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.     

Nanomechanical Properties of Tenascin-X Revealed by Single-Molecule Force Spectroscopy

Tenascin-X is an extracellular matrix protein and binds a variety of molecules in extracellular matrix and on cell membrane. Tenascin-X plays important roles in regulating the structure and mechanical properties of connective tissues. Using single-molecule atomic force microscopy, we have investigated the mechanical properties of bovine tenascin-X in detail. Our results indicated that tenascin-X is an elastic protein and the fibronectin type III (FnIII) domains can unfold under a stretching force and refold to regain their mechanical stability upon the removal of the stretching force. All the 30 FnIII domains of tenascin-X show similar mechanical stability, mechanical unfolding kinetics, and contour length increment upon domain unfolding, despite their large sequence diversity. In contrast to the homogeneity in their mechanical unfolding behaviors, FnIII domains fold at different rates. Using the 10th FnIII domain of tenascin-X (TNXfn10) as a model system, we constructed a polyprotein chimera composed of alternating TNXfn10 and GB1 domains and used atomic force microscopy to confirm that the mechanical properties of TNXfn10 are consistent with those of the FnIII domains of tenascin-X. These results lay the foundation to further study the mechanical properties of individual FnIII domains and establish the relationship between point mutations and mechanical phenotypic effect on tenascin-X. Moreover, our results provided the opportunity to compare the mechanical properties and design of different forms of tenascins. The comparison between tenascin-X and tenascin-C revealed interesting common as well as distinguishing features for mechanical unfolding and folding of tenascin-C and tenascin-X and will open up new avenues to investigate the mechanical functions and architectural design of different forms of tenascins.     

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.     

Tenascin in the developing and adult human intestine

The tenascins are a family of multifunctional extracellular matrix glycoproteins subject to complex spatial and temporal patterns of expression in the course of various organogenetic processes, namely those involving epithelial-mesenchymal interactions. In the intestine, the tenascins, in particular tenascin-C, have been found to be differentially expressed in the developing and adult small intestinal and colonic mucosa as well as in neoplasm. While tenascin-C emerges as a key player likely to be involved in intestinal mucosa development, maintenance and disease, its exact role in the regulation of fundamental intestinal cell function(s) such as proliferation, migration and tissue-specific gene expression remains however to be established.     

Adhesion-modulating/matricellular ECM protein families: a structural, functional and evolutionary appraisal

The thrombospondins are a family of secreted, oligomeric glycoproteins that interact with cell surfaces, multiple components of the extracellular matrix, growth factors and proteases. These interactions underlie complex roles in cell interactions and tissue homeostasis in animals. Thrombospondins have been grouped functionally with SPARCs, tenascins and CCN proteins as adhesion-modulating or matricellular components of the extracellular milieu. Although all these multi-domain proteins share various commonalities of domains, the grouping is not based on structural homologies. Instead, the terms emphasise the general observations that these proteins do not form large-scale ECM structures, yet act at cell surfaces and function in coordination with the structural ECM and associated extracellular proteins. The designation of adhesion-modulation thus depends on observed tissue and cell culture ECM distributions and on experimentally identified functional properties. To date, the evolutionary relationships of these proteins have not been critically compared: yet, knowledge of their evolutionary histories is clearly relevant to any consideration of functional similarities. In this article, we survey briefly the structural and functional knowledge of these protein families, consider the evolution of each family, and outline a perspective on their functional roles.     

Identification and characterization of a conformational heparin-binding site involving two fibronectin type III modules of bovine tenascin-X

Tenascin-X is known as a heparin-binding molecule, but the localization of the heparin-binding site has not been investigated until now. We show here that, unlike tenascin-C, the recombinant fibrinogen-like domain of tenascin-X is not involved in heparin binding. On the other hand, the two contiguous fibronectin type III repeats b10 and b11 have a predicted positive charge at physiological pH, hence a set of recombinant proteins comprising these domains was tested for interaction with heparin. Using solid phase assays and affinity chromatography, we found that interaction with heparin was conformational and involved both domains 10 and 11. Construction of a three-dimensional model of domains 10 and 11 led us to predict exposed residues that were then submitted to site-directed mutagenesis. In this way, we identified the basic residues within each domain that are crucial for this interaction. Blocking experiments using antibodies against domain 10 were performed to test the efficiency of this site within intact tenascin-X. Binding was significantly reduced, arguing for the activity of a heparin-binding site involving domains 10 and 11 in the whole molecule. Finally, the biological significance of this site was tested by cell adhesion studies. Heparan sulfate cell surface receptors are able to interact with proteins bearing domains 10 and 11, suggesting that tenascin-X may activate different signals to regulate cell behavior.     

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.     

Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture

Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.     

Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo

Lung fibrosis involves the overexpression of ECM proteins, primarily collagen, by alpha-smooth muscle actin (ASMA)-positive cells. Caveolin-1 is a master regulator of collagen expression by cultured lung fibroblasts and of lung fibrosis in vivo. A peptide equivalent to the caveolin-1 scaffolding domain (CSD peptide) inhibits collagen and tenascin-C expression by normal lung fibroblasts (NLF) and fibroblasts from the fibrotic lungs of scleroderma patients (SLF). CSD peptide inhibits ASMA expression in SLF but not NLF. Similar inhibition of collagen, tenascin-C, and ASMA expression was also observed when caveolin-1 expression was upregulated using adenovirus. These observations suggest that the low caveolin-1 levels in SLF cause their overexpression of collagen, tenascin-C, and ASMA. In mechanistic studies, MEK, ERK, JNK, and Akt were hyperactivated in SLF, and CSD peptide inhibited their activation and altered their subcellular localization. These studies and experiments using kinase inhibitors suggest many differences between NLF and SLF in signaling cascades. To validate these data, we determined that the alterations in signaling molecule activation observed in SLF also occur in fibrotic lung tissue from scleroderma patients and in mice with bleomycin-induced lung fibrosis. Finally, we demonstrated that systemic administration of CSD peptide to bleomycin-treated mice blocks epithelial cell apoptosis, inflammatory cell infiltration, and changes in tissue morphology as well as signaling molecule activation and collagen, tenascin-C, and ASMA expression associated with lung fibrosis. CSD peptide may be a prototype for novel treatments for human lung fibrosis that act, in part, by inhibiting the expression of ASMA and ECM proteins.     

Non-steroidal anti-inflammatory drugs target the pro-tumorigenic extracellular matrix of the postpartum mammary gland

Breast cancer patients diagnosed postpartum have poor prognosis. The postpartum mammary gland undergoes tissue regression to return to the pre-pregnant state. This involution is characterized by wound healing programs known to be tumor promotional in other contexts. Previous studies have shown that mammary extracellular matrix (ECM) from nulliparous rats has tumor suppressive attributes, while mammary ECM from involuting mammary glands is promotional. In models of pregnancy-associated breast cancer, non-steroidal anti-inflammatory drug (NSAID) treatment targeted to postpartum involution inhibits tumor progression, in part by suppressing COX-2 dependent collagen deposition. Because mammary ECM proteins are coordinately regulated, NSAID treatment is anticipated to result in additional protective changes in the mammary extracellular matrix. Here, systemic NSAID treatment was utilized during postpartum involution to reduce mammary COX-2 activity. ECM was isolated from actively involuting glands of rats treated with NSAIDs and compared to ECM isolated from control-involution and nulliparous rats in 3D cell culture and xenograft assays. Compositional changes in ECM between groups were identified by proteomics. In four distinct 3D culture assays, normal and transformed mammary epithelial cells plated in NSAID-involution ECM, phenocopied cells plated in ECM from nulliparous rats rather than ECM from control-involution rats. Tumor cells mixed with NSAID-involution ECM and injected orthotopically in mice formed smaller tumors than cells mixed with control-involution ECM. Proteomic analyses identified and 3D culture assays implicated the ECM protein tenascin-C as a potential mediator of tumor progression during involution that is decreased by NSAID treatment. In summary, NSAID treatment decreases tumor-promotional attributes of postpartum involution mammary ECM.     

The Expression of Tenascin-X in Developing and Adult Rat and Human Eye

Tenascin-X has been studied in developing and adult rat eye and in foetal and adult human eyes, using immunohistochemistry and frozen sections. The data were compared with the distribution of tenascin-C. The immunoreactivity for tenascin-X was seen in a basement membrane-like feature in different structures of embryonic (E) day 16–17 rat eyes. Postnatal (P) day 2 and older rat eyes showed immunoreactivity for tenascin-X in different connective tissues. In the epithelial basement membrane zone of the cornea, immunostaining was positive in P5 eyes, negative in P10 and P15 eyes and again positive in P30 and adult eyes. In the 20-week-old human foetus, immunoreactivity for the tenascin was seen in the posterior parts of the conjunctival stroma adjacent to the sclera and in a basement membrane-like fashion in anterior conjunctiva. In the adult human eye, immunoreactivity for tenascin-X was seen in the anterior one-third stroma of cornea as thin fibrils, in the stroma of the limbus and conjunctiva, and in blood vessels. Immunostaining for tenascin-C was seen in the posterior aspect of the further cornea, and in mesenchyme adjacent to cornea in E16–17 rat eyes. Corneal keratocytes and Descemet's membrane showed immunoreactivity for tenascin-C in P2–P15 rat eyes. Sclera and the junction of the cornea, and sclera expressed tenascin-C in P2 and older rat eyes. In human foetal eyes, immunostaining for tenascin-C was seen in the anterior parts of the corneal stroma, in the basement membrane zone and Bowman's membrane of the corneal epithelium, in the posterior one-fifth of the corneal stroma and the sclera starting from the junction of the cornea and sclera. In normal human adult eyes, immunostaining for tenascin-X was seen in the anterior one-third stroma of cornea, in the stroma of limbus and conjunctiva, and in blood vessels. The association of tenascin-X and basement membranes in early development evokes a question of its potential function in the development of the basement membrane. The results also suggest the association of tenascin-X with connective tissue development as well as the association of tenascin-C with the migration of keratocytes during the development of the corneal stroma.     

Cleavage of extracellular matrix in periodontitis: Gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C

Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease.     

The evolution of tenascins and fibronectin

Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes.     

Role of Integrins and Evidence for two Distinct Mechanisms Mediating Human Colorectal Carcinoma Cell Interaction with Peritoneal Mesothelial Cells and Extracellular Matrix

Peritoneal carcinomatosis involves a series of events including tumor cell interactions with mesothelial cells and the extracellular matrix (ECM). We have studied the adhesive and invasive properties of four human colorectal carcinoma cell lines (Co115, HT29, SW480, SW620) confronted in vitro with a human mesothelial cell monolayer or with the ECM proteins collagen IV, laminin-1, fibronectin, tenascin-C and vitronectin. Quantitation was achieved following staining of tumor cells with the calcein-AM fluorescent dye. We found that all four cell lines rapidly adhered to a mesothelial cell monolayer. This adhesion event was not inhibitable by anti-integrin and anti-CD44 antibodies. Following initial attachment, the SW480 and SW620 cells invaded the mesothelial cell monolayer more aggressively than HT29 and Col 15 cells. All cell lines adhered to ECM proteins with each one exhibiting an individual adhesion pattern. Adhesion to matrix was completely integrin-dependent. When tested in an invasion assay, HT29 and Co115 cells crossed Matrigel-coated filters while SW480 and SW620 cells did not. This invasion was inhibited by anti-β1 integrin antibodies. Taken together, our results demonstrate that the initial colorectal tumor cell—mesothelial cell interaction occurs through an integrin-independent mechanism while adhesion to matrix proteins and invasion through Matrigel are integrin-dependent events. Furthermore, the different invasive capacity of SW480 and SW620 versus HT29 and Co115 cells upon interaction with a mesothelial cell monolayer or Matrigel suggests that these two invasion events may be mediated by distinct mechanisms.     

Role of Integrins and Evidence for two Distinct Mechanisms Mediating Human Colorectal Carcinoma Cell Interaction with Peritoneal Mesothelial Cells and Extracellular Matrix

Peritoneal carcinomatosis involves a series of events including tumor cell interactions with mesothelial cells and the extracellular matrix (ECM). We have studied the adhesive and invasive properties of four human colorectal carcinoma cell lines (Co115, HT29, SW480, SW620) confronted in vitro with a human mesothelial cell monolayer or with the ECM proteins collagen IV, laminin-1, fibronectin, tenascin-C and vitronectin. Quantitation was achieved following staining of tumor cells with the calcein-AM fluorescent dye. We found that all four cell lines rapidly adhered to a mesothelial cell monolayer. This adhesion event was not inhibitable by anti-integrin and anti-CD44 antibodies. Following initial attachment, the SW480 and SW620 cells invaded the mesothelial cell monolayer more aggressively than HT29 and Col 15 cells. All cell lines adhered to ECM proteins with each one exhibiting an individual adhesion pattern. Adhesion to matrix was completely integrin-dependent. When tested in an invasion assay, HT29 and Co115 cells crossed Matrigel-coated filters while SW480 and SW620 cells did not. This invasion was inhibited by anti-β1 integrin antibodies. Taken together, our results demonstrate that the initial colorectal tumor cell—mesothelial cell interaction occurs through an integrin-independent mechanism while adhesion to matrix proteins and invasion through Matrigel are integrin-dependent events. Furthermore, the different invasive capacity of SW480 and SW620 versus HT29 and Co115 cells upon interaction with a mesothelial cell monolayer or Matrigel suggests that these two invasion events may be mediated by distinct mechanisms.     

Opposite regulation of tenascin-C and tenascin-X in MeLiM swine heritable cutaneous malignant melanoma

Interactions between tumour cells and surrounding extracellular matrix (ECM) influence the growth of tumour cells and their ability to metastasise. It is thus interesting to compare ECM composition in tumours and healthy tissues. Using the recently described MeLiM miniature pig model of heritable cutaneous malignant melanoma, we studied the expression of two ECM glycoproteins, the tenascin-C (TN-C) and tenascin-X (TN-X), in normal skin and melanoma. Using semiquantitative RT-PCR, we observed a 3.6-fold mean increase of TN-C RNAs in melanoma compared to normal skin. Both stromal and tumour cells synthesise TN-C. On the contrary, TN-X RNAs decreased 30-fold on average in melanoma. This opposite regulation of TN-C and TN-X RNAs was confirmed at the protein level by indirect immunofluorescence. Whereas pig normal skin displayed a discrete TN-C signal at the dermo-epidermal junction, around blood vessels and hair bulbs, the swine tumour showed enhanced expression of TN-C in these areas and around stromal and tumour cells. In contrast, normal skin showed a strong TN-X staining at the dermo-epidermal junction and in the dermis, whereas this signal almost completely disappeared in the tumour. The results presented here describe a dramatic alteration of the ECM composition in swine malignant melanoma which might have a large influence on tumourigenesis or invasion and metastasis of melanoma cells.     

Degradation of extracellular matrix protein tenascin-C by cathepsin B: an interaction involved in the progression of gliomas

Degradation of extracellular matrix proteins by proteases such as the cysteine protease cathepsin B is critical to malignant progression. We have established that procathepsin B presents on the surface of tumor cells through its interaction with the annexin II tetramer [Mai et al., J. Biol. Chem. 275 (2000),12806-12812]. Cathepsin B activity can also be detected on the tumor cell surface and in their culture medium. Interestingly, the annexin II tetramer also interacts with extracellular matrix proteins, such as collagen I, fibrin and tenascin-C. Both cathepsin B and tenascin-C are expressed at high levels in malignant tumors, especially at the invasive edges of tumors, and are implicated in tumor angiogenesis. In this study, we report that tenascin-C can be degraded by cathepsin B in vitro. We demonstrate by immunohistochemistry that both cathepsin B and tenascin-C are expressed highly in malignant anaplastic astrocytomas and glioblastomas as compared to normal brain tissues. Interestingly, cathepsin B and tenascin-C were also detected in association with tumor neovessels. We suggest that interactions between cathepsin B and tenascin-C are involved in the progression of gliomas including the angiogenesis that is a hallmark of anaplastic astrocytomas.