Plasticity of Cadherin–Catenin Expression in the Melanocyte Lineage

Cadherins are calcium‐dependent cell adhesion receptors with strong morphoregulatory functions. To mediate functional adhesion, cadherins must interact with actin cytoskeleton. Catenins are cytoplasmic proteins that mediate the interactions between cadherins and the cytoskeleton. In addition to their role in cell–cell adhesion, catenins also participate in signaling pathways that regulate cell growth and differentiation. Cadherins and catenins appear to be involved in melanocyte development and transformation. Here, we investigated the function of cadherin–catenin complexes in the normal development and transformation of melanocytes by studying the patterns of expression of the cell–cell adhesion molecules, E‐, N‐ and P‐cadherin, and the expression of their cytoplasmic partners, α‐, β‐ and Γ‐catenin, during murine development. Similar analyses were performed in vitro using murine melanoblast, melanocyte, and melanoma cell lines in the presence and absence of keratinocytes, the cells with which melanocytes interact in vivo. Overall, the results suggest that the expression of cadherins and catenins is very plastic and depends on their environment as well as the transformation status of the cells. This plasticity is important in fundamental cellular mechanisms associated with normal and pathological ontogenesis, as well as with tumorigenesis.     

μ2‐Dependent endocytosis of N‐cadherin is regulated by β‐catenin to facilitate neurite outgrowth

Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N‐cadherin, a calcium‐dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N‐cadherin internalizes through clathrin‐mediated endocytosis (CME). Two tyrosine‐based motifs in the cytoplasmic domain of N‐cadherin recognized by the μ2 subunit of the AP‐2 adaptor complex are responsible for CME of N‐cadherin. Moreover, β‐catenin, a core component of the N‐cadherin adhesion complex, inhibits N‐cadherin endocytosis by masking the 2 tyrosine‐based motifs. Removal of β‐catenin facilitates μ2 binding to N‐cadherin, thereby increasing clathrin‐mediated N‐cadherin endocytosis and neurite outgrowth without affecting the steady‐state level of surface N‐cadherin. These results identify and characterize the mechanism controlling N‐cadherin endocytosis through β‐catenin‐regulated μ2 binding to modulate neurite outgrowth.      

Lysophosphatidic acid modulates the association of PTP1B with N‐cadherin/catenin complex in SKOV3 ovarian cancer cells

LPA (lysophosphatidic acid) is a natural phospholipid that plays important roles in promoting cancer cell proliferation, invasion and metastases. We previously reported that LPA induces ovarian cancer cell dispersal and disruption of AJ (adherens junction) through the activation of SFK (Src family kinases). In this study, we have investigated the regulatory mechanisms during the early phase of LPA‐induced cell dispersal. An in vitro model of the ovarian cancer cell line SKOV3 for cell dispersal was used. LPA induces rapid AJ disruption by increasing the internalization of N‐cadherin‐β‐catenin. By using immunoprecipitations, LPA was shown to induce increased tyrosine phosphorylation of β‐catenin and alter the balance of β‐catenin‐bound SFK and PTP1B (phosphotyrosine phosphatase 1B). The altered balance of tyrosine kinase/phosphatase correlated with a concomitant disintegration of the β‐catenin‐α‐catenin, but not the β‐catenin—N‐cadherin complex. This disintegration of β‐catenin from α‐catenin and the cell dispersal caused by LPA can be rescued by blocking SFK activity with the chemical inhibitor, PP2. More importantly, PP2 also restores the level of PTP1B bound to β‐catenin. We propose that LPA signalling alters AJ stability by changing the dynamics of tyrosine kinase/phosphatase bound to AJ proteins. This work provides further understanding of the early signalling events regulating ovarian cancer cell dispersal and AJ disruption induced by LPA.     

E‐, P‐, and N‐cadherin are co‐expressed in the nasopharyngeal carcinoma cell line TW‐039

The cadherin/catenin complex plays a key role in the initiation of cell‐cell recognition, and adhesion, and the elaboration of structural and functional organization in multicellular tissues and organs. It is associated with tumor metastasis and also acts as an “invasion suppressor” of cancer cells. Nasopharyngeal carcinoma (NPC) is notorious for its highly metastatic nature. The expression of the E‐cadherin/catenin complex is down‐regulated in NPC tumor specimens. To obtain better insight into the intercellular adhesive property of NPC cells, we used immunofluorescence microscopy, immunoprecipitation, and immunoblot analysis to examine the expression of the classical cadherins and β‐catenin in a NPC cell line, TW‐039. The results demonstrate a change in the distribution of E‐cadherin from cytosolic flakes to cell‐cell contacts with increasing time in culture. Between days 1 and 5 after plating, the detergent‐insoluble fraction of E‐cadherin increased from 20% to 37% of total E‐cadherin, and that for P‐cadherin increased from 33% to 40%. By contrast, the values for β‐catenin remained unchanged (26% and 25%). Both immunofluorescence and immunoblot studies suggested that P‐cadherin may be involved in pioneer contact adhesion of TW‐039 cells. Interestingly, E‐, P‐, and N‐cadherin are co‐expressed in this cell line. Immunoprecipitation studies also showed that other members of the cadherin family may be involved in the contact adhesion of TW‐039 cells. J. Cell. Biochem. 76:161–172, 1999. © 1999 Wiley‐Liss, Inc.     

Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin

Cadherins are Ca(2+)-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, transmembrane cadherins such as E- , P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intracellular proteins termed catenins, including alpha-catenin, beta- catenin, and plakoglobin, are tightly associated with these cadherins and serve to link them to the cytoskeleton. Here, we present evidence that in fibroblasts alpha-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. We further show that alpha- actinin, but not vinculin, coimmunoprecipitates specifically with alpha- and beta-catenin from N- and E-cadherin-expressing cells, but only if alpha-catenin is present. Moreover, we show that alpha-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin- independent manner. We therefore propose that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between alpha-actinin and alpha-catenin.     

Postsynaptic Y654 dephosphorylation of β‐catenin modulates presynaptic vesicle turnover through increased n‐cadherin‐mediated transsynaptic signaling

Synaptic adhesion molecules, which coordinately control structural and functional changes at both sides of synapses, are important for synaptogenesis and synaptic plasticity. Because they physically form homophilic or heterophilic adhesions across synaptic junctions, these molecules can initiate transsynaptic communication in both anterograde and retrograde directions. Using optical imaging approaches, we investigated whether an increase in postsynaptic N‐cadherin could correspondingly alter the function of connected presynaptic terminals. Postsynaptic expression of β‐catenin Y654F, a phosphorylation‐defective form with enhanced binding to N‐cadherin, is sufficient to increase postsynaptic surface levels of N‐cadherin and consequently promote presynaptic reorganizations. Such reorganizations include increases in the densities of the synaptic vesicle protein, Synaptotagmin 1 and the active zone scaffold protein, Bassoon, the number of active boutons and the size of the total recycling vesicle pool. In contrast, synaptic vesicle turnover is significantly impaired, preventing the exchange of synaptic vesicles with adjacent boutons. Together, N‐cadherin‐mediated retrograde signaling, governed by phosphoregulation of postsynaptic β‐catenin Y654, coordinately modulates presynaptic vesicle dynamics to enhance synaptic communication in mature neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 61–74, 2017     

E‐cadherin can limit the transforming properties of activating β‐catenin mutations

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in β‐catenin (CTNNB1). We have compared the dynamics and the potency of β‐catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β‐catenin took much longer to achieve Wnt deregulation and acquire a crypt‐progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β‐catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β‐catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E‐cadherin and a higher number of E‐cadherin:β‐catenin complexes at the membrane. Reduction in E‐cadherin synergised with an activating mutation of β‐catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β‐catenin that is required to drive transformation, and E‐cadherin can act as a buffer to sequester mutated β‐catenin.     

Protein kinase C modifications of VE-cadherin,p120, and beta-catenin contribute to endothelial barrier dysregulation induced by thrombin

The adherens junction is a multiprotein complex consisting of the transmembrane vascular endothelial cadherin (VEC) and cytoplasmic catenins (p120, beta-catenin, plakoglobin, alpha-catenin) responsible for the maintenance of endothelial barrier function. Junctional disassembly and modifications in cadherin/catenin complex lead to increased paracellular permeability of the endothelial barrier. However, the mechanisms of junctional disassembly remain unclear. In this study, we used the proinflammatory mediator thrombin to compromise the barrier function and test the hypothesis that phosphorylation-induced alterations of VEC, beta-catenin, and p120 regulate junction disassembly and mediate the increased endothelial permeability response. The study showed that thrombin induced dephosphorylation of VEC, which is coupled to disassembly of cell-cell contacts, but VEC remained in aggregates at the plasma membrane. The cytoplasmic catenins dissociated from the VEC cytoplasmic domain in thin membrane projections formed in interendothelial gaps. We also showed that thrombin induced dephosphorylation of beta-catenin and phosphorylation of p120. Thrombin-induced interendothelial gap formation and increased endothelial permeability were blocked by protein kinase C inhibition using chelerythrine and G?-6976 but not by LY-379196. Chelerythrine also prevented thrombin-induced phosphorylation changes of the cadherin/catenin complex. Thus the present study links posttranslational modifications of VEC, beta-catenin, and p120 to the mechanism of thrombin-induced increase in endothelial permeability.     

GSKIP,an inhibitor of GSK3β, mediates the N‐cadherin/β‐catenin pool in the differentiation of SH‐SY5Y cells

Emerging evidence has shown that GSK3β plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3β interaction protein (GSKIP) able to negatively regulate GSK3β in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH‐SY5Y cells treated with retinoic acid (RA) to differentiate to neuron‐like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH‐SY5Y cells. GSKIP may affect GSK3β activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases β‐catenin in the nucleus and raises the level of cyclin D1 to promote cell‐cycle progression in SH‐SY5Y cells. Additionally, overexpression of GSKIP downregulates N‐cadherin expression, resulting in decreased recruitment of β‐catenin. Moreover, depletion of β‐catenin by small interfering RNA, neurite outgrowth is blocked in SH‐SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3β/β‐catenin, β‐catenin/cyclin D1, and β‐catenin/N‐cadherin pool during RA signaling in SH‐SY5Y cells. J. Cell. Biochem. 108: 1325–1336, 2009. © 2009 Wiley‐Liss, Inc.     

Urocortin increased LPS‐induced endothelial permeability by regulating the cadherin–catenin complex via corticotrophin‐releasing hormone receptor 2

Urocortin (Ucn1), a member of corticotrophin‐releasing hormone (CRH) family, has been reported to be upregulated in inflammatory diseases and function as an autocrine or paracrine inflammatory mediator. Growing evidence shows that Ucn1 increases the endothelial permeability in inflammatory conditions; however, the detailed mechanisms are not clear. In the present study, we investigated the mechanisms of increased endothelial permeability by Ucn1 in human umbilical vein endothelial cells (HUVECs) exposed to lipopolysaccharide (LPS). Pretreatment of HUVECs with Ucn1 increased the endothelial cell permeability, which was augmented by LPS synergistically. Significant downregulation of VE‐cadherin expression was also observed. Moreover, Ucn1 increased phosphorylation of protein kinase D (PKD) and heat shock protein 27 (HSP27) in a time‐ and CRHR₂‐dependent manner. Inhibition of PKD and HSP27 drastically attenuated Ucn1‐induced downregulation of VE‐cadherin expression. Further investigations demonstrated that Ucn1 phosphorylated β‐catenin at Ser552 to disrupt the cadherin–catenin complex and hence promote the disassociation of β‐catenin and VE‐cadherin. Disassociation of β‐catenin and VE‐cadherin resulted in decreased VE‐cadherin expression while on the contrary β‐catenin was increased, which may due to the inactivation of GSK‐3β. Increased β‐catenin translocated into the nucleus and subsequently bound to TCF/LEF site, contributing to the elevated expression of vascular endothelial growth factor (VEGF). The above effects of Ucn1 were completely reversed by CRHR₂ receptor blocker, antisauvagine‐30. Taken together, our data suggest that Ucn1 increase LPS‐induced endothelial permeability by disrupting the VE‐cadherin–β‐catenin complex via activation of CRHR₂ and PKD‐HSP27 signaling pathway. J. Cell. Physiol. 228: 1295–1303, 2013. © 2012 Wiley Periodicals, Inc.     

Activation of Smad‐mediated TGF‐β signaling triggers epithelial–mesenchymal transitions in murine cloned corneal progenitor cells

Epithelial–mesenchymal transition (EMT), via activation of Wnt signaling, is prevailing in embryogenesis, but postnatally it only occurs in pathological processes, such as in tissue fibrosis and tumor metastasis. Our prior studies led us to speculate that EMT might be involved in the loss of limbal epithelial stem cells in explant cultures. To examine this hypothesis, we successfully grew murine corneal/limbal epithelial progenitors by prolonging the culture time and by seeding at a low density in a serum‐free medium. Single cell‐derived clonal growth was accompanied by a gradient of Wnt signaling activity, from the center to the periphery, marked by a centrifugal loss of E‐cadherin and β‐catenin from intercellular junctions, coupled with nuclear translocation of β‐catenin and LEF‐1. Large‐colony‐forming efficiency at central location of colony was higher than peripheral location. Importantly, there was also progressive centrifugal differentiation, with positive K14 keratin expression and the loss of p63 and PCNA nuclear staining, and irreversible EMT, evidenced by cytoplasmic expression of α‐SMA and nuclear localization of S100A4; and by nuclear translocation of Smad4. Furthermore, cytoplasmic expression of α‐SMA was promoted by high‐density cultures and their conditioned media, which contained cell density‐dependent levels of TGF‐β1, TGF‐β2, GM‐CSF, and IL‐1α. Exogenous TGF‐β1 induced α‐SMA positive cells in a low‐density culture, while TGF‐β1 neutralizing antibody partially inhibited α‐SMA expression in a high‐density culture. Collectively, these results indicate that irreversible EMT emerges in the periphery of clonal expansion where differentiation and senescence of murine corneal/limbal epithelial progenitors occurs as a result of Smad‐mediated TGF‐β‐signaling. J. Cell. Physiol. 228: 225–234, 2013. © 2012 Wiley Periodicals, Inc.     

MicroRNA‐300 promotes apoptosis and inhibits proliferation,migration, invasion and epithelial‐mesenchymal transition via the Wnt/β‐catenin signaling pathway by targeting CUL4B in pancreatic cancer cells

The study aims to verify the hypothesis that up‐regulation of microRNA‐300 (miR‐300) targeting CUL4B promotes apoptosis and suppresses proliferation, migration, invasion, and epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells by regulating the Wnt/β‐catenin signaling pathway. Pancreatic cancer tissues and adjacent tissues were collected from 110 pancreatic cancer patients. Expression of miR‐300, CUL4B, Wnt, β‐catenin, E‐cadherin, N‐cadherin, Snail, GSK‐3β, and CyclinD1 were detected using qRT‐PCR and Western blot. CFPAC‐1, Capan‐1, and PANC‐1 were classified into blank, negative control (NC), miR‐300 mimics, miR‐300 inhibitors, siRNA‐CUL4B, and miR‐300 inhibitors + siRNA‐CUL4B groups. The proliferation, migration, invasion abilities, the cell cycle distribution, and apoptosis rates were measured in CCK‐8 and Transwell assays. Pancreatic cancer tissues showed increased CUL4B expression but decreased miR‐300 expression. When miR‐300 was lowly expressed, CUL4B was upregulated which in‐turn activated the Wnt/β‐catenin pathway to protect the β‐catenin expression and thus induce EMT. When miR‐300 was highly expressed, CUL4B was downregulated which in‐turn inhibited the Wnt/β‐catenin pathway to prevent EMT. Weakened cell migration and invasion abilities and enhanced apoptosis were observed in the CUL4B group. The miR‐300 inhibitors group exhibited an evident increase in growth rate accompanied the largest tumor volume. Smaller tumor volume and slower growth rate were observed in the miR‐300 mimics and siRNA‐CUL4B group. Our study concludes that lowly expressed miR‐300 may contribute to highly expressed CUL4B activating the Wnt/β‐catenin signaling pathway and further stimulating EMT, thus promoting proliferation and migration but suppressing apoptosis of pancreatic cancer cells.     

Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain.

Differential adhesion between embryonic cells has been proposed to be mediated by a family of closely related glycoproteins called the cadherins. The cadherins mediate adhesion in part through an interaction between the cadherin cytoplasmic domain and intracellular proteins, called the catenins. To determine whether these interactions could regulate cadherin function in embryos, a form of N-cadherin was generated that lacks an extracellular domain. Expression of this mutant in Xenopus embryos causes a dramatic inhibition of cell adhesion. Analysis of the mutant phenotype shows that at least two regions of the N-cadherin cytoplasmic domain can inhibit adhesion and that the mutant cadherin can inhibit catenin binding to E-cadherin. These results suggest that cadherin-mediated adhesion can be regulated by cytoplasmic interactions and that this regulation may contribute to morphogenesis when emerging tissues coexpress several cadherin types.     

IQGAP1 mediates the disruption of adherens junctions to promote Escherichia coli K1 invasion of brain endothelial cells

The transcellular entry of Escherichia coli K1 through human brain microvascular endothelial cells (HBMEC) is responsible for tight junction disruption, leading to brain oedema in neonatal meningitis. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with its receptor, Ecgp96, to induce PKC‐α phosphorylation, adherens junction (AJ) disassembly (by dislodging β‐catenin from VE‐cadherin), and remodelling of actin in HBMEC. We report here that IQGAP1 mediates β‐catenin dissociation from AJs to promote actin polymerization required for E. coli K1 invasion of HBMEC. Overexpression of C‐terminal truncated IQGAP1 (IQΔC) that cannot bind β‐catenin prevents both AJ disruption and E. coli K1 entry. Of note, phospho‐PKC‐α interacts with the C‐terminal portion of Ecgp96 as well as with VE‐cadherin after IQGAP1‐mediated AJ disassembly. HBMEC overexpressing either C‐terminal truncated Ecgp96 (Ecgp96Δ200) or IQΔC upon infection with E. coli showed no interaction ofphospho‐PKC‐α with Ecgp96. These data indicate that the binding of OmpA to Ecgp96 induces PKC‐α phosphorylation and association of phospho‐PKC‐α with Ecgp96, and then signals IQGAP1 to detach β‐catenin from AJs. Subsequently, IQGAP1/β‐catenin bound actin translocates to the site of E. coli K1 attachment to promote invasion.     

Resveratrol promotes trophoblast invasion in pre‐eclampsia by inducing epithelial‐mesenchymal transition

Impairment spiral arteries remodelling was considered to be the underlying cause of pathogenesis of pre‐eclampsia (PE). Resveratrol (RE) was reported that it could modulate cellar phenotype to ameliorate diverse human diseases. However, the biological function of RE in PE remains poorly understood. In this report, we investigated the effect of RE on trophoblast phenotype both in vivo and in vitro. We conducted MTT and transwell assays to explore cell proliferation and invasion events in HTR‐8/SVneo. In mice model, the clinical characteristics of PE were established through the injection of NG‐nitro‐l ‐arginine methyl ester (L‐NAME). Furthermore, related experiments were performed to detect cellar phenotype‐associated signalling pathway, including epithelial‐mesenchymal transition (EMT) and Wnt/β‐catenin. Cell assays indicated that RE could increase trophoblasts migration and invasion. In addition, hypertension and proteinuria were markedly ameliorated by RE compared with the controls in PE mice model. Moreover, treatment by RE in trophoblasts or in PE model, we found that RE activated EMT progress through the regulation of E‐cadherin, β‐catenin, N‐cadherin, vimentin expression, and further altered the WNT‐related gene expression, including WNT1, WNT3 and WNT5B. Our findings demonstrated that RE might stimulate the invasive capability of human trophoblasts by promoting EMT and mediating the Wnt/β‐catenin pathway in PE.     

Cadherin-catenin complex: Protein interactions and their implications for cadherin function

Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. In epithelial cells, E-cadherin represents a key molecule in the establishment and stabilization of cellular junctions. On the cellular level, E-cadherin is concentrated at the adherens junction and interacts homophilically with E-cadherin molecules of adjacent cells. Significant progress has been made in understanding the extra- and intracellular interactions of E-cadherin. Recent success in solving the three-dimensional structure of an extracellular cadherin domain provides a structural basis for understanding the homophilic interaction mechanism and the calcium requirement of cadherins. According to the crystal structure, individual cadherin molecules cooperate to form a linear cell adhesion zipper. The intracellular anchorage of cadherins is regulated by the dynamic association with cytoplasmic proteins, termed catenins. The cytoplasmic domain of E-cadherin is complexed with either β-catenin or plakoglobin (γ-catenin). β-catenin and plakoglobin bind directly to α-catenin, giving rise to two distinct cadherin-catenin complexes (CCC). α-catenin is thought to link both CCC's to actin filaments. The anchorage of cadherins to the cytoskeleton appears to be regulated by tyrosine phosphorylation. Phosphorylation-induced junctional disassembly targets the catenins, indicating that catenins are components of signal transduction pathways. The unexpected association of catenins with the product of the tumor suppressor gene APC has led to the discovery of a second, cadherin-independent catenin complex. Two separate catenin complexes are therefore involved in the cross-talk between cell adhesion and signal transduction. In this review we focus on protein interactions regulating the molecular architecture and function of the CCC. In the light of a fundamental role of the CCC during mammalian development and tissue morphogenesis, we also discuss the phenotypes of embryos lacking E-cadherin or β-catenin. © 1996 Wiley-Liss, Inc.