Cadherin function in junctional complex rearrangement and posttranslational control of cadherin expression

1999 ◽  
Vol 276 (2) ◽  
pp. C404-C418 ◽  
Author(s):  
Megan L. Troxell ◽  
Yih-Tai Chen ◽  
Nicole Cobb ◽  
W. James Nelson ◽  
James A. Marrs
Keyword(s):  
Tight Junction ◽  
Adherens Junction ◽  
Junctional Complex ◽  
Adhesion Protein ◽  
Cell Monolayers ◽  
Calcium Dependent ◽  
Protein Levels ◽  
Epithelial Junctions ◽  
Junctional Complexes ◽  
E Cadherin

The role of E-cadherin, a calcium-dependent adhesion protein, in organizing and maintaining epithelial junctions was examined in detail by expressing a fusion protein (GP2-Cad1) composed of the extracellular domain of a nonadherent glycoprotein (GP2) and the transmembrane and cytoplasmic domains of E-cadherin. All studies shown were also replicated using an analogous cell line that expresses a mutant cadherin construct (T151) under the control of tet repressor. Mutant cadherin was expressed at ∼10% of the endogenous E-cadherin level and had no apparent effect on tight junction function or on distributions of adherens junction, tight junction, or desmosomal marker proteins in established Madin-Darby canine kidney cell monolayers. However, GP2-Cad1 accelerated the disassembly of epithelial junctional complexes and delayed their reassembly in calcium switch experiments. Inducing expression of GP2-Cad1 to levels approximately threefold greater than endogenous E-cadherin expression levels in control cells resulted in a decrease in endogenous E-cadherin levels. This was due in part to increased protein turnover, indicating a cellular mechanism for sensing and controlling E-cadherin levels. Cadherin association with catenins is necessary for strong cadherin-mediated cell-cell adhesion. In cells expressing low levels of GP2-Cad1, protein levels and stoichiometry of the endogenous cadherin-catenin complex were unaffected. Thus effects of GP2-Cad1 on epithelial junctional complex assembly and stability were not due to competition with endogenous E-cadherin for catenin binding. Rather, we suggest that GP2-Cad1 interferes with the packing of endogenous cadherin-catenin complexes into higher-order structures in junctional complexes that results in junction destabilization.

scholarly journals Differential regulation of junctional complex assembly in renal epithelial cell lines

2003 ◽  
Vol 285 (1) ◽  
pp. C102-C111 ◽  
Author(s):  
Shobha Gopalakrishnan ◽  
Mark A. Hallett ◽  
Simon J. Atkinson ◽  
James. A. Marrs
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Rho Gtpase ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Stress Fiber ◽  
Cell Junctions ◽  
Junctional Complex ◽  
Complex Assembly ◽  
E Cadherin

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and β-catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner.

Mislocalization of Adherens Junction- Associated Proteins in a Patient with Darier Disease

2018 ◽  
Vol 2 (3) ◽  
pp. 184-201
Author(s):  
George D Glinos ◽  
Irena Pastar ◽  
Marjana Tomic-Canic ◽  
Rivka C Stone
Keyword(s):  
Adherens Junction ◽  
Cellular Adhesion ◽  
Calcium Flux ◽  
Keratinocyte Differentiation ◽  
Calcium Dependent ◽  
Endoplasmic Reticulum Calcium ◽  
Darier Disease ◽  
Associated Proteins ◽  
Attachment Proteins ◽  
E Cadherin

Darier disease (DD) is an autosomal dominant keratinizing genodermatosis that manifests clinically with red-brown pruritic papules in a seborrheic distribution often in association with palmoplantar pits and dystrophic nail changes. It is caused by mutation in ATP2A2 which encodes a sarco/endoplasmic reticulum calcium ATPase isoform 2 (SERCA2) pump that regulates calcium flux. Consequent alteration of intracellular calcium homeostasis is thought to impair trafficking of cellular adhesion proteins and to lead to aberrant keratinocyte differentiation, contributing to the characteristic histopathologic features of acantholysis and dyskeratosis in DD, though the precise mechanisms are incompletely understood. Previous studies have identified defective localization of desmosomal attachment proteins in skin biopsies and cultured keratinocytes from DD patients, but reports of effects on adherens junction proteins (including calcium-dependent E-cadherin) are conflicting. Here we describe a case of DD presenting with characteristic clinical and histologic features in which we performed immunofluorescence staining of four adherens junction-associated proteins (E-cadherin, α-catenin, β-catenin, and vinculin). In lesional (acantholytic) DD skin, we identified loss of distinctive bright membranous staining that was present at the periphery of keratinocytes throughout the epidermis in the healthy skin of a matched donor. Perilesional (non-acantholytic) portions of DD skin partially recapitulated the normal phenotype. Our findings support a role for SERCA2 dysfunction in impaired assembly of adherens junctions, which together with defective desmosomes contribute to acantholysis in DD.

scholarly journals Junctional Adhesion Molecule-A Is Critical for the Formation of Pseudocanaliculi and Modulates E-cadherin Expression in Hepatic Cells

2007 ◽  
Vol 282 (38) ◽  
pp. 28137-28148 ◽  
Author(s):  
Genevieve Konopka ◽  
Jackie Tekiela ◽  
Moriah Iverson ◽  
Clive Wells ◽  
Stephen A. Duncan
Keyword(s):  
Tight Junction ◽  
Adhesion Molecule ◽  
Cell Morphology ◽  
Adherens Junction ◽  
Tight Junction Proteins ◽  
Hepatic Cells ◽  
Junction Protein ◽  
Striking Change ◽  
Junction Proteins ◽  
E Cadherin

Hepatocytes are polarized epithelial cells whose function depends upon their ability to distinguish between the apical and basolateral surfaces that are located at intercellular tight junctions. It has been proposed that the signaling cascades originating at these junctions influence cellular activity by controlling gene expression in the cell nucleus. To assess the validity of this proposal with regard to hepatocytes, we depleted expression of the tight junction protein junctional adhesion molecule-A (JAM-A) in the HepG2 human hepatocellular carcinoma cell line. Reduction of JAM-A resulted in a striking change in cell morphology, with cells forming sheets 1-2 cells thick instead of the normal multilayered clusters. In the absence of JAM-A, other tight junction proteins were mislocalized, and pseudocanaliculi, which form the apical face of the hepatocyte, were consequently absent. There was a strong transcriptional induction of the adherens junction protein E-cadherin in cells with reduced levels of JAM-A. This increase in E-cadherin was partially responsible for the observed alterations in cell morphology and mislocalization of tight junction proteins. We therefore propose the existence of a novel mechanism of cross-talk between specific components of tight and adherens junctions that can be utilized to regulate adhesion between hepatic cells.

scholarly journals Lactobacillus casei and Epidermal Growth Factor Prevent Osmotic Stress-Induced Tight Junction Disruption in Caco-2 Cell Monolayers

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3578
Author(s):  
Geetha Samak ◽  
Rupa Rao ◽  
Radhakrishna Rao
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Osmotic Stress ◽  
Tight Junction ◽  
Lactobacillus Casei ◽  
Kinase Inhibitor ◽  
Adherens Junction ◽  
Barrier Dysfunction ◽  
Cell Monolayers ◽  
Epidermal Growth

Osmotic stress plays a crucial role in the pathogenesis of many gastrointestinal diseases. Lactobacillus casei and epidermal growth factor (EGF) effects on the osmotic stress-induced epithelial junctional disruption and barrier dysfunction were investigated. Caco-2 cell monolayers were exposed to osmotic stress in the presence or absence of L. casei or EGF, and the barrier function was evaluated by measuring inulin permeability. Tight junction (TJ) and adherens junction integrity were assessed by immunofluorescence confocal microscopy. The role of signaling molecules in the L. casei and EGF effects was determined by using selective inhibitors. Data show that pretreatment of cell monolayers with L. casei or EGF attenuates osmotic stress-induced TJ and adherens junction disruption and barrier dysfunction. EGF also blocked osmotic stress-induced actin cytoskeleton remodeling. U0126 (MEK1/2 inhibitor), the MAP kinase inhibitor, blocked EGF-mediated epithelial protection from osmotic stress. In contrast, the L. casei-mediated epithelial protection from osmotic stress was unaffected by U0126, AG1478 (EGFR tyrosine kinase inhibitor), SP600125 (JNK1/2 inhibitor), or SB202190 (P38 MAP kinase inhibitor). On the other hand, Ro-32-0432 (PKC inhibitor) blocked the L. casei-mediated prevention of osmotic stress-induced TJ disruption and barrier dysfunction. The combination of EGF and L. casei is more potent in protecting the barrier function from osmotic stress. These findings suggest that L. casei and EGF ameliorate osmotic stress-induced disruption of apical junctional complexes and barrier dysfunction in the intestinal epithelium by distinct signaling mechanisms.

scholarly journals Structure-Based Virtual Screening Allows the Identification of Efficient Modulators of E-Cadherin-Mediated Cell–Cell Adhesion

2019 ◽  
Vol 20 (14) ◽  
pp. 3404 ◽  
Author(s):  
Andrea Dalle Vedove ◽  
Federico Falchi ◽  
Stefano Donini ◽  
Aurelie Dobric ◽  
Sebastien Germain ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Virtual Screening ◽  
Adherens Junction ◽  
Large Family ◽  
Calcium Dependent ◽  
Molecule Inhibitor ◽  
Dependent Cell ◽  
Cell Adhesion Proteins ◽  
E Cadherin ◽  
Cell Cell

Cadherins are a large family of transmembrane calcium-dependent cell adhesion proteins that orchestrate adherens junction formation and are crucially involved in tissue morphogenesis. Due to their important role in cancer development and metastasis, cadherins can be considered attractive targets for drug discovery. A recent crystal structure of the complex of a cadherin extracellular portion and a small molecule inhibitor allowed the identification of a druggable interface, thus providing a viable strategy for the design of cadherin dimerization modulators. Here, we report on a structure-based virtual screening approach that led to the identification of efficient and selective modulators of E-cadherin-mediated cell–cell adhesion. Of all the putative inhibitors that were identified and experimentally tested by cell adhesion assays using human pancreatic tumor BxPC-3 cells expressing both E-cadherin and P-cadherin, two compounds turned out to be effective in inhibiting stable cell–cell adhesion at micromolar concentrations. Moreover, at the same concentrations, one of them also showed anti-invasive properties in cell invasion assays. These results will allow further development of novel and selective cadherin-mediated cell–cell adhesion modulators for the treatment of a variety of cadherin-expressing solid tumors and for improving the efficiency of drug delivery across biological barriers.

scholarly journals PALS1 Regulates E-Cadherin Trafficking in Mammalian Epithelial Cells

2007 ◽  
Vol 18 (3) ◽  
pp. 874-885 ◽  
Author(s):  
Qian Wang ◽  
Xiao-Wei Chen ◽  
Ben Margolis
Keyword(s):  
Tight Junction ◽  
Adherens Junction ◽  
Cell Periphery ◽  
Madin Darby Canine Kidney ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Evolutionarily Conserved ◽  
Junction Formation ◽  
Darby Canine Kidney ◽  
E Cadherin

Protein Associated with Lin Seven 1 (PALS1) is an evolutionarily conserved scaffold protein that targets to the tight junction in mammalian epithelia. Prior work in our laboratory demonstrated that the knockdown of PALS1 in Madin Darby canine kidney cells leads to tight junction and polarity defects. We have created new PALS1 stable knockdown cell lines with more profound reduction of PALS1 expression, and a more severe defect in tight junction formation was observed. Unexpectedly, we also observed a severe adherens junction defect, and both defects were corrected when PALS1 wild type and certain PALS1 mutants were expressed in the knockdown cells. We found that the adherens junction structural component E-cadherin was not effectively delivered to the cell surface in the PALS1 knockdown cells, and E-cadherin puncta accumulated in the cell periphery. The exocyst complex was also found to be mislocalized in PALS1 knockdown cells, potentially explaining why E-cadherin trafficking is disrupted. Our results suggest a broad and evolutionarily conserved role for the tight junction protein PALS1 in the biogenesis of adherens junction.

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

2000 ◽  
Vol 113 (6) ◽  
pp. 985-996 ◽  
Author(s):  
M.L. Troxell ◽  
S. Gopalakrishnan ◽  
J. McCormack ◽  
B.A. Poteat ◽  
J. Pennington ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Tight Junction ◽  
Mdck Cells ◽  
Freeze Fracture ◽  
Junctional Complex ◽  
Negative Effects ◽  
Tight Junction Formation ◽  
Freeze Fracture Electron Microscopy ◽  
E Cadherin ◽  
Cell Cell

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.

scholarly journals Glucocorticoids Increase VE-Cadherin Expression and Cause Cytoskeletal Rearrangements in Murine Brain Endothelial cEND Cells

2008 ◽  
Vol 28 (6) ◽  
pp. 1139-1149 ◽  
Author(s):  
Kinga G Blecharz ◽  
Detlev Drenckhahn ◽  
Carola Y Förster
Keyword(s):  
Protein Synthesis ◽  
Tight Junction ◽  
Transcriptional Activation ◽  
Adherens Junction ◽  
Barrier Properties ◽  
Positive Influence ◽  
Brain Capillary Endothelial Cell ◽  
Protein Levels ◽  
Junction Protein ◽  
Adherens Junction Proteins

Recent studies have shown the influence of glucocorticoids on the expression of the tight junction protein occludin in the brain capillary endothelial cell line cEND, contributing to improvement in endothelial barrier functions. In this study, we investigated glucocorticoid effects on the expression of the adherens junction proteins VE- (vascular-endothelial) cadherin, α-catenin and β-catenin as well as that of ZO-1, the plaque protein shared by both adherens and tight junctions on stimulation with dexamethasone. We were able to show a positive influence of dexamethasone administration on VE-cadherin protein levels as well as a rearrangement of VE-cadherin protein to the cytoskeleton after dexamethasone treatment. Investigation of transcriptional activation of the VE-cadherin promoter by dexamethasone, however, did not point to direct glucocorticoid-mediated VE-cadherin gene induction but rather suggested indirect steroid effects leading to increased VE-cadherin protein synthesis. Dexamethasone was further shown to induce cellular differentiation into a cobblestone cellular morphology and reinforcement of adherens junctions concomitant with the increased anchorage of VE-cadherin to the actin cytoskeleton. We thus propose that glucocorticoid effects on VE-cadherin protein synthesis and organization are important for the formation of both adherens and tight junction, and for improved barrier properties in microvascular brain endothelial cells.

Phage Display Screening of Epithelial Cell Monolayers Treated with EGTA: Identification of Peptide FDFWITP that Modulates Tight Junction Activity

2007 ◽  
Vol 12 (8) ◽  
pp. 1092-1101 ◽  
Author(s):  
Richard E. Herman ◽  
Ekaterina G. Makienko ◽  
Mary G. Prieve ◽  
Mark Fuller ◽  
Michael E. Houston ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Phage Display ◽  
Tight Junctions ◽  
Phage Library ◽  
Junctional Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Monolayers ◽  
Lysine Residues

Phage display was used to screen for peptides that modulate the activity of epithelial cell tight junctions. Panning with a phage library that displays random 7-mers was performed using monolayers of human bronchial epithelial cells (16HBE14o—) treated with a calcium chelator, ethylene glycol-bis(2-aminoethylether)- N, N, N′, N′-tetraacetic acid (EGTA), to increase accessibility to the junctional complex/paracellular space, followed by subtractive panning. A novel peptide, FDFWITP, identified as a potential tight junction modulator, was synthesized in linear and cyclic forms with lysine residues added to improve solubility. The cyclic form of the peptide reduced transepithelial electrical resistance (TER) in a concentration-dependent manner (80% reduction at 100 µM and 95% reduction at 500 µM) and was reversible within 2 h; the linear form only affected TER at the highest concentration. Interestingly, the constrained peptide did not increase permeation of the model small molecule, fluorescein. The highly selective activity of FDFWITP supports the hypothesis that ions and small molecules may be transported paracellularly across tight junctions by separate pathways. ( Journal of Biomolecular Screening 2007:1092-1101)

scholarly journals Cyclic stretch disrupts apical junctional complexes in Caco-2 cell monolayers by a JNK-2-, c-Src-, and MLCK-dependent mechanism

2014 ◽  
Vol 306 (11) ◽  
pp. G947-G958 ◽  
Author(s):  
Geetha Samak ◽  
Ruchika Gangwar ◽  
Lynn M. Crosby ◽  
Leena P. Desai ◽  
Kristina Wilhelm ◽  
...  
Keyword(s):  
Tight Junction ◽  
Actin Cytoskeleton ◽  
Tight Junctions ◽  
Src Kinase ◽  
Adherens Junction ◽  
Paracellular Permeability ◽  
Cyclic Stretch ◽  
Cell Monolayers ◽  
Dependent Mechanism ◽  
Junction Proteins

The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 h resulted in a dramatic modulation of tight junction protein distribution from a linear organization into wavy structure. Continuation of cyclic stretch for 6 h led to redistribution of tight junction proteins from the intercellular junctions into the intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and β-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src, and myosin light-chain kinase (MLCK). Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions, and actin cytoskeleton. Paracellular permeability measured by a novel method demonstrated that cyclic stretch increases paracellular permeability by a JNK, Src kinase, and MLCK-dependent mechanism. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin, and β-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC colocalizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src colocalizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src, and MLCK-dependent mechanism.

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