Disassembly Of Adherens Junctions Research Articles

Src-Induced Disassembly of Adherens Junctions Requires Localized Phosphorylation and Degradation of the Rac Activator Tiam1

The Rac activator Tiam1 is required for adherens junction (AJ) maintenance, and its depletion results in AJ disassembly. Conversely, the oncoprotein Src potently induces AJ disassembly and epithelial-mesenchymal transition (EMT). Here, we show that Tiam1 is phosphorylated on Y384 by Src. This occurs predominantly at AJs, is required for Src-induced AJ disassembly and cell migration, and creates a docking site on Tiam1 for Grb2. We find that Tiam1 is associated with ERK. Following recruitment of the Grb2-Sos1 complex, ERK becomes activated and triggers the localized degradation of Tiam1 at AJs, likely involving calpain proteases. Furthermore, we demonstrate that, in human tumors, Y384 phosphorylation positively correlates with Src activity, and total Tiam1 levels are inversely correlated. Thus, our data implicate Tiam1 phosphorylation and consequent degradation in Src-mediated EMT and resultant cell motility and establish a paradigm for regulating local concentrations of Rho-GEFs.

Identification of a small molecule class to enhance cell-cell adhesion and attenuate prostate tumor growth and metastasis

Expression of calcitonin (CT) and its receptor (CTR) is elevated in advanced prostate cancer, and activated CT-CTR autocrine axis plays a pivotal role in tumorigenicity and metastatic potential of multiple prostate cancer cell lines. Recent studies suggest that CT promotes prostate cancer metastasis by reducing cell-cell adhesion through the disassembly of tight and adherens junctions and activation of beta-catenin signaling. We attempted to identify a class of molecules that enhances cell-cell adhesion of prostate cells and reverses the disruptive actions of CT on tight and adherens junctions. Screening several compounds led to the emergence of phenyl-methylene hydantoin (PMH) as a lead candidate that can augment cell-cell adhesion and abolish disruptive actions of CT on junctional complexes. PMH reduced invasiveness of PC-3M cells and abolished proinvasive actions of CT. Importantly, PMH did not display significant cytotoxicity on PC-3M cells at the tested doses. I.p. administered PMH and its S-ethyl derivative remarkably decreased orthotopic tumor growth and inhibited the formation of tumor micrometastases in distant organs of nude mice. PMH treatment also reduced the growth of spontaneous tumors in LPB-Tag mice to a significant extent without any obvious cytotoxic effects. By virtue of its ability to stabilize cell junctions, PMH could reverse the effect of CT on junctional disruption and metastasis, which strengthens the possibility of using PMH as a potential drug candidate for CT-positive androgen-independent prostate cancers.

Cleavage of Armadillo/beta-catenin by the caspase DrICE in Drosophilaapoptotic epithelial cells

BackgroundDuring apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases. In epithelial tissues, apoptotic cells loose their apical/basal polarity and are extruded from the epithelium. We used the Drosophila embryo as a system to investigate the regulation of components of the zonula adherens during apoptosis. Since Armadillo/beta-catenin (Arm) is a major regulator of cadherin-mediated adhesion, we analyzed the mechanisms of Arm proteolysis in apoptosis.ResultsWe define early and late apoptotic stages and find that early in apoptosis Dα-catenin remains relatively stable, while Arm and DE-cadherin protein levels are strongly reduced. Arm is cleaved by caspases in embryo extracts and we provide evidence that the caspase-3 homolog drICE cleaves Arm in vitro and in vivo. Cleavage by drICE creates a stable protein fragment that remains associated with the plasma membrane early in apoptosis. To further understand the role of caspase-mediated cleavage of Arm, we examined potential caspase cleavage sites and found that drICE cleaves Arm at a unique DQVD motif in the N-terminal domain of the protein. Mutation of the drICE cleavage site in Arm results in a protein that is not cleaved in vitro and in vivo. Furthermore we provide evidence that cleavage of Arm plays a role in the removal of DE-cadherin from the plasma membrane during apoptosis.ConclusionThis study defines the specificity of caspase cleavage of Arm in Drosophila apoptotic cells. Our data suggest that N-terminal truncation of Arm by caspases is evolutionarily conserved and thus might provide a principal mechanism involved in the disassembly of adherens junctions during apoptosis.

Lymphostatin – ein Multitalent

Gastrointestinal infections are a significant cause of diarrhea and a worldwide problem with annually one billion illnesses and 3 to 4 million deaths. Gram negative bacteria like Enteropathogenic E. coli (EPEC) in developing countries and Enterohemorrhagic E. coli (EHEC) in the developed world are responsible for the majority of acute diarrheal episodes, especially among children less than three years of age. Pathogenic E. coli are supplied with an arsenal of effector proteins to modify and neutralize specific cellular functions of the host organism.Based on personal and extensively published results we provide a selected overview of Gram negative virulence functions with a focus on lymphostatin.Lymphostatin is an effector protein encoded by lifA/efa-1 (lymphocyte inhibitory factor A/ EHEC factor for adherence). lifA/efa-1 and homologous genes have been identified in EPEC, EHEC, and mouse pathogen Citrobacter rodentium, as well as various Chlamydia strains. Multiple groups have shown that in various EHEC strains lifA/efa-1 is part of a larger pathogenicity island responsible for increased virulence. Statistically, DNA Microarray analysis associated lifA/efa-1 as the single most imortant gene with diarrhea caused by EPEC. Interestingly, lifA/efa-1 encodes for two critical enzymatic activities that have been identified in other pathogenic bacteria: glucosyltransferase- in Clostridium- and protease activity in Yersinia strains. In vitro studies identified lymphostatin as an effector protein with an immunosuppressive effect on peripheral blood and gastrointestinal mucosa T lymphocytes. Further, lymphostatin regulates the barrier function of epithelial monolayer cultures: activation of small GTPase RhoA and inhibition of Cdc42 lead to disassembly of adherens junctions and tight junctions, respectively. Besides an effect on immune and epithelial barrier function, lymphostatin also functions as an adhesion factor for EPEC and EHEC, is essential for colonization of mouse and calf intestine, and regulates bacterial effector proteins.Lymphostatin is a common toxin in Gram negative bacteria with multiple functions: cell adhesion, immunosuppression, disruption of epithelial barrier function, and intestinal colonization.

Protein kinase C-Fyn kinase cascade mediates the oleic acid-induced disassembly of neonatal rat cardiomyocyte adherens junctions

Oleic acid (OA) affects assembly of gap junctions in neonatal cardiomyocytes. Adherens junction (AJ) regulates the stability of gap junction integrity; however, the effect of OA on AJ remains largely unexplored. The distribution of N-cadherin and catenins at cell–cell junction was decreased by OA. OA induced activation of protein kinase C(PKC)-α and -ɛ and Src family kinase, and all three kinases were involved in the oleic acid-induced disassembly of the adherens junction, since it was blocked by pretreatment with Gö6976 (a PKCα inhibitor), ɛV1–2 (a PKCɛ inhibitor), or PP2 (a Src family kinase inhibitor). Src family kinase appeared to be the downstream of PKC-α and -ɛ, as blockade of either PKC-α or -ɛ activity prevented the OA-induced activation of Src family kinase. Immunoprecipitation analyses showed that OA activated Fyn and Fer. OA promoted the association of p120 catenin/β-catenin with Fyn and Fer and caused increased tyrosine phosphorylation of p120 catenin and β-catenin, resulting in decreased binding of the former to N-cadherin and of the latter to α-catenin. Pretreatment with PP2 abrogated this OA-induced tyrosine phosphorylation of p120 catenin and β-catenin and restored the association of N-cadherin with p120 catenin and that of β-catenin with α-catenin. In conclusion, these results show that OA activates the PKC-Fyn signaling pathway, leading to the disassembly of the AJ. Therefore, inhibitors of PKC-α/-ɛ and Src family kinase are potential candidates as cardioprotection agents against OA-induced heart injury during ischemia-reperfusion.

Letting go: modification of cell adhesion during apoptosis

Apoptosis appears to be a carefully orchestrated process for the ordered dismantling of cells. A recent paper in BMC Developmental Biology shows that the disassembly of adherens junctions during apoptosis in Drosophila is progressive and requires the amino-terminal cleavage of the β-catenin Armadillo by the apoptotic effector caspase DrICE.

Breaking down EMT

Epithelial-mesenchymal transition, in which epithelial cells lose their polarity and become motile mesenchymal cells, occurs during development and marks a key step in tumour progression towards metastasis. Most studies of this process have focused on the disassembly of adherens junctions, but regulation of basement membrane breakdown by a pathway involving RhoA and microtubules may be equally important.

Activation of protein kinase C disrupts epithelial apical junctions via ROCK‐dependent stimulation of myosin II contractility

Activation of protein kinase C (PKC) destabilizes epithelial cell‐cell adhesions and therefore promotes cancer cell dissemination via unknown mechanism. The aim of this study was to investigate mechanisms underlying disassembly of epithelial tight junctions (TJ) and adherens junctions (AJ) induced by PKC activation. Exposure of HPAFII human pancreatic adenocarcinoma cell monolayers to either Octylindolactam V (OI) or phorbol ester caused a rapid (within 3–5 h) and orchestrated disassembly and internalization of AJ and TJ. Junctional disassembly was accompanied by translocation of the F‐actin motor myosin II to the lateral plasma membrane and by myosin II activation (phosphorylation). The OI‐induced disruption of AJ/TJ was significantly attenuated by either pharmacological inhibition of myosin II with blebbistatin or by siRNA‐mediated knockdown of the myosin IIA isoform. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho‐associated kinase (ROCK) but was insensitive to blockage of MLCK, ERK, calmodulin or Rac‐1. These findings support a role of PKC in disassembly of epithelial junctions by stimulating ROCK‐dependent contractility of myosin II which in turn can contribute to cancer cell dissociation and metastasis. Supported by the Crohn's and Colitis Foundation of America (AII and SNS) and NIH (AN: DK59888 and CAP: DK61379).

Deletion of PKCalpha in mice attenuates the thrombin‐induced increase in lung vascular permeability

We addressed the role of protein kinase Cα (PKCα) in signaling the increase in lung vascular permeability induced by the mediator thrombin. We used PKCα knockout mice to determine the effect of PKCα deletion on changes in endothelial permeability and adherens junction proteins. We determined microvessel filtration coefficient (Kfc) using the isolated‐perfused mouse lung preparation. Stimulation with PAR‐1 agonist peptide (10 μM) increased Kfc 50% less in the knockout mice than control wild type mice. We quantified vascular endothelial (VE) cadherin levels in mouse lung endothelial cells (MLECs) by Western blot analysis. Thrombin rapidly diminished VE‐cadherin levels in MLECs. PKCα deletion abrogated the VE‐cadherin disruption induced by thrombin. As PKCα activation was known to inhibit Akt, we observed higher Akt activity in PKCα‐deleted MLECs. We also observed that activated PKCζ was markedly increased in PKCα‐deleted MLECs, suggesting compensatory activation of PKCζ. The data demonstrate that PKCα is required to elicit the increase in lung vascular permeability. The mechanism of PKCα‐mediated increase in vascular permeability may involve signaling at the level of VE‐cadherin that promotes the disassembly of adherens junctions.This study is supported by NIH # HL45638

Actin motors that drive formation and disassembly of epithelial apical junctions

Tight junctions (TJ) and adherens junctions (AJ) are the most characteristic morphological features of differentiated epithelia which mediate cell-cell adhesions, establishment of the paracellular barrier and development of apico-basal cell polarity. In polarized epithelial cells, TJ and AJ associate with the prominent apical actin cytoskeleton, which is known to stabilize junctional structure and to tighten the epithelial barrier. Furthermore, plasticity of the actin cytoskeleton is thought to be critical for the remodeling of epithelial junctions. Two major molecular motors such as myosin II and actin filament turnover provide driving forces for reorganizations of the actin cytoskeleton. The review focuses on the roles of these actin motors in the establishment, maintenance and disassembly of epithelial TJ and AJ during tissue morphogenesis and in pathology.

Regulation of cerebral endothelial cell morphology by extracellular calcium

Cerebral endothelial cells interconnected by tight and adherens junctions constitute the structural basis of the blood-brain barrier. Extracellular calcium ions have been reported to play an important role in the formation and maintenance of the junctional complex. However, little is known about the action of calcium depletion on the structural characteristics of cerebral endothelial cells. Using atomic force microscopy we analyzed the effect of calcium depletion and readdition on the shape and size of living brain endothelial cells. It was found that the removal of extracellular calcium from confluent cell cultures induced the dissociation of the cells from each other accompanied by an increase in their height. After readdition of calcium a gradual recovery was observed until total confluency was regained. We have also demonstrated that Rho-kinase plays an important role in the calcium-depletion-induced disassembly of endothelial tight and adherens junctions. The Rho-kinase inhibitor Y27632 could prevent the morphological changes induced by a lack of calcium as well. Our results suggest that calcium depletion induces Rho-kinase-dependent cytoskeletal changes that may be partly responsible for the disassembly of the junctional complex.

Id-1 promotes TGF-β1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-β1 (transforming growth factor β1). Here we demonstrate a novel role of Id-1 in promoting TGF-β1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-β1, which was associated with increased cell–substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-β1-induced cell motility was mediated through activation of MEK–ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-β1-treated cells through down-regulation of E-cadherin, redistribution of β-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell–cell adhesion in response to TGF-β1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-β1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.

Polarity of Response to Transforming Growth Factor-β1 in Proximal Tubular Epithelial Cells Is Regulated by β-Catenin

Transforming growth factor-beta1 (TGF-beta1)-mediated loss of proximal tubular epithelial cell-cell interaction is regulated in a polarized fashion. The aim of this study was to further explore the polarity of the TGF-beta1 response and to determine the significance of R-Smad-beta-catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-beta1 was assessed by activity of the Smad3-responsive reporter gene construct (SBE)(4)-Lux and by immunoblotting for phospho-Smad proteins. Similar results were obtained with both methods. Apical application of TGF-beta1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with beta-catenin was greater following basolateral TGFbeta-1 stimulation, as was the expression of cytoplasmic Triton-soluble beta-catenin. Inhibition of beta-catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of beta-catenin and attenuated TGF-beta1-dependent Smad3 signaling. Lithium chloride did not influence degradation of Smad3 but resulted in decreased nuclear translocation. Smad2 activation as assessed by Western blot analysis and activity of the Smad2-responsive reporter constructs ARE/MF1 was also greater following apical as compared with basolateral TGFbeta-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-beta1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-beta1 and increased expression of beta-catenin. We used this cell line to address the general applicability of our observations. Inhibition of beta-catenin in this cell line by small interfering RNA resulted in increased TGF-beta1-dependent Smad3 phosphorylation and restoration of TGF-beta1 anti-proliferative effects.

Adherens junction remodeling by the Notch pathway in Drosophila melanogaster oogenesis

Identifying genes involved in the control of adherens junction (AJ) remodeling is essential to understanding epithelial morphogenesis. During follicular epithelium development in Drosophila melanogaster, the main body follicular cells (MBFCs) are displaced toward the oocyte and become columnar. Concomitantly, the stretched cells (StCs) become squamous and flatten around the nurse cells. By monitoring the expression of epithelial cadherin and Armadillo, I have discovered that the rate of AJ disassembly between the StCs is affected in follicles with somatic clones mutant for fringe or Delta and Serrate. This results in abnormal StC flattening and delayed MBFC displacement. Additionally, accumulation of the myosin II heavy chain Zipper is delayed at the AJs that require disassembly. Together, my results demonstrate that the Notch pathway controls AJ remodeling between the StCs and that this role is crucial for the timing of MBFC displacement and StC flattening. This provides new evidence that Notch, besides playing a key role in cell differentiation, also controls cell morphogenesis.

A unique role for the nonmuscle myosin IIA in regulation of epithelial apical junctions

The integrity of epithelial monolayers depends on the formation of tight junctions (TJ) and adherens junctions (AJ). TJ and AJ associate with underlying actomyosin bundles that are critical for their biogenesis and functions. We investigated roles of three nonmuscle myosin II (NMM II) isoforms, A, B, and C in the regulation of epithelial TJ and AJ. NMM IIA, NMM IIB and NMM IIC were all expressed in SK-CO15 and Caco-2 colonic epithelial cell lines, and all isoforms colocalized with perijunctional F-actin. Using isoform-specific siRNAs, we selectively down-regulated expression of the NMM IIA, NMM IIB, or NMM IIC in SK-CO15 cells. Down-regulation of the NMM IIA caused the following morphological and functional changes: transformation from an orthogonal epithelial to a protrusive fibroblast-like cell shape; a three fold decrease in transepithelial electrical resistance; attenuation of TJ and AJ disassembly and cell contraction induced by extracellular calcium depletion; inhibition of TJ and AJ reassembly after calcium repletion. None of the above changes were observed after down-regulation of either the NMM IIB or NMM IIC. These findings suggest that the NMM IIA plays a unique role in regulation of the dynamics and barrier properties of epithelial apical junctions. Supported by the Crohn’s and Colitis Foundation of America (AII) and NIH (AN and CAP).

Endosomes generate localized Rho–ROCK–MLC2–based contractile signals via Endo180 to promote adhesion disassembly

The regulated assembly and disassembly of focal adhesions and adherens junctions contributes to cell motility and tumor invasion. Pivotal in this process is phosphorylation of myosin light chain-2 (MLC2) by Rho kinase (ROCK) downstream of Rho activation, which generates the contractile force necessary to drive disassembly of epithelial cell–cell junctions and cell–matrix adhesions at the rear of migrating cells. How Rho–ROCK–MLC2 activation occurs at these distinct cellular locations is not known, but the emerging concept that endocytic dynamics can coordinate key intracellular signaling events provides vital clues. We report that endosomes containing the promigratory receptor Endo180 (CD280) can generate Rho–ROCK–MLC2–based contractile signals. Moreover, we provide evidence for a cellular mechanism in which Endo180-containing endosomes are spatially localized to facilitate their contractile signals directly at sites of adhesion turnover. We propose migration driven by Endo180 as a model for the spatial regulation of contractility and adhesion dynamics by endosomes.

Effect of Heparin II Domain of Fibronectin on Actin Cytoskeleton and Adherens Junctions in Human Trabecular Meshwork Cells

To determine whether the heparin II (HepII) domain of fibronectin previously shown to increase outflow facility affects the formation and assembly of actin cytoskeleton and adherens junctions in human trabecular meshwork (HTM) cells. Normal HTM cells and two transformed HTM cell lines were treated for 24 hours with increasing concentrations of the HepII domain. Disruptions in adherens junctions and the actin cytoskeleton were determined using immunofluorescence microscopy and Western blot analysis of immunoprecipitated cadherin/catenin complexes. Actin filaments were detected with phalloidin. Catenin (alpha and beta) and cadherin antibodies were used to detect adherens junctions. Treatments of cultures with the HepII domain caused cadherin/beta-catenin complexes in adherens junctions and actin filaments to disassemble. The disruption of adherens junctions and actin filaments occurred in a dose-dependent and temporal fashion. The disassembly of actin filaments occurred first, followed by the disassembly of adherens junctions. Dissociation of adherens junctions, but not actin filaments, was reversible if the HepII domain was removed. Reassembly of actin filaments required the addition of serum. Serum, however, could not trigger the reassembly of actin filaments if the HepII domain was present, suggesting that the HepII domain acted downstream of the serum stimulated RhoA activity. The exposure of HTM cells to the HepII domain triggers the disassembly of actin filaments and the subsequent destabilization of adherens junctions in HTM cells. This suggests that the HepII domain may increase outflow facility in cultured anterior segments by altering the organization of the TM cytoarchitecture.

E-cadherin endocytosis regulates the activity of Rap1: a traffic light GTPase at the crossroads between cadherin and integrin function

The coordinate modulation of cadherin and integrin functions plays an essential role in fundamental physiological and pathological processes, including morphogenesis and cancer. However, the molecular mechanisms underlying the functional crosstalk between cadherins and integrins are still elusive. Here, we demonstrate that the small GTPase Rap1, a crucial regulator of the inside-out activation of integrins, is a target for E-cadherin-mediated outside-in signaling. In particular, we show that a strong activation of Rap1 occurs upon adherens junction disassembly that is triggered by E-cadherin internalization and trafficking along the endocytic pathway. By contrast, Rap1 activity is not influenced by integrin outside-in signaling. Furthermore, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and controlled by an increased Src kinase activity, and is paralleled by the colocalization of Rap1 and E-cadherin at the perinuclear Rab11-positive recycling endosome compartment, and the association of Rap1 with a subset of E-cadherin-catenin complexes that does not contain p120ctn. Conversely, Rap1 activity is suppressed by the formation of E-cadherin-dependent cell-cell junctions as well as by agents that inhibit either Src activity or E-cadherin internalization and intracellular trafficking. Finally, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and is required for the formation of integrin-based focal adhesions. Our findings provide the first evidence of an E-cadherin-modulated endosomal signaling pathway involving Rap1, and suggest that cadherins may have a novel modulatory role in integrin adhesive functions by fine-tuning Rap1 activation.

Possible mechanism of metastasis in lung adenocarcinomas with a micropapillary pattern

Micropapillary differentiation in adenocarcinomas has recently been associated with poor prognosis because these tumors are more likely to metastasize. However, no clear explanation exists as to why the presence of a micropapillary pattern is associated with metastasis. A case of primary lung adenocarcinoma with a prominent micropapillary pattern is presented here, with special reference to the immunohistochemical expression of the E-cadherin-mediated system and IQGAP1. Histologically, the tumor was diagnosed as a moderately differentiated papillary adenocarcinoma, showing an extensive micropapillary pattern, with intrapulmonary metastases, pulmonary disseminations, lymphovascular invasions, and lymph node metastases. Immunohistochemically, positive staining for the adhesion molecules E-cadherin, alpha-catenin, and beta-catenin was detected in both the micropapillary and non-micropapillary areas, whereas IQGAP1 was detected in the micropapillary, but not in the non-micropapillary, area. The adhesive function of E-cadherin depends on the integrity of the entire cadherin-catenin-actin network, and thus the expression of IQGAP1 may lead to adherens junction disassembly, and consequently, the release of carcinoma cells organizing in a micropapillary pattern. This is the first report to suggest correlation between adenocarcinoma with a micropapillary pattern and the presence of adhesion molecules, and offers an intriguing first glimpse on the role of the micropapillary pattern in the process of metastasis.

Crosstalk between Rab GTPases and cell junctions

For the past several years, studies from other laboratories, as well as ours, have begun to unravel the mechanism of germ cell movement in the testis by using several in vitro and in vivo models of tight and adherens junction assembly and disassembly, two cellular phenomena that confer cell movement. However, for cell movement to be fully appreciated, the importance of “intracellular” cell movements, such as those involving actin and microtubule filaments, must be better understood. Recent research on Rab GTPases has shown that members of this superfamily function in the trafficking of vesicles containing cargo to distinct subcellular sites such as the plasma membrane while utilizing actin and microtubule filaments as tracks. In this mini-review, we provide an overview of Rab GTPase structure, function, and regulation, while placing added emphasis on the role of Rabs in cell junction dynamics in the testis.