Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats

The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats.     

J1-160 and J1-180 are oligodendrocyte-secreted nonpermissive substrates for cell adhesion

The glia-derived J1 extracellular matrix glycoproteins have been referred to as J1-160/J1-180 (the developmentally late appearing lower molecular weight group) and J1-200/J1-220 (the developmentally early appearing higher molecular group immunochemically related to tenascin). Members of the two groups show distinct cross-reactivities. To characterize the structural and functional differences between these J1 glycoproteins, two monoclonal antibodies were generated which recognize only the members of the lower molecular weight group. The two antibodies detect immunochemical similarities among the members of the lower molecular weight group, but do not react with J1/tenascin. J1-160 and J1-180 are specifically expressed by differentiated oligodendrocytes in culture and by myelin of the central nervous system and have not been found in the peripheral nervous system nor in any other organ of the adult mice tested. Electron microscopic examination of rotary-shadowed J1-160 and J1-180 reveals, respectively, dimeric and trimeric (tribrachion) kink-armed rodlike structures, which are linked by disulfide bridges. J1-160/J1-180 are nonpermissive substrates for the attachment and spreading of early postnatal small cerebellar neurons, astrocytes, and fibroblasts. In a mixture with laminin, J1- 160/J1-180 are nonpermissive substrates for neurons, but not for astrocytes or fibroblasts. The repulsive effect toward neurons can be neutralized by one of the monoclonal antibodies, but not by the other. These observations are discussed in the context of cell interactions during regeneration in the mammalian nervous system.     

Amino acid sequence of mouse tenascin and differential expression of two tenascin isoforms during embryogenesis

We have isolated cDNA clones for mouse tenascin and analyzed expression of tenascin mRNAs during embryonic development of the kidney and gut. The deduced amino acid sequence of the mouse tenascin cDNAs shows a modular structure of repeats similar to chicken and human tenascin. In mouse there are 14.5 cysteine-rich repeats with similarity to the EGF repeat, followed by several repeats with similarity to the type III repeat of fibronectin. A longer variant contains 13 fibronectin type III repeats, whereas a shorter splice variant of mouse tenascin lacks the 5 type III repeats that occur directly after the fifth repeat in the longer variant. Contrary to the chicken and human sequences, mouse tenascin does not contain an RGD sequence in the third type III repeat implicated in cell attachment, or in any other positions. In Northern hybridizations to RNA from primary embryonic fibroblasts, the cDNA clone M 20/1 detects two mRNAs with sizes close to 6 and 8 kb. This, and the other data presented here suggest that the two major mouse tenascin polypeptides arise through an alternative RNA splicing. The two major mRNAs are differentially expressed during development. The 8-kb mRNA is more prominent than the 6-kb mRNA throughout prenatal kidney development, but during postnatal development the ratio of the two mRNAs changes. A different expression pattern is seen in the developing gut where the 6-kb mRNA predominates during embryogenesis with the 8-kb mRNA appearing later. The mRNA data of the developing gut correspond with previous protein data, which showed that the shorter Mr 210,000 polypeptide predominates during earlier developmental stages and the larger Mr 260,000 polypeptide appears later in the embryonic gut (Aufderheide, E., and P. Ekblom. 1988. J. Cell Biol. 107:2341-2349).     

Localization of a cryptic binding site for tenascin on fibronectin

Fibronectin and tenascin are large extracellular matrix proteins that interact with each other and with integrin receptors to regulate cell growth and movement. They are both modular proteins composed of independently folded domains (modules) that are arranged in linear fashion. Fibronectin is a covalent dimer and tenascin is a hexamer. The site on tenascin to which fibronectin binds has been localized to type III modules 3-5. In this study we use surface plasmon resonance to examine the interaction between various fragments of fibronectin and tenascin to further characterize and localize the binding sites. We found that tenascin fragments that contain type III modules 3-5 bind primarily to the N-terminal 29-kDa hep-1/fib-1 domain, which contains the first five type I modules of fibronectin. The dissociation constant, K(d), is approximately 1 microm. The binding site on fibronectin appears to be cryptic in the whole molecule in solution but is exposed on the proteolytic fragments and probably when fibronectin is in the extended conformation.     

A novel tenascin type III repeat is part of a complex of tenascin mRNA alternative splices.

Sequence analysis of two human tenascin encoding cDNA clones from a cDNA library of the U251 glioblastoma cell line revealed the presence of a novel 276 bp tenascin type III fibronectin like repeat. This alternatively spliced type III repeat designated AD1 is located between the previously identified repeats 10 and 11 and has sequence homology with human, chicken and mouse tenascin type III repeats. These results show that tenascin has at least 16 consecutive fibronectin like type III repeats. PCR amplification of random primed mRNA with specific type III repeat primers revealed a pattern of multiple alternative splices of AD1 and flanking type III repeats. The alternative splice variants were confirmed by direct sequencing. Differences were observed in the expression of the various alternative splices of tenascin mRNA between tumor and normal cells and may thus indicate differences in tenascin isoform expression and function in normal and tumor cells. PCR and Southern analysis of genomic DNA indicate that AD1 is coded by a single exon present in both human and mouse genome.     

J1/tenascin in substrate-bound and soluble form displays contrary effects on neurite outgrowth

The influence of J1/tenascin adsorbed to polyornithine-conditioned plastic (substrate-bound J1/tenascin) and J1/tenascin present in the culture medium (soluble J1/tenascin) on neurite outgrowth was studied with cultured single cells from hippocampus and mesencephalon of embryonic rats. Neurons at low density grew well on J1/tenascin substrates and extended neurites that were approximately 40% longer than on the polyornithine control substrate after 24 h in vitro. The neurite outgrowth promoting effect of substrate bound J1/tenascin was largely abolished in the presence of mAb J1/tn2, but not by mAb J1/tn1. In contrast to the neurite growth-promoting effects of substrate bound J1/tenascin, neurite outgrowth on polyornithine, laminin, fibronectin, or J1/tenascin as substrates was inhibited by addition of soluble J1/tenascin to the cultures. Neither of the two mAbs neutralized the neurite outgrowth-inhibitory properties of soluble J1/tenascin. In contrast to their opposite effects on neurite outgrowth, both substrate-bound and soluble J1/tenascin reduced spreading of the neuronal cell bodies, suggesting that the neurite outgrowth-promoting and antispreading effects are mediated by two different sites on the molecule. This was further supported by the inability of the mAb J1/tn2 to neutralize the antispreading effect. The J1/tn2 epitope localizes to a fibronectin type III homology domain that is presumably distinct from the putative Tn68 cell-binding domain of chicken tenascin for fibroblasts, as shown by electronmicroscopic localization of antibody binding sites. We infer from these experiments that J1/tenascin contains a neurite outgrowth promoting domain that is distinguishable from the cell-binding site and presumably not involved in the inhibition of neurite outgrowth or cell spreading. Our observations support the notion that J1/tenascin is a multifunctional extracellular matrix molecule.     

Nerve Growth Factor Mediates Monosialoganglioside-Induced Release of Fibronectin and J1/Tenascin from C6 Glioma Cells

C6 rat glioma cells incubated in serum-free medium with D-[14C]glucosamine secrete, on stimulation with nerve growth factor (NGF) or monosialogangliosides (MSGs), several glycoproteins (Gps), the most prominent of which are a 270-, 220-, and 69-kDa Gp. Several growth factors, hormones, phorbol ester, and disialo- and trisialogangliosides did not stimulate secretion. Western blot analysis of the conditioned medium from C6 cells stimulated with NGF or MSG identified one distinct band of approximately 220 kDa for fibronectin and J1/tenascin, which comigrated. Antiserum to NGF prevented NGF-stimulated release and also blocked MSG-evoked release. The 220-kDa band was labeled after pulse labeling with [35S]methionine in the presence of NGF, and by a 15-min chase period radioactively labeled J1/tenascin could be immunoprecipitated. Tunicamycin drastically inhibited almost completely release of the 220-kDa Gp labeled by D-[14C]glucosamine or [35S]methionine. These results extend the range of neurotrophic properties attributed to NGF to cells of glial origin and suggest that NGF regulates secretion of extracellular matrix proteins. MSG stimulation of fibronectin and J1/tenascin secretion may be mediated by NGF or an NGF-like molecule also secreted by the C6 glioma cells.     

The effect of catecholamines and adenosine on the induction of morphological alterations and depigmentation of newt iris epithelial cells in vitro

The J1 glycoproteins have been shown to mediate neuron-astrocyte adhesion and appear in the nervous system as four species of Mr 160,000 (J1-160), 180,000 (J1-180), 200,000 (J1-200), and 220,000 (J1-220), respectively. Tenascin is a disulfide-linked oligomeric, extracellular matrix glycoprotein of subunit Mr 170,000, 190,000, 200,000, and 220,000, which has been proposed to promote epithelial cell proliferation. In view of the structural similarities of the molecules we have used immunohistochemical and immunochemical techniques to compare them. Immunohistochemically, polyclonal J1 and tenascin antibodies yielded identical staining patterns in non-nervous-system tissues, and staining could be completely blocked by preincubating the sera with purified tenascin. In the central nervous system all structures expressing tenascin immunoreactivity were also recognized by J1 antibodies. However, not all J1-positive structures were also tenascin-positive, indicating that J1 antibodies recognized additional epitopes not present on tenascin. Western-blot experiments performed with affinity-purified polyclonal J1 antibodies showed that J1 glycoproteins can be subdivided into two separate pairs, J1-160/180 and J1-200/220, which share a small degree of homology. Western-blot experiments and sequential immunoprecipitations on biosynthetically [35S]methionine- or 125I-radiolabeled J1 glycoproteins carried out with polyclonal J1 and tenascin antibodies demonstrated that J1-200/220 is immunochemically indistinguishable from tenascin. These observations suggest that one set of extracellular glycoproteins is associated with processes as different as neural histogenesis and carcinogenesis of mammary glands.     

Cluster of fibronectin type III repeats found in the human major histocompatibility complex class III region shows the highest homology with the repeats in an extracellular matrix protein, tenascin.

Walking and sequencing a genome portion centromeric of CYP21B in the human MHC class III region disclosed a cluster of fibronectin type III repeats in an approximately 50-kb DNA segment. Fibronectin type III repeats are known to consist of ca. 90 amino acid residues and exist in a wide range of protein species. Homology searches in protein databases showed that the repeats found had the highest homology with the repeats of human tenascin, an extracellular matrix protein. One cDNA sequence located immediately centromeric of CYP21B, the 3' portion of which is transcribed by the opposite strand of CYP21B, was found also to have six type III repeats followed by a fibrinogen domain. Pairwise homology comparison of these repeats in the MHC locus with those of human tenascin showed a general parallelism in their gene organization, indicating that the newly found repeats are elements of certain tenascin-like genea.     

Neuronal cell adhesion molecule contactin/F11 binds to tenascin via its immunoglobulin-like domains

Adhesive interactions between neurons and extracellular matrix (ECM) play a key role in neuronal pattern formation. The prominent role played by the extracellular matrix protein tenascin/cytotactin in the development of the nervous system, tied to its abundance, led us to speculate that brain may contain yet unidentified tenascin receptors. Here we show that the neuronal cell adhesion molecule contactin/F11, a member of the immunoglobulin(Ig)-superfamily, is a cell surface ligand for tenascin in the nervous system. Through affinity chromatography of membrane glycoproteins from chick brain on tenascin-Sepharose, we isolated a major cell surface ligand of 135 kD which we identified as contactin/F11 by NH2-terminal sequencing. The binding specificity between contactin/F11 and tenascin was demonstrated in solid-phase assays. Binding of immunopurified 125I-labeled contactin/F11 to immobilized tenascin is completely inhibited by the addition of soluble tenascin or contactin/F11, but not by fibronectin. When the fractionated isoforms of tenascin were used as substrates, contactin/F11 bound preferentially to the 190-kD isoform. This isoform differs in having no alternatively spliced fibronectin type III domains. Our results imply that the introduction of these additional domains in some way disrupts the contactin/F11 binding site on tenascin. To localize the binding site on contactin/F11, proteolytic fragments were generated and characterized by NH2-terminal sequencing. The smallest contactin/F11 fragment which binds tenascin is 45 kD and also begins with the contactin/F11 NH2-terminal sequence. This implies that contactin/F11 binds to tenascin through a site within the first three Ig-domains.     

Zebrafish tenascin-W,a new member of the tenascin family

A cDNA clone encoding tenascin-W, a novel member of the tenascin family, was isolated from a 20- to 28-h postfertilization (hpf) zebrafish cDNA library on the basis of the conserved epidermal growth factor-like domains represented in all tenascin molecules. An open reading frame of 2796 base pairs encodes a mature protein consisting of heptad repeats, a cysteine-rich amino terminal region, 3.5 epidermal growth factor-like repeats, five fibronectin type III homologous repeats, and a domain homologous to fibrinogen. These domains are the typical modular elements of molecules of the tenascin family. Sequence comparison demonstrated that TN-W shares homologies with the members of the tenascin family but is not a species homolog of any identified tenascin. The expression pattern of tn-w was analyzed by in situ hybridization in 1-day-old embryos, in 3-day-old larvae, and in juvenile zebrafish. At 24–25 hpf, tn-w mRNA was expressed in the lateral plate mesoderm, most conspicuously in the presumptive sclerotome. Migrating cells of sclerotomal and neural crest origins also showed high levels of expression. At 3 days, expression by sclerotomal and neural crest cells continued to be observed while expression in the somitic mesoderm was decreased. In juvenile fish, tn-w was expressed weakly by cells in the myosepta and, more strongly, by presumably nonneuronal cells in the dorsal root ganglia. In these tissues and at the same developmental stages, the expression of tn-w partially overlapped with the distribution of tn-c mRNA. In addition, tn-c was expressed in the central nervous system (CNS) and in the axial mesoderm, neither of which expressed tn-w at any of the age stages examined. The expression pattern of tn-w suggests an involvement in neural crest and sclerotome cell migration and in the formation of the skeleton. Similar and possibly overlapping functions could also be performed by tn-c, which appears to have additional functions during the development of the CNS. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 1–16, 1998     

Differential effects of cytotactin/tenascin fusion proteins on intracellular pH and cell morphology

Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH (designated pH_i), we have measured pH_i in NIH3T3 and U251MG cells in the presence of the cytotactin/tenascin fusion proteins and intact cytototactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pH_i lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pH_i values than control cells. When two fragments were simultaneously presented, one of which alone increased pH_i and the other of which alone decreased pH_i, the predominant effect was that of lowered pH_i. Incubation with an RGD-containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pH_i changes. The activity of protein kinase C appeared to be important for the changes in pH_i mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pH_i elicited by the adhesive fusion proteins and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pH_i in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pH_i are correlated with the phenomenon of counteradhesion whereas the ability to increase pH_i is associated with cell attachment via at least two different types of cell surface receptors. The data raise the possibility that binding of cytotactin/tenascin may influence primary cellular processes such as migration and proliferation through the differential regulation of pH_i. © 1994 Wiley-Liss, Inc.     

Binding of the J 1 Adhesion Molecules to Extracellular Matrix Constituents

The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1-160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1-220) from developing brain. J1-160 and J1-220 share common epitopes but are considered as separate entities, with J1-220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1-160 and J1-220 bound to collagens type I-VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1-220 bound to all collagen types, whereas J1-160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1-220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10(-9) and 1.8 and 5.5 X 10(-8) M, respectively, under hypotonic buffer conditions and a single KD of 2.1-8.0 X 10(-8) M under isotonic buffer conditions. Binding of J1-160 to collagens had an apparent KD of 1.9-8.0 X 10(-9) M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1-160 to collagen types V and VI were approximately 2 X 10(-8) M. Binding of J1-220 to collagen type I could be inhibited by J1-220, J1-160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1-160 was inhibited by J1-220, J1-160, and collagen type VI (in order of decreasing efficacy of competition). J1-160 and J1-220 were retained on a heparin-agarose column and eluted in a salt gradient at approximately 0.5 M NaCl. The formation of the J1-heparin complexes was inhibited 100-fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor.     

Human cellular fibronectin: comparison of the carboxyl-terminal portion with rat identifies primary structural domains separated by hypervariable regions

We report the isolation and characterization of four overlapping cDNA clones coding for human cellular fibronectin which continuously cover more than 3 kilobases in length. The nucleotide sequence of these cDNAs has been determined, thus elucidating the amino acid sequence of the C-terminal 794 residues of human fibronectin, which cover the edge of cellular-, heparin-, and fibrin-binding domains of this protein. Comparisons of the nucleotide sequences and the deduced amino acid sequences with those of rat [Schwarzbauer, J. E., Tamkun, J. W., Lemischka, I. R., & Hynes, R. O. (1983) Cell (Cambridge, Mass.) 35, 421] indicate a high degree of conservation at both nucleotide and amino acid levels. Comparison with previously published data on amino acid sequences of bovine fibronectin made it possible to identify structurally important features of the protein during the evolution of human, calf, and rat. The deduced human amino acid sequences contain five type III and three type I repeats of internal homologies. The interspecies conservation in amino acids is more pronounced in regions containing the internal repeats and within each functional domain. The implications of these interspecies conservation and divergence are discussed.     

Tenascin in the human intervertebral disc: alterations with aging and disc degeneration

Our objective for this study was to determine the presence and distribution of tenascin in the human intervertebral disc. The tenascins are a family of extracellular matrix proteins with repeated structural domains homologous to epidermal growth factor, fibronectin type III and the fibrinogens. Little is known about the presence of this protein in the disc. Ten normal human discs donated from subjects newborn to 15 years old, 10 control discs from adult donors aged 24-41 years, and 11 surgical disc specimens from patients aged 26-76 years were examined for immunolocalization of tenascin. In young discs, tenascin was localized throughout the annulus; in the nucleus, localization was confined to pericellular matrix. In adult control and degenerating disc specimens, tenascin in the annulus was localized primarily in pericellular matrix regions encircling either single cells or clusters of disc cells; in rare instances localization was more diffuse in the intraterritorial matrix. In young, healthy disc, tenascin was abundant throughout the annulus. In contrast, degenerating discs in adults showed a localization restricted to the pericellular, and rarely, more restricted intraterritorial matrix. These observations indicate that changes in the amount and distribution of tenascin may have a role in disc aging and degeneration, possibly by modulating fibronectin-disc-cell interactions, and causing alterations in the shape of disc cells.     

Evolution of the fibronectin gene. Exon structure of cell attachment domain

Genomic DNA coding for human fibronectin was identified from a human genomic library by screening with a cDNA clone that specifies the cell attachment domain in human fibronectin. Two clones which together provided more than 22 kilobase pairs of the fibronectin gene were isolated. The exons in this region correspond to approximately 40% of the coding region in the fibronectin gene. They code for the middle region of the polypeptide which consists of homologous repeating segments of about 90 amino acids called type III homologies. Nucleotide sequence of the portion of the gene corresponding to the cell attachment domain showed that the Arg-Gly-Asp-Ser cell attachment site is encoded within a 165-base pair exon. This exon, together with a 117-base pair exon codes for a homology unit. Analysis of the exon/intron organization in some of the neighboring homology units indicated a similar 2-exon structure. An exception to this pattern is that a single large exon codes for a type III homology unit that, due to alternative mRNA splicing, exists in some but not all fibronectin polypeptides. The introns separating the coding sequences for the type III homology units are located in conserved positions whereas the introns that interrupt the coding sequence within the units are in a variable position generating variations in the size of the homologous exons. This exon/intron organization suggests that the type III homology region of the fibronectin gene has evolved by a series of gene duplications of a primordial gene consisting of two exons. Specification of one of these homology units to the cell attachment domain has occurred within this exon/intron arrangement.     

Fibroblasts that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan

Four adhesive molecules, tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan, accumulate in interstitial spaces near synaptic sites after denervation of rat skeletal muscle (Sanes, J. R., M. Schachner, and J. Covault. 1986. J. Cell Biol. 102:420-431). We have now asked which cells synthesize these molecules, and how this synthesis is regulated. Electron microscopy revealed that mononucleated cells selectively accumulate in perisynaptic interstitial spaces beginning 2 d after denervation. These cells were identified as fibroblasts by ultrastructural and immunohistochemical criteria; [3H]thymidine autoradiography revealed that their accumulation results from local proliferation. Electron microscopic immunohistochemistry demonstrated that N-CAM is associated with the surface of the fibroblasts, while tenascin(J1) is associated with collagen fibers that abut fibroblasts. Using immunofluorescence and immunoprecipitation methods, we found that fibroblasts isolated from perisynaptic regions of denervated muscle synthesize N-CAM, tenascin(J1), fibronectin, and a heparan sulfate proteoglycan in vitro. Thus, fibroblasts that selectively proliferate in interstitial spaces near synaptic sites are likely to be the cellular source of the interstitial deposits of adhesive molecules in denervated muscle. To elucidate factors that might regulate the accumulation of these molecules in vivo, we analyzed the expression of tenascin(J1) and fibronectin by cultured fibroblasts. Fibroblasts from synapse-free regions of denervated muscle, as well as skin, lung, and 3T3 fibroblasts accumulate high levels of tenascin(J1) and fibronectin in culture, showing that perisynaptic fibroblasts are not unique in this regard. However, when they are first placed in culture, fibroblasts from denervated muscle bear more tenascin(J1) than fibroblasts from innervated muscle, indicating that expression of this molecule by fibroblasts is regulated by the muscle's state of innervation; this difference is no longer apparent after a few days in culture. In 3T3 cells, accumulation of tenascin(J1) is high in proliferating cultures, depressed in confluent cultures, and reactivated in cells stimulated to proliferate by replating at low density or by wounding a confluent monolayer. Thus, synthesis of tenascin(J1) is regulated in parallel with mitotic activity. In contrast, levels of fibronectin, which increase less dramatically after denervation in vivo, are similar in fibroblasts from innervated and denervated muscle and in proliferating and quiescent 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)