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Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search of functions
Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search of functions Harold Duke
University
P Erickson
Medical
Center,
Durham,
USA
“Mice develop normally without tenascin”, was a shock to biologists studying the extracellular matrix. Could tenascin be a useless protein? This seems most improbable, as it is conserved in every vertebrate species. Moreover, two new proteins have been discovered in the last year that are closely related to the original tenascin, providing evidence for a tenascin family. Speculations on functions are ripe for re-evaluation. Current
Opinion
in Cell Biology
Introduction
alogous sequences, which are derived from a gene duplication, typically before separation of the species [ 21. For example, a- and P-hemoglobin are products of a gene duplication that was fixed long before mammals arose. All mammals have both forms of hemoglobin, and each chain has been evolving independently in all species. The a-hemoglobins of different species are orthologous to each other, and are paralogous to the P-hemoglobins.
Tenascin (TN)-C, the first member of the tenascin family to be discovered, is a very large, complex protein of the extracellular matrix. Each subunit comprises -30 independently folding domains, each of which could have an independent function and hence many talents. TNC is expressed in developing brain, cartilage and mesenchyme, and is re-expressed in tumors, bound healing and inflammation. This apparently regulated and specific expression implied crucial roles in embryonic development and tissue restructuring. Numerous activities have been demonstrated in cell culture experiments. It was therefore most surprising that mice, genetically engineered by homologous recombination to knock out the TN-Cgene, developed normally and showed no apparent defect [I**].
TN-C was discovered independently in several laboratories in the early 1980s. Each discovery was in a different context and brought with it a new name: myotendinous antigen, glioma mesenchymal extracellular matrix, hexabrachion, tenascin, Jl-200/220 and cytotactin all describe the same protein (see [3] for a review and references to the earlier papers). The name tenascin has now been adopted by almost all laboratories working in this area and will be extended here to cover the entire family of related proteins, as suggested by Bristow et al. [4*-l. The original tenascin is designated TN-C, the C indicating cytotactin, the name associated with the first published sequence [ 51, and the two newer family members (paralogues) as TN-R and TN-X. (The name tenascin, without a letter, will continue to be used for TN-C in research papers dealing exclusively with this most studied family member, but the more precise nomenclature will be useful as the other family members become better known.)
It would be premature, however, to conclude that TNC has no important function. TN-C is conserved in every vertebrate species, which argues strongly for a fundamental role, probably the same in all species, that contributes to survival. The lack of a phenotype in the knockout mouse simply means that the function may be subtle, rather than vital. It also brings into question all of the functions previously proposed. Remarkably, just as we are faced with this crisis of linding a function for TN-C, two new proteins have been discovered that are related to TN-C. We now have a family of tenascins, each member with similar multiple domains and potentially similar functions. In this review I will first describe the tenascin family, and return to the question of function.
TN-R was originally discovered under the names restrictin in chicken [6] and Jl-160/180 in rat [7]; both chicken and rat TN-R have recently been sequenced [8**,9”]. These proteins are as closely related to each other as are the TN-Cs from chicken and rat, but are more distantly related to the TN-Cs of all species.
Protein sequences that appear to be related are referred to as homologous, with the understanding that they are derived from a common ancestral sequence. Homologous sequences may be classified into two types: orthologous sequences, which are found in different species where the differences reflect species divergence; and par-
ECL-epidermal
growth
factor;
fbgfibrinogen; @
Current
TN-X was originally reported as a partial sequence encoded by gene X [lo]. The same gene was identied by Matsumoto et al. [ 111, who provided additional sequences important in the analysis of the gene. An al-
Abbreviations FN-fibronectin; Biology
1993, 5:869-876
Ltd
TN--tenascin; ISSN
0955-0674
UEP-unit
evoltitionary
period.
870
Cell-to-cell,
contact
and extracellular
matrix
most complete sequence of this very large’ protein has now been reported by Bristow ,et al [4**].
Teriascin
domain
structure
The tenascins are modular proteins [ 121 in which the polypeptide chain shows repeating sequence motifs that fold independently into small globular domains or modules. The known tenascin family members share a very distinctive pattern of four types of domains (Figs 1 and 2).
The amino-terminal - 150 ammo acids contain oligomerization sites where the subunits are connected to form trimers and hexamers; this region is unique to the tenascins. All tenascins contain an a-helical segment of 22 amino acids that could form a triple-stranded coiled coil, flanked by pairs of cysteines that could form disulfide rings [ 131. TN-C and TN-R contain a single cysteine amino-terminal to the trimer-forming segment, which could connect two trimers into the characteristic hexabrachion structure. Although TN-R protein isolated from tissues has only been found in the form of dimers and trimers [7,8*=], it is likely that TN-R can also form hexabrachions; the smaller oligomers may result from proteolysis. TN-X, however, diverges in sequence aminoterminal to the trimer-forming segment, and is missing the cysteine needed to form a hexabrachion. TN-X can apparently form only tribrachions. The next segment comprises a series of 4i/r18i/r epidermal growth factor (EGF) domains, each 31 amino acids long. The common ancestry of this EGF segment is suggested by the curious i/z repeat that begins each segment, and by the fact that it is encoded by a single exon in both TN-C [14] and TN-X [4**], the two proteins with a known gene structure. The individual EGF
domains have 70-80 % amino acid identity, making it difficult to map the domains of TN-R and TN-X to precise equivalents in TN-C. The next segment comprises a series of libronectin (FN)III domains [ 15.1, each about 91 amino acids long. In contrast to the EGF domains, most of the FN-III domains share only 25-30% amino acid identity, and are no more similar to each other than they are to those of other proteins, like libronectin. However, FN-III domains l-5 of TN-C and TN-R, and domains 68 of all three tenascins, correspond to each other, with 34-43 % amino acid identity between family members. The alternatively spliced domains, indicated by letters, are inserted between the universal domains 5 and 6 in TN-C and TNR. Eight alternatively spliced domains have been identified in human TN-C, including the newly discovered ad1 [ 161. Only three of these, A, B and D, have been found in chicken. The 23 domains indicated as X-type FN-III in Fig. 1 are remarkable in being > 50 % identical to each other [4**], similar to the recently duplicated domains At-4 in human TN-C [ 171. They are also more closely related to the alternatively spliced domains of TN-C than to the universal domains. It seems a good guess that the 23 Xtype FN-III domains are alternatively spliced, perhaps in a complex pattern. The atomic structure of TNfn3, the third FN-III domain of human TN-C, was determined recently by X-ray crystallography [ 18**]. An atomic model was proposed for the thick segment of the hexabrachion arm that involves a tight packing of adjacent FN-III domains to make an extended filament. The carboxyl-terminal domain, corresponding to the terminal knob visualized by electron microscopy, consists of 215 amino acids with homology to the carboxy-terminal domain of p- and y-fibrinogen. It is abbreviated here TNfbg. This domain is well conserved in TN-C,
Fig. 1. An
electron micrograph of a molecule of human TN-C, showing the hexabrachion structure. The domain segments are indicated: CN, central nodule; ECF, the segment of 14 epiderma1 growth factor like domains; FN-III, the segment of 15 fibronectin type-Ill domains; and fbg, the fibrinogen-like terminal knob. An atomic resolution structure of one FN-III domain, and a model for the FN-III segment have been determined [I89
Tenascin-C,
Tenascin-C
(human,
pig,
mouse,
tenascin-R
and tenascin-X
Erickson
chicken, newt)
A COOH
Tenascin-R
(chicken.
mouse)
COOH
Tenascin-X
(human)
NH,
COOH
0
0
ECF
domains
0
Universal
FN-III
domains
l
Alternatively
spliced
FN-III
domains
1993 Current
TNfbg
Opinion
in Cell Biolom
domain
Fig. 2. The domain structure of the tenascin family. The hexabrachions or tribrachions are indicated at the central nodule. The universal FN-III domains, present in all transcripts of TN-C and TN-R, are indicated by l-5 and 6-8; the alternatively spliced domains are indicated by letters [32-l. The human TN-C shown here has the largest number of alternatively spliced domains of the five known vertebrate species. The domain Rl of TN-R is not obviously related to any of the domains of TN-C. The 23 X-type FN-III domains of TN-X may be alternatively spliced.
TNR and TN-X, the three sharing 4148% identity.
amino acid TN-X.hum TN-C.hum TN-C.pig TN-C.mus
A phylogenetic
tree of the tenascins
The carboxy-terminal segment, comprising the last three FN-III domains (TNfnG8) and TNfl~g, is clearly conserved among all family members. This region was selected for further analysis of the relation between the tenascins. The multiple alignment algorithm in the GeneWorks @program (Intelligenetics, Mountain View, California) generates a convincing alignment with virtuallly no insertions, and gives the phylogenetic tree shown in Fig. 3. The TN-Cs of the five species (human, pig, mouse, chicken and newt) all align with the expected order of relation among the species. The two TN-Rs form a group related to each other but much more distantly to the TN-& TN-X is most distant. 1 have made a separate pair-wise alignment of human TN-C with TN-C of other species, to estimate the rate of evolution of the tenascins. The TN-C of human, mouse and pig differ by only 10 %, while human TN-C differs from that of chicken and newt by 25-30 %. Humans separated from mice and pigs 75-95 million years (MYrsj ago, and from birds and amphibians 300-350MYrs ago. One can estimate the unit evolutionary period (UEP, time required for a 1% divergence in sequence [2] ) for TN-
TN-C.chk TN-C.nwI TN-R.chk
1
TN-R.rat 0 1993 Current
Opinion
in Cell Biology
Fig. 3. A family tree of the tenascins constructed by the multiple alignment program of GeneWorks. The alignment is based only on the carboxyl-terminal segment, TNfnG3 plus TNfbg, which is well conserved in all family members. The lengths of the lines indicate the degree of similarity of the sequences. The accession numbers and references for the tenascin sequences are: TN-Chum M55618 [451 and X56160 [461; TN-C.pig X61599 (471; TN-C.mus X56304 I481 and D90343 [491; TN-Cchk JO4519 [51 and M23121 1131; TN-Cnwt M76615 [311; TN-R.chk X64649 I8**1; TNR.rat 218630 [9*-l; TN-X.hum X71923-38 (each piece of genomic DNA has a separate entry) [4*-l.
C to be 7-10 MYrs. Different proteins diverge at widely different rates, illustrated by the following LJEP ranges: 60-400 MYrs for histones; 14 for insulin; 5 for prolactin; 3.7 and 3.3 for hemoglobin, and 3 for serum albumin. Thus TN-C is evolving at a modest rate, equivalent to some growth factors that are considered functionally important and conserved.
871
872
Cell-to-cell
contact
and extracellular
matrix
I have extended this analysis to speculate bn the age of divergence of TN-R, TN-X and TN-C. Assuming that all members of the tenascin family are evolving at the same rate, with UEP * 9, the divergence of the three tenascins would date to 800 MYrs ago. This extrapolation should not be taken too quantitatively, but does suggest that a primordial tenascin existed in early Cambrian or Pre-Cambrian periods, before the divergence of insects, mollusks, annelids, cnidarians, etc. Thus we might expect to find tenascins in invertebrate phyla.
Invertebrate
tenascin?
Two proteins from Drosophila, identified by cDNA sequencing, have domains related to those of tenascins. Scabrous, a diffusible protein with probable growth factor activity in development, has a carboxyl-terminal domain homologous to TNfbg [ 191. More recently, a protein designated Tena was identified in a low stringency screen for Drmophikz proteins related to the tenascin EGF domains. Tena has eight EGF domains with a size and cysteine spacing similar to those of tenascins [ 201. A related molecule, Tenm, identified in the same screen, shows a similar set of eight EGF domains, followed by 11 domains that might be related to FN-III (S Baumgartner, D Martin, C Hagios, R Chiquet-Ehrismann, unpublished data). The relationship of Ten”, Tenm and Scabrous to the corresponding domains in tenascin may provide interesting documentation on domain shuffling of modular proteins [ 121, and perhaps on the evolution of the tenascin family. A molecule visualized by electron microscopy in extracts from leech is a potential invertebrate tenascin [ 211. It has a hexabrachion structure, with a central knob linking the six arms, a thin proximal segment and a very long thick segment, which could correspond to EGF and FNIII domains. Protein biochemistry and cloning data are obviously essential to determine whether this invertebrate protein is really related to tenascin. Several other reports of invertebrate tenascins have been based on cross-reaction with polyclonal antibodies, but these are not convincing without more extensive biochemistry. An invertebrate tenascin should probably have three or all four types of domains in the proper order to be included ln the tenascin family. For example, Drosophila laminin closely matches the domain structure of vertebrate laminins in all three subunits, Ig superfamily adhesion molecules and integrins are also conserved in Drosophila [22]. However, it should be recalled that Dmsopbika apparently has no fibronectin [22], so it is quite possible that it has no tenascin. Extending the search to other phyla, such as annelids, may reveal unmistakeable tenascin relatives. This would provide exciting new opportunities to explore the function of tenascins, as well as their evolution.
Tenascin expression in developing tissues, tumors and wound healing
and adult
For more than a decade, the patterns of expression of newly discovered proteins have been carehllly researched for clues to their functions. Several careful surveys of TNC, already reviewed in [ 31, were accompanied by the expectation that TN-C would have an important function wherever it was expressed. This expectation has recently been challenged by the hypothesis that proteins might be expressed superfluously in some tissues where they are non-functional [23*]. Thus the high level of TNC in brain or healing wounds might be an accident of gene regulation, tolerated because TN-C is an innocuous molecule in these locations. This warning that expression in a particular tissue does not necessarily imply function is important. However, it is also important to know where TN-C is expressed because it should have a function in at least some tissues. Expression of TN-C in the brain has always attracted interest. A very detailed study of mouse cerebellum showed TN-C mRNA primarily in Golgi epithelial cells, which form a narrow band beneath the Purkinje cells [ 241. It is not clear how the TN-C protein becomes widely distributed through the layers of the developing cerebellum. The recent study by Rettig et al. [ 25*] revises the earlier conclusion that TN-C was expressed only in developing brain. They demonstrated prominent TN-C protein in at least some regions of adult brain of five mammalian species. A remarkable finding was the variation among species in regional patterning at specific developmental stages. For example, TN-C is highly expressed in all areas of the bovine brain, is most prominent in the molecular layer of the cerebellum in rat and mouse, but is completely missing from the cerebellum of human. This variation is consistent with the idea that TN-C may not be serving an important function in brain. TN-R is apparently restricted to the central nervous system, and is most prominent in development. It is found in retina, cerebellum and spinal cord of embryonic chicken [800], and in cerebellum, hippocampus and olfactory bulb of postnatal rat, peaking at 7-14 days [ 9=*]. Expression continued in adult rat cerebellum. TN-X mRNA is prominently expressed in fetal muscle (smooth muscle of the gut, skeletal muscle and heart) and testis, with a lower level in fetal adrenal gland, kidney and lung [4**]. TN-X was virtually absent from 22 week human fetal brain, and also from 2 day postnatal mouse brain [1*-l, where TN-C and TN-R are very prominent. Expression of TN-C in a wide variety of tumors was one of the most exciting early findings. Reports that TNC might be associated with the most malignant tumors gave promise of clinical importance. Recent studies [26] confirmed the abundant expression of TN-C in malignant breast carcinomas, but noted that it is much more widespread than previously thought. TN-C is conspicuous in lactating-gestational breast, fibrocystic disease and benign tumors. In carcinomas, TN-C is localized primarily in the
Tenascin-C,
stroma around the islands of epithelial cells, raising the question of which cells secrete it. Earlier studies concluded that TN-C was secreted primarily by mesenchymal cells, presumably induced by growth factors from the carcinoma cells. It now seems that the reverse scenario is also important. Human carcinoma cells injected into nude mice first caused secretion of mouse TN-C by surrounding libroblasts, but after 2 days the carcinoma cells secreted substantial amounts of human TN-C [27]. The carcinoma cells seemed to be stimulated by growth factors from the stroma. The growth factors that stimulate several types of cells to secrete TN-C have been studied most extensively by Rettig et al [28] (this paper has been lost to the tenascin literature because it was published under the name neuronectin, now known to be TN-C [ 25.1). Expression of TN-C in wound healing is weU established, but has been studied in greatest detail in comparative studies of fetal and adult wounds [29,30]. A most remarkable observation in these studies was the rapid time course of expression, in l-4 h, of TN-C in fetal wounds, which also healed without scarring. A study of TN-C expression during limb regeneration in newt [31] used in situ hybridization to demonstrate that TN-C mRNA was first expressed by the wound epithelial cells and later by mesenchymal cells, again demonstrating the capacity of several ceU types to secrete TN-C.
TN-C in cell culture: specific domains
functions
mapped
to
The search for TN-C function has led to specific assays in cell culture of cell adhesion, neurite outgrowth and mitogenesis, with the controversial findings that TNC can either promote or inhibit any of these activities. An important development in the past year has been the production of sets of expression proteins that replicate specific domains of TN-C. The most extensive set is that of Aukhil et al. [32*], which covers all the FN-III domains and TNfbg in 10 defined segments. These proteins have all been highly purified and demonstrated to be soluble and monomeric. The bacterial expression proteins studied by Prieto et al. [33*] cover different and overlapping domains, including the EGF domains. Two proteolytic fragments isolated by Chiquet et al. [34] have provided important indications of activity. CeU adhesion to TN-C has long been controversial, but a recent study has demonstrated that it can be either nonadhesive or adhesive depending on how it is coated on plastic [35]. Cells adhere to the third FN-III domain of chicken [ 33.1 and human [35] TN-C; adhesion is blocked by RGD peptides and appears to be mediated by the aVj33 integrin [35,36]. A second adhesion site has been mapped to the fbg domain of TN-C and probably also TN-R [8”,32*,33*,35]. Adhesion to TN-Cfbg is mediated by a ceU surface proteoglycan [32*] on fibroblasts, and an integrin, probably a2p1, on endothelial ceUs [ 35,361. A third cell adhesion site may exist in TN-Cfn7-8, but this is
tenascin-R
and tenascin-X
Erickson
complicated by competing anti-adhesion activities of this domain [33-l. Anti-cell adhesion activity is perhaps the most popular activity ascribed to TN-C in recent years. The best established mechanism is steric blocking, in which the large hexabrachion molecules bind to the substrate (plastic), straddle the Iibronectin molecules and cover up the adhesion sites [ 371. Lightner and Erickson [37] reported no anti-adhesion effect of soluble TN. In contrast, Prieto et al. (33*] reported anti-adhesion activity of both substrate-bound and soluble TN-C, and mapped the activities to the EGF domains and to TN-Cfn7-8. Chiquet et al. [34] reported anti-adhesion of a proteolytic fragment containing TN-Cfn7-8. Neurite outgrowth is a more complex phenomenon than cell adhesion and is affected by many different molecules. Tenascin can either stimulate or inhibit neurite outgrowth depending on the mode of presentation. Antibodies have been used to map the active domains [38-40]. There has long been an interest in whether TN-C might stimulate ceU growth by acting as a mitogen. Two recent studies report completely contradictory results. Crossin [41] reported that TN-C inhibited cell growth, while End et al. [42*] reported mitogenic stimulation. Further work will be needed to determine which of these is correct or how the result depends on particular experimental conditions. The availability of domain-specific expression proteins should provide an important tool in this work. AU of the above effects on ceU behavior imply the existence of cell surface receptors for tenascins. An important discovery in the past year was that TN-C binds to Fll/contactin [43*]. Fll is one of the Ig super-family cell surface/adhesion molecules, and is abundant on neurons Remarkably, TN-R was originally identified as a ligand for Fll [ 61. It seems hardly a coincidence that both of these tenascins, which are prominent in developing brain, bind this neuronal ceU surface molecule.
Is there
life without
tenascins?
The most important test of function of a protein is to identify mutants with a defective or completely disrupted gene. This is exactly what was undertaken by Saga et al. [l**], who created a mouse completely lacking TN-C. This is clearly the most important paper of the year, if not the decade, on tenascin. The expectation was that these mice would be severely crippled in several aspects of development. Remarkably, the knockout mice appeared completely normal. AU tissues that normally express high levels of TN-C in development, including the brain, lung and thymus, were histologically normal without TN-C. The mice behaved normally (but they have not been carefully analyzed for intelligence or memory), they gave birth, and their wounds healed normally. How can this absence of phenotype in TN-C knockout mice be reconciled with the dramatic functions inferred from tissue culture assays?
873
874
Cell-to-cell
contact
and extracellular
matrix
The word redundancy arises quickly in disoussions, with suggestions that the functions of TN-C might be duplicated by other proteins. Interest is focused on the paralogues, but they can only duplicate functions in the tissues where they are co-expressed (see above). Nevertheless, knockouts of TN-R and TN-X will be eagerly awaited to see whether there are effects of the individual knockouts, and additional effects of double knockouts. Bristow et al [4**]# suggested that a knockout of TN-X may already have been achieved as a deletion mutation in the mouse steroid hydroxylase region. Shiroishi et al [44] identiiied the deletion of an 80 kb fragment extending from the C4B gene to P45Oc21A and presumably including the mouse TN-XA This deletion was lethal in the homozygous state. However, Shiroishi et al. [44] reported that the homozygous mice did not die until 5-15 days after birth, so the lethality was probably due to the P45Oc21A deletion (21 hydroxylase is not needed until after birth). That these mice could complete fetal development and live for 5-15 days suggests that TN-XA is not needed for development. It is not known for mice whether TN-XA or TN-XB is the active gene (remember, in humans TN-XB is active and TN-XA is like a pseudogene). If TN-XA is the active gene, then the TN-X knock-out appears to give a subtle phenotype much like TN-C. This is a complicated gene locus but some careful genetics might pm down some important information without engineering a de novo gene knockout. It should be emphasized that TN-C cannot be totally redundant [23*]; a protein must have a unique function in at least some tissue, or it would revert to a pseudogene. An immediate challenge is then to determine the tissue where TN-C is expressing its unique function. Close examination of the knockout mice for subtle defects in specific tissues may be productive, and the clues provided by cell culture assays may be helpful. We need to remember, however, that a gene can be iixed in a population if it confers only a 1% survival advantage. Defects in the knockout mice may not be obvious even when closely examined. We also have to consider the possibility that the true function of tenascins in vivo is completely different from all previous suggestions. We should perhaps look for a function that would be common to the three tenascins, and consider closely the structural and biochemical features that are conserved in the three tenascin paralogues. Most striking to me is the carboxyl-terminal segment, comprising IN-III domains 6-g and the fibrinogen-like terminal knob. These domains are conserved in all three tenascins, as is their positioning at the end of long am-is of a multivalent tri- or hexabrachion molecule. If these domains bound other extracellular matrix molecules, they might be ideally positioned to organize them into a network, and to maintain a spacing of 5ck200~1 between the binding sites. Binding studies to identify candidate proteins should now be renewed with the available domainspecilic expression proteins.
Conclusion The past year has seen an expansion of tenascin from an elabomte protein consened in all vertebrates, to a family of three related proteins. At the same time the founding member of the family was submitted to the ultimate test of function a genetic knockout. The failure to iind any abnormality in TN-C-deficient mice dashes our hopes that a dramatic phenotype or failure in specific tissues would con&n-~ important functions. Some functions demonstrated in cell culture assays may be irrelevant in vivo, but others may provide clues as to which tissues should be examined more closely in the knockout mice. Even less is known about the two paralogues, TN-R and TN-X, but it is attractive to think that their similar structures are engaged in similar functions in the different tissues. While awaiting results of additional knockouts, the field is now open for totally new hypotheses on the functions of these molecules.
Acknowledgements I thank H Onda, Ohio State University, J Bristow and W Miller, University of California, San Francisco, and S Baumgarten, Friedrich Miescher Institut, Basel, for sharing with me information on newt TNC, human TN-X and Drosophila Tenm, respectively, prior to publication.
References
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest .. of outstanding interest 1.
SAGAY, YAGI T. IIWWAY, SAIUKURAT, AIZAWAS: Mice Develop Normally Without Tenascin. Genes Deu 1992, 6:1821-1831. xe creation of mice with a genetic knockout of TN-C is the most important test of function ever. All tissues normally expressing TN-C in development appeared histologically normal in its absence. 2.
WIIX)N AC, CAIUON SS, WHITE TJ: Biochemical Annu Rev Bicxbem 1977, 46573639.
Evolution.
3.
ERICKSONHP, BOURBONMA Tenascm: Art ExttaceUular Matrbt Rotein Prominent in Specialized Embryonic Tissues and Tumors. Annu Rev Cell Biol 1989, 5:71-92.
4. ..
BRISTOWJ, TEE MK, GITELMANSE, MELLON SH, MUIR WI Tenascio-X: A Novel Extracelhtlat Matrix Protein Encoded by the Human XB Gene Overlapping p450cZlB. J Ceil Biol 1993, 122:265-278. The third member of the tenascin family was sequenced mostly from genomic DNA, showing the structure of the gene. mRNA expression is demonstrated in several embryonic tissues. 5.
JONESFS, HOFFMANS, CUNNINGHAMBA, EDELMANGM: A Detailed Sttuctural Model of Cytotactio: Roteio Homologies, Alternative RNA Spliciog, and Binding Regions. Proc Nurl Acud Sci USA 1989, 86:1905-1909.
6.
RATHJENFG, WOLFFJM, CHIQUET-EHRISMANN R Restrictin: A Chick Neural ExtraceRuku Matrbt Protein Involved in Cell Attachment Co-Purities with the Cell Recognition Molecule Fll. Deuekpnenf 1991, 113:151-K%.
Tenascin-C, 7.
k%SHEVA P. SPIESSE, SCHACHNERM: Jl-160 and Jl-180 are OUgodendrocyte-Secreted Nonpermissive Substrates for CeII Adhesion. J Cell Biol 1989, 109:1765-1778.
NORENBERGU, Wrll~ H, WOLFFJM, FRANKR, R~THJENFG: The Chicken Neural Extracellular Matrix Molecule Restrictin: SimiIarIty with EGF-, Fibronectin Type III-, and FibrinogenLie Motifs. Neuron 1992, 8:84+863. The complete cDNA sequence of chick TN-R is presented and compared with that of TN-C. Protein biochemistry and cell adhesion to the carboxyl-terminal domain are presented.
8.
..
Fuss B, WINTERGERST E.S, BAR%H U, SCHACHNERM: Molecu.. Iar Characterization and In Situ mRNA Localization of the Neural Recognition Molecule Jl-160/180: A Modular Structure Similar to Tenascin. J Cell Biol 1993, 120:1237-1249. This paper presents the complete cDNA sequence of rat TN-R, and de.scribeclthe in situ labeling of the cells in developing brain that express it. 9.
10.
MORELY, BRISTOWJ, GITE&IAN SE, MIUER WL Transcript Encoded on the Opposite Strand of the Human Steroid 21Hydroxylase/Complement Component of C4 Gene Locus. Proc
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X-ray ClystaUography provides the atomic structure of an FN-III domain from TN-C. The cell adhesion RGD sequence is localized, and a model for an extended filament of FN.III domains is presented. 19.
BAKER NE, MLODZIK M, RUBIN GM: Spacing Differentiation in the Developing Drosophila Eye: a Fibrinogen-Related Lateral Inhibitor Encoded by scabrous. Science 1991, 250:137@1377. S, CHIQUET-EHRISMANN R: Ten”, a Drosophila Gene Related to Tenascin, Shows Selective Transcript Localization. Mech Dev 1993, 40:16%176.
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N, KIDA H, SAKAKURA T, KU%KABE M: Induction of Tenascin in Cancer CeUs by Interactions with Embryonic Mesenchyme Mediated by a Diisible Factor. J cell Sci 1993, 104289-296.
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RITITG WJ, TR&ZHE TJ, GARINCHESAP: StimuIatIon of Human Neuronecdn Secretion by Brain-Derived Growth Factors.
29.
DJ, FERGU~ON MWJ: The ExtraceUuIar Mat& of Lip Wounds In Fetal, Neonatal and AduIt Mice. Deve@ment 1991, 112:651-668.
30.
Wwrsv DJ, LONGAKERMT, I&RRISONMR. ADZICKNS, FERGKJ~ON MWJ: Rapid EpitheUaUsation of Fetal Wounds is Associated with tbe Early Deposition of Tenascin. J Cell Sci 1991, 99:583586. ONDA H, POULIN MI+ TASSAVARA, CHIU I-M: Characterization of a Newt TenascIn cDNA and Localization of Tenascin mRNA During Newt Limb Regeneration by in Situ Hybridization. Dev Bio. 1991, 148:219-232.
HIRAI~A
Brain
31.
32. .
SRIRAMARAO P, BOURDON MA A Novel Tenascin Type III Repeat is Part of a Complex of Tenascin mRNA Alternative Splices. Nucleic Acid Res 1993, 21:163168.
U,
REITIG WJ, HOFFMANS, Su SL, GA~UN.CHESAP: Species Diversity of Neuronectin and Cytotactin Expression Patterns in the Vertebrate Central Nervous System. Brain Res 1992, 5903219-228. An extensive survey of TN-C expression in the brains of different vertebrates.
BORK P, Dooum RF: Proposed Acquisition of an Animal Protein Domain by Bacteria. Proc Nat1 Acud Sci USA 1992,
A thorough analysis of all the FN-III domains in animal proteins.
S, B~H
.
GUL~HER JR, NIES DE, ALEXAKOS MJ, RAWKANT NA, SIIJRGILL ME, MARTON IS, STEFAN%ON K: Structure of the Human
Gene. Proc
BAUTXH EKBU)M
25.
Biol
R: Two Contrary Functions of Tenascin: Dissection of the Active Site by Recombinant Tenascin Fragments. Cell 1989, 59325-334.
ERICKSON
P, S~HACHNER M: Expression of TenascIn in tbe Developing and Adult CerebeUar Cortex. J Neurosci 1992, 12:736-749.
SPRING J, BECK K, CHIQUET-EHRISMANN
Hexabrachion (Tenascin) 1991, 88:943%9442. 15. .
Struct
2:413421.
Erickson
.
36:40&403. 12.
and tenascin-X
HP: Gene Knockouts of c-src, TGFBI, and Tenascin Suggest Superlluous, Non-Functional Expression of Proteins. J Cell Biol 1993, 120:1079-1081. Expression of a protein in a tissue does not necessadly mean that it is functional there.
23.
T:
Extracellular Matrix Protein Tenascin-Like Gene Found in Human MHC Class III Region. Immunogenetics 1992,
tenascind
Res 1989, 4873171-177.
WHITBY
1, JOSHI P, YAN Y, ERICKSON HP: CeU- and HeparinBiding Domains of tbe Hexabrachion Arm Identified by Tenascin Expression Proteins. J Biol C&m 1993,
AUKHIL
268:2542-2553.
Ten bacterial and three mammalian expression proteins covering specif~cdomains of human TN-C. These proteins are available to scientists for research purposes. AL, ANDERSSON-FISONE C, CROSSIN KL: Characterization of Multiple Adhesive and Counteradhesive Domains in the Extracellular Mati Protein Cytotacdn. J Cell Bill 1992, 119663-678. In this paper an independent set of bacterial expression proteins was used to map cell adhesion activities to chicken TN-C.
33.
PRIETO
.
34.
CHIQUET M, VRUCINIC-FIUPIN, SCHENKS, BECK K, CHIQUET. EHRISMANNR Isolation of Chick Tenascin Variants and Fragments. A C-Terminal HeparIn-Binding Fragment Produced by Cleavage of the Extra Domain f?om the Largest Subunit Splicing Variant. Eur J Biocbem 1991, 199:37%388.
35.
JOSHIP, CHUNGC-Y, AUKHILI. ERICKSONHP: EndotbeUaI CeUs Adhere to the RGD Domain and the Fibrinogen-Like Termid Knob of Tenascin. J Cell Sci 1993, in pre.%-
20.
BAUMGARTNER
36.
S-0 P, MENDLER M, BOURDON MA: EndotbeUaI CeU Attachment and Spreading on Human Tenascin Is Mediated by a2-01 and aV-p3 Integrins. J Cell Sci 1993, in press.
21.
MASUDA-NAKAGAWA LM, NlCHOUS JG: ExtraceUuIar Matrix Molecules in Development and Regeneration of the Leech CNS. Pbilos Trans Roy Sot Land [B] 1991, 331:323335.
37.
LIGHTNER VA, ERICKSON HP: Biding of Hexabracbion (Tenascin) to the ExtraceUuIar Matrix and Substratum and its Effect on CeU Adhesion. J Cell Sci 1990, 95:263277.
22.
HORTXH M, GOODMAN CS: CeU and Substrate Adhesion Molecules in Drosophila. Annu Rev Cell Biol 1991,
38.
WEHRLEB, CHIQUETM: Tenascin is Accumulated Along Developing Peripheral Nerves and AUows NeurIte Outgrowth
7:505-557.
in Vitm
Develcgnnent
1990,
110:401-415.
875
876
Cell-to-cell 39.
contact
and extracellular
LLXH~FR A, VAUGHAN I, Khp~ow M, FAISSNER A: Jlflenascin in
matrix A, P~~c~wvrt
A, SCHACHNER
45.
Substrate-Bound and Soluble Form Displays Contrary Effects on Neurite Outgrowth. J Cell Eiol 1991, 113:1159-1172.
40.
Hushww K, FNSSNER A, SCHACHNER M: Tenascin Promotes CerebeIlar Granule CeU Migration and Neurite Outgrowth by Diierent Domains in the Fibronectin Type 111Repeats. J Cell ,Biol 1992, 116:1475-1486.
41.
CRCX~IN
m Cytotactin Binding: lnhibition of Stimulated Proliferation and lntracelhdar AIkaIinization in Fibroblasts. Proc Nut1 Acud Sci US,4 1991, 88:11403-11407.
42.
END P, PANAYOTOU
G, E~STLE
A, WATERFIELD
MD,
46.
ZI%H AH, D’AIZX~ANDRI k WINTERHALTER KH, VAUGHAN
FLwcm
B,
FALCHETTO
47.
44.
SHIROISHI T. SAGN
T, NATXUUME-SAKAI
S, MORIWAKI
GULCHER
JR, S’IFFANSSON
SIRI A,
CARNEMOUA
B,
SAGINATI
M,
IXPIUNI
4
NISHI
T, WEINSTEIN
J, GILIISI’IE
WM,
PAULSON
Primary Structure of Porcine Tenascin Tenascin Transcripts in Adult Submaxillary Biocbem 1991, 20264348. 48.
WEUER
49.
SAGA Y,
R,
L: Neuronal CeU Adhesion Molecule Contactin/Fll Binds to Tenascin Via its lmmunoglobuIin-Lie Domains. J Cell Biol 1992. 119~203-213. Identilication of Fll as a cell surface ligand for TN-C. Fll also binds TN-R [6]. .
‘Q, KIM J-H,
K:
CA%RI
G,
BARAUE F. ~XRDI Lz Human Tenascin: Primary Structure, Pre-mRNi Splicing Patterns and Localization of the Epitopes Recognized by Two Monoclonal Antibodies. Nucleic Acid Res 1991, 19:525-531.
.
43.
HEMESA-IH
The Complete cDNA Sequence of Human Hexabrachion (Tenascin). A Multidomain Protein Containing Unique Epidermal Growth Factor Repeats. J Biol Cbem 1991, 266:2818-2823.
CHIQUET
M: Tenascin: A Modulator of CeU Growth. Ew J Biocbem 1992, 209:1041-1051. TN-C stimulates mitogenesis (but see [41] for contradictory results).
NIE~ DE,
JC: Complete Detection of Glands. kw J
4 BECK S, EKBLOM P: Amino Acid Sequence of Mouse Tenascin and Differential Expression of Two Tenascin Isoforms During Embryogenesis. J Cell Biol 1991, 112:355-362. TSUKAMOTO
T, JING
N,
KUSAKAL%
M,
SAKAKURA T:
Murine Tenascin: cDNA Cloning, Structure and Temporal Expression of lsoforms. Gene 1991, 104:177-185.
K: Lethal
Deletion of the Complement Component C4 and Steroid 21-Hydroxylase Genes in the Mouse H-2 Class III Region, Caused by a Meiotic Recombination. Proc Natl Acud Sci USA 1987, 8428192823.
HP Erickson, Drive, Duke
USA
Department of Cell Biology, 365 Sands Bldg, Research University Medical Center, Durham, North Carolina 27710,
University
P Erickson
Medical
Center,
Durham,
USA
“Mice develop normally without tenascin”, was a shock to biologists studying the extracellular matrix. Could tenascin be a useless protein? This seems most improbable, as it is conserved in every vertebrate species. Moreover, two new proteins have been discovered in the last year that are closely related to the original tenascin, providing evidence for a tenascin family. Speculations on functions are ripe for re-evaluation. Current
Opinion
in Cell Biology
Introduction
alogous sequences, which are derived from a gene duplication, typically before separation of the species [ 21. For example, a- and P-hemoglobin are products of a gene duplication that was fixed long before mammals arose. All mammals have both forms of hemoglobin, and each chain has been evolving independently in all species. The a-hemoglobins of different species are orthologous to each other, and are paralogous to the P-hemoglobins.
Tenascin (TN)-C, the first member of the tenascin family to be discovered, is a very large, complex protein of the extracellular matrix. Each subunit comprises -30 independently folding domains, each of which could have an independent function and hence many talents. TNC is expressed in developing brain, cartilage and mesenchyme, and is re-expressed in tumors, bound healing and inflammation. This apparently regulated and specific expression implied crucial roles in embryonic development and tissue restructuring. Numerous activities have been demonstrated in cell culture experiments. It was therefore most surprising that mice, genetically engineered by homologous recombination to knock out the TN-Cgene, developed normally and showed no apparent defect [I**].
TN-C was discovered independently in several laboratories in the early 1980s. Each discovery was in a different context and brought with it a new name: myotendinous antigen, glioma mesenchymal extracellular matrix, hexabrachion, tenascin, Jl-200/220 and cytotactin all describe the same protein (see [3] for a review and references to the earlier papers). The name tenascin has now been adopted by almost all laboratories working in this area and will be extended here to cover the entire family of related proteins, as suggested by Bristow et al. [4*-l. The original tenascin is designated TN-C, the C indicating cytotactin, the name associated with the first published sequence [ 51, and the two newer family members (paralogues) as TN-R and TN-X. (The name tenascin, without a letter, will continue to be used for TN-C in research papers dealing exclusively with this most studied family member, but the more precise nomenclature will be useful as the other family members become better known.)
It would be premature, however, to conclude that TNC has no important function. TN-C is conserved in every vertebrate species, which argues strongly for a fundamental role, probably the same in all species, that contributes to survival. The lack of a phenotype in the knockout mouse simply means that the function may be subtle, rather than vital. It also brings into question all of the functions previously proposed. Remarkably, just as we are faced with this crisis of linding a function for TN-C, two new proteins have been discovered that are related to TN-C. We now have a family of tenascins, each member with similar multiple domains and potentially similar functions. In this review I will first describe the tenascin family, and return to the question of function.
TN-R was originally discovered under the names restrictin in chicken [6] and Jl-160/180 in rat [7]; both chicken and rat TN-R have recently been sequenced [8**,9”]. These proteins are as closely related to each other as are the TN-Cs from chicken and rat, but are more distantly related to the TN-Cs of all species.
Protein sequences that appear to be related are referred to as homologous, with the understanding that they are derived from a common ancestral sequence. Homologous sequences may be classified into two types: orthologous sequences, which are found in different species where the differences reflect species divergence; and par-
ECL-epidermal
growth
factor;
fbgfibrinogen; @
Current
TN-X was originally reported as a partial sequence encoded by gene X [lo]. The same gene was identied by Matsumoto et al. [ 111, who provided additional sequences important in the analysis of the gene. An al-
Abbreviations FN-fibronectin; Biology
1993, 5:869-876
Ltd
TN--tenascin; ISSN
0955-0674
UEP-unit
evoltitionary
period.
870
Cell-to-cell,
contact
and extracellular
matrix
most complete sequence of this very large’ protein has now been reported by Bristow ,et al [4**].
Teriascin
domain
structure
The tenascins are modular proteins [ 121 in which the polypeptide chain shows repeating sequence motifs that fold independently into small globular domains or modules. The known tenascin family members share a very distinctive pattern of four types of domains (Figs 1 and 2).
The amino-terminal - 150 ammo acids contain oligomerization sites where the subunits are connected to form trimers and hexamers; this region is unique to the tenascins. All tenascins contain an a-helical segment of 22 amino acids that could form a triple-stranded coiled coil, flanked by pairs of cysteines that could form disulfide rings [ 131. TN-C and TN-R contain a single cysteine amino-terminal to the trimer-forming segment, which could connect two trimers into the characteristic hexabrachion structure. Although TN-R protein isolated from tissues has only been found in the form of dimers and trimers [7,8*=], it is likely that TN-R can also form hexabrachions; the smaller oligomers may result from proteolysis. TN-X, however, diverges in sequence aminoterminal to the trimer-forming segment, and is missing the cysteine needed to form a hexabrachion. TN-X can apparently form only tribrachions. The next segment comprises a series of 4i/r18i/r epidermal growth factor (EGF) domains, each 31 amino acids long. The common ancestry of this EGF segment is suggested by the curious i/z repeat that begins each segment, and by the fact that it is encoded by a single exon in both TN-C [14] and TN-X [4**], the two proteins with a known gene structure. The individual EGF
domains have 70-80 % amino acid identity, making it difficult to map the domains of TN-R and TN-X to precise equivalents in TN-C. The next segment comprises a series of libronectin (FN)III domains [ 15.1, each about 91 amino acids long. In contrast to the EGF domains, most of the FN-III domains share only 25-30% amino acid identity, and are no more similar to each other than they are to those of other proteins, like libronectin. However, FN-III domains l-5 of TN-C and TN-R, and domains 68 of all three tenascins, correspond to each other, with 34-43 % amino acid identity between family members. The alternatively spliced domains, indicated by letters, are inserted between the universal domains 5 and 6 in TN-C and TNR. Eight alternatively spliced domains have been identified in human TN-C, including the newly discovered ad1 [ 161. Only three of these, A, B and D, have been found in chicken. The 23 domains indicated as X-type FN-III in Fig. 1 are remarkable in being > 50 % identical to each other [4**], similar to the recently duplicated domains At-4 in human TN-C [ 171. They are also more closely related to the alternatively spliced domains of TN-C than to the universal domains. It seems a good guess that the 23 Xtype FN-III domains are alternatively spliced, perhaps in a complex pattern. The atomic structure of TNfn3, the third FN-III domain of human TN-C, was determined recently by X-ray crystallography [ 18**]. An atomic model was proposed for the thick segment of the hexabrachion arm that involves a tight packing of adjacent FN-III domains to make an extended filament. The carboxyl-terminal domain, corresponding to the terminal knob visualized by electron microscopy, consists of 215 amino acids with homology to the carboxy-terminal domain of p- and y-fibrinogen. It is abbreviated here TNfbg. This domain is well conserved in TN-C,
Fig. 1. An
electron micrograph of a molecule of human TN-C, showing the hexabrachion structure. The domain segments are indicated: CN, central nodule; ECF, the segment of 14 epiderma1 growth factor like domains; FN-III, the segment of 15 fibronectin type-Ill domains; and fbg, the fibrinogen-like terminal knob. An atomic resolution structure of one FN-III domain, and a model for the FN-III segment have been determined [I89
Tenascin-C,
Tenascin-C
(human,
pig,
mouse,
tenascin-R
and tenascin-X
Erickson
chicken, newt)
A COOH
Tenascin-R
(chicken.
mouse)
COOH
Tenascin-X
(human)
NH,
COOH
0
0
ECF
domains
0
Universal
FN-III
domains
l
Alternatively
spliced
FN-III
domains
1993 Current
TNfbg
Opinion
in Cell Biolom
domain
Fig. 2. The domain structure of the tenascin family. The hexabrachions or tribrachions are indicated at the central nodule. The universal FN-III domains, present in all transcripts of TN-C and TN-R, are indicated by l-5 and 6-8; the alternatively spliced domains are indicated by letters [32-l. The human TN-C shown here has the largest number of alternatively spliced domains of the five known vertebrate species. The domain Rl of TN-R is not obviously related to any of the domains of TN-C. The 23 X-type FN-III domains of TN-X may be alternatively spliced.
TNR and TN-X, the three sharing 4148% identity.
amino acid TN-X.hum TN-C.hum TN-C.pig TN-C.mus
A phylogenetic
tree of the tenascins
The carboxy-terminal segment, comprising the last three FN-III domains (TNfnG8) and TNfl~g, is clearly conserved among all family members. This region was selected for further analysis of the relation between the tenascins. The multiple alignment algorithm in the GeneWorks @program (Intelligenetics, Mountain View, California) generates a convincing alignment with virtuallly no insertions, and gives the phylogenetic tree shown in Fig. 3. The TN-Cs of the five species (human, pig, mouse, chicken and newt) all align with the expected order of relation among the species. The two TN-Rs form a group related to each other but much more distantly to the TN-& TN-X is most distant. 1 have made a separate pair-wise alignment of human TN-C with TN-C of other species, to estimate the rate of evolution of the tenascins. The TN-C of human, mouse and pig differ by only 10 %, while human TN-C differs from that of chicken and newt by 25-30 %. Humans separated from mice and pigs 75-95 million years (MYrsj ago, and from birds and amphibians 300-350MYrs ago. One can estimate the unit evolutionary period (UEP, time required for a 1% divergence in sequence [2] ) for TN-
TN-C.chk TN-C.nwI TN-R.chk
1
TN-R.rat 0 1993 Current
Opinion
in Cell Biology
Fig. 3. A family tree of the tenascins constructed by the multiple alignment program of GeneWorks. The alignment is based only on the carboxyl-terminal segment, TNfnG3 plus TNfbg, which is well conserved in all family members. The lengths of the lines indicate the degree of similarity of the sequences. The accession numbers and references for the tenascin sequences are: TN-Chum M55618 [451 and X56160 [461; TN-C.pig X61599 (471; TN-C.mus X56304 I481 and D90343 [491; TN-Cchk JO4519 [51 and M23121 1131; TN-Cnwt M76615 [311; TN-R.chk X64649 I8**1; TNR.rat 218630 [9*-l; TN-X.hum X71923-38 (each piece of genomic DNA has a separate entry) [4*-l.
C to be 7-10 MYrs. Different proteins diverge at widely different rates, illustrated by the following LJEP ranges: 60-400 MYrs for histones; 14 for insulin; 5 for prolactin; 3.7 and 3.3 for hemoglobin, and 3 for serum albumin. Thus TN-C is evolving at a modest rate, equivalent to some growth factors that are considered functionally important and conserved.
871
872
Cell-to-cell
contact
and extracellular
matrix
I have extended this analysis to speculate bn the age of divergence of TN-R, TN-X and TN-C. Assuming that all members of the tenascin family are evolving at the same rate, with UEP * 9, the divergence of the three tenascins would date to 800 MYrs ago. This extrapolation should not be taken too quantitatively, but does suggest that a primordial tenascin existed in early Cambrian or Pre-Cambrian periods, before the divergence of insects, mollusks, annelids, cnidarians, etc. Thus we might expect to find tenascins in invertebrate phyla.
Invertebrate
tenascin?
Two proteins from Drosophila, identified by cDNA sequencing, have domains related to those of tenascins. Scabrous, a diffusible protein with probable growth factor activity in development, has a carboxyl-terminal domain homologous to TNfbg [ 191. More recently, a protein designated Tena was identified in a low stringency screen for Drmophikz proteins related to the tenascin EGF domains. Tena has eight EGF domains with a size and cysteine spacing similar to those of tenascins [ 201. A related molecule, Tenm, identified in the same screen, shows a similar set of eight EGF domains, followed by 11 domains that might be related to FN-III (S Baumgartner, D Martin, C Hagios, R Chiquet-Ehrismann, unpublished data). The relationship of Ten”, Tenm and Scabrous to the corresponding domains in tenascin may provide interesting documentation on domain shuffling of modular proteins [ 121, and perhaps on the evolution of the tenascin family. A molecule visualized by electron microscopy in extracts from leech is a potential invertebrate tenascin [ 211. It has a hexabrachion structure, with a central knob linking the six arms, a thin proximal segment and a very long thick segment, which could correspond to EGF and FNIII domains. Protein biochemistry and cloning data are obviously essential to determine whether this invertebrate protein is really related to tenascin. Several other reports of invertebrate tenascins have been based on cross-reaction with polyclonal antibodies, but these are not convincing without more extensive biochemistry. An invertebrate tenascin should probably have three or all four types of domains in the proper order to be included ln the tenascin family. For example, Drosophila laminin closely matches the domain structure of vertebrate laminins in all three subunits, Ig superfamily adhesion molecules and integrins are also conserved in Drosophila [22]. However, it should be recalled that Dmsopbika apparently has no fibronectin [22], so it is quite possible that it has no tenascin. Extending the search to other phyla, such as annelids, may reveal unmistakeable tenascin relatives. This would provide exciting new opportunities to explore the function of tenascins, as well as their evolution.
Tenascin expression in developing tissues, tumors and wound healing
and adult
For more than a decade, the patterns of expression of newly discovered proteins have been carehllly researched for clues to their functions. Several careful surveys of TNC, already reviewed in [ 31, were accompanied by the expectation that TN-C would have an important function wherever it was expressed. This expectation has recently been challenged by the hypothesis that proteins might be expressed superfluously in some tissues where they are non-functional [23*]. Thus the high level of TNC in brain or healing wounds might be an accident of gene regulation, tolerated because TN-C is an innocuous molecule in these locations. This warning that expression in a particular tissue does not necessarily imply function is important. However, it is also important to know where TN-C is expressed because it should have a function in at least some tissues. Expression of TN-C in the brain has always attracted interest. A very detailed study of mouse cerebellum showed TN-C mRNA primarily in Golgi epithelial cells, which form a narrow band beneath the Purkinje cells [ 241. It is not clear how the TN-C protein becomes widely distributed through the layers of the developing cerebellum. The recent study by Rettig et al. [ 25*] revises the earlier conclusion that TN-C was expressed only in developing brain. They demonstrated prominent TN-C protein in at least some regions of adult brain of five mammalian species. A remarkable finding was the variation among species in regional patterning at specific developmental stages. For example, TN-C is highly expressed in all areas of the bovine brain, is most prominent in the molecular layer of the cerebellum in rat and mouse, but is completely missing from the cerebellum of human. This variation is consistent with the idea that TN-C may not be serving an important function in brain. TN-R is apparently restricted to the central nervous system, and is most prominent in development. It is found in retina, cerebellum and spinal cord of embryonic chicken [800], and in cerebellum, hippocampus and olfactory bulb of postnatal rat, peaking at 7-14 days [ 9=*]. Expression continued in adult rat cerebellum. TN-X mRNA is prominently expressed in fetal muscle (smooth muscle of the gut, skeletal muscle and heart) and testis, with a lower level in fetal adrenal gland, kidney and lung [4**]. TN-X was virtually absent from 22 week human fetal brain, and also from 2 day postnatal mouse brain [1*-l, where TN-C and TN-R are very prominent. Expression of TN-C in a wide variety of tumors was one of the most exciting early findings. Reports that TNC might be associated with the most malignant tumors gave promise of clinical importance. Recent studies [26] confirmed the abundant expression of TN-C in malignant breast carcinomas, but noted that it is much more widespread than previously thought. TN-C is conspicuous in lactating-gestational breast, fibrocystic disease and benign tumors. In carcinomas, TN-C is localized primarily in the
Tenascin-C,
stroma around the islands of epithelial cells, raising the question of which cells secrete it. Earlier studies concluded that TN-C was secreted primarily by mesenchymal cells, presumably induced by growth factors from the carcinoma cells. It now seems that the reverse scenario is also important. Human carcinoma cells injected into nude mice first caused secretion of mouse TN-C by surrounding libroblasts, but after 2 days the carcinoma cells secreted substantial amounts of human TN-C [27]. The carcinoma cells seemed to be stimulated by growth factors from the stroma. The growth factors that stimulate several types of cells to secrete TN-C have been studied most extensively by Rettig et al [28] (this paper has been lost to the tenascin literature because it was published under the name neuronectin, now known to be TN-C [ 25.1). Expression of TN-C in wound healing is weU established, but has been studied in greatest detail in comparative studies of fetal and adult wounds [29,30]. A most remarkable observation in these studies was the rapid time course of expression, in l-4 h, of TN-C in fetal wounds, which also healed without scarring. A study of TN-C expression during limb regeneration in newt [31] used in situ hybridization to demonstrate that TN-C mRNA was first expressed by the wound epithelial cells and later by mesenchymal cells, again demonstrating the capacity of several ceU types to secrete TN-C.
TN-C in cell culture: specific domains
functions
mapped
to
The search for TN-C function has led to specific assays in cell culture of cell adhesion, neurite outgrowth and mitogenesis, with the controversial findings that TNC can either promote or inhibit any of these activities. An important development in the past year has been the production of sets of expression proteins that replicate specific domains of TN-C. The most extensive set is that of Aukhil et al. [32*], which covers all the FN-III domains and TNfbg in 10 defined segments. These proteins have all been highly purified and demonstrated to be soluble and monomeric. The bacterial expression proteins studied by Prieto et al. [33*] cover different and overlapping domains, including the EGF domains. Two proteolytic fragments isolated by Chiquet et al. [34] have provided important indications of activity. CeU adhesion to TN-C has long been controversial, but a recent study has demonstrated that it can be either nonadhesive or adhesive depending on how it is coated on plastic [35]. Cells adhere to the third FN-III domain of chicken [ 33.1 and human [35] TN-C; adhesion is blocked by RGD peptides and appears to be mediated by the aVj33 integrin [35,36]. A second adhesion site has been mapped to the fbg domain of TN-C and probably also TN-R [8”,32*,33*,35]. Adhesion to TN-Cfbg is mediated by a ceU surface proteoglycan [32*] on fibroblasts, and an integrin, probably a2p1, on endothelial ceUs [ 35,361. A third cell adhesion site may exist in TN-Cfn7-8, but this is
tenascin-R
and tenascin-X
Erickson
complicated by competing anti-adhesion activities of this domain [33-l. Anti-cell adhesion activity is perhaps the most popular activity ascribed to TN-C in recent years. The best established mechanism is steric blocking, in which the large hexabrachion molecules bind to the substrate (plastic), straddle the Iibronectin molecules and cover up the adhesion sites [ 371. Lightner and Erickson [37] reported no anti-adhesion effect of soluble TN. In contrast, Prieto et al. (33*] reported anti-adhesion activity of both substrate-bound and soluble TN-C, and mapped the activities to the EGF domains and to TN-Cfn7-8. Chiquet et al. [34] reported anti-adhesion of a proteolytic fragment containing TN-Cfn7-8. Neurite outgrowth is a more complex phenomenon than cell adhesion and is affected by many different molecules. Tenascin can either stimulate or inhibit neurite outgrowth depending on the mode of presentation. Antibodies have been used to map the active domains [38-40]. There has long been an interest in whether TN-C might stimulate ceU growth by acting as a mitogen. Two recent studies report completely contradictory results. Crossin [41] reported that TN-C inhibited cell growth, while End et al. [42*] reported mitogenic stimulation. Further work will be needed to determine which of these is correct or how the result depends on particular experimental conditions. The availability of domain-specific expression proteins should provide an important tool in this work. AU of the above effects on ceU behavior imply the existence of cell surface receptors for tenascins. An important discovery in the past year was that TN-C binds to Fll/contactin [43*]. Fll is one of the Ig super-family cell surface/adhesion molecules, and is abundant on neurons Remarkably, TN-R was originally identified as a ligand for Fll [ 61. It seems hardly a coincidence that both of these tenascins, which are prominent in developing brain, bind this neuronal ceU surface molecule.
Is there
life without
tenascins?
The most important test of function of a protein is to identify mutants with a defective or completely disrupted gene. This is exactly what was undertaken by Saga et al. [l**], who created a mouse completely lacking TN-C. This is clearly the most important paper of the year, if not the decade, on tenascin. The expectation was that these mice would be severely crippled in several aspects of development. Remarkably, the knockout mice appeared completely normal. AU tissues that normally express high levels of TN-C in development, including the brain, lung and thymus, were histologically normal without TN-C. The mice behaved normally (but they have not been carefully analyzed for intelligence or memory), they gave birth, and their wounds healed normally. How can this absence of phenotype in TN-C knockout mice be reconciled with the dramatic functions inferred from tissue culture assays?
873
874
Cell-to-cell
contact
and extracellular
matrix
The word redundancy arises quickly in disoussions, with suggestions that the functions of TN-C might be duplicated by other proteins. Interest is focused on the paralogues, but they can only duplicate functions in the tissues where they are co-expressed (see above). Nevertheless, knockouts of TN-R and TN-X will be eagerly awaited to see whether there are effects of the individual knockouts, and additional effects of double knockouts. Bristow et al [4**]# suggested that a knockout of TN-X may already have been achieved as a deletion mutation in the mouse steroid hydroxylase region. Shiroishi et al [44] identiiied the deletion of an 80 kb fragment extending from the C4B gene to P45Oc21A and presumably including the mouse TN-XA This deletion was lethal in the homozygous state. However, Shiroishi et al. [44] reported that the homozygous mice did not die until 5-15 days after birth, so the lethality was probably due to the P45Oc21A deletion (21 hydroxylase is not needed until after birth). That these mice could complete fetal development and live for 5-15 days suggests that TN-XA is not needed for development. It is not known for mice whether TN-XA or TN-XB is the active gene (remember, in humans TN-XB is active and TN-XA is like a pseudogene). If TN-XA is the active gene, then the TN-X knock-out appears to give a subtle phenotype much like TN-C. This is a complicated gene locus but some careful genetics might pm down some important information without engineering a de novo gene knockout. It should be emphasized that TN-C cannot be totally redundant [23*]; a protein must have a unique function in at least some tissue, or it would revert to a pseudogene. An immediate challenge is then to determine the tissue where TN-C is expressing its unique function. Close examination of the knockout mice for subtle defects in specific tissues may be productive, and the clues provided by cell culture assays may be helpful. We need to remember, however, that a gene can be iixed in a population if it confers only a 1% survival advantage. Defects in the knockout mice may not be obvious even when closely examined. We also have to consider the possibility that the true function of tenascins in vivo is completely different from all previous suggestions. We should perhaps look for a function that would be common to the three tenascins, and consider closely the structural and biochemical features that are conserved in the three tenascin paralogues. Most striking to me is the carboxyl-terminal segment, comprising IN-III domains 6-g and the fibrinogen-like terminal knob. These domains are conserved in all three tenascins, as is their positioning at the end of long am-is of a multivalent tri- or hexabrachion molecule. If these domains bound other extracellular matrix molecules, they might be ideally positioned to organize them into a network, and to maintain a spacing of 5ck200~1 between the binding sites. Binding studies to identify candidate proteins should now be renewed with the available domainspecilic expression proteins.
Conclusion The past year has seen an expansion of tenascin from an elabomte protein consened in all vertebrates, to a family of three related proteins. At the same time the founding member of the family was submitted to the ultimate test of function a genetic knockout. The failure to iind any abnormality in TN-C-deficient mice dashes our hopes that a dramatic phenotype or failure in specific tissues would con&n-~ important functions. Some functions demonstrated in cell culture assays may be irrelevant in vivo, but others may provide clues as to which tissues should be examined more closely in the knockout mice. Even less is known about the two paralogues, TN-R and TN-X, but it is attractive to think that their similar structures are engaged in similar functions in the different tissues. While awaiting results of additional knockouts, the field is now open for totally new hypotheses on the functions of these molecules.
Acknowledgements I thank H Onda, Ohio State University, J Bristow and W Miller, University of California, San Francisco, and S Baumgarten, Friedrich Miescher Institut, Basel, for sharing with me information on newt TNC, human TN-X and Drosophila Tenm, respectively, prior to publication.
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