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b-Catenin is an essential element for the transcriptional activation of target genes in the Wnt signaling cascade and is also a cell adhesion molecule that couples with cadherins. Although plakoglobin (c-catenin), a closely related homologue of b-catenin, is also known to be a cell adhesion molecule, its function as a transcriptional factor has not been revealed in detail. Using a human malignant mesothelioma cell line, NCI-H28, in which we have identified a homozygous deletion of the b-catenin gene, we studied whether plakoglobin has a T-cell factor/ lymphocyte enhancer factor (TCF/LEF) family-dependent transcriptional activity. Transfection with the wildtype plakoglobin expression vector induced accumulation of plakoglobin in the nucleus. Immunoprecipitation assay with cotransfection of plakoglobin and either TCF-4 or LEF-1 detected binding of plakoglobin to TCF-4 or LEF-1. Luciferase reporter assay demonstrated transcriptional activity of the wild-type plakoglobin when transfected with TCF/LEF, although plakoglobin showed less activity than b-catenin. Exogenous plakoglobin was also shown to promote entrance of exogenous b-catenin into the nuclei. Furthermore, small interfering RNA directed against plakoglobin suppressed expression of endogenous plakoglobin and its transcriptional activity, suggesting that endogenous plakoglobin has a weak transcriptional activity. These results suggest that plakoglobin can activate the Wnt signaling cascade directly without interaction of b-catenin, and that plakoglobin has multiple functions as a transcriptional activator and a cell adhesion molecule like b-catenin.

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 is a transmembrane heparan sulfate proteoglycan belonging to the syndecan family. Following intraperitoneal injection of lipopolysaccharide (LPS), syndecan-4-deficient mice exhibited high mortality compared with wild-type controls. Severe endotoxin shock was observed in the deficient mice: systolic blood pressure and left ventricular fractional shortening were lower in the deficient mice than in the wild-type controls 9 h after LPS injection. Although histological examinations revealed no apparent differences between two groups, the plasma level of interleukin (IL)-1␤ was higher in the deficient mice than in the wild-type controls 9 h after LPS injection. Consistent with the regulatory roles of syndecan-4, its expression in monocytes and endothelial cells of microvasculature increased in the wild-type mice after LPS administration. Although IL-1␤ was produced to the same extent by macrophages from syndecan-4-deficient and wild-type mice after LPS stimulation, inhibition of its production by transforming growth factor-␤1 was impaired in the syndecan-4-deficient macrophages. These results indicate that syndecan-4 could be involved in prevention of endotoxin shock, at least partly through the inhibitory action of transforming growth factor-␤1 on IL-1␤ production.