Distinct roles of cadherin-6 and E-cadherin in tubulogenesis and lumen formation

Classic cadherins are important regulators of tissue morphogenesis. The predominant cadherin in epithelial cells, E-cadherin, has been extensively studied because of its critical role in normal epithelial development and carcinogenesis. Epithelial cells may also coexpress other cadherins, but their roles are less clear. The Madin Darby canine kidney (MDCK) cell line has been a popular mammalian model to investigate the role of E-cadherin in epithelial polarization and tubulogenesis. However, MDCK cells also express relatively high levels of cadherin-6, and it is unclear whether the functions of this cadherin are redundant to those of E-cadherin. We investigate the specific roles of both cadherins using a knockdown approach. Although we find that both cadherins are able to form adherens junctions at the basolateral surface, we show that they have specific and mutually exclusive roles in epithelial morphogenesis. Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation. Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity. In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.     

The Fat-like cadherin CDH-4 controls axon fasciculation, cell migration and hypodermis and pharynx development in Caenorhabditis elegans

Cadherins are one of the major families of adhesion molecules with diverse functions during embryonic development. Fat-like cadherins form an evolutionarily conserved subgroup characterized by an unusually large number of cadherin repeats in the extracellular domain. Here we describe the role of the Fat-like cadherin CDH-4 in Caenorhabditis elegans development. Cdh-4 mutants are characterized by hypodermal defects leading to incompletely penetrant embryonic or larval lethality with variable morphogenetic defects. Independently of the morphogenetic defects cdh-4 mutant animals also exhibit fasciculation defects in the ventral and dorsal cord, the major longitudinal axon tracts, as well as migration defects of the Q neuroblasts. In addition CDH-4 is essential for establishing and maintaining the attachment between the buccal cavity and the pharynx. Cdh-4 is expressed widely in most affected cells and tissues during embryogenesis suggesting that CDH-4 functions to ensure that proper cell contacts are made and maintained during development.     

Mice lacking the giant protocadherin mFAT1 exhibit renal slit junction abnormalities and a partially penetrant cyclopia and anophthalmia phenotype

While roles in adhesion and morphogenesis have been documented for classical cadherins, the nonclassical cadherins are much less well understood. Here we have examined the functions of the giant protocadherin FAT by generating a transgenic mouse lacking mFAT1. These mice exhibit perinatal lethality, most probably caused by loss of the renal glomerular slit junctions and fusion of glomerular epithelial cell processes (podocytes). In addition, some mFAT1(-/-) mice show defects in forebrain development (holoprosencephaly) and failure of eye development (anophthalmia). In contrast to Drosophila, where FAT acts as a tumor suppressor gene, we found no evidence for abnormalities of proliferation in two tissues (skin and central nervous system [CNS]) containing stem and precursor cell populations and in which FAT is expressed strongly. Our results confirm a necessary role for FAT1 in the modified adhesion junctions of the renal glomerular epithelial cell and reveal hitherto unsuspected roles for FAT1 in CNS development.     

R-cadherin influences cell motility via Rho family GTPases

Classical cadherins are the transmembrane proteins of the adherens junction and mediate cell-cell adhesion via homotypic interactions in the extracellular space. In addition, they mediate connections to the cytoskeleton by means of their association with catenins. Decreased cadherin-mediated adhesion has been implicated as an important component of tumorigenesis. Cadherin switching is central to the epithelial to mesenchymal transitions that drive normal developmental processes. Cadherin switching has also been implicated in tumorigenesis, particularly in metastasis. Recently, cadherins have been shown to be engaged in cellular activities other than adhesion, including motility, invasion, and signaling. In this study, we show that inappropriate expression of R-cadherin in tumor cells results in decreased expression of endogenous cadherins (cadherin switching) and sustained signaling through Rho GTPases. In addition, we show that R-cadherin induces cell motility when expressed in epithelial cells and that this increased motility is dependent upon Rho GTPase activity.     

Desmosomal adhesion regulates epithelial morphogenesis and cell positioning

Desmosomes are intercellular junctions of epithelia and are of widespread importance in the maintenance of tissue architecture. We provide evidence that desmosomal adhesion has a function in epithelial morphogenesis and cell-type-specific positioning. Blocking peptides corresponding to the cell adhesion recognition (CAR) sites of desmosomal cadherins block alveolar morphogenesis by epithelial cells from mammary lumen. Desmosomal CAR-site peptides also disrupt positional sorting of luminal and myoepithelial cells in aggregates formed by the reassociation of isolated cells. We demonstrate that desmosomal cadherins and E-cadherin are comparably involved in epithelial morphoregulation. The results indicate a wider role for desmosomal adhesion in morphogenesis than has previously been considered.     

Cadherins and catenins in dendrite and synapse morphogenesis

Neurons are highly polarized specialized cells. Neuronal integrity and functional roles are critically dependent on dendritic architecture and synaptic structure, function and plasticity. The cadherins are glycosylated transmembrane proteins that form cell adhesion complexes in various tissues. They are associated with a group of cytosolic proteins, the catenins. While the functional roles of the complex have been extensively investigates in non-neuronal cells, it is becoming increasingly clear that components of the complex have critical roles in regulating dendritic and synaptic architecture, function and plasticity in neurons. Consistent with these functional roles, aberrations in components of the complex have been implicated in a variety of neurodevelopmental disorders. In this review, we discuss the roles of the classical cadherins and catenins in various aspects of dendrite and synapse architecture and function and their relevance to human neurological disorders. Cadherins are glycosylated transmembrane proteins that were initially identified as Ca²⁺-dependent cell adhesion molecules. They are present on plasma membrane of a variety of cell types from primitive metazoans to humans. In the past several years, it has become clear that in addition to providing mechanical adhesion between cells, cadherins play integral roles in tissue morphogenesis and homeostasis. The cadherin family is composed of more than 100 members and classified into several subfamilies, including classical cadherins and protocadherins. Several of these cadherin family members have been implicated in various aspects of neuronal development and function.^1-3 The classical cadherins are associated with a group of cytosolic proteins, collectively called the catenins. While the functional roles of the cadherin-catenin cell adhesion complex have been extensively investigated in epithelial cells, it is now clear that components of the complex are well expressed in central neurons at different stages during development.^4,5 Recent exciting studies have shed some light on the functional roles of cadherins and catenins in central neurons. In this review, we will provide a brief overview of the cadherin superfamily, describe cadherin family members expressed in central neurons, cadherin-catenin complexes in central neurons and then focus on role of the cadherin-catenin complex in dendrite morphogenesis and synapse morphogenesis, function and plasticity. The final section is dedicated to discussion of the emerging list of neural disorders linked to cadherins and catenins. While the roles of cadherins and catenins have been examined in several different types of neurons, the focus of this review is their role in mammalian central neurons, particularly those of the cortex and hippocampus. Accompanying this review is a series of excellent reviews targeting the roles of cadherins and protocadherins in other aspects of neural development.     

Cadherin superfamily proteins in Caenorhabditis elegans and Drosophila melanogaster

The ability to form selective cell-cell adhesions is an essential property of metazoan cells. Members of the cadherin superfamily are important regulators of this process in both vertebrates and invertebrates. With the advent of genome sequencing projects, determination of the full repertoire of cadherins available to an organism is possible and here we present the identification and analysis of the cadherin repertoires in the genomes of Caenorhabditis elegans and Drosophila melanogaster. Hidden Markov models of cadherin domains were matched to the protein sequences obtained from the translation of the predicted gene sequences. Matches were made to 21 C. elegans and 18 D. melanogaster sequences. Experimental and theoretical work on C. elegans sequences, and data from ESTs, show that three pairs of genes, and two triplets, should be merged to form five single genes. It also produced sequence changes at one or both of the 5' and 3' termini of half the sequences. In D. melanogaster it is probable that two of the cadherin genes should also be merged together and that three cadherin genes should be merged with other neighbouring genes.Of the 15 cadherin proteins found in C. elegans, 13 have the features of cell surface proteins, signal sequences and transmembrane helices; the other two have only signal sequences. Of the 17 in D. melanogaster, 11 at present have both features and another five have transmembrane helices. The evidence currently available suggests about one-third of the cadherins in the two organisms can be grouped into subfamilies in which all, or parts of, the molecules are conserved. Each organism also has a approximately 980 residue protein (CDH-11 and CG11059) with two cadherin domains and whose sequences match well over their entire length two proteins from human brain. Two proteins in C. elegans, HMR-1A and HMR-1B, and three in D. melanogaster, CadN, Shg and CG7527, have cytoplasmic domains homologous to those of the classical cadherin genes of chordates but their extracellular regions have different domain structures. Other common subclasses include the seven-helix membrane cadherins, Fat-like protocadherins and the Ret-like cadherins. At present, the remaining cadherins have no obvious similarities in their extracellular domain architecture or homologies to their cytoplasmic domains and may, therefore, represent species-specific or phylum-specific molecules.     

Differential expression of E-cadherin,N-cadherin and beta-catenin in proximal and distal segments of the rat nephron.

Background  

The classical cadherins such as E- and N-cadherin are Ca²⁺-dependent cell adhesion molecules that play important roles in the development and maintenance of renal epithelial polarity. Recent studies have shown that a variety of cadherins are present in the kidney and are differentially expressed in various segments of the nephron. However, the interpretation of these findings has been complicated by the fact that the various studies focused on different panels of cadherins and utilized different species. Moreover, since only a few of the previous studies focused on the rat, information regarding the expression and localization of renal cadherins in this important species is lacking. In the present study, we have employed dual immunofluorescent labeling procedures that utilized specific antibodies against either E- or N-cadherin, along with antibodies that target markers for specific nephron segments, to characterize the patterns of cadherin expression in frozen sections of adult rat kidney.     

The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy

We previously identified a 220-kD constitutive protein of the plasma membrane undercoat which colocalizes at the immunofluorescence microscopic level with cadherins and occurs not only in epithelial M., S. Yonemura, A. Nagafuchi, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). To clarify the nature and possible functions of this protein, we cloned its full-length cDNA and sequenced it. Unexpectedly, we found mouse 220-kD protein to be highly homologous to rat protein ZO-1, only a part of which had been already sequenced. This relationship was confirmed by immunoblotting with anti-ZO-1 antibody. As protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, we analyzed the distribution of cadherins and the 220-kD protein by ultrathin cryosection immunoelectron microscopy. We found that in non-epithelial cells lacking tight junctions cadherins and the 220-kD protein colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions cadherins and the 220-kD protein are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, which tight junctions are not so well developed, the 220-kD protein is detected not only in the tight junction zone but also at adherens junctions. Furthermore, we show in mouse L cells transfected with cDNAs encoding N-, P-, E-cadherins that cadherins interact directly or indirectly with the 220-kD protein. Possible functions of the 220-kD protein (ZO-1) are discussed with special reference to the molecular mechanism for adherens and tight junction formation.     

p120 catenin is essential for mesenchymal cadherin-mediated regulation of cell motility and invasiveness

During epithelial tumor progression, the loss of E-cadherin expression and inappropriate expression of mesenchymal cadherins coincide with increased invasiveness. Reexpression experiments have established E-cadherin as an invasion suppressor. However, the mechanism by which E-cadherin suppresses invasiveness and the role of mesenchymal cadherins are poorly understood. We show that both p120 catenin and mesenchymal cadherins are required for the invasiveness of E-cadherin-deficient cells. p120 binding promotes the up-regulation of mesenchymal cadherins and the activation of Rac1, which are essential for cell migration and invasiveness. p120 also promotes invasiveness by inhibiting RhoA activity, independently of cadherin association. Furthermore, association of endogenous p120 with E-cadherin is required for E-cadherin-mediated suppression of invasiveness and is accompanied by a reduction in mesenchymal cadherin levels. The data indicate that p120 acts as a rheostat, promoting a sessile cellular phenotype when associated with E-cadherin or a motile phenotype when associated with mesenchymal cadherins.     

Association of an A-kinase-anchoring protein signaling scaffold with cadherin adhesion molecules in neurons and epithelial cells

A-kinase-anchoring protein (AKAP) 79/150 organizes a scaffold of cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and protein phosphatase 2B/calcineurin that regulates phosphorylation pathways underlying neuronal long-term potentiation and long-term depression (LTD) synaptic plasticity. AKAP79/150 postsynaptic targeting requires three N-terminal basic domains that bind F-actin and acidic phospholipids. Here, we report a novel interaction of these domains with cadherin adhesion molecules that are linked to actin through beta-catenin (beta-cat) at neuronal synapses and epithelial adherens junctions. Mapping the AKAP binding site in cadherins identified overlap with beta-cat binding; however, no competition between AKAP and beta-cat binding to cadherins was detected in vitro. Accordingly, AKAP79/150 exhibited polarized localization with beta-cat and cadherins in epithelial cell lateral membranes, and beta-cat was present in AKAP-cadherin complexes isolated from epithelial cells, cultured neurons, and rat brain synaptic membranes. Inhibition of epithelial cell cadherin adhesion and actin polymerization redistributed intact AKAP-cadherin complexes from lateral membranes to intracellular compartments. In contrast, stimulation of neuronal pathways implicated in LTD that depolymerize postsynaptic F-actin disrupted AKAP-cadherin interactions and resulted in loss of the AKAP, but not cadherins, from synapses. This neuronal regulation of AKAP79/150 targeting to cadherins may be important in functional and structural synaptic modifications underlying plasticity.     

N-cadherin-mediated cell motility requires cis dimers

Cadherins are expressed on the cell surface as a dimer in the membrane of one cell (cis dimer) that interacts with a cis dimer on an adjacent cell to form an adhesive trans dimer. It is well established that both cis and trans dimers must form for the cadherin to be an effective adhesion protein. In addition to their adhesive activity cadherins also play an important role in modulating cell behavior by regulating cell motility and signal transduction. Whether or not cis or trans dimers are necessary for the nonadhesive functions of cadherins has not been addressed. Here we show that N-cadherin cis dimers are necessary to induce cell motility in epithelial cells and that N-cadherin's ability to modulate the steady state levels of activated small GTPases requires both cis and trans dimers.     

N-Cadherin-Mediated Cell Motility Requires Cis Dimers

Cadherins are expressed on the cell surface as a dimer in the membrane of one cell (cis dimer) that interacts with a cis dimer on an adjacent cell to form an adhesive trans dimer. It is well established that both cis and trans dimers must form for the cadherin to be an effective adhesion protein. In addition to their adhesive activity cadherins also play an important role in modulating cell behavior by regulating cell motility and signal transduction. Whether or not cis or trans dimers are necessary for the nonadhesive functions of cadherins has not been addressed. Here we show that N-cadherin cis dimers are necessary to induce cell motility in epithelial cells and that N-cadherin's ability to modulate the steady state levels of activated small GTPases requires both cis and trans dimers.     

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.     

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.     

Rac activation upon cell-cell contact formation is dependent on signaling from the epidermal growth factor receptor

Cadherins are transmembrane receptors that mediate cell-cell adhesion. They play an essential role in embryonic development and maintenance of tissue architecture. The Rho family small GTPases regulate actin cytoskeletal dynamics in different cell types. The function of two family members, Rho and Rac, is required for the stability of cadherins at cell-cell contacts. Consistent with the published data we have found that Rac is activated upon induction of intercellular adhesion in epithelial cells. This activation is dependent on functional cadherins (Nakagawa, M., Fukata, M., Yamaga, M., Itoh, N., and Kaibuchi, K. (2001) J. Cell Sci. 114, 1829-1838; Noren, N. K., Niessen, C. M., Gumbiner, B. M., and Burridge, K. (2001) J. Biol. Chem. 276, 3305-3308). Here we show for the first time that clustering of cadherins using antibody-coated beads is sufficient to promote Rac activation. In the presence of Latrunculin B, Rac can be partially activated by antibody-clustered cadherins. These results suggest that actin polymerization is not required for initial Rac activation. Contrary to what has been described before, phosphatidylinositol 3-kinases are not involved in Rac activation following cell-cell adhesion in keratinocytes. Interestingly, inhibition of epidermal growth factor receptor signaling efficiently blocks the increased Rac-GTP levels observed after contact formation. We conclude that cadherin-dependent adhesion can activate Rac via epidermal growth factor receptor signaling.     

Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules

A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E- cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.     

Identification of a cadherin cell adhesion recognition sequence

The molecular mechanisms by which the cadherins interact with one another to promote cell adhesion have not been elucidated. In particular, the amino acid sequences of the cadherin cell adhesion recognition sites have not been determined. Here we demonstrate that synthetic peptides containing the sequence HAV, which is common to all of the cadherins, inhibit two processes (compaction of eight-cell-stage mouse embryos and rat neurite outgrowth on astrocytes) that are known to be mediated by cadherins. The data suggest that the tripeptide HAV is a component of a cadherin cell adhesion recognition sequence.     

The classic cadherins in synaptic specificity

During brain development, billions of neurons organize into highly specific circuits. To form specific circuits, neurons must build the appropriate types of synapses with appropriate types of synaptic partners while avoiding incorrect partners in a dense cellular environment. Defining the cellular and molecular rules that govern specific circuit formation has significant scientific and clinical relevance because fine scale connectivity defects are thought to underlie many cognitive and psychiatric disorders. Organizing specific neural circuits is an enormously complicated developmental process that requires the concerted action of many molecules, neural activity, and temporal events. This review focuses on one class of molecules postulated to play an important role in target selection and specific synapse formation: the classic cadherins. Cadherins have a well-established role in epithelial cell adhesion, and although it has long been appreciated that most cadherins are expressed in the brain, their role in synaptic specificity is just beginning to be unraveled. Here, we review past and present studies implicating cadherins as active participants in the formation, function, and dysfunction of specific neural circuits and pose some of the major remaining questions.