Selective degradation of E-cadherin and dissolution of E-cadherin-catenin complexes in epithelial ischemia

2000 ◽  
Vol 278 (5) ◽  
pp. F847-F852 ◽  
Author(s):  
Kevin T. Bush ◽  
Tatsuo Tsukamoto ◽  
Sanjay K. Nigam
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Adherens Junction ◽  
Intercellular Junctions ◽  
Atp Depletion ◽  
Cell Phenotype ◽  
Western Blots ◽  
Selective Degradation ◽  
E Cadherin

Ischemic epithelial cells are characterized by disruption of intercellular junctions and loss of apical-basolateral protein polarity, which are normally dependent on the integrity of the adherens junction (AJ). Biochemical analysis of both whole ischemic kidneys and ATP-depleted Madin-Darby canine kidney (MDCK) cells demonstrated a striking loss of E-cadherin (the transmembrane protein of the AJ) with the appearance and accumulation of an ∼80-kDa fragment reactive with anti-E-cadherin antibodies on Western blots of ATP-depleted MDCK cells. This apparent ischemia-induced degradation of E-cadherin was not blocked by either inhibitors of the major proteolytic pathways (i.e., proteasome, lysosome, or calpain), or by chelation of intracellular calcium, suggesting the involvement of a protease capable of functioning at low ATP and low calcium levels. Immunocytochemistry revealed the movement of several proteins normally comprising the AJ, including E-cadherin and β-catenin, away from lateral portions of the plasma membrane to intracellular sites. Moreover, rate-zonal centrifugation and immunoprecipitation with anti-E-cadherin and anti-β-catenin antibodies indicated that ATP depletion disrupted normal E-cadherin-catenin interactions, resulting in the dissociation of α- and γ-catenin from E-cadherin and β-catenin-containing complexes. Because the generation and maintenance of polarized epithelial cells are dependent upon E-cadherin-mediated cell-cell adhesion and normal AJ function, we propose that the rapid degradation of E-cadherin and dissolution of the AJ is a key step in the development of the ischemic epithelial cell phenotype. Furthermore, we hypothesize that the reassembly of the AJ after ischemia/ATP depletion may require a novel bioassembly mechanism involving recombination of newly synthesized and sorted E-cadherin with preexisting pools of catenins that have (temporally) redistributed intracellularly.

scholarly journals Polycystin-1 and Gα12 regulate the cleavage of E-cadherin in kidney epithelial cells

2015 ◽  
Vol 47 (2) ◽  
pp. 24-32 ◽  
Author(s):  
Jen X. Xu ◽  
Tzong-Shi Lu ◽  
Suyan Li ◽  
Yong Wu ◽  
Lai Ding ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Signaling Pathway ◽  
Cell Polarity ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Renal Epithelial Cells ◽  
Extracellular Milieu ◽  
Kidney Epithelial Cells ◽  
Autosomal Dominant Polycystic Kidney ◽  
E Cadherin

Interaction of polycystin-1 (PC1) and Gα12 is important for development of kidney cysts in autosomal dominant polycystic kidney disease (ADPKD). The integrity of cell polarity and cell-cell adhesions (mainly E-cadherin-mediated adherens junction) is altered in the renal epithelial cells of ADPKD. However, the key signaling pathway for this alteration is not fully understood. Madin-Darby canine kidney (MDCK) cells maintain the normal integrity of epithelial cell polarity and adherens junctions. Here, we found that deletion of Pkd1 increased activation of Gα12, which then promoted the cystogenesis of MDCK cells. The morphology of these cells was altered after the activation of Gα12. By using liquid chromatography-mass spectrometry, we found several proteins that could be related this change in the extracellular milieu. E-cadherin was one of the most abundant peptides after active Gα12 was induced. Gα12 activation or Pkd1 deletion increased the shedding of E-cadherin, which was mediated via increased ADAM10 activity. The increased shedding of E-cadherin was blocked by knockdown of ADAM10 or specific ADAM10 inhibitor GI254023X. Pkd1 deletion or Gα12 activation also changed the distribution of E-cadherin in kidney epithelial cells and caused β-catenin to shift from cell membrane to nucleus. Finally, ADAM10 inhibitor, GI254023 X, blocked the cystogenesis induced by PC1 knockdown or Gα12 activation in renal epithelial cells. Our results demonstrate that the E-cadherin/β-catenin signaling pathway is regulated by PC1 and Gα12 via ADAM10. Specific inhibition of this pathway, especially ADAM10 activity, could be a novel therapeutic regimen for ADPKD.

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.

Cadmium is more toxic to LLC-PK1cells than to MDCK cells acting on the cadherin-catenin complex

1998 ◽  
Vol 275 (1) ◽  
pp. F143-F153 ◽  
Author(s):  
L. B. Zimmerhackl ◽  
F. Momm ◽  
G. Wiegele ◽  
M. Brandis
Keyword(s):  
Mdck Cells ◽  
Morphological Changes ◽  
Collecting Duct ◽  
Cadmium Toxicity ◽  
Distal Tubule ◽  
Insoluble Fraction ◽  
Atp Depletion ◽  
Rhodamine Phalloidin ◽  
Apical Side ◽  
E Cadherin

Cadmium toxicity to renal cells was investigated in Madin-Darby canine kidney (MDCK) and LLC-PK1cells as models of the distal tubule/collecting duct and proximal tubule, respectively. Cells were grown on two-compartment filters and exposed to 0.1–50 μM Cd2+. In MDCK cells, Cd2+was more toxic from the basolateral than from the apical side and dependent on the extracellular Ca2+concentration. Toxicity was evident within 24 h, as shown by a decrease in transepithelial resistance (TER), reduced proliferation (bromodeoxyuridine incorporation), reduction in ATP concentration, and morphological changes. On confocal microscopy, E-cadherin and α-catenin staining patterns indicated interference with the cadherin-catenin complex. LLC-PK1cells showed a similar toxicity pattern, which was evident at lower Cd2+concentrations. An increase of E-cadherin and α-catenin molecules in the Triton X-100-insoluble fraction was detectable at high Cd2+concentrations in LLC-PK1cells but not in MDCK cells. Lactate dehydrogenase release indicated membrane leakage in LLC-PK1cells. Rhodamine-phalloidin staining, a probe for F-actin filaments, demonstrated alterations of the actin cytoskeleton in both cell lines. In conclusion, cadmium caused ATP depletion and interfered with the cadherin-catenin complex and probably the tight junctions changing renal cell morphology and function.

scholarly journals Novel Membrane Protein shrew-1 Targets to Cadherin-Mediated Junctions in Polarized Epithelial Cells

2004 ◽  
Vol 15 (1) ◽  
pp. 397-406 ◽  
Author(s):  
Sanita Bharti ◽  
Heike Handrow-Metzmacher ◽  
Silvia Zickenheiner ◽  
Andreas Zeitvogel ◽  
Rudolf Baumann ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Membrane Protein ◽  
Transmembrane Protein ◽  
Adherens Junctions ◽  
Direct Interaction ◽  
Potential Candidate ◽  
Putative Open Reading Frame ◽  
Polarized Epithelial Cells ◽  
E Cadherin

While searching for potential candidate molecules relevant for the pathogenesis of endometriosis, we discovered a 2910-base pair cDNA encoding a novel putative 411-amino acid integral membrane protein that we called shrew-1. The putative open-reading frame was confirmed with antibodies against shrew-1 peptides that labeled a protein of ∼48 kDa in extracts of shrew-1 mRNA-positive tissue and also detected ectopically expressed shrew-1. Expression of epitope-tagged shrew-1 in epithelial cells and analysis by surface biotinylation and immunoblots demonstrated that shrew-1 is indeed a transmembrane protein. Shrew-1 is able to target to E-cadherin-mediated adherens junctions and interact with the E-cadherin–catenin complex in polarized MCF7 and Madin-Darby canine kidney cells, but not with the N-cadherin–catenin complex in nonpolarized epithelial cells. Direct interaction of shrew-1 with β-catenin in in vitro pull-down assay suggests that β-catenin might be one of the proteins that targets and/or retains shrew-1 in the adherens junctions. Interestingly, shrew-1 was partially translocated in response to scatter factor (ligand of receptor tyrosine kinase c-met) from the plasma membrane to the cytoplasm where it still colocalized with endogenous E-cadherin. In summary, we introduce shrew-1 as a novel component of adherens junctions, interacting with E-cadherin–β-catenin complexes in polarized epithelial cells.

scholarly journals E–N-cadherin heterodimers define novel adherens junctions connecting endoderm-derived cells

2011 ◽  
Vol 195 (5) ◽  
pp. 873-887 ◽  
Author(s):  
Beate K. Straub ◽  
Steffen Rickelt ◽  
Ralf Zimbelmann ◽  
Christine Grund ◽  
Caecilia Kuhn ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Adherens Junctions ◽  
Tumor Biology ◽  
Intercellular Junctions ◽  
Pivotal Role ◽  
Major Portion ◽  
Tissue Development ◽  
Cadherin Switch ◽  
And Function ◽  
E Cadherin

Intercellular junctions play a pivotal role in tissue development and function and also in tumorigenesis. In epithelial cells, decrease or loss of E-cadherin, the hallmark molecule of adherens junctions (AJs), and increase of N-cadherin are widely thought to promote carcinoma progression and metastasis. In this paper, we show that this “cadherin switch” hypothesis does not hold for diverse endoderm-derived cells and cells of tumors derived from them. We show that the cadherins in a major portion of AJs in these cells can be chemically cross-linked in E–N heterodimers. We also show that cells possessing E–N heterodimer AJs can form semistable hemihomotypic AJs with purely N-cadherin–based AJs of mesenchymally derived cells, including stroma cells. We conclude that these heterodimers are the major AJ constituents of several endoderm-derived tissues and tumors and that the prevailing concept of antagonistic roles of these two cadherins in developmental and tumor biology has to be reconsidered.

scholarly journals Cross-Talk between Adherens Junctions and Desmosomes Depends on Plakoglobin

1997 ◽  
Vol 136 (4) ◽  
pp. 919-934 ◽  
Author(s):  
Jani E. Lewis ◽  
James K. Wahl ◽  
Kristin M. Sass ◽  
Pamela J. Jensen ◽  
Keith R. Johnson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Adherens Junctions ◽  
Adherens Junction ◽  
Epithelial Cell Line ◽  
Cell Contact ◽  
Common Component ◽  
Classical Cadherins ◽  
E Cadherin

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either β-catenin or plakoglobin, which is linked to α-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell–cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of β-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization.

Rho-kinase regulates myosin II activation in MDCK cells during recovery after ATP depletion

2001 ◽  
Vol 281 (5) ◽  
pp. F810-F818 ◽  
Author(s):  
Timothy A. Sutton ◽  
Henry E. Mang ◽  
Simon J. Atkinson
Keyword(s):  
Epithelial Cells ◽  
Actin Cytoskeleton ◽  
Mdck Cells ◽  
Myosin Ii ◽  
Atp Depletion ◽  
Stress Fibers ◽  
Tubular Epithelial Cells ◽  
Renal Tubular Epithelial Cells ◽  
Renal Tubular ◽  
Actin Stress Fibers

Alterations in the actin cytoskeleton of renal tubular epithelial cells during periods of ischemic injury and recovery have important consequences for normal cell and kidney function. Myosin II has been demonstrated to be an important effector in organizing basal actin structures in some cell types. ATP depletion in vitro has been demonstrated to recapitulate alterations of the actin cytoskeleton in renal tubular epithelial cells observed during renal ischemia in vivo. We utilized this reversible cell culture model of ischemia to examine the correlation of the activation state and cellular distribution of myosin II with disruption of actin stress fibers in Madin-Darby canine kidney (MDCK) cells during ATP depletion and recovery from ATP depletion. We found that myosin II inactivation occurs rapidly and precedes dissociation of myosin II from actin stress fibers during ATP depletion. Myosin II activation temporally correlates with colocalization of myosin II to reorganizing stress fibers during recovery from ATP depletion. Furthermore, myosin activation and actin stress fiber formation were found to be Rho-associated Ser/Thr protein kinase dependent during recovery from ATP depletion.

scholarly journals The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity

2015 ◽  
Vol 210 (2) ◽  
pp. 333-346 ◽  
Author(s):  
Pierre-Olivier Strale ◽  
Laurence Duchesne ◽  
Grégoire Peyret ◽  
Lorraine Montel ◽  
Thao Nguyen ◽  
...  
Keyword(s):  
Adherens Junction ◽  
Intercellular Junctions ◽  
Cell Contact ◽  
Collective Cell Migration ◽  
Contact Stability ◽  
Junction Formation ◽  
Mediate Cell ◽  
The Stability ◽  
E Cadherin ◽  
Cell Cell

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell–cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell–cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell–cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell–cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell–cell contact fluidity.

scholarly journals Hydrolysis of Epithelial Junctional Proteins by Porphyromonas gingivalis Gingipains

2002 ◽  
Vol 70 (5) ◽  
pp. 2512-2518 ◽  
Author(s):  
Jannet Katz ◽  
Qiu-Bo Yang ◽  
Ping Zhang ◽  
Jan Potempa ◽  
James Travis ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Porphyromonas Gingivalis ◽  
Catalytic Domain ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Cell Junction ◽  
Critical Threshold ◽  
Hydrolysis Of ◽  
E Cadherin ◽  
Cell Cell

ABSTRACT Porphyromonas gingivalis has been implicated as an etiologic agent of adult periodontitis. We have previously shown that P. gingivalis can degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains of P. gingivalis (i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation of P. gingivalis cells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure of P. gingivalis cells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue by P. gingivalis.

scholarly journals Disruption of Tight Junctions and Induction of Proinflammatory Cytokine Responses in Colonic Epithelial Cells by Campylobacter jejuni

2006 ◽  
Vol 74 (12) ◽  
pp. 6581-6589 ◽  
Author(s):  
Ming L. Chen ◽  
Zhongming Ge ◽  
James G. Fox ◽  
David B. Schauer
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Campylobacter Jejuni ◽  
Transmembrane Protein ◽  
Intercellular Junctions ◽  
Subcellular Fractionation ◽  
Intestinal Epithelial ◽  
Intracellular Location ◽  
Mitogen Activated Protein ◽  
Triton X

ABSTRACT Campylobacter jejuni is a leading cause of human enterocolitis and is associated with postinfectious complications, including irritable bowel syndrome and Guillain-Barré syndrome. However, the pathogenesis of C. jejuni infection remains poorly understood. Paracellular pathways in intestinal epithelial cells are gated by intercellular junctions (tight junctions and adherens junctions), providing a functional barrier between luminal microbes and host immune cells in the lamina propria. Here we describe alterations in tight junctions in intestinal epithelial monolayers following C. jejuni infection. Apical infection of polarized T84 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TER). Immunofluorescence microscopy revealed a redistribution of the tight junctional transmembrane protein occludin from an intercellular to an intracellular location. Subcellular fractionation using equilibrium sucrose density gradients demonstrated decreased hyperphosphorylated occludin in lipid rafts, Triton X-100-soluble fractions, and the Triton X-100-insoluble pellet following apical infection. Apical infection with C. jejuni also caused rapid activation of NF-κB and AP-1, phosphorylation of extracellular signal-regulated kinase, Jun N-terminal protein kinase, and p38 mitogen-activated protein kinases, and basolateral secretion of the CXC chemokine interleukin-8 (IL-8). Basolateral infection with C. jejuni caused a more rapid decrease in TER, comparable redistribution of tight-junction proteins, and secretion of more IL-8 than that seen with apical infection. These results suggest that compromised barrier function and increased chemokine expression contribute to the pathogenesis of C. jejuni-induced enterocolitis.

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