Dynamic expression of the cell adhesion molecule fasciclin I during embryonic development in Drosophila.

A number of different cell surface glycoproteins expressed in the central nervous system (CNS) have been identified in insects and shown to mediate cell adhesion in tissue culture systems. The fasciclin I protein is expressed on a subset of CNS axon pathways in both grasshopper and Drosophila. It consists of four homologous 150-amino acid domains which are unrelated to other sequences in the current databases, and is tethered to the cell surface by a glycosyl-phosphatidylinositol linkage. In this paper we examine in detail the expression of fasciclin I mRNA and protein during Drosophila embryonic development. We find that fasciclin I is expressed in several distinct patterns at different stages of development. In blastoderm embryos it is briefly localized in a graded pattern. During the germ band extended period its expression evolves through two distinct phases. Fasciclin I mRNA and protein are initially localized in a 14-stripe pattern which corresponds to segmentally repeated patches of neuroepithelial cells and neuroblasts. Expression then becomes confined to CNS and peripheral sensory (PNS) neurons. Fasciclin I is expressed on all PNS neurons, and this expression is stably maintained for several hours. In the CNS, fasciclin I is initially expressed on all commissural axons, but then becomes restricted to specific axon bundles. The early commissural expression pattern is not observed in grasshopper embryos, but the later bundle-specific pattern is very similar to that seen in grasshopper. The existence of an initial phase of expression on all commissural bundles helps to explain the loss-of-commissures phenotype of embryos lacking expression of both fasciclin I and of the D-abl tyrosine kinase. Fasciclin I is also expressed in several nonneural tissues in the embryo.     

Leukocyte adhesion molecule expression during intense resistance exercise

We hypothesized that expression of L-selectinand very late antigen-4 (VLA-4) integrin adhesionmolecules would influence cell type-specific redistribution duringexercise. Women subjects performed six sets of 10-repetition maximumsquats. L-selectin and VLA-4 integrin were measured by using flowcytometry pre- and postexercise on peripheral blood neutrophils andlymphocytes (n = 29 subjects) andlymphocyte subsets (n = 70 subjects),respectively. Neutrophil concentration increased 41.8%(P < 0.001), whereas the percentexpressing L-selectin was unchanged (79%). Lymphocyte concentrationincreased 61.8% (P < 0.001). Thepercent of T cells expressing L-selectin decreased from 73.5 ± 8.9 to 68.2 ± 11.4% (P < 0.001); the combined population of natural killer and B cells expressing L-selectin decreased from 80.4 ± 22.5 to 62.7 ± 25.8% (P < 0.001).VLA-4 integrin was expressed by nearly all lymphocytes both pre- andpostexercise. The proportional decrease in L-selectin positive cellscould have resulted from 1) sheddingof L-selectin, 2) selective entry ofL-selectin-negative subsets, or 3)selective removal of L-selectin-positive subsets.

     

Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons

The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system.     

Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development

The rodent neural cell adhesion molecule (N-CAM) consists of three glycoprotein chains of 180, 140, and 120 kD in their adult forms. Although the proportions of the three components are known to change during development and differ between brain regions, their individual distribution and function are unknown. Here we report studies carried out with a monoclonal antibody that specifically recognizes the 180-kD component of mouse N-CAM (N-CAM180) in its highly sialylated embryonic and less glycosylated adult forms. In primary cerebellar cell cultures, N-CAM180 antibody reacts intracellularly with all types of neural cells including astrocytes, oligodendrocytes, and neurons. During cerebellar, telencephalic, and retinal development N-CAM180 is detectable by indirect immunohistology in differentiated neural cells, but, in contrast to total N-CAM, not in their proliferating precursors in the ventricular zone and primordial and early postnatal external granular layer. In monolayer cultures of C1300 neuroblastoma cells, N-CAM180 appears by immunofluorescence more concentrated at contact points between adjacent cells, while N-CAM comprising the 180- and 140-kD component shows a more uniform distribution at the plasma membrane. Treatment of neuroblastoma cells with dimethylsulfoxide, which promotes differentiation, induces a shift toward the predominant expression of N- CAM180. These observations support the notion that N-CAM180 is expressed selectively in more differentiated neural cells and suggest a differential role of N-CAM180 in the stabilization of cell contacts.     

BEN/DM-GRASP/SC1 expression during mouse facial development: differential expression and regulation in molars and incisors

The cell adhesion molecule BEN/DM-GRASP/SC1 is expressed in a variety of tissues during embryogenesis. Here, we studied the expression pattern of BEN/DM-GRASP/SC1 in different organs involved in facial mouse development, especially in the developing teeth. BEN/DM-GRASP/SC1 was expressed in nose, whisker, gland, and tongue epithelia, as well as in myogenic mesenchyme. In molars, BEN/DM-GRASP/SC1 was firstly expressed in the condensed mesenchyme and thereafter expression was confined to mesenchymal cells of the dental follicle. In contrast, in incisors, transient BEN/DM-GRASP/SC1 expression was restricted to epithelium. In tissue recombination experiments, BEN/DM-GRASP/SC1 expression in mesenchyme was activated by molar, but not incisor epithelium.     

Cytokine regulation of cellular adhesion molecule expression in inflammation.

Cellular adhesion molecules (CAMs) play an essential role in tethering circulating leukocytes to the vascular endothelium at sites of inflammation. They are also instrumental in enabling leukocytes to transmigrate from blood vessels into adjacent inflamed tissues. In the absence of signals to stimulate expression of CAMs, the adhesive forces between leukocytes and the vascular endothelium are below the threshold level required to tether leukocytes. Research in the last decade has shown that several cytokines, including tumour necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1beta), potently increase the expression of many CAMs and thus increase the adhesiveness between leukocytes and the endothelium. The CAM-inducing activity of these cytokines is therefore crucial to the regulation of inflammatory processes. Overactivation of CAM expression is linked to a number of acute and chronic inflammatory conditions, and has led to the rationale of antagonising cytokine activity and or CAM expression in order to treat these conditions. The potential application of 'adhesion' antagonists for the therapy of acute chronic inflammatory conditions is briefly discussed.     

Ubiquitination and endocytosis of cell adhesion molecule DM-GRASP regulate its cell surface presence and affect its role for axon navigation

DM-GRASP, cell adhesion molecule of the immunoglobulin superfamily, has been shown to promote growth and navigation of axons. We here demonstrate that clustering of DM-GRASP in the plasma membrane induces its rapid internalization via dynamin- and clathrin-dependent endocytosis, which is controlled by phosphatidylinositol 3-kinase and mitogen-activated protein kinase ERK. The clustering of DM-GRASP activates ERK; the intensity and duration of ERK activation by DM-GRASP do not depend on rapid clathrin-mediated internalization of DM-GRASP. Moreover, the preference of retinal ganglion cell axons for DM-GRASP-coated micro-lanes requires clathrin-mediated endocytosis for the appropriate axonal turning reactions at substrate borders. Because the intracellular domain of DM-GRASP does not contain motifs for direct interactions with the endocytosis machinery, we performed a yeast two-hybrid screen to identify intracellular proteins mediating the uptake of DM-GRASP and isolated ubiquitin. Immunoprecipitation of DM-GRASP coexpressed with ubiquitin revealed that one or two ubiquitin(s) are attached to the intracellular domain of cell surface-resident DM-GRASP. Furthermore, elevated ubiquitination levels result in a decrease of cell surface-resident DM-GRASP as well as in the amount of total DM-GRASP. The endocytosis rate is not affected, but the delivery to multivesicular bodies is increased, indicating that DM-GRASP ubiquitination enhances its sorting into the degradation pathway. Together, our data show that ubiquitination and endocytosis of DM-GRASP in concert regulate its cell surface concentration, which is crucial for its function in axon navigation.     

Molecular cloning and expression of a novel adhesion molecule, SC1

SC1, an integral membrane glycoprotein of 100 kd, is uniquely and transiently expressed on spinal cord motoneurons early in development and appears in peripheral neurons and several other tissues during development. SC1 has been purified by immunoaffinity techniques, and SC1 cDNA clones have been obtained by screening an E4 chick embryo phage expression library with a rabbit polyclonal antibody produced against purified SC1. The deduced protein sequence of 588 amino acids consists of a signal peptide, five immunoglobulin-like domains, a transmembrane region, and a short cytoplasmic tail. The sequence is most similar to MUC18, reported as a tumor progression marker in human melanoma. Transfection of SC1 cDNA into mammalian cells leads to cell surface expression of SC1 antigen and a subsequent increase in cell-cell adhesion. SC1 molecules bind to each other via a homophilic adhesion mechanism, independently of calcium or magnesium ions. SC1 may have a role in lateral motor column formation or neurite growth or fasciculation.     

Grainyhead-like 2 regulates neural tube closure and adhesion molecule expression during neural fold fusion

Defects in closure of embryonic tissues such as the neural tube, body wall, face and eye lead to severe birth defects. Cell adhesion is hypothesized to contribute to closure of the neural tube and body wall; however, potential molecular regulators of this process have not been identified. Here we identify an ENU-induced mutation in mice that reveals a molecular pathway of embryonic closure. Line2F homozygous mutant embryos fail to close the neural tube, body wall, face, and optic fissure, and they also display defects in lung and heart development. Using a new technology of genomic sequence capture and high-throughput sequencing of a 2.5 Mb region of the mouse genome, we discovered a mutation in the grainyhead-like 2 gene (Grhl2). Microarray analysis revealed Grhl2 affects the expression of a battery of genes involved in cell adhesion and E-cadherin protein is drastically reduced in tissues that require Grhl2 function. The tissue closure defects in Grhl2 mutants are similar to that of AP-2α null mutants and AP-2α has been shown to bind to the promoter of E-cadherin. Therefore, we tested for a possible interaction between these genes. However, we find that Grhl2 and AP-2α do not regulate each other's expression, E-cadherin expression is normal in AP-2α mutants during neural tube closure, and Grhl2;AP-2α trans-heterozygous embryos are morphologically normal. Taken together, our studies point to a complex regulation of neural tube fusion and highlight the importance of comparisons between these two models to understand more fully the molecular pathways of embryonic tissue closure.     

Function of Neurolin (DM-GRASP/SC-1) in guidance of motor axons during zebrafish development.

Neurolin (zf DM-GRASP), a transmembrane protein with five extracellular immunoglobulin domains, is expressed by secondary but not primary motoneurons during zebrafish development. The spatiotemporally restricted expression pattern suggests that Neurolin plays a role in motor axon growth and guidance. To test this hypothesis, we injected zebrafish embryos with function-blocking Neurolin antibodies. In injected embryos, secondary motor axons form a broadened bundle along the common path and ectopic branches leave the common path at right angles. Moreover, the formation of the ventral and the rostral projection of secondary motor axons is inhibited during the second day of development. Pathfinding errors, resulting in secondary motor axons growing through ectopic regions of the somites, occur along the common path and in the dorsal and rostral projection. Our data are compatible with the view that Neurolin is involved in the recognition of guidance cues and acts as a receptor on secondary motor axons. Consistent with this idea is the binding pattern of a soluble Neurolin-Fc construct showing that putative ligands are distributed along the common path, the ventral projection, and in the area where the rostral projection develops.     

N-myc down regulates neural cell adhesion molecule expression in rat neuroblastoma.

In human neuroblastoma, amplification of the N-myc oncogene is correlated with increased metastatic ability. We recently showed that transfection of the rat neuroblastoma cell line B104 with an N-myc expression vector resulted in an increase in metastatic ability and a significant reduction in the expression of major histocompatibility complex class I antigens. We examined whether N-myc causes additional phenotypic changes in these cells. We showed that expression of N-myc leads to a dramatic reduction in the levels of neural cell adhesion molecule (NCAM) polypeptides and mRNAs. Spontaneous revertants of the high N-myc phenotype were found to have regained significant levels of NCAM expression, indicating that the continued expression of N-myc is required to maintain the low NCAM phenotype. NCAM was not reduced in B104 cells transfected with the neomycin resistance vector alone, and other neuronal markers were not specifically reduced in N-myc-transfected B104 cells. As NCAM functions in cell-cell adhesion, decreased NCAM expression could contribute significantly to the increased metastatic potential of N-myc-amplified neuroblastomas.     

Specific regulation of N-CAM/D2-CAM cell adhesion molecule during skeletal muscle development

The expression of the N-CAM/D2-CAM cell adhesion molecule was studied in skeletal muscle. In cell cultures derived from adult human muscle N-CAM/D2-CAM was found at the cell surface of myoblasts and myotubes but not fibroblasts, showing that N-CAM/D2-CAM is a specific gene product of muscle. Western blots showed that the anti N-CAM/D2-CAM antibody reacted with a single protein band of 180 000 daltons in these cultures that differed in mobility from the broad band of 150 000-200 000 daltons found in brain. N-CAM/D2-CAM is also expressed by muscle at certain stages of development. Human foetal muscle of 10 and 20 weeks gestation showed N-CAM/D2-CAM around developing myofibres while both fast and slow adult muscle fibres did not express N-CAM/D2-CAM, suggesting that the protein is down regulated during myofibre maturation. This was studied further in developing rat muscle where N-CAM/D2-CAM was found on myofibres in the day 1 neonate, but had disappeared by day 9. N-CAM/D2-CAM is, however, re-expressed in human muscle disease where there is muscle regeneration such as in polymyositis, and here is associated with classic regenerating myofibres. N-CAM/D2-CAM expression is temporally regulated and is expressed only at times of synapse formation consistent with the idea that it may be involved in early nerve-muscle interactions.     

Differential expression of neural cell adhesion molecule and cadherins in pancreatic islets, glucagonomas, and insulinomas.

The endocrine cells of the pancreas develop from the endoderm and yet display several characteristics of a neuronal phenotype. During embryonic life, ductal epithelial cells give rise to first the glugagon-producing cells (alpha-cells) and then cells that express insulin (beta-cells), somatostatin (delta-cells), and pancreatic polypeptide (PP-cells) in a sequential order. The endocrine cells are believed to arise from a stem cell with neuronal traits. The developmental lineage from a common neuron-like progenitor is evidenced by: transient coexpression of more than one cell type-specific hormone in immature cells, expression of neuronal markers during islet cell development, and the pluripotentiality of clones of insulinoma cells to develop into cells expressing other islet cell hormones. The four mature endocrine cell types assume a particular organization within the islets of Langerhans in a process where cell adhesion molecules are involved. In this study we have analyzed the expression of neural cell adhesion molecule (NCAM) and cadherin molecules in neonatal, young, and adult rat islet cells as well as in glucagonomas and insulinomas derived from a pluripotent rat islet cell tumor. Whereas primary islet cells at all ages express unsialylated NCAM and E-cadherin, as do insulinomas, the glucagonomas express the polysialylated NCAM, which is characteristic for developing neurons. The glucagonomas also lose E-cadherin expression and instead express a cadherin which is similar to N-cadherin in brain. Insulinoma cells express E-cadherin but differ from primary islet cells by expressing a second cadherin molecule, which is similar to N-cadherin. The expression of NCAM and cadherin isoforms in the glucagonoma suggest that this transformed alpha-cell type has converted to an immature phenotype with strong neuronal traits, reflecting the early palce of glucagon-producing cells in the islet cell lineage. In contrast, insulinoma cells are more islet-like in their phenotype and show less neuronal traits.     

Homeoprotein Hbx4 represses the expression of the adhesion molecule DdCAD-1 governing cytokinesis and development

We investigated the function of homeodomain-containing protein Hbx4 in Dictyostelium discoideum. Hbx4-overexpressing cells (Hbx4(OE)) displayed defects in growth rate and cytokinesis and showed differences in slug motility and cell-type proportioning from KAx3. Furthermore, the overexpression of Hbx4 inhibited the induction of cadA, which encoded the Ca(2+)-dependent cell adhesion molecule DdCAD-1, despite expression of csaA and gpaB. The electrophoretic mobility shift assay showed that the promoter of cadA contained the Hbx4-binding site. Moreover, constitutively expressed DdCAD-1 in Hbx4(OE) rescued the defects in cytokinesis and development. These results suggest that Hbx4 modulates DdCAD-1-mediated cytokinesis and cell-type proportioning.     

Role of galectin-3 as an adhesion molecule for neutrophil extravasation during streptococcal pneumonia

Recruitment of neutrophils from blood vessels to sites of infection represents one of the most important elements of innate immunity. Movement of neutrophils across blood vessel walls to the site of infection first requires that the migrating cells firmly attach to the endothelial wall. Generally, neutrophil extravasation is mediated at least in part by two classes of adhesion molecules, beta(2) integrins and selectins. However, in the case of streptococcal pneumonia, recent studies have revealed that a significant proportion of neutrophil diapedesis is not mediated by the beta(2) integrin/selectin paradigm. Galectin-3 is a beta-galactoside-binding lectin implicated in inflammatory responses as well as in cell adhesion. Using an in vivo streptococcal pneumonia mouse model, we found that accumulation of galectin-3 in the alveolar space of streptococcus-infected lungs correlates closely with the onset of neutrophil extravasation. Furthermore, immunohistological analysis of infected lung tissue revealed the presence of galectin-3 in the lung tissue areas composed of epithelial and endothelial cell layers as well as of interstitial spaces. In vitro, galectin-3 was able to promote neutrophil adhesion to endothelial cells. Promotion of neutrophil adhesion by galectin-3 appeared to result from direct cross-linking of neutrophils to the endothelium and was dependent on galectin-3 oligomerization. Together, these results suggest that galectin-3 acts as an adhesion molecule that can mediate neutrophil adhesion to endothelial cells. However, accumulation of galectin-3 in lung was not observed during neutrophil emigration into alveoli induced by Escherichia coli infection, where the majority of neutrophil emigration is known to be beta(2) integrin dependent. Thus, based on our results, we propose that galectin-3 plays a role in beta(2) integrin-independent neutrophil extravasation, which occurs during alveolar infection with Streptococcus pneumoniae.     

HEMCAM, an adhesion molecule expressed by c-kit+ hemopoietic progenitors

We have characterized the adhesion molecule HEMCAM, which is expressed by hemopoietic progenitors of embryonic bone marrow. HEMCAM belongs to the immunoglobulin superfamily and consists of the V-V-C2-C2-C2 Ig domains. There are three mRNA splice variants. One has a short cytoplasmic tail; another has a long tail; while the third seems to lack transmembrane and cytoplasmic regions. Except for the NH2-terminal sequence, HEMCAM is identical to gicerin, a molecular involved in neurite outgrowth and Wilm's kidney tumor progression in the chicken and it is significantly homologous with MUC18 a molecule involved in melanoma progression and metastasis in human beings. In the bone marrow the HEMCAM+ cell population contains c-kit+ subsets. HEMCAM+ cells coexpressing the receptor tyrosine kinase c-kit give rise to T cells at a frequency of 0.17 when injected intrathymically in congenic animals. As HEMCAM+, c-kit+ cells differentiate into myeloid and erythroid CFU's the double-positive cell population seems to contain precursors for multiple lineages. HEMCAM promotes cell-cell adhesion of transfected cells. Cross-linking of murine HEMCAM leads to cell spreading of T- lymphocyte progenitors adhering to the vascular adhesion molecules, PECAM-1 and VCAM-1. Thus, HEMCAM is likely to be involved in cellular adhesion and homing processes.     

Human CD38, a surface receptor, an enzyme, an adhesion molecule and not a simple marker.

Human CD38 is a 45 kDa type II trans-membrane glycoprotein with a peculiar discontinuous pattern of expression in leukocytes, although evidence is accumulating of its quite widespread expression outside of the hematopoietic system. CD38 is a member of an emerging family of cytosolic and membrane-bound enzymes whose substrate is nicotinamide adenine dinucleotide (NAD), a coenzyme ubiquitously distributed in nature. CD38 is a multifunctional molecule able to exert not only an enzymatic activity but also to mobilize calcium, to transduce signals, to adhere to hyaluronan and to other ligands. Interaction with CD38 on various leukocyte subpopulation has profound though diverse effects on their life-span, however, the immunoregulatory activities seem to be independent of the enzymatic functions of the molecule.