Component Of Adherens Junctions Research Articles

The Mammalian Crumbs Complex Defines a Distinct Polarity Domain Apical of Epithelial Tight Junctions

Epithelial apico-basal polarity is established through the asymmetric cortical distribution of the Par, Crumbs and Scribble polarity modules. Apical (Par and Crumbs) and basolateral (Scribble) polarity modules overlap at the apical-lateral border, which, in mammals, is defined by the apical junctional complex (AJC). The AJC is composed of tight junctions (TJ) and adherens junctions (AJ) and plays fundamental roles in epithelial morphogenesis and plasticity. However, the molecular composition and precise sub-junctional organization of the AJC and its associated polarity regulators are not well defined. Here, we used the peroxidase APEX2 for quantitative proximity proteomics (QPP) and electron microscopy (EM) imaging to dissect the architecture of the AJC in fully polarized MDCK-II cells. We present a high-confidence proteome of the apical-lateral border in which TJ and AJ components and apical and lateral compartment markers are spatially resolved. We further demonstrate that the Crumbs complex (Pals1, PatJ, Lin7c, and Crumbs3) defines a hitherto unidentified membrane compartment apical of TJ, which we coin the vertebrate marginal zone (VMZ). QPP, imaging, and immunoprecipitation assays showed that the HOMER scaffolding proteins, PKN2 and PTPN13, and the membrane-proximal HIPPO pathway proteins ARHGAP29 and STXBP4 are recruited to the VMZ via the PDZ domains of PatJ. Taken together, our work defines the spatial and molecular organization of the apical-lateral border in mammalian epithelial cells, reveals an intriguing molecular and spatial conservation of invertebrate and vertebrate cell polarity protein domains, and identifies a VMZ-associated protein network implicated in HIPPO signaling and the control of the cortical actin cytoskeleton.

Dual effects of JNK activation in blood-milk barrier damage induced by zinc oxide nanoparticles

Zinc oxide nanoparticles (ZnO-NPs) have been extensively applied in our daily life. Humans are at high risk of being exposed to ZnO-NPs, which induce potentially adverse health effects. Although a growing number of studies have investigated the toxic effects of ZnO-NPs, the available data concerning ZnO-NP interactions with the blood-milk barrier (BMB) remain highly limited. Herein, we systematically investigated the damage to BMB integrity induced by ZnO-NPs and the mechanisms involved. ZnO-NPs that were intravenously injected into lactating dams accumulated in the mammary gland and entered into the breast milk, inducing disruption to BMB integrity and changes in the tight junction (TJ) and adherens junction (AJ) components. Furthermore, using an in vitro BMB model composed of EpH4-Ev cells, we verified that ZnO-NP-triggered ROS generation and the activation of MKK4 and JNK are the main mechanism of cell-cell junction damage. More interestingly, JNK activation played different roles in inducing changes in the TJ and AJ complex, and these effects did not need to activate the downstream c-Jun. These data provide more information for understanding ZnO-NP interactions with the BMB and raise concern for the daily use and the intravenous use of ZnO-NPs by lactating mothers.

Predominant Distribution of the RNAi Machinery at Apical Adherens Junctions in Colonic Epithelia Is Disrupted in Cancer.

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function.

Light and electron microscopic studies on the pecten oculi showing blood-retina barrier properties in Turkey's native Gerze chicken.

The pecten oculi is a highly vascularized and pigmented organ that projects from the optic disc into the vitreous body in the avian eye. In this study, the pecten oculi of Turkey's native Gerze chicken was examined by light and scanning electron microscopy. Furthermore, the localization of some adherens junction components (E-cadherin and pan-cadherin) in intact vessels of the blood-retina barrier was investigated by immunohistochemistry. In the Gerze chicken, the pecten oculi was a thin structure, which was located over the head of the discus nervi optici and projected from the retina into the corpus vitreum. The pecten oculi consisted of 18-21 highly vascularized pleats, joined apically by a bridge and resembled an accordion in appearance. Hyalocytes and melanocytes were observed around the small and large vessels. The morphometric data of the pecten oculi showed that there were no statistical differences in terms of sex. The immunohistochemical analysis of the pecten oculi, which is used as a model for the investigation of the formation and maturation of the barrier properties in the central nervous system, revealed cytoplasmic E-cadherin and pan-cadherin immunoreactivity in the endothelial cells of the small, large and capillary vessels. These observations suggest that while the morphological and histological structure of the Gerze chicken's pecten oculi was generally similar to that of other diurnal domestic birds, the pecten oculi, a model system for vascular differentiation and the blood-retina barrier, expressed different cadherins.

AJAP1 affects behavioral changes and GABABR1 level in epileptic mice

Adherens junction-associated protein-1 (AJAP1), also called SHREW1, was first discovered as a novel component of adherens junctions in 2004. In later studies, AJAP1 was found to suppress invasion and predict recurrence of some tumors. Apart from its function as a putative tumor suppressor, AJAP1 is still poorly understood. Schwenk et al. recently found that AJAP1 was tightly associated with the γ-Aminobutyric acid type B receptor subunit 1(GABABR1). It is well known that GABABR plays a vital role in epilepsy as an inhibitory transmitter receptor. Structurally adjacent, possibly functionally interacting, therefore, we hypothesize that AJAP1 participates in the onset and progression of epilepsy. We designed this experiment to investigate the expression and location of AJAP1 in temporal lobe epilepsy (TLE) patients and kainic acid(KA)-induced epilepsy animal models by immunofluorescence and Western blot analyses. We overexpressed and inhibited AJAP1 through lentiviruses in KA-induced models and observed the corresponding effects on epileptic animals. Double-label immunofluorescence showed that AJAP1 was expressed mainly in neurons. Western blot analysis revealed that AJAP1 expression was downregulated in the neocortex of TLE patients and the hippocampus and neocortex of epileptic animal models. The overexpression of AJAP1 can reduce the frequency of spontaneous seizures, whereas the inhibition of AJAP1 expression can increase the incidence rate. Our study demonstrated that AJAP1 may be involved in the pathogenic process of epilepsy and may represent a novel antiepileptic target.

Epidermal structure and differentiation.

In this Primer, Moreci and Lechler follow the lifetime of an epidermal cell from its birth to its ultimate death, and detail how this journey is necessary for epidermal function.

Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish.

The epidermis, a multilayered epithelium, surrounds and protects the vertebrate body. It develops from a bilayered epithelium formed of the outer periderm and underlying basal epidermis. How apicobasal polarity is established in the developing epidermis has remained poorly understood. We show that both the periderm and the basal epidermis exhibit polarised distribution of adherens junctions in zebrafish. aPKC, an apical polarity regulator, maintains the robustness of polarisation of E-cadherin- an adherens junction component- in the periderm. E-cadherin in one layer controls the localisation of E-cadherin in the second layer in a layer non-autonomous manner. Importantly, E-cadherin controls the localisation and levels of Lgl, a basolateral polarity regulator, in a layer autonomous as well non-autonomous manner. Since periderm formation from the enveloping layer precedes the formation of the basal epidermis, our analyses suggest that peridermal polarity, initiated by aPKC, is transduced in a stepwise manner by E-cadherin to the basal layer.

Monoubiquitination of p120-catenin is essential for TGFβ-induced epithelial-mesenchymal transition and tumor metastasis.

Disassembly of intercellular junctions is a hallmark of epithelial-mesenchymal transition (EMT). However, how the junctions disassemble remains largely unknown. Here, we report that E3 ubiquitin ligase Smurf1 targets p120-catenin, a core component of adherens junction (AJ) complex, for monoubiquitination during transforming growth factor β (TGFβ)-induced EMT, thereby leading to AJ dissociation. Upon TGFβ treatment, activated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylates T900 of p120-catenin to promote its interaction with Smurf1 and subsequent monoubiquitination. Inhibition of T900 phosphorylation or ubiquitination of p120-catenin abrogates TGFβ-induced AJ dissociation and consequent tight junction (TJ) dissociation and cytoskeleton rearrangement, hence markedly blocking lung metastasis of murine breast cancer. Moreover, the T900 phosphorylation level of p120-catenin is positively correlated with malignancy of human breast cancer. Hence, our study reveals the underlying mechanism by which TGFβ induces dissociation of AJs during EMT and provides a potential strategy to block tumor metastasis.

A Simple Method to Test Mechanical Strain on Epithelial Cell Monolayers Using a 3D-Printed Stretcher.

With the realization that mechanical forces mediate many biological processes and contribute to disease progression, researchers are focusing on developing new methods to understand the role of mechanotransduction in biological systems. Despite recent advances in stretching devices that analyze the effects of mechanical strain in vitro, there are still possibilities to develop new equipment. For example, many of these devices tend be expensive, whereas few have been designed to assess the effects of mechanical strain driven by the extracellular matrix (ECM) to epithelial cell monolayers and to cell-cell adhesion. In this chapter, we introduce a cost-efficient, user-friendly, 3D-printed stretching device that can be used to test the effects of mechanical strain on cultured epithelial cells. Evaluation of the device using speckle-tracking shows homogeneous strain distribution along the horizontal plane of membranes at 2.5% and 5% strains, supporting the reliability of the device. Since cell-cell junctions are mechanosensitive protein complexes, we hereby used this device to examine effects on cell-cell adhesion. For this, we used colon epithelial Caco2 cell monolayers that well-differentiate in culture and form mature adherens junctions. Subjecting Caco2 cells to 2.5% and 5% strain using our device resulted in significant reduction in the localization of the core adherens junction component E-cadherin at areas of cell-cell contact and its increased translocation to the cytoplasm, which in agreement with other methodologies showing that increased ECM-driven strain negatively affects cell-cell adhesion. In summary, we here present a new, cost-effective, homemade device that can be reliably used to examine effects of mechanical strain on epithelial cell monolayers and cell-cell adhesion, in vitro.

Proteomic Analysis Reveals a Role for RSK in p120-catenin Phosphorylation and Melanoma Cell-Cell Adhesion.

The RAS/mitogen-activated protein kinase (MAPK) signaling pathway regulates various biological functions, including cell survival, proliferation and migration. This pathway is frequently deregulated in cancer, including melanoma, which is the most aggressive form of skin cancer. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its function and the nature of its cellular partners. In this study, we used a proximity-based labeling approach to identify RSK proximity partners in cells. We identified many potential RSK-interacting proteins, including p120ctn (p120-catenin), which is an essential component of adherens junction (AJ). We found that RSK phosphorylates p120ctn on Ser320, which appears to be constitutively phosphorylated in melanoma cells. We also found that RSK inhibition increases melanoma cell-cell adhesion, suggesting that constitutive RAS/MAPK signaling negatively regulates AJ integrity. Together, our results indicate that RSK plays an important role in the regulation of melanoma cell-cell adhesion.

Oral variant of acantholytic squamous cell carcinoma—Histochemical and immunohistochemical features

Acantholytic squamous cell carcinoma (ASCC) is an uncommon variant of squamous cell carcinoma (SCC). It is characterized by a combination of typical SCC and pseudoglandular structures, dyskeratotic cells and prominent acantholysis.The purpose of this study was to analyze the histochemical and immunohistochemical characteristics of the intraoral variant of ASCC. Cases of intraoral ASCC were retrieved from the English language literature. Four new cases from our files were added.In total, 35 cases were included and analyzed in this study. The mean age of the patients was 61.5 + 13 years (age range 38–92 years), with a male-to-female ratio of 1.7:1. According to the available data, histochemical and immunohistochemical stains for mucins were found to be consistently negative. E- cadherin, a marker of adherens junctions, was usually reported to be expressed in areas of “typical” (non acantholytic) SCC, but reduced in the acantholytic areas. We examined for the first time the expression of claudin 1, a marker of tight junctions, and found it to be reduced in the acantholytic areas, similar to E-cadherin. Several cases of oral ASCC also expressed vimentin and cytokeratin (CK) 19, markers associated with epithelial-mesenchymal transition. A wide range of non-epithelial markers yielded negative immunoreactions.In conclusion, ASCC is an uncommon variant of squamous cell carcinoma. The acantholytic process appears to involve reduced expression of molecular components of both adherens junctions and tight junctions. These findings could suggest a relation to the epithelial mesenchymal transition process and therefore further studies are needed in order to establish such a link and the subsequent possible impact on the clinical outcome of the patients.

Increased Expression of Adherens Junction Components in Mouse Liver following Bile Duct Ligation.

Adherens junctions, consisting of cadherins and catenins, are a group of cell-to-cell junctions that mediate mechanistic linkage between neighboring cells. By doing so, adherens junctions ensure direct intercellular contact and play an indispensable role in maintaining tissue architecture. Considering these critical functions, it is not surprising that adherens junctions are frequently involved in disease. In the present study, the effects of bile duct ligation—a surgical procedure to experimentally induce cholestatic and fibrotic liver pathology—on hepatic adherens junctions were investigated in mice. In essence, it was found that liver mRNA and protein levels of E-cadherin, β-catenin and γ-catenin drastically increase following bile duct ligation. These results could suggest a cytoprotective role for hepatic adherens junctions following bile duct ligation.

An ensemble of flexible conformations underlies mechanotransduction by the cadherin–catenin adhesion complex

The cadherin-catenin adhesion complex is the central component of the cell-cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-catenin•β-catenin•epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and β-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of β-catenin forms a flexible "tongue" that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin-catenin adhesion complex in mechanotransduction.

P120 and E-cadherin: Double-edged swords in tumor metastasis.

Cell-cell adhesion by adherens junctions controls proliferation and cell polarization and is crucial to maintain epithelial architecture and homeostasis. Downregulation of two of the main components of adherens junctions, E-cadherin and p120, is an often recurring hallmark of carcinomas, causing loss of polarity and increased proliferation, survival and invasion of epithelial cells. On the other hand, tumor-promoting effects of both E-cadherin and p120 have been reported, substantiated by sustained, or even elevated expression of these molecules in many cancers. In this review, we will discuss how expression regulation by EMT, E-cadherin cleavage or p120 isoform expression can contribute to either tumor-supressing or tumor-promoting processes. Furthermore, we will focus on the contradictory functions of E-cadherin and p120 in the different phases of tumor progression, from carcinoma in situ up to the formation of distant metastasis. Finally, we will discuss the possibilities and challenges when using either protein as a biomarker.

Sidekick Is a Key Component of Tricellular Adherens Junctions that Acts to Resolve Cell Rearrangements

Tricellular adherens junctions are points of high tension that are central to the rearrangement of epithelial cells. However, the molecular composition of these junctions is unknown, making it difficult to assess their role in morphogenesis. Here, we show that Sidekick, an immunoglobulin family cell adhesion protein, is highly enriched at tricellular adherens junctions in Drosophila. This localization is modulated by tension, and Sidekick is itself necessary to maintain normal levels of cell bond tension. Loss of Sidekick causes defects in cell and junctional rearrangements in actively remodeling epithelial tissues like the retina and tracheal system. The adaptor proteins Polychaetoid and Canoe are enriched at tricellular adherens junctions in a Sidekick-dependent manner; Sidekick functionally interacts with both proteins and directly binds to Polychaetoid. We suggest that Polychaetoid and Canoe link Sidekick to the actin cytoskeleton to enable tricellular adherens junctions to maintain or transmit cell bond tension during epithelial cell rearrangements.

Vitamin D (1,25-(OH)2D3) regulates the gene expression through competing endogenous RNAs networks in high glucose-treated endothelial progenitor cells

Vitamin D (vit-D) supplementation can improve endothelial cell function in type 2 diabetes mellitus patients with vit-D insufficiency or deficiency. In the present study, we aimed to compare the expression profiles of circRNAs, lncRNAs, miRNAs, and mRNAs between 1,25-(OH)2D3-treated endothelial progenitor cells (EPCs) and control cells, and to further construct the 1,25-(OH)2D3-regulated ceRNA networks in EPCs. RNA sequencing was performed on the 1,25-(OH)2D3-treated EPCs and control cells derived from the bone marrow (BM). Bioinformatics analyses were performed to identify differentially expressed (DE) microRNAs (miRNAs), circular RNAs (circRNAs), mRNAs, and long non-coding RNAs (lncRNAs). Then Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predict the function of genes. Competing endogenous RNA (ceRNA) networks were constructed with Cytoscape software. 1,25-(OH)2D3 application induced changes in the expression profiles of 1791 mRNAs, 2726 lncRNAs, 205 circRNAs, and 45 miRNAs in EPCs treated with high levels of glucose. These DE RNAs were associated with MMP and GTPase activities, specific signaling pathways, and components of actin, extracellular matrix, or adherens junction. DE circRNAs, which functioned independently of their linear host genes, interacted with miRNAs to serve as miRNA sponges in complex ceRNA networks. The data indicated that circRNAs and lncRNAs comprised ceRNAs to sponge effects of miRNAs on the expressions of mRNAs following 1,25-(OH)2D3 application in EPCs. 1,25-(OH)2D3 improved the function of EPCs via associated ceRNA interaction networks in diabetes patients.

E-Cadherin is Dispensable to Maintain Langerhans Cells in the Epidermis

The cell adhesion molecule E-cadherin is a major component of adherens junctions and marks Langerhans cells (LC), the only dendritic cell (DC) population of the epidermis. LC form a dense network and attach themselves to the surrounding keratinocytes via homophilic E-cadherin binding. LC activation, mobilization, and migration require a reduction in LC E-cadherin expression. To determine whether E-cadherin plays a role in regulating LC homeostasis and function, we generated CD11c-specific E-cadherin knockout mice (CD11c-Ecaddel). In the absence of E-cadherin-mediated cell adhesion, LC numbers remained stable and similar as in control mice, even in aged animals. Intriguingly, E-cadherin-deficient LC displayed a dramatically changed morphology characterized by a more rounded cell body and fewer dendrites than wild-type cells. Nevertheless, maturation and migration of LC lacking E-cadherin was not altered, neither under steady-state nor inflammatory conditions. Accordingly, CD11c-Ecaddel and control mice developed comparable contact hypersensitivity reactions and imiquimod-triggered psoriatic skin inflammation, indicating that E-cadherin on LC does not influence their ability to orchestrate T cell-mediated immunity. In conclusion, our data demonstrate that E-cadherin is dispensable to maintain LC in the epidermis and does not regulate LC maturation, migration, and function.

E‐cadherin is downregulated in benign prostatic hyperplasia and required for tight junction formation and permeability barrier in the prostatic epithelial cell monolayer

We previously reported the presence of prostate-specific antigen (PSA) in the stromal compartment of benign prostatic hyperplasia (BPH). Since PSA is expressed exclusively by prostatic luminal epithelial cells, PSA in the BPH stroma suggests increased tissue permeability and the compromise of epithelial barrier integrity. E-cadherin, an important adherens junction component and tight junction regulator, is known to exhibit downregulation in BPH. These observations suggest that the prostate epithelial barrier is disrupted in BPH and E-cadherin downregulation may increase epithelial barrier permeability. The ultra-structure of cellular junctions in BPH specimens was observed using transmission electron microscopy (TEM) and E-cadherin immunostaining analysis was performed on BPH and normal adjacent specimens from BPH patients. In vitro cell line studies using benign prostatic epithelial cell lines were performed to determine the impact of small interfering RNA knockdown of E-cadherin on transepithelial electrical resistance and diffusion of fluorescein isothiocyanate (FITC)-dextran in transwell assays. The number of kiss points in tight junctions was reduced in BPH epithelial cells as compared with the normal adjacent prostate. Immunostaining confirmed E-cadherin downregulation and revealed a discontinuous E-cadherin staining pattern in BPH specimens. E-cadherin knockdown increased monolayer permeability and disrupted tight junction formation without affecting cell density. Our results indicate that tight junctions are compromised in BPH and loss of E-cadherin is potentially an important underlying mechanism, suggesting targeting E-cadherin loss could be a potential approach to prevent or treat BPH.

E-cadherin Beyond Structure: A Signaling Hub in Colon Homeostasis and Disease.

E-cadherin is the core component of epithelial adherens junctions, essential for tissue development, differentiation, and maintenance. It is also fundamental for tissue barrier formation, a critical function of epithelial tissues. The colon or large intestine is lined by an epithelial monolayer that encompasses an E-cadherin-dependent barrier, critical for the homeostasis of the organ. Compromised barriers of the colonic epithelium lead to inflammation, fibrosis, and are commonly observed in colorectal cancer. In addition to its architectural role, E-cadherin is also considered a tumor suppressor in the colon, primarily a result of its opposing function to Wnt signaling, the predominant driver of colon tumorigenesis. Beyond these well-established traditional roles, several studies have portrayed an evolving role of E-cadherin as a signaling epicenter that regulates cell behavior in response to intra- and extra-cellular cues. Intriguingly, these recent findings also reveal tumor-promoting functions of E-cadherin in colon tumorigenesis and new interacting partners, opening future avenues of investigation. In this Review, we focus on these emerging aspects of E-cadherin signaling, and we discuss their implications in colon biology and disease.

High expression of CDH3 predicts a good prognosis for colon adenocarcinoma patients.

Colon adenocarcinoma (COAD) is one of the most common types of malignancy of the digestive system, and a better understanding of the molecular mechanisms will contribute to an improvement in the quality of life for COAD patients. Cadherin 3 (CDH3), a gene encoding P-cadherin, is a major component of adherens junctions and is closely associated with the occurrence and development of a variety of tumor types. However, the current knowledge regarding the role of CDH3 in COAD is limited. The present study aimed to identify the relative mRNA and protein expression levels of CDH3 in COAD tissues, and whether CDH3 had any influence on the survival rate of patients with COAD. Analysis of differentially expressed genes using the UALCAN database revealed that CDH3 was significantly upregulated in COAD tissues, and reverse transcription-quantitative PCR analysis further confirmed that CDH3 was upregulated in 48 COAD tissues compared with that in their paired normal tissues (n=48). Consistent with this, analysis of the Human Protein Atlas database indicated that the expression levels of the CDH3 protein were upregulated in COAD tissues (n=11) compared with those in normal tissues (n=3; P=0.0245). Next, the association between the mRNA levels of CDH3 and the survival rate of the COAD patients was analyzed using the UALCAN database, and the Kaplan-Meier curves revealed that the CDH3 high expression group (n=69) had a better overall survival compared with that of the CDH3 medium/low expression group (n=210; P=0.037). Furthermore, analysis of clinical data of a cohort from our hospital indicated that the median survival time for COAD patients with high (n=20) and low (n=20) CDH3 levels was 55.5 and 43.5 months, respectively, and there was a significant difference in the survival time between the two groups (P=0.0078). The above results verified that CDH3 was significantly upregulated in the COAD tissues and that high expression of CDH3 predicts a good prognosis for COAD patients.