Extracellular Domain Of N-cadherin Research Articles

N-cadherin directs the collective Schwann cell migration required for nerve regeneration through Slit2/3 mediated contact inhibition of locomotion.

Collective cell migration is fundamental for the development of organisms and in the adult, for tissue regeneration and in pathological conditions such as cancer. Migration as a coherent group requires the maintenance of cell-cell interactions, while contact inhibition of locomotion (CIL), a local repulsive force, can propel the group forward. Here we show that the cell-cell interaction molecule, N-cadherin, regulates both adhesion and repulsion processes during rat Schwann cell (SC) collective migration, which is required for peripheral nerve regeneration. However, distinct from its role in cell-cell adhesion, the repulsion process is independent of N-cadherin trans-homodimerisation and the associated adherens junction complex. Rather, the extracellular domain of N-cadherin is required to present the repulsive Slit2/Slit3 signal at the cell-surface. Inhibiting Slit2/Slit3 signalling inhibits CIL and subsequently collective Schwann cell migration, resulting in adherent, nonmigratory cell clusters. Moreover, analysis of ex vivo explants from mice following sciatic nerve injury showed that inhibition of Slit2 decreased Schwann cell collective migration and increased clustering of Schwann cells within the nerve bridge. These findings provide insight into how opposing signals can mediate collective cell migration and how CIL pathways are promising targets for inhibiting pathological cell migration.

Fbxo45 Binds SPRY Motifs in the Extracellular Domain of N-Cadherin and Regulates Neuron Migration during Brain Development.

Several events during the normal development of the mammalian neocortex depend on N-cadherin, including the radial migration of immature projection neurons into the cortical plate. Remarkably, radial migration requires the N-cadherin extracellular domain but not N-cadherin-dependent homophilic cell-cell adhesion, suggesting that other N-cadherin-binding proteins may be involved. We used proximity ligation and affinity purification proteomics to identify N-cadherin-binding proteins. Both screens detected MycBP2 and SPRY domain protein Fbxo45, two components of an intracellular E3 ubiquitin ligase. Fbxo45 appears to be secreted by a nonclassical mechanism, not involving a signal peptide and not requiring transport from the endoplasmic reticulum to the Golgi apparatus. Fbxo45 binding requires N-cadherin SPRY motifs that are not involved in cell-cell adhesion. SPRY mutant N-cadherin does not support radial migration in vivo Radial migration was similarly inhibited when Fbxo45 expression was suppressed. The results suggest that projection neuron migration requires both Fbxo45 and the binding of Fbxo45 or another protein to SPRY motifs in the extracellular domain of N-cadherin.

Niche-mimicking interactions in peptide-functionalized 3D hydrogels amplify mesenchymal stromal cell paracrine effects

Cells encounter complex environments in vivo where they interact with the extracellular matrix, neighboring cells, and soluble cues, which together influence their fate and function. However, the interplay of these interactions and their collective impact on the regenerative effects of mesenchymal stromal cells (MSCs) remains insufficiently explored. Here, we show that 3D culture in microporous (~125 μm) hydrogels that passively promote cell-cell interactions sensitizes MSCs to growth factors, particularly to IGF-1. IGF-1 enhances MSC paracrine secretion activity, and application of secreted factors to myoblasts potently stimulates their migration and differentiation. In contrast, the paracrine activity of MSCs encapsulated in nanoporous (~10 nm) hydrogels remain unchanged. Blocking N-cadherin on MSCs abrogates the stimulatory effects of IGF-1 in microporous but not nanoporous hydrogels. The role of N-cadherin in regulating MSC function is further clarified by functionalizing alginates with the HAVDI peptide sequence that is derived from the extracellular domain of N-cadherin and that acts to mimic cell-cell interactions. MSCs encapsulated in nanoporous HAVDI-gels, but not in gels functionalized with a scrambled sequence, show heightened paracrine activity in response to IGF-1. These findings reveal how interactions with the matrix, neighboring cells, and soluble factors impact and maximize the regenerative potential of MSCs.

Overexpression of Cathepsin E Interferes with Neuronal Differentiation of P19 Embryonal Teratocarcinoma Cells by Degradation of N-cadherin.

Cathepsin E (CatE), an aspartic protease, has a limited distribution in certain cell types such as gastric cells. CatE is not detectable in the normal brain, whereas it is increasingly expressed in damaged neurons and activated microglia of the pathological brain. Neurons expressing high levels of CatE showed apparent morphological changes, including a marked shrinkage of the cytoplasmic region and beading of neurites, suggesting neuronal damage. The intracellular level of CatE in neurons is strictly regulated at both transcriptional and translational levels. Although the up-regulation of CatE may cause pathological changes in neurons, little information is available about the precise outcome of the increased expression of CatE in neurons. In this study, we have attempted to clarify the outcome of up-regulated CatE gene expression in neurons using the P19 cell neuronal differentiation after the overexpression of CatE. We unexpectedly found that the overexpression of CatE interfered with neuronal differentiation of P19 cells through an impairment of cell aggregate formation. Pepstatin A, an aspartic protease inhibitor, restored the impaired cell aggregation of P19/CatE cells. The small number of P19 cells differentiated into neurons had abnormal morphology characterized by their fusiform cell bodies with short processes. Furthermore, CatE proteolytically cleaved the extracellular domain of N-cadherin. These observations suggest that the overexpression of CatE interferes with neuronal differentiation of P19 cells through an impairment of cell aggregate formation, possibly through proteolytic degradation of N-cadherin.

Extraordinary Stability of Domain 1 of Neural-Cadherin

Cadherins are transmembrane proteins responsible for calcium dependent cell-cell adhesion. Classical cadherins have a common structure with five extracellular domains, connected by a 7-residue sequence linker region. Cadherins mediate adhesion via Adherens Junctions by forming a strand-swapped structure between identical protomers from apposing cells. The adhesive interface has been characterized structurally, and biophysical studies have been used to elucidate the forces that stabilize the strand-swapped structure. Upon calcium binding, the swapping of the N-terminal strands between protomers allows for the symmetrical docking of a tryptophan residue required for adhesion. Fundamental questions still remain regarding the striking difference in the calcium-dependent kinetics of dimerization between N- and E-cadherin. This study compares the first extracellular domain of N-cadherin (NCAD1) and E-cadherin (ECAD1). With NCAD1, the binding pocket for calcium is not complete, so dimerization was not expected. However, we observed a dimeric form that was not in exchange with monomer. This dimer was reversibly converted to monomer by heating the protein. Noticeably, NCAD1 was found to unfold at an uncharacteristically high temperature. Results for NCAD1 construct were similar to results for domain 1 in studies of the two-domain construct, NCAD12. Similar studies of ECAD1 show that it is significantly lower stability. In summary, our results indicate that the difference in kinetics of dimerization may be due to a difference in the intrinsic stability of domain 1.GAANN P200A120046

Abstract 3310: N-cadherin promotes docetaxel resistance through upregulated TLR4 signaling in castration resistant prostate cancers

Abstract Introduction: Docetaxel is a common therapeutic agent given to men with castration-resistant prostate cancer (CRPC). However, a number of patients show poor response or eventually develop resistance. Previously, different groups have suggested an association between chemoresistance and epithelial to mesenchimal transition (EMT) in certain advanced cancers. N-cadherin, a calcium-dependent cell-cell adhesion surface protein associated with EMT, is implicated in castration resistance and metastasis in prostate cancer. We have shown in previous studies that monoclonal antibodies raised against the extracellular domain of N-cadherin have potential therapeutic effects in castration-resistant tumor growth and metastasis. In this study, our objective is to determine the role of N-cadherin expression in chemoresistance and whether treatment with monoclonal antibodies restores chemosensitivity. Methods: Tissue microarrays of CRPC and high grade prostate cancer clinical samples were stained for TLR4. N-cadherin positive and negative cell lines were evaluated for sensitivity against docetaxel. N-cadherin and TLR4 were ectopically expressed in N-cadherin negative prostate cancer cells (LNCaP, VCaP). Docetaxel resistant cell lines (LNCaP-DocR and VCaP-DocR) were established through prolonged exposure to 10-20uM Docetaxel. For in vitro experiments, these models were used to evaluate cytotoxicity and apoptosis in the presence of docetaxel. N-cadherin expressing cell lines were treated with 2A9 monoclonal antibody (mAb), which was raised against the extracellular domain of N-cadherin, and by N-cadherin siRNA knockdown. For in vivo studies, N-cadherin positive xenografts were treated with 2A9 mAb and docetaxel, and tumor growth monitored over time. Results: Tissue microarray results showed significant TLR4 expression in CRPC samples (p < 0.005). In vitro studies showed TLR4 induction correlated with N-cadherin expression. This upregulation of N-cadherin and TLR4 expression promoted docetaxel resistance in CRPC by the activation of NF-kB signaling. Monoclonal antibodies against N-cadherin and TLR4 potentiated docetaxel induced cytotoxicity. Docetaxel resistant cells exhibited EMT phenotypes with increased invasion and castration resistant growth. Treatment with 2A9 antibody restored sensitivity to docetaxel. Combination therapy with 2A9 monoclonal antibody and docetaxel significantly affected tumor growth in N-cadherin positive CRPC xenograft models. Conclusions: These studies suggest that N-cadherin promotes TLR4-dependent docetaxel resistance in CRPC. Although further investigation will be needed, therapeutic targeting of N-cadherin with monoclonal antibodies in combination with therapeutic agents such as docetaxel may have significant clinical benefit. Citation Format: Tatsuya Shimomura, Evelyn Kono, Chau P. Tran, Joyce Yamashiro, Shu Lin, Sean Hyung-Kwon Lee, Zev A. Wainberg, Robert E. Reiter. N-cadherin promotes docetaxel resistance through upregulated TLR4 signaling in castration resistant prostate cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3310. doi:10.1158/1538-7445.AM2014-3310

Characterization of N‐cadherin unbinding properties in non‐malignant (HCV29) and malignant (T24) bladder cells

The expression of N-cadherin, characteristic of various cancers, very often leads to changes in the cells' adhesive properties. Thus, we sought to find out if N-cadherin expressed in various, but cancer-related cells, differs in its functional properties that could contribute to variations in cells' phenotypes. In our work, measurements of an unbinding force of a single N-cadherin molecule, probed with the same antibody both on a surface of living non-malignant (HCV29) and malignant cells (T24) of bladder cancer, were carried out with the use of an atomic force microscopy. The results show the enhanced N-cadherin level in T24 malignant cells (8.7% vs. 3.6% obtained for non-malignant one), confirmed by the Western blot and the immunohistochemical staining. The effect was accompanied by changes in unbinding properties of an individual N-cadherin molecule. Lower unbinding force values (26.1 ± 7.1 pN) in non-malignant cells reveal less stable N-cadherin complexes, as compared to malignant cells (61.7 ± 14.6 pN). This suggests the cancer-related changes in a structure of the binding site of the antibody, located at the extracellular domain of N-cadherin.

Abstract 5052: N-cadherin monoclonal antibody as a therapeutic agent against chemoresistance in prostate cancer

Abstract Introduction: The development of chemoresistance is a formidable obstacle in realizing effective therapy against cancer. Docetaxel is a common therapeutic agent given to men with castration-resistant prostate cancer (CRPC). However, a number of patients show poor response or eventually develop resistance to the treatment. N-cadherin, a calcium-dependent cell-cell adhesion surface protein associated with Epithelial to mesenchymal transition (EMT), is implicated in castration resistance and metastasis in prostate cancer. We have shown in previous studies that monoclonal antibodies raised against the extracellular domain of N-cadherin have potential therapeutic effects in castration-resistant tumor growth and metastasis. In this study, our objective is to determine the role of N-cadherin expression in chemoresistance and whether treatment with monoclonal antibodies restores chemosensitivity. Methods: N-cadherin was ectopically expressed in N-cadherin negative prostate cancer cells (LNCaP, MDA PCa 2b). For in vitro experiments, these models were used to evaluate cytotoxicity and apoptosis in the presence of docetaxel. Endogenously expressing N-cadherin were either treated with monoclonal antibodies raised against the extracellular domain of N-cadherin or N-cadherin expression was reduced by siRNA knockdown. For in vivo studies, monoclonal antibodies raised against N-cadherin were used in combination with docetaxel to treat N-cadherin positive xenografts, and tumor growth monitored over time. Results: Ectopic expression of N-cadherin in N-cadherin negative cells resulted in >25-fold increase in resistance to cytotoxicity by docetaxel treatment when compared with control cells. N-cadherin knockdown by siRNA of cells endogenously expressing N-cadherin resulted in an increase of cytotoxicity in response to docetaxel when compared with cells transfected with scramble siRNA. Treatment with monoclonal antibody also resulted in increase of cytotoxicity in response to docetaxel. In vitro studies also show that increase in N-cadherin leads to upregulation in NFkB (>2.5-fold ± 0.17 increase in activity) and Toll-like receptor signaling, which are implicated in chemoresistance. Preliminary in vivo studies show some additive effect monoclonal antibodies against N-cadherin in combination with docetaxel on growth of xenograft tumors expressing N-cadherin. Conclusions: These studies suggest that N-cadherin is involved in the development of chemoresistance in prostate cancers. Therapeutic targeting of N-cadherin with monoclonal antibodies, in combination with therapeutic agents such as docetaxel may have significant clinical benefit. Future experiments involve further elucidating the pathways involved and downstream targets, such as those involved in NFkB and Toll-like receptor signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5052. doi:10.1158/1538-7445.AM2011-5052

Abstract 3623: Monoclonal antibody against the 4th extracellular domain of N-cadherin affects FGF2 mediated pathways in prostate cancer

Abstract Introduction: Fibroblast growth factor 2 (FGF2) has been reported to synergize with N-cadherin, a cell surface marker of epithelial mesenchymal transition (EMT), to enhance migration and invasion in breast cancer and other cancers. Recent studies from our group suggested that N-cadherin promotes castration resistance and metastasis in prostate cancer. In addition, our studies showed that antibodies against N-cadherin ectodomains blocked prostate cancer invasion and castration resistant growth. To investigate the mechanism of action of N-cadherin antibodies in prostate cancer cells, we seek to determine the contribution of the different N-cadherin domains in promoting invasion and growth in prostate cancer cells, with particular focus on interaction with FGF2. Methods: Chimeric constructs, in which a N-cadherin domain was swapped with an E-cadherin domain, were generated and ectopically expressed in androgen dependent prostate cancer cell line LNCaP. The following combinations were made: 1/ NE: N-cadherin ectodomains linked to E-cadherin cytoplasmic domain (NE), 2/ EN: the reverse of NE, 3/ NEN: swapping N-cadherin ectodomain 4 for that of E-cadherin. Full length N-cadherin and empty vector sublines served as positive and negative controls. All cell lines were assayed for invasion and growth in androgen depleted media in combination with recombinant FGF2, FGF2 neutralizing antibody and N-cadherin antibodies. Results: Ectopic expression of N-cadherin in N-cadherin negative cells result in a 5-fold increase of FGF2 secreted in the media. In vitro studies have shown that the addition of recombinant FGF2 enhanced invasion by 56%± 0.04 in N-cadherin positive cells while treatment with FGF2 neutralizing antibody resulted in a 65%± 0.03 reduction in invasion. Chimeric cell lines which harbor extracellular domain 4 of N-cadherin, N and NE cell lines, showed >56%± 0.11 enhanced invasion to the addition of recombinant FGF2 while EN, NEN, and control cell lines did not. Monoclonal antibodies developed against regions in the extracellular domain of N-cadherin block the response to recombinant FGF2 with 2A9 (antibody raised against extracellular domain 4) being the least responsive. Conclusions: These studies suggest that the relationship between N-cadherin and FGF2 is involved in prostate cancer progression. Therapeutic targeting of N-cadherin with monoclonal antibodies, alone or in combination with other drugs targeting those potentially involved in downstream pathways may have significant clinical benefit. Future experiments include in vivo models where chimeric cell lines will be implanted in mice as xenografts, and will be monitored for tumor growth, local invasion, and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3623. doi:10.1158/1538-7445.AM2011-3623

A fusion protein N-cadherin-Fc as an artificial extracellular matrix surface for maintenance of stem cell features

N-cadherin is a cell–cell adhesion molecule and plays important roles in neural development. With conventionally used extracellular matrices (ECMs), maintenance of undifferentiated state of stem cells and regulation of their neural differentiation process is very difficult due to the colony formation through intercellular interactions. To overcome the above-mentioned problems, we developed a new artificial ECM to mimic N-cadherin-mediated cell adhesion. In this study, we constructed a chimeric protein (N-cadherin fused to IgG-Fc, abbreviated as N-cad-Fc), which contains extracellular domain of N-cadherin and Fc domain of IgG. We confirmed that N-cad-Fc can stably adsorb to hydrophobic surface. We checked maintenance of undifferentiated state and neural differentiation ability of stem cells cultured on N-cad-Fc-coated surface. Both P19 and MEB5 cells cultured on N-cad-Fc-coated surface showed scattering morphologies without colony formation and higher proliferating potency than conventional culture systems with maintenance of undifferentiated state. Both of two cell lines cultured on N-cad-Fc-coated surface differentiated into neural cells at a single cell level when induced with proper conditions. Furthermore, the expression of neuron-related gene Neurog1 in two cell lines cultured on N-cad-Fc-coated surface was promoted. Therefore, it will be expected that the constructed N-cad-Fc can be used as an artificial ECM for stem cells.

Stimulation of N-cadherin-dependent neurite outgrowth by small molecule peptide mimetic agonists of the N-cadherin HAV motif

N-cadherin is a cell adhesion molecule that promotes axon outgrowth and synapse formation during the development of the central nervous system. In addition, N-cadherin promotes glial cell adhesion and myelination of axons. Therefore, stimulating N-cadherin function with N-cadherin agonists could be used therapeutically to promote regeneration of the nervous system and remyelination after injury or disease. In the extracellular domain of N-cadherin, the amino acid sequence HAV is required for N-cadherin-mediated adhesion and neurite outgrowth. The ADH-1 cyclic peptide, derived from the N-cadherin HAV site, is an effective antagonist of N-cadherin-mediated neurite outgrowth and is currently being tested in clinical trials for cancer chemotherapy. Of interest, a dimeric version of this cyclic peptide, N-Ac- CHAVDINGHAVDIC-NH 2, functions as an N-cadherin agonist. This dimeric peptide agonist and the peptide antagonist ADH-1 both have limitations as drugs due to their metabolic instability and lack of oral delivery. To address this issue Adherex Technologies Inc. generated a small molecule library of peptidomimetics to the HAV region of N-cadherin, which would be more amenable to therapeutic use. We screened the Adherex library for compounds that altered neurite outgrowth and identified eight N-cadherin agonists that stimulated N-cadherin-dependent neurite outgrowth. Five of these agonists also stimulated retinal cell migration. These small molecule agonists may be effective reagents for promoting axon growth and remyelination after injury or disease.

Novel peptide mimetic small molecules of the HAV motif in N-cadherin inhibit N-cadherin-mediated neurite outgrowth and cell adhesion

The cell adhesion molecule, N-cadherin, stabilizes cell–cell junctions and promotes cellular migration during tissue morphogenesis in development. N-cadherin is also implicated in mediating tumor progression and metastasis in cancer. Therefore, developing antagonists of N-cadherin adhesion may be of therapeutic value in cancer treatment. The amino acid sequence HAV in the extracellular domain of N-cadherin is required for N-cadherin-mediated adhesion and migration. A cyclic peptide, ADH-1, derived from the N-cadherin HAV site is an effective antagonist of N-cadherin-mediated processes and is now in clinical trials for cancer chemotherapy. Because it is a peptide, ADH-1 has certain limitations as a drug, namely its metabolic instability and lack of oral delivery. Adherex set out to identify small molecule antagonists of N-cadherin, which would be more amenable to therapeutic use. Using three-dimensional computational screening, Adherex identified a set of small molecules as potential antagonists with sufficient structural similarity to the HAV region of N-cadherin. We tested the ability of these small molecules to interfere with two N-cadherin-dependent processes: neurite outgrowth (axonal migration) and N-cadherin-dependent cell adhesion. We identified 21 N-cadherin antagonists of varying potency. More importantly, our studies demonstrate that these compounds are significantly more potent than ADH-1 at perturbing N-cadherin-mediated processes. The IC 50 of ADH-1 is 2.33 mM while the IC 50 of the small molecules ranges from 4.5 to 30 μM. Given the efficacy of ADH-1 for treating cancer, these small molecule antagonists will be highly effective in treatment of cancer metastasis and conditions of aberrant neurite outgrowth, such as neuropathic pain.

Soluble N-cadherin fragment promotes angiogenesis

Endothelial cells express two dependent intercellular adhesion molecules: vascular endothelial (VE)-cadherin, specific for endothelial cells, and N-cadherin, also present in neuronal, lens, skeletal and heart muscle cells, osteoblasts, pericytes and fibroblasts. While there exists a vast amount of evidence that VE-cadherin promotes angiogenesis, the role of N-cadherin still remains to be elucidated. We found that a soluble 90-kDa fragment N-cadherin promotes angiogenesis in the rabbit cornea assay and in the chorioallantoic assay when cleaved enzymatically from the extracellular domain of N-cadherin. Soluble N-cadherin stimulates migration of endothelial cells in the wound healing assay and stimulates phosphorylation of extracellular regulated kinase. In vitro experiments with PD173074 and knock-down of N-cadherin and fibroblast growth factor (FGF)-receptor, showed that the pro-angiogenic effect of soluble N-cadherin is N-cadherin- and FGF-receptor-dependent. Our results suggest that soluble N-cadherin stimulates migration of endothelial cells through the FGF-receptor.

N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells

The molecular mechanisms underlying the contact behavior of Schwann cells (SCs) and SC–axon association are poorly understood. SC–SC and SC–axon interactions were studied using purified adult rat SCs and cocultures of SCs with embryonic dorsal root ganglion neurons. After contact of SCs with axons, SCs start to extend processes in alignment with axons. This unique alignment was quantitated using a new assay. SC–axon alignment and SC–SC band formation were disrupted in medium containing low extracellular calcium, indicating the involvement of calcium-dependent adhesion molecules. N-cadherin expression was strong in developing rat sciatic nerves but weak in adult sciatic nerves. In purified adult-derived rat SCs, N-cadherin expression was increased by mitogens (neuregulins) and decreased by high cell density. High-resolution confocal images show intense N-cadherin signals in SC process tips. Subcellular N-cadherin was accumulated in bands at intercellular junctions between SCs and was clustered at axon–SC contact sites. Blocking antibodies (rabbit and guinea pig IgG directed against the first extracellular domain of N-cadherin) and cyclic pentapeptides (including the HAV motif) were used to perturb N-cadherin function. All blocking agents reduced the number of N-cadherin-positive SC–SC junctions and perturbed axon-aligned growth of SC processes. Averaging over all N-cadherin-perturbation experiments, in controls 67–86% of SCs exhibited axon-aligned process growth, whereas in treated cultures only 41% of the SCs aligned with axons. These results are evidence that in mammals N-cadherin is important for formation of SC–SC junctions and SC process growth in alignment with axons.

N-cadherin mediates axon-aligned process growth and cell-cell interaction in rat Schwann cells.

The molecular mechanisms underlying the contact behavior of Schwann cells (SCs) and SC-axon association are poorly understood. SC-SC and SC-axon interactions were studied using purified adult rat SCs and cocultures of SCs with embryonic dorsal root ganglion neurons. After contact of SCs with axons, SCs start to extend processes in alignment with axons. This unique alignment was quantitated using a new assay. SC-axon alignment and SC-SC band formation were disrupted in medium containing low extracellular calcium, indicating the involvement of calcium-dependent adhesion molecules. N-cadherin expression was strong in developing rat sciatic nerves but weak in adult sciatic nerves. In purified adult-derived rat SCs, N-cadherin expression was increased by mitogens (neuregulins) and decreased by high cell density. High-resolution confocal images show intense N-cadherin signals in SC process tips. Subcellular N-cadherin was accumulated in bands at intercellular junctions between SCs and was clustered at axon-SC contact sites. Blocking antibodies (rabbit and guinea pig IgG directed against the first extracellular domain of N-cadherin) and cyclic pentapeptides (including the HAV motif) were used to perturb N-cadherin function. All blocking agents reduced the number of N-cadherin-positive SC-SC junctions and perturbed axon-aligned growth of SC processes. Averaging over all N-cadherin-perturbation experiments, in controls 67-86% of SCs exhibited axon-aligned process growth, whereas in treated cultures only 41% of the SCs aligned with axons. These results are evidence that in mammals N-cadherin is important for formation of SC-SC junctions and SC process growth in alignment with axons.

VE-Cadherin Mediates Endothelial Cell Capillary Tube Formation in Fibrin and Collagen Gels1

Various cell adhesion molecules mediate the diverse functions of the vascular endothelium, such as cell adhesion, neutrophil migration, and angiogenesis. In order to identify cell adhesion molecules important for angiogenesis, we used anin vitromodel (Chalupowicz, Chowdhury, Bach, Barsigian, and Martinez,J. Cell Biol.130, 207–215, 1995) in which human umbilical vein endothelial cell monolayers are induced to form capillary-like tubes when a second gel, composed of either fibrin or collagen, is formed overlying the apical surface. In the present investigation, we observed that a monoclonal antibody directed against the first extracellular domain of human vascular endothelial cadherin (VE-cadherin, cadherin 5) inhibited the formation of capillary tubes formed between either fibrin or collagen gels. Moreover, when added to preformed capillary tubes, this antibody disrupted the capillary network. In contrast, monoclonal antibodies directed against the extracellular domain of N-cadherin, the αvβ3integrin, and PECAM-1 failed to inhibit capillary tube formation. During capillary tube formation, Western blot and RT-PCR analysis revealed no marked change in VE-cadherin expression. Immunocytochemical studies demonstrated that VE-cadherin was concentrated at intercellular junctions in multicellular capillary tubes. Thus, VE-cadherin plays a specific role in fibrin-induced or collagen-induced capillary tube formation and is localized at areas of intercellular contact where it functions to maintain the tubular architecture. Moreover, its function at tubular intercellular junctions is distinct from that at intercellular junctions present in confluent monolayers, since only the former was inhibited by monoclonal antibodies.

Differentiation and survival of rat olfactory epithelial neurons in dissociated cell culture

Olfactory epithelial neurons in mammals are unique in that they continue to differentiate from precursor cells in the adult. These neurons extend long axons into the olfactory bulb. Previous attempts to grow these cells in dissociated cell cultures at low density, have not been entirely satisfactory. We report that when plated at very low density, rat olfactory epithelial neurons will differentiate morphologically and biochemically when cultured on astrocytes, but not on non-cellular substrata, such as polylysine, laminin or fibronectin. We demonstrate with antibodies, that these olfactory epithelial neurons require N-CAM, N-cadherin and L1 for neurite extension. Furthermore, synthetic cadherin-peptides containing the tripeptide HAV which is found in the first extracellular domain of N-cadherin, as well as the amino acids flanking this region, appear to be important in cadherin-mediated neurite growth on astrocytes. Astrocytes also appear to enhance the survivial and differentiation of olfactory epithelial neurons from embryonic day 15 and 4–5 week post-natal rats, but this effect is not sustained beyond 5 days in cultures of postnatal epithelium.