Neuronal Cadherin Research Articles

N-cadherin expression in malignant germ cell tumours of the testis

BackgroundTesticular germ cell tumours (TGCTs) are the most common malignancy in young men aged 18–35 years. They are clinically and histologically subdivided into seminomas and non-seminomas. Cadherins are calcium-dependent transmembrane proteins of the group of adhesion proteins. They play a role in the stabilization of cell-cell contacts, the embryonic morphogenesis, in the maintenance of cell polarity and signal transduction. N-cadherin (CDH2), the neuronal cadherin, stimulates cell-cell contacts during migration and invasion of cells and is able to suppress tumour cell growth.MethodsTumour tissues were acquired from 113 male patients and investigated by immunohistochemistry, as were the three TGCT cell lines NCCIT, NTERA-2 and Tcam2. A monoclonal antibody against N-cadherin was used.ResultsTumour-free testis and intratubular germ cell neoplasias (unclassified) (IGCNU) strongly expressed N-cadherin within the cytoplasm. In all seminomas investigated, N-cadherin expression displayed a membrane-bound location. In addition, the teratomas and yolk sac tumours investigated also differentially expressed N-cadherin. In contrast, no N-cadherin could be detected in any of the embryonal carcinomas and chorionic carcinomas examined. This expression pattern was also seen in the investigated mixed tumours consisting of seminomas, teratomas, and embryonal carcinoma.ConclusionsN-cadherin expression can be used to differentiate embryonal carcinomas and chorionic carcinomas from other histological subtypes of TGCT.

Notch-mediated induction of N-cadherin and α9-integrin confers higher invasive phenotype on rhabdomyosarcoma cells

Background:Rhabdomyosarcoma (RMS) is the commonest type of soft-tissue sarcoma in children. Patients with metastatic RMS continue to have very poor prognosis. Recently, several works have demonstrated a connection between Notch pathway activation and the regulation of cell motility and invasiveness. However, the molecular mechanisms of this possible relationship remain unclear.Methods:The Notch pathway was manipulated pharmacologically and genetically. The mRNA changes were analysed by quantitative PCR and protein variations by western blot and immunofluorescence. Finally, the capabilities of RMS cells to adhere, heal a wound and invade were assessed in the presence of neuronal cadherin (N-cadherin)- and α9-integrin-blocking antibodies.Results:Cells treated with γ-secretase inhibitor showed lower adhesion capability and downregulation of N-cadherin and α9-integrin. Genetic manipulation of the Notch pathway led to concomitant variations in N-cadherin and α9-integrin. Treatment with anti-N-cadherin-blocking antibody rendered marked inhibition of cell adhesion and motility, while anti-α9-integrin-blocking antibody exerted a remarkable effect on cell adhesion and invasiveness.Conclusion:Neuronal cadherin and α9-integrin are postulated as leading actors in the association between the Notch pathway and promotion of cell adhesion, motility and invasion, pointing to these proteins and the Notch pathway itself as interesting putative targets for new molecular therapies against metastases in RMS.

Neuronal cadherin (NCAD) increases sensory neurite formation and outgrowth on astrocytes

We examined the neurite outgrowth of sensory neurons on astrocytes following the genetic deletion of N-cadherin (NCAD). Deletion abolished immunostaining for NCAD and the other classical cadherins, indicating that NCAD is likely the only classical cadherin expressed by astrocytes. Only 38% of neurons grown on NCAD-deficient astrocytes for 24h produced neurites, as compared to 74% of neurons grown on NCAD-expressing astrocytes. Of the neurons that produced neurites, those grown on NCAD-deficient astrocytes had a mean total length of 378μm, as compared to 1093μm for neurons grown on NCAD-expressing astrocytes. Thus, the loss of NCAD greatly impairs the formation and extension neurites on astrocytes.

Ectopic expression of cadherin 8 is sufficient to cause cyst formation in a novel 3D collagen matrix renal tubule culture

While a variety of genetic mutations have been shown to be associated with renal cyst formation, mechanisms of renal cyst formation are largely unknown. In prior communications we described alterations in E-cadherin assembly in cultured cystic epithelial cells (Charron AJ, Nakamura S, Bacallao R, Wandinger-Ness A. J Cell Biol 149: 111-124, 2000). Using the same cell line we assayed cadherin expression by RT-PCR using primer pairs that anneal to highly conserved sequences of cadherin genes but flank informative regions of cadherins. Using this approach we found that autosomal dominant polycystic kidney disease (ADPKD) cells express cadherin 8, a neuronal cadherin with limited expression in the kidney. Immunohistochemistry confirmed cadherin 8 expression in cystic epithelia. To test the functional significance of cadherin 8 expression in renal epithelial cells, we adapted a three-dimensional collagen culture method in which HK-2 cells form tubule structures and microinjected adenovirus into the matrix space surrounding tubule structures. Adenovirus expressing cadherin 8 under the control of a tet promoter caused cyst structures to grow out of the tubules when coinjected with adenovirus expressing a tet transactivator. Microinjection of single adenovirus expressing either tet transactivator or cadherin 8 failed to cause cyst formation. When doxycycline was added to the culture, following coinjection of adenovirus, there was a dose-response reduction in cadherin 8 expression and cyst formation. Similarly, HK-2 cells transfected with Flag-tagged cadherin 8 form cysts in addition to tubular structures. HK-2 cells transfected with Flag-tagged N-cadherin do not form cysts. These data suggest that ectopic expression of cadherin 8 in renal epithelial cells is sufficient to cause the morphogenic pattern of cyst formation.

Assembly of Connexin43 into Gap Junctions Is Regulated Differentially by E-Cadherin and N-Cadherin in Rat Liver Epithelial Cells

Cadherins have been thought to facilitate the assembly of connexins (Cxs) into gap junctions (GJs) by enhancing cell-cell contact, however the molecular mechanisms involved in this process have remained unexplored. We examined the assembly of GJs composed of Cx43 in isogenic clones derived from immortalized and nontransformed rat liver epithelial cells that expressed either epithelial cadherin (E-Cad), which curbs the malignant behavior of tumor cells, or neuronal cadherin (N-Cad), which augments the invasive and motile behavior of tumor cells. We found that N-cad expression attenuated the assembly of Cx43 into GJs, whereas E-Cad expression facilitated the assembly. The expression of N-Cad inhibited GJ assembly by causing endocytosis of Cx43 via a nonclathrin-dependent pathway. Knock down of N-Cad by ShRNA restored GJ assembly. When both cadherins were simultaneously expressed in the same cell type, GJ assembly and disassembly occurred concurrently. Our findings demonstrate that E-Cad and N-Cad have opposite effects on the assembly of Cx43 into GJs in rat liver epithelial cells. These findings imply that GJ assembly and disassembly are the down-stream targets of the signaling initiated by E-Cad and N-Cad, respectively, and may provide one possible explanation for the disparate role played by these cadherins in regulating cell motility and invasion during tumor progression and invasion.

Spatiotemporal distribution and function of N‐cadherin in postnatal Schwann cells: A matter of adhesion?

During embryonic development of the peripheral nervous system (PNS), the adhesion molecule neuronal cadherin (N-cadherin) is expressed by Schwann cell precursors and associated with axonal growth cones. N-cadherin expression levels decrease as precursors differentiate into Schwann cells. In this study, we investigated the distribution of N-cadherin in the developing postnatal and adult rat peripheral nervous system. N-cadherin was found primarily in ensheathing glia throughout development, concentrated at neuron-glial or glial-glial contacts of the sciatic nerve, dorsal root ganglia (DRG), and myenteric plexi. In the sciatic nerve, N-cadherin decreases with age and progress of myelination. In adult animals, N-cadherin was found exclusively in nonmyelinating Schwann cells. The distribution of N-cadherin in developing E17 DRG primary cultures is similar to what was observed in vivo. Functional studies of N-cadherin in these cultures, using the antagonist peptide INPISGQ, show a disruption of the attachment between Schwann cells, but no interference in the initial or long-term contact between Schwann cells and axons. We suggest that N-cadherin acts primarily in the adhesion between glial cells during postnatal development. It may form adherents/junctions between nonmyelinating glia, which contribute to the stable tubular structure encapsulating thin caliber axons and thus stabilize the nerve structure as a whole.

TGF-beta3 inhibits chondrogenesis by suppressing precartilage condensation through stimulation of N-cadherin shedding and reduction of cRREB-1 expression.

Transforming growth factor-beta (TGF-beta) plays crucial roles in controlling cell differentiation and maintaining tissue integrity. Previously we reported that TGF-beta3 treatment decreased the mRNA expression of the gap junction protein, connexin 43 as well as cell number, which lead to the inhibition of chondrogenic condensation in cultured chick leg bud mesenchymal cells. The present study demonstrates that TGF-beta3 can induce cleavage in the ectodomain of neuronal cadherin (N-cadherin) at the initiation stage of chondrogenesis and reduce cell numbers, cellular adhesion and the expression level of connexin 43. Differential displayed PCR (DD-PCR) comparison of adherent- and non-adherent chick leg chondrogenic progenitor cells showed increased expression of the chick ras-responsive element binding transcription factor, cRREB-1, in adherent cells. In chick leg bud mesenchymal cells, cRREB-1 transcription was inhibited by TGF-beta3 at the early stage of chondrogenesis. Small interfering RNA (siRNA)-mediated knockdown of cRREB-1 reduced cell numbers, cellular adhesion, and the expression level of connexin 43 resulting in the inhibition of precartilage condensation. Taken together, these findings indicate that TGF-beta3 mediates the inhibitory signal necessary for precartilage condensation by stimulating N-cadherin shedding and reducing cRREB-1 expression levels.

Probing Intercellular Interactions between Vascular Endothelial Cadherin Pairs at Single-molecule Resolution and in Living Cells

Vascular endothelial (VE) cadherin is the surface glycoprotein cadherin specific to the endothelium that mediates cell-cell adhesion and plays a major role in the remodeling, gating, and maturation of vascular vessels. To investigate the contribution of individual VE-cadherins to endothelial cell-cell interactions and investigate whether different classical cadherins display different kinetics and micromechanical properties, we characterize the binding properties of VE-cadherin/VE-cadherin bonds at single-molecule resolution and in living human umbilical vein endothelial cells (HUVECs). Our single-molecule force spectroscopy measurements reveal that type II VE-cadherin molecules form bonds that are less prone to rupture and display a higher tensile strength than bonds formed by classical type I neuronal (N) cadherin and epithelial (E) cadherin. The equilibrium lifetime of the VE-cadherin/VE-cadherin bond is significantly longer than formed by N-cadherin/N-cadherin bonds and E-cadherin/E-cadherin bonds. These results indicate that VE-cadherins form bonds that have kinetics and mechanical properties that are significantly different from those formed by classical type I cadherins, properties that are particularly well adapted to the barrier and adhesive functions of VE-cadherin in endothelial cell-cell junctions.

Astroblastoma: Immunohistochemical and ultrastructural study of distinctive epithelial and probable tanycytic differentiation

We report the clinicopathological findings of astroblastoma found in an 8-year-old girl who was subsequently treated for 11 years. The primary superficially circumscribed tumor was located in the frontoparietal lobe, while the recurrent and the second recurrent tumor were restricted to the same region 11 years later. The tumors obtained on these three occasions showed fundamentally the same histological, immunohistochemical and fine structural features. They exhibited astrocytic as well as ependymal tanycytic features with apparent epithelial cell lineage. The tumor cells showed typical features of astroblastoma comprising prominent perivascular pseudorosettes with remarkable vascular sclerosis. The immunohistochemical study revealed intensive positivity of GFAP, vimentin, epithelial membrane antigen (EMA), cytokeratin, connexin 26 and 32, desmocollin 1 and neuronal cadherin. The fine structure revealed divergent types of junctional complexes, some of which were connected with tonofilament bundles. Numerous microvilli protruded and basal lamina abutted on the tumor cell surface. We report these unique histological features, and stress that astroblastoma should be categorized as a specific type of neuroepithelial tumor.

ADAM10 cleavage of N-cadherin and regulation of cell–cell adhesion and β-catenin nuclear signalling

Cadherins are critically involved in tissue development and tissue homeostasis. We demonstrate here that neuronal cadherin (N-cadherin) is cleaved specifically by the disintegrin and metalloproteinase ADAM10 in its ectodomain. ADAM10 is not only responsible for the constitutive, but also for the regulated, shedding of this adhesion molecule in fibroblasts and neuronal cells directly regulating the overall levels of N-cadherin expression at the cell surface. The ADAM10-induced N-cadherin cleavage resulted in changes in the adhesive behaviour of cells and also in a dramatic redistribution of beta-catenin from the cell surface to the cytoplasmic pool, thereby influencing the expression of beta-catenin target genes. Our data therefore demonstrate a crucial role of ADAM10 in the regulation of cell-cell adhesion and on beta-catenin signalling, leading to the conclusion that this protease constitutes a central switch in the signalling pathway from N-cadherin at the cell surface to beta-catenin/LEF-1-regulated gene expression in the nucleus.

DN-Cadherin Is Required for Spatial Arrangement of Nerve Terminals and Ultrastructural Organization of Synapses

We studied roles of DN-cadherin, the Drosophila major neuronal cadherin, in neuronal connections in the visual system. In DN-cadherin mutants, axon terminals of a large subset of photoreceptor cells reached and associated with their target interneurons, but their characteristic spatial arrangement was disrupted as synaptogenesis proceeded. Although synapses were formed at contact sites between the axon terminals and target neurons, underlying cytoplasmic structures were not fully specialized at both pre- and postsynaptic terminals and synaptic vesicles appeared to accumulate at the presynapses. These results suggest that the cadherin adhesion system is required for interaction between pre- and postsynaptic terminals and for generation of the mature synaptic structures.

Mutational analysis of the neuronal cadherin gene CELSR1 and exclusion as a candidate for catatonic schizophrenia in a large family.

The cadherin gene CELSR1 is specifically expressed in the brain and located on chromosome 22q13.33, a region that has recently been shown to be involved in the etiopathogenesis of familial catatonic schizophrenia. The gene is a strong positional candidate and was considered for mutational analysis. A total of 17 allelic variants of CELSR1 was found by sequencing all 35 exons, intron-exon junctions, and the putative promoter region by screening two patients from a large family mainly supporting this locus, and three control subjects in a first step. No variant exclusively co-segregates with the disease in the large pedigree, providing evidence that CELSR1 is not causative for the pathogenesis of catatonic schizophrenia in this family.

Expressed cadherin pseudogenes are localized to the critical region of the spinal muscular atrophy gene.

Low-copy repeats have been associated with genomic rearrangements and have been implicated in the generation of mutations in several diseases. Here we characterize a subset of low-copy repeats in the spinal muscular atrophy (SMA) region in human chromosome 5q13. We show that this repeated sequence, named c41-cad, is a highly expressed pseudogene derived from an intact neuronal cadherin gene, Br-cadherin, situated on 5p13-14. Br-cadherin is expressed specifically in the brain, whereas the c41-cad transcripts are 10-15 times more abundant and are present in all tissues examined. We speculate that the c41-cad repeats, separately or in concert with other repeats in the SMA region, are involved in the pathogenesis of SMA by promoting rearrangements and deletions.