of Biomedical Sciences and Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark). All cells were maintained in DMEM with 10% FCS, 100 units/ml penicillin, and 100 units/ml streptomycin at 37°C in 5% CO 2 .…”