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PubMed2006

The secreted autotransporter toxin, Sat, functions as a virulence factor in Afa/Dr diffusely adhering Escherichia coli by promoting lesions in tight junction of polarized epithelial cells.

Afa/Dr diffusely adhering Escherichia coli (DAEC) strains are responsible for urinary tract and intestinal infections. Both in intestine and kidney, the epithelial cells forming epithelium are sealed by junctional domains. We provide evidence that the Secreted autotransporter toxin, Sat, belonging to the subfamily of serine protease autotransporters of Enterobacteriaceae (SPATEs), acts as a virulence factor in Afa/Dr DAEC by promoting lesions in the tight junctions (TJs) of polarized epithelial Caco-2/TC7 cells. Southern blot analysis reveals that the prototype strains of the subclass-1 and subclass-2 typical Afa/Dr DAEC strains, hybridize with a sat probe. Using the wild-type IH11128 strain, the recombinant E. coli AAEC185 strain that expresses Sat, the recombinant E. coli that expresses both Dr adhesin and Sat, we report that Sat in monolayers of cultured enterocyte-like Caco-2/TC7 cells, induces rearrangements of the TJs-associated proteins ZO-1, ZO-3 and occludin, and increases the formation of domes as the result of an increase in the paracellular permeability without affecting the transepithelial electrical resistance of the cell monolayers. Moreover, we observe that Sat-induced disassembly of TJs-associated proteins is dependent on the serine protease motif. Finally, an analysis of the prevalence of the sat gene in three collections of Afa/Dr DAEC strains collected from the stools of children with and without diarrhoea, and from the urine of patients with urinary tract infection (UTI) shows that: (i) the sat gene is highly prevalent in UTI-associated Afa/Dr DAEC strains (88% positive), (ii) the sat gene is generally absent from Afa/Dr DAEC strains collected from the stools of children without diarrhoea (16% positive); whereas (iii) it is present in about half of the strains collected from the stools of children with diarrhoea (46% positive).

Actins↗

PubMed2005

Tight junction proteins and perineurial cells in neurofibromas.

Cutaneous neurofibromas consist of axonal processes, Schwann cells, fibroblasts, perineurial cells, mast cells, and abundant extracellular matrix. The distribution and role of perineurial cells in neurofibromas has been uncertain, partly because there has not been a specific immunohistochemical marker for perineurial cells. In this study, tight junctions (TJs) of 16 neurofibromas from 12 patients with neurofibromatosis type 1 (NF1) were analyzed using electron microscopy, immunohistochemistry, and Western transfer analysis. Cell-cell contacts with typical ultrastructural morphology of TJs were seen between adjacent perineurial cells surrounding the small nerves and between contacting perineurial cell processes embedded in tumor stroma. Immunohistochemistry showed expression of claudin-1, claudin-3, and ZO-1 in the intercellular junctions of a subpopulation of tumor cells. Occludin was present mainly in perineurium and claudin-5 localized to the blood vessels. Double immunolabelings were used to identify the cell types expressing claudin-1. The results showed that claudin-1 positive cells were also positive for type IV collagen and epithelial membrane antigen but not for S-100 protein. This labeling pattern is consistent with perineurial cell phenotype. Using claudin-1 as a marker, our results showed that clusters of perineurial cells are distributed around the rudimentary nerves within cutaneous neurofibromas and at the periphery of some neurofibromas.

Blotting, Western↗

PubMed2004

Analysis of invasion-metastasis mechanism in pancreatic cancer: involvement of tight junction transmembrane protein occludin and MEK/ERK signal transduction pathway in cancer cell dissociation.

Mitogen-activated protein kinase kinase 2 (MEK2) was detected as an invasion-metastasis related factor between highly invasive (PC-1.0) and weakly invasive (PC-1) pancreatic cancer cell lines in our previous study. On the other hand, tight junction (TJ) was found to be correlated with carcino-genesis and tumor development. In this study, the expressions and correlation of TJ transmembrane protein occludin and MEK/extracellular signal-regulated kinase (ERK) signaling pathway were analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and human (AsPC-1 and CAPAN-2) pancreatic cancer cell lines were analyzed immunocytochemically with anti-occludin, phosphorylated MEK1/2 (p-MEK1/2), phosphorylated ERK1/2 (p-ERK1/2) antibodies. MEK1/2 inhibitor U0126 significantly induced the expression of occludin at the cell-cell junction and substantially suppressed the p-MEK1/2 and p-ERK1/2 expressions in PC-1.0 and AsPC-1 cells. In contrast, dissociation factor (DF) treatment obviously disrupted the occludin expressions at the sites of cell-cell junction and markedly induced the p-MEK1/2 and p-ERK1/2 expressions in PC-1 and CAPAN-2 cells. In addition, occludin expressions at cell-cell junction were restored and p-MEK1/2 and p-ERK1/2 expressions were suppressed by subsequent U0126-treatment in DF treated PC-1 and CAPAN-2 cells. Correspondingly, light microscopic images showed that DF induced the dissociation of cell island-like colonies in PC-1 and CAPAN-2 cells, and U0126-treatment induced cell aggregation in these pancreatic cancer cells. Occludin is involved in the cell dissociation in pancreatic cancer cells. Moreover, MEK/ERK signaling pathway probably regulates the cell dissociation status of pancreatic cancer through influencing the intracellular localization and expression of occludin.

Animals↗

PubMed2003

NZO-3 expression causes global changes to actin cytoskeleton in Madin-Darby canine kidney cells: linking a tight junction protein to Rho GTPases.

We previously demonstrated that exogenous expression of a truncated form of the tight junction protein ZO-3 affected junctional complex assembly and function. Current results indicate that this ZO-3 construct influences actin cytoskeleton dynamics more globally. We show that expression of the amino-terminal half of ZO-3 (NZO-3) in Madin-Darby canine kidney cells results in a decreased number of stress fibers and focal adhesions and causes an increased rate of cell migration in a wound healing assay. We also demonstrate that RhoA activity is reduced in NZO-3-expressing cells. We determined that ZO-3 interacts with p120 catenin and AF-6, proteins localized to the junctional complex and implicated in signaling pathways important for cytoskeleton regulation and cell motility. We also provide evidence that NZO-3 interacts directly with the C terminus of ZO-3, and we propose a model where altered interactions between ZO-3 and p120 catenin in NZO-3-expressing cells affect RhoA GTPase activity. This study reveals a potential link between ZO-3 and RhoA-related signaling events.

Actins↗

PubMed2005

Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane.

Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.

Actins↗

PubMed2006

Control of tight junctional sealing: role of epidermal growth factor.

Epithelia can adjust the permeability of their paracellular permeation route to physiological requirements, pathological conditions, and pharmacological challenges. This is reflected by a transepithelial electrical resistance (TER) ranging from a few tenth to several thousands Omega.cm(2), depending on the degree of sealing of the tight junction (TJ). The present work is part of an effort to understand the causes and mechanisms underlying these adaptations. We observed that an extract of human urine (hDLU) increases TER in a concentration- and time-dependent manner and is more effective when added from the basolateral side of cultured monolayers of Madin-Darby canine kidney cells than from the apical one. We found that its main TER-increasing component is epidermal growth factor (hEGF), as depletion of this peptide with specific antibodies, or inhibition of its receptor with PD153035, abolishes its effect. Since the permeability of the TJ depends on the expression of several species of membrane proteins, chiefly claudins, we explored whether hDLU can affect five members of the claudin family, the three known members of the ZO family, and occludin. EGF present in hDLU decreases the content of claudins-1 and -2 as well as delocalizes them from the TJ and increases the content of claudin-4. As expected from the fact that the degree of sealing of the TJ must be a physiologically regulated parameter, besides of hEGF, we also found that hDLU appears to contain also other components that decrease TER, claudin-4 and -7, and that seem to act with different kinetics than the TER-increasing ones.

Adolescent↗

PubMed2004

Arrangement of expression and distribution of tight junction protein claudin-1 in cell dissociation of pancreatic cancer cells.

Mitogen-activated protein kinase kinase 2 (MEK2) was isolated previously as a potential factor related to cancer cell dissociation in highly (PC-1.0) and weakly (PC-1) invasive pancreatic cancer cells. On the other hand, changes of structure and function of tight junction (TJ) are reported to be correlated with carcinogenesis and tumor development. In this study, immunocytochemistry and Western blot analysis were performed in pancreatic cancer cells using anti-claudin-1, MEK2 and phosphorylated MEK1/2 (p-MEK1/2) antibodies to reveal the correlation between TJ and cancer cell dissociation, as well as the involvement of MEK2 in regulation of TJ in cell dissociation of pancreatic cancer. After incubation with conditioned medium of PC-1.0 cells, plasma membrane distribution of claudin-1 was obviously disrupted, and expressions of MEK2 and p-MEK1/2, as well as dissociation of cell colonies, were significantly induced in PC-1 and CAPAN-2 cells. However, U0126 (a MEK1/2 inhibitor) treatment apparently induced the plasma membrane distribution of claudin-1 and aggregation of single cells in PC-1.0 and AsPC-1 cells, synchronously seriously suppressed MEK2 and p-MEK1/2 expression. Arrangement of expression and distribution of claudin-1 is closely related to cell dissociation status in pancreatic cancer cells through MEK2 activation.

Blotting, Western↗

PubMed2004

[Effect of borneol on the intercellular tight junction and pinocytosis vesicles in vitro blood-brain barrier model].

OBJECTIVE: To explore the mechanism of borneol in opening the blood-brain barrier (BBB). METHODS: Borneol contained serum was prepared and using Matin-Darby canine kidney epithelium (MDCKE) cell line as the in vitro BBB model to observe the effects of borneol on intercellular tight junction (ICTJ) and pinocytosis vesicles of BBB model. RESULTS: Borneol reduced the ICTJ and caused increase of the number and enlarged the diameter of vesicles. The ICTJ was opened firstly 4 hrs after borneol treatment, then the pinocytosis was affected 24 hrs later. The effects disappeared 24 hrs after removal of the borneol contained serum, indicating that the above-mentioned effects were reversible. CONCLUSION: Borneol could obviously loosen the ICTJ in BBB, accelerate the transportation of substance through the intercellular passage, it also could increase the number and volume of pinocytosis vesicles in BBB cells, thus to accelerate the transportation of substance by way of cell pinocytosis.

Animals↗

PubMed2004

Tight junction proteins ZO-1, occludin, and claudins in developing and adult human perineurium.

In peripheral nerves, groups of Schwann cell-axon units are isolated from the adjacent tissues by the perineurium, which creates a diffusion barrier responsible for the maintenance of endoneurial homeostasis. The perineurium is formed by concentric layers of overlapping, polygonal perineurial cells that form tight junctions at their interdigitating cell borders. In this study, employing indirect immunofluorescence and immunoelectron microscopy, we demonstrate that claudin-1 and -3, ZO-1, and occludin, but not claudin-2, -4, and -5, are expressed in the perineurium of adult human peripheral nerve. We also describe the expression of occludin, ZO-1, claudin-1, -3, and -5 in the developing human perineurium, showing that the expressions of claudin-1 and -3, ZO-1, and occludin follow similar spatial developmental expression patterns but follow different timetables in achieving their respective adult distributions. Specifically, claudin-1 is already largely restricted to perineurium-derived structures at 11 fetal weeks, whereas claudin-3 and occludin are weakly expressed in the perineurial structures at this age and acquire a well-defined perineurial distribution only between 22 and 35 fetal weeks. ZO-1 appears to acquire its mature profile even later during the third trimester. The results of the present and previous studies show that the perineurial diffusion barrier matures relatively late during human peripheral nerve development.

Adolescent↗

PubMed1999

Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions.

House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergen's own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.

Allergens↗

PubMed2004

Formation and barrier function of tight junctions in human ovarian surface epithelium.

The normal ovarian surface epithelium (OSE) is a primitive epithelium made up by a single layer of mesothelial-type epithelial cells. When these cells get trapped in the ovarian stroma, expression of epithelial specific markers, such as E-cadherin, are induced. Most epithelial cells are also characterized by the ability to form tight junctions (TJ). Incomplete TJ have earlier been demonstrated in the OSE by electron microscopy studies. We have investigated expression and localization of the TJ proteins ZO-1, occludin, and claudin-1 in tissue biopsies from normal human ovaries and OSE in culture. The dynamics of TJ formation were studied in human OSE cultured on porous filters in culture inserts by measuring trans epithelial resistance (TER) including Ca(2+) switch experiments. Confluent OSE cells were also analyzed by electron microscopy. The results show that normal human OSE has expression of all three TJ proteins investigated. These proteins, ZO-1, occludin, and claudin-1, were localized to OSE cell borders both in ovarian biopsies and in cultured OSE. There was no difference in this regard between fertile and postmenopausal women. Cells in culture were polarized and presented junctional complexes seen by electron microscopy. In the Ca(2+) switch experiments, removing free Ca(2+) transiently, TER decreased significantly (P < 0.05) in the Ca(2+)-free group compared with nontreated OSE. TER was fully restored after 24 h. N-cadherin but not E-cadherin was expressed in the OSE and localized to the cell borders. We conclude that normal human OSE express and form functional TJ both in vivo and vitro. This report also describes a method to study the influence of ovarian-derived mediators on TJ in cultured OSE.

Adult↗

PubMed2003

Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation.

The effects of pathogenic organisms on host intestinal epithelial cells are vast. Innumerable signalling pathways are triggered leading ultimately to drastic changes in physiological functions. Here, the ways in which enteric bacterial pathogens utilise and impact on the three major physiological functions of the intestinal epithelium are discussed: alterations in the structure and function of the tight junction barrier, induction of fluid and electrolyte secretion, and activation of the inflammatory cascade. This field of investigation, which was virtually non-existent a decade ago, has now exploded, thus rapidly expanding our understanding of bacterial pathogenesis. Through increased delineation of the ways in which microbes alter host physiology, we simultaneous gain insight into the normal regulatory mechanisms of the intestinal epithelium.

Bacterial Infections↗

PubMed2006

Tight junctions and cell-cell interactions.

Chronic inflammation in mucosal tissues can influence epithelial barrier function via pro-inflammatory cytokines such as interferon (IFN)-gamma and tumor necrosis factor-alpha. Increased mucosal levels of these cytokines have been observed in mucosal biopsies from patients with a chronic inflammatory condition referred to as inflammatory bowel disease. Paracellular permeability across epithelial cells is regulated by tight junctions (TJs), which are the apical most junctions in epithelial cells. Given that pro-inflammatory cytokines modulate the epithelial barrier and that TJs regulate epithelial permeability, we analyzed the influence of IFN-gamma on U function/structure. Our results suggest that IFN-gamma induced a time-dependent increase in paracellular permeability that was associated with internalization of TJ transmembrane proteins, occludin, junction adhesion molecule A, and claudin-1. In this chapter, we focus on selected methods used to investigate the influence of IFN-gamma on epithelial barrier function.

Animals↗

PubMed2004

Indigestible disaccharides open tight junctions and enhance net calcium, magnesium, and zinc absorption in isolated rat small and large intestinal epithelium.

The effects of three indigestible disaccharides on net calcium (Ca), magnesium (Mg), and zinc (Zn) transport in isolated rat jejunal, ileal, cecal, and colonic epithelium were determined. Permeability of fluorescein isothiocynate-dextran-4 (FD4) and transepithelial electrical resistance (TEER), which vary according to tight junction (TJ) activity in the intestinal mucosa, were also determined. The addition of 1-100 mM melibiose, difructose anhydride (DFA) III, or DFA IV to the mucosal medium increased the net absorption of the three minerals and FD4 permeability, while decreasing TEER dose dependently in the four intestinal portions. Positive linear relations were found between the net transport of the three minerals and FD4 passage in all portions of the intestine, whereas negative linear relations were observed between net absorption of the three minerals and TEER. We concluded that the three indigestible saccharides directly affect the epithelial tissue and open TJs, thereby promoting Ca, Mg, and Zn absorption in the small and large intestine in vitro.

Animals↗

PubMed2000

Characterization of huJAM: evidence for involvement in cell-cell contact and tight junction regulation.

Cell-cell interactions of the mucosal epithelia are important for the maintenance and establishment of epithelial barrier function. During events of inflammation, such cell-cell interactions are often disrupted, resulting in a leaky epithelial barrier, which in turn can lead to various inflammatory and infective dysfunctions. Human junctional adhesion molecule (huJAM), found on the mucosal epithelia and vascular endothelia of many major organ systems, is a membrane glycoprotein which resolves to a doublet band of approximately 40 and approximately 37 kDa under SDS-PAGE analysis, representing differentially glycosylated forms of the same protein. huJAM was localized to the lateral membrane of Caco-2 cells (a human colonic epithelial cell line) monolayers, in an area basolateral of the epithelial tight junctions (TJ). Through functional and biochemical assays, we show huJAM to be able to homotypically associate and to participate in TJ restitution after trypsin-EDTA disruption. Furthermore, we also observed a migration of huJAM expression toward areas of cell-cell contacts during events of cell adhesion and monolayer formation. These qualities makes huJAM a likely player in the regulation of cell-cell contacts and the subsequent formation of TJs.

Animals↗

PubMed1993

Deposition of BaSO4 in the tight junctions of amphibian epithelia causes their opening; apical Ca2+ reverses this effect.

Selective deposition of BaSO4 in the tight junctions (TJs) of frog skins led to profound and reversible functional alterations of these structures, as revealed by changes of tissue conductance (G), clamping current (I), and fluxes of extracellular markers (sulfate (JSO4) and sucrose (JSUC)). Experiments were performed with nominally Ca(2+)-free simple salt solutions on the apical side (usually KCl) and Na2SO4-Ringer on the inner side of skins. The deposition of BaSO4 in the TJs was obtained by diffusion and/or migration through the paracellular path of Ba2+ from the apical solution and SO4(2-) from the inner solution. A brief presence (2 to 6 min) of apical Ba2+ (Ba2+ pulse) is followed (i.e., when Ba2+ is removed from the apical fluid) by a large increase of G, I, JSO4 and JSUC, above pre-Ba2+ levels. These attain a steady state within 15 to 30 min (overshoot phase), characterizing a conspicuous increase of the paracellular permeability. During the overshoot phase, a second Ba2+ pulse blocks the paracellular route while apical Ba2+ is present, leading to a new and larger overshoot when the Ba2+ pulse is terminated. Addition of apical Ca2+ triggers the resealing of the TJs, resulting in a full recovery of G, I, JSO4 and JSUC. This Ca(2+)-induced recovery persists when apical Ca2+ is removed. The presence of a normal Ca2+ concentration in the inner bathing Ringer does not induce the recovery process. Tissues remain viable after being submitted to the Ba2+ treatment and the subsequent overshoot. Experiments performed in the urinary bladder of Rana catesbeiana and skins and urinary bladders of Bufo marinus indicate that Ba2+ effect can also be elicited in these tissues. The above results seem to report general properties of the TJs. Incidentally, they warn about the use of Ba2+ as an ion channel blocker in epithelial membranes in association with SO4(2-)-containing solutions on the contralateral side.

Animals↗

PubMed1980

Interlamellar tight junctions of central myelin. II. A freeze fracture and cytochemical study on their arrangement and composition.

The interlamellar tight junctions (ITJ) of central myelin (white matter from the parietal lobe and the medulla oblongata of the rat) were analyzed electron microscopically, making use of a wide range of different preparatory techniques. Freeze-fracture observations indicate that the ITJ are composed of rows of particulate subunits in glutaraldehyde-fixed or formaldehyde-fixed material, and in the unfixed state. The particulate subunits of the ITJ are preferentially associated with the protoplasmic (P) face in the aldehyde-fixed state, and no shift in the binding characteristics of the particles was observed after omission of aldehyde fixation. Tracer studies in conjunction with the dissociated appearance of the junctional globules suggest that the ITJ represent a leaky type of zonula occludens. It is assumed that the ITJ particles represent an "integral-type protein" that preferentially serves as a mechanical device maintaining the structural integrity of the central myelin sheath. By means of cytochemical experiments, the proteinaceous character of the ITJ subunits is established. An attempt is made, based on results from lipid extraction and protein digestion, to define certain cytochemical parameters of the ITJ proteins and to compare them with the current collection of chemically identified proteins of central myelin.

Animals↗

PubMed2006

Shoichiro Tsukita: a life exploring the molecular architecture of the tight junction.

On December 11, 2005, Shoichiro Tsukita died at the young age of 52, after 14 months of treatment for cancer. Early in his career, Tsukita succeeded in isolating and purifying the adherens junction with his wife Sachiko, an accomplishment that he followed up with an impressive series of discoveries of cell adhesion and cytoskeletal molecules, including what may have been his greatest contribution to the field, the identification of occludin and the claudin family of molecules, which were watershed discoveries in the study of the molecular nature of tight junctions.

Adherens Junctions↗