Schwann cell type V collagen inhibits axonal outgrowth and promotes Schwann cell migration via distinct adhesive activities of the collagen and noncollagen domains.

Previously, we reported the cloning of alpha4 type V collagen, a novel member of the collagen type V gene family that is expressed by Schwann cells in developing peripheral nerves (Chernousov et al., 2000). The present study was performed to investigate the effects of this collagen on the adhesion and migration of premyelinating Schwann cells and neurite outgrowth from embryonic dorsal root ganglion neurons. Purified alpha4(V)-containing collagen isolated from Schwann cell conditioned medium (collagen type V(SC)) promoted migration of Schwann cells but inhibited outgrowth of axons from rat embryo dorsal root ganglia. Collagen type V(SC) blocked axonal outgrowth in the presence of otherwise active substrates such as collagen type IV, indicative of active inhibition. The noncollagen N-terminal domain of alpha4(V) promoted Schwann cell adhesion, spreading, and migration. These processes were inhibited by soluble heparin but not by function-blocking antibodies against alpha1- and alpha2-integrins. The collagen domain of pepsin-digested collagen type V was poorly adhesive for Schwann cells. The type V collagen domain but not the alpha4(V) N-terminal domain blocked neurite outgrowth from dorsal root ganglion neurons. In cocultures of dorsal root ganglion neurons and Schwann cells, collagen type V(SC) promoted axon fasciculation and association of axons with Schwann cells. These results suggest that in embryonic peripheral nerves, collagen type V(SC) plays a dual role in regulating cell migration. This represents a heretofore unrecognized function of peripheral nerve collagen fibrils in regulating patterns of peripheral nerve growth during development.

Schwann cells synthesize type V collagen that contains a novel alpha 4 chain. Molecular cloning, biochemical characterization, and high affinity heparin binding of alpha 4(V) collagen.

Previously, we reported the isolation of a heparan sulfate-binding collagenous protein, p200, that is expressed by Schwann cells in developing peripheral nerves ((1996) J. Biol. Chem. 271, 13844-13853; (1999) J. Neurosci. Res. 56, 284-294). Here, we report the cloning of p200 cDNA from a Schwann cell cDNA library. The deduced amino acid sequence identifies p200 as a novel member of the collagen type V gene family. This polypeptide, which we have named alpha4 type V (alpha4(V)) collagen, contains an uninterrupted Gly-X-X collagen domain of 1011 amino acids that shows 82% sequence identity to human alpha3(V) collagen and 71% identity to rat alpha1(V) collagen. alpha4(V) is secreted by Schwann cells as a collagen heterotrimer that also contains alpha1(V) chains. alpha4(V)-containing collagen molecules synthesized by Schwann cells retain their amino-terminal non-collagenous domains. alpha4(V) mRNA was detected by reverse transcriptase-linked polymerase chain reaction amplification in neonatal and adult brain and neonatal peripheral nerve. alpha4(V) mRNA and protein were not detected in most other tissues, including the placenta and heart, which are known to contain alpha3(V). This pattern of alpha4(V) expression contrasted with that of alpha1(V) mRNA and protein, which were ubiquitously expressed. The isolated alpha4(V) chain demonstrated an unusually high affinity for heparin. The restricted expression and unusual properties of alpha4(V)-containing collagen type V molecules suggest a unique and important role for these molecules in peripheral nerve development.

Schwann cell extracellular matrix molecules and their receptors.

The major cellular constituents of the mammalian peripheral nervous system are neurons (axons) and Schwann cells. During peripheral nerve development Schwann cells actively deposit extracellular matrix (ECM), comprised of basal lamina sheets that surround individual axon-Schwann cell units and collagen fibrils. These ECM structures are formed from a diverse set of macromolecules, consisting of glyco-proteins, collagens and proteoglycans. To interact with ECM, Schwann cells express a number of integrin and non-integrin cell surface receptors. The expression of many Schwann cell ECM proteins and their receptors is developmentally regulated and, in some cases, dependent on axonal contact. Schwann cell ECM acts as an organizer of peripheral nerve tissue and strongly influences Schwann cell adhesion, growth and differentiation and regulates axonal growth during development and regeneration.

p200, a collagen secreted by Schwann cells, is expressed in developing nerves and in adult nerves following axotomy.

Previously we reported that cultured rat Schwann cells secrete p200, a collagen-like heparin-binding adhesive glycoprotein with a restricted pattern of expression. Here we report that p200 is secreted as a stable trimer, but only after treatment of Schwann cells with ascorbic acid, and was deposited in the fibrillar extracellular matrix. Heparin and heparitinase treatment inhibited incorporation of p200 into extracellular matrix, suggesting the involvement of Schwann cell heparan sulfate proteoglycans in this process. Pepsin digestion revealed that p200 secreted by ascorbate-treated cells contains a collagenous domain of approximately 140 kDa. Immunofluorescent staining of rat embryos at different ages showed that p200 first appeared between embryonic days 15 and 18, and was confined to peripheral nerves. Staining of adult peripheral nerve was negative, but p200 expression was induced in adult sciatic nerve following nerve transection. These data suggest that p200 carries out unique functions during peripheral nerve development and regeneration and that its expression by Schwann cells is regulated by axon-Schwann cell interaction.

Schwann cells use a novel collagen-dependent mechanism for fibronectin fibril assembly.

Cultured rat Schwann cells were stimulated to deposit fibrillar extracellular matrix by treatment with ascorbic acid in the absence of nerve cells. Immunofluoresence staining of the matrix showed that it contains collagens types I and IV, fibronectin and perlecan but not laminin. Collagen type IV, fibronectin and perlecan co-distributed completely in the matrix fibrils, whereas collagen type I was present in only a subset of these fibrils. Time course studies indicated that collagen type I fibrils appear at late stages of matrix formation. Digestion of Schwann cell extracellular matrix with collagenase effectively disrupted most of the matrix including fibronectin fibrils. This was in contrast with fibroblasts, where collagenase treatment removed collagen with no visible effect on fibronectin fibrils. alpha5 integrin was expressed on the cell surface of Schwann cells and partially codistributed with fibronectin-containing fibrils. This suggests that the inability of Schwann cells to deposit fibronectin-containing matrix through a conventional, collagen-independent mechanism was not due to the lack of fibronectin-binding integrins on their cell surface. Polyclonal anti-fibronectin antibodies inhibited the deposition of fibronectin into the matrix fibrils, whereas collagen type IV fibrils were generally unaffected. Growth of Schwann cells on collagen type IV-coated substrate in the absence of ascorbate induced deposition of fine fibronectin fibrils. These results suggest that Schwann cells use an apparently novel, collagen type IV-dependent mechanism for the deposition of fibronectin into their extracellular matrix.

Schwann cells secrete a novel collagen-like adhesive protein that binds N-syndecan.

A heparin-binding glycoprotein was purified from conditioned medium of cultured rat Schwann cells. The protein, p200, which has an apparent molecular mass of approximately 200 kDa, was identified by its ability to bind the cell surface heparan sulfate proteoglycan N-syndecan (syndecan-3) in a membrane overlay assay. Soluble heparin but not chondroitin sulfate inhibited the binding, suggesting the involvement of heparan sulfate chains of proteoglycan in the interaction. Purified p200 promoted the attachment and spreading of Schwann cells. Adhesion to p200 was blocked by heparin, suggesting that heparan sulfate proteoglycans are cell surface receptors for p200. The tissue distribution of p200 was determined by immunoblot analysis with anti-p200 antibodies. Among neonatal rat tissues examined p200 was detected only in sciatic nerve and, at lower levels, in skeletal muscle. p200 expression in sciatic nerve was detectable only during the first 2-3 weeks of postnatal development and was not detected in adult rats. Immunofluorescent staining of rat sciatic nerve showed that p200 was localized in the extracellular matrix surrounding individual Schwann cells-axon units. Two tryptic peptides from p200 were purified and sequenced. These contained multiple GXX collagen-like repeats. Bacterial collagenase digestion of p200 produced a product with an apparent molecular mass of approximately 90 kDa. These data suggest that Schwann cells secrete an apparently novel collagen-like adhesive protein that interacts with cells through cell surface heparan sulfate proteoglycans.

Isolation of a neuronal cell surface receptor of heparin binding growth-associated molecule (HB-GAM). Identification as N-syndecan (syndecan-3).

HB-GAM (heparin binding growth-associated molecule; pleiotrophin) is a secretory, extracellular matrix-associated protein that is strongly expressed in developing nervous tissues and belongs to a novel family of differentiation/growth factors. It promotes axonal growth from perinatal rat brain neurons and is suggested to be mitogenic for some cell types and to display cell-transforming activity. Since the receptors of HB-GAM in cells are unknown, we have started isolation of putative cell surface receptors from brain neurons and from perinatal rat brain. For this purpose, recombinant HB-GAM was produced with the aid of a baculovirus vector and used as an affinity matrix in receptor isolation. A detergent-solubilized component from cultured brain neurons and from brain was identified that binds specifically to HB-GAM and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad smear with an apparent molecular mass of about 200 kDa. This cell surface component was found to contain heparan sulfate chains, which are bound to a core protein with an apparent molecular mass of 120 kDa. Gel electrophoretic characteristics, immunochemical analysis, and partial peptide sequencing revealed that the cell surface component isolated as an HB-GAM receptor is N-syndecan (syndecan-3). In a solid phase binding assay, N-syndecan was found to bind to HB-GAM in a similar manner as to basic fibroblast growth factor (KD = 0.6 nM). Immunofluorescence microscopy indicated that in brain neurons, N-syndecan occurs at the surface of the cell soma and of the neurites that grow along HB-GAM-coated substrates. Anti-N-syndecan antibodies added to culture media had an inhibitory effect on HB-GAM-induced neurite outgrowth. We suggest that N-syndecan mediates the neurite outgrowth-promoting signal from HB-GAM to the cytoskeleton of growing neurites.

N-syndecan (syndecan 3) from neonatal rat brain binds basic fibroblast growth factor.

The cell surface proteoglycan N-syndecan (syndecan 3) was isolated from neonatal rat brain. Purified brain N-syndecan had biochemical properties very similar to N-syndecan previously identified in Schwann cells: it contained mainly, if not exclusively, heparan sulfate glycosaminoglycan chains and had a core protein with an apparent molecular mass of 120 kDa by sodium dodecyl sulfate-gel electrophoresis. We examined the interactions between purified N-syndecan and extracellular ligands using a solid phase binding assay. It was found that among all proteins tested, including a variety growth factors and extracellular matrix molecules, only basic fibroblast growth factor (bFGF) exhibited significant N-syndecan binding. N-syndecan binding to bFGF was saturable and exhibited a KD = 0.5 nM. Soluble bFGF effectively competed with immobilized bFGF for binding to N-syndecan, indicating that these two proteins also interact in solution. Heparin and heparan sulfate, but not chondroitin sulfate, inhibited N-syndecan-bFGF binding. Isolated N-syndecan core protein did not exhibit significant binding to bFGF. Thus, the heparan sulfate chains of N-syndecan, rather than its core protein, appear to be responsible for binding to bFGF. Interestingly, acidic fibroblast growth factor, which is structurally similar to bFGF, did not exhibit significant N-syndecan binding. N-syndecan also did not bind to several other heparin-binding proteins used in this study, indicating a high degree of specificity for the N-syndecan-bFGF interaction. Both N-syndecan and bFGF are abundant in neonatal brain, suggesting that N-syndecan may function as a co-receptor for bFGF during nerve tissue development.

Distinct regions of human fibronectin are essential for fibril assembly in an in vivo developing system.

In early vertebrate development, the proper assembly of fibronectin into fibrils is crucial for embryonic cells to adhere and to migrate on the extracellular matrix. The molecular mechanisms by which such a process occurs in vivo are poorly understood. In the amphibian embryo Pleurodeles waltl fibronectin fibrils appear first at the blastula stage. They form a fibrillar matrix on the basal surface of animal cells facing the blastocoel. Using competition and perturbation experiments with purified proteolytic fragments and domain-specific monoclonal antibodies, we demonstrate that at least three fibronectin sites are essential for assembly of fibronectin fibrils in the blastula of Pleurodeles waltl. Two sites, the RGDS sequence and the synergistic domain in the 10th type III repeat, are both involved in receptor recognition. A third site that spans the 9th type I and 1st type III homology sequences is also likely to participate in fibronectin-fibronectin interactions.

Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix.

Cultured fibroblasts bind soluble protomeric fibronectin and mediate its conversion to insoluble disulfide-bonded multimers. The disulfide-bonded multimers are deposited in fibrillar pericellular matrix. Antifibronectin monoclonal antibodies were analyzed to identify domains of fibronectin required for assembly into matrix. Two antibodies, L8 and 9D2, inhibited binding and insolubilization of 125I-labeled plasma fibronectin by fibroblasts but did not inhibit binding of labeled amino-terminal 70-kDa fragment of fibronectin to matrix assembly sites. Immunoblotting of fibronectin fragments showed that the epitope for 9D2 is in the first type III homology sequence (III-1) whereas the epitope for L8 requires that the last type I sequence of the gelatin binding region (I-9) be contiguous to III-1 and is sensitive to reduction of disulfides in I-9. A 56-kDa gelatin-binding thermolysin fragment of fibronectin that contains III-1 and the L8 and 9D2 epitopes inhibited binding of fibronectin to cell layers 10-fold better than a 40-kDa gelatin-binding fragment that lacks III-1 and the antigenic sites. This 56-kDa fragment, however, did not bind specifically to cell layers. These results indicate that the I-9 and III-1 modules of fibronectin form a functional unit that mediates an interaction, perhaps between protomers, important in the assembly of fibronectin.

Assembly of fibronectin into extracellular matrix.

The results summarized above suggest that assembly of fibronectin is a fundamental biological process and that knowledge of the process of assembly may reveal new ways by which cells interact with extracellular molecules. Deposition of a fibronectin matrix seems to be regulated as tightly as synthesis of fibronectin or expression of adhesion receptors for fibronectin and is influenced profoundly by two products of blood coagulation--TGF-beta released from platelets and factor XIII activated by thrombin. Fibronectin assembly may be important in all sorts of physiological and pathophysiological processes. Cell A--for instance, a stromal cell--can influence the behavior of cell B--for instance, a lymphocyte--by assembling fibronectin made by cell C--for instance, a hepatocyte. We hope that the testable models of assembly presented in this paper will lead to new understanding of the process of assembly and suggest new modalities for treatment of diseases that result in fibrosis, damaged tissues, and neoplastic growth.

Monoclonal antibody to fibronectin which inhibits extracellular matrix assembly.

A monoclonal antibody L8 specific to fibronectin was shown to inhibit fibronectin incorporation into the fibroblast extracellular matrix. Antibody L8 could not interact with fibronectin complexed with gelatin. The results suggest the existence of a specific site on the fibronectin molecule playing a critical role in the assembly of the fibronectin extracellular matrix. This site is located near the collagen-binding domain.

[Immunomorphological study of fibronectin and collagen types I, III, IV and V in the myocardium in cardiosclerosis]. / Immunomorfologicheskoe issledovanie fibronektina i kollagena I, III, IV, V tipov v miokarde pri kardioskleroze.

Fibronectin and collagen, types I, III, IV and V, in the granulation tissue replacing the myocardial infarction, in the focal and diffuse cardiosclerosis were studied by means of the immunofluorescent method. The extracellular matrix in the granulation tissue contained fibronectin and collagen of the above types. Intracellular fibronectin was also found in the necrotized cardiomyocytes. Fibronectin and collagen of type IV were not detected in the postinfarction scars. The extracellular matrix consisted mainly of collagen, types III and V. Fibronectin and collagen, types I, III and V, are observed in the connective tissue in diffuse cardiosclerosis in the presence of the coronary arteries stenosis. No prevalence of the collagen of any type was found.

Studies of extracellular fibronectin matrix formation with fluoresceinated fibronectin and fibronectin fragments.

Fluorescein isothiocyanate conjugated human plasma fibronectin, 70-kDa collagen-binding, 60-kDa central, 60-kDa heparin-binding, 180-kDa heparin, collagen-binding fibronectin fragments and gelatin were used to study extracellular fibronectin matrix formation. Exogenous fibronectin, gelatin, 70-kDa collagen-binding and 180-kDa heparin, collagen-binding fragments were shown to be able to bind specifically to preexisting extracellular matrix of living fibroblasts. The results suggest that: (i) Fibronectin matrix formation may occur through a self-assembly process; (ii) the NH2-terminal part of fibronectin is responsible for fibronectin-fibronectin interaction during fibronectin fibril formation; (iii) plasma fibronectin may be the source for tissue fibronectin.

The role of actin-binding proteins vinculin, filamin, and fibronectin in intracellular and intercellular linkages in cardiac muscle.

The localization in cardiac muscle and the biochemical properties of fibronectin, filamin, and vinculin were studied. Fibronectin was localized between cardiomyocytes. Filamin was identified in the Z-line region of sarcomers and in the intercalated disks of heart muscle. Vinculin was found to be present in intercalated disks and near the plasma membrane at the cell periphery between external myofibrils and sarcolemma. It was suggested that fibronectin, filamin, and vinculin play an important role in intercellular and intracellular linkages in cardiac muscle.

[Study of the structural and functional properties of fibronectin--a high molecular weight plasma glycoproteins--by using monoclonal antibodies]. / Izuchenie strukturno-funktsional'nykh svoistv fibronektina-- vysokomolekuliarnogo glikoproteida iz plazmy--pri pomoshchi monoklonal'nykh antitel.

Functional and structural properties of fibronectin--high molecular weight glyco-protein from human plasma--were studied by monoclonal antibodies against fibronectin. It was shown that monoclonal antibodies against human plasma fibronectin exhibit a certain species specificity. Antigenic determinant for our monoclonal antibody is located in the central part of the protein polypeptide chain--in the structural domain. The monoclonal antibodies studied do not inhibit any tested functions of fibronectin. In contrast, polyclonal antibodies are not species specific and inhibit all fibronectin functions.

Distribution of secondary structure along the fibronectin molecule.

30-kDa, 50-kDa and 70-kDa gelatin-binding, 60-kDa central and 60-65-kDa heparin-binding fragments were produced by trypsin digestion of fibronectin. The secondary structure of the fragments was studied by circular dichroism and quantitative infrared spectroscopy. The structure of the 70-kDa gelatin-binding, 60-kDa central and 60-65-kDa heparin-binding fragments in solution appeared to be very close to that in the intact fibronectin. The content of the antiparallel beta-form, the only element of the secondary structure in all the fragments studied, was shown to be 30-35%.