Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification.

Cranial neural crest (CNC) cells migrate extensively, typically in a pattern of cell streams. In Xenopus, these cells express the adhesion molecule Xcadherin-11 (Xcad-11) as they begin to emigrate from the neural fold. In order to study the function of this molecule, we have overexpressed wild-type Xcad-11 as well as Xcad-11 mutants with cytoplasmic (deltacXcad-11) or extracellular (deltaeXcad-11) deletions. Green fluorescent protein (GFP) was used to mark injected cells. We then transplanted parts of the fluorescent CNC at the premigratory stage into non-injected host embryos. This altered not only migration, but also the expression of neural crest markers. Migration of transplanted cranial neural crest cells was blocked when full-length Xcad-11 or its mutant lacking the beta-catenin-binding site (deltacXcad-11) was overexpressed. In addition, the expression of neural crest markers (AP-2, Snail and twist) diminished within the first four hours after grafting, and disappeared completely after 18 hours. Instead, these grafts expressed neural markers (2G9, nrp-I and N-Tubulin). Beta-catenin co-expression, heterotopic transplantation of CNC cells into the pharyngeal pouch area or both in combination failed to prevent neural differentiation of the grafts. By contrast, deltaeXcad-11 overexpression resulted in premature emigration of cells from the transplants. The AP-2 and Snail patterns remained unaffected in these migrating grafts, while twist expression was strongly reduced. Co-expression of deltaeXcad-11 and beta-catenin was able to rescue the loss of twist expression, indicating that Wnt/beta-catenin signalling is required to maintain twist expression during migration. These results show that migration is a prerequisite for neural crest differentiation. Endogenous Xcad-11 delays CNC migration. Xcad-11 expression must, however, be balanced, as overexpression prevents migration and leads to neural marker expression. Although Wnt/beta-catenin signalling is required to sustain twist expression during migration, it is not sufficient to block neural differentiation in non-migrating grafts.     

Cadherin-11 Mediates Contact Inhibition of Locomotion during Xenopus Neural Crest Cell Migration

Collective cell migration is an essential feature both in embryonic development and cancer progression. The molecular mechanisms of these coordinated directional cell movements still need to be elucidated. The migration of cranial neural crest (CNC) cells during embryogenesis is an excellent model for collective cell migration in vivo. These highly motile and multipotent cells migrate directionally on defined routes throughout the embryo. Interestingly, local cell-cell interactions seem to be the key force for directionality. CNC cells can change their migration direction by a repulsive cell response called contact inhibition of locomotion (CIL). Cell protrusions collapse upon homotypic cell-cell contact and internal repolarization leads to formation of new protrusions toward cell-free regions. Wnt/PCP signaling was shown to mediate activation of small RhoGTPase RhoA and inhibition of cell protrusions at the contact side. However, the mechanism how a cell recognizes the contact is poorly understood. Here, we demonstrate that Xenopus cadherin-11 (Xcad-11) mediated cell-cell adhesion is necessary in CIL for directional and collective migration of CNC cells. Reduction of Xcad-11 adhesive function resulted in higher invasiveness of CNC due to loss of CIL. Additionally, transplantation analyses revealed that CNC migratory behaviour in vivo is non-directional and incomplete when Xcad-11 adhesive function is impaired. Blocking Wnt/PCP signaling led to similar results underlining the importance of Xcad-11 in the mechanism of CIL and directional migration of CNC.     

Xenopus cadherin-6 regulates growth and epithelial development of the retina

Cadherins are crucial for tissue cohesion, separation of cell layers and cell migration during embryogenesis. To investigate the role of classical type II Xcadherin-6 (Xcad-6), we performed loss-of-function studies by morpholino oligonucleotide injections. This resulted in severe eye defects which could be rescued with murine cadherin-6. In the absence of Xcadherin-6, morphological alterations and a decrease in cell proliferation were observed with eye cup formation. Eye field transplantations of Xcadherin-6 depleted donors yielded grafts that failed to form a proper neuroepithelium in a wildtype environment. At later developmental stages Xcadherin-6 deficient eyes showed lamination defects in the outer neural retina, a reduced thickness of the ganglion cell layer (GCL) and a fragmented retina pigment epithelium (RPE). Thus, Xcadherin-6 is essential early in eye development for structural organization and growth of the neuroepithelium before it differentiates into neural retina and RPE.     

Cadherin-6 Mediates the Heterotypic Interactions between the Hemopoietic Osteoclast Cell Lineage and Stromal Cells in a Murine Model of Osteoclast Differentiation

Osteoclasts are multinucleated cells of hemopoietic origin that are responsible for bone resorption during physiological bone remodeling and in a variety of bone diseases. Osteoclast development requires direct heterotypic cell–cell interactions of the hemopoietic osteoclast precursors with the neighboring osteoblast/stromal cells. However, the molecular mechanisms underlying these heterotypic interactions are poorly understood. We isolated cadherin-6 isoform, denoted cadherin-6/2 from a cDNA library of human osteoclast-like cells. The isolated cadherin-6/2 is 3,423 bp in size consisting of an open reading frame of 2,115 bp, which encodes 705 amino acids. This isoform lacks 85 amino acids between positions 333 and 418 and contains 9 different amino acids in the extracellular domain compared with the previously described cadherin-6. The human osteoclast-like cells also expressed another isoform denoted cadherin-6/1 together with the cadherin-6. Introduction of cadherin-6/2 into L-cells that showed no cell–cell contact caused evident morphological changes accompanied with tight cell–cell association, indicating the cadherin-6/2 we isolated here is functional. Moreover, expression of dominant-negative or antisense cadherin-6/2 construct in bone marrow–derived mouse stromal ST2 cells, which express only cadherin-6/2, markedly impaired their ability to support osteoclast formation in a mouse coculture model of osteoclastogenesis. Our results suggest that cadherin-6 may be a contributory molecule to the heterotypic interactions between the hemopoietic osteoclast cell lineage and osteoblast/bone marrow stromal cells required for the osteoclast differentiation. Since both osteoclasts and osteoblasts/bone marrow stromal cells are the primary cells controlling physiological bone remodeling, expression of cadherin-6 isoforms in these two cell types of different origin suggests a critical role of these molecules in the relationship of osteoclast precursors and cells of osteoblastic lineage within the bone microenvironment.     

The human cadherin-10 gene: complete coding sequence, predominant expression in the brain, and mapping on chromosome 5p13-14.

In a quest for novel cadherin gene family members in the human dbEST database, an interesting EST clone was identified and chosen for subsequent analysis. Using the technique of 5' rapid amplification of cDNA ends, we isolated the complete coding sequence and a large part of the UTRs of a novel gene. The sequence appeared to correspond to the human cadherin-10 gene, whose sequence was only partially known before. The expression pattern of this cadherin was found to be largely brain-specific, with additional expression in both adult and fetal kidney, and with minor expression in prostate and fetal lung. By FISH analysis the genomic location was determined at human chromosome 5p13-14, which is nearby the reported positions of the human cadherin-6, -12, and cadherin-14 (CDH18) genes. Cadherin-10 shows high relationship to the human cadherin-6 gene.     

Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development

In ventricular cells of the mouse telencephalon, differential expression of cadherin cell adhesion molecules defines neighbouring regions; R-cadherin delineates the future cerebral cortex, while cadherin-6 delineates the lateral ganglionic eminence. By using cell labelling analyses in the whole embryo culture system, we demonstrated that the interface between R-cadherin and cadherin-6 expression is a boundary for cell lineage restriction at embryonic day 10.5. Interestingly, when a group of cells with exogenous cadherin-6 were generated to straddle the cortico-straital boundary by electroporation at embryonic day 11.0, ectopic cadherin-6-expressing cortical cells were sorted into the striatal compartment, and the reverse was the trend for ectopic R-cadherin-expressing striatal cells. Although cadherin-6 gene knockout mice engineered in this study showed no obvious phenotype in telencephalic compartmentalisation, the preferential sorting of ectopic cadherin-6-expressing cells was abolished in this mutant background. Thus, the differential expression pattern of cadherins in the embryonic telencephalon is responsible for maintaining the cortico-striatal compartment boundary.     

Differential regulation of endogenous cadherin expression in Madin-Darby canine kidney cells by cell-cell adhesion and activation of beta -catenin signaling

Cadherins mediate cell-cell adhesion, but little is known about how their expression is regulated. In Madin-Darby canine kidney (MDCK) cells, the cadherin-associated cytoplasmic proteins alpha- and beta-catenin form high molecular weight protein complexes with two glycoproteins (Stewart, D. B., and Nelson, W. J. (1997) J. Biol. Chem. 272, 29652-29662), one of which is E-cadherin and the other we show here is the type II cadherin, cadherin-6 (K-cadherin). In low density, motile MDCK cells, the steady-state level of cadherin-6 is low, but protein is synthesized. However, following cell-cell adhesion, cadherin-6 becomes stabilized and accumulates by >50-fold at cell-cell contacts while the E-cadherin level increases only 5-fold during the same period. To investigate a role of beta-catenin in regulation of cadherin expression in MDCK cells, we examined the effects of expressing signaling-active beta-catenin mutants (DeltaGSK, DeltaN90, and DeltaN131). In these cells, while levels of E-cadherin, alpha- and beta-catenin are similar to those in control cells, levels of cadherin-6 are significantly reduced due to rapid degradation of newly synthesized protein. Additionally, these cells appeared more motile and less cohesive, as expression of DeltaGSK-beta-catenin delayed the establishment of tight confluent cell monolayers compared with control cells. These results indicate that the level of cadherin-6, but not that of E-cadherin, is strictly regulated post-translationally in response to Wnt signaling, and that E-cadherin and cadherin-6 may contribute different properties to cell-cell adhesion and the epithelial phenotype.     

Characterization of three novel human cadherin genes (CDH7, CDH19, and CDH20) clustered on chromosome 18q22-q23 and with high homology to chicken cadherin-7

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5' and 3' rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3' RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse "cadherin-7" cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22-q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.     

Regulation of cadherin expression in the chicken neural crest by the Wnt/ β-catenin signaling pathway

In neural crest cell development, the expression of the cell adhesion proteins cadherin-7 and cadherin-11 commences after delamination of the neural crest cells from the neuroepithelium. The canonical Wnt signaling pathway is known to drive this delamination step and is a candidate for inducing expression of these cadherins at this time. This project was initiated to investigate the role of canonical Wnt signaling in the expression of cadherin-7 and cadherin-11 by treating neural crest cells with Wnt3a ligand. Expression of cadherin-11 was first confirmed in the neural crest cells for the chicken embryo. The changes in the expression level of cadherin-7 and -11 following the treatment with Wnt3a ligand were studied using real-time RT-PCR and immunostaining. Statistically significant up-regulation in the mRNA expression of cadherin-7 and cadherin-11 and in the amount of cadherin-7 and cadherin-11 protein found in cell-cell interfaces between neural crest cells was observed in response to Wnt, demonstrating that cadherin-7 and cadherin-11 expressed by the migrating neural crest cells can be regulated by the canonical Wnt pathway.     

Cadherin-11, a marker of the mesenchymal phenotype, regulates glioblastoma cell migration and survival in vivo

Glioblastoma multiforme (GBM) is the most malignant and lethal form of astrocytoma. The GBM patient survival time of approximately 1 year necessitates the identification of novel molecular targets and more effective therapeutics. Cadherin-11, a calcium-dependent cell-cell adhesion molecule and mesenchymal marker, plays a role in both normal tissue development and in cancer cell migration. The functional significance of cadherin-11 in GBM has not been investigated. Here, we show that cadherin-11 is expressed in human GBM tumors and human glioma stem-like cells by immunohistochemical labeling. In addition, we show that cadherin-11 is expressed in human glioma cell lines by immunoblotting. Short hairpin RNA-mediated knockdown of cadherin-11 expression in human glioma cell lines results in decreased migration and growth factor-independent cell survival in vitro. More importantly, knockdown of cadherin-11 inhibits glioma cell survival in heterotopic and orthotopic mouse xenograft models. Together, our results show the functional significance of cadherin-11 expression in GBM and provide evidence for a novel role of cadherin-11 in promoting glioma cell survival in an in vivo environment. Thus, our studies suggest cadherin-11 is a viable molecular target for therapeutic intervention in GBM.     

Interactions between cadherin-11 and platelet-derived growth factor receptor-alpha signaling link cell adhesion and proliferation

Cadherins are homophilic cell-to-cell adhesion molecules that help cells respond to environmental changes. Newly formed cadherin junctions are associated with increased cell phosphorylation, but the pathways driving this signaling response are largely unknown. Since cadherins have no intrinsic signaling activity, this phosphorylation must occur through interactions with other signaling molecules. We previously reported that cadherin-11 engagement activates joint synovial fibroblasts, promoting inflammatory and degradative pathways important in rheumatoid arthritis (RA) pathogenesis. Our objective in this study was to discover interacting partners that mediate cadherin-11 signaling. Protein array screening showed that cadherin-11 extracellular binding domains linked to an Fc domain (cad11Fc) induced platelet-derived growth factor (PDGFR)-α phosphorylation in synovial fibroblasts and glioblastoma cells. PDGFRs are growth factor receptor tyrosine kinases that promote cell proliferation, survival, and migration in mesodermally derived cells. Increased PDGFR activity is implicated in RA pathology and associates with poor prognosis in several cancers, including sarcoma and glioblastoma. PDGFRα activation by cadherin-11 signaling promoted fibroblast proliferation, a signaling pathway independent from cadherin-11-stimulated IL-6 or matrix metalloproteinase (MMP)-3 release. PDGFRα phosphorylation mediated most of the cad11Fc-induced phosphatidyl-3-kinase (PI3K)/Akt activation, but only part of the mitogen-activated protein kinase (MAPK) response. PDGFRα-dependent signaling did not require cell cadherin-11 expression. Rather, cad11Fc immunoprecipitated PDGFRα, indicating a direct interaction between cadherin-11 and PDGFRα extracellular domains. This study is the first to report an interaction between cadherin-11 and PDGFRα and adds to our growing understanding that cadherin-growth factor receptor interactions help balance the interplay between tissue growth and adhesion.     

Biochemical characterization and functional analysis of two type II classic cadherins, cadherin-6 and -14, and comparison with E-cadherin

Classic cadherins can be grouped based on their deduced primary structures. Among them the type I cadherins have been well characterized; however, little is known about non-type I cadherins. In this study we characterized two human type II cadherins, cadherin-6 and cadherin-14, using a cDNA transfection system. They were each detected as two bands electrophoretically, were expressed on the external cell surface at cell-cell contact sites, and were associated with caten- ins. Direct sequencing of the N-terminal amino acids showed that the two bands of cadherin-14 corresponded to precursor and mature forms, whereas the two bands of cadherin-6 both had the N-terminal sequence of the mature form. Unlike type I cadherins, both cadherin-6 and -14 were not protected from trypsin degradation by Ca2+. We evaluated their adhesive functions by a long term cell aggregation method. The results suggest that both cadherin-6 and -14 have cell-cell binding strengths virtually equivalent to that of E-cadherin and that their binding specificities are distinct from that of E-cadherin. Cadherin-6 and -14 interacted with each other in an incomplete manner. They have a QAI tripeptide in the first extracellular subdomain instead of the HAV motif that is characteristic of type I cadherins and is intimately involved in the adhesive function. The QAI tripeptide, however, appeared not to be involved in the adhesive functions of cadherin-6 and -14.     

Regulation of cadherin expression in the chicken neural crest by the Wnt/β-catenin signaling pathway

In neural crest cell development, the expression of the cell adhesion proteins cadherin-7 and cadherin-11 commences after delamination of the neural crest cells from the neuroepithelium. The canonical Wnt signaling pathway is known to drive this delamination step and is a candidate for inducing expression of these cadherins at this time. This project was initiated to investigate the role of canonical Wnt signaling in the expression of cadherin-7 and cadherin-11 by treating neural crest cells with Wnt3a ligand. Expression of cadherin-11 was first confirmed in the neural crest cells for the chicken embryo. The changes in the expression level of cadherin-7 and -11 following the treatment with Wnt3a were studied using real-time RT-PCR and immunostaining. Statistically significant upregulation in the mRNA expression of cadherin-7 and cadherin-11 and in the amount of cadherin-7 and cadherin-11 protein found in cell-cell interfaces between neural crest cells was observed in response to Wnt, demonstrating that cadherin-7 and cadherin-11 expressed by the migrating neural crest cells can be regulated by the canonical Wnt pathway.Key words: neural crest, Wnt, cadherin-7, cadherin-11     

Dynamic testicular adhesion junctions are immunologically unique. II. Localization of classic cadherins in rat testis

In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact and likely transmit signals between contacting cells. Defining the molecular composition of testicular cell-cell junctions is an important step in determining their function. Proteins belonging to the cadherin superfamily are important mediators of cell-cell adhesion, as well as cell signaling. Here, we determined the spatial and temporal protein expression of four classic cadherins in rat testis: N-cadherin, cadherin-6, cadherin-11, and a cadherin defined by an antiserum generated against a conserved classic cadherin peptide (L4). Through Western blot analysis, all antibodies recognized unique proteins. Similarly, each cadherin displayed unique, cell-type specific immunostaining patterns. Whereas N-cadherin, cadherin-11, and L4-positive cadherin were expressed from Postnatal Day 7 through adulthood, cadherin-6 protein was not present at Postnatal Day 7 and first appeared at Day 21. Immunostaining of testis cryosections on Postnatal Days 7, 21, 31, 43, and those of adults indicated that cadherin-11 localized to peritubular cell junctions. N-cadherin immunostaining localized to basal inter-Sertoli junctions, Sertoli-spermatocyte junctions, and at about stages I-VII in Sertoli-elongate spermatid junctions. Cadherin-6 immunostaining was restricted to Sertoli-round spermatid and in Sertoli-elongate spermatid junctions at approximately stages XII-I. Finally, L4-positive immunostaining also detected Sertoli-round spermatid junctions in addition to Sertoli-elongate spermatid junctions at approximately stages XII-I. These data show that the various testicular cell-cell junctions are molecularly unique and dynamic complexes.     

Regulated expression of cadherin-11 in human extravillous cytotrophoblasts undergoing aggregation and fusion in response to transforming growth factor beta 1

Transforming growth factor beta 1 is believed to be a key regulator of extravillous cytotrophoblast invasion during the first trimester of pregnancy. In addition, this growth factor has been shown to regulate cellular differentiation and fusion in cultured extravillous cytotrophoblasts. To date, the cellular mechanisms by which transforming growth factor beta 1 promotes these developmental processes remain poorly understood. Recent studies indicate that the expression of the novel cadherin subtype, known as cadherin-11, is associated with the terminal differentiation and fusion of villous cytotrophoblasts isolated from the human term placenta and human myoblasts in vitro. In this study, cadherin-11 mRNA and protein expression were examined in primary cultures of human extravillous cytotrophoblasts cultured in the presence of increasing concentrations of transforming growth factor beta 1 using northern and western blot analysis, respectively. Transforming growth factor beta 1 was shown to increase cadherin-11 mRNA and protein expression in these cultured extravillous cytotrophoblasts in a dose-dependent manner. Cadherin-11 was further localized to the large cellular aggregates and multinucleated cells that formed in response to increasing concentrations of transforming growth factor beta 1 using immunocytochemistry. Collectively, these observations suggest that the morphogenetic effects of transforming growth factor beta 1 on cultured extravillous cytotrophoblasts are mediated, at least in part, by an increase in cadherin-11 expression. This study not only adds to the understanding of the cellular mechanisms by which transforming growth factor beta 1 promotes trophoblast differentiation and fusion but provides useful insight into the cell biology of the cadherins.     

Cadherin-11 promotes the metastasis of prostate cancer cells to bone

Bone is the most common site of metastases from prostate cancer. The mechanism by which prostate cancer cells metastasize to bone is not fully understood, but interactions between prostate cancer cells and bone cells are thought to initiate the colonization of metastatic cells at that site. Here, we show that cadherin-11 (also known as osteoblast-cadherin) was highly expressed in prostate cancer cell line derived from bone metastases and had strong homophilic binding to recombinant cadherin-11 in vitro. Down-regulation of cadherin-11 in bone metastasis-derived PC3 cells with cadherin-11-specific short hairpin RNA (PC3-shCad-11) significantly decreased the adhesion of those cells to cadherin-11 in vitro. In a mouse model of metastasis, intracardiac injection of PC3 cells led to metastasis of those cells to bone. However, the incidence of PC3 metastasis to bone in this model was reduced greatly when the expression of cadherin-11 by those cells was silenced. The clinical relevance of cadherin-11 in prostate cancer metastases was further studied by examining the expression of cadherin-11 in human prostate cancer specimens. Cadherin-11 was not expressed by normal prostate epithelial cells but was detected in prostate cancer, with its expression increasing from primary to metastatic disease in lymph nodes and especially bone. Cadherin-11 expression was not detected in metastatic lesions that occur in other organs. Collectively, these findings suggest that cadherin-11 is involved in the metastasis of prostate cancer cells to bone.     

Beta-Actin Is Required for Proper Mouse Neural Crest Ontogeny

The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11.