Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule,N-cadherin

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2mM Ca⁺⁺ or 0.2 mM Ca⁺⁺, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca⁺⁺, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca⁺⁺ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca⁺⁺ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca⁺⁺ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca⁺⁺ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca⁺⁺, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca⁺⁺. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca⁺⁺ levels had similar effects as lowering the Ca⁺⁺ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.     

Nerve growth cone migration onto Schwann cells involves the calcium-dependent adhesion molecule, N-cadherin

The role of calcium-dependent adhesion molecules in the migration of nerve growth cones onto the top of Schwann cells was probed by examination of sensory growth cone-Schwann cell interactions in medium containing either 1.0 mM Ca2+ or 0.1 mM Ca2+. In the presence of 1.0 mM Ca2+ growth cones rapidly migrated onto Schwann cells, spread, and remained for extended periods. However, in 0.1 mM Ca2+ growth cones still made frequent contacts with Schwann cells, but migration onto the upper cell surface was much reduced. This contrast in growth cone-Schwann cell interactions could be switched rapidly by changing the Ca2+ concentration of the culture medium. Growth cones of retinal neurons showed similar calcium-dependence in their migration onto Schwann cells. Antibodies to the calcium-dependent adhesion molecule, N-cadherin, also blocked growth cone migration onto Schwann cells, but antibodies to another neuronal adhesion molecule, L1, had no effect on growth cone-Schwann cell interactions. Immunocytochemical staining for N-cadherin and L1 indicated that growth cones and Schwann cells have N-cadherin on their surfaces, while L1 is present only on axons and growth cones. These results provide two kinds of evidence that N-cadherin is important in the initial interactions of growth cones and Schwann cells.     

Regulation of neural cell adhesion molecule expression on cultured mouse Schwann cells by nerve growth factor.

Schwann cells from early postnatal mouse sciatic nerve were obtained as a homogenous population and shown by indirect immunofluorescence to express the neural cell adhesion molecules L1, N-CAM and J1 and their common carbohydrate epitope L2/HNK-1. L1 and N-CAM are synthesized in molecular forms that are slightly different from those expressed by small cerebellar neurons or astrocytes. As in astrocytes, the J1 antigen is expressed by Schwann cells in multiple forms generally ranging from 160 to 230 kd in the reduced state. J1 is secreted by Schwann cells in a 230-kd mol. wt form. Expression of L1 by Schwann cells can be regulated by nerve growth factor (NGF). L1 expression on the cell surface is increased 1.6-fold in the presence of NGF after 3 days of maintenance in vitro and 3-fold after 16 days. NGF does not change expression of N-CAM. The glia-derived neurite-promoting factor (GdNPF) increases L1 expression by a factor of 1.9 and decreases N-CAM expression by a factor of 0.4 after 3 days in vitro. J1 expression on Schwann cell surfaces remains unchanged in the presence of NGF or GdNPF. Antibodies to NGF abolish the influence of NGF on L1 expression. Addition of NGF antibodies to the Schwann cell cultures without exogenously added NGF decreases L1 expression, indicating that Schwann cells secrete NGF that may influence L1 expression by an autocrine mechanism. Our experiments show for the first time that cell adhesion molecule expression on a non-neuronal cell, the Schwann cell, can be directly regulated by the neurotrophic factor NGF. These observations indicate a considerable degree of 'plasticity' of peripheral glia in regulating cell adhesion molecule expression.     

A role for the Ca2(+)-dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis

The formation of multinucleate skeletal muscle cells (myotubes) is a Ca2(+)-dependent process involving the interaction and fusion of mononucleate muscle cells (myoblasts). Specific cell-cell adhesion precedes lipid bilayer union during myoblast fusion and has been shown to involve both Ca2(+)-independent (CI)2 and Ca2(+)-dependent (CD) mechanisms. In this paper we present evidence that CD myoblast adhesion involves a molecule similar or identical to two known CD adhesion glycoproteins, N-cadherin and A-CAM. These molecules were previously identified by other laboratories in brain and cardiac muscle, respectively, and are postulated to be the same molecule. Antibodies to N-cadherin and A-CAM immunoblotted a similar band with a molecular weight of approximately 125,000 in extracts of brain, heart, and pectoral muscle isolated from chick embryos and in extracts of muscle cells grown in vitro at Ca2+ concentrations that either promoted or inhibited myotube formation. In assays designed to measure the interaction of fusion-competent myoblasts in suspension, both polyclonal and monoclonal anti-N-cadherin antibodies inhibited CD myoblast aggregation, suggesting that N-cadherin mediates the CD aspect of myoblast adhesion. Anti-N-cadherin also had a partial inhibitory effect on myotube formation likely due to the effect on myoblast-myoblast adhesion. The results indicate that N-cadherin/A-CAM plays a role in myoblast recognition and adhesion during skeletal myogenesis.     

N-cadherin mediates retinal lamination,maintenance of forebrain compartments and patterning of retinal neurites

The complex, yet highly ordered and predictable, structure of the neural retina is one of the most conserved features of the vertebrate central nervous system. In all vertebrate classes, retinal neurons are organized into laminae with each neuronal class adopting specific morphologies and patterns of connectivity. Using genetic analyses in zebrafish, we demonstrate that N-cadherin (Ncad) has several distinct and crucial functions during the establishment of retinal organization. Although the location of cell division is disorganized in embryos with reduced or no Ncad function, different classes of retinal neurons are generated. However, these neurons fail to organize into correct laminae, most probably owing to compromised adhesion between retinal cells. In addition, amacrine cells exhibit exuberant and misdirected outgrowth of neurites that contributes to severe disorganization of the inner plexiform layer. Retinal ganglion cells also exhibit defects in process outgrowth, with axons exhibiting fasciculation defects and adopting incorrect ipsilateral trajectories. At least some of these defects are likely to be due to a failure to maintain compartment boundaries between eye, optic nerve and brain. Although in vitro studies have implicated Fgf receptors in modulating the axon outgrowth promoting properties of Ncad, most aspects of the Ncad mutant phenotype are not phenocopied by treatments that block Fgf receptor function.     

Preferential outgrowth of central nervous system neurites on astrocytes and Schwann cells as compared with nonglial cells in vitro

I have compared central nervous system (CNS) neurite outgrowth on glial and nonglial cells. Monolayers of glial cells (astrocytes and Schwann cells) or nonglial cells (e.g., fibroblasts) were prepared and were shown to be greater than 95% pure as judged by cell type-specific markers. These monolayers were then tested for their ability to support neurite outgrowth from various CNS explants. While CNS neurites grew vigorously on the glial cells, most showed little growth on nonglial cell monolayers. Neurites grew singly or in fine fascicles on the glial cells at rates greater than 0.5 mm/d. The neurite outgrowth on astrocytes was investigated in detail. Scanning and transmission electron microscopy showed that the neurites were closely apposed to the astrocyte surface and that the growth cones were well spread with long filopodia. There was no evidence of significant numbers of explant- derived cells migrating onto the monolayers. Two types of experiments indicated that factors associated with the astrocyte surface were primarily responsible for the vigorous neurite outgrowth seen on these cells: (a) Conditioned media from either astrocytes or fibroblasts had no effect on the pattern of outgrowth on fibroblasts and astrocytes, and conditioned media factors from either cell type did not promote neurite outgrowth when bound to polylysine-coated dishes. (b) When growing CNS neurites encountered a boundary between astrocytes and fibroblasts, they stayed on the astrocytes and did not encroach onto the fibroblasts. These experiments strongly suggest that molecules specific to the surfaces of astrocytes make these cells particularly attractive substrates for CNS neurite outgrowth, and they raise the possibility that similar molecules on embryonic glial cells may play a role in guiding axonal growth during normal CNS development.     

Identification of mammalian and invertebrate analogues of the avian calcium-dependent cell adhesion protein N-cadherin with synthetic-peptide directed antibodies against a conserved cytoplasmic domain

N-cadherin, a 130kD transmembrane adhesive glycoprotein, is a mediator of specific cellular interactions during development. Analysis of N-cadherin at the protein level, to date, has been largely dependent upon monoclonal antibody NCD-2 which recognizes only avian N-cadherin. We produced a monospecific polyclonal antiserum, C-NCAD(838-856), to a synthetic peptide corresponding to a portion of the highly conserved c-terminal cytoplasmic domain of chick N-cadherin. Using polyacrylamide gel electrophoresis and immunoblotting to map tissue distribution we show that the antiserum detects chick N-cadherin with a similar tissue distribution as NCD-2. Unlike NCD-2, however, anti-C-NCAD(838-856) recognizes N-cadherin analogues in a wide variety of species, including mouse, human, fish and drosophila. The results of comparative immunoblot studies demonstrate similar tissue-specific patterns and apparent molecular weight variation in the chick, mouse and human. This indicates that N-cadherin structure and expression, and most likely function as well, have been highly conserved in evolution. The antiserum recognizes an epitope unique to N-cadherin which is conserved among N-cadherins from a variety of species but is absent from other members of the cadherin gene family, as no immunoreactivity was detected with tissues bearing these other cadherins. The antiserum is thus a useful tool for the phylogenetic and biochemical investigation of N-cadherin from a variety of tissue sources.     

3T3 cell surface galactosyltransferase is a calcium-dependent adhesion molecule for collagen type IV.

Cell surface galactosyltransferase (GalTase) has been previously shown to mediate cell spreading or migration on laminin matrices. This work demonstrates that 3T3 cell surface GalTase also mediates cell attachment to collagen type IV. Attachment to collagen type IV was blocked by perturbations of GalTase or substrate pregalactosylation on cells possessing only calcium-dependent mechanisms of adhesion. Cells with both calcium-dependent and calcium-independent systems were not affected by GalTase perturbation. Collagen type IV was shown to possess GalTase substrates since matrices could be galactosylated by both soluble enzyme and 3T3 cells.     

Effects of N-cadherin overexpression on the adhesion properties of embryonic stem cells

Constitutive overexpression of N-cadherin in mouse embryonic stem cells led to marked changes in the phenotype and adhesion properties of these cells. The changes included the formation of smaller embryonic bodies, elevated mRNA and total protein levels of N-cadherin, and increased amounts of p120 catenin and connexin-43. N-cadherin cells exhibited decreased attachment to non-cell surfaces, while their adhesiveness to each other and to rat neonatal cardiomyocytes was significantly elevated. The findings suggest that N-cadherin overexpression can facilitate electromechanical integration of stem cells into excitable tissues with endogenously high levels of N-cadherin, such as the heart and brain.Key words: stem cells, cardiomyocytes, N-cadherin, connexin 43, gap junctions     

Effects of N-cadherin overexpression on the adhesion properties of embryonic stem cells

Constitutive overexpression of N-cadherin in mouse embryonic stem cells led to marked changes in the phenotype and adhesion properties of these cells. The changes included the formation of smaller embryonic bodies, elevated mRNA and total protein levels of N-cadherin, and increased amounts of p120 catenin and connexin-43. N-cadherin cells exhibited decreased attachment to non-cell surfaces, while their adhesiveness to each other and to rat neonatal cardiomyocytes was significantly elevated. The findings suggest that N-cadherin overexpression can facilitate electromechanical integration of stem cells into excitable tissues with endogenously high levels of N-cadherin, such as the heart and brain.     

Glycosyl phosphatidylinositol--anchored T-cadherin mediates calcium-dependent, homophilic cell adhesion.

Cadherins are a family of cell adhesion molecules that exhibit calcium-dependent, homophilic binding. Their function depends on both an HisAlaVal sequence in the first extracellular domain, EC1, and the interaction of a conserved cytoplasmic region with intracellular proteins. T-cadherin is an unusual member of the cadherin family that lacks the HisAlaVal motif and is anchored to the membrane through a glycosyl phosphatidylinositol moiety (Ranscht, B., and M. T. Dours-Zimmermann. 1991. Neuron. 7:391-402). To assay the function of T-cadherin in cell adhesion, we have transfected T-cadherin cDNA into CHO cells. Two proteins, mature T-cadherin and the uncleaved T-cadherin precursor, were produced from T-cadherin cDNA. The T-cadherin proteins differed from classical cadherins in several aspects. First, the uncleaved T-cadherin precursor was expressed, together with mature T-cadherin, on the surface of the transfected cells. Second, in the absence of calcium, T-cadherin was more resistant to proteolytic cleavage than other cadherins. Lastly, in contrast to classical cadherins, T-cadherin was not concentrated into cell-cell contacts between transfected cells in monolayer cultures. In cellular aggregation assays, T-cadherin induced calcium-dependent, homophilic adhesion which was abolished by treatment of T-cadherin-transfected cells with phosphatidylinositol-specific phospholipase C. These results demonstrate that T-cadherin is a functional cadherin that differs in several properties from classical cadherins. The function of T-cadherin in homophilic cell recognition implies that the mechanism of T-cadherin-induced adhesion is distinct from that of classical cadherins.     

Antibodies to the L1 adhesion molecule inhibit Schwann cell ensheathment of neurons in vitro

To investigate whether neural adhesion molecules are involved in neuron- induced Schwann cell differentiation, cocultures of pure dorsal root ganglion neurons, and Schwann cells were maintained in the presence of antibodies to evaluate possible perturbing effects. Several parameters characteristic of differentiating Schwann cells were studied, such as transition of spindle-shaped to flattened, i.e., more epithelioid morphology, association with neuronal cell bodies, ensheathment of neurites, production of basal lamina and collagen fibrils, and expression of the myelin associated glycoprotein (MAG). A complete ablation of Schwann cell differentiation in all features studied was seen with antibodies to the neural adhesion molecule L1. Antibodies to N-CAM did not reduce the association of Schwann cells with neurites but abolished the interdigitation of Schwann cell processes into neurite bundles, while leaving the other parameters studied unaffected. Fab fragments of antibodies to J1, MAG, and mouse liver membranes did not interfere with the manifestation of any of these parameters. None of the antibodies changed incorporation of [3H]thymidine into Schwann cells.     

Dynamic adhesion and separation of cells in vitro. II. Interactions of cells with hydrophilic and hydrophobic surfaces

Experiments made on the passage of cells through untreated and siliconized glass beads, and on the adhesion and spread of cultured cells on glass and Teflon surfaces show that, in the absence of serum and in its presence in low concentrations, cell adhesion and spread is sensitive to substratum wettability. On the other hand, in the presence of 100% serum, no differences in adhesive parameters are detectable. It is concluded that arguments correlating cell adhesion to surface wettability, and, by inference, surface free energy, are unsubstantiated in 100% serum, which may well approximate to the in vivo situation. The results also show no correlation between parameters of cell adhesion and cell separation, and thereby support the hypothesis that these are different processes.     

Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase

The calcium-dependent tyrosine kinase (CADTK), also known as Pyk2/RAFTK/CAKbeta/FAK2, is a cytoskeleton-associated tyrosine kinase. We compared CADTK regulation with that of the highly homologous focal adhesion tyrosine kinase (FAK). First, we generated site-specific CADTK mutants. Mutation of Tyr402 eliminated autophosphorylation and significantly decreased kinase activity. Mutation of Tyr881, a putative Src kinase phosphorylation site predicted to bind Grb2, had little effect on CADTK regulation. Src family tyrosine kinases resulted in CADTK tyrosine phosphorylation even when co-expressed with the Tyr402/Tyr881 double mutant, suggesting that Src/Fyn etc. phosphorylate additional tyrosine residues. Interestingly, CADTK tyrosine-phosphorylated FAK when both were transiently expressed, but FAK did not phosphorylate CADTK. Biochemical experiments confirmed direct CADTK phosphorylation of FAK. This phosphorylation utilized tyrosine residues other than Tyr397, Tyr925, or Tyr576/Tyr577, suggesting that new SH2-binding sites might be created by CADTK-dependent FAK phosphorylation. Last, expression of the CADTK carboxyl terminus (CRNK) abolished CADTK but not FAK autophosphorylation. In contrast, FAK carboxyl terminus overexpression inhibited both FAK and CADTK autophosphorylation, suggesting that a FAK-dependent cytoskeletal function may be necessary for CADTK activation. Thus, CADTK and FAK, which both bind to some, but not necessarily the same, cytoskeletal elements, may be involved in coordinate regulation of cytoskeletal structure and signaling.     

N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling

Cell migration is a process which is essential during embryonic development, throughout adult life and in some pathological conditions. Cadherins, and more specifically the neural cell adhesion molecule N-cadherin, play an important role in migration. In embryogenesis, N-cadherin is the key molecule during gastrulation and neural crest development. N-cadherin mediated contacts activate several pathways like Rho GTPases and function in tyrosine kinase signalling (for example via the fibroblast growth factor receptor). In cancer, cadherins control the balance between suppression and promotion of invasion. E-cadherin functions as an invasion suppressor and is downregulated in most carcinomas, while N-cadherin, as an invasion promoter, is frequently upregulated. Expression of N-cadherin in epithelial cells induces changes in morphology to a fibroblastic phenotype, rendering the cells more motile and invasive. However in some cancers, like osteosarcoma, N-cadherin may behave as a tumour suppressor. N-cadherin can have multiple functions: promoting adhesion or induction of migration dependent on the cellular context.     

The fibroblast growth factor receptor acid box is essential for interactions with N-cadherin and all of the major isoforms of neural cell adhesion molecule

Interactions between the neural cell adhesion molecules NCAM and N-cadherin with the fibroblast growth factor receptor (FGFR) are important for a number of developmental events and have also been implicated in tumor progression. The factors regulating these interactions are not known. We have used co-immunoprecipitation and co-clustering paradigms to show that both adhesion molecules can interact with the 3Ig IIIC isoform of the FGFR1 in a number of cell types. Interestingly, whereas the interaction can be seen over most of the cell surface, it is not seen at points of cell-cell contact where the adhesion molecules accumulate at stable junctions. We also demonstrate for the first time that all of the major isoforms of NCAM can interact with the FGFR. Using deletion mutagenesis we have found that the adhesion molecule/FGFR interaction can withstand the removal of most of any one of the FGFR immunoglobulin-like domains (D1-D3). In contrast, the FGFR interaction with N-cadherin and NCAM (but not FGF) is absolutely dependant on the presence of the acid box motif that can be found in the linker region between D1 and D2. As this motif can be spliced out of all four FGFRs, it suggests that this is one mechanism that can regulate the interaction of the receptor with different ligand classes.     

Interactions of bilirubin and other ligands with ligandin.

Circular dichroism methods were used to study the structure of rat ligandin and the binding of organic anions to the protein. Ligandin has a highly ordered secondary structure with about 40%alpha helix, 15% beta structure, and 45% random coil. Bilirubin binding occurred primarily at a single high affinity site on the protein. The binding constant for bilirubin (5 X 10-7 Mminus 1) was the highest among the ligands studied. The bilirubin-ligandin complex exhibited a well-defined circular dichroic spectrum with two major overlapping ellipticity bands of opposite sign in the bilirubin absorption region. This spectrum was virtually a mirror image of that of human or rat serum albumin-bilirubin complexes. Studies on the direct transfer of bilirubin from ligandin to rat serum albumin showed that sasociation constants of bilirubin-ligandin complexes were approximately tenfold less than those of the bilirubin-albumin system. Ligandin exhibited a broad specificity with respect to the typeof ligand bond. A series of organic anions inclucing dyes used clinically for liver function tests, fatty acids, hormones, heme derivatives, bile acids, and other ligands that were considered likely to interact with ligandin, were examined. Most induced ellipticity changes consistent with competitive displacement of bilirubin from ligandin and relative affinities of these compounds for ligandin were determined based on their effectiveness in desplacing the bilirubin. Some substances such as glutathione, conjugated sulfobromophthaleins and lithocholic acid bound to ligandin but induced anomalous spectral shifts, when added to ligandin-bilirubin complexes. Other compounds, including some that act as substrates for the glutathione transferase activity exhibited by ligandin, revealed no apparent competitive effects with respect to the bilitubin binding site.     

Gicerin,a cell adhesion molecule,promotes the metastasis of lymphoma cells of the chicken

Gicerin is an immunoglobulin superfamily cell adhesion molecule purified from chicken gizzards. This molecule displays an adhesive interaction with a laminin-like protein as well as with gicerin itself. Gicerin appears in embryonic tissues and plays a role in chick development through its cell adhesive properties. An increase in gicerin expression is found in some sporadic tumors of the chicken. To elucidate the possible role of gicerin in tumor progression in chickens, we introduced gicerin cDNA into an endogenous gicerin negative lymphoma MDCC-MSB1 cell line, and subsequently analyzed them for changes in their metastatic potentials. After intravenous implantation of the gicerin transfectants into chickens, the metastatic potential to the lung, liver and kidney was enhanced compared with parental MDCC-MSB1 cells. Self-aggregation activity was increased in gicerin transfectants. In addition, adhesive and migratory activities of the gicerin transfectants to the gicerin ligands were enhanced in vitro. These findings indicate that gicerin can contribute to the malignancy and metastatic properties of lymphoma.This work was supported in part by a Grant-in-Aid for Scientific Research (No. 13760210), and a grant for Scientific Research on Priority Areas "Cancer" (No. 12215133), from the Ministry of Education, Science, Sports and Culture, Japan, grants from the Uehara Memorial Foundation and Senri Life Science and a Grant-in-Aid for Advanced Scientific Research from Osaka Prefecture University