Functional studies and cellular distribution of the F3 GPI-anchored adhesion molecule

Many adhesion molecules of the immunoglobulin superfamily expressed in the nervous system are attached to the neuronal membrane by a glycan-phosphatidylinositol. Using neuronal glycoprotein F3 as a model we will discuss how this lipid modification might confer on molecules specific properties which may be particularly well suited to a role in modulating neuronal interactions. In particular, the following data dealing with the question of how the glycosylphosphatidylinositol (GPI) anchor influences the function, transport and localization of this molecule will be presented. 1) When anchored to the plasma membrane, F3 fulfills the operational criteria of an adhesion molecule while its soluble form is able to stimulate neurite outgrowth of sensory neurons in culture. 2) In the hypothalamo-hypophyseal system, immunoblot analysis indicates that there is more F3 in the neurohypophysis where secretory axons terminate than in the hypothalamic nuclei where the molecule is synthesized. In addition, GPI-linked forms predominate in the nuclei while there are mainly soluble forms in the neurohypophysis, suggesting that there is conversion of the GPI-bearing form to the soluble form during axonal transport. 3) In the cerebellum, F3 is polarized to the tips of the axons of granule cells, the major neuronal population in this system, as an indication that indeed GPI might be a signal for targeting molecules to axons. However, some neurons such as Golgi cells express F3 over all their surface.     

In vitro oligodendrogliotrophic properties of cell adhesion molecules in the immunoglobulin superfamily: myelin-associated glycoprotein and N-CAM

The occurrence of conserved epitopes in the immune system was investigated on the leukocytes of cattle, river buffalo, sheep, camel, swine and humans by indirect immunofluorescence and flow cytometry. The distribution of the most conservative epitopes on leukocyte sub-populations suggests that the expression pattern of the proteins is similar. Western blotting experiments indicate that the recognized antigens are structural homologues.     

Biliary glycoprotein (CD66a), a cell adhesion molecule of the immunoglobulin superfamily, on human lymphocytes: structure, expression and involvement in T cell activation

The biliary glycoproteins (BGP or CD66a), a group of different splice variants of a single gene, are members of the carcinoembryonic antigen family and the immunoglobulin superfamily. Recently, we detected CD66a on IL-2 activated lymphocytes. In this study we characterized the structure and the expression pattern of BGP on human lymphocytes and investigated its role in T cell activation. Lymphocytes express 2 of the 13 known splice variants, i.e. BGPa and BGPb, which are glycosylated in a lymphocyte-specific manner. Both BGPa and BGPb have the long cytoplasmic tail, which contains two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like motifs, but differ in their extracellular region containing 4 and 3 immunoglobulin-like domains, respectively. On PBL BGP is expressed in small amounts only on B cells and Th cells. Stimulation with IL-2 leads to a strong up-regulation of BGP by these cells, and induces de novo BGP expression on gammabeta T cells, CD8+ and CD56+ cells, but not on CD16+ lymphocytes. This up-regulation of BGP seems to be part of the physiological process of T cell activation, since stimulation with anti-CD3 mAb is sufficient to induce BGP up-regulation. Based on the presence of the two ITIM-like motifs, one may expect that BGP inhibits T cell activation, but surprisingly, engagement of BGP enhances the proliferation of anti-CD3-stimulated T cells.     

Influence of adhesion molecule expression by human brain microvessel endothelium on cancer cell adhesion

Cultures of endothelial (En) cells derived from human brain microvessels were established in order to characterize adhesion molecule expression and to assay the adhesion properties of neoplastic cell lines to monolayers of En cells. Low constitutive expression of beta1 integrin (CD29), and ICAM-2 (CD102) was detected on human brain microvessel En cells. The beta1 chain of the VLA integrin family, ICAM-1, E-selectin (CD62E) and VCAM-1 (CD106) but not ICAM-2 and PECAM-1 (CD31) expression was upregulated by IL1-alpha, and TNF-alpha proinflammatory cytokines. High expression of PECAM-1 was found on non-activated human brain EN cells. In order to study the potential role of adhesion molecules in neoplastic cell adhesion two tumor cell lines were chosen. Adhesion of a cell line (DU145) derived from a cerebral metastasis of prostate carcinoma to human brain microvessel En cell monolayers was less pronounced compared to adhesion of a primary prostate carcinoma cell line (ND1). Adhesion of cerebral metastatic neoplastic cell line (DU145) was not significantly influenced by incubation of endothelial cells with different proinflammatory cytokines. The adhesion capability of primary prostate carcinoma line (NDI) was significantly upregulated by TNF-alpha proinflammatory cytokine. Furthermore, the adhesion of ND1 was partly inhibited using anti-E-selectin and VCAM-1 monoclonal antibodies. There was no significant effect of anti-adhesion antibodies on the adhesion characteristics of the cerebral metastatic (DU145) cell line. Our data demonstrate that different mechanisms are involved in the adhesion of neoplastic cells to cerebral En cells and turn our attention to the importance of adhesion molecule expression in the formation of metastases.     

Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule

Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of alpha-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with alpha-actinin. Binding of alpha-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for alpha-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via alpha-actinin.     

Expression of cellular adhesion regulatory molecule in hepatocellular carcinoma

OBJECTIVE: Studying gender differences in fat mass and distribution in a homogeneous group of children. DESIGN: Cross-sectional study. SUBJECTS: 610 children aged 5-7 y in Kiel, Germany. METHODS: Anthropometric measures, bioelectrical impedance analysis (BIA). RESULTS: Although boys had increased body weights (P<0.05), body mass indexes (BMI's) (P<0.001) and waist/hip ratios (WHRs) (P<0.001), the %fat mass as assessed by BIA (P<0.05) was increased in girls. Although the increased BMI in boys was independent of the percentile used, gender differences (that is, lower values for boys than for girls at the same age) in WHR, the sum of four skinfolds and %fat were seen up to the 90th percentile. By contrast, above the 90th percentile there were no differences in skinfold thickness and %fat between boys and girls. Studying 42 BMI-matched pairs (boys and girls) also showed that the %fat estimated by BIA (P<0.001) was increased in girls. Plotting the average of %fat as obtained from skinfold- and BAI-measurements against the difference between data obtained by the use of the two methods shows that BIA %fat overestimates skinfold %fat at low or normal percent fat mass (that is, up to 20%) in both genders. By contrast, at increased fat mass, BIA %fat seems to underestimate skinfold %fat in both genders. CONCLUSIONS: Gender differences in fat mass and fat distribution are obvious in children aged 5-7 y. These differences are independent of gender differences in body weight. However, the nutritional state has an influence and gender differences cannot be detected in overweight and obese children. Our data also suggest that a children-specific formula used to calculate %fat from skinfold measurements is inappropriate.     

Expression of E-cadherin adhesion molecule in vocal cord carcinomas

E-cadherin is a 120 kDa transmembrane protein that plays a major role in the maintenance of cell-cell adhesion in epithelial tissues. In the present study its expression was examined immunohistochemically for the first time using paraffin-embedded archival tissues of 38 vocal cord carcinomas. Ten cases of vocal cord polyps were used as positive controls. According to our criteria, results showed that all but one of the polyps, in which staining was lost, had strong or moderate E-cadherin expression, whereas 32% of the cases with tumors showed preserved staining, and 68% of cases showed a reduced staining. E-cadherin expression was significantly correlated with histological stages, with a greater expression in carcinoma in situ and microinvasive carcinoma than in invasive carcinoma (P = 0.010). A significant correlation was also found between E-cadherin expression and the degree of tumor differentiation (P = 0.018). Some of these findings were consistent with previously published data using fresh tissues. Our study provides evidence that E-cadherin may be linked to progression and differentiation of laryngeal cancer and could play a role as a tumor-suppressor gene in this cancer.     

Studying receptor-mediated cell adhesion at the single molecule level

Cell adhesion is essentially mediated by specific interactions between membrane receptors and ligands. It is now apparent that the mere knowledge of the on- and off-rate of association of soluble forms of these receptors and ligands is not sufficient to yield accurate prediction of cell adhesive behavior. During the last few years, a variety of complementary techniques relying on the use of hydrodynamic flow, atomic force microscopy, surface forces apparatus or soft vesicles yielded accurate information on i) the dependence of the lifetime of individual bonds on applied forces and ii) the distance dependence of the association rate of bound receptors and ligands. The purpose of this review is, first to recall the physical significance of these parameters, and second to describe newly obtained results. It is emphasized that molecular size and flexibility may be a major determinant of the efficiency of receptor mediated adhesion, and this cannot be studied by conventional methods dealing with soluble molecules.     

Expression of polysialylated neural cell adhesion molecule by proliferating cells in the subependymal layer of the adult rat, in its rostral extension and in the olfactory bulb

The highly sialylated isoform of the neural cell adhesion molecule is thought to be expressed predominantly in the developing nervous system, where it is implicated in a variety of dynamic events linked to neural morphogenesis. It has become increasingly evident, however, that this "embryonic" neural cell adhesion molecule isoform continues to be expressed in certain adult neuronal systems, and in particular, in those that can undergo structural plasticity. In the present study, we performed light microscopic immunocytochemistry with an antibody specific for polysialylated neural cell adhesion molecule and confirmed our earlier observations [Bonfanti L. et al. (1992) Neuroscience 49, 419-436] showing polysialylated neural cell adhesion molecule-immunoreactive cells in the subependymal layer of the lateral ventricle of the adult rat, a region where cell proliferation continues into the postnatal period. In addition, we used an antibody raised against the proliferating cell nuclear antigen and found that proliferating cells continue to be visible in this area, even in the adult. Double immunolabeling showed that many of these newly generated cells displayed high polysialylated neural cell adhesion molecule immunoreactivity. Cells from a portion of the subependymal layer migrate to the olfactory bulb and contribute to the continual replacement of its granule neurons [Luskin M. B. (1993) Neuron 11, 173-189]. We found polysialylated neural cell adhesion molecule-immunoreactive cells all along the pathway purported to be followed by the newly generated cells to their final destination and in neurons corresponding to granular and periglomerular cells in the olfactory bulb. Our present observations thus support the contention that polysialylation is a feature of neurons capable of dynamic change and may contribute to the molecular mechanisms permitting cell proliferation and migration not only during development but also in the adult.     

Regulated expression of the cell adhesion glycoprotein F3 in adult hypothalamic magnocellular neurons

F3, a glycoprotein of the immunoglobulin superfamily implicated in axonal growth, occurs in oxytocin (OT)-secreting and vasopressin (AVP)-secreting neurons of the adult hypothalamo-neurohypophysial system (HNS) whose axons undergo morphological changes in response to stimulation. Immunocytochemistry and immunoblot analysis showed that during basal conditions of HNS secretion, there are higher levels of this glycosylphosphatidyl inositol-anchored protein in the neurohypophysis, where their axons terminate, than in the hypothalamic nuclei containing their somata. Physiological stimulation (lactation, osmotic challenge) reversed this pattern and resulted in upregulation of F3 expression, paralleling that of OT and AVP under these conditions. In situ hybridization revealed that F3 expression in the hypothalamus is restricted to its magnocellular neurons and demonstrated a more than threefold increase in F3 mRNA levels in response to stimulation. Confocal and electron microscopy localized F3 in secretory granules in all neuronal compartments, a localization confirmed by detection of F3 immunoreactivity in granule-enriched fractions obtained by sucrose density gradient fractionation of rat neurohypophyses. F3 was not visible on any cell surface in the magnocellular nuclei. In contrast, in the neurohypophysis, it was present not only in secretory granules but also on the surface of axon terminals and glia and in extracellular spaces. Taken together, our observations reveal that the cell adhesion glycoprotein F3 is colocalized with neurohypophysial peptides in secretory granules. It follows, therefore, the regulated pathway of secretion in HNS neurons to be released by exocytosis at their axon terminals in the neurohypophysis, where it may intervene in activity-dependent structural axonal plasticity.     

Homophilic synaptic target recognition mediated by immunoglobulin-like cell adhesion molecule Fasciclin III

We demonstrate that the cell adhesion molecule Fasciclin III (FAS3) mediates synaptic target recognition through homophilic interaction. FAS3 is expressed by the RP3 motoneuron and its target muscles during synaptic target recognition. The RP3 growth cone can form synapses on muscles that ectopically express FAS3. This mistargeting is dependent on FAS3 expression in the motoneurons. In addition, when the FAS3-negative aCC and SNa motoneuron growth cones ectopically express FAS3, they gain the ability to recognize FAS3-expressing muscles as alternative targets. We propose that homophilic synaptic target recognition serves as a basic mechanism of neural network formation.     

Spatio-temporal diversity in the microenvironments for neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1-cell adhesion molecule expression by sensory neurons and their targets during cochleo-vestibular innervation

Sixteen phases in the microenvironments were defined for the structural development and innervation of the cochleo-vestibular ganglion and its targets. In each phase the cell adhesion molecules, neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1-cell adhesion molecule, were expressed differentially by cochleo-vestibular ganglion cells, their precursors, and the target cells on which they synapse. Detected by immunocytochemistry in staged chicken embryos, in the otocyst, neural cell adhesion molecule, but not L1-cell adhesion molecule, was localized to the ganglion and hair cell precursors. Ganglionic precursors, migrating from the otocyst, only weakly expressed neural cell adhesion molecule. Epithelial hair cell precursors, remaining in the otocyst, expressed neural cell adhesion molecule, but not L1-cell adhesion molecule. Post-migratory ganglion cell processes expressed both molecules in all stages. The cell adhesion molecules were most heavily expressed by axons penetrating the otic epithelium and accumulated in large amounts in the basal lamina. In the basilar papilla (cochlea), cell adhesion molecule expression followed the innervation gradient. Neural cell adhesion molecule and L1 were heavily concentrated on axonal endings peripherally and centrally. In the rhombencephalon, primitive epithelial cells expressed neural cell adhesion molecule, but not L1-cell adhesion molecule, except in the floorplate. The neuroblasts and their axons expressed L1-cell adhesion molecule, but not neural cell adhesion molecule, when they began to migrate and form the dorsal commissure. There was a stage-dependent, differential distribution of the cell adhesion molecules in the floorplate. Commissural axons expressed both cell adhesion molecules, but their polysialic acid disappeared within the floorplate at later stages. In conclusion, the cell adhesion molecules are expressed by the same cells at different times and places during their development. They are positioned to play different roles in migration, target penetration, and synapse formation by sensory neurons. A multiphasic model provides a morphological basis for experimental analyses of the molecules critical for the changing roles of the microenvironment in neuronal specification.     

Ammonia inhibits neural cell adhesion molecule polysialylation in Chinese hamster ovary and small cell lung cancer cells

Contrasts between implicit and explicit knowledge in the serial reaction time (SJRT) paradigm have been challenged because they have depended on a single dissociation; intact implicit knowledge in the absence of corresponding explicit knowledge. In the SRT task, subjects respond with a corresponding keypress to a cue that appears in one of four locations. The cue follows a repeating sequence of locations, and subjects can exhibit knowledge of the repeating sequence through increasingly rapid performance (an implicit test) or by being able to recognize the sequence (an explicit test). In our study, amnesic patients were given extensive SRT training. Their implicit and explicit test performance was compared to the performance of control subjects who memorized the training sequence. Compared with control subjects, amnesic patients exhibited superior performance on the implicit task and impaired performance on the explicit task. This crossover interaction suggests that implicit and explicit knowledge of the embedded sequence are separate and encapsulated and that they presumably depend on different brain systems.     

Epithelial V-like antigen (EVA), a novel member of the immunoglobulin superfamily, expressed in embryonic epithelia with a potential role as homotypic adhesion molecule in thymus histogenesis

Granule cells in the rat hippocampal dentate gyrus contain intracellular receptors for the adrenal hormone corticosterone. Activation of these receptors seems essential for granule cell viability, since removal of the adrenal gland (adrenalectomy) results within three days in apoptotic-like degeneration of granule cells. In the present study we used extracellular in vitro recording methods to study the synaptic transmission in the dentate gyrus of adrenalectomized animals, in sham-operated controls and adrenalectomized rats treated with a low dose of corticosterone. We found that particularly three days after adrenalectomy orthodromic field responses in the dentate gyrus were reduced in amplitude. Corticosterone-treated rats did not show this impairment of synaptic transmission. Antidromically-evoked field responses were also reduced after adrenalectomy, which indicates that postsynaptic cell properties rather than signal transduction in the synapses are under steroid control. Responses to paired pulse stimulation were only marginally affected, suggesting that interneuronal networks may be less affected by the hormones than the principal cells. These electrophysiological data indicate that adrenalectomy induced apoptotic-like degeneration in the hippocampal dentate gyrus is clearly associated with impaired processing of incoming information.     

Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.