Involvement of gicerin, a cell adhesion molecule, in development and regeneration of oviduct and metastasis of oviductal adenocarcinomas of the chicken

Gicerin is a novel cell adhesion molecule in the immunoglobulin superfamily and has both homophilic adhesion and heterophilic adhesive activity to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family. We investigated the possible involvement of gicerin in oviductal development, regeneration, and metastasis of oviductal adenocarcinomas of the chicken. In the oviductal epithelium, gicerin was expressed strongly during development, disappeared after maturation, and reappeared during regeneration. NOF was constitutively expressed in the basement membrane of the epithelium. These molecules were expressed strongly in oviductal adenocarcinomas in both primary and metastatic lesions in the mesentery. An anti-gicerin antibody inhibited the attachment of adenocarcinoma cells to the mesentery in vitro. Many cells migrated from adenocarcinoma tissues on NOF, which were inhibited by an anti-gicerin antibody. These results suggest that gicerin might play a role in oviductal development and regeneration and also in the metastasis of adenocarcinomas.     

Gicerin,a cell adhesion molecule,participates in the histogenesis of retina

Gicerin is a novel cell adhesion molecule that belongs to the immunoglobulin superfamily. Gicerin protein adheres to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family, and also exhibits homophilic adhesion. Heterophilic adhesion of gicerin to NOF is thought to play an active role in neurite outgrowth of developing retinal cells in vitro. In this study, we examined the adhesion activity of gicerin during the retinal development of Japanese quail using an antibody directed against gicerin, to elucidate the biological importance of gicerin in retinal histogenesis. Immunohistochemical and Western blot analysis showed that gicerin was highly expressed in the developing retina but suppressed in the mature retina. The aggregation of neural retinal cells from 5-day embryonic quail retina was significantly inhibited when incubated with a polyclonal antibody to gicerin, suggesting that gicerin protein participates in the adhesion of neural retinal cells of the developing retina. Furthermore, histogenesis of retina both in the organ cultures and in ovo embryos was severely disrupted by incubation with a gicerin antibody. These findings provide evidence that gicerin plays an important role in retinal histogenesis. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 769–780, 1997     

Expression and involvement of gicerin, a cell adhesion molecule, in the development of chick optic tectum

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It has both a homophilic binding activity and a heterophilic binding activity to neurite outgrowth factor (NOF) a molecule belonging to the laminin family. We have reported many studies on the heterophilic activity of gicerin and NOF, but the function of its homophilic binding activity in vivo had been unclear. In the retina, gicerin is expressed in retinal ganglion cells only when they extend neurites to the optic tectum. In this report we have found that gicerin is also transiently expressed in the optic tectum during this time. First, cell aggregation assays were used to show that gicerin expressed in the optic tectum displays homophilic binding activity. Then, explant cultures of embryonic day 6 chick optic tectum on gicerin-Fc chimeric protein-coated dishes and NOF-coated dishes were carried out. It was found that gicerin-gicerin homophilic interactions promoted cell migration, whereas heterophilic interactions with NOF induced neurite formation. Furthermore, when anti-gicerin antibodies were injected in order to examine the effect of gicerin protein in the formation of the tectal layer in ovo, cell migration was strongly inhibited. These data suggest that homophilic interaction of gicerin participates in the migration of neural cells during the layer formation and plays a crucial role in the organization of the optic tectum.     

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of chick sciatic nerve

We have examined the role of gicerin, an immunoglobulin superfamily cell adhesion molecule, in chick sciatic nerves during development and regeneration. Gicerin was expressed in the spinal cord, dorsal root ganglion (DRG) and sciatic nerves in embryos, but declined after hatching. Neurite extensions from explant cultures of the DRG were promoted on gicerin's ligands, which were inhibited by an anti-gicerin antibody. Furthermore, gicerin expression was upregulated in the regenerating sciatic nerves, DRG and dorsal horn of the spinal cord after injury to the sciatic nerve. These results indicate that gicerin might participate in the development and regeneration of sciatic nerves.     

Ethanol causes the redistribution of L1 cell adhesion molecule in lipid rafts

Fetal alcohol spectrum disorder is estimated to affect 1% of live births. The similarities between children with fetal alcohol syndrome and those with mutations in the gene encoding L1 cell adhesion molecule (L1) implicates L1 as a target of ethanol developmental neurotoxicity. Ethanol specifically inhibits the neurite outgrowth promoting function of L1 at pharmacologic concentrations. Emerging evidence shows that localized disruption of the lipid rafts reduces L1-mediated neurite outgrowth. We hypothesize that ethanol impairment of the association of L1 with lipid rafts is a mechanism underlying ethanol's inhibition of L1-mediated neurite outgrowth. In this study, we examine the effects of ethanol on the association of L1 and lipid rafts. We show that, in vitro, L1 but not N-cadherin shifts into lipid rafts following treatment with 25 mM ethanol. The ethanol concentrations causing this effect are similar to those inhibiting L1-mediated neurite outgrowth. Increasing chain length of the alcohol demonstrates the same cutoff as that previously shown for inhibition of L1-L1 binding. In addition, in cerebellar granule neurons in which lipid rafts are disrupted with methyl-beta-cyclodextrin, the rate of L1-mediated neurite outgrowth on L1-Fc is reduced to background rate and that this background rate is not ethanol sensitive. These data indicate that ethanol may inhibit L1-mediated neurite outgrowth by retarding L1 trafficking through a lipid raft compartment.     

Proteolytic cleavage of the neural cell adhesion molecule by ADAM17/TACE is involved in neurite outgrowth

The transmembrane and multidomain neural cell adhesion molecule (NCAM) plays important functional roles in the developing and adult nervous system. NCAM is proteolytically processed and appears in soluble forms in the cerebrospinal fluid and in serum under normal and pathological conditions. In this report, we present evidence that the metalloprotease a disintegrin and a metalloprotease (ADAM)17/tumour necrosis factor alpha converting enzyme (TACE) cleaves the polysialylated as well as the non-polysialylated transmembrane isoforms of NCAM, whereas the glycophosphatidylinositol-linked isoform of NCAM is not proteolytically cleaved. A truncated, enzymatically inactive mutant of TACE did not result in release of the NCAM110 cleavage product. Proteolytic cleavage was enhanced by a calmodulin-specific inhibitor and the actin-destabilizing agents cytochalasin D and latrunculin B. In contrast, the microtubule-stabilizing agent colchicine or microtubule-destabilizing agent paclitaxel did not affect the release of the 110-kDa fragment of NCAM. Neurite outgrowth from cerebellar microexplants was inhibited in the presence of the metalloprotease inhibitor GM 6001 on substrate-coated NCAM, but not on poly-l-lysine. Upon transfection of hippocampal neurones with an enzymatically inactive mutant of TACE, NCAM-stimulated neurite outgrowth was inhibited without affecting neurite outgrowth on poly-l-lysine, showing that proteolytic processing of NCAM by the metalloprotease TACE is involved in NCAM-mediated neurite outgrowth.     

Sensitivity of neurite outgrowth to microfilament disruption varies with adhesion molecule substrate

Interactions between the cytoskeleton and cell adhesion molecules are presumed responsible for neurite extension. We have examined the role of microfilaments in neurite outgrowth on the cell adhesion molecules L1, P84, N-CAM, and on laminin. Cerebellar neurons growing on each substrate exhibited differing growth cone morphologies and rates of neurite extension. Growth of neurites in the presence of cytochalasin B (CB) was not inhibited on substrates of L1 or P84 but was markedly inhibited on N-CAM. Neurons on laminin were initially unable to extend neurites in the presence of CB but recovered this ability within 9 h. These studies suggest that neurite outgrowth mediated by different cell adhesion molecules proceeds via involvement of distinct cytoskeletal interactions. © 1993 John Wiley & Sons, Inc.     

The zebrafish down syndrome cell adhesion molecule is involved in cell movement during embryogenesis

The Down syndrome cell adhesion molecule (Dscam) is a protein overexpressed in the brains of Down syndrome patients and implicated in mental retardation. Dscam is involved in axon guidance and branching in Drosophila, but cellular roles in vertebrates have yet to be elucidated. To understand its role in vertebrate development, we cloned the zebrafish homolog of Dscam and showed that it shares high amino acid identity and structure with the mammalian homologs. Zebrafish dscam is highly expressed in developing neurons, similar to what has been described in Drosophila and mouse. When dscam expression is diminished by morpholino injection, embryos display few neurons and their axons do not enter stereotyped pathways. Zebrafish dscam is also present at early embryonic stages including blastulation and gastrulation. Its loss results in early morphogenetic defects. dscam knockdown results in impaired cell movement during epiboly as well as in subsequent stages. We propose that migrating cells utilize dscam to remodel the developing embryo.     

A direct interaction of axonin-1 with NgCAM-related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.     

Crystallization and preliminary crystallographic analysis of the N-terminal two domain fragment of vascular cell adhesion molecule-1 (VCAM-1)

A molecular fragment comprising the first two domains of the human vascular cell adhesion molecule-l (VCAM-l) has been crystallized by the vapor diffusion method. Two crystal forms have been examined by X-ray analysis: One crystal form belongs to the space group C2 with two molecules in the asymmetric unit and cell parameters: a = 122.1 Å, b = 48.9 Å, c = 73.4 Å, and β = 117.4°. The other crystal form belongs to the space group P2₁ with one molecule in the asymmetric unit and cell parameters: a = 40.4 Å, b = 45.7 Å, c = 54.7 Å, and β = 100.5°. Diffraction data up to 1.9 Å resolution have been collected for the C2 crystal form. © 1994 Wiley-Liss, Inc.     

Polysialic acid as an antigen for monoclonal antibody HIgM12 to treat multiple sclerosis and other neurodegenerative disorders

CNS regeneration is a desirable goal for diseases of brain and spinal cord. Current therapeutic strategies for the treatment of multiple sclerosis (MS) aim to eliminate detrimental effects of the immune system, so far without reversing disability or affecting long‐term prognosis in patients. Approachable molecular targets that stimulate CNS repair are not part of the clinical praxis or have not been identified yet. The purpose of this study was to identify the molecular target of the human monoclonal antibody HIgM12. HIgM12 reverses motor deficits in chronically demyelinated mice, a model of MS. Here, we identified polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) as the antigen for HIgM12 by using different NCAM knockout strains and through PSA removal from the NCAM protein core. Antibody binding to CNS tissue and primary cells, antibody‐mediated cell adhesion, and neurite outgrowth on HIgM12‐coated nitrocellulose was detected only in the presence of PSA as assessed by western blotting, immunoprecipitation, immunocytochemistry, and histochemistry. We conclude that HIgM12 mediates it's in vivo and in vitro effects through binding to PSA and has the potential to be an effective therapy for MS and neurodegenerative diseases.

     

Neurotractin, a novel neurite outgrowth-promoting Ig-like protein that interacts with CEPU-1 and LAMP.

The formation of axon tracts in nervous system histogenesis is the result of selective axon fasciculation and specific growth cone guidance in embryonic development. One group of proteins implicated in neurite outgrowth, fasciculation, and guidance is the neural members of the Ig superfamily (IgSF). In an attempt to identify and characterize new proteins of this superfamily in the developing nervous system, we used a PCR-based strategy with degenerated primers that represent conserved sequences around the characteristic cysteine residues of Ig-like domains. Using this approach, we identified a novel neural IgSF member, termed neurotractin. This GPI-linked cell surface glycoprotein is composed of three Ig-like domains and belongs to the IgLON subgroup of neural IgSF members. It is expressed in two isoforms with apparent molecular masses of 50 and 37 kD, termed L-form and S-form, respectively. Monoclonal antibodies were used to analyze its biochemical features and histological distribution. Neurotractin is restricted to subsets of developing commissural and longitudinal axon tracts in the chick central nervous system. Recombinant neurotractin promotes neurite outgrowth of telencephalic neurons and interacts with the IgSF members CEPU-1 (KD = 3 x 10(-8) M) and LAMP. Our data suggest that neurotractin participates in the regulation of neurite outgrowth in the developing brain.     

Crystal structure of the Ig1 domain of the neural cell adhesion molecule NCAM2 displays domain swapping

The crystal structure of the first immunoglobulin (Ig1) domain of neural cell adhesion molecule 2 (NCAM2/OCAM/RNCAM) is presented at a resolution of 2.7 Å. NCAM2 is a member of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). In the structure, two Ig domains interact by domain swapping, as the two N-terminal β-strands are interchanged. β-Strand swapping at the terminal domain is the accepted mechanism of homophilic interactions amongst the cadherins, another class of CAMs, but it has not been observed within the IgCAM superfamily. Gel-filtration chromatography demonstrated the ability of NCAM2 Ig1 to form dimers in solution. Taken together, these observations suggest that β-strand swapping could have a role in the molecular mechanism of homophilic binding for NCAM2.     

Gicerin,an Ig-superfamily cell adhesion molecule,promotes the invasive and metastatic activities of a mouse fibroblast cell line

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and plays an important role during development through its adhesive properties. Gicerin has two isoforms that differ in their cytoplasmic domains; s-gicerin is the shorter and l-gicerin the longer form of the protein. Gicerin is over-expressed in some sporadic tumors as well as in developing tissues. To provide direct evidence that gicerin has the potential to participate in malignant aspects of tumor cell behavior, a gicerin cDNA was introduced into L-929 cells, an endogenous gicerin-negative mouse fibroblast and subsequently analyzed for changes in their invasive and metastatic potential by implantation into nude mice and chick embryos. Compared with parental cells, both gicerin isoform transfectants showed an enhanced cell growth and invaded deeply into surrounding tissues from implanted sites in both animal models. Furthermore, l-gicerin transfectants markedly enhanced metastasis to the lung. These findings suggest that gicerin promotes the tumor growth and invasion, and the isoform bearing the longer cytoplasmic domain may play a role in metastasis.     

A peptide from the first fibronectin domain of NCAM acts as an inverse agonist and stimulates FGF receptor activation, neurite outgrowth and survival

Neural cell adhesion molecule (NCAM) contributes to axon growth and guidance during development and learning and memory in adulthood. Although the Ig domains mediate homophilic binding, outgrowth activity localizes to two membrane proximal fibronectin-like domains. The first of these contains a site identified as a potential FGF receptor (FGFR) activation motif (FRM) important for NCAM stimulation of neurite outgrowth, but its activity has hitherto remained hypothetical. Here, we have tested the effects of a domain-specific antibody and peptides corresponding to the FRM in cellular assays in vitro. The first fibronectin domain antibody inhibited NCAM-stimulated outgrowth, indicating the importance of the domain for NCAM function. Monomeric FRM peptide behaved as an inverse agonist; low concentrations specifically inhibited neurite outgrowth stimulated by NCAM and cellular responses to FGF2, while saturating concentrations stimulated FGFR-dependent neurite outgrowth equivalent to NCAM itself. Dendrimeric FRM peptide was 125-fold more active and stimulated FGFR activation, FGFR-dependent and FGF-mimetic neurite outgrowth and cell survival (but not proliferation). We conclude that the FRM peptide contains NCAM-mimetic bioactivity accounted for by stimulation of FGF signalling pathways at the level of or upstream from FGF receptors, and discuss the possibility that FRM comprises part of an FGFR activation site on NCAM.     

Neural cell adhesion molecule-180-mediated homophilic binding induces epidermal growth factor receptor (EGFR) down-regulation and uncouples the inhibitory function of EGFR in neurite outgrowth

The neural cell adhesion molecule (NCAM) plays important roles in neuronal development, regeneration, and synaptic plasticity. NCAM homophilic binding mediates cell adhesion and induces intracellular signals, in which the fibroblast growth factor receptor plays a prominent role. Recent studies on axon guidance in Drosophila suggest that NCAM also regulates the epidermal growth factor receptor (EGFR) (Molecular and Cellular Neuroscience, 28 , 2005, 141). A possible interaction between NCAM and EGFR in mammalian cells has not been investigated. The present study demonstrates for the first time a functional interaction between NCAM and EGFR in mammalian cells and investigates the molecular mechanisms underlying this interaction. First, NCAM and EGFR are shown to play opposite roles in neurite outgrowth regulation in cerebellar granular neurons. The data presented indicate that negative regulation of EGFR is one of the mechanisms underlying the neuritogenic effect of NCAM. Second, it is demonstrated that expression of the NCAM-180 isoform induces EGFR down-regulation in transfected cells and promotes EGFR down-regulation induced by EGF stimulation. It is demonstrated that the mechanism underlying this NCAM-180-induced EGFR down-regulation involves increased EGFR ubiquitination and lysosomal EGFR degradation. Furthermore, NCAM-180-mediated EGFR down-regulation requires NCAM homophilic binding and interactions of the cytoplasmic domain of NCAM-180 with intracellular interaction partners, but does not require NCAM-mediated fibroblast growth factor receptor activation.     

3,4-dihydroxy-L-phenylalanine as a cell adhesion molecule in serum-free cell culture

In this article, we examined the feasibility of using 3,4‐dihydroxy‐L ‐phenylalanine (DOPA) as a cell adhesion molecule in serum‐free cultures of anchorage‐dependent mammalian cells. DOPA is a critical, functional element in mussel adhesive proteins and is known to bind strongly to various natural or synthetic materials. DOPA coating on culture plates was confirmed using X‐ray photoelectron spectroscopy and energy‐dispersive spectroscopy. Human dermal fibroblasts (HDFs) were cultured on DOPA‐coated, fibronectin‐coated, or no material‐coated culture plates in serum‐free medium. HDFs cultured on DOPA showed the highest cell adhesion ratio, spreading, and viability but the lowest apoptotic activity. Therefore, DOPA may be a useful cell‐adhesion molecule for serum‐free culture. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1055–1060, 2012     

Alternative Splicing of the Cytoplasmic Domain of Neural Cell Adhesion Molecule Alters Its Ability to Act as a Substrate for Neurite Outgrowth

A full-length cDNA encoding 180-kDa neural cell adhesion molecule (NCAM 180) has been transfected into mouse NIH-3T3 fibroblasts, and stable clones expressing the transgene have been isolated and characterised. Transfection was associated with the expression of a major protein band of 180 kDa and a minor related band of 140 kDa. Antibodies reactive exclusively with human NCAM immunoprecipitated both proteins but failed to coprecipitate any other proteins. The ability of transfected NCAM to stimulate neurite outgrowth was determined by culturing rat cerebellar neurons on top of confluent monolayers of parental 3T3 cells or clones of transfected 3T3 cells expressing either NCAM 140 or NCAM 180. The results show that NCAM 180 is less able to act as a substrate for neurite outgrowth than NCAM 140.     

Metalloprotease‐induced ectodomain shedding of neural cell adhesion molecule (NCAM)

Transmembrane forms of neural cell adhesion molecule (NCAM140, NCAM180¹) are key regulators of neuronal development. The extracellular domain of NCAM can occur as a soluble protein in normal brain, and its levels are elevated in neuropsychiatric disorders, such as schizophrenia; however the mechanism of ectodomain release is obscure. Ectodomain shedding of NCAM140, releasing a fragment of 115 kD, was found to be induced in NCAM‐transfected L‐fibroblasts by the tyrosine phosphatase inhibitor pervanadate, but not phorbol esters. Pervanadate‐induced shedding was mediated by a disintegrin metalloprotease (ADAM), regulated by ERK1/2 MAP kinase. In primary cortical neurons, NCAM was shed at high levels, and the metalloprotease inhibitor GM6001 significantly increased NCAM‐dependent neurite branching and outgrowth. Moreover, NCAM‐dependent neurite outgrowth and branching were inhibited in neurons isolated from a transgenic mouse model of NCAM shedding. These results suggest that regulated metalloprotease‐induced ectodomain shedding of NCAM down‐regulates neurite branching and neurite outgrowth. Thus, increased levels of soluble NCAM in schizophrenic brain have the potential to impair neuronal connectivity. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006