Differential effects of NgCAM and N-cadherin on the development of axons and dendrites by cultured hippocampal neurons

A fundamental step in neuronal development is the acquisition of a polarized form, with distinct axons and dendrites. Although the ability to develop a polarized form appears to be largely an intrinsic property of neurons, it can be influenced by environmental cues. For example, in cell cultures substrate and diffusible factors can enhance and orient axonal development. In this study we examine the effects of growth on each of two cell adhesion molecules (CAMs), NgCAM and N-cadherin, on the development of polarity by cultured hippocampal neurons. We find that although the same pattern of development occurs on control substrates and the CAMs, the CAMs greatly accelerate the rate and extent of development of axons—axons form sooner and grow longer on the CAMs than on the control substrate. In contrast, the CAMs have opposite effects on dendritic development—N-cadherin enhances, but NgCAM reduces dendritic growth compared to control. These results provide further evidence that the development of polarity is largely determined by a cell-autonomous program, but that environmental cues can independently regulate axonal and dendritic growth.     

Calcium-dependent cell adhesion molecules

The adhesive function of Ca2(+)-dependent CAMS has in the past been studied only indirectly, mainly using immunological techniques. The molecular cloning and information about the primary structure of several CAMs has been an important step in a more detailed molecular analysis. If there is a homophilic interaction between CAMs of neighbouring cells, an important question concerns the specificity of each CAM-mediated adhesiveness. Has each CAM a unique specificity and can this specificity be linked to a defined amino acid sequence? It will be important to elucidate the molecular mechanism of how each CAM interacts with the other. The experiments of Volk et al. (1987) suggest that an interaction of two different CAMs can occur. Since during development a given cell can express more than one CAM such an heterophilic interaction could play some regulatory role. Alternative splicing mechanisms or different protein forms during development or on different cell types have not yet been observed for Ca2(+)-dependent CAMs. However, uvomorulin is assumed to have a slightly different function during development and in adult tissues. During development uvomorulin is involved in the condensation, the pattern formation, and the sorting out of cells. In these processes the uvomorulin-mediated adhesiveness should be controlled, since cells reorganize and migrate during development. For the maintenance of the histoarchitecture in adult tissues uvomorulin might act more as a glue. This argues for the existence of mechanisms to regulate the strength of adhesiveness, and the cytoplasmic domain might be involved in these processes. The association of the cytoplasmic domain of uvomorulin with catenins could be an important observation in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synapse adhesion: a dynamic equilibrium conferring stability and flexibility

Cell adhesion molecules (CAMs) linked to cytoskeleton generate stable cell-cell junctions. Cadherins provide a canonical example, but paradoxically, they participate in a multitude of transient and regulatable interactions. Their extracellular binding generates weak adhesion that is modified by clustering; interactions with F-actin are regulated, can be transient, and can alter F-actin dynamics. Additionally, cadherin recycling from the cell surface can modify the size and location of junctions and strength of adhesion. In epithelial cells, this ongoing dynamic behavior is important for maintaining stable junctions. Recent work supports that cadherins act similarly at synapses where their actions are likely to be shared by integrins and other actin-linked CAMs. Together the collaborative activities of such CAMs provide a stable, but flexible structure that can promote and support changes in synapse shape and size while maintaining stable junctions to permit information flow.     

Cell-CAM 105-an adhesive cell surface glycoprotein

Summary Cell recognition and adhesion are important events in embryonic development as well as in adult physiology. In recent years several cell adhesion molecules (CAMs), that mediate adhesive interactions between vertebrate cells, have been identified and characterized. These CAMs are in general cell surface-associated high molecular weight glycoproteins. Two groups of CAMs have been classified: primary CAMs, that appear early in development; secondary CAMs, that become expressed later and with a more restricted tissue distribution. One example of a secondary CAM is cellCAM 105. This glycoprotein was originally identified in rat hepatocytes, and was shown to be involved in the reaggregation of freshly isolated hepatocytesin vitro. Physico-chemical studies on pure cellCAM 105 have demonstrated that it has adhesive properties and can bind to itself in a homophilic, calcium-independent reaction. Immunochemical and immunohistochemical investigations have shown that cellCAM 105 occurs in liver, several epithelia, vessel endothelia, platelets and polymorphonuclear leukocytes, and that it is expressed primarily in terminally differentiated cells or cells that are undergoing terminal differentiation. Available information suggests that cellCAM 105 has different functions in different cell types, and that the common functional denominator might be membrane-membrane binding. Recent data indicate that cellCAM 105 is a calmodulin-binding protein, suggesting that cellCAM-mediated cell binding could be involved in transmembrane signalling.Abbreviation CAM cell adhesion molecule     

Tumor cell/endothelial cell tight contact upregulates endothelial adhesion molecule expression mediated by NFκB: Differential role of the shear stress

Cancer metastasis is a multistep process involving cell-cell interactions, but little is known about the adhesive interactions and signaling events during extravasation of tumor cells (TCs). In this study, cell adhesion molecule (CAM) expression was investigated using an in vitro assay, in which TCs were seeded onto an endothelial cell (ECs) monolayer and cocultured during 5 h. Flow cytometry, confocal microscopy as well as western blot analysis indicated that endothelial ICAM-1 (Inter Cellular Adhesion Molecule-1), VCAM-1 (Vascular Adhesion Molecule-1) and E-selectin were up-regulated after TC-EC coculture, whereas no change was observed for CAMs expression in tumor cells. This increased CAMs expression required tight contact between TCs and ECs. Incubation of ECs with the pyrrolidine-dithiocarbamate NFκB inhibitor prior to coculture, fully prevented coculture-induced expression of endothelial CAMs. Using specific blocking antibodies we showed an implication of ICAM-1 and VCAM-1 for TCs extravasation and VCAM-1 for adhesion. Moreover, fluid flow experiments revealed that high shear stress totally abolished coculture-induced as well as TNFα-induced CAMs over-expression. This study suggests that TCs could act as a potent inflammatory stimulus on ECs by inducing CAMs expression via NFκB activation, and that this action can be modulated by shear stress.     

In vivo invasion of modified chorioallantoic membrane by tumor cells: the role of cell surface-bound urokinase

The ability of the chick embryo chorioallantoic membrane (CAM) to withstand invasion by tumor cells can be intentionally compromised by altering its morphological integrity. Using a newly developed quantitative assay of invasion we showed that intact CAMs were completely resistant to invasion by tumor cells, wounded CAMs did not pose a barrier to penetration, and CAMs that were wounded and then allowed to reseal displayed partial susceptibility to invasion. The invasion of resealed CAMs required catalytically active plasminogen activator (PA) of the urokinase type (uPA); the invasive efficiency of tumor cells was reduced by 75% when tumor uPA activity or tumor uPA production was inhibited. The invasive ability of human tumor cells, which have surface uPA receptors but which do not produce the enzyme, could be augmented by saturating their receptors with exogenous uPA. The mere stimulation of either uPA or tissue plasminogen activator production, in absence of binding to cell receptors, did not result in an enhancement of invasiveness. These findings suggest that the increased invasive potential of tumor cells is correlated with cell surface-associated proteolytic activity stemming from the interaction between uPA and its surface receptor.     

Simvastatin modulates cytokine-mediated endothelial cell adhesion molecule induction: involvement of an inhibitory G protein

Endothelial cell adhesion molecules (CAMs) E-selectin, ICAM-1, and VCAM-1 play variably important roles in immune-mediated processes. They are induced by the proinflammatory cytokines IL-1 and TNF-alpha, and NF-kappaB is required for the regulated expression of all three genes. Regulators of this pathway could potentially be potent immune modulators. We studied the effect of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin, on cytokine-induced expression of CAMs in HUVEC. Unexpectedly, pretreatment with simvastatin potentiated the induction of all three endothelial CAMs by IL-1 and TNF, but not LPS or PMA, as detected by flow cytometry. Northern blot analysis demonstrated an increase in steady state IL-1-induced E-selectin mRNA levels in cells pretreated with simvastatin. This was associated with an increase in nuclear translocation of NF-kappaB, as detected by EMSA. The effect of simvastatin was reversed by mevalonate and geranylgeranyl pyrophosphate but not squalene, indicating that an inhibitory prenylated protein is involved in endothelial responses to proinflammatory cytokines. Pertussis toxin mimicked the effect of simvastatin, and the G protein activator NaF inhibited the cytokine-induced expression of endothelial CAMs, indicating that a Gialpha protein is involved. These results demonstrate that cytokine-mediated activation of the endothelium, and specifically CAM induction, can be modulated by a heterotrimeric G protein-coupled pathway. This may represent a "basal tone" of endothelial inactivation, which can either be disinhibited or amplified, depending on the stimulus.     

The Production of Arachidonic Acid Can Account for Calcium Channel Activation in the Second Messenger Pathway Underlying Neurite Outgrowth Stimulated by NCAM, N-Cadherin, and L1

Abstract: We have used monolayers of control 3T3 fibroblasts and 3T3 fibroblasts expressing transfected cell adhesion molecules (CAMs)—NCAM, N-cadherin, and L1—as a culture substrate for cerebellar neurones. The transfected CAMs promote neurite outgrowth by activating a second messenger pathway that culminates in calcium influx into neurones through N-and l -type calcium channels. We show that the same neurite outgrowth response can be directly induced by arachidonic acid (10 μ M ) and that this response can be inhibited by N-and l -type calcium channel antagonists. In cells, arachidonic acid can be generated by phospholipase A₂ or by the sequential activities of a phospholipase C (to generate diacylglycerol) and diacylglycerol lipase. In the present study we show the neurite outgrowth stimulated by CAMs (but not by various other agents) can be abolished by an inhibitor of diacylglycerol lipase acting at a site upstream from calcium channel activation. The results suggest that arachidonic acid and/or one of its metabolites is the second messenger that activates calcium channels in the CAM signalling pathway leading to axonal growth, and this is supported by recent evidence that shows the same concentrations of arachidonic acid can increase voltage-dependent calcium currents in cardiac myocytes.     

Intrinsic differences in the response of the human lutropin receptor versus the human follitropin receptor to activating mutations

In contrast to the human lutropin receptor (hLHR), very few naturally occurring activating mutations of the structurally related human follitropin receptor (hFSHR) have been identified. The present study was undertaken to determine if one aspect underlying this discrepancy might be a general resistance of the hFSHR to mutation-induced constitutive activity. Five different mutations were introduced into both the hLHR and hFSHR (four based on activating mutations of the hLHR gene, one based on an activating mutation of the hFSHR gene). Our results demonstrate that hFSHR constitutively activating mutants (CAMs) were not as active as hLHR CAMs containing the comparable mutation. Furthermore, although all hFSHR CAMs exhibited strong promiscuous activation by high concentrations of the other glycoprotein hormone receptors, hLHR CAMs showed little or no promiscuous activation. Our in vitro findings are consistent with in vivo observations of known pathophysiological conditions associated with hLHR CAMs, but not hFSHR CAMs, and with promiscuous activation of hFSHR CAMs, but not hLHR CAMs. Computational experiments suggest that the mechanisms through which homologous mutations increase the basal activity of the hLHR and the hFSHR are similar. This is particularly true for the strongest CAMs like L460(3.43)R. Disparate properties of the hLHR versus hFSHR CAMs may, therefore, be due to differences in shape and electrostatics features of the solvent-exposed cytosolic receptor domains involved in the receptor-G protein interface rather than to differences in the nature of local perturbation at the mutation site or in the way local perturbation is transferred to the putative G protein binding domains.     

Constitutive internalization of constitutively active agiotensin II AT(1A) receptor mutants is blocked by inverse agonists.

As constitutively active mutants (CAMs) mimic an active conformation, they can be used to characterize the process of G protein-coupled receptor activation. Here, we used CAMs to study the link between activation and internalization of the angiotensin II AT(1A) receptor. The cellular localization of fluorescently tagged N111A, I245T, and L305Q mutants was determined by confocal microscopy. In the absence of ligand, CAMs were mostly located in intracellular vesicles, whereas the wild-type AT(1A) was found at the cell surface. After 2 h incubation with inverse agonist, losartan, CAMs were translocated to the plasma membrane. Similar observations were made in H295, a human adrenocortical cell line which expresses physiologically the AT(1) receptor. This phenomenon, which was not dependent on protein synthesis and the pharmacology and kinetics of which were similar to the recycling of the wild-type receptor, was called "externalization". After externalization and losartan removal, the L305Q CAM underwent rapid ligand-independent endocytosis, with the same kinetics and temperature sensitivity as the angiotensin II-induced internalization of the wild-type AT(1A). Moreover, the addition of a second mutation known to block internalization (Delta 329 truncation) prevented intracellular localization of the CAM. These data show that AT(1A) CAMs are constitutively and permanently internalized and recycled. This mechanism is different from the down-regulation observed for CAMs of other G protein-coupled receptors and thus defines a new paradigm for the cellular regulation of CAMs.     

Tyrosine kinase inhibitors can differentially inhibit integrin- dependent and CAM-stimulated neurite outgrowth

We have used monolayers of parental 3T3 cells and 3T3 cells expressing one of three transfected cell adhesion molecules (CAMs) (NCAM, N- cadherin, and L1) as a culture substrate for rat cerebellar neurons. A number of tyrosine kinase inhibitors have been tested for their ability to inhibit neurite outgrowth over parental 3T3 monolayers which we show to be partly dependent on neuronal integrin receptor function, as compared with neurite outgrowth stimulated by the above three CAMs. Whereas genistein (100 microM), lavendustin A (20 microM), and tyrphostins 34 and 47 (both at 150 microM) had no effect on integrin dependent or CAM stimulated neurite outgrowth, the erbstatin analogue (10-15 micrograms/ml) and tyrphostins 23 and 25 (both at 150 microM) specifically inhibited the response stimulated by all three CAMs. CAM stimulated neurite outgrowth can be accounted for by a G-protein- dependent activation of neuronal calcium channels; experiments with agents that directly activate this pathway localized the erbstatin analogue site of action upstream of the G-protein and calcium channels, whereas tyrphostins have sites of action downstream from calcium channel activation. These data suggest that activation of an erbstatin sensitive tyrosine kinase is an important step upstream of calcium channel activation in the second messenger pathway underlying the neurite outgrowth response stimulated by a variety of CAMs, and that this kinase is not required for integrin-dependent neurite outgrowth.     

The mechanism of axon growth

Cell adhesion molecules (CAMs) are not just an inert glue that mediates static cell-cell and cell-extracellular matrix (ECM) adhesion; instead, their adhesivity is dynamically controlled to enable a cell to migrate through complex environmental situations. Furthermore, cell migration requires distinct levels of CAM adhesivity in various subcellular regions. Recent studies on L1, a CAM in the immunoglobulin superfamily, demonstrate that cell adhesion can be spatially regulated by the polarized internalization and recycling of CAMs. This article examines the molecular mechanism of axon growth, with a particular focus on the role of L1 trafficking in the polarized adhesion and migration of neuronal growth cones.     

Ligand rescue of constitutively active mutant receptors

Constitutively active mutants (CAMs) of G protein-coupled receptors are found naturally in disease states and they can be generated by point mutation. As these mutants are able to activate G proteins in the absence of a ligand, they are useful tools in the study of conformational changes leading to receptor activation and in the drug discovery process. Early studies on CAMs noted that they are often expressed at lower levels than their wild-type forms. In this review we discuss the mechanisms responsible for this reduced expression and also provide an explanation for the observation that challenging cells with receptor ligands can increase the CAM expression level. The application of these observations to the development of a high-throughput reporter assay suitable for ligand identification is also discussed.     

Activation of EGF receptor kinase by L1-mediated homophilic cell interactions

Neural cell adhesion molecules (CAMs) are important players during neurogenesis and neurite outgrowth as well as axonal fasciculation and pathfinding. Some of these developmental processes entail the activation of cellular signaling cascades. Pharmacological and genetic evidence indicates that the neurite outgrowth-promoting activity of L1-type CAMs is at least in part mediated by the stimulation of neuronal receptor tyrosine kinases (RTKs), especially FGF and EGF receptors. It has long been suspected that neural CAMs might physically interact with RTKs, but their activation by specific cell adhesion events has not been directly demonstrated. Here we report that gain-of-function conditions of the Drosophila L1-type CAM Neuroglian result in profound sensory axon pathfinding defects in the developing Drosophila wing. This phenotype can be suppressed by decreasing the normal gene dosage of the Drosophila EGF receptor gene. Furthermore, in Drosophila S2 cells, cell adhesion mediated by human L1-CAM results in the specific activation of human EGF tyrosine kinase at cell contact sites and EGF receptors engage in a physical interaction with L1-CAM molecules. Thus L1-type CAMs are able to promote the adhesion-dependent activation of EGF receptor signaling in vitro and in vivo.     

The mechanism of axon growth: what we have learned from the cell adhesion molecule L1

Cell adhesion molecules (CAMs) are not just an inert glue that mediates static cell-cell and cell-extracellular matrix (ECM) adhesion; instead, their adhesivity is dynamically controlled to enable a cell to migrate through complex environmental situations. Furthermore, cell migration requires distinct levels of CAM adhesivity in various subcellular regions. Recent studies on L1, a CAM in the immunoglobulin superfamily, demonstrate that cell adhesion can be spatially regulated by the polarized internalization and recycling of CAMs. This article examines the molecular mechanism of axon growth, with a particular focus on the role of L1 trafficking in the polarized adhesion and migration of neuronal growth cones.     

Cell adhesion molecules, signal transduction and cell growth

Signals from dynamic cellular interactions between the extracellular matrix and neighboring cells ultimately input into the cellular decision-making process. These interactions form the basis of anchorage-dependent growth. Recent advances have provided the mechanistic details behind the ability of integrins, and other cell adhesion molecules (CAMs), to regulate both early signal transduction events initiated by soluble factors and downstream events more proximally involved in cell cycle progression. These actions appear to depend on the ability of CAMs to initiate the formation of organized structures that permit the efficient flow of information.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

Adhesion molecule signalling: not always a sticky business

The signalling activity of cell adhesion molecules (CAMs) such as cadherins, immunoglobulin-like CAMs or integrins has long been considered to be a direct consequence of their adhesive properties. However, there are physiological and pathological processes that reduce or even abrogate the adhesive properties of CAMs, such as cleavage, conformational changes, mutations and shedding. In some cases these 'adhesion deficient' CAMs still retain signalling properties through their cytoplasmic domains and/or their mutated or truncated extracellular domains. The ability of CAMs to activate signal transduction cascades in the absence of cell adhesion significantly extends their range of biological activities.     

The role of the cytoskeleton in cellular adhesion molecule expression in tumor necrosis factor-stimulated endothelial cells

Leukocyte infiltration is a hallmark of the atherosclerotic lesion. These cells are captured by cellular adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cell adhesion molecule (PECAM), and E-selectin, on endothelial cells (EC). We examined the role of the actin cytoskeleton in tumor necrosis factor-alpha (TNF-alpha)-induced translocation of CAMs to the cell surface. Human aortic EC were grown on 96-well plates and an ELISA was used to assess surface expression of the CAMs. TNF-alpha increased VCAM-1, ICAM-1, and E-selectin by 4 h but had no affect on the expression of PECAM. A functioning actin cytoskeleton was important for VCAM-1 and ICAM-1 expression as both cytochalasin D, an actin filament disruptor, and jasplakinolide, an actin filament stabilizer, attenuated the expression of these CAMs. These compounds were ineffective in altering E-selectin surface expression. Myosin light chains are phosphorylated in response to TNF-alpha and this appears to be regulated by Rho kinase instead of myosin light chain kinase. However, the Rho kinase inhibitor, Y27632, had no affect on TNF-alpha-induced CAM expression. ML-7, a myosin light chain kinase inhibitor, had a modest inhibitory effect on the translocation of VCAM-1 but not on ICAM-1 or E-selectin. These data suggest that the surface expression of VCAM-1 and ICAM-1 is dependent on cycling of the actin cytoskeleton. Nevertheless, modulation of actin filaments via myosin light chain phosphorylation is not necessary. The regulation of E-selectin surface expression differs from that of the other CAMs.