Dislocation of tight junction proteins without F-actin disruption in inactive Crohn's disease

Crohn's disease is associated with increased permeability of the intestinal barrier even in quiescent patients. Increased intestinal permeability may cause dysregulated immunological responses in the intestinal mucosa that leads to chronic intestinal inflammation. We have studied the expression of tight junction proteins (occludin and zonula occludens), alpha2-smooth muscle actin, TGF-beta with a cytoskeletal protein (F-actin) in the intestinal epithelium of patients with inflammatory bowel disease. Surgical samples were obtained from 6 controls (individuals without inflammatory bowel disease), 8 patients with ulcerative colitis and 7 patients with Crohn's disease. F-actin was visualized with fluorescein phalloidin. Tight junction proteins, alpha2 smooth muscle actin, and TGFbeta were visualized by the immunofluorescent method. Occludin and zonula occludens found in apical tight junctions in normal epithelium were dislocated to the basolateral position and in the lamina propria extracellular matrix in patients with Crohn's disease, while the structure of F-actin was maintained in inactive or minimally inflamed mucosa. TGF-beta positive inflammatory cells were increased in ulcerative colitis and Crohn's disease mucosa. Subepithelial myofibroblasts were constitutively found in controls, ulcerative colitis, and Crohn's disease mucosa. Latent dislocation of tight junction proteins, without disturbance of the cytoskeleton in the inactive mucosa of patients with Crohn's disease, may permit the invasion of gut antigens because the functional disruption of tight junctions could initiate an altered immune response.     

Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression

Numerous intestinal diseases are characterized by immune cell activation and compromised epithelial barrier function. We have shown that cytokine treatment of epithelial monolayers increases myosin II regulatory light chain (MLC) phosphorylation and decreases barrier function and that these are both reversed by MLC kinase (MLCK) inhibition. The aim of this study was to determine the mechanisms by which interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha regulate MLC phosphorylation and disrupt epithelial barrier function. We developed a model in which both cytokines were required for barrier dysfunction. Barrier dysfunction was also induced by TNF-alpha addition to IFN-gamma-primed, but not control, Caco-2 monolayers. TNF-alpha treatment of IFN-gamma-primed monolayers caused increases in both MLCK expression and MLC phosphorylation, suggesting that MLCK is a TNF-alpha-inducible protein. These effects of TNF-alpha were not mediated by nuclear factor-kappaB. However, at doses below those needed for nuclear factor-kappaB inhibition, sulfasalazine was able to prevent TNF-alpha-induced barrier dysfunction, MLCK up-regulation, and MLC phosphorylation. Low-dose sulfasalazine also prevented morphologically evident tight junction disruption induced by TNF-alpha. These data show that IFN-gamma can prime intestinal epithelial monolayers to respond to TNF-alpha by disrupting tight junction morphology and barrier function via MLCK up-regulation and MLC phosphorylation. These TNF-alpha-induced events can be prevented by the clinically relevant drug sulfasalazine.     

Differential expression of endothelial beta-catenin and plakoglobin during development and maturation of the blood-brain and blood-retina barrier in the chicken.

The development of the blood-brain barrier depends upon the formation of a closely regulated system of adherens and tight junctions. A prerequisite for a functional junction system is the linkage of transmembrane adhesion receptors (cadherins) to the cytoskeleton via catenins. The localization of specific catenins at the adherens junction correlates with the stability of interendothelial contacts in vitro, but in vivo data are lacking thus far. Investigating brain angiogenesis in the chicken, we demonstrated that beta-catenin, but not plakoglobin, initially codistributed with N-cadherin at the ablumenal endothelial membrane at contact sites to perivascular cells, from where both antigens disappeared during blood-brain barrier maturation. In contrast, plakoglobin was most prominent at the interendothelial junction where only small amounts of beta-catenin were present. Western-blot analysis revealed a stronger developmental decrease of beta-catenin than plakoglobin, whereas N-cadherin was completely lost. beta-Catenin but not N-cadherin was reinduced in brain endothelial cells during dedifferentiation in vitro and localized to the interendothelial junctions. These first in vivo data support the hypothesis that endothelial beta-catenin and N-cadherin are transiently relevant for the contact of brain endothelial to perivascular cells. Plakoglobin seems not to interact with N-cadherin but is exclusively localized at interendothelial junctions providing evidence for its role in the formation of stable adherens junctions, which may play a role for the initiation, and/or stabilization of tight junctions. Dev Dyn 2000;217:86-98.     

Junctional complexes of the blood-testis barrier in the Japanese quail (Coturnix Coturnix japonica)

This study investigated the developmental changes in the adherens junctions, gap junctions, as well as tight junctions forming the blood-testis barrier (BTB) in Japanese quail (Coturnix Coturnix japonica) testis. Testicular tissue from pre-pubertal, pubertal, adult, and aged Japanese quail were examined by immunohistochemistry and transmission electron microscopy (TEM). The tight junction proteins claudin-3, claudin-11, occludin and zonula occludens-1 (ZO-1), were generally localised in the cytoplasm of Sertoli cells, spermatogonia, and spermatocytes of pre-pubertal, pubertal, some adult birds. The adherens junction protein E-cadherin had a similar distribution pattern. During pre-pubertal development, the gap junction protein connexin-43 (Cx43) was only localised between Leydig cells in the testicular interstitium. However, TEM revealed the presence of gap junctions between cells of the seminiferous epithelium as early as the pre-pubertal stage. Furthermore, TEM confirmed the presence of tight and adherens junctions in the seminiferous epithelia of all age groups. The findings of this study document age-related differences in the immunolocalisation and intensity of the junctional proteins and the ultrastructure of the junctional complexes forming the BTB in quail testes. Additionally, the junctional complexes forming the BTB in the Japanese quail are well established prior to puberty. This study provides baseline information for the future evaluation of pathological changes in the BTB of avian species at different developmental stages.     

Mind the gap: mechanisms regulating the endothelial barrier

The endothelial barrier consists of intercellular contacts localized in the cleft between endothelial cells, which is covered by the glycocalyx in a sievelike manner. Both types of barrier‐forming junctions, i.e. the adherens junction (AJ) serving mechanical anchorage and mechanotransduction and the tight junction (TJ) sealing the intercellular space to limit paracellular permeability, are tethered to the actin cytoskeleton. Under resting conditions, the endothelium thereby builds a selective layer controlling the exchange of fluid and solutes with the surrounding tissue. However, in the situation of an inflammatory response such as in anaphylaxis or sepsis intercellular contacts disintegrate in post‐capillary venules leading to intercellular gap formation. The resulting oedema can cause shock and multi‐organ failure. Therefore, maintenance as well as coordinated opening and closure of interendothelial junctions is tightly regulated. The two principle underlying mechanisms comprise spatiotemporal activity control of the small GTPases Rac1 and RhoA and the balance of the phosphorylation state of AJ proteins. In the resting state, junctional Rac1 and RhoA activity is enhanced by junctional components, actin‐binding proteins, cAMP signalling and extracellular cues such as sphingosine‐1‐phosphate (S1P) and angiopoietin‐1 (Ang‐1). In addition, phosphorylation of AJ components is prevented by junction‐associated phosphatases including vascular endothelial protein tyrosine phosphatase (VE‐PTP). In contrast, inflammatory mediators inhibiting cAMP/Rac1 signalling cause strong activation of RhoA and induce AJ phosphorylation finally leading to endocytosis and cleavage of VE‐cadherin. This results in dissolution of TJs the outcome of which is endothelial barrier breakdown.     

Hepatic immunohistochemical localization of the tight junction protein ZO-1 in rat models of cholestasis.

Structural alterations in hepatocyte tight junctions accompanying cholestasis were investigated using immunolocalization of ZO-1, the first known protein component of the tight junction. Disruption in the paracellular barrier function of the tight junction has been proposed to allow reflux of bile into the blood. Cholestasis was induced in 210 to 235 g male Sprague-Dawley rats either by five consecutive daily subcutaneous injections of 17-alpha-ethinyl estradiol (0.5 mg/kg/d in propylene glycol) or ligation of the common bile duct for 72 hours. The structural organization of the tight junction was assessed in each model by indirect immunofluorescent and immunoperoxidase staining for ZO-1 on frozen sections of liver and compared with controls. In control, sham-operated, and estradiol-injected animals, ZO-1 localizes in a uniform continuous manner along the margins of the canaliculi. In contrast, bile duct ligation results in the appearance of numerous discontinuities in ZO-1 staining accompanied by dilation or collapse of the lumenal space. Tissue content of the ZO-1 protein, as determined by quantitative immunoblotting, was unaffected in either cholestatic model compared with controls. These findings indicate that the molecular organization of the tight junction can be assessed from immunostaining patterns of ZO-1 in frozen sections of cholestatic livers. Under these experimental conditions, the organization of the tight junction at the level of the ZO-1 protein is altered by bile duct obstruction but not by ethinyl estradiol.     

Regional differentiation of cell junctions in the excurrent duct epithelium of the rat testis as revealed by freeze-fracture

Intercellular junctions of the excurrent duct system of the adult rat testis were studied by freeze-fracture. In the terminal segment of the seminiferous tubule, where there are no spermatogenic cells, the tight junctions of Sertoli cells consist of many parallel strands of particles. The particles of the junctions predominantly appear on the E face instead of the P face, similar to those of the seminiferous tubules reported previously. From the rete testis to the ductus deferens, the tight junctional particles or smooth strands are mainly found on the P face, and the tight junctions show anastomosing networks. In the ductuli efferentes, whose epithelial lining consists of ciliated and nonciliated cells, the tight junctions between two adjacent nonciliated cells and between nonciliated and ciliated cells are poorly developed. In the former, belt-like gap junctions are often associated with segmented tight junctions. In such area, there are tiny regions, where no junctional elements are observed. Between two ciliated cells, several strands of the tight junctions can be seen. The result of the tracer experiment suggests that the barrier of the ductuli efferentes is weak. In the epididymis, the tight junctions are well developed throughout the duct. Corresponding with the regional variation of the epi-didymal epithelium, the geometrical organization of the tight junction networks varies along the duct. In the ductus deferens, many strands of the tight junctions are scattered throughout the lateral cell surface in addition to the belt-like network of the tight junction in the adluminal area. The number of the tight junctional strands is presented graphically in the various segments of the excurrent duct of the testis.     

Localization of actin in the retina of the crayfish Procambarus clarkii.

Summary The distribution of actin in the retina of the crayfish was investigated at the LM level using FITC-phalloidin. Fluorescent staining was associated with the main rhabdom and eighth cell rhabdom, the zonula adherens junctions between retinula cells, and the basement membrane of the retina. EM and S1 decoration were used to confirm the presence of actin and identify its structural relationships. Phalloidin staining of the rhabdom and S1 decoration of actin filaments in the rhabdom microvilli confirmed earlier findings that actin is a component of the microvillus cytoskeleton in the crayfish. At the zonula adherens junctions, actin filaments, identified by S1 decoration, run longitudinally within the plaque of the junction. At the extreme proximal end of the rhabdom, actin filaments associated with the junctions fill each small area of retinula cell cytoplasm. In the basement membrane, EM and S1 decoration show that basilar cells contain large bundles of actin filaments which are associated with cell-matrix adherens junctions. Foot cells which lie immediately below the rhabdom also contain similar junctions and actin is tentatively identified in these cells. The functional role of actin at these various locations is discussed in relation to retinal organization in the crayfish and other invertebrates.     

Cytoskeletal Regulation of Epithelial Barrier Function During Inflammation

Increased epithelial permeability is a common and important consequence of mucosal inflammation that results in perturbed body homeostasis and enhanced exposure to external pathogens. The integrity and barrier properties of epithelial layers are regulated by specialized adhesive plasma membrane structures known as intercellular junctions. It is generally believed that inflammatory stimuli increase transepithelial permeability by inducing junctional disassembly. This review highlights molecular events that lead to disruption of epithelial junctions during inflammation. We specifically focus on key mechanisms of junctional regulation that are dependent on reorganization of the perijunctional F-actin cytoskeleton. We discuss critical roles of myosin-II–dependent contractility and actin filament turnover in remodeling of the F-actin cytoskeleton that drive disruption of epithelial barriers under different inflammatory conditions. Finally, we highlight signaling pathways induced by inflammatory mediators that regulate reorganization of actin filaments and junctional disassembly in mucosal epithelia.The epithelium plays an important role in inflammation by serving as an interface between invading pathogens and the immune system of the host. Under physiological conditions, polarized epithelia form a protective barrier that allows regulated paracellular fluxes of solutes and nutrients as well as antigen sampling and surveillance by mucosal immune cells. However, during inflammation, this protective mechanism becomes compromised by various stimuli that originate on both sides of the epithelial barrier. On the apical (luminal) side, invading pathogenic microorganisms increase epithelial permeability to gain access into host tissue. The pathogens release a variety of epithelial barrier-disrupting agents that include pore-forming toxins, cytoskeleton-modifying proteins, and bacterial lipopolysaccharide (LPS). On the basal (tissue) side of the epithelial layer, activated immune cells also induce barrier disruption to facilitate their movement to sites of pathogen invasion. Mucosal immune cells increase epithelial permeability by secreting proinflammatory cytokines such as interferon (IFN) γ, tumor necrosis (TNF) α, and interleukin (IL)−1β or by releasing proteases and reactive oxygen species (ROS). As a result, mucosal inflammation commonly leads to sustained epithelial barrier compromise, which increases body exposure to external noxious agents, thereby further exaggerating the inflammatory response. Consequently it is believed that decreasing epithelial permeability may have beneficial effects by limiting inflammatory responses. Thus, understanding mechanisms that control the epithelial barrier disruption is important in identifying novel molecular targets for pharmacological modulation of mucosal inflammation.Properties of the epithelial barrier are regulated by specialized plasma membrane structures referred to as apical junctions. These structures are composed of adhesive and scaffolding proteins that are anchored into different cytoskeletal structures such as actin filaments, intermediate filaments, and microtubules. During inflammation, it is known that reorganizations of apical junctions mediate epithelial barrier dysfunction. Mounting evidence suggests that the actin cytoskeleton plays a pivotal role in regulating junctional integrity and remodeling under physiological and pathological states.In this review, we will discuss the role of actin filaments in the regulation of epithelial barrier integrity and its breakdown during inflammation. How inflammatory processes in different mucosal tissues induce remodeling of junction-associated filaments (F) actin and alter structure and barrier properties of epithelial cell-cell adhesions will be discussed. While epithelial junctions will be the major focus of this review, we will occasionally refer to examples of junctional regulation in the vascular endothelium. Likewise, we limit the discussion to simple columnar epithelia and exclude complex stratified epithelia such as the epidermis. Finally, we specifically focus on the role of actin filaments in maintenance and disassembly of epithelial junctions without discussing junctional reassembly during epithelial wound healing.     

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.     

Extracellular matrix,junctional integrity and matrix metalloproteinase interactions in endothelial permeability regulation

Vascular endothelial permeability is maintained by the regulated apposition of adherens and tight junctional proteins whose organization is controlled by several pharmacological and physiological mediators. Endothelial permeability changes are associated with: (1) the spatial redistribution of surface cadherins and occludin, (2) stabilization of focal adhesive bonds and (3) the progressive activation of matrix metalloproteinases (MMPs). In response to peroxide, histamine and EDTA, endothelial cells sequester VE-cadherin and alter its cytoskeletal binding. Simultaneously, these mediators enhance focal adhesion to the substratum. Oxidants, cytokines and pharmacological mediators also trigger the activation of matrix metalloproteinases (MMPs) in a cytoskeleton and tyrosine phosphorylation dependent manner to degrade occludin, a well-characterized tight junction element. These related in vitro phenomena appear to co-operate during inflammation, to increase endothelial permeability, structurally stabilize cells while also remodelling cell junctions and substratum.     

Regulation of the blood–biliary barrier: interaction between gap and tight junctions in hepatocytes

Hepatocytes tightly connect with each other by intercellular junctions to form liver cell plates. The junctions composed of gap, tight, and adherens junctions and desmosomes concentrate around bile canaliculi. In particular, tight junctions serve as a barrier to keep bile in bile canaliculi away from the blood circulation. Thus, it is very reasonable to call tight junctions of hepatocytes the blood–biliary barrier. On the other hand, gap junctions of hepatocytes are considered to enable ordered contraction of bile canaculi from centrizonal to periportal hepatocytes by their function of intercellular communication. Gap and tight junctions may thus play a crucial role in bile secretion, one of the most differentiated functions of the liver. In intrahepatic cholestasis, a common pathological condition of the liver, downregulation of gap and tight junctional functions is seen, which results in impaired intercellular communication and in leaky tight junctions. Although the changes in gap and tight junctions had been considered to be independent of each other, recent findings that the tight junction-associated proteins ZO-1 and occludin bind to connexins indicate the possibility of either coordinate or reciprocal regulation of macromolecular complexes containing gap- and tight-junction proteins. In this review, we introduce the interaction and regulation between gap and tight junctions of hepatocytes in vitro and discuss the regulatory mechanisms of the blood–biliary barrier to study the molecular pathogenesis of cholestasis.     

Tight junctions and human diseases

Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the barrier (or gate) function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. Of the proteins comprising tight junctions, integral membrane proteins occludin, claudins, and JAMs have been recently discovered. Of these molecules, claudins are exclusively responsible for the formation of tight-junction strands and are connected with the actin cytoskeleton mediated by ZO-1. Thus, both functions of tight junctions are dependent on the integrity of the actin cytoskeleton as well as ATP. Mutations in the claudin14 and the claudin16 genes result in hereditary deafness and hereditary hypomagnesemia, respectively. Some pathogenic bacteria and viruses target and affect the tight-junction function, leading to diseases. In this review, the relationship between tight junctions and human diseases is summarized.     

Redistribution of Tight Junction Proteins During EPEC Infection In Vivo

Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea among infants. Tight junction plays a vital role in intestinal paracellular permeability by forming physical intercellular barriers in epithelial cells. However, the impact of this enteric pathogen on tight junctions in vivo has not been fully investigated. In the present study, the alterations in tight junctions following EPEC infection in vivo were investigated. Western blot analysis revealed that the tight junction proteins, occludin and claudin-1, were displaced from tight junction membrane microdomains to Triton X-100 soluble fractions after EPEC infection. Changes in intestinal paracellular permeability were determined using the molecular tracer biotin, which was observed to penetrate the epithelia and extended into the lamina propria, indicating disruption in tight junction barrier function. Our results suggested that redistribution of tight junction proteins plays an important role in the disruption of epithelial barrier function induced by EPEC infection, which may provide new insight into the pathogenesis of diarrhea caused by EPEC.     

Non-junctional human desmoglein 3 acts as an upstream regulator of Src in E-cadherin adhesion, a pathway possibly involved in the pathogenesis of pemphigus vulgaris

E-cadherin, a classical cadherin, is an adhesion receptor in adherens junctions and has important functions in cell-cell adhesion and cell signalling. Recently we reported that a desmosomal cadherin, desmoglein 3 (Dsg3), an autoantigen in pemphigus vulgaris (PV), associates with E-cadherin and activates Src, which results in tyrosine phosphorylation of adherens junction proteins. However, the nature of such an interaction and its role in cell-cell adhesion remain unclear. In this report, we provide direct evidence that it is the detergent-soluble, non-desmosomal Dsg3 that regulates the activity of Src and its association with E-cadherin in adherens junction formation. Modulation of Dsg3 levels, either by Dsg3 silencing or over-expression, alters Src activity and its association with E-cadherin. Dsg3 silencing caused retardation of calcium-induced E-cadherin junction assembly and a reduction of desmosomal protein expression. Furthermore, we provide evidence that this signalling pathway is involved, at least in part, in the pathophysiology of pemphigus. Along with the reduced expression of Dsg3, loss and disruption of E-cadherin and a concomitant decreased pSrc signalling was identified in the basal keratinocytes surrounding the blisters in PV. These findings suggest a novel function for Dsg3 in the control of E-cadherin-Src signalling and cell-cell adhesion.     

Local thermal injury induces general endothelial cell contraction through p38 MAP kinase activation

Endothelial cells (ECs) of thin‐walled blood vessels form a barrier between blood and tissue. As a response to inflammation, the EC junctions widen and gaps form, resulting in compromised barrier functions. Although the mechanisms behind the establishment of these changes are still incompletely understood, one known reason is actomyosin‐dependent actin rearrangement. Here, by using atomic force microscopy and a combination of confocal microscopy methods, we are the first to report that thermal injury induces general venular hyperpermeability and that serum from burned rats induces EC actin rearrangement, contraction, as well as tight‐junction damage. Inhibition of the p38 mitogen‐activated protein kinase (p38MAPK) largely ameliorates resulting vascular dysfunction by significantly reducing EC stress‐fiber formation, contraction, volume changes and tight‐junction damage, thereby greatly reducing the appearance of EC gaps. The findings may be of importance for the design of future pharmacotherapies aiming to ease the severe general vascular dysfunction that follows extensive burns.     

Similar and differential behaviour between the nectin-afadin-ponsin and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells

BACKGROUND: We have recently identified a novel cell-cell adhesion system, named NAP system, which is localized at cadherin-based cell-cell adherens junctions (AJs). The NAP system is composed of at least nectin, afadin and ponsin. Nectin is an immunoglobulin-like cell adhesion molecule. Afadin is an actin filament-binding protein which associates nectin with the actin cytoskeleton. Ponsin is an afadin-binding protein which furthermore binds to vinculin and provides a possible linkage of nectin-afadin to cadherin-catenin through vinculin. We compared here the behaviour of the NAP and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells. RESULTS: At the early stage of the formation of the polarized junctional alignment in MTD-1 A cells, primordial spot-like junctions were formed at the tips of thin cellular protrusions radiating from adjacent cells. Nectin, afadin, ponsin, cadherin and catenin were simultaneously recruited to these junctions. As the cell polarization proceeded, the spot-like junctions were gradually fused to form belt-like AJs where all these proteins were concentrated. The disruption of cell-cell AJs in MDCK cells by culturing at a low Ca2+ concentration caused rapid endocytosis of cadherin, but not that of nectin or afadin. Addition of 12-O-tetradecanoylphorbol-13-acetate to the cells formed a tight junction-like structure where nectin and afadin, but not cadherin, accumulated. CONCLUSION: These results indicate that the NAP and cadherin-catenin systems show similar and differential behaviour during the formation and disruption of the polarized junctional alignment in epithelial cells.     

The Impact of Environmental Pollutants on Barrier Dysfunction in Respiratory Disease

Respiratory epithelial cells form a selective barrier between the outside environment and underlying tissues. Epithelial cells are polarized and form specialized cell-cell junctions, known as the apical junctional complex (AJC). Assembly and disassembly of the AJC regulates epithelial morphogenesis and remodeling processes. The AJC consists of tight and adherens junctions, functions as a barrier and boundary, and plays a role in signal transduction. Endothelial junction proteins play important roles in tissue integrity and vascular permeability, leukocyte extravasation, and angiogenesis. Air pollutants such as particulate matter, ozone, and biologic contaminants penetrate deep into the airways, reaching the bronchioles and alveoli before entering the bloodstream to trigger airway inflammation. Pollutants accumulating in the lungs exacerbate the symptoms of respiratory diseases, including asthma and chronic obstructive lung disease. Biological contaminants include bacteria, viruses, animal dander and cat saliva, house dust mites, cockroaches, and pollen. Allergic inflammation develops in tissues such as the lung and skin with large epithelial surface areas exposed to the environment. Barrier dysfunction in the lung allows allergens and environmental pollutants to activate the epithelium and produce cytokines that promote the induction and development of immune responses. In this article, we review the impact of environmental pollutants on the cell barrier in respiratory diseases.     

Localization of dysfunctional tight junctions in Salmonella enterica serovar typhimurium-infected epithelial layers

Infection of polarized MDCK epithelial layers by Salmonella enterica serovar Typhimurium is accompanied by increased tight junction permeability and by contraction of perijunctional actinomyosin. We localized dysfunctional tight junctions in serovar Typhimurium-infected MDCK layers by imaging apical-basolateral intramembrane diffusion of fluorescent lipid and found that loss of the apical-basolateral diffusion barrier (tight junction fence function) was most marked in areas of prominent perijunctional contraction. The protein kinase inhibitor staurosporine prevented perijunctional contraction but did not reverse the effects of serovar Typhimurium on tight junction barrier function. Hence, perijunctional contraction is not required for Salmonella-induced tight junction dysfunction and this epithelial response to infection may be multifactorial.     

CCL2 disrupts the adherens junction: implications for neuroinflammation

Alterations to blood-brain barrier (BBB) adhesion molecules and junctional integrity during neuroinflammation can promote central nervous system (CNS) pathology. The chemokine CCL2 is elevated during CNS inflammation and is associated with endothelial dysfunction. The effects of CCL2 on endothelial adherens junctions (AJs) have not been defined. We demonstrate that CCL2 transiently induces Src-dependent disruption of human brain microvascular endothelial AJ. β-Catenin is phosphorylated and traffics from the AJ to PECAM-1 (platelet endothelial cell adhesion molecule-1), where it is sequestered at the membrane. PECAM-1 is also tyrosine-phosphorylated, an event associated with recruitment of the phosphatase SHP-2 (Src homology 2 domain-containing protein phosphatase) to PECAM-1, β-catenin release from PECAM-1, and reassociation of β-catenin with the AJ. Surface localization of PECAM-1 is increased in response to CCL2. This may enable the endothelium to sustain CCL2-induced alterations in AJ and facilitate recruitment of leukocytes into the CNS. Our novel findings provide a mechanism for CCL2-mediated disruption of endothelial junctions that may contribute to BBB dysfunction and increased leukocyte recruitment in neuroinflammatory diseases.