Meet the tenascins: multifunctional and mysterious.

The tenascins are a highly conserved family of large oligomeric glycoproteins found in the extracellular matrix (ECM) 1 of vertebrate organisms. Two decades ago, the molecule now known as tenascin-C was among the first proteins shown to have an adhesion modulatory role antagonizing cell attachment to fibronectin (1). Cells that normally demonstrate a stationary phenotype on the fibronectin-containing matrix by spreading out and forming cortical actin stress fibers (Fig. 1A) will show morphological changes when tenascin-C is included in the matrix by extending membrane protrusions and exhibiting decreased stress fiber formation (Fig. 1B), characteristics more typical of a migratory phenotype (2). The presence of ECM proteins such as tenascins during developmental and pathological states is now well recognized if still not well understood. Although each type has a distinctive expression pattern, the tenascins share the characteristic of having tightly regulated expression during development and throughout an organism's life. Tenascins also share the characteristic of modulating cell-matrix interactions and mediating a state of matrix attachment that promotes motility while also influencing other cell functions. As such, this protein family has important functions not only during development but also during pathological states in the adult such as tissue injury and tumorigenesis where remodeling processes are prominent. Reviews in recent years have covered pertinent aspects of tenascin biology including domain structure, modulation of cell functions, in vivo null deletion phenotypes, and contributions to human pathology (3-10). This minireview will provide a concise summary of information from these reviews, which the reader is encouraged to refer to for greater detail, and will also discuss a few of the more recent developments in the field.

The tenascins are a highly conserved family of large oligomeric glycoproteins found in the extracellular matrix (ECM) 1 of vertebrate organisms. Two decades ago, the molecule now known as tenascin-C was among the first proteins shown to have an adhesion modulatory role antagonizing cell attachment to fibronectin (1). Cells that normally demonstrate a stationary phenotype on the fibronectin-containing matrix by spreading out and forming cortical actin stress fibers (Fig. 1A) will show morphological changes when tenascin-C is included in the matrix by extending membrane protrusions and exhibiting decreased stress fiber formation (Fig. 1B), characteristics more typical of a migratory phenotype (2). The presence of ECM proteins such as tenascins during developmental and pathological states is now well recognized if still not well understood. Although each type has a distinctive expression pattern, the tenascins share the characteristic of having tightly regulated expression during development and throughout an organism's life. Tenascins also share the characteristic of modulating cell-matrix interactions and mediating a state of matrix attachment that promotes motility while also influencing other cell functions. As such, this protein family has important functions not only during development but also during pathological states in the adult such as tissue injury and tumorigenesis where remodeling processes are prominent. Reviews in recent years have covered pertinent aspects of tenascin biology including domain structure, modulation of cell functions, in vivo null deletion phenotypes, and contributions to human pathology (3)(4)(5)(6)(7)(8)(9)(10). This minireview will provide a concise summary of information from these reviews, which the reader is encouraged to refer to for greater detail, and will also discuss a few of the more recent developments in the field.

About the Family
Nomenclature and Structure-Tenascins are considered unique to vertebrates. Although Drosophila proteins such as ten-m/Odz were once thought to be possible orthologues due to shared motifs, the identification of more closely related vertebrate equivalents has left the tenascin family without any known invertebrate orthologue (11,12). There are currently four tenascin paralogues that have been identified in mammals, each designated with a letter derived, for the most part, from earlier eponyms: C, R, X, and W. The primary structures of tenascin family proteins have common motifs all ordered in the same consecutive sequence (Fig. 2): amino-terminal heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats, and a carboxyl-terminal fibrinogen-like globular domain. The heptad repeats lie in a highly conserved amino-terminal oligomerization region allowing individual subunits to assemble, usually into trimers. In some tenascins, additional cysteine residues allow the assembly of two trimers into a hexamer. Each protein member is associated with typical variations among different species in the number and nature of EGF-like and fibronectin type III repeats. Isoform variants produced through alternative splicing within the fibronectin type III repeats have been described across the family; however to date, only tenascin-C has shown splice variants being expressed in significant numbers and diversity (5,7).
Tenascin-C-Assembly into hexamers is a classic feature of tenascin-C. Glycosylation differences can cause the sizes of individual subunits to be quite variable, but most are generally around 200 kDa, although some human tenascin-C monomers have been estimated to be over 300 kDa (5,9). Mammalian protein subunits typically have 14.5 EGF-like repeats with 8 fibronectin type III repeats that are shared by all tenascin-C isoforms. Alternative splicing of an additional 9 distinctive repeats that can be independently included or excluded in a combinatorial manner allows tenascin-C to show the greatest number and diversity in isoforms with as many as 27 different mRNA variants having been identified in the developing mouse brain (13). Although much data exist demonstrating that splice variants are differentially expressed during tissue morphogenesis and tumorigenesis under the influence of growth factors and cytokines such as transforming growth factor-␤ and basic fibroblast growth factor, the determining mechanisms underlying tenascin-C alternative splicing are still not known (9). As the first tenascin to be identified, tenascin-C remains the best studied member of the family, accounting for most reports examining the component domains shared by tenascin family proteins. These domains and their functions, however, also remain poorly understood. Although it is believed that the different regions of tenascin-C have distinct actions and functions, it is also probable that the overall effects of tenascin-C on cells and their interactions with the ECM require the concerted action of multiple domains (14). A multiplicity of binding sites have been identified for integrin cell surface receptors, proteoglycans, and cell adhesion molecules of the immunoglobulin family, as well as annexin II receptor proteins and ECM components such as heparin, fibronectin, and collagen. Almost all known binding sites lie in the fibronectin type III repeats or the fibrinogen globe, but evidence has been reported suggesting that the EGF-like repeats act as low affinity ligands for EGF receptors (15). As a consequence, cell interactions with tenascin-C are quite complex, and several signaling mechanisms have been identified, including recent observations that tenascin-C blocks focal adhesion kinase-and Rho-mediated signaling pathways activated by fibronectin (2,16,17) as well as stimulating Wnt and other growth-promoting pathways (18). For a detailed discussion of these mechanisms, please refer to Gertrude Orend's recent review (4).
In the developing embryo, tenascin-C is expressed during neural, skeletal, and vascular morphogenesis. It then virtually disappears in the adult organism with continued basal expression detectable only in tendon-associated tissues; however, sharp up-regulation in expression occurs in tissues undergoing remodeling processes seen during wound repair and neovascularization or in pathological states such as inflammation or tumorigenesis. Given this expression pattern and that its structure has been well conserved among vertebrates and appears to be capable of numerous interactions and potential functions, reports during the 1990s demonstrating that mice with complete tenascin-C gene deletions are viable and appear to develop normally were unexpected (19,20). Later reports have since noted more subtle abnormalities, and the issue of tenascin knock-out phenotypes will be discussed further below.
Tenascin-R-Having subunits 160 -180 kDa in size that can form oligomers of 2 or 3 polypeptide chains, tenascin-R shares a high degree of structural homology with tenascin-C. Its expression seems limited exclusively to the central nervous system, although one report of expression in a cell line originating from the peripheral nervous system has been published (21). Subunits typically have 4.5 EGF-like repeats and 8 -9 fibronectin type III repeats. Alternative splicing at the sixth fibronectin type III repeat produces two isoforms that, even when compared with other tenascins, are very highly conserved across different species (3,22). Although the 160-kDa isoform tends to form dimers and the larger 180-kDa isoform trimers, the functional significance of the two isoforms is not understood (3). Tenascin-R expression has some degree of overlap with tenascin-C expression in the developing nervous system, but unlike tenascin-C, the onset of tenascin-R expression occurs at later time points (23). In vitro studies have demonstrated that the protein influences neural pattern formation through adhesive and anti-adhesive effects on cell-matrix interactions (3); however, like their tenascin-C counterparts, tenascin-R knock-out mice are viable and fertile and only recently has it been demonstrated that they also show behavioral differences (24 -26).
Tenascin-X-Over 400 kDa in size, tenascin-X is the largest known member of the family and widely expressed during development. Adult expression, however, is mostly limited to musculoskeletal, cardiac, and dermis tissue. Individual subunits have 18.5 EGF-like repeats and 29 or more fibronectin type III repeats depending on the species (27)(28)(29). Although tenascin-X appears capable of forming trimers, it differs from other family members in that it lacks the amino-terminal cysteine residues involved in hexamer formation. Although alternative splicing of the fibronectin type III repeats has not been described for tenascin-X in humans, splice variants have been reported for the mouse homologue (28, 30, 31). A close relative sharing similar expression patterns, but smaller in size (170 -220 kDa subunits) with significantly fewer EGF-like repeats, was originally identified in chickens as a new tenascin paralogue called tenascin-Y (32). It contains among the fibronectin type III repeats a region rich in amino acid triplets of serine and proline, an unusual domain that has been found in some variants of tenascin-X (5). More recently, although the two proteins differ greatly in their respective numbers of EGF-like repeats, their many other similarities have led to the suggestion that tenascin-Y is an avian orthologue of tenascin-X (6, 7).
Tenascin-X is the first tenascin whose deficiency has been clearly associated with a pathological disorder in humans, a variant of a heritable connective tissue disorder known as Ehler-Danlos Syndrome, which is associated with fibrillar collagen defects. The tenascin-X gene overlaps on human chromosome 6 with the gene coding for the steroid 21-hydroxylase whose deficiency results in congenital adrenal hyperplasia. The first instances of human tenascin-X deficiency were found in patients exhibiting clinical signs of both congenital adrenal hyperplasia and Ehler-Danlos Syndrome who were found to have a contiguous deletion encompassing both genes (33,34).
Tenascin-W-First identified in zebrafish (35), tenascin-W remains the least well characterized member of the tenascin family. Trimers of 130-kDa subunits have been isolated from zebrafish tissues with each subunit containing 3.5 EGF-like repeats and 5 fibronectin type III repeats. Isoforms from alternative splicing have not been reported for zebrafish tenascin-W. Expression has been found in developing skeletal tissue and neural crest cells with a pattern that partially overlaps tenascin-C expression patterns. Although descriptions of tenascin-W expression have been almost exclusively limited to zebrafish, recent reports have identified possible mammalian orthologues (see below and Fig. 2).

The Search for Function
Tenascin-C and -R Knock-out Phenotypes-Observations that tenascin-C-and tenascin-R-null mice (including single and double knock-outs) have grossly normal phenotypes belied the presumed essential, albeit unknown, role suggested by their highly regulated expression and well conserved presence in vertebrate genomes. Subsequent studies, however, have revealed subtle abnormalities in behavior and wound healing, suggesting that whereas tenascins may be redundant for gross organismal development, they may have subtle morphologic and/or physiologic actions that play essential roles in adult survival (10). This view of a critical impact on adult survival has been reinforced by recent reports describing reduced airway branching in the lungs of fetal tenascin-C-null mice (36) and confirming that tenascin-R-null mice show behavioral differences (25,26). Although the mechanisms are not yet clear, it has been suggested that tenascins may play an important role in extrasynaptic communication between neurons as well as with glial cells based on observations that neuroactive substances in tenascin-R-deficient mice show altered diffusion through the central nervous system extracellular space (37).
Tenascin-R in Postnatal Central Nervous System Growth and Function-The subtle behavioral abnormalities but grossly normal appearance of tenascin-R-knock-out mice have led to speculation that the protein may be more important for postnatal and adult central nervous system development and function (25,26). Observed behaviors differing from wild-type mice include decreased motor coordination, decreased willingness to explore, increased anxiety when confronted with new stimuli, and impairments in spatial and associative learning, suggesting that tenascin-R is important for adaptive behavior responses and, therefore, a possible reason for its highly conserved structure. Patterns of tenascin-R post-translational modification with distinct sulfated oligosaccharides change in dramatic fashion during postnatal development in rat cerebel- FIG. 1. Cell attachment to fibronectin is antagonized by tenascin-C. On matrix containing fibrin and fibronectin, fibroblasts from wild-type rats take on a stationary phenotype by spreading and forming cortical actin stress fibers (A). The same cells will behave differently on a fibrin-fibronectin matrix incorporating tenascin-C by extending membrane protrusions and showing decreased stress fiber formation (B), features that are consistent with a migratory phenotype. Scale bar, 20 m.

Minireview: Tenascins 26642
lum, suggesting a mechanism by which the modulatory functions of tenascin-R might change during the life of an organism (38).
Recent evidence has suggested potential significant roles for the protein in adult neural growth. In zebrafish, tenascin-R has been shown to act as a repellent guidance molecule for regenerating axons after optic nerve injury in adults (39) and to initiate migration of neuroblasts in adult mice olfactory bulbs (40). In addition to direct effects on neuronal cells, tenascin-R may play a neuroprotective role through its ability to modulate the function of microglia, cells that are activated after central nervous system injury (41). It has been reported recently that this modulation may be the result of distinct but coordinated actions by the EGF-like and fibronectin type III domains on microglia receptors (42).
Mammalian Tenascin-W-Recent reports have described mammalian proteins with close similarities to zebrafish tenascin-W (43,44). Their domain structures are depicted in Fig. 2. Although specific functions have yet to be characterized, the presence of such proteins in mammals suggests a compensating mechanism that could explain the relatively mild developmental phenotypes seen for mice deficient in other tenascin family proteins (44).
Tenascin-X Deficiency and Ehler-Danlos Syndrome-Patients suffering from Ehler-Danlos Syndrome show clinical symptoms consistent with ECM structural defects including skin and joint laxity, vascular fragility, and poor wound healing. Until recently, the syndrome has been associated with either dominant negative genetic defects in collagen structure (collagen types I, III, V) or recessive deficiencies in collagenprocessing enzymes such as lysyl hydroxylase and procollagen peptidase (45). The association of tenascin-X deficiency with Ehler-Danlos Syndrome, in addition to demonstrating the first clinical relevance for a tenascin family protein, has established for the first time that causative gene defects for this and similar syndromes can extend beyond those directly related to the collagens and their metabolism (45,46). The tenascin-X-associated form is transmitted in recessive fashion with collagen fibrils that have subtle morphologic alterations, such as lower degrees of packing density and co-alignment, but otherwise appear normal. Mice deficient in tenascin-X demonstrate alter-ations in collagen deposition that mimic Ehler-Danlos Syndrome, confirming that tenascin-X plays a significant role in collagen fibrillogenesis (47). The underlying mechanisms are still not well understood, but recent evidence suggests that tenascin-X can affect the rate of collagen fibril formation either by direct binding to collagen or through regulating the synthesis of type VI collagen, which is known to affect fibril formation (48). Patients deficient in tenascin-X also have abnormal elastic fibers (49), suggesting another pathophysiological mechanism although it is not clear how tenascin-X deficiency would affect elastic fiber structure. Finally, recent evidence also suggests that tenascin-X deficiency results in alterations in triglyceride synthesis and the composition of triglyceride-associated fatty acids in mouse skin in such a way as to potentially affect its physical properties (50).

Conclusion
Tenascin biology seems to be embedded in a web of complexity that mirrors human biology as a whole. The multifunctional nature of tenascins seems obvious from the shared similarities of their repeating domains with proteins such as EGF, fibronectin, and fibrinogen. Their highly conserved structures and tightly regulated expression patterns only reinforce the perception that tenascins must have some critical purpose in vertebrate biology. The inability to identify any clear and specific role, however, has led to tenascins being labeled over a decade ago as "talented proteins in search of functions" (12). Although a great deal of knowledge has been gained about these proteins in the interim including an association with a specific human disease, they remain frustratingly mysterious entities whose specific functions are still not well understood. Nonetheless, it is evident that tenascins represent an important class of extracellular proteins that have a clear ability to regulate cell behavior and therefore carry significant ramifications in our understanding and treatment of a broad and diverse range of human disease.
FIG. 2. Schematic representations of tenascin family proteins. All tenascin proteins share an amino-terminal oligomerization region followed by consecutively arranged domains of heptad repeats, EGF-like and fibronectin type III repeats, and a fibrinogen globe. Human protein versions have been depicted unless otherwise indicated. There is general agreement regarding the primary structures of tenascin-C and -R, which show a high degree of homology to each other. It has been suggested that recently described mammalian proteins similar to tenascin-W (tenascin-N and mouse tenascin-W) may provide a compensating mechanism in mice with null deletions for other tenascins. Although tenascin-X and -Y have generally been considered separate paralogues, several similarities including the presence in some tenascin-X variants of a tenascin-Y domain rich in serine and proline residues (represented as an open diamond) has led to the suggestion that the two proteins are orthologues. Please refer to text for further explanation and references.