Hydrolysis of epithelial junctional proteins by Porphyromonas gingivalis gingipains

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.     

Apically exposed, tight junction-associated beta1-integrins allow binding and YopE-mediated perturbation of epithelial barriers by wild-type Yersinia bacteria

Using polarized epithelial cells, primarily MDCK-1, we assessed the mode of binding and effects on epithelial cell structure and permeability of Yersinia pseudotuberculosis yadA-deficient mutants. Initially, all bacteria except the invasin-deficient (inv) mutant adhered apically to the tight junction areas. These contact points of adjacent cells displayed beta1-integrins together with tight junction-associated ZO-1 and occludin proteins. Indeed, beta1-integrin expression was maximal in the tight junction area and then gradually decreased along the basolateral membranes. Wild-type bacteria also opened gradually the tight junction to paracellular permeation of different-sized markers, viz., 20-, 40-, and 70-kDa dextrans and 45-kDa ovalbumin, as well as to their own translocation between adjacent cells in intimate contact with beta1-integrins. The effects on the epithelial cells and their barrier properties could primarily be attributed to expression of the Yersinia outer membrane protein YopE, as the yopE mutant bound but caused no cytotoxicity. Moreover, the apical structure of filamentous actin (F-actin) was disturbed and tight junction-associated proteins (ZO-1 and occludin) were dispersed along the basolateral membranes. It is concluded that the Yersinia bacteria attach to beta1-integrins at tight junctions. Via this localized injection of YopE, they perturb the F-actin structure and distribution of proteins forming and regulating tight junctions. Thereby they promote paracellular translocation of bacteria and soluble compounds.     

Role of nectin in organization of tight junctions in epithelial cells

BACKGROUND: In polarized epithelial cells, cell-cell adhesion forms specialized membrane structures comprised of claudin-based tight junctions (TJs) and of E-cadherin-based adherens junctions (AJs). These structures are aligned from the apical to the basal side of the lateral membrane, but the mechanism of this organization remains unknown. Nectin is a Ca2+ independent immunoglobulin-like cell-cell adhesion molecule which localizes at AJs. Nectin is associated with E-cadherin through their respective cytoplasmic tail-binding proteins, afadin and catenins, and involved in the formation of AJs in cooperation with E-cadherin. We show here that nectin is also involved in the formation of TJs. RESULTS: During the formation of the junctional complex consisting of AJs and TJs in Madin-Darby canine kidney (MDCK) cells, claudin and occludin accumulated at the apical sites of the nectin-based cell-cell adhesion sites. This accumulation of claudin and occludin was inhibited by inhibitors acting on the trans interaction of nectin. The barrier function of TJs was also impaired by the nectin inhibitors. It has been shown that a phorbol ester promotes the formation of a TJ-like structure in an E-cadherin-independent manner. This phorbol ester-induced formation of the TJ-like structure was also inhibited by the nectin inhibitors. CONCLUSIONS: These results suggest a role of the nectin-afadin system in the organization of TJs as well as AJs in epithelial cells.     

Requirement of the actin cytoskeleton for the association of nectins with other cell adhesion molecules at adherens and tight junctions in MDCK cells

Nectins, Ca(2+)-independent immunoglobulin-like cell adhesion molecules (CAMs), first form cell-cell adhesion where cadherins are recruited, forming adherens junctions (AJs) in epithelial cells and fibroblasts. In addition, nectins recruit claudins, occludin, and junctional adhesion molecules (JAMs) to the apical side of AJs, forming tight junctions (TJs) in epithelial cells. Nectins are associated with these CAMs through peripheral membrane proteins (PMPs), many of which are actin filament-binding proteins. We examined here the roles of the actin cytoskeleton in the association of nectins with other CAMs in MDCK cells stably expressing exogenous nectin-1. The nectin-1-based cell-cell adhesion was formed and maintained irrespective of the presence and absence of the actin filament-disrupting agents, such as cytochalasin D and latrunculin A. In the presence of these agents, only afadin remained at the nectin-1-based cell-cell adhesion sites, whereas E-cadherin and other PMPs at AJs, alpha-catenin, beta-catenin, vinculin, alpha-actinin, ADIP, and LMO7, were not concentrated there. The CAMs at TJs, claudin-1, occludin and JAM-1, or the PMPs at TJs, ZO-1 and MAGI-1, were not concentrated there, either. These results indicate that the actin cytoskeleton is required for the association of the nectin-afadin unit with other CAMs and PMPs at AJs and TJs.     

Recruitment of E-cadherin associated with alpha- and beta-catenins and p120ctn to the nectin-based cell-cell adhesion sites by the action of 12-O-tetradecanoylphorbol-13-acetate in MDCK cells

The formation of tight junctions (TJs) is dependent on the formation of adherens junctions (AJs) in MDCK cells. E-Cadherin and nectin are major cell-cell adhesion molecules (CAMs) at AJs, whereas claudin, occludin and junctional adhesion molecule (JAM) are major CAMs at TJs. When MDCK cells precultured at 2 microm Ca(2+) are cultured at 2 mm Ca(2+), nectin first forms cell-cell adhesion and recruits E-cadherin to the nectin-based cell-cell adhesion sites to form AJs. Thereafter, nectin recruits first JAM-A and then claudin-1 and occludin to the apical side of AJs to form TJs. In contrast, when MDCK cells precultured at 2 microm Ca(2+) are cultured at 2 microm Ca(2+) in the presence of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a TJ-like structure is formed without the formation of the E-cadherin-based AJs. We showed here that GFP-E-cadherin, which did not trans-interact due to 2 microm Ca(2+) but associated with alpha- and beta-catenins and p120(ctn), was recruited to the nectin-based cell-cell adhesion sites by the action of TPA. The nectin inhibitors, which inhibited the trans-interaction of nectin, inhibited the recruitment of GFP-E-cadherin and their associating catenins by the action of TPA. Microbeads coated with the extracellular fragment of nectin recruited not only cellular nectin but also GFP-E-cadherin and their associating catenins by the action of TPA. These results indicate that when the TJ-like structure is formed by the action of TPA, non-trans-interacting E-cadherin and its associating catenins are recruited to the nectin-based cell-cell adhesion sites and that the trans-interaction of E-cadherin is not essential for the formation of TJs.     

Cleavage of E-cadherin: a mechanism for disruption of the intestinal epithelial barrier by Candida albicans

To investigate how intestinal epithelial cells respond to contact with Candida albicans, an organism able to invade the bloodstream via the gastrointestinal tract, we focused on the junction proteins occludin, E-cadherin, and desmoglein-2. The levels of these 3 junction proteins were reduced in lysates of human intestinal epithelial monolayers (Caco-2) after a 24-h inoculation with C. albicans, compared with lysates from Saccharomyces cerevisiae-inoculated monolayers. Treatment with pepstatin A did not change the effect of C. albicans on full-length occludin, desmoglein-2, and E-cadherin; however, pepstatin A enhanced the accumulation of a 35-kDa fragment derived from the intracellular portion of E-cadherin. This 35-kDa fragment also accumulated in the presence of gamma-secretase inhibitors. These observations suggest that enhancement of E-cadherin cleavage by C. albicans generates an intracellular E-cadherin fragment that can serve as a substrate for gamma-secretase. An 89-kDa extracellular fragment of E-cadherin was detected in supernatants of C. albicans-inoculated monolayers; this cleavage event was insensitive to both pepstatin A and gamma-secretase inhibitors. Transepithelial electrical resistance, a measure of monolayer integrity, decreased significantly and synchronously with increased generation of the 89-kDa extracellular E-cadherin fragment. Cleavage of E-cadherin may destabilize the homotypic interactions between adjacent epithelial cells and could contribute to loss of monolayer integrity. These experiments identify 2 E-cadherin cleavage events that are enhanced by contact with C. albicans: an intracellular cleavage event that generates a substrate for gamma-secretase and an extracellular cleavage event that is temporally associated with an increase in monolayer permeability.     

Disruption of tight junctions and induction of proinflammatory cytokine responses in colonic epithelial cells by Campylobacter jejuni

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-kappaB 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.     

Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis

Crohn's disease is characterized by a defect in intestinal barrier function, where bacteria are considered the most important inflammation-driving factor. Enteric bacteria, including E. coli and Yersinia spp, affect tight junctions in enterocytes, but little is known about bacterial effects on the transcellular pathway. Our objective was to study the short-term effects of Y. pseudotuberculosis on uptake of nanoparticles across human villus epithelium. Monolayers of human colon epithelium-derived Caco-2 cells and biopsies of normal human ileum were studied after 2 h exposure to Y. pseudotuberculosis expressing (inv+) or lacking (inv-) the bacterial adhesion molecule, invasin. Transepithelial transport of fluorescent nanoparticles (markers of transcytosis) was quantified by flow cytometry, and mechanisms explored by using inhibitors of endocytosis. Epithelial expressions of beta1-integrin and particle uptake pathways were studied by confocal microscopy. The paracellular pathway was assessed by electrical resistance (TER), mannitol flux, and expression of tight junction proteins occludin and caludin-4 by confocal microscopy. Inv+ Y. pseudotuberculosis adhered to the apical surface of epithelial cells and induced transcytosis of exogenous nanoparticles across Caco-2 monolayers (30-fold increase, P<0.01) and ileal mucosa (268+/-47% of control; P<0.01), whereas inv bacteria had no effect on transcytosis. The transcytosis was concentration-, particle size- and temperature-dependent, and possibly mediated via macropinocytosis. Y. pseudotuberculosis also induced apical expression of beta1-integrin on epithelial cells. A slight drop in TER was seen after exposure to inv+ Y. pseudotuberculosis, whereas mannitol flux and tight junction protein expression was unchanged. In summary, Y. pseudotuberculosis induced apical expression of beta1-integrin and stimulated uptake of nanoparticles via invasin-dependent transcytosis in human intestinal epithelium. Our findings suggest that bacterial factors may initiate transcytosis of luminal exogenous particles across human ileal mucosa, thus presenting a novel mechanism of intestinal barrier dysfunction.     

Intercellular junctions in Ewing sarcoma/primitive neuroectodermal tumor: additional evidence of epithelial differentiation.

Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) has recently been shown to frequently express cytokeratins, suggesting partial epithelial differentiation. Older ultrastructural studies have documented primitive cell-cell junctions in ES/PNET, reportedly resembling poorly formed desmosomes. Recently, paraffin-reactive antibodies have become available to proteins found in a variety of intercellular junctions indicative of epithelial differentiation, including tight junctions, desmosomes and adherens junctions. We examined intercellular junction protein expression in a large number of genetically confirmed ES/PNET. Formalin-fixed, paraffin-embedded sections from 23 primary and seven recurrent or metastatic cases of genetically confirmed ES/PNET were immunostained for claudin-1 and occludin (tight junction structural proteins), zonula occludens-1 (ZO-1, tight junction linker protein), desmoglein 1/2 (desmosomal adherens protein), desmoplakin (desmosomal structural protein) and E-cadherin (epithelial adherens junction protein), using steam heat-induced epitope retrieval and the Dako Envision system. Cases with >5% positive cells were scored as 'positive'. Normal colonic epithelium and skin served as external positive controls. Claudin-1 was expressed by 19 of 30 specimens (63%), ZO-1 was expressed by 15 of 29 specimens (51%), and occludin was expressed by three of 28 specimens (11%). In 28 specimens all three tight junction markers were evaluable. In all, 15 samples (54%) expressed only one tight junction marker, and 10 samples (36%) expressed two tight junction markers. No case expressed all three tight junction markers. Desmoglein was expressed in one of 30 (3%) samples. Desmoplakin was expressed in two of 28 (7%) samples. E-cadherin was negative in all cases. Our data suggest that many of the previously described cell-cell junctions in ES/PNET are poorly formed tight junctions, given the high frequency of claudin-1 and ZO-1 expression. This may underestimate the true frequency of tight junction protein expression in ES/PNET, as there are at least 20 different claudins and other ZO proteins. These tight junctions are almost certainly abnormal, given the absence of occludin expression in most cases. Desmosomal and adherens junction protein expression was rare to absent. Our findings provide additional evidence that ES/PNET frequently show partial epithelial differentiation.     

Interaction between the transforming growth factor-beta type II receptor/Smad pathway and beta-catenin during transforming growth factor-beta1-mediated adherens junction disassembly

The aim of the current study was to examine the influence of transforming growth factor (TGF)-beta 1 on proximal tubular epithelial cell-cell interaction, with particular emphasis on the regulation of adherens junction complex formation. Stimulation of the proximal tubular cell line HK-2 cells by TGF-beta 1 led to loss of cell-cell contact and disassembly of both adherens and tight junctional complexes. Adherens junction disassembly was associated with reduction of both Triton-soluble and Triton-insoluble E-cadherin, and an increase in detergent-soluble beta-catenin. Under these conditions, immunoprecipitation and Western analysis demonstrated decreased association of beta-catenin, both with E-cadherin, alpha-catenin, and the cell cytoskeleton. Confocal microscopy after immunostaining, showed decreased intensity of peripheral E-cadherin staining, and redistribution of beta-catenin expression to a perinuclear location. Tight junction disassembly was manifest by a reduction in the expression of Triton-soluble occludin and ZO-1 by Western analysis and their disassociation manifested by immunostaining and confocal microscopy. Loss of cell-cell contact and disassembly of adherens junctions were seen after addition of TGF-beta 1 to the basolateral aspect of the cells. Immunoprecipitation experiments demonstrated co-localization of E-cadherin, beta-catenin, and TGF-beta 1 RII in unstimulated cells. After TGF-beta 1 stimulation, the TGF-beta 1 RII no longer associated with either E-cadherin or beta-catenin. Dissociation of the adherens junction protein from the TGF-beta 1 receptor was associated with increased beta-catenin tyrosine phosphorylation and decreased threonine phosphorylation. Furthermore after receptor ligand binding, beta-catenin became associated with the TGF-beta 1-signaling molecules Smad3 and Smad4.     

Neutrophil Transmigration in Inflammatory Bowel Disease Is Associated with Differential Expression of Epithelial Intercellular Junction Proteins

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohn's (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohn's disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.     

Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro

We have established a coculture system of human distal lung epithelial cells and human microvascular endothelial cells in order to study the cellular interactions of epithelium and endothelium at the alveolocapillary barrier in both pathogenesis and recovery from acute lung injury. The aim was to determine conditions for the development of functional cellular junctions and the formation of a tight epithelial barrier similar to that observed in vivo. The in vitro coculture system consisted of monolayers of human lung epithelial cell lines (A549 or NCI H441) and primary human pulmonary microvascular endothelial cells (HPMEC) on opposite sides of a permeable filter membrane. A549 failed to show sufficient differentiation with respect to formation of a tight epithelial barrier with intact cell-cell junctions. Stimulated with dexamethasone, the cocultures of NCI H441 and HPMEC established contact-inhibited differentiated monolayers, with NCI H441 showing a continuous, circumferential immunostaining of the tight junctional protein, ZO-1 and the adherens junction protein, E-cadherin. The generation of a polarized epithelial cell monolayer with typical junctional structures was confirmed by transmission electron microscopy. Dexamethasone treatment resulted in average transbilayer electrical resistance (TER) values of 500 Omega cm(2) after 10-12 days of cocultivation and correlated with a reduced flux of the hydrophilic permeability marker, sodium-fluorescein. In addition, basolateral distribution of the proinflammatory cytokine tumour necrosis factor-alpha caused a significant reduction of TER-values after 24 h exposure. This decrease in TER could be re-established to control level by removal of the cytokine within 24 h. Thus, the coculture system of the NCI H441 with HPMEC should be a suitable in vitro model system to examine epithelial and endothelial interactions in the pathogenesis of acute lung injury, infectious lung diseases and toxic lung injury. In addition, it could be used to improve techniques of lung drug delivery that also requires a functional barrier.     

Decreased distribution of lung epithelial junction proteins after intratracheal antigen or lipopolysaccharide challenge: correlation with neutrophil influx and levels of BALF sE-cadherin

Distribution of airway junctional complex proteins after antigen or lipopolysaccharide challenge in sensitized or naive mice, respectively, was investigated. E-cadherin immunoreactivity was detected continuously along neighboring epithelial cell borders and between adjacent alveolar epithelial cells in naive and saline-challenged mice. Occludin and ZO-1 immunoreactivity were observed in the tight junction areas. Both challenges induced changes in epithelial morphology and phenotype, accompanied initially by focal loss of epithelial E-cadherin that increased in size with time and number of allergen challenges. Allergen challenge also led to focal loss of occludin and ZO-1. Western blot analysis revealed increased levels of sE-cadherin in lavage fluid after either challenge, and this increase correlated with lavage neutrophil numbers (P = 0.002). Immunocytochemistry of lavage cells 6 h after either challenge revealed E-cadherin epitopes within cytoplasmic vacuoles of neutrophils, the major cell type. In contrast, peripheral blood neutrophils or tissue neutrophils before epithelial transmigration were negative, suggesting that in airway inflammation, E-cadherin extracellular domain is cleaved by neutrophils during epithelial penetration, instigating the destabilization of adherens and tight junctions. This junctional deterioration could lead to a progressive decrease in epithelial integrity and induce alterations in epithelial morphology, with consequent enhanced paracellular transit of antigens and pathogens.     

Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa invasion and cytotoxicity.

Intact tissues are relatively resistant to Pseudomonas aeruginosa-induced disease, and injury predisposes tissue to infection. Intact epithelia contain polarized cells that have distinct apical and basolateral membranes with unique lipids and proteins. In this study, the role of cell polarity in epithelial cell susceptibility to P. aeruginosa virulence mechanisms was tested. Madin-Darby canine kidney (MDCK) cells, human corneal epithelial cells, and primary cultures of two different types of airway epithelial cells were grown on Transwell filters or in plastic tissue culture wells. P. aeruginosa invasion of cells was quantified by gentamicin survival assays with two isolates that invade epithelial cells (6294 and PAO1). Cytotoxic activity was assessed by trypan blue exclusion assays with two cytotoxic strains (6206 and PA103). Basolateral surfaces of cells were exposed by one of two methods: EGTA pretreatment of epithelial cells or growth of cells in low-calcium medium. Both methods of exposing basolateral membranes increased epithelial cell susceptibility to P. aeruginosa invasion and cytotoxicity. Migrating cells were also found to be more susceptible to P. aeruginosa invasion than confluent monolayers that had established membrane polarity. Monolayers of MDCK cells that had been selected for resistance to killing by concanavalin A were resistant to both cytotoxicity and invasion by P. aeruginosa because they were more efficiently polarized for their susceptibility to P. aeruginosa virulence factors than regular MDCK cells and not because they were defective in glycosylation. These results suggest that there are factors on the basolateral surfaces of epithelial cells that promote interaction with P. aeruginosa or that there are inhibitory factors on the apical cell surface. Thus, cell polarity of intact epithelia is likely to contribute to defense against P. aeruginosa infection.     

Matrilysin (matrix metalloproteinase-7) mediates E-cadherin ectodomain shedding in injured lung epithelium

Matrilysin (matrix metalloproteinase-7) is highly expressed in lungs of patients with pulmonary fibrosis and other conditions associated with airway and alveolar injury. Although matrilysin is required for closure of epithelial wounds ex vivo, the mechanism of its action in repair is unknown. We demonstrate that matrilysin mediates shedding of E-cadherin ectodomain from injured lung epithelium both in vitro and in vivo. In alveolar-like epithelial cells, transfection of activated matrilysin resulted in shedding of E-cadherin and accelerated cell migration. In vivo, matrilysin co-localized with E-cadherin at the basolateral surfaces of migrating tracheal epithelium, and the reorganization of cell-cell junctions seen in wild-type injured tissue was absent in matrilysin-null samples. E-cadherin ectodomain was shed into the bronchoalveolar lavage fluid of bleomycin-injured wild-type mice, but was not shed in matrilysin-null mice. These findings identify E-cadherin as a novel substrate for matrilysin and indicate that shedding of E-cadherin ectodomain is required for epithelial repair.     

Disruption of cell polarity by enteropathogenic Escherichia coli enables basolateral membrane proteins to migrate apically and to potentiate physiological consequences

Enteropathogenic Escherichia coli (EPEC) disrupts the structure and barrier function of host intestinal epithelial tight junctions (TJs). The impact of EPEC on TJ "fence function," i.e., maintenance of cell polarity, has not been investigated. In polarized cells, proteins such as beta(1)-integrin and Na(+)/K(+) ATPase are restricted to basolateral (BL) membranes. The outer membrane EPEC protein intimin possesses binding sites for the EPEC translocated intimin receptor (Tir) and beta(1)-integrin. Restriction of beta(1)-integrin to BL domains, however, precludes opportunity for interaction. We hypothesize that EPEC perturbs TJ fence function and frees BL proteins such as beta(1)-integrin to migrate to apical (AP) membranes of host cells, thus allowing interactions with bacterial adhesins such as intimin. The aim of this study was to determine whether EPEC alters the polar distribution of BL proteins, in particular beta(1)-integrin, and if such redistribution contributes to pathogenesis. Human intestinal epithelial T84 cells and EPEC strain E2348/69 were used. Selective biotinylation of AP or BL membrane proteins and confocal microscopy showed the presence of beta(1)-integrin and Na(+)/K(+) ATPase on the AP membrane following infection. beta(1)-Integrin antibody afforded no protection against the initial EPEC-induced decrease in transepithelial electrical resistance (TER) but halted the progressive decrease at later time points. While the effects of EPEC on TJ barrier and fence function were Tir dependent, disruption of cell polarity by calcium chelation allowed a tir mutant to be nearly as effective as wild-type EPEC. In contrast, deletion of espD, which renders the type III secretory system ineffective, had no effect on TER even after calcium chelation, suggesting that the putative beta(1)-integrin-intimin interaction serves to provide intimate contact, like that of Tir and intimin, making translocation of effector molecules more efficient. We conclude that the initial alterations of TJ barrier and fence function by EPEC are Tir dependent but that later disruption of cell polarity and accessibility of EPEC to BL membrane proteins, such as beta(1)-integrin, potentiates the physiological perturbations.     

E-Cadherin and tight junctions between epithelial cells of different animal species

The assembly and permanent sealing of tight junctions (TJs) depend crucially on cell-cell contacts containing E-cadherin. This poses a puzzling problem because, while TJs can be established between epithelial cells from different tissues and even different animal species ("heterotypic TJs"; Gonzalez-Mariscal et al. 1989, J Membr Biol 107:43), the cell-cell binding mediated by E-cadherin is a highly specific one (Takeichi 1995, Curr Opin Cell Biol 7:619). Yet the demonstration that TJs can be established at heterotypic borders is open to two distinct challenges. First, it is based on transepithelial electrical resistance (TER) and restriction to ruthenium red permeation only, which today are known to be just two of the many characteristics of TJs; and second some attributes of the TJs (e.g. the presence of specific molecules) have been found even in cells that do not establish these structures. This raised the question of whether heterotypic TJs were not true or full TJs. In the present work we demonstrate that heterotypic TJs in mixed monolayers of MDCK cells with a different cell type (LLC-PK1) are true TJs through several criteria, such as TER, the ability to stop the membrane diffusion of fluorescent sphingomyelin from the apical to the lateral domain, the presence of ZO-1, ZO-2, occludin, claudin-1 and claudin-2. We then turn to the presence of E-cadherin at heterotypic borders, and observe that it cannot be detected by the highly specific DECMA-1 antibody, in spite of the fact that this antibody does reveal the presence of E-cadherin at homotypic contacts of the same cell. Yet, ECCD-2, an antibody against another domain of E-cadherin, reveals that this molecule may be present at both types of borders. Thus, E-cadherin is present at heterotypic borders, yet it seems to be in a conformation unable to bind DECMA-1. Our results suggest: (1) that heterotypic borders can establish fully developed TJs; (2) that the sealing of these heterotypic TJs depends on E-cadherin; (3) but that this dependence is mediated through a cascade of chemical reactions involving two different G-proteins, PLC, PKC and calmodulin, which we have characterized elsewhere (Balda et al. 1991, J Membr Biol 122:193); and (4) hence molecules of E-cadherin that trigger junction formation can act from a distant homotypic contact.     

Basolateral junctions are sufficient to suppress epithelial invasion during Drosophila oogenesis.

Epithelial junctions play crucial roles during metazoan evolution and development by facilitating tissue formation, maintenance, and function. Little is known about the role of distinct types of junctions in controlling epithelial transformations leading to invasion of neighboring tissues. Discovering the key junction complexes that control these processes and how they function may also provide mechanistic insight into carcinoma cell invasion. Here, using the Drosophila ovary as a model, we show that four proteins of the basolateral junction (BLJ), Fasciclin-2, Neuroglian, Discs-large, and Lethal-giant-larvae, but not proteins of other epithelial junctions, directly suppress epithelial tumorigenesis and invasion. Remarkably, the expression pattern of Fasciclin-2 predicts which cells will invade. We compared the apicobasal polarity of BLJ tumor cells to border cells (BCs), an epithelium-derived cluster that normally migrates during mid-oogenesis. Both tumor cells and BCs differentiate a lateralized membrane pattern that is necessary but not sufficient for invasion. Independent of lateralization, derepression of motility pathways is also necessary, as indicated by a strong linear correlation between faster BC migration and an increased incidence of tumor invasion. However, without membrane lateralization, derepression of motility pathways is also not sufficient for invasion. Our results demonstrate that spatiotemporal patterns of basolateral junction activity directly suppress epithelial invasion by organizing the cooperative activity of distinct polarity and motility pathways.     

The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus

There have been only a few studies of how allergens cross the airway epithelium to cause allergic sensitization. House dust mite fecal pellets (HDMFP) contain several proteolytic enzymes. Group 1 allergens are cysteine peptidases, whilst those of groups 3, 6 and 9 have catalytic sites indicative of enzymes that mechanistically behave as serine peptidases. We have previously shown that the group 1 allergen Der p 1 leads to cleavage of tight junctions (TJs), allowing allergen delivery to antigen presenting cells. In this study we determined whether HDMFP serine peptidases similarly compromise the airway epithelium by attacking TJs, desmosomes and adherens junctions. Experiments were performed in monolayers of MDCK, Calu-3 or 16HBE14o-epithelial cells. Cell junction morphology was examined by 2-photon molecular excitation microscopy and digital image analysis. Barrier function was measured as mannitol permeability. Cleavage of cell adhesion proteins was studied by immunoblotting and mass spectrometry. HDMFP serine peptidases led to a progressive cleavage of TJs and increased epithelial permeability. Desmosomal puncta became more concentrated. Cleavage of TJs involved proteolysis of the TJ proteins, occludin and ZO-1. This was associated with activation of intracellular proteolysis of ZO-1. In contrast to occludin, E-cadherin of adherens junctions was cleaved less extensively. Although Calu-3 and 16HBE14o-cells expressed tethered ligand receptors for serine peptidases, these were not responsible for transducing the changes in TJs. HDMFP serine peptidases cause cleavage of TJs. This study identifies a second general class of HDM peptidase capable of increasing epithelial permeability and thereby creating conditions that would favour transepithelial delivery of allergens.     

Immunohistological characterization of intercellular junction proteins in rhesus macaque intestine

Epithelial junctions play an important role in regulating paracellular permeability and intercellular adhesion. It has been reported that changes in the density of epithelial junctions and/or distribution pattern can contribute to various gastrointestinal (GI) disorders. In this study, we investigated the distribution of the tight junction (Claudins. 1, 3, 4, 5, 7, 10, Zonula Occludens (ZO-1), Occludin), adherens junction (E-cadherin), desmosome (Desmoglein 2, Desmocollin 2) and gap junction (Connexin 43) proteins in the jejunum, ileum and colonic epithelium of healthy rhesus macaques (RM) using immunofluorescence labeling. While proteins in these respective junctions were expressed throughout the jejunum, ileum and colon of RM, we observed differential labeling in epithelial cells from these sites. Claudins 1, 3, 4, 7, E-cadherin and Desmoglein 2 were distributed in the respective intercellular junctions with additional labeling in the lateral membrane of epithelial cells in both small and large intestine. However, claudin 5, claudin 10, ZO-1 and occludin showed uniform distribution in the intercellular junctions of crypt and surface epithelial cells of the intestine. Desmocollin 2 localized predominantly in the upper two thirds along the lateral membrane while desmoglein 2 was distributed along the entire lateral membrane of intestinal epithelial cells. In contrast, connexin 43 exhibited punctate lateral labeling in crypt epithelial cells of the small and large intestine. Our results show diverse localization of epithelial intercellular junction proteins along the intestinal tract of RM. These findings may correlate with differences in paracellular permeability and adhesion along the intestinal tract and could correlate with pathologic disease in these regions of the intestine.