RESUMEN
The syndecans make up a family of transmembrane heparan sulfate proteoglycans that act as coreceptors with integrins and growth factor tyrosine kinase receptors. Syndecan-4 is upregulated in skin dermis after wounding, and, in cultured fibroblasts adherent to the ECM protein fibronectin, this proteoglycan signals cooperatively with beta1 integrins. In this study, we generated mice in which the syndecan-4 gene was disrupted by homologous recombination in embryonic stem cells to test the hypothesis that syndecan-4 contributes to wound repair. Mice heterozygous or homozygous for the disrupted syndecan-4 gene are viable, fertile, and macroscopically indistinguishable from wild-type littermates. Compared with wild-type littermates, mice heterozygous or homozygous for the disrupted gene have statistically significant delayed healing of skin wounds and impaired angiogenesis in the granulation tissue. These results indicate that syndecan-4 is an important cell-surface receptor in wound healing and angiogenesis and that syndecan-4 is haplo-insufficient in these processes.
Asunto(s)
Glicoproteínas de Membrana/deficiencia , Neovascularización Patológica/genética , Proteoglicanos/deficiencia , Enfermedades de la Piel/genética , Cicatrización de Heridas/genética , Animales , Femenino , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Neovascularización Patológica/patología , Proteoglicanos/genética , Enfermedades de la Piel/patología , Sindecano-4RESUMEN
Syndecan-4 is a cell surface heparan sulfate proteoglycan which, in cooperation with integrins, transduces signals for the assembly of focal adhesions and actin stress fibers in cells plated on fibronectin. The regulation of these cellular events is proposed to occur, in part, through the interaction of the cytoplasmic domains of these transmembrane receptors with intracellular proteins. To identify potential intracellular proteins that interact with the cytoplasmic domain of syndecan-4, we carried out a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait. As a result of this screen, we have identified a novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three syndecan family members. The interaction involves both the membrane proximal and variable central regions of the cytoplasmic domain. We have named this cDNA and encoded protein syndesmos. Syndesmos is ubiquitously expressed and can be myristylated. Consistent with its myristylation and syndecan-4 association, syndesmos colocalizes with syndecan-4 in the ventral plasma membranes of cells plated on fibronectin. When overexpressed in NIH 3T3 cells, syndesmos enhances cell spreading, actin stress fiber and focal contact formation in a serum-independent manner.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Adhesión Celular/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteoglicanos/metabolismo , Células 3T3 , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Citoesqueleto/metabolismo , ADN Complementario/genética , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/química , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteoglicanos/química , Sindecano-4 , Distribución Tisular , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The formation of focal adhesions and actin stress fibers on fibronectin is dependent on signaling through (&bgr;)1 integrins and the heparan sulfate proteoglycan syndecan-4, and we have analyzed the requirement of the glycosaminoglycan chains of syndecan-4 during these events. Chinese hamster ovary cells with mutations in key enzymes of the glycanation process do not synthesize glycosaminoglycan chains and are unable to assemble actin stress fibers and focal contacts when cultured on fibronectin. Transfection of the mutant cells with a cDNA that encodes the core protein of chicken syndecan-4 leads to the production of unglycanated core protein. The overexpression of syndecan-4 core protein in these mutant cells increases cell spreading and is sufficient for these cells to assemble actin stress fibers and focal adhesions similar to wild-type cells seeded on fibronectin and vitronectin matrices. Syndecan-4 core protein colocalizes to focal contacts in mutant cells that have been transfected with the syndecan-4 core protein cDNA. These data indicate an essential role for the core protein of syndecan-4 in the generation of signals leading to actin stress fiber and focal contact assembly.
Asunto(s)
Actinas/fisiología , Adhesión Celular/fisiología , Glicoproteínas de Membrana/fisiología , Proteoglicanos/fisiología , Animales , Células CHO , Pollos , Cricetinae , Glicosaminoglicanos/fisiología , Glicosilación , Glicoproteínas de Membrana/genética , Proteoglicanos/genética , Proteínas Recombinantes/metabolismo , Estrés Mecánico , Sindecano-4 , Transfección , Vinculina/fisiologíaRESUMEN
The assembly of focal adhesions and actin stress fibers by cells plated on fibronectin depends on adhesion-mediated signals involving both integrins and cell-surface heparan sulfate proteoglycans. These two cell-surface receptors interact with different domains of fibronectin. To attempt to identify the heparan sulfate proteoglycans involved, we used fibronectin-null (FN-/-) mouse fibroblasts to eliminate the contribution of endogenous fibronectin during the analysis. FN-/- fibroblasts plated on the cell-binding domain of fibronectin or on antibodies directed against mouse beta1 integrin chains attach but fail to spread and do not form focal adhesions or actin stress fibers. When such cells are treated with antibodies directed against the ectodomain of mouse syndecan-4, they spread fully and assemble focal adhesions and actin stress fibers indistinguishable from those seen in cells plated on intact fibronectin. These results identify syndecan-4 as a heparan sulfate proteoglycan involved in the assembly process. The antibody-stimulated assembly of focal adhesions and actin stress fibers in cells plated on the cell-binding domain of fibronectin can be blocked with C3 exotransferase, an inhibitor of the small GTP-binding protein Rho. Treatment of cells with lysophosphatidic acid, which activates Rho, results in full spreading and assembly of focal adhesions and actin stress fibers in fibroblasts plated on the cell-binding domain of fibronectin. We conclude that syndecan-4 and integrins can act cooperatively in generating signals for cell spreading and for the assembly of focal adhesions and actin stress fibers. We conclude further that these joint signals are regulated in a Rho-dependent manner.