Coregulation of fibronectin signaling and matrix contraction by tenascin-C and syndecan-4

Syndecan-4 is a ubiquitously expressed heparan sulfate proteoglycan that modulates cell interactions with the extracellular matrix. It is transiently up-regulated during tissue repair by cells that mediate wound healing. Here, we report that syndecan-4 is essential for optimal fibroblast response to the three-dimensional fibrin-fibronectin provisional matrix that is deposited upon tissue injury. Interference with syndecan-4 function inhibits matrix contraction by preventing cell spreading, actin stress fiber formation, and activation of focal adhesion kinase and RhoA mediated-intracellular signaling pathways. Tenascin-C is an extracellular matrix protein that regulates cell response to fibronectin within the provisional matrix. Syndecan-4 is also required for tenascin-C action. Inhibition of syndecan-4 function suppresses tenascin-C activity and overexpression of syndecan-4 circumvents the effects of tenascin-C. In this way, tenascin-C and syndecan-4 work together to control fibroblast morphology and signaling and regulate events such as matrix contraction that are essential for efficient tissue repair.     

The complexity in regulating the expression of tenascins

The tenascins are a growing family of extracellular matrix proteins of typical multidomain structure. The prototype to be discovered was tenascin-C. It shows a highly regulated expression pattern during embryonic development and is often transiently associated with morphogenetic tissue interactions during organogenesis. In the adult organism reexpression of tenascin-C occurs in tumors and many other pathological conditions. Tenascin-C expression can be regulated by many different growth factors and hormones. Furthermore, mechanical strain exerted by fibroblasts seems to induce the expression of tenascin-C. This could represent a mechanism of translating mechanical forces into protein patterns, a step of potential relevance in the organization of embryogenesis. Tenascin-C as well as tenascin-R are believed to counteract the cell adhesion and spreading activity of fibronectin, thereby facilitating cell movement.     

Tenascins

Tenascins are a family of large multimeric extracellular matrix (ECM) proteins. Vertebrates express four tenascins termed tenascin-C, -R, -X and -W present in their connective tissues. Each tenascin has a specific expression pattern. To the contrary of many other ECM proteins, tenascins promote only weak cell adhesion and do not activate cell spreading. They have been classified as anti-adhesive, adhesion-modulating or even repellent ECM proteins. Tenascin-C and tenascin-R deficient mice show abnormalities in the nervous system and tenascin-C deficient mice, in addition, have defects in several regenerative processes. Mice lacking tenascin-X display hyperelastic skin much like Ehlers Danlos patients with mutations in their tenascin-X gene. Since tenascin-C is highly overexpressed in tumor stroma antibodies against tenascin-C have been used in tumor diagnosis and therapy. Since tenascins are known to influence cell shape, migration and growth they represent good candidate molecules for inclusion in artificial bioengineered tissue implants.     

Cryptic domains of tenascin-C differentially control fibronectin fibrillogenesis

The three-dimensional organization of the ubiquitous extracellular matrix glycoprotein fibronectin regulates cell fate and morphogenesis during development; in particular tubule formation that constitutes the vasculature, lung and kidney. Tenascin-C is a matrix protein with a restricted expression pattern; it is specifically up-regulated at sites of fibronectin fibril assembly during development and in remodeling adult tissues. Here we demonstrate that specific domains of tenascin-C inhibit fibronectin matrix assembly whereas full-length tenascin-C does not. These domains act via distinct mechanisms: TNfn1-8 blocks fibrillogenesis by binding to fibronectin fibrils and preventing intermolecular fibronectin interactions whilst FBG acts independently of binding to fibronectin and instead is internalized and causes cytoskeletal re-organization. We also show that TNfn1-8 disrupts epithelial cell tubulogenesis. Our data demonstrate that tenascin-C contains cryptic sites which can control tissue levels of fibrillar fibronectin either by preventing de novo fibril assembly or reducing levels of deposited fibronectin. Exposure of these domains during tissue remodeling may provide a novel means of controlling fibronectin assembly and tubulogenic processes dependent on the assembly of this matrix.     

Interaction between cell and extracellular matrix in heart disease: multiple roles of tenascin-C in tissue remodeling

The heart remodels myocardial tissue in physiological and pathological response. The cell-extracellular matrix (ECM) interaction provides not only structural and mechanical support but also important biological signaling during tissue remodeling. Among various ECM molecules, tenascin-C (TNC) is well known as a regulator of multiple cellular functions during embryogenesis, wound healing or cancer progression. In the heart, TNC appears in several important steps of embryonic development such as the initial differentiation of cardiomyocytes or coronary vasculo/angiogenesis, but it is not detected in a normal adult myocardium. However, TNC is found to re-express after myocardial injury and may regulate cellular behavior during tissue remodeling by modulating the attachment of cardiomyocytes to connective tissue, by enhancing migration and differentiation of myofibroblasts, and by inducing matrix metallo-proteinases. TNC also interacts with other ECM molecules and may modulate progression of fibrosis. Furthermore, transient and site specific expression of TNC closely associated with myocardial injury and inflammation suggests not only its key roles during tissue remodeling but also that TNC can be a marker for myocardial disease activity.     

Tenascin-C induction by the diffusible factor epidermal growth factor in stromal-epithelial interactions

Tenascin-C, a six-armed extracellular matrix glycoprotein, is expressed in a temporally and spatially restricted pattern during carcinogenesis and invasion or metastasis of carcinoma cells in association with stromal-epithelial interactions. The human epidermoid carcinoma-derived cell lines, A431 and HEp-2, which do not express tenascin-C by themselves in vitro, do express tenascin-C after transplantation into nude mice, and transforming growth factor β1 (TGF-β1) induces them to express tenascin-C in vitro. Epidermal growth factor (EGF) induced tenascin-C in these cells more effectively (about 3.5-fold greater) than did TGF-β1. Hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) had little effect on the induction of tenascin-C. EGF also induced other extracellular matrix components, fibronectin and laminin. Tenascin-C was also induced when the carcinoma cells were co-cultured with embryonic fibroblasts from mice which were homozygous for a null mutation in the tenascin-C gene, or when the conditioned medium from these cells was added. The induction of tenascin-C in the co-culture was reduced by treating the cells with antibodies against EGF or its receptor. The addition of EGF caused both cell types to disrupt their cytoskeleton and focal contacts as evidenced by the loss of stress fibers and vinculin plaques. EGF did neither induce tenascin-C nor affect the morphology in tenascin-C-nonproducing A549 carcinoma cells, which did not produce tenascin-C after transplantation. Thus, EGF induces tenascin-C in tenascin-C-nonproducing human carcinoma cells through EGF receptors. Furthermore, in stromalepithelial interactions, the diffusible factor EGF participates in the induction of human tenascin-C in these cells through EGF receptors. © 1995 Wiley-Liss Inc.     

Involvement of tenascin-C and PG-M/versican in flexor tenosynovial pathology of idiopathic carpal tunnel syndrome

Increased intra-carpal-tunnel pressure due to swelling of the flexor tenosynovium is the most probable pathological mechanism of idiopathic carpal tunnel syndrome (CTS). To clarify the role of tenascin-C and PG-M/versican, which have often been found to be involved in tissue remodeling and vascular stenosis in the pathogenesis of CTS, we histologically and biochemically examined the production of extracellular matrix in the flexor tenosynovium from 40 idiopathic CTS patients. Tenascin-C was temporarily expressed in the vessel wall, synovial lining and fibrous tissue, with expression regulated differently in each tissue. Tenascin-C expression by vessels correlated with disease duration and appeared to be involved in vascular lesion pathology. Morphometric analysis showed that tenascin-C expression by small arteries is correlated with PG-M/versican expression in surrounding connective tissue. PG-M/versican was also present at the neointima of severely narrowed vessels. Although tenascin-C expression by synovial lining and connective tissue shows marked regional variation and seems inconsistent, in vitro examination suggested that tenascin-C production by these tissues is regulated in response to mechanical strain on the flexor tenosynovium.     

Tenascin-C splice variant adhesive/anti-adhesive effects on chondrosarcoma cell attachment to fibronectin

Tenascin-C is an oligomeric glycoprotein of the extracellular matrix that has been found to have both adhesive and anti-adhesive properties for cells. Recent elucidation of the two major TNC splice variants (320 kDa and 220 kDa) has shed light on the possibility of varying functions of the molecule based on its splicing pattern. Tenascin-C is prominently expressed in embryogenesis and in pathologic conditions such as tumorogenesis and wound healing. Fibronectin is a prominent adhesive molecule of the extracellular matrix that is often co-localized with tenascin-C in these processes. We studied the chondrosarcoma cell line JJ012 with enzyme-linked immunoabsorbance assays, cell attachment assays and antibody-blocking assays to determine the adhesive/anti-adhesive properties of the two major tenascin-C splice variants with respect to fibronectin and their effect on chondrosarcoma cell attachment. We found that the small tenascin-C splice variant (220 kDa) binds to fibronectin, whereas the large tenascin-C splice variant (320 kDa) does not. In addition, the small tenascin-C splice variant was found to decrease adhesion for cells when bound to fibronectin, but contributed to adhesion when bound to plastic in fibronectin-coated wells. Antibody blocking experiments confirmed that both the small tenascin-C splice variant and fibronectin contribute to cell adhesion when bound to plastic. The large tenascin-C splice variant did not promote specific cell attachment. We hypothesize that the biologic activity of tenascin-C is dependent on the tissue-specific splicing pattern. The smaller tenascin-C isoform likely plays a structural and adhesive role, whereas the larger isoform, preferentially expressed in malignant tissue, likely plays a role in cell egress and metastasis.     

TN-C与MDA-7在增生性瘢痕中表达与调控

增生性瘢痕是以皮肤损伤后成纤维细胞过度增殖为特征的一种病理改变,其发病机制尚不明确,目前没有有效的治疗方法。当皮肤组织损伤时,腱糖蛋白C(Tenascin-C,TN-C)具有多种不同的作用介导炎症和纤维化进程,并使组织有效修复。TN-C是细胞外基质中一个具有独特的六聚体结构的寡聚糖蛋白家族,TN-C一过性表达在器官形成期,在大多数成人组织不表达或表达极少。然而,在病理条件下TN-C表达增加,诸如炎症,伤口愈合和纤维化。TN-C参与胚胎形成、肿瘤发生及损伤修复过程有关,参与细胞黏附、增殖、迁徙、分化、细胞间相互作用以及细胞凋亡。黑色素瘤分化相关基因7/白介素24(MDA-7/IL-24)能选择性抑制瘢痕疙瘩中成纤维细胞的增殖,并诱导瘢痕疙瘩中成纤维细胞的凋亡,而对正常细胞无任何作用。MDA-7/IL-24很可能与瘢痕的形成有关。     

Tenascin-C suppresses Rho activation

Cell binding to extracellular matrix (ECM) components changes cytoskeletal organization by the activation of Rho family GTPases. Tenascin-C, a developmentally regulated matrix protein, modulates cellular responses to other matrix proteins, such as fibronectin (FN). Here, we report that tenascin-C markedly altered cell phenotype on a three-dimensional fibrin matrix containing FN, resulting in suppression of actin stress fibers and induction of actin-rich filopodia. This distinct morphology was associated with complete suppression of the activation of RhoA, a small GTPase that induces actin stress fiber formation. Enforced activation of RhoA circumvented the effects of tenascin. Effects of active Rho were reversed by a Rho inhibitor C3 transferase. Suppression of GTPase activation allows tenascin-C expression to act as a regulatory switch to reverse the effects of adhesive proteins on Rho function. This represents a novel paradigm for the regulation of cytoskeletal organization by ECM.     

Connective tissues: signalling by tenascins

Different connective tissue cells secrete different types of tenascins. These glycoproteins contribute to extracellular matrix (ECM) structure and influence the physiology of the cells in contact with the tenascin containing environment. Tenascin-C expression is regulated by mechanical stress. It shows highest expression in connective tissue surrounding tumors, in wounds and in inflamed tissues where it may regulate cell morphology, growth, and migration by activating diverse intracellular signalling pathways. Thus, integrin and syndecan signalling is influenced by tenascin-C and the levels and/or activies of several proteins involved in intracellular signalling pathways are regulated by its presence. Tenascin-X is important for the proper deposition of collagen fibers in dermis and patients with a tenascin-X deficiency suffer from Ehlers Danlos syndrome. Tenascin-R (and -C) is prominent in the nervous system and has an impact on neurite outgrowth and synaptic functions, and tenascin-W is found in the extracellular matrix of bone, muscle, and kidney. Cell facts:bone: osteoblasts produce tenascin-C, -W cartilage: perichondrial cells produce tenascin-C tendon: fibroblasts produce tenascin-C smooth muscle cells produce tenascin-W, -C skeletal muscle: endo-, peri-, and epimysial fibroblasts produce tenascin-X dermal fibroblasts produce tenascin-X tumors: stromal fibroblasts produce tenascin-C wounds: fibroblasts produce tenascin-C nervous system: glial cells produce tenascin-R, -C, -X.     

Tenascins and the Importance of Adhesion Modulation

Tenascins are a family of extracellular matrix proteins that evolved in early chordates. There are four family members: tenascin-X, tenascin-R, tenascin-W, and tenascin-C. Tenascin-X associates with type I collagen, and its absence can cause Ehlers-Danlos Syndrome. In contrast, tenascin-R is concentrated in perineuronal nets. The expression of tenascin-C and tenascin-W is developmentally regulated, and both are expressed during disease (e.g., both are associated with cancer stroma and tumor blood vessels). In addition, tenascin-C is highly induced by infections and inflammation. Accordingly, the tenascin-C knockout mouse has a reduced inflammatory response. All tenascins have the potential to modify cell adhesion either directly or through interaction with fibronectin, and cell-tenascin interactions typically lead to increased cell motility. In the case of tenascin-C, there is a correlation between elevated expression and increased metastasis in several types of tumors.     

Tenascin-C modulates matrix contraction via focal adhesion kinase- and Rho-mediated signaling pathways

A provisional matrix consisting of fibrin and fibronectin (FN) is deposited at sites of tissue damage and repair. This matrix serves as a scaffold for fibroblast migration into the wound where these cells deposit new matrix to replace lost or damaged tissue and eventually contract the matrix to bring the margins of the wound together. Tenascin-C is expressed transiently during wound repair in tissue adjacent to areas of injury and contacts the provisional matrix in vivo. Using a synthetic model of the provisional matrix, we have found that tenascin-C regulates cell responses to a fibrin-FN matrix through modulation of focal adhesion kinase (FAK) and RhoA activation. Cells on fibrin-FN+tenascin-C redistribute their actin to the cell cortex, downregulate focal adhesion formation, and do not assemble a FN matrix. Cells surrounded by a fibrin-FN+tenascin-C matrix are unable to induce matrix contraction. The inhibitory effect of tenascin-C is circumvented by downstream activation of RhoA. FAK is also required for matrix contraction and the absence of FAK cannot be overcome by activation of RhoA. These observations show dual requirements for both FAK and RhoA activities during contraction of a fibrin-FN matrix. The effects of tenascin-C combined with its location around the wound bed suggest that this protein regulates fundamental processes of tissue repair by limiting the extent of matrix deposition and contraction to fibrin-FN-rich matrix in the primary wound area.     

Tenascin-C mRNA and Tenascin-C Protein Immunoreactivity Increase in Astrocytes after Activation by bFGF

Tenascin-C is an extracellular matrix glycoprotein with trophic and repulsive properties, involved in migratory processes in CNS. Previous reports demonstrated that this molecule is produced and secreted by astrocytes. Preliminary data on fibroblasts and astrocytes have suggested that bFGF may modulate tenascin-C expression. bFGF is a mitogenic growth factor, involved in cell differentiation and neovascularization. In the present study, we ex amined whether bFGF modulates the expression of tenascin-C in hippocampal astrocytes from newborn rats. Our results suggest that bFGF increases the production of tenascin-C by cultured hippocampal astrocytes. We found that both tenascin-C mRNA and protein immunoreactivity were increased after bFGF treatment. Our results also demonstrated that tenas cin-C polypeptides were secreted into the extracellular medium. In agreement with previous studies, we suggest that secreted tenascin-C is mainly the high molecular weight form. In addition, our results suggest that a cleavage of the high molecular weight form may occur in the extracellular medium causing production of proteolytic fragments, that may modify the biological properties of tenascin-C. The present results may be relevant to the understanding of lesion scarring and regeneration process.     

The adhesion modulating properties of tenascin-W

Tenascins are extracellular matrix glycoproteins associated with cell motility, proliferation and differentiation. Tenascin-C inhibits cell spreading by binding to fibronectin; tenascin-R and tenascin-X also have anti-adhesive properties in vitro. Here we have studied the adhesion modulating properties of the most recently characterized tenascin, tenascin-W. C2C12 cells, a murine myoblast cell line, will form broad lamellipodia with stress fibers and focal adhesion complexes after culture on fibronectin. In contrast, C2C12 cells cultured on tenascin-W fail to spread and form stress fibers or focal adhesion complexes, and instead acquire a multipolar shape with short, actin-tipped pseudopodia. The same stellate morphology is observed when C2C12 cells are cultured on a mixture of fibronectin and tenascin-W, or on fibronectin in the presence of soluble tenascin-W. Tenascin-W combined with fibronectin also inhibits the spreading of mouse embryo fibroblasts when compared with cells cultured on fibronectin alone. The similarity between the adhesion modulating effects of tenascin-W and tenascin-C in vitro led us to study the possibility of tenascin-W compensating for tenascin-C in tenascin-C knockout mice, especially during epidermal wound healing. Dermal fibroblasts harvested from a tenascin-C knockout mouse express tenascin-W, but dermal fibroblasts taken from a wild type mouse do not. However, there is no upregulation of tenascin-W in the dermis of tenascin-C knockout mice, or in the granulation tissue of skin wounds in tenascin-C knockout animals. Similarly, tenascin-X is not upregulated in early wound granulation tissue in the tenascin-C knockout mice. Thus, tenascin-W is able to inhibit cell spreading in vitro and it is upregulated in dermal fibroblasts taken from the tenascin-C knockout mouse, but neither it nor tenascin-X are likely to compensate for missing tenascin-C during wound healing.     

TN—C与MDA-7在增生性瘢痕中表达与调控

增生性瘢痕是以皮肤损伤后成纤维细胞过度增殖为特征的一种病理改变,其发病机制尚不明确,目前没有有效的治疗方法。当皮肤组织损伤时,腱糖蛋白C（Tenascin-C,TN-C）具有多种不同的作用介导炎症和纤维化进程,并使组织有效修复。TN—C是细胞外基质中一个具有独特的六聚体结构的寡聚糖蛋白家族,TN—C一过性表达在器官形成期,在大多数成人组织不表达或表达极少。然而,在病理条件下TN—C表达增加,诸如炎症,伤口愈合和纤维化。TN—C参与胚胎形成、肿瘤发生及损伤修复过程有关,参与细胞黏附、增殖、迁徙、分化、细胞间相互作用以及细胞凋亡。黑色素瘤分化相关基因7／白介素24（MDA-7／IL-24）能选择性抑制瘢痕疙瘩中成纤维细胞的增殖,并诱导瘢痕疙瘩中成纤维细胞的凋亡,而对正常细胞无任何作用。MDA-7／IL-24很可能与瘢痕的形成有关。     

Tenascin-C inhibits beta1 integrin-dependent cell adhesion and neurite outgrowth on fibronectin by a disialoganglioside-mediated signaling mechanism

We have previously shown that the extracellular matrix molecule tenascin-C inhibits fibronectin-mediated cell adhesion and neurite outgrowth by an interaction with a cellular RGD-independent receptor which interferes with the adhesion and neurite outgrowth promoting activities of the fibronectin receptor(s). Here we demonstrate that the inhibitory effect of tenascin-C on beta1integrin-dependent cell adhesion and neurite outgrowth is mediated by the interaction of the protein with membrane-associated disialogangliosides, which interferes with protein kinase C-related signaling pathways. First, in substratum mixtures with fibronectin, an RGD sequence-containing fragment of the molecule or synthetic peptide, tenascin-C inhibited cell adhesion and spreading by a disialoganglioside-dependent, sialidase-sensitive mechanism leading to an inhibition of protein kinase C. Second, the interaction of intact or trypsinized, i.e., cell surface glycoprotein- free, cells with immobilized tenascin-C was strongly inhibited by gangliosides or antibodies to gangliosides and tenascin-C. Third, preincubation of immobilized tenascin-C with soluble disialogangliosides resulted in a delayed cell detachment as a function of time. Similar to tenascin-C, immobilized antibody to GD2 (3F8) or sphingosine, a protein kinase C inhibitor, strongly inhibited RGD- dependent cell spreading. Finally, the degree of tenascin-C-induced inhibition of cell adhesion was proportional to the degree of disialoganglioside levels of expression by different cells suggesting the relevance of such mechanism in modulating integrin-mediated cell- matrix interactions during pattern formation or tumor progression.      

Location and Distribution of Non-collagenous Matrix Proteins in Musculoskeletal Tissues of Rat

The study assessed immunohistochemically the location and distribution of various non-collagenous matrix proteins (fibronectin, laminin, tenascin-C, osteocalcin, thrombospondin-1, vitronectin and undulin) in musculoskeletal tissues of rat. Fibronectin and thrombospondin-1 were found to be ubiquitous in the studied tissues. High immunoreactivity of these proteins was found in the extracellular matrix of the anatomical sites where firm bindings are needed, i.e. between muscle fibres and fibre bundles, between the collagen fibres of a tendon and at myotendinous junctions, osteotendinous junctions and articular cartilage. Tenascin-C was found in the extracellular matrix of regions where especially high forces are transmitted from one tissue component to the other, such as myotendinous junctions and osteotendinous junctions. Laminin was demonstrated in the basement membranes of the muscle cells and capillaries of the muscle–tendon units. Osteocalc in immunoreactivity concentrated in the extracellular matrix of areas of newly formed bone tissue, i.e. in the subperiosteal and subchondral regions, osteoid tissue and mineralized fibrocartilage zone of the osteotendinous junction. Mild vitronectin activity could be seen in the extracellular matrix of the osteotendinous and myotendinous junctions, and high activity around the bone marrow cells. Undulin could be demonstrated in the extracellular matrix (i.e. on the collagen fibres) of the tendon and epimysium only. However, it was co-distributed with fibronectin and tenascin-C. Together, these findings on the normal location and distribution of these non-collagenous proteins in the musculoskeletal tissues help to form the basis of knowledge against which the location and distribution of the these proteins in various pathological processes could be compared.