Disassembly Of Adherens Junctions Research Articles

Regulation of epithelial polarity by the E3 ubiquitin ligase Neuralized and the Bearded inhibitors in Drosophila

Understanding how epithelial polarity is established and regulated during tissue morphogenesis is a major issue. Here, we identify a regulatory mechanism important for mesoderm invagination, germ-band extension and transepithelial migration in the Drosophila melanogaster embryo. This mechanism involves the inhibition of the conserved E3 ubiquitin ligase Neuralized by proteins of the Bearded family. First, Bearded mutant embryos exhibited a loss of epithelial polarity associated with an early loss of the apical domain. Bearded regulated epithelial polarity by antagonizing neuralized. Second, repression of Bearded gene expression by Snail was required for the Snail-dependent disassembly of adherens junctions in the mesoderm. Third, neuralized was strictly required to promote the downregulation of the apical domain in the midgut epithelium and to facilitate the transepithelial migration of primordial germ cells across this epithelium. This function of Neuralized was independent of its known role in Notch signalling. Thus, Neuralized has two distinct functions in epithelial cell polarity and Notch signalling.

Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through β-arrestin and dishevelled-2 scaffolds

Protease-activated receptor-1 (PAR1) is a guanine nucleotide-binding (G) protein-coupled receptor that elicits cellular responses to coagulant and anticoagulant proteases. Activation of PAR1 by the coagulant protease thrombin results in Ras homolog gene family member A (RhoA) activation, disassembly of adherens junctions, and disruption of the endothelial barrier. In contrast, activation of PAR1 with the anticoagulant protease activated protein C (APC) results in activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and endothelial barrier protection. We previously showed that APC cytoprotective signaling requires the compartmentalization of PAR1 in caveolar microdomains. However, the mechanism by which APC-activated PAR1 promotes cytoprotective signaling in human endothelial cells remains poorly understood. Here we show that APC-activated PAR1 cytoprotective signaling is mediated by β-arrestin recruitment and activation of the dishevelled-2 (Dvl-2) scaffold and not by G protein α inhibiting activity polypeptide 2 (Gα(i)) signaling. In human endothelial cells, PAR1 and β-arrestins form a preassembled complex and cosegregate in caveolin-1-enriched fractions. Remarkably, we found that depletion of β-arrestin expression by RNA interference resulted in the loss of APC-induced Rac1 activation but not of thrombin-stimulated RhoA signaling. APC also failed to protect against thrombin-induced endothelial barrier permeability in cells deficient in β-arrestin expression. We further demonstrate that APC activation of PAR1 results in β-arrestin-dependent recruitment of Dvl-2, which is critical for Rac1 signaling and endothelial barrier protection but not for thrombin-induced RhoA signaling. Our findings identify a role for β-arrestin and Dvl-2 scaffolds in APC-activated PAR1 cytoprotective signaling in human endothelial cells.

Cigarette smoke causes lung vascular barrier dysfunction via oxidative stress-mediated inhibition of RhoA and focal adhesion kinase

Cigarette smoke (CS) is a major cause of chronic lung and cardiovascular diseases. Recent studies indicate that tobacco use is also a risk factor for acute lung injury (ALI) associated with blunt trauma. Increased endothelial cell (EC) permeability is a hallmark of ALI. CS increases EC permeability in vitro and in vivo; however, the underlying mechanism is not well understood. In this study, we found that only 6 h of exposure to CS impaired endothelial barrier function in vivo, an effect associated with increased oxidative stress in the lungs and attenuated by the antioxidant N-acetylcysteine (NAC). CS also exacerbated lipopolysaccharide (LPS)-induced increase in vascular permeability in vivo. Similar additive effects were also seen in cultured lung EC exposed to cigarette smoke extract (CSE) and LPS. We further demonstrated that CSE caused disruption of focal adhesion complexes (FAC), F-actin fibers, and adherens junctions (AJ) and decreased activities of RhoA and focal adhesion kinase (FAK) in cultured lung EC. CSE-induced inhibition of RhoA and FAK, endothelial barrier dysfunction, and disassembly of FAC, F-actin, and AJ were prevented by NAC. In addition, the deleterious effects of CSE on FAC, F-actin fibers, and AJ were blunted by overexpression of constitutively active RhoA and of FAK. Our data indicate that CS causes endothelial barrier dysfunction via oxidative stress-mediated inhibition of RhoA and FAK.

Helicobacter pylori Activates Calpain via Toll-Like Receptor 2 To Disrupt Adherens Junctions in Human Gastric Epithelial Cells

Helicobacter pylori is a risk factor for the development of gastritis, gastroduodenal ulcers, and gastric adenocarcinoma. H. pylori-induced disruption of epithelial adherens junctions (AJs) is thought to promote the development of severe disease; however, the mechanisms whereby H. pylori alters AJ structure remain incompletely understood. The present study demonstrates that H. pylori infection in human patients is associated with elevated serum levels of an 80-kDa E-cadherin ectodomain, whose presence is independent of the presence of serum antibodies against CagA. In vitro, a heat-labile H. pylori surface component activates the host protease calpain in human gastric MKN45 cells independently of the virulence factors CagA and VacA. H. pylori-induced calpain activation results in cleavage of E-cadherin to produce a 100-kDa truncated form and induce relocalization of E-cadherin and β-catenin. Stimulation of MKN45 cells with the toll-like receptor 2 (TLR2) ligand P3C activated calpain and disrupted E-cadherin and β-catenin in a pattern similar to that induced by H. pylori. Inhibition of TLR2 prevented H. pylori-induced calpain activation and AJ disassembly. Together, these findings identify a novel pathway whereby H. pylori activates calpain via TLR2 to disrupt gastric epithelial AJ structure.

EPLIN downregulation promotes epithelial-mesenchymal transition in prostate cancer cells and correlates with clinical lymph node metastasis.

Epithelial-mesenchymal transition (EMT) is a crucial mechanism for the acquisition of migratory and invasive capabilities by epithelial cancer cells. By conducting quantitative proteomics in experimental models of human prostate cancer (PCa) metastasis, we observed strikingly decreased expression of EPLIN (epithelial protein lost in neoplasm; or LIM domain and actin binding 1, LIMA-1) upon EMT. Biochemical and functional analyses demonstrated that EPLIN is a negative regulator of EMT and invasiveness in PCa cells. EPLIN depletion resulted in the disassembly of adherens junctions, structurally distinct actin remodeling, and activation of β-catenin signaling. Microarray expression analysis identified a subset of putative EPLIN target genes associated with EMT, invasion and metastasis. By immunohistochemistry EPLIN downregulation was also demonstrated in lymph node metastases of human solid tumors including PCa, breast cancer, colorectal cancer and squamous cell carcinoma of the head and neck. This study reveals a novel molecular mechanism for converting cancer cells into a highly invasive and malignant form, and has important implications in prognosing and treating metastasis at early stages.

Caveolin-1–eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability

Endothelial barrier function is regulated by adherens junctions (AJs) and caveolae-mediated transcellular pathways. The opening of AJs that is observed in caveolin-1(-/-) (Cav-1(-/-)) endothelium suggests that Cav-1 is necessary for AJ assembly or maintenance. Here, using endothelial cells isolated from Cav-1(-/-) mice, we show that Cav-1 deficiency induced the activation of endothelial nitric oxide synthase (eNOS) and the generation of nitric oxide (NO) and peroxynitrite. We assessed S-nitrosylation and nitration of AJ-associated proteins to identify downstream NO redox signaling targets. We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation. Inhibition of eNOS or RhoA restored AJ integrity and diminished endothelial hyperpermeability in Cav-1(-/-) mice. Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A. Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

Teasing a(PAR)t biased‐agonism: Protease‐activated Receptor‐1 at signaling crossroads in endothelial barrier function

Protease‐activated G protein‐coupled receptors (PARs) elicit cellular responses to extracellular proteases. The coagulant protease thrombin contributes to inflammatory responses associated with vascular injury and thrombotic disease, whereas the anti‐coagulant protease activated protein C (APC) diminishes thrombin generation, promotes cytoprotective and anti‐inflammatory signaling in endothelial cells. Both thrombin and APC signal through PAR1 but elicit opposite effects on endothelial barrier function. Activation of PAR1 by thrombin stimulates RhoA and disassembly of adherens junctions. Remarkably, incubation with APC stimulates activation of Rac, and not RhoA, and ablates thrombin‐induced endothelial barrier permeability. The mechanism by which APC protects endothelial barrier from thrombin signaling remains poorly understood. We examined PAR1 distribution with cytoprotective and inflammatory signaling mediators in caveolin‐enriched microdomains using density gradient fractionation. We found that β‐arrestins co‐sediment with PAR1 and are essential for APC‐stimulated Rac activation and endothelial barrier protection. Interestingly, APC induces translocation of Disheveled, a signaling scaffold that may function to distinguish differential regulation of endothelial barrier permeability. Supported by NIH R01 HL073328, UC TRDRP and AHA Postdoctoral Fellowship Award.

VEGFR2 and Src Kinase Inhibitors Suppress Andes Virus-Induced Endothelial Cell Permeability

Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ∼70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC(50)s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.

The 19-Amino Acid Insertion in the Tumor-associated Splice Isoform Rac1b Confers Specific Binding to p120 Catenin

The Rac1b splice isoform contains a 19-amino acid insertion not found in Rac1; this insertion leads to decreased GTPase activity and reduced affinity for GDP, resulting in the intracellular predominance of GTP-bound Rac1b. Here, using co-precipitation and proteomic methods, we find that Rac1b does not bind to many common regulators of Rho family GTPases but that it does display enhanced binding to SmgGDS, RACK1, and p120 catenin (p120(ctn)), proteins involved in cell-cell adhesion, motility, and transcriptional regulation. We use molecular modeling and structure analysis approaches to determine that the interaction between Rac1b and p120(ctn) is dependent upon protein regions that are predicted to be unstructured in the absence of molecular complex formation, suggesting that the interaction between these two proteins involves coupled folding and binding. We also find that directed cell movement initiated by Rac1b is dependent upon p120. These results define a distinct binding functionality of Rac1b and provide insight into how the distinct phenotypic program activated by this protein may be implemented through molecular recognition of effectors distinct from those of Rac1.

Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.

Abstract 598: EMT and nectin-1 mediate sensitivity to HSV-1 oncolysis in human HCC cell lines

Abstract Background: Oncolytic viral therapy mediated by herpes simplex virus 1 (HSV-1) is an emerging and promising cancer therapy. The entry of HSV-1 into cells is mediated in part by cell surface co-receptors. One of these co-receptors, nectin-1, is highly expressed in epithelial cells. Nectin-1 is localized to adherens junctions (AJs) where it interacts with E-cadherin through their respective peripheral membrane proteins, afadin and catenin. The loss of E-cadherin is a hallmark of epithelial-to-mesenchymal transition (EMT) and results in the disassembly of AJs. Here, we investigate the effects of EMT on cell surface nectin-1 expression, HSV infection and viral oncolysis. Methods: Western blot analysis was performed to determine E-cadherin expression in human HCC cell lines. Cytotoxicity to HSV-1 KOS strain was assessed by MTT analysis. KOS replication was assessed by a single-step burst assay, while viral infectivity was examined using the mutant bM24-BAC#3 virus which encodes GFP. Cell surface levels of nectin-1 and herpesvirus entry mediator (HVEM) were determined by flow cytometry and the role of nectin-1 in HSV-1 infectivity was further assessed using a nectin-1 neutralizing antibody. Results: Four of the human HCC cell lines were epithelial and four were mesenchymal. KOS oncolysis resulted in 50% cell death in the epithelial cell lines at multiplicities of infection (MOIs) 2-3 log orders less than the mesenchymal HCC cell lines. This difference was not attributed to cell growth rates as determined by population doubling times or to viral replication as assessed by burst assay. Instead, the epithelial cell lines had higher rates of infectivity which were attributed to greater cell surface expression of nectin-1 and not HVEM. Finally, blocking of nectin-1 with a neutralizing antibody inhibited viral oncolysis in all four epithelial HCC cell lines. Conclusions: EMT in human HCC cells results in the loss of nectin-1 cell surface expression and resistance to HSV-1 oncolysis. E-cadherin and nectin-1 are markers of sensitivity to HSV-1 oncolysis in human HCC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 598.

Adducins regulate remodeling of intercellular junctions in model human epithelia

Epithelial adherens junctions (AJs) and tight junctions (TJs) are dynamic structures readily undergoing disintegration and reassembly. Remodeling of AJs and TJs depends on orchestrated dynamics of the plasma membrane and the actin cytoskeleton, which implicates the membrane/cytoskeleton interface in junctional regulation. Spectrin‐adducin polymers link the plasma membrane to the actin cytoskeleton. We investigated roles of adducin in the remodeling of epithelial junctions. We used SK‐CO15 human colonic and HPAF‐II human pancreatic epithelial cells, which possess well‐formed AJs and TJs and express α‐ and γ isoforms of adducin. Both adducins colocalized with E‐cadherin and β‐catenin at mature and newly‐assembled AJs. siRNA‐mediated downregulation of α‐ and γ‐adducin expression in SK‐CO15 cells attenuated AJ reformation without affecting TJ assembly. Depletion of adducins decreased protein level of βII spectrin and dewnregulation of βII spectrin mimicked effects of adducins knock‐down on AJ reassembly. In HPAF‐II cell monolayers, disruption of apical junctions induced by phorbol ester was paralleled by phophorylation of adducins and their disappearance from intercellular contacts. Downregulation of α‐ and γ‐adducins accelerated phorbol ester‐induced AJ disassembly. These finding suggest novel roles for adducins in the establishment and stabilization of epithelial adherens junctions.

Formation and Disassembly of Adherens and Tight Junctions in the Corneal Endothelium: Regulation by Actomyosin Contraction

Purpose. To determine the role of actin cytoskeleton in the disassembly and reformation of adherens junctions (AJs) and tight junctions (TJs) in bovine corneal endothelial monolayers. Methods. Disassembly and reformation of AJs and TJs were induced by extracellular Ca(2+) depletion and subsequent add-back of Ca(2+), respectively. Resultant changes in the transendothelial electrical resistance (TER), an indicator of integrity of TJs, were measured based on electrical cell-substrate impedance. Phosphorylated myosin light chain (ppMLC), a biochemical measure of actomyosin contraction, and activation of its upstream regulatory molecule RhoA-GTP were assessed by Western blot analysis. Results. Extracellular Ca(2+) depletion led to activation of RhoA, increase in ppMLC, decrease in TER, contraction of the perijunctional actomyosin ring (PAMR), and redistribution of zonula occludens-1 (ZO-1) and cadherins. These effects were reversed on Ca(2+) add-back. Pretreatment with Y-27632 and blebbistatin (as inhibitors of actomyosin contraction) reduced the rate of decline in TER, opposed the contraction of the PAMR, and blocked the redistribution of ZO-1 and cadherins. Both drugs reduced the recovery in TER and opposed the normal redistribution of ZO-1 and cadherins on Ca(2+) add-back. Cytochalasin D, which led to dissolution of the PAMR, also reduced the recovery of TER on Ca(2+) add-back. Conclusions. The (Ca(2+) depletion)-induced disassembly of AJs accelerates the breakdown of TJs through a concomitant increase in the actomyosin contraction of the PAMR. However, these data on reassembly show that a contractile tone of the PAMR is essential for assembly of the apical junctional complex.

Bone marrow-derived progenitor cells prevent thrombin-induced increase in lung vascular permeability

Since thrombin activation of endothelial cells (ECs) is well-known to increase endothelial permeability by disassembly of adherens junctions (AJs) and actinomyosin contractility mechanism involving myosin light chain (MLC) phosphorylation, we investigated the effects of bone marrow-derived progenitor cells (BMPCs) on the thrombin-induced endothelial permeability response. We observed that addition of BMPCs to endothelial monolayers at a fixed ratio prevented the thrombin-induced decrease in transendothelial electrical resistance, a measure of AJ integrity, and increased mouse pulmonary microvessel filtration coefficient, a measure of transvascular liquid permeability. The barrier protection was coupled to increased vascular endothelial cadherin expression and increased Cdc42 activity in ECs. Using small interfering RNA (siRNA) to deplete Cdc42 in ECs, we demonstrated a key role of Cdc42 in signaling the BMPC-induced endothelial barrier protection. Endothelial integrity induced by BMPCs was also secondary to inhibition of MLC phosphorylation in ECs. Thus BMPCs interacting with ECs prevent thrombin-induced endothelial hyperpermeability by a mechanism involving AJ barrier annealing, inhibition of MLC phosphorylation, and activation of Cdc42.

An acidic extracellular pH disrupts adherens junctions in HepG2 cells by Src kinases‐dependent modification of E‐cadherin

We have previously shown that culturing HepG2 cells in pH 6.6 culture medium increases the c-Src-dependent tyrosine phosphorylation of beta-catenin and induces disassembly of adherens junctions (AJs). Here, we investigated the upstream mechanism leading to this pH 6.6-induced modification of E-cadherin. In control cells cultured at pH 7.4, E-cadherin staining was linear and continuous at cell-cell contact sites. Culturing cells at pH 6.6 was not cytotoxic, and resulted in weak and discontinuous junctional E-cadherin staining, consistent with the decreased levels of E-cadherin in membrane fractions. pH 6.6 treatment activated c-Src and Fyn kinase and induced tyrosine phosphorylation of p120 catenin (p120ctn) and E-cadherin. Inhibition of Src family kinases by PP2 attenuated the pH 6.6-induced tyrosine phosphorylation of E-cadherin and p120ctn, and prevented the loss of these proteins from AJs. In addition, E-cadherin was bound to Hakai and ubiquitinated. Furthermore, pH 6.6-induced detachment of E-cadherin from AJs was blocked by pretreatment with MG132 or NH(4)Cl, indicating the involvement of ubiquitin-proteasomal/lysosomal degradation of E-cadherin. An early loss of p120ctn prior to E-cadherin detachment from AJs was noted, concomitant with a decreased association between p120ctn and E-cadherin at pH 6.6. PP2 pretreatment prevented the dissociation of these two proteins. In conclusion, pH 6.6 activated Src kinases, resulting in tyrosine phosphorylation of E-cadherin and p120ctn and a weakening of the association of E-cadherin with p120ctn and contributing to the instability of E-cadherin at AJs.

Deubiquitinase Activities Required for Hepatocyte Growth Factor-Induced Scattering of Epithelial Cells

SummaryThe scattering response of epithelial cells to activation of the Met receptor tyrosine kinase represents one facet of an “invasive growth” program [1, 2]. It is a complex event that incorporates loss of cell-cell adhesion, morphological changes, and cell motility. Ubiquitination is a reversible posttranslational modification that may target proteins for degradation or coordinate signal transduction pathways [3, 4]. There are ∼79 active deubiquitinating enzymes (DUBs) predicted in the human genome [5, 6]. Here, via a small interfering RNA (siRNA) library approach, we have identified 12 DUBs that are necessary for aspects of the hepatocyte growth factor (HGF)-dependent scattering response of A549 cells. Different phenotypes are evident that range from full loss of scattering, similar to receptor knockdown (e.g., USP30, USP33, USP47), to loss of cell-cell contacts even in the absence of HGF but defective motility (e.g., USP3, ATXN3L). The knockdowns do not incur defective receptor, phosphatidylinositol 3-kinase, or MAP kinase activation. Our data suggest widespread involvement of the ubiquitin system at multiple stages of the Met activation response, implying significant crosstalk with phosphorylation-based transduction pathways. Development of small-molecule inhibitors of particular DUBs may offer a therapeutic approach to contain metastasis.

Caveolae Mediate Growth Factor-induced Disassembly of Adherens Junctions to Support Tumor Cell Dissociation

Remodeling of cell-cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell-cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell-cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.

C-Jun N-Terminal kinase mediates disassembly of apical junctions in model intestinal epithelia

Dynamic remodeling of intercellular junctions is a critical determinant of epithelial barrier function in both physiological and pathophysiological states. While the disassembly of epithelial tight junctions (TJ) and adherens junctions (AJ) has been well-described in response to pathogens and other external stressors, the role of stress-related signaling in TJ/AJ regulation remains poorly understood. The aim of this study was to define the role of stress-activated c-Jun N-terminal kinase (JNK) in disruption of intercellular junctions in model intestinal epithelia. We show that rapid AJ/TJ disassembly triggered by extracellular calcium depletion of T84 and SK-CO15 cell monolayers was accompanied by activation (phosphorylation) of JNK, and prevented by pharmacological inhibitors of JNK. The opposite process, TJ/AJ reassembly, was accelerated by JNK inhibition and suppressed by the JNK activator anisomycin. JNK1 but not JNK2 was found to colocalize with intercellular junctions, and siRNA-mediated down-regulation of JNK1 attenuated the TJ/AJ disruption caused by calcium depletion. JNK inhibition also blocked formation of characteristic contractile F-actin rings in calcium-depleted epithelial cells, suggesting that JNK regulates junctions by remodeling the actin cytoskeleton. In this role JNK acts downstream of the actin-reorganizing Rho-dependent kinase (ROCK), since ROCK inhibition abrogated JNK phosphorylation and TJ/AJ disassembly after calcium depletion. Furthermore, JNK acts upstream of F-actin-membrane linker proteins of the ERM (ezrin-radixin-moesin) family, but in a complex relationship yet to be fully elucidated. Taken together, our findings suggest a novel role for JNK in the signaling pathway that links ROCK and F-actin remodeling during disassembly of epithelial junctions.

Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility

BackgroundDisruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.ResultsExposure of HPAF-II human pancreatic adenocarcinoma cell monolayers to either OI-V or 12-O-tetradecanoylphorbol-13-acetate caused rapid disruption and internalization of AJs and TJs. Activity of classical PKC isoenzymes was responsible for the loss of cell-cell contacts which was accompanied by cell rounding, phosphorylation and relocalization of the F-actin motor nonmuscle myosin (NM) II. The OI-V-induced disruption of AJs and TJs was prevented by either pharmacological inhibition of NM II with blebbistatin or by siRNA-mediated downregulation of NM IIA. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho-associated kinase (ROCK) II, but was insensitive to blockage of MLCK, calmodulin, ERK1/2, caspases and RhoA GTPase.ConclusionOur data suggest that stimulation of PKC disrupts epithelial apical junctions via ROCK-II dependent activation of NM II, which increases contractility of perijunctional actin filaments. This mechanism is likely to be important for cancer cell dissociation and tumor metastasis.

PKCalpha‐mediated signaling in regulating adherens junction integrity

The aim of this study was to determine the mechanism of protein kinase C‐α (PKCα)‐mediated disruption of adherens junctions (AJs). We have shown that depletion of PKCα in human microvascular endothelial cells reduces the thrombin‐induced permeability increase by 50%, as assessed by change in transendothelial resistance. PKCα‐depleted endothelial cells formed fewer interendothelial gaps and retained more vascular endothelial (VE)‐cadherin at AJs after thrombin stimulation compared to control cells. VE‐cadherin stability at AJs has been shown to be dependent on p120‐catenin (p120) binding. Dissociation of p120 from VE‐cadherin results in VE‐cadherin internalization and disassembly of AJs. Since activation of PKCα after thrombin stimulation leads to changes in phosphorylation of p120 in endothelial cells, we tested the hypothesis that PKCα‐mediated phosphorylation of p120 causes dissociation of p120 from VE‐cadherin, subsequent disassembly of AJs and increased permeability of the endothelial barrier. Our data demonstrate that thrombin stimulation of endothelial cells results in translocation of PKCα to AJs and interaction with p120. Furthermore, the level of p120 remains unchanged at AJs in PKCα‐depleted cells after thrombin stimulation, supporting our hypothesis that PKCα may induce p120 dissociation from VE‐cadherin, resulting in AJ disassembly.Supported by NIH Grants HL045638 and HL007829