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.     

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 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.     

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.     

The structure and function of tenascins in the nervous system.

The tenascins are a family of large extracellular matrix glycoproteins that comprise five known members. Three of these, tenascin-C (TN-C) tenascin-R (TN-R) and tenascin-Y (TN-Y) are expressed in specific patterns during nervous system development and are down-regulated after maturation. The expression of TN-C, the best studied member of the family, persists in restricted areas of the nervous system that exhibit neuronal plasticity and is reexpressed after lesion. Numerous studies in vitro suggest specific roles for tenascins in the nervous system involving precursor cell migration, axon growth and guidance. TN-C has been shown to occur in a large number of isoform variants generated by combinatorial variation of alternatively spliced fibronectin type III (FNIII) repeats. This finding indicates that TN-C might specify neural microenvironments, a hypothesis supported by recent analysis of TN-C knockout animals, which has begun to reveal subtle nervous system dysfunctions.     

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.     

Tenascin-C: Its functions as an integrin ligand

This review summarizes the experimental evidence of tenascin-C/integrin interactions, emphasizing the identification of integrin binding sites and the effects of specific interactions on cell behavior. At least four integrins appear to bind to the third fibronectin-type 3 domain of tenascin-C: α9β1, αVβ3, α8β1 and αVβ6. The α9β1 integrin recognizes a highly conserved IDG motif in this domain, while the others recognize an RGD motif. There is also significant evidence that the collagen receptor α2β1 can bind to tenascin-C, but the interacting site is unknown. Tenascin-C interactions with α9β1 and αVβ3 can promote cell proliferation and interactions with αVβ3 can also inhibit apoptosis. Interactions with α7β1 integrin, which may bind to the alternatively spliced domain of tenascin-C, and α9β1 integrin are able to influence the differentiation of mesenchymal stem cells into the neuronal lineage. This illustrates the potential for using our knowledge of tenascins and their integrin receptors in stem cell-based therapies.     

Extracellular matrix alterations in brains lacking four of its components

The organization of the brain extracellular matrix appears to be based on aggregates of hyaluronan and proteoglycans, connected by oligomeric glycoproteins. Mild phenotypical consequences were reported from several mouse strains lacking components of this matrix such as neurocan, brevican, tenascin-R, and tenascin-C. To further challenge the flexibility of the extracellular matrix network of the brain, mice lacking all four brain extracellular matrix molecules were generated, which were found to be viable and fertile. Analysis of the brains of 1-month-old quadruple KO mice revealed increased protein levels of fibulin-1 and fibulin-2. Histochemical analysis showed an unusual parenchymal deposition of these fibulins. The quadruple KO mice also displayed obvious changes in the pattern of deposition of hyaluronan. Further, an almost quadruple knockout like extracellular environment was noticed in the brains of triple knockout mice lacking both tenascins and brevican, since these brains had strongly reduced levels of neurocan.     

Tenascins and inflammation in disorders of the nervous system

In vitro and in vivo studies on the role of tenascins have shown that the two paradigmatic glycoproteins of the tenascin family, tenascin-C (TnC) and tenascin-R (TnR) play important roles in cell proliferation and migration, fate determination, axonal pathfinding, myelination, and synaptic plasticity. As components of the extracellular matrix, both molecules show distinct, but also overlapping dual functions in inhibiting and promoting cell interactions depending on the cell type, developmental stage and molecular microenvironment. They are expressed by neurons and glia as well as, for TnC, by cells of the immune system. The functional relationship between neural and immune cells becomes relevant in acute and chronic nervous system disorders, in particular when the blood brain and blood peripheral nerve barriers are compromised. In this review, we will describe the functional parameters of the two molecules in cell interactions during development and, in the adult, in synaptic activity and plasticity, as well as regeneration after injury, with TnC being conducive for regeneration and TnR being inhibitory for functional recovery. Although not much is known about the role of tenascins in neuroinflammation, we will describe emerging knowledge on the interplay between neural and immune cells in autoimmune diseases, such as multiple sclerosis and polyneuropathies. We will attempt to point out the directions of experimental approaches that we envisage would help gaining insights into the complex interplay of TnC and TnR with the cells that express them in pathological conditions of nervous and immune systems.     

Specific processing of tenascin-C by the metalloprotease meprinβ neutralizes its inhibition of cell spreading

The metalloprotease meprin has been implicated in tissue remodelling due to its capability to degrade extracellular matrix components. Here, we investigated the susceptibility of tenascin-C to cleavage by meprinβ and the functional properties of its proteolytic fragments. A set of monoclonal antibodies against chicken and human tenascin-C allowed the mapping of proteolytic fragments generated by meprinβ. In chicken tenascin-C, meprinβ processed all three major splicing variants by removal of 10 kDa N-terminal and 38 kDa C-terminal peptides, leaving a large central part of subunits intact. A similar cleavage pattern was found for large human tenascin-C variant where two N-terminal peptides (10 or 15 kDa) and two C-terminal fragments (40 and 55 kDa) were removed from the intact subunit. N-terminal sequencing revealed the exact amino acid positions of cleavage sites. In both chicken and human tenascin-C N-terminal cleavages occurred just before and/or after the heptad repeats involved in subunit oligomerization. In the human protein, an additional cleavage site was identified in the alternative fibronectin type III repeat D. Whereas all these sites are known to be attacked by several other proteases, a unique cleavage by meprinβ was located to the 7th constant fibronectin type III repeat in both chicken and human tenascin-C, thereby removing the C-terminal domain involved in its anti-adhesive activity. In cell adhesion assays meprinβ-digested human tenascin-C was not able to interfere with fibronectin-mediated cell spreading, confirming cleavage in the anti-adhesive domain. Whereas the expression of meprinβ and tenascin-C does not overlap in normal colon tissue, inflamed lesions of the mucosa from patients with Crohn's disease exhibited many meprinβ-positive leukocytes in regions where tenascin-C was strongly induced. Our data indicate that, at least under pathological conditions, meprinβ might attack specific functional sites in tenascin-C that are important for its oligomerization and anti-adhesive activity.     

The role of tenascin-C in tissue injury and tumorigenesis

The extracellular matrix molecule tenascin-C is highly expressed during embryonic development, tissue repair and in pathological situations such as chronic inflammation and cancer. Tenascin-C interacts with several other extracellular matrix molecules and cell-surface receptors, thus affecting tissue architecture, tissue resilience and cell responses. Tenascin-C modulates cell migration, proliferation and cellular signaling through induction of pro-inflammatory cytokines and oncogenic signaling molecules amongst other mechanisms. Given the causal role of inflammation in cancer progression, common mechanisms might be controlled by tenascin-C during both events. Drugs targeting the expression or function of tenascin-C or the tenascin-C protein itself are currently being developed and some drugs have already reached advanced clinical trials. This generates hope that increased knowledge about tenascin-C will further improve management of diseases with high tenascin-C expression such as chronic inflammation, heart failure, artheriosclerosis and cancer.     

Potential oncogenic action of tenascin-C in tumorigenesis

The prominent expression of tenascin-C in the stroma of most solid tumors, first observed in the mid 1980s, implicates tenascin-C in tumorigenesis. This is also supported by in vitro experiments that demonstrate the capacity of tenascin-C to stimulate tumor growth by various mechanisms including promotion of proliferation, escaping immuno-surveillance and positively influencing angiogenesis. However, tumorigenesis in tenascin-C knock-out mice is not significantly different from that observed in control animals. Perhaps this is not unexpected if one considers that tenascin-C may act as an oncogene. The potential role of tenascin-C in tumorigenesis through its oncogenic action on cellular signaling will be discussed in this review, including how tenascin-C mediated tumor cell detachment might affect genome stability.     

Tenascin-C mRNA is expressed in cranial neural crest cells,in some placodal derivatives,and in discrete domains of the embryonic zebrafish brain

A partial zebrafish tenascin-C cDNA clone was isolated from an embryonic zebrafish cDNA library on the basis of homology to mouse tenascin-C. The expression pattern in the head of embryonic zebrafish was analyzed by in situ hybridization. Tenascin-C mRNA was detected in neural crest cells during the period of their migration and differentiation. Expression also occurred in differentiating placodal tissues and in mesodermal cells. In the developing brain, tenascin-C mRNA was expressed in specific domains. In the hindbrain the pattern of the domains was dynamic. At 18 to 22 h postfertilization, expression was widespread in rhombomeres 3, 5, and 6, confined to periventricular cells in rhombomere 2, and not detectable in rhombomere 4. At 32 h postfertilization, tenascin-C was expressed at the rhombomere boundaries. In contrast to the hindbrain, the pattern in the forebrain and midbrain did not show any major changes between 22 and 32 h postfertilization. Domains expressing tenascin-C alternated with regions devoid of it. The most anterior domain of expression was observed at the telencephalic-diencephalic border, surrounding the optic recess. A second domain, at the border between the diencephalon and the midbrain, and a third domain, in the caudal midbrain tegmentum, appeared restricted to the basal plate. Additionally, expression of tenascin-C mRNA was detected in the hypothalamus and in the developing epiphysis. These expression patterns suggest that tenascin-C may play a role in neural crest cell migration and during the differentiation of neural crest, placodal, and mesodermal derivatives. In the developing brain, tenascin-C may be involved in the consolidation of different regional identities. © 1995 John Wiley & Sons, Inc.     

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.     

Regulated binding of the fibrinogen-like domains of tenascin-R and tenascin-C to the neural EGF family member CALEB

The neural transmembrane protein CALEB was discovered in a screen for novel molecules implicated in neuronal differentiation processes and was found to bind to two proteins of the extracellular matrix, tenascin-C and tenascin-R. The expression of different isoforms of CALEB in axon- and synapse-rich areas in the nervous system is regulated during development. Here we show that an unusual acidic peptide segment of CALEB is sufficient to mediate the binding of CALEB to the fibrinogen-like globes of both tenascin family members as well as to native tenascin-C. We identify a small sequence element within the acidic peptide segment of CALEB as important for this binding. Interestingly, the interactions of CALEB and tenascin-C and -R seem to be regulated during development. We demonstrate that only CALEB-80, the expression of which is up-regulated in the chicken retina during synaptogenesis, but not CALEB-140, expressed later on in development, can bind to the fibrinogen-like domains of tenascin-R or tenascin-C and to native tenascin-C. While both CALEB-80 and CALEB-140 are expressed in the plexiform layers and the optic fiber layer of embryonic chicken retina, CALEB-140 labeling is more intense in the optic fiber layer in comparison to the inner plexiform layer.