Endogenous Cadherins Research Articles

Transcriptome analysis reveals synergistic modulation of E-cadherin/N-cadherin in hMSC aggregates chondrogenesis.

N-cadherin-mediated cell adhesion is a vital inductor for mesenchymal condensation in chondrogenesis. Recent studies have revealed the involvement of E-cadherin in enhancing the multipotency of mesenchymal stem cells (MSCs) and limb development; however, the signaling crosstalk of E/N-cadherin remains unclear. This study aimed to explore the synergistic modulation of E/N-cadherin in the chondrogenic differentiation of MSC aggregates. Human E/N-cadherin-functionalized (hE/N-cad-Fc) poly (lactic-co-glycolic acid) (PLGA) microparticles (hE/N-cad-PLGA) were incorporated into the human MSC (hMSC) aggregates to upregulate the expression of the corresponding endogenous cadherin. The chondrogenic differentiation of the hMSC aggregates was initiated by hE/N-cad-PLGA, controlling the release of transforming growth factor-β (TGF-β). A transcriptome analysis was used to assess differentially expressed genes (DEGs) modulated by hE/N-cad-Fc in hMSC aggregate chondrogenesis. Gene functions and signaling pathways were assessed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The associated biological pathways were assessed by a protein-protein interaction (PPI) network analysis, and the results were further confirmed by real-time quantitative PCR (qPCR) and western blotting. A total of 1083 DEGs, comprising 111 upregulated and 972 downregulated genes, were discovered to be related to the enhanced chondrogenic differentiation modulated by hE/N-cad-Fc. The GO and KEGG functional enrichment analyses revealed that hE/N-cad-Fc synergistically regulated the p53-related survival signaling pathway. PPI analysis revealed that mitogen-activated protein kinases (MAPK) caspase regulation is a core aspect of the chondrogenic differentiation process, confirmed by western blotting. To the best of our knowledge, our study is the first to reveal that the synergistic modulation of E/N-cadherin enhances the chondrogenic differentiation of hMSCs via the ERK1/2-p53 signaling axis.

Extracellular Vesicle Transfer from Endothelial Cells Drives VE-Cadherin Expression in Breast Cancer Cells, Thereby Causing Heterotypic Cell Contacts.

Cadherins mediate cohesive contacts between isotypic cells by homophilic interaction and prevent contact between heterotypic cells. Breast cancer cells neighboring endothelial cells (ECs) atypically express vascular endothelial (VE)-cadherin. To understand this EC-induced VE-cadherin expression in breast cancer cells, MCF7 and MDA-MB-231 cells expressing different endogenous cadherins were co-cultured with ECs and analyzed for VE-cadherin at the transcriptional level and by confocal microscopy, flow cytometry, and immunoblotting. After losing their endogenous cadherins and neo-expression of VE-cadherin, these cells integrated into an EC monolayer without compromising the barrier function instantly. However, they induced the death of nearby ECs. EC-derived extracellular vesicles (EVs) contained soluble and membrane-anchored forms of VE-cadherin. Only the latter was re-utilized by the cancer cells. In a reporter gene assay, EC-adjacent cancer cells also showed a juxtacrine but no paracrine activation of the endogenous VE-cadherin gene. This cadherin switch enabled intimate contact between cancer and endothelial cells in a chicken chorioallantoic membrane tumor model showing vasculogenic mimicry (VM). This EV-mediated, EC-induced cadherin switch in breast cancer cells and the neo-expression of VE-cadherin mechanistically explain the mutual communication in the tumor microenvironment. Hence, it may be a target to tackle VM, which is often found in breast cancers of poor prognosis.

Cadherin 26 is an alpha integrin-binding epithelial receptor regulated during allergic inflammation.

Cadherins mediate diverse processes critical in inflammation including cell adhesion, migration, and differentiation. Herein, we report that the uncharacterized cadherin 26 (CDH26) is highly expressed by epithelial cells in human allergic gastrointestinal tissue. In vitro, CDH26 promotes calcium-dependent cellular adhesion of cells lacking endogenous cadherins by a mechanism involving homotypic binding and interaction with catenin family members (alpha, beta, and p120), as assessed by biochemical assays. Additionally, CDH26 enhances cellular adhesion to recombinant integrin α4β7 in vitro; conversely, recombinant CDH26 binds αE and α4 integrins in biochemical and cellular functional assays, respectively. Interestingly, CDH26-Fc inhibits activation of human CD4+ T cells in vitro including secretion of IL-2. Taken together, we have identified a novel functional cadherin regulated during allergic responses with unique immunomodulatory properties, as it binds α4 and αE integrins and regulates leukocyte adhesion and activation, and may thus represent a novel checkpoint for immune regulation and therapy via CDH26-Fc.

E-cadherin cytoplasmic domain inhibits cell surface localization of endogenous cadherins and fusion of C2C12 myoblasts.

ABSTRACTMyoblast fusion is a highly regulated process that is essential for skeletal muscle formation during muscle development and regeneration in mammals. Much remains to be elucidated about the molecular mechanism of myoblast fusion although cadherins, which are Ca2+-dependent cell–cell adhesion molecules, are thought to play a critical role in this process. Mouse myoblasts lacking either N-cadherin or M-cadherin can still fuse to form myotubes, indicating that they have no specific function in this process and may be functionally replaced by either M-cadherin or N-cadherin, respectively. In this study, we show that expressing the E-cadherin cytoplasmic domain ectopically in C2C12 myoblasts inhibits cell surface localization of endogenous M-cadherin and N-cadherin, as well as cell–cell fusion. This domain, however, does not inhibit myoblast differentiation according to microarray-based gene expression analysis. In contrast, expressing a dominant-negative β-catenin mutant ectopically, which suppresses Wnt/β-catenin signaling, did not inhibit cell–cell fusion. Therefore, the E-cadherin cytoplasmic domain inhibits cell–cell fusion by inhibiting cell surface localization of endogenous cadherins and not by inhibiting Wnt/β-catenin signaling.

P‐cadherin functional role is dependent on E‐cadherin cellular context: a proof of concept using the breast cancer model

P-cadherin overexpression is associated with worse breast cancer survival, being a poor prognostic marker as well as a putative therapeutic target for the aggressive triple-negative and basal-like carcinomas (TNBCs). Previously, we have shown that P-cadherin promotes breast cancer invasion of cells where membrane E-cadherin was maintained; however, it suppresses invasion in models without endogenous cadherins, like melanomas. Here, we investigated if P-cadherin expression would interfere with the normal adhesion complex and which were the cellular/molecular consequences, constituting, in this way, a new mechanism by which E-cadherin invasive-suppressor function was disrupted. Using breast TNBC models, we demonstrated, for the first time, that P-cadherin co-localizes with E-cadherin, promoting cell invasion due to the disruption caused in the interaction between E-cadherin and cytoplasmic catenins. P-cadherin also induces cell migration and survival, modifying the expression profile of cells expressing wild-type E-cadherin and contributing to alter their cellular behaviour. Additionally, E- and P-cadherin co-expressing cells significantly enhanced in vivo tumour growth, compared with cells expressing only E- or only P-cadherin. Finally, we still found that co-expression of both molecules was significantly correlated with high-grade breast carcinomas, biologically aggressive, and with poor patient survival, being a strong prognostic factor in this disease. Our results show a role for E- and P-cadherin co-expression in breast cancer progression and highlight the potential benefit of targeting P-cadherin in the aggressive tumours expressing high levels of this protein.

A Positive Role of Cadherin in Wnt/β-Catenin Signalling during Epithelial-Mesenchymal Transition

The Wnt/β-catenin signalling pathway shares a key component, β-catenin, with the cadherin-based adhesion system. The signalling function of β-catenin is conferred by a soluble cytoplasmic pool that is unstable in the absence of a Wnt signal, whilst the adhesion function is based on a cadherin-bound, stable pool at the membrane. The cadherin complex is dynamic, allowing for cell-cell rearrangements such as epithelial-mesenchymal transition (EMT), where the complex turns over through internalisation. Potential interplay between the two pools remains poorly understood, but cadherins are generally considered negative regulators of Wnt signalling because they sequester cytoplasmic β-catenin. Here we explore how cellular changes at EMT affect the signalling capacity of β-catenin using two models of EMT: hepatocyte growth factor (HGF) treatment of MDCK cells, and gastrulation in embryonic development. We show that EMT not only provides a pool of signalling-competent β-catenin following internalisation of cadherin, but also significantly facilitates activation of the Wnt pathway in response to both Wnt signals and exogenous β-catenin. We further demonstrate that availability of β-catenin in the cytoplasm does not necessarily correlate with Wnt/β-catenin pathway activity, since blocking endocytosis or depleting endogenous cadherin abolishes pathway activation despite the presence of β-catenin in the cytoplasm. Lastly we present data suggesting that cadherins are required for augmented activation of the Wnt/β-catenin pathway in vivo. This suggests that cadherins play a crucial role in β-catenin-dependent transcription.

RET Modulates Cell Adhesion via Its Cleavage by Caspase in Sympathetic Neurons

RET is a tyrosine kinase receptor involved in numerous cellular mechanisms including proliferation, neuronal navigation, migration, and differentiation upon binding with glial cell derived neurotrophic factor family ligands. RET is an atypical tyrosine kinase receptor containing four cadherin domains in its extracellular part. Furthermore, it has been shown to act as a dependence receptor. Such a receptor is active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. However, different data suggest that RET is not always associated with the cell death/survival balance but rather provides positional information. We demonstrate here that caspase cleavage of RET is involved in the regulation of adhesion in sympathetic neurons. The cleavage of RET generates an N-terminal truncated fragment that functions as a cadherin accessory protein, modifying cadherin environment and potentiating cadherin-mediated cell aggregation. Thus, the caspase cleavage of RET generates two RET fragments: one intracellular domain that can trigger cell death in apoptotic permissive settings, and one membrane-anchored ectodomain with cadherin accessory activity. We propose that this latter function may notably be important for the adequate development of the superior cervical ganglion.

Spontaneous assembly and active disassembly balance adherens junction homeostasis

The homeostasis of adherens junctions was studied using E-cadherin and its two mutants tagged by the photoconvertible protein Dendra2 in epithelial A-431 cells and in CHO cells lacking endogenous cadherin. The first mutant contained point mutations of two elements, Lys738 and the dileucine motif that suppressed cadherin endocytosis. The second mutant contained, in addition, an extensive truncation that uncoupled the mutant from beta-catenin and p120. Surprisingly, the intact cadherin and its truncated mutant were recruited into the junctions with identical kinetics. The full-size cadherin was actively removed from the junctions by a process that was unaffected by the inactivation of its endocytic elements. The cadherin's apparent half-residence time in the junction was about 2 min. Cadherin clusters made of the truncated mutant exhibited much slower but ATP-independent junctional turnover. Taken together, our experiments showed that adherens junction homeostasis consists of three distinctive steps: cadherin spontaneous recruitment, its lateral catenin-dependent association, and its active release from the resulting clusters. The latter process, whose mechanism is not clear, may play an important role in various kinds of normal and abnormal morphogenesis.

GSK3β Activity Modifies the Localization and Function of Presenilin 1

Presenilin 1, a causative gene product of familial Alzheimer disease, has been reported to be localized mainly in the endoplasmic reticulum and Golgi membranes. However, endogenous Presenilin 1 also localizes at the plasma membrane as a biologically active molecule. Presenilin 1 interacts with N-cadherin/beta-catenin to form a trimeric complex at the synaptic site through its loop domain, whose serine residues (serine 353 and 357) can be phosphorylated by glycogen synthase kinase 3beta. Here, we demonstrate that cell-surface expression of Presenilin 1/gamma-secretase is enhanced by N-cadherin-based cell-cell contact. Physical interaction between Presenilin 1 and N-cadherin/beta-catenin plays an important role in this process. Glycogen synthase kinase 3beta-mediated phosphorylation of Presenilin 1 reduces its binding to N-cadherin, thereby down-regulating its cell-surface expression. Moreover, reduction of the Presenilin 1.N-cadherin.beta-catenin complex formation leads to an impaired activation of contact-mediated phosphatidylinositol 3-kinase/Akt cell survival signaling. Furthermore, phosphorylation of Presenilin 1 hinders epsilon-cleavage of N-cadherin, whereas epsilon-cleavage of APP remained unchanged. This is the first report that clarifies the regulatory mechanism of Presenilin 1/gamma-secretase with respect to its subcellular distribution and its differential substrate cleavage. Because the cleavage of various membrane proteins by Presenilin 1/gamma-cleavage is involved in cellular signaling, glycogen synthase kinase 3beta-mediated phosphorylation of Presenilin 1 should be deeply associated with signaling functions. Our findings indicate that the abnormal activation of glycogen synthase kinase 3beta can reduce neuronal viability and synaptic plasticity via modulating Presenilin 1/N-cadherin/beta-catenin interaction and thus have important implications in the pathophysiology of Alzheimer disease.

Expression of inappropriate cadherins by epithelial tumor cells promotes endocytosis and degradation of E-cadherin via competition for p120ctn

Cadherin cell-cell adhesion proteins play an important role in modulating the behavior of tumor cells. E-cadherin serves as a suppressor of tumor cell invasion, and when tumor cells turn on the expression of a non-epithelial cadherin, they often express less E-cadherin, enhancing the tumorigenic phenotype of the cells. Here, we show that when A431 cells are forced to express R-cadherin, they dramatically downregulate the expression of endogenous E- and P-cadherin. In addition, we show that this downregulation is owing to increased turnover of the endogenous cadherins via clathrin-dependent endocytosis. p120(ctn) binds to the juxtamembrane domain of classical cadherins and has been proposed to regulate cadherin adhesive activity. One way p120(ctn) may accomplish this is to serve as a rheostat to regulate the levels of cadherin. Here, we show that the degradation of E-cadherin in response to expression of R-cadherin is owing to competition for p120(ctn).

N-Cadherin Induces Hematopoietic Stem Cells in a Quiescent State in the Bone Marrow Niche.

Hematopoietic stem cells (HSCs) are responsible for blood cell production throughout the lifetime of individuals. Interaction of HSCs with their particular microenvironments, known as stem cell niches, is critical for maintaining the stem cell properties, including self-renewal capacity and the ability of differentiation into single or multiple lineages. The niche cells produce signaling molecules, extracellular matrix, and cell adhesion molecules, and regulate stem cell fates. Recently, it was clarified that long-term bone marrow (BM) repopulating (LTR) HSCs exist frequently in BM trabecular bone surface, and that N-cadherin + spindle-shaped osteoblasts (OBs) are identified as a major niche component. We found that side-population (SP) in c-Kit +Sca-1 +Lin −(KSL) fraction, which is the quiescent HSC in the OB niche, expressed N-cadherin. Expression of N-cadherin in both of the quiescent HSCs and OBs thought to be essential for an adherens junction between HSCs and OBs in the niche. However, the role of N-cadherin in hematopoiesis is still unclear.In this study, we focused on the function of N-cadherin in the maintenance of the stem cell specific property, such as cell adhesion, quiescence, and LTR-activity. To clarify the function of N-cadherin in hematopoiesis, we prepared the retroviruses expressing wild-type N-cadherin, transfected retroviruses into OP9 stromal cell line and KSL cells, and performed the coculture. After coculture of KSL cells with OP9 cells, long-term culture-initiating cells (LTC-ICs) were maintained on OP9 cells overexpressing WT-N-cadherin (OP9/WT-NCAD). In addition, overexpression of WT-N-cadherin in both of the KSL cells and stromal cells enhanced cobblestone formation. N-cadherin overexpressing KSL cell showed slow-cell division from the single cell, when they cultured on OP9/WT-N-cedherin or N-cadherin-Fc protein coated plates, suggesting that N-cadherin-mediated cell-cell adhesion between HSCs and stromal cells enhances the quiescence of HSCs and keeps HSCs in immature state in in vitro. To clarify the role of N-cadherin in the BM reconstitution ability of HSC, we transfected control-IRES-GFP, WT-N-cadherin-IRES-GFP and N-cedherin/390Δ-IRES-GFP retrovirus into the Ly5.1 BM mononuclear cells and transplanted into lethally irradiated Ly5.2 mice. N-cedherin/390Δ, which is a mutant N-cadherin with a deletion at the extracellular domain, exhibits a dominant negative effect on the activity of endogenous cadherins. Control and WT-N-cadherin expressing cell reconstitute the recipient mice BM, while N-cadherin/390Δ expressing cells did not. It suggests that the adhesion between HSCs and BM niche cell is indispensable for the LTR-activity. In addition, we found that WT-N-cadherin overexpressing HSCs were enriched in the SP fraction after 4 months of BM transplantation, indicating that N-cadherin-mediated cell adhesion induced HSCs in the quiescent and kept quiescent HSCs in the niche.Altogether, these observations suggest that N-cadherin is a critical niche factor for the maintenance of the quiescence and self-renewal activity of HSCs. N-cadherin promotes tight adhesion of HSCs to the niche and keeps HSCs in the quiescent state

Modulating the strength of cadherin adhesion: evidence for a novel adhesion complex

Adherens junctions and desmosomes are critical for embryogenesis and the integrity of adult tissues. To form these junctions, classical cadherins interact via alpha- and beta-catenin with the actin cytoskeleton, whereas desmosomal cadherins interact with the intermediate filament system. Here, we used a hormone-activated mutant N-cadherin expressed in fibroblasts to show the existence of a novel classical cadherin adhesion system. N-cadherin was fused at its C-terminus to a modified estrogen receptor ligand-binding domain (NcadER) that binds 4-hydroxytamoxifen (4OHT) and expressed in L cells, which lack an endogenous cadherin. Cells with the mutant cadherin (LNER cells) aggregated in the absence of 4OHT, but only in its presence formed tightly compacted aggregates like those formed by L cells expressing wild-type N-cadherin (LN cells). Compaction of LNER cells treated with 4OHT was accompanied by elevated levels of p120ctn in NcadER immunoprecipitates, compared to immunoprecipitates of non-treated cells, but without changes in alpha- and beta-catenin, or actin. Compaction induced by 4OHT was also accompanied by increased interaction of the NcadER with the cytoskeleton and increased vimentin organization. Vimentin co-immunoprecipitated with the NcadER/catenin complex, suggesting an interaction between cadherin and vimentin. The mechanism by which vimentin interacts with the cadherin appears to involve p120ctn as it co-immunoprecipitates and colocalizes with vimentin in the parent L cells, which lack a cadherin and alpha- and beta-catenins. Disrupting the actin cytoskeleton with cytochalasin B inhibited aggregation, whereas knocking down vimentin with specific siRNAs inhibited compaction. Based on our results we propose that a vimentin-based classical cadherin complex functions together with the actin-based complex to promote strong cell-cell adhesion in fibroblasts.

Adherens Junctions in Myelinating Schwann Cells Stabilize Schmidt-Lanterman Incisures via Recruitment of p120 Catenin to E-Cadherin

Schwann cell myelin contains highly compacted layers of membrane as well as noncompacted regions with a visible cytoplasm. One of these cytoplasmic compartments is the Schmidt-Lanterman incisure, which spirals through the compacted layers and is believed to help sustain the growth and function of compact myelin. Incisures contain adherens junctions (AJs), the key components of which are E-cadherin, its cytoplasmic partners called catenins, and F-actin. To explore in vivo the role of cadherin and catenins in incisures, E-cadherin mutant proteins that completely replace endogenous cadherin have been delivered to the cells using adenovirus. When the introduced cadherin lacked its extracellular domain, association of p120 catenin (p120ctn) with the cadherin did not occur, and incisures disappeared. Remarkably, the additional replacement of two phosphorylatable tyrosines by phenylalanine in the cytoplasmic tail of the mutant cadherin restored both p120ctn binding and incisure architecture, indicating that p120ctn recruitment is critical for incisures maintenance and might be regulated by phosphorylations. In addition, the ability of the p120ctn/cadherin complex to support incisures was blocked by mutation of the Rho GTPase regulatory region of the p120ctn, and downregulation of Rac1 activity at the junction reversed this inhibition. Because Rho GTPases regulate the state of the actin filaments, these findings suggest that one role of p120ctn in incisures is to organize the cytoskeleton at the AJ. Finally, developmental studies of Schwann cells demonstrated that p120ctn recruitment from the cytoplasm to the AJ occurs before the appearance of Rac1 GTPase and F-actin at the junction.

Regulation of cadherin stability and turnover by p120ctn: implications in disease and cancer

The strength of cadherin based cell-cell adhesion is modulated by signaling events that control the amount of cadherin present at the cell surface, and the clustering of cadherins into strong adhesive junctions. p120ctn has been indirectly implicated in clustering for some time, but it now appears that its main function is to regulate cadherin turnover. Forced p120 downregulation (e.g., by siRNA targeting) results in a striking dose-dependant loss of endogenous cadherins, indicating that p120 is essential for cadherin stability. These data challenge some important paradigms and suggest novel interpretations of existing data. For example, most of the effects of DN-cadherin expression can be accounted for by sequestration of p120. Thus, DN-cadherins phenocopy p120-downregulation, and a significant literature exists already that suggests consequences of p120-deficiency in disease and cancer. Moreover, p120 downregulation occurs frequently in essentially all of the major carcinoma types. Thus, it is possible that the classic observation of E-cadherin-deficiency in metastatic cancer may in some cases be due to p120 downregulation rather than better understood mechanisms acting at the level of E-cadherin transcription.

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.

Exogenous cadherin microdisplay can interfere with endogenous signaling and reprogram gene expression in cultured hepatocytes

We recently found that the basal micro substrate presentation of E-cadherin, a key cell-cell adhesion molecule in the liver, can modulate hepatocellular proliferative potential and differentiated function (Brieva and Moghe, in press). In the current study, we established a similar experimental model involving rat hepatocytes cultured on collagen and incorporated 5 microm polystyrene microbeads functionalized with Protein A-anchored E-cadherin/human lgG Fc chimeric fusion constructs. We investigated the cadherin governed dose-response of cell proliferative potential and quantified the underlying changes in intracellular gene signaling processes. Hepatocellular proliferative potential was found to be intensified with an increase in the microdisplay of acellular cadherins and this effect was offset by increased cell seeding density. Notably, we report that following overnight exposure to acellular cadherins, the expression of genes known to mediate the control of cell proliferation, cyclin D1 and c-myc, was upregulated, while the expression of differentiation-related genes, namely albumin and cytochrome p450 II B1, was reduced. The exposure of cell cultures to exogenous cadherins was found to markedly disrupt the localization of endogenous E-cadherin and beta-catenin to junctions at cell-cell contacts and cause a quantitative decrease in the endogenous cadherin protein levels. Based on all of our observations, we propose that the acellular presentation of E-cadherin chimeras competitively disrupts endogenous cadherin containing complexes at cell-cell junctions and increases intracellular cadherin turnover, thereby promoting beta-catenin mediated signaling, which ultimately engenders an increase in cell proliferative potential and a decrease in differentiated function.

Differential Regulation of Endogenous Cadherin Expression in Madin-Darby Canine Kidney Cells by Cell-Cell Adhesion and Activation of β-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.

Inhibiting cadherin function by dominant mutant E-cadherin expression increases the extent of tight junction assembly.

Previous studies have shown that induction of cadherin-mediated cell-cell adhesion leads to tight junction formation, and that blocking cadherin-mediated cell-cell adhesion inhibits tight junction assembly. Here we report analysis of tight junction assembly in MDCK cells overexpressing a mutant E-cadherin protein that lacks an adhesive extracellular domain (T151 cells). Mutant E-cadherin overexpression caused a dramatic reduction in endogenous cadherin levels. Despite this, tight junction assembly was extensive. The number of tight junction strands observed by freeze-fracture electron microscopy significantly increased in T151 cells compared to that in control cells. Our data indicate that the hierarchical regulation of junctional complex assembly is not absolute, and that inhibition of cadherin function has both positive and negative effects on tight junction assembly.

Mechanism of extracellular domain-deleted dominant negative cadherins.

The cadherin/catenin complex mediates Ca2+-dependent cell-cell interactions that are essential for normal developmental processes. It has been proposed that sorting of cells during embryonic development is due, at least in part, to expression of different cadherin family members or to expression of differing levels of a single family member. Expression of dominant-negative cadherins has been used experimentally to decrease cell-cell interactions in whole organisms and in cultured cells. In this study, we elucidated the mechanism of action of extracellular domain-deleted dominant-negative cadherin, showing that it is not cadherin isotype-specific, and that it must be membrane-associated but the orientation within the membrane does not matter. In addition, membrane-targeted cytoplasmic domain cadherin with the catenin-binding domain deleted does not function as a dominant-negative cadherin. Expression of extracellular domain-deleted dominant-negative cadherin results in down-regulation of endogenous cadherins which presumably contributes to the non-adhesive phenotype.

Activation of Transfected M1or M3Muscarinic Acetylcholine Receptors Induces Cell–Cell Adhesion of Chinese Hamster Ovary Cells Expressing Endogenous Cadherins

Expression of endogenous cadherins by Chinese hamster ovary (CHO) cells has not been previously reported. However, we observed that CHO cells adhere to one another upon activation of transfected muscarinic acetylcholine receptors (mAChR), suggesting that the cells express endogenous cadherins. A 160-base pair RT-PCR product with 100% homology to the cytoplasmic domain of human E-cadherin was amplified from CHO cells. A second RT-PCR product amplified from these cells has 92% homology to the cytoplasmic domain of human cadherin-9 and 86% homology to the cytoplasmic domain of human cadherin-6. Western blotting indicates that CHO cells express a 165-kDa protein recognized by E-cadherin antibodies and a 120-kDa protein recognized by an antibody to the cadherin C-terminus sequence. The ability of transfected mAChR subtypes to regulate cadherin-mediated adhesion of CHO cells was tested by measuring the permeation of horseradish peroxidase across confluent CHO cell monolayers, by microscopic examination of the cells, and by aggregation assays. Cell–cell adhesion is induced within 15 min of activating transfected M1or M3mAChR which functionally couple to protein kinase C (PKC). In contrast, CHO cell adhesion is not affected by activating transfected M2mAChR which functionally couple to other effectors. Activation of PKC with phorbol esters also induces cell–cell adhesion of all CHO sublines tested. Immunofluorescence assays reveal that endogenous cadherins redistribute on the plasma membrane of CHO cells following mAChR or PKC activation. Inactivation of cadherins by removal of extracellular Ca2+abrogates adhesion induced by mAChR or PKC activation. Our demonstration that activation of only odd-numbered mAChR subtypes induces cadherin-mediated adhesion suggests that the unique responses of cells to M1or M3mAChR stimulation may involve cadherin activation.