In tight junctions, claudins regulate the interactions between occludin, tricellulin and marvelD3, which, inversely, modulate claudin oligomerization.

PubMed

Cording, Jimmi; Berg, Johanna; Käding, Nadja; Bellmann, Christian; Tscheik, Christian; Westphal, Julie K; Milatz, Susanne; Günzel, Dorothee; Wolburg, Hartwig; Piontek, Jörg; Huber, Otmar; Blasig, Ingolf Ernst

2013-01-15

Tight junctions seal the paracellular cleft of epithelia and endothelia, form vital barriers between tissue compartments and consist of tight-junction-associated marvel proteins (TAMPs) and claudins. The function of TAMPs and the interaction with claudins are not understood. We therefore investigated the binding between the TAMPs occludin, tricellulin, and marvelD3 and their interaction with claudins in living tight-junction-free human embryonic kidney-293 cells. In contrast to claudins and occludin, tricellulin and marvelD3 showed no enrichment at cell-cell contacts indicating lack of homophilic trans-interaction between two opposing cell membranes. However, occludin, marvelD3 and tricellulin exhibited homophilic cis-interactions, along one plasma membrane, as measured by fluorescence resonance energy transfer. MarvelD3 also cis-interacted with occludin and tricellulin heterophilically. Classic claudins, such as claudin-1 to -5 may show cis-oligomerization with TAMPs, whereas the non-classic claudin-11 did not. Claudin-1 and -5 improved enrichment of occludin and tricellulin at cell-cell contacts. The low mobile claudin-1 reduced the membrane mobility of the highly mobile occludin and tricellulin, as studied by fluorescence recovery after photobleaching. Co-transfection of claudin-1 with TAMPs led to changes of the tight junction strand network of this claudin to a more physiological morphology, depicted by freeze-fracture electron microscopy. The results demonstrate multilateral interactions between the tight junction proteins, in which claudins determine the function of TAMPs and vice versa, and provide deeper insights into the tight junction assembly.

Transmembrane proteins of tight junctions.

PubMed

Chiba, Hideki; Osanai, Makoto; Murata, Masaki; Kojima, Takashi; Sawada, Norimasa

2008-03-01

Tight junctions contribute to the paracellular barrier, the fence dividing plasma membranes, and signal transduction, acting as a multifunctional complex in vertebrate epithelial and endothelial cells. The identification and characterization of the transmembrane proteins of tight junctions, claudins, junctional adhesion molecules (JAMs), occludin and tricellulin, have led to insights into the molecular nature of tight junctions. We provide an overview of recent progress in studies on these proteins and highlight their roles and regulation, as well as their functional significance in human diseases.

Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2

PubMed Central

Aijaz, Saima; Sanchez-Heras, Elena; Balda, Maria S; Matter, Karl

2007-01-01

Background Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis. Results We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures. Conclusion Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation. PMID:18028534

Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2.

PubMed

Aijaz, Saima; Sanchez-Heras, Elena; Balda, Maria S; Matter, Karl

2007-11-20

Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis. We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures. Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation.

Cellular entry of G3.5 poly (amido amine) dendrimers by clathrin- and dynamin-dependent endocytosis promotes tight junctional opening in intestinal epithelia.

PubMed

Goldberg, Deborah S; Ghandehari, Hamidreza; Swaan, Peter W

2010-08-01

This study investigates the mechanisms of G3.5 poly (amido amine) dendrimer cellular uptake, intracellular trafficking, transepithelial transport and tight junction modulation in Caco-2 cells in the context of oral drug delivery. Chemical inhibitors blocking clathrin-, caveolin- and dynamin-dependent endocytosis pathways were used to investigate the mechanisms of dendrimer cellular uptake and transport across Caco-2 cells using flow cytometry and confocal microscopy. Dendrimer cellular uptake was found to be dynamin-dependent and was reduced by both clathrin and caveolin endocytosis inhibitors, while transepithelial transport was only dependent on dynamin- and clathrin-mediated endocytosis. Dendrimers were quickly trafficked to the lysosomes after 15 min of incubation and showed increased endosomal accumulation at later time points, suggesting saturation of this pathway. Dendrimers were unable to open tight junctions in cell monolayers treated with dynasore, a selective inhibitor of dynamin, confirming that dendrimer internalization promotes tight junction modulation. G3.5 PAMAM dendrimers take advantage of several receptor-mediated endocytosis pathways for cellular entry in Caco-2 cells. Dendrimer internalization by dynamin-dependent mechanisms promotes tight junction opening, suggesting that dendrimers act on intracellular cytoskeletal proteins to modulate tight junctions, thus catalyzing their own transport via the paracellular route.

3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells.

PubMed

Matsusaki, Michiya; Hikimoto, Daichi; Nishiguchi, Akihiro; Kadowaki, Koji; Ohura, Kayoko; Imai, Teruko; Akashi, Mitsuru

2015-02-13

Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

MicroRNA-205 targets tight junction-related proteins during urothelial cellular differentiation.

PubMed

Chung, Pei-Jung Katy; Chi, Lang-Ming; Chen, Chien-Lun; Liang, Chih-Lung; Lin, Chung-Tzu; Chang, Yu-Xun; Chen, Chun-Hsien; Chang, Yu-Sun

2014-09-01

The mammalian bladder urothelium classified as basal, intermediate, and terminally differentiated umbrella cells offers one of the most effective permeability barrier functions known to exist in nature because of the formation of apical uroplakin plaques and tight junctions. To improve our understanding of urothelial differentiation, we analyzed the microRNA (miRNA) expression profiles of mouse urinary tissues and by TaqMan miRNA analysis of microdissected urothelial layers and in situ miRNA-specific hybridization to determine the dependence of these miRNAs on the differentiation stage. Our in situ hybridization studies revealed that miR-205 was enriched in the undifferentiated basal and intermediate cell layers. We then used a quantitative proteomics approach to identify miR-205 target genes in primary cultured urothelial cells subjected to antagomir-mediated knockdown of specific miRNAs. Twenty-four genes were reproducibly regulated by miR-205; eleven of them were annotated as cell junction- and tight junction-related molecules. Western blot analysis demonstrated that antagomir-induced silencing of miR-205 in primary cultured urothelial cells elevated the expression levels of Tjp1, Cgnl1, and Cdc42. Ectopic expression of miR-205 in MDCK cells inhibited the expression of tight junction proteins and the formation of tight junctions. miR-205- knockdown urothelial cells showed alterations in keratin synthesis and increases of uroplakin Ia and Ib, which are the urothelial differentiation products. These results suggest that miR-205 may contribute a role in regulation of urothelial differentiation by modulating the expression of tight junction-related molecules. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells.

PubMed

Buckley, Alysia G; Looi, Kevin; Iosifidis, Thomas; Ling, Kak-Ming; Sutanto, Erika N; Martinovich, Kelly M; Kicic-Starcevich, Elizabeth; Garratt, Luke W; Shaw, Nicole C; Lannigan, Francis J; Larcombe, Alexander N; Zosky, Graeme; Knight, Darryl A; Rigby, Paul J; Kicic, Anthony; Stick, Stephen M

2018-01-01

Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. Here, we assessed four fixation methods including; (i) 4% ( v /v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.

Cell-cell interactions of isolated and cultured oligodendrocytes: formation of linear occluding junctions and expression of peculiar intramembrane particles.

PubMed

Massa, P T; Szuchet, S; Mugnaini, E

1984-12-01

Oligodendrocytes were isolated from lamb brain. Freshly isolated cells and cultured cells, either 1- to 4-day-old unattached or 1- to 5-week-old attached, were examined by thin section and freeze-fracture electron microscopy. Freeze-fracture of freshly isolated oligodendrocytes showed globular and elongated intramembrane particles similar to those previously described in oligodendrocytes in situ. Enrichment of these particles was seen at sites of inter-oligodendrocyte contact. Numerous gap junctions and scattered linear tight junctional arrays were apparent. Gap junctions were connected to blebs of astrocytic plasma membrane sheared off during isolation, whereas tight junctions were facing extracellular space or blebs of oligodendrocytic plasma membrane. Thin sections of cultured, unattached oligodendrocytes showed rounded cell bodies touching one another at points without forming specialized cell junctions. Cells plated on polylysine-coated aclar dishes attached, emanated numerous, pleomorphic processes, and expressed galactocerebroside and myelin basic protein, characteristic markers for oligodendrocytes. Thin sections showed typical oligodendrocyte ultrastructure but also intermediate filaments not present in unattached cultures. Freeze-fracture showed intramembrane particles similar to but more numerous, and with a different fracture face repartition, than those seen in oligodendrocytes, freshly isolated or in situ. Gap junctions were small and rare. Apposed oligodendrocyte plasma membrane formed linear tight junctions which became more numerous with time in culture. Thus, cultured oligodendrocytes isolated from ovine brains develop and maintain features characteristic of mature oligodendrocytes in situ and can be used to explore formation and maintenance of tight junctions and possibly other classes of cell-cell interactions important in the process of myelination.

Cadmium disorganises the scaffolding of gap and tight junction proteins in the hepatic cell line WIF B9.

PubMed

Boucherie, Sylviane; Decaens, Catherine; Verbavatz, Jean-Marc; Grosse, Brigitte; Erard, Marie; Merola, Fabienne; Cassio, Doris; Combettes, Laurent

2013-12-01

Hepatocytes, which perform the main functions of the liver, are particularly vulnerable to toxic agents such as cadmium, an environmental pollutant. To identify the molecular targets for cadmium in hepatocytes, we have studied the effects of CdCl2 on the hybrid cell line WIF-B9 that exhibits stable structural and functional hepatocytic polarity. We showed that the toxicity of CdCl2 (1 µM, 24 h) resulted in a reduction in direct intercellular communication (via gap junctions) and in an increase in paracellular permeability (decrease in the sealing of tight junctions). These effects were not related to changes in the expression of the key proteins involved, Cx32 and claudin 2, the first being constitutive of gap junctions and the second of tight junctions in this cell line. Using immunofluorescence experiments, we observed a change in the location of Cx32 and claudin 2: these two proteins were less often found in the tight junction network that closes the bile canaliculi (BC). In control cells, 'Proximity Ligation Assay' (PLA Duolink®) has confirmed in situ that molecules of claudin 2 and Cx32 are very close to each other at the BC (probably less than 16 nm). This was no longer the case after treatment with CdCl2 . Localisation of occludin and Cx32 relative to each other was not modified by CdCl2 , but CdCl2 increased the PLA signal between molecules of JAM-A and Cx32. Finally, examination of freeze-fracture replicas obtained from cultures treated with CdCl2 showed the disruption of the network of tight junctions and the depletion or the disintegration of the junctional plaques associated with tight junctions. This study demonstrates in situ the changes induced by cadmium on the organisation of cell-cell junctions and points out the importance of the association Cx32/claudin 2 for the maintenance of normal hepatocyte functions. © 2013 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma

PubMed Central

Naz, Anam; Obaid, Ayesha; Awan, Faryal M.; Ikram, Aqsa; Ahmad, Jamil; Ali, Amjad

2017-01-01

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells. PMID:28932213

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma.

PubMed

Naz, Anam; Obaid, Ayesha; Awan, Faryal M; Ikram, Aqsa; Ahmad, Jamil; Ali, Amjad

2017-01-01

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells.

Disruptions of occludin and claudin-5 in brain endothelial cells in vitro and in brains of mice with acute liver failure

PubMed Central

Chen, Florence; Ohashi, Norifumi; Li, Wensheng; Eckman, Christopher; Nguyen, Justin H.

2010-01-01

Brain edema in acute liver failure (ALF) remains lethal. The role of vasogenic mechanisms of brain edema has not been explored. We previously demonstrated that matrix metalloproteinase-9 (MMP-9) contributes to the pathogenesis of brain edema. Here, we show that MMP-9 mediates disruptions in tight junction proteins in vitro and in brains of mice with ALF. We transfected murine brain endothelial cells with MMP-9 cDNA using pc DNA3.1 (+)/Myc-His A expression vector. Tissue inhibitor of matrix metalloproteinases (TIMP-1) cDNA transfection or GM6001 was used to inhibit MMP-9. ALF was induced in mice with azoxymethane. Endogenous overexpression of MMP-9 in brain endothelial cells resulted in significant degradation of tight junction proteins occludin and claudin-5. The alterations in tight junction proteins correlated with increased permeability to FITC-dextran molecules. The degradation of tight junction proteins and the increased permeability were reversed by TIMP-1 and GM6001. Similar results were found when MMP-9 was exogenously added to brain EC. We also found that tight junction proteins degradation was reversed with GM6001 in brains of mice with ALF. Conclusions Tight junction proteins are significantly perturbed in brains of mice with ALF. These data corroborate the important role of MMP-9 in the vasogenic mechanism of brain edema in ALF. PMID:19821483

Proteomic and cellular localisation studies suggest non-tight junction cytoplasmic and nuclear roles for occludin in astrocytes.

PubMed

Morgan, Sarah V; Garwood, Claire J; Jennings, Luke; Simpson, Julie E; Castelli, Lydia M; Heath, Paul R; Mihaylov, Simeon R; Vaquéz-Villaseñor, Irina; Minshull, Thomas C; Ince, Paul G; Dickman, Mark J; Hautbergue, Guillaume M; Wharton, Stephen B

2018-05-08

Occludin is a component of tight junctions, which are essential structural components of the blood-brain barrier. However, occludin is expressed in cells without tight junctions, implying additional functions. We determined the expression and localisation of occludin in astrocytes in cell culture and in human brain tissue, and sought novel binding partners using a proteomic approach. Expression was investigated by immunocytochemistry and immunoblotting in the 1321N1 astrocytoma cell line and ScienCell human primary astrocytes, and by immunohistochemistry in human autopsy brain tissue. Recombinant N- and C-terminal occludin was used to pull-down proteins from 1321N1 cell lysates and protein-binding partners identified by mass spectrometry analysis. Occludin was expressed in both the cytoplasm and nucleus of astrocytes in vitro and in vivo. Mass spectrometry identified binding to nuclear and cytoplasmic proteins, particularly those related to RNA metabolism and nuclear function. Occludin is expressed in several subcellular compartments of brain cell-types that do not form tight junctions and the expression patterns in cell culture reflect those in human brain tissue, indicating they are suitable model systems. Proteomic analysis suggests that occludin has novel functions in neuroepithelial cells that are unrelated to tight junction formation. Further research will establish the roles of these functions in both cellular physiology and in disease states. © 2018 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Epithelial-mesenchymal transition abolishes the susceptibility of polarized epithelial cell lines to measles virus.

PubMed

Shirogane, Yuta; Takeda, Makoto; Tahara, Maino; Ikegame, Satoshi; Nakamura, Takanori; Yanagi, Yusuke

2010-07-02

Measles virus (MV), an enveloped negative-strand RNA virus, remains a major cause of morbidity and mortality in developing countries. MV predominantly infects immune cells by using signaling lymphocyte activation molecule (SLAM; also called CD150) as a receptor, but it also infects polarized epithelial cells, forming tight junctions in a SLAM-independent manner. Although the ability of MV to infect polarized epithelial cells is thought to be important for its transmission, the epithelial cell receptor for MV has not been identified. A transcriptional repressor, Snail, induces epithelial-mesenchymal transition (EMT), in which epithelial cells lose epithelial cell phenotypes, such as adherens and tight junctions. In this study, EMT was induced by expressing Snail in a lung adenocarcinoma cell line, II-18, which is highly susceptible to wild-type MV. Snail-expressing II-18 cells lost adherens and tight junctions. Microarray analysis confirmed the induction of EMT in II-18 cells and suggested a novel function of Snail in protein degradation and distribution. Importantly, wild-type MV no longer entered EMT-induced II-18 cells, suggesting that the epithelial cell receptor is down-regulated by the induction of EMT. Other polarized cell lines, NCI-H358 and HT-29, also lost susceptibility to wild-type MV when EMT was induced. However, the complete formation of tight junctions rather reduced MV entry into HT-29 cells. Taken together, these data suggest that the unidentified epithelial cell receptor for MV is involved in the formation of epithelial intercellular junctions.

Exploiting the Gastric Epithelial Barrier: Helicobacter pylori's Attack on Tight and Adherens Junctions.

PubMed

Backert, Steffen; Schmidt, Thomas P; Harrer, Aileen; Wessler, Silja

2017-01-01

Highly organized intercellular tight and adherens junctions are crucial structural components for establishing and maintenance of epithelial barrier functions, which control the microbiota and protect against intruding pathogens in humans. Alterations in these complexes represent key events in the development and progression of multiple infectious diseases as well as various cancers. The gastric pathogen Helicobacter pylori exerts an amazing set of strategies to manipulate these epithelial cell-to-cell junctions, which are implicated in changing cell polarity, migration and invasive growth as well as pro-inflammatory and proliferative responses. This chapter focuses on the H. pylori pathogenicity factors VacA, CagA, HtrA and urease, and how they can induce host cell signaling involved in altering cell-to-cell permeability. We propose a stepwise model for how H. pylori targets components of tight and adherens junctions in order to disrupt the gastric epithelial cell layer, giving fresh insights into the pathogenesis of this important bacterium.

Monocytic cell junction proteins serve important roles in atherosclerosis via the endoglin pathway

PubMed Central

Chen, Lina; Chen, Zhongliang; Ge, Menghua; Tang, Oushan; Cheng, Yinhong; Zhou, Haoliang; Shen, Yu; Qin, Fengming

2017-01-01

The formation of atherosclerosis is recognized to be caused by multiple factors including pathogenesis in monocytes during inflammation. The current study provided evidence that monocytic junctions were significantly altered in patients with atherosclerosis, which suggested an association between cell junctions and atherosclerosis. Claudin-1, occludin-1 and ZO-1 were significantly enhanced in atherosclerosis, indicating that the tight junction pathway was activated during the pathogenesis of atherosclerosis. In addition, the gene expression of 5 connexin members involved in the gap junction pathway were quantified, indicating that connexin 43 and 46 were significantly up-regulated in atherosclerosis. Furthermore, inflammatory factors including endoglin and SMAD were observed, suggesting that immune regulative factors were down-regulated in this pathway. Silicon-based analysis additionally identified that connexins and tight junctions were altered in association with monocytic inflammation regulations, endoglin pathway. The results imply that reduced expression of the immune regulation pathway in monocytes is correlated with the generation of gap junctions and tight junctions which serve important roles in atherosclerosis. PMID:28901429

Decursin inhibits VEGF-mediated inner blood-retinal barrier breakdown by suppression of VEGFR-2 activation.

PubMed

Kim, Jin Hyoung; Kim, Jeong Hun; Lee, You Mie; Ahn, Eun-Mi; Kim, Kyu-Won; Yu, Young Suk

2009-09-01

The blood-retinal barrier (BRB) is essential for the normal structural and functional integrity of the retina, whose breakdown could cause the serious vision loss. Vascular endothelial growth factor (VEGF), as a permeable factor, induces alteration of tight junction proteins to result in BRB breakdown. Herein, we demonstrated that decursin inhibits VEGF-mediated inner BRB breakdown through suppression of VEGFR-2 signaling pathway. In retinal endothelial cells, decursin inhibited VEGF-mediated hyperpermeability. Decursin prevented VEGF-mediated loss of tight junction proteins including zonula occludens-1 (ZO-1), ZO-2, and occludin in retinal endothelial cells, which was also supported by restoration of tight junction proteins in intercellular junction. In addition, decursin significantly inhibited VEGF-mediated vascular leakage from retinal vessels, which was accompanied by prevention of loss of tight junction proteins in retinal vessels. Decursin significantly suppressed VEGF-induced VEGFR-2 phosphrylation that consequently led to inhibition of extracellular signal-regulated kinase (ERK) 1/2 activation. Moreover, decursin induced no cytotoxicity to retinal endothelial cells and no retinal toxicity under therapeutic concentrations. Therefore, our results suggest that decursin prevents VEGF-mediated BRB breakdown through blocking of loss of tight junction proteins, which might be regulated by suppression of VEGFR-2 activation. As a novel inhibitor to BRB breakdown, decursin could be applied to variable retinopathies with BRB breakdown.

Presence of claudins mRNA in the brain. Selective modulation of expression by kindling epilepsy.

PubMed

Lamas, Mónica; González-Mariscal, Lorenza; Gutiérrez, Rafael

2002-08-15

In the central nervous system, the junctional types that establish and maintain tissue architecture include gap junctions, for cytoplasmic connectivity, and tight junctions, for paracellular and/or cell polarity barriers. Connexins are the integral membrane proteins of gap junctions, whereas occludin and members of the multigene family of claudins form tight junctions. In the brain, there are no transendothelial pathways, as continuous tight junctions are present between the endothelial cells. Thus, they provide a continuous cellular barrier between the blood and the insterstitial fluid. However, several brain pathologies, including epilepsy, are known to alter the permeability of the blood-brain barrier and to cause edema. Therefore, since claudins, as constitutive proteins of tight junctions are likely candidates for modulation under pathological states, we explored their normal pattern of expression in the brain and its modulation by seizures. We found that several members of this family are normally expressed in the hippocampus and cortex. Interestingly, claudin-7 is expressed in the hippocampus but not in the cortex. On the other hand, the expression of claudin-8 is selectively down-regulated in the hippocampus as kindling evolves. These results link for the first time the modulation of expression of a tight junction protein to abnormal neuronal synchronization that could probably be reflected in permeability changes of the blood-brain barrier or edema.

House Dust Mite Der p 1 Effects on Sinonasal Epithelial Tight Junctions

PubMed Central

Henriquez, Oswaldo A.; Beste, Kyle Den; Hoddeson, Elizabeth K.; Parkos, Charles A.; Nusrat, Asma; Wise, Sarah K.

2013-01-01

Background Epithelial permeability is highly dependent upon the integrity of tight junctions, cell-cell adhesion complexes located at the apical aspect of the lateral membrane of polarized epithelial cells. We hypothesize that sinonasal epithelial exposure to Der p 1 house dust mite antigen decreases expression of tight junction proteins (TJPs), representing a potential mechanism for increased permeability and presentation of antigens across the sinonasal epithelial layer. Methods Confluent cultured primary human sinonasal epithelial cells were exposed to recombinant Der p 1 antigen versus control, and transepithelial resistance measurements were performed over 24 hours. Antibody staining for a panel of tight junction proteins was examined with immunofluorescence/confocal microscopy and Western blotting. Tissue for these experiments was obtained from 4 patients total. Results Der p 1 exposed sinonasal cells showed a marked decrease in transepithelial resistance when compared to control cells. In addition, results of Western immunoblot and immunofluorescent labeling demonstrated decreased expression of TJPs claudin-1 and junction adhesion molecule-A (JAM-A) in Der p 1 exposed cultured sinonasal cells versus controls. Conclusion Der p 1 antigen exposure decreases sinonasal epithelium TJP expression, most notably seen in JAM-A and claudin-1 in these preliminary experiments. This decreased TJP expression likely contributes to increased epithelial permeability and represents a potential mechanism for transepithelial antigen exposure in allergic rhinitis. PMID:23592402

CAVEOLIN-1 REGULATES HIV-1 TAT-INDUCED ALTERATIONS OF TIGHT JUNCTION PROTEIN EXPRESSION VIA MODULATION OF THE RAS SIGNALING

PubMed Central

Zhong, Yu; Smart, Eric J.; Weksler, Babette; Couraud, Pierre-Olivier; Hennig, Bernhard; Toborek, Michal

2009-01-01

The blood-brain barrier (BBB) is the critical structure for preventing HIV trafficking into the brain. Specific HIV proteins, such as Tat protein, can contribute to the dysfunction of tight junctions at the BBB and HIV entry into the brain. Tat is released by HIV-1 infected cells and can interact with a variety of cell surface receptors activating several signal transduction pathways, including those localized in caveolae. The present study focused on the mechanisms of Tat-induced caveolae-associated Ras signaling at the level of the BBB. Treatment with Tat activated the Ras pathway in human brain microvascular endothelial cells (HBMEC). However, caveolin-1 silencing markedly attenuated these effects. Because the integrity of the brain endothelium is regulated by intercellular tight junctions, these structural elements of the BBB were also evaluated in the present study. Exposure to Tat diminished the expression of several tight junction proteins, namely, occludin, zonula occludens (ZO)-1, and ZO-2 in the caveolar fraction of HBMEC. These effects were effectively protected by pharmacological inhibition of the Ras signaling and by silencing of caveolin-1. The present data indicate the importance of caveolae-associated signaling in the disruption of tight junctions upon Tat exposure. They also demonstrate that caveolin-1 may constitute an early and critical modulator that controls signaling pathways leading to the disruption of tight junction proteins. Thus, caveolin-1 may provide an effective target to protect against Tat-induced HBMEC dysfunction and the disruption of the BBB in HIV-1-infected patients. PMID:18667611

Progressive Hearing Loss in Mice Carrying a Mutation in Usp53

PubMed Central

Kazmierczak, Marcin; Harris, Suzan L.; Kazmierczak, Piotr; Shah, Prahar; Starovoytov, Valentin; Ohlemiller, Kevin K.

2015-01-01

Disordered protein ubiquitination has been linked to neurodegenerative disease, yet its role in inner ear homeostasis and hearing loss is essentially unknown. Here we show that progressive hearing loss in the ethylnitrosourea-generated mambo mouse line is caused by a mutation in Usp53, a member of the deubiquitinating enzyme family. USP53 contains a catalytically inactive ubiquitin-specific protease domain and is expressed in cochlear hair cells and a subset of supporting cells. Although hair cell differentiation is unaffected in mambo mice, outer hair cells degenerate rapidly after the first postnatal week. USP53 colocalizes and interacts with the tight junction scaffolding proteins TJP1 and TJP2 in polarized epithelial cells, suggesting that USP53 is part of the tight junction complex. The barrier properties of tight junctions of the stria vascularis appeared intact in a biotin tracer assay, but the endocochlear potential is reduced in adult mambo mice. Hair cell degeneration in mambo mice precedes endocochlear potential decline and is rescued in cochlear organotypic cultures in low potassium milieu, indicating that hair cell loss is triggered by extracellular factors. Remarkably, heterozygous mambo mice show increased susceptibility to noise injury at high frequencies. We conclude that USP53 is a novel tight junction-associated protein that is essential for the survival of auditory hair cells and normal hearing in mice, possibly by modulating the barrier properties and mechanical stability of tight junctions. SIGNIFICANCE STATEMENT Hereditary hearing loss is extremely prevalent in the human population, but many genes linked to hearing loss remain to be discovered. Forward genetics screens in mice have facilitated the identification of genes involved in sensory perception and provided valuable animal models for hearing loss in humans. This involves introducing random mutations in mice, screening the mice for hearing defects, and mapping the causative mutation. Here, we have identified a mutation in the Usp53 gene that causes progressive hearing loss in the mambo mouse line. We demonstrate that USP53 is a catalytically inactive deubiquitinating enzyme and a novel component of tight junctions that is necessary for sensory hair cell survival and inner ear homeostasis. PMID:26609154

Tight junction physiology of pleural mesothelium

PubMed Central

Markov, Alexander G.; Amasheh, Salah

2014-01-01

Pleura consists of visceral and parietal cell layers, producing a fluid, which is necessary for lubrication of the pleural space. Function of both mesothelial cell layers is necessary for the regulation of a constant pleural fluid volume and composition to facilitate lung movement during breathing. Recent studies have demonstrated that pleural mesothelial cells show a distinct expression pattern of tight junction proteins which are known to ubiquitously determine paracellular permeability. Most tight junction proteins provide a sealing function to epithelia, but some have been shown to have a paracellular channel function or ambiguous properties. Here we provide an in-depth review of the current knowledge concerning specific functional contribution of these proteins determining transport and barrier function of pleural mesothelium. PMID:25009499

Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells With Tight Junction Modulator Peptides.

PubMed

Bocsik, Alexandra; Walter, Fruzsina R; Gyebrovszki, Andrea; Fülöp, Lívia; Blasig, Ingolf; Dabrowski, Sebastian; Ötvös, Ferenc; Tóth, András; Rákhely, Gábor; Veszelka, Szilvia; Vastag, Monika; Szabó-Révész, Piroska; Deli, Mária A

2016-02-01

The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, β-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

TNF-α Signals Through PKCζ/NF-κB to Alter the Tight Junction Complex and Increase Retinal Endothelial Cell Permeability

PubMed Central

Aveleira, Célia A.; Lin, Cheng-Mao; Abcouwer, Steven F.; Ambrósio, António F.; Antonetti, David A.

2010-01-01

OBJECTIVE Tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) are elevated in the vitreous of diabetic patients and in retinas of diabetic rats associated with increased retinal vascular permeability. However, the molecular mechanisms underlying retinal vascular permeability induced by these cytokines are poorly understood. In this study, the effects of IL-1β and TNF-α on retinal endothelial cell permeability were compared and the molecular mechanisms by which TNF-α increases cell permeability were elucidated. RESEARCH DESIGN AND METHODS Cytokine-induced retinal vascular permeability was measured in bovine retinal endothelial cells (BRECs) and rat retinas. Western blotting, quantitative real-time PCR, and immunocytochemistry were performed to determine tight junction protein expression and localization. RESULTS IL-1β and TNF-α increased BREC permeability, and TNF-α was more potent. TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins. Dexamethasone prevented TNF-α–induced cell permeability through glucocorticoid receptor transactivation and nuclear factor-kappaB (NF-κB) transrepression. Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α–stimulated permeability. Finally, inhibiting protein kinase C zeta (PKCζ) using both a peptide and a novel chemical inhibitor reduced NF-κB activation and completely prevented the alterations in the tight junction complex and cell permeability induced by TNF-α in cell culture and rat retinas. CONCLUSIONS These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy. PMID:20693346

Holding Tight: Cell Junctions and Cancer Spread.

PubMed

Knights, Alexander J; Funnell, Alister P W; Crossley, Merlin; Pearson, Richard C M

2012-01-01

Cell junctions are sites of intercellular adhesion that maintain the integrity of epithelial tissue and regulate signalling between cells. These adhesive junctions are comprised of protein complexes that serve to establish an intercellular cytoskeletal network for anchoring cells, in addition to regulating cell polarity, molecular transport and communication. The expression of cell adhesion molecules is tightly controlled and their downregulation is essential for epithelial-mesenchymal transition (EMT), a process that facilitates the generation of morphologically and functionally diverse cell types during embryogenesis. The characteristics of EMT are a loss of cell adhesion and increased cellular mobility. Hence, in addition to its normal role in development, dysregulated EMT has been linked to cancer progression and metastasis, the process whereby primary tumors migrate to invasive secondary sites in the body. This paper will review the current understanding of cell junctions and their role in cancer, with reference to the abnormal regulation of junction protein genes. The potential use of cell junction molecules as diagnostic and prognostic markers will also be discussed, as well as possible therapies for adhesive dysregulation.

The role of apical cell-cell junctions and associated cytoskeleton in mechanotransduction.

PubMed

Sluysmans, Sophie; Vasileva, Ekaterina; Spadaro, Domenica; Shah, Jimit; Rouaud, Florian; Citi, Sandra

2017-04-01

Tissues of multicellular organisms are characterised by several types of specialised cell-cell junctions. In vertebrate epithelia and endothelia, tight and adherens junctions (AJ) play critical roles in barrier and adhesion functions, and are connected to the actin and microtubule cytoskeletons. The interaction between junctions and the cytoskeleton is crucial for tissue development and physiology, and is involved in the molecular mechanisms governing cell shape, motility, growth and signalling. The machineries which functionally connect tight and AJ to the cytoskeleton comprise proteins which either bind directly to cytoskeletal filaments, or function as adaptors for regulators of the assembly and function of the cytoskeleton. In the last two decades, specific cytoskeleton-associated junctional molecules have been implicated in mechanotransduction, revealing the existence of multimolecular complexes that can sense mechanical cues and translate them into adaptation to tensile forces and biochemical signals. Here, we summarise the current knowledge about the machineries that link tight and AJ to actin filaments and microtubules, and the molecular basis for mechanotransduction at epithelial and endothelial AJ. © 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Irsogladine maleate regulates gap junctional intercellular communication-dependent epithelial barrier in human nasal epithelial cells.

PubMed

Miyata, Ryo; Nomura, Kazuaki; Kakuki, Takuya; Takano, Ken-Ichi; Kohno, Takayuki; Konno, Takumi; Sawada, Norimasa; Himi, Tetsuo; Kojima, Takashi

2015-04-01

The airway epithelium of the human nasal mucosa acts as the first physical barrier that protects against inhaled substances and pathogens. Irsogladine maleate (IM) is an enhancer of gastric mucosal protective factors via upregulation of gap junctional intercellular communication (GJIC). GJIC is thought to participate in the formation of functional tight junctions. However, the effects of IM on GJIC and the epithelial barrier in human nasal epithelial cells (HNECs) remain unknown. To investigate the effects of IM on GJIC and the tight junctional barrier in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with IM and the GJIC inhibitors oleamide and 18β-GA. Some cells were pretreated with IM before treatment with TLR3 ligand poly(I:C) to examine whether IM prevented the changes via TLR3-mediated signal pathways. In hTERT-HNECs, GJIC blockers reduced the expression of tight junction molecules claudin-1, -4, -7, occludin, tricellulin, and JAM-A. IM induced GJIC activity and enhanced the expression of claudin-1, -4, and JAM-A at the protein and mRNA levels with an increase of barrier function. GJIC blockers prevented the increase of the tight junction proteins induced by IM. Furthermore, IM prevented the reduction of JAM-A but not induction of IL-8 and TNF-α induced by poly(I:C). In conclusion, IM can maintain the GJIC-dependent tight junctional barrier via regulation of GJIC in upper airway nasal epithelium. Therefore, it is possible that IM may be useful as a nasal spray to prevent the disruption of the epithelial barrier by viral infections and exposure to allergens in human nasal mucosa.

Modulation of Intestinal Paracellular Transport by Bacterial Pathogens.

PubMed

Roxas, Jennifer Lising; Viswanathan, V K

2018-03-25

The passive and regulated movement of ions, solutes, and water via spaces between cells of the epithelial monolayer plays a critical role in the normal intestinal functioning. This paracellular pathway displays a high level of structural and functional specialization, with the membrane-spanning complexes of the tight junctions, adherens junctions, and desmosomes ensuring its integrity. Tight junction proteins, like occludin, tricellulin, and the claudin family isoforms, play prominent roles as barriers to unrestricted paracellular transport. The past decade has witnessed major advances in our understanding of the architecture and function of epithelial tight junctions. While it has been long appreciated that microbes, notably bacterial and viral pathogens, target and disrupt junctional complexes and alter paracellular permeability, the precise mechanisms remain to be defined. Notably, renewed efforts will be required to interpret the available data on pathogen-mediated barrier disruption in the context of the most recent findings on tight junction structure and function. While much of the focus has been on pathogen-induced dysregulation of junctional complexes, commensal microbiota and their products may influence paracellular permeability and contribute to the normal physiology of the gut. Finally, microbes and their products have become important tools in exploring host systems, including the junctional properties of epithelial cells. © 2018 American Physiological Society. Compr Physiol 8:823-842, 2018. Copyright © 2018 American Physiological Society. All rights reserved.

Bile duct epithelial tight junctions and barrier function

PubMed Central

Rao, R.K.; Samak, G.

2013-01-01

Bile ducts play a crucial role in the formation and secretion of bile as well as excretion of circulating xenobiotic substances. In addition to its secretory and excretory functions, bile duct epithelium plays an important role in the formation of a barrier to the diffusion of toxic substances from bile into the hepatic interstitial tissue. Disruption of barrier function and toxic injury to liver cells appear to be involved in the pathogenesis of a variety of liver diseases such as primary sclerosing cholangitis, primary biliary cirrhosis and cholangiocarcinoma. Although the investigations into understanding the structure and regulation of tight junctions in gut, renal and endothelial tissues have expanded rapidly, very little is known about the structure and regulation of tight junctions in the bile duct epithelium. In this article we summarize the current understanding of physiology and pathophysiology of bile duct epithelium, the structure and regulation of tight junctions in canaliculi and bile duct epithelia and different mechanisms involved in the regulation of disruption and protection of bile duct epithelial tight junctions. This article will make a case for the need of future investigations toward our understanding of molecular organization and regulation of canalicular and bile duct epithelial tight junctions. PMID:24665411

A Homozygous Mutation in the Tight-Junction Protein JAM3 Causes Hemorrhagic Destruction of the Brain, Subependymal Calcification, and Congenital Cataracts

PubMed Central

Mochida, Ganeshwaran H.; Ganesh, Vijay S.; Felie, Jillian M.; Gleason, Danielle; Hill, R. Sean; Clapham, Katie Rose; Rakiec, Daniel; Tan, Wen-Hann; Akawi, Nadia; Al-Saffar, Muna; Partlow, Jennifer N.; Tinschert, Sigrid; Barkovich, A. James; Ali, Bassam; Al-Gazali, Lihadh; Walsh, Christopher A.

2010-01-01

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans. PMID:21109224

miR-Let7A Controls the Cell Death and Tight Junction Density of Brain Endothelial Cells under High Glucose Condition

PubMed Central

Song, Juhyun; Yoon, So Ra

2017-01-01

Hyperglycemia-induced stress in the brain of patients with diabetes triggers the disruption of blood-brain barrier (BBB), leading to diverse neurological diseases including stroke and dementia. Recently, the role of microRNA becomes an interest in the research for deciphering the mechanism of brain endothelial cell damage under hyperglycemia. Therefore, we investigated whether mircoRNA Let7A (miR-Let7A) controls the damage of brain endothelial (bEnd.3) cells against high glucose condition. Cell viability, cell death marker expressions (p-53, Bax, and cleaved poly ADP-ribose polymerase), the loss of tight junction proteins (ZO-1 and claudin-5), proinflammatory response (interleukin-6, tumor necrosis factor-α), inducible nitric oxide synthase, and nitrite production were confirmed using MTT, reverse transcription-PCR, quantitative-PCR, Western blotting, immunofluorescence, and Griess reagent assay. miR-Let7A overexpression significantly prevented cell death and loss of tight junction proteins and attenuated proinflammatory response and nitrite production in the bEnd.3 cells under high glucose condition. Taken together, we suggest that miR-Let7A may attenuate brain endothelial cell damage by controlling cell death signaling, loss of tight junction proteins, and proinflammatory response against high glucose stress. In the future, the manipulation of miR-Let7A may be a novel solution in controlling BBB disruption which leads to the central nervous system diseases. PMID:28680530

miR-Let7A Controls the Cell Death and Tight Junction Density of Brain Endothelial Cells under High Glucose Condition.

PubMed

Song, Juhyun; Yoon, So Ra; Kim, Oh Yoen

2017-01-01

Hyperglycemia-induced stress in the brain of patients with diabetes triggers the disruption of blood-brain barrier (BBB), leading to diverse neurological diseases including stroke and dementia. Recently, the role of microRNA becomes an interest in the research for deciphering the mechanism of brain endothelial cell damage under hyperglycemia. Therefore, we investigated whether mircoRNA Let7A (miR-Let7A) controls the damage of brain endothelial (bEnd.3) cells against high glucose condition. Cell viability, cell death marker expressions (p-53, Bax, and cleaved poly ADP-ribose polymerase), the loss of tight junction proteins (ZO-1 and claudin-5), proinflammatory response (interleukin-6, tumor necrosis factor- α ), inducible nitric oxide synthase, and nitrite production were confirmed using MTT, reverse transcription-PCR, quantitative-PCR, Western blotting, immunofluorescence, and Griess reagent assay. miR-Let7A overexpression significantly prevented cell death and loss of tight junction proteins and attenuated proinflammatory response and nitrite production in the bEnd.3 cells under high glucose condition. Taken together, we suggest that miR-Let7A may attenuate brain endothelial cell damage by controlling cell death signaling, loss of tight junction proteins, and proinflammatory response against high glucose stress. In the future, the manipulation of miR-Let7A may be a novel solution in controlling BBB disruption which leads to the central nervous system diseases.

The role of JAM-A in inflammatory bowel disease: unrevealing the ties that bind.

PubMed

Vetrano, Stefania; Danese, Silvio

2009-05-01

Tight junctions (TJ) are junctional proteins whose function is to maintain an intact intestinal epithelial barrier and regulate the paracellular movement of water and solutes. Altered TJ structure and epithelial permeability are observed in inflammatory bowel disease and seem to have an important role in the pathogenesis of these diseases. Junctional adhesion molecule-A (JAM-A) is a protein expressed at tight junctions of epithelial and endothelial cells, as well as on circulating leukocytes. Its function at tight junctions appears to be crucial as an extracellular adhesive molecule in the direct regulation of intestinal barrier function. This review focuses on the role of JAM-A in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function.

Dlg3 Trafficking and Apical Tight Junction Formation Is Regulated by Nedd4 and Nedd4-2 E3 Ubiquitin Ligases

PubMed Central

Van Campenhout, Claude A.; Eitelhuber, Andrea; Gloeckner, Christian J.; Giallonardo, Patrizia; Gegg, Moritz; Oller, Heide; Grant, Seth G.N.; Krappmann, Daniel; Ueffing, Marius; Lickert, Heiko

2011-01-01

Summary The Drosophila Discs large (Dlg) scaffolding protein acts as a tumor suppressor regulating basolateral epithelial polarity and proliferation. In mammals, four Dlg homologs have been identified; however, their functions in cell polarity remain poorly understood. Here, we demonstrate that the X-linked mental retardation gene product Dlg3 contributes to apical-basal polarity and epithelial junction formation in mouse organizer tissues, as well as to planar cell polarity in the inner ear. We purified complexes associated with Dlg3 in polarized epithelial cells, including proteins regulating directed trafficking and tight junction formation. Remarkably, of the four Dlg family members, Dlg3 exerts a distinct function by recruiting the ubiquitin ligases Nedd4 and Nedd4-2 through its PPxY motifs. We found that these interactions are required for Dlg3 monoubiquitination, apical membrane recruitment, and tight junction consolidation. Our findings reveal an unexpected evolutionary diversification of the vertebrate Dlg family in basolateral epithelium formation. PMID:21920314

The tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tkachuk, Natalia; Tkachuk, Sergey; Patecki, Margret

2011-07-08

Highlights: {yields} The tight junction protein ZO-2 associates with Jak1 in vascular smooth muscle cells via ZO-2 N-terminal fragment. {yields} Jak1 mediates ZO-2 tyrosine phosphorylation and ZO-2 localization to the sites of homotypic intercellular contacts. {yields} The urokinase receptor uPAR regulates ZO-2/Jak1 functional association. {yields} The ZO-2/Jak1/uPAR signaling complex is required for vascular smooth muscle cells functional network formation. -- Abstract: Recent evidence points to a multifunctional role of ZO-2, the tight junction protein of the MAGUK (membrane-associated guanylate kinase-like) family. Though ZO-2 has been found in cell types lacking tight junction structures, such as vascular smooth muscle cells (VSMC),more » little is known about ZO-2 function in these cells. We provide evidence that ZO-2 mediates specific homotypic cell-to-cell contacts between VSMC. Using mass spectrometry we found that ZO-2 is associated with the non-receptor tyrosine kinase Jak1. By generating specific ZO-2 constructs we further found that the N-terminal fragment of ZO-2 molecule is responsible for this interaction. Adenovirus-based expression of Jak1 inactive mutant demonstrated that Jak1 mediates ZO-2 tyrosine phosphorylation. By means of RNA silencing, expression of Jak1 mutant form and fluorescently labeled ZO-2 fusion protein we further specified that active Jak1, but not Jak1 inactive mutant, mediates ZO-2 localization to the sites of intercellular contacts. We identified the urokinase receptor uPAR as a pre-requisite for these cellular events. Functional requirement of the revealed signaling complex for VSMC network formation was confirmed in experiments using Matrigel and in contraction assay. Our findings imply involvement of the ZO-2 tight junction independent signaling complex containing Jak1 and uPAR in VSMC intercellular communications. This mechanism may contribute to vascular remodeling in occlusive cardiovascular diseases and in arteriogenesis.« less

Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin

NASA Astrophysics Data System (ADS)

Gordon, Geoffrey; Lo, Chun-Min

2007-03-01

Both in vitro and animal studies in breast, prostate, and ovarian cancers have shown that clostridium perfringens enterotoxin (CPE), which binds to CLDN4, may have an important therapeutic benefit, as it is rapidly cytotoxic in tissues overexpressing CLDN4. This study sought to evaluate the ability of C-terminal clostridium perfringens enterotoxin (C-CPE), a CLDN4-targetting molecule, to disrupt tight junction barrier function. Electric cell-substrate impedance sensing (ECIS) was used to measure both junctional resistance and average cell-substrate separation of ovarian cancer cell lines after exposure to C-CPE. A total of 14 ovarian cancer cell lines were used, and included cell lines derived from serous, mucinous, and clear cells. Our results showed that junctional resistance increases as CLDN4 expression increases. In addition, C-CPE is non-cytotoxic in ovarian cancer cells expressing CLDN4. However, exposure to C-CPE results in a significant (p<0.05) dose- and CLDN4-dependent decrease in junctional resistance and an increase in cell-substrate separation. Treatment of ovarian cancer cell lines with C-CPE disrupts tight junction barrier function.

[The effect of 18β-glycyrrhetinic acid on tight junctions of the nasal mucosa epithelial cells in rat models with allergic rhinitis].

PubMed

Ma, Yi; Gui, Yan; Wang, Youhu; Xi, Kehu; Chen, Xiaowan; Zhang, Fuhong; Ma, Chunxia; Hong, Hao; Liu, Xiangyi; Jiang, Ying; Dong, Ming; Yang, Guijun; Zhang, Xiaobing

2014-10-01

To observe 18β-glycyrrhetinic acid (GA) impact on ultrastructure of tight junctions (TJs) of nasal mucosa epithelial cells in rats models of allergic rhinitis (AR). Ninety-six Wistar rats were randomly divided into control group, model group, loratadine group, and 18β-glycyrrhetinic acid group, and each group had 24 rats. Ovalbumin was used to establish a rat AR model. The behavioral changes and the tight junctions of nasal epithelial were observed and compared in different groups after 2,4,6 and 10 weeks intervention. The length of TJs in allergic rhinitis model became shorter, electron-high-density plasma membrane became thicker, number of the integration loci reduced and gap of TJs widened or even ruptured. With the consistent effect of allergens,the changes of TJs in the model group aggravated gradually,and the changes of ultrastructure of TJs in 18β-glycyrrhetinic acid group was relieved apparently compared to model group and even were close to the control model with time. 18β-glycyrrhetinic acid can recover the ultrastructure of the tight junctions of AR rat nasal epithelial cells.

Tight junctions in cancer metastasis.

PubMed

Martin, Tracey A; Mason, Malcolm D; Jiang, Wen G

2011-01-01

Tight Junctions (TJ) are well known to function as a control for the paracellular diffusion of ions and certain molecules, it has however, become evident that the TJ has a vital role in maintaining cell to cell integrity. Loss of cohesion of the TJ structure can lead to invasion and ultimately to the metastasis of cancer cells. This review will discuss how modulation of expression of TJ molecules results in key changes in TJ barrier function leading to the progression of cancer and progression of metastasis.

Anti-mouse CD52 monoclonal antibody ameliorates intestinal epithelial barrier function in interleukin-10 knockout mice with spontaneous chronic colitis.

PubMed

Wang, Honggang; Dong, Jianning; Shi, Peiliang; Liu, Jianhui; Zuo, Lugen; Li, Yi; Gong, Jianfeng; Gu, Lili; Zhao, Jie; Zhang, Liang; Zhang, Wei; Zhu, Weiming; Li, Ning; Li, Jieshou

2015-02-01

Intestinal inflammation causes tight junction changes and death of epithelial cells, and plays an important role in the development of Crohn's disease (CD). CD52 monoclonal antibody (CD52 mAb) directly targets the cell surface CD52 and is effective in depleting mature lymphocytes by cytolytic effects in vivo, leading to long-lasting changes in adaptive immunity. The aim of this study was to investigate the therapeutic effect of CD52 mAb on epithelial barrier function in animal models of IBD. Interleukin-10 knockout mice (IL-10(-/-) ) of 16 weeks with established colitis were treated with CD52 mAb once a week for 2 weeks. Severity of colitis, CD4(+) lymphocytes and cytokines in the lamina propria, epithelial expression of tight junction proteins, morphology of tight junctions, tumour necrosis factor-α (TNF-α)/TNF receptor 2 (TNFR2) mRNA expression, myosin light chain kinase (MLCK) expression and activity, as well as epithelial apoptosis in proximal colon were measured at the end of the experiment. CD52 mAb treatment effectively attenuated colitis associated with decreased lamina propria CD4(+) lymphocytes and interferon-γ/IL-17 responses in colonic mucosa in IL-10(-/-) mice. After CD52 mAb treatment, attenuation of colonic permeability, increased epithelial expression and correct localization of tight junction proteins (occludin and zona occludens protein-1), as well as ameliorated tight junction morphology were observed in IL-10(-/-) mice. CD52 mAb treatment also effectively suppressed the epithelial apoptosis, mucosa TNF-α mRNA expression, epithelial expression of long MLCK, TNFR2 and phosphorylation of MLC. Our results indicated that anti-CD52 therapy may inhibit TNF-α/TNFR2-mediated epithelial apoptosis and MLCK-dependent tight junction permeability by depleting activated T cells in the gut mucosa. © 2014 John Wiley & Sons Ltd.

Paracellular tightness and the functional expression of efflux transporters P-gp and BCRP in bEnd3 cells.

PubMed

Yang, Shu; Jin, Hong; Zhao, Zhigang

2018-04-23

Objective The blood-brain barrier (BBB), regulating brain homeostasis and limiting the entry of most drugs, is characterized by intercellular tight junctions and the presence of transporters. In this study, the paracellular tightness and functional expression of efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) were evaluated in mouse brain immortalized cell line bEnd3 to prove it as a useful BBB-mimicking system for biological and pharmacological research. Methods The presence of P-gp, BCRP and tight junction proteins occludin, claudin-5 and ZO-1 were validated by RT-PCR and Western blot. The tightness of bEnd3 monolayers was evaluated by measuring the permeability of hydrophilic marker Lucifer yellow. The P-gp functionality was identified by intracellular uptake assay using Rhodamine 123 (R123) as P-gp substrate and verapamil as P-gp inhibitor. The BCRP functionality was identified by flow cytometric analysis of mitoxantrone accumulation and fluorescence microscopic analysis of Hoechst 33342 accumulation using Ko-143 as BCRP inhibitor. Results The bEnd3 cells demonstrated the expression of P-gp, BCRP and tight junction proteins occludin, claudin-5 and ZO-1 at mRNA and protein levels. The permeability coefficient of Lucifer yellow was 1.3 ± 0.13 × 10 -3  cm/min, indicating the moderate paracellular tightness barrier formed by bEnd3 cells. The verapamil induced a higher cellular uptake of Rhodamine 123, and Ko-143 significantly elevated cellular accumulation of mitoxantrone and Hoechst 33342, suggesting the P-gp and BCRP functionality shown by bEnd3 cells. Conclusions The bEnd3 cell line represents a useful in vitro tool for studying BBB characteristics and drug transport mechanisms at the BBB.

Regulation of tight junction permeability with switch-like speed.

PubMed

Beyenbach, Klaus W

2003-09-01

The case is made that tight junctions can undergo large reversible conductance changes in a matter of seconds and yet preserve their permselectivity. The diuretic peptide leucokinin transforms (renal) Malpighian tubules of the yellow fever mosquito from a moderately tight epithelium to a leaky epithelium by increasing the chloride-conductance of the paracellular shunt pathway. The nine-fold increase in the paracellular chloride-conductance brings about a non-selective stimulation of transepithelial sodium chloride and potassium chloride secretion, as expected from a conductance increase in the pathway taken by the counterion of sodium and potassium. The leucokinin signaling pathway consists in part of a receptor coupled G-protein, phospholipase C, inositol-1,4,5-trisphosphate, and increased intracellular calcium concentration that bring about the increase in the paracellular, tight junction chloride-conductance. As the conductance of the tight junction pathway increases it becomes more selective for the transepithelial passage of chloride. Epithelial cells in Malpighian tubules taper to tight junctions at their lateral edges exposing them directly to apical and serosal solutions. Furthermore, evolutionary pressures to excrete salt and water at high rates without the aid of glomerular filtration have led to powerful mechanisms of tubular secretion, capable of diuresis when the mosquito is challenged with the volume expansion of a blood meal. The tubular diuresis is mediated in part by increasing the paracellular chloride conductance. Thus, anatomical and physiological specializations in Malpighian tubules combine to yield the evidence for the dynamic hormonal regulation of the tight junction pathway.

Glucocorticoids induce transactivation of tight junction genes occludin and claudin-5 in retinal endothelial cells via a novel cis-element.

PubMed

Felinski, Edward A; Cox, Amy E; Phillips, Brett E; Antonetti, David A

2008-06-01

Tight junctions between vascular endothelial cells help to create the blood-brain and blood-retinal barriers. Breakdown of the retinal tight junction complex is problematic in several disease states including diabetic retinopathy. Glucocorticoids can restore and/or preserve the endothelial barrier to paracellular permeability, although the mechanism remains unclear. We show that glucocorticoid treatment of primary retinal endothelial cells increases content of the tight junction proteins occludin and claudin-5, co-incident with an increase in barrier properties of endothelial monolayers. The glucocorticoid receptor antagonist RU486 reverses both the glucocorticoid-stimulated increase in occludin content and the increase in barrier properties. Transcriptional activity from the human occludin and claudin-5 promoters increases in retinal endothelial cells upon glucocorticoid treatment, and is dependent on the glucocorticoid receptor (GR) as demonstrated by siRNA. Deletion analysis of the occludin promoter reveals a 205bp sequence responsible for the glucocorticoid response. However, this region does not possess a canonical glucocorticoid response element and does not bind to the GR in a chromatin immunoprecipitation (ChIP) assay. Mutational analysis of this region revealed a novel 40bp occludin enhancer element (OEE), containing two highly conserved regions of 10 and 13 base pairs, that is both necessary and sufficient for glucocorticoid-induced gene expression in retinal endothelial cells. These data suggest a novel mechanism for glucocorticoid induction of vascular endothelial barrier properties through increased occludin and claudin-5 gene expression.

GLUCOCORTICOIDS INDUCE TRANSACTIVATION OF TIGHT JUNCTION GENES OCCLUDIN AND CLAUDIN-5 IN RETINAL ENDOTHELIAL CELLS VIA A NOVEL CIS-ELEMENT

PubMed Central

Felinski, Edward A.; Cox, Amy E.; Phillips, Brett E.; Antonetti, David A.

2008-01-01

Tight junctions between vascular endothelial cells help to create the blood-brain and blood-retinal barriers. Breakdown of the retinal tight junction complex is problematic in several disease states including diabetic retinopathy. Glucocorticoids can restore and/or preserve the endothelial barrier to paracellular permeability, although the mechanism remains unclear. We show that glucocorticoid treatment of primary retinal endothelial cells increases content of the tight junction proteins occludin and claudin-5, co-incident with an increase in barrier properties of endothelial monolayers. The glucocorticoid receptor antagonist RU486 reverses both the glucocorticoid-stimulated increase in occludin content and the increase in barrier properties. Transcriptional activity from the human occludin and claudin-5 promoters increases in retinal endothelial cells upon glucocorticoid treatment, and is dependent on the glucocorticoid receptor (GR) as demonstrated by siRNA. Deletion analysis of the occludin promoter reveals a 205 bp sequence responsible for the glucocorticoid response. However, this region does not posses a canonical glucocorticoid response element and does not bind to the GR in a chromatin immunoprecipitation (ChIP) assay. Mutational analysis of this region revealed a novel 40 bp occludin enhancer element (OEE), containing two highly-conserved regions of 10 and 13 base pairs, that is both necessary and sufficient for glucocorticoid-induced gene expression in retinal endothelial cells. These data suggest a novel mechanism for glucocorticoid induction of vascular endothelial barrier properties through increased occludin and claudin-5 gene expression. PMID:18501346

A nuclear factor-κB signaling pathway via protein kinase C δ regulates replication of respiratory syncytial virus in polarized normal human nasal epithelial cells

PubMed Central

Masaki, Tomoyuki; Kojima, Takashi; Okabayashi, Tamaki; Ogasawara, Noriko; Ohkuni, Tsuyoshi; Obata, Kazufumi; Takasawa, Akira; Murata, Masaki; Tanaka, Satoshi; Hirakawa, Satoshi; Fuchimoto, Jun; Ninomiya, Takafumi; Fujii, Nobuhiro; Tsutsumi, Hiroyuki; Himi, Tetsuo; Sawada, Norimasa

2011-01-01

Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma, and severe lower respiratory tract disease in infants and young children. The airway epithelium, which has a well-developed barrier regulated by tight junctions, is the first line of defense during respiratory virus infection. In upper airway human nasal epithelial cells (HNECs), however, the primary site of RSV infection, the mechanisms of replication and budding of RSV, and the epithelial cell responses, including the tight junctional barrier, remain unknown. To investigate the detailed mechanisms of replication and budding of RSV in HNECs and the epithelial cell responses, we established an RSV-infected model using human telomerase reverse transcriptase–-transfected HNECs. We first found that the expression and barrier function of tight junction molecules claudin-4 and occludin were markedly induced together with production of proinflammatory cytokines interleukin 8 and tumor necrosis factor-α in HNECs after RSV infection, and the induction of tight junction molecules possibly contributed to budding of RSV. Furthermore, the replication and budding of RSV and the epithelial cell responses in HNECs were regulated via a protein kinase C δ/hypoxia-inducible factor-1α/nuclear factor-κB pathway. The control of this pathway in HNECs may be useful not only for prevention of replication and budding of RSV, but also in therapy for RSV-induced respiratory pathogenesis. PMID:21562222

Modulation of Tight Junction Structure and Function by Kinases and Phosphatases Targeting Occludin

PubMed Central

Dörfel, Max Johannes; Huber, Otmar

2012-01-01

Tight junctions (TJs) typically represent the most apical contacts in epithelial and endothelial cell layers where they play an essential role in the separation of extracellular or luminal spaces from underlying tissues in the body. Depending on the protein composition, TJs define the barrier characteristics and in addition maintain cell polarity. Two major families of integral membrane proteins form the typical TJ strand network, the tight junction-associated MARVEL protein (TAMP) family members occludin, tricellulin, and MarvelD3 as well as a specific set of claudins. Occludin was the first identified member of these tetraspanins and is now widely accepted as a regulator of TJ assembly and function. Therefore, occludin itself has to be tightly regulated. Phosphorylation of occludin appears to be of central importance in this context. Here we want to summarize current knowledge on the kinases and phosphatases directly modifying occludin, and their role in the regulation of TJ structure, function, and dynamics. PMID:22315516

Effects of Osmolality on Paracellular Transport in MDCK II Cells

PubMed Central

Hirai, Toyohiro; Furuse, Mikio

2016-01-01

Epithelia separate apical and basal compartments, and movement of substances via the paracellular pathway is regulated by tight junctions. Claudins are major constituents of tight junctions and involved in the regulation of tight junction permeability. On the other hand, the osmolality in the extracellular environment fluctuates in association with life activity. However, effects of osmotic changes on the permeaibility of claudins are poorly understood. Therefore, we investigated the effects of osmotic changes on the paracellular transport in MDCK II cells. Interestingly, apical hyposmolality decreased cation selectivity in the paracellular pathway gradually with time, and the elimination of the osmotic gradient promptly restored the cation selectivity. Apical hyposmolality also induced bleb formation at cell-cell contacts and changed the shape of cell-cell contacts from a jagged pattern to a slightly linear pattern. In claudin-2 knockout MDCK II cells, the decrease of cation selectivity, the bleb formation, nor the changes in the shape of cell-cell contacts was observed under the apical hyposmolality. Our findings in this study indicate that osmotic gradient between apical and basal sides is involved in the acute regulation of the cation selective property of claudin-2 channels. PMID:27855213

Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers

NASA Astrophysics Data System (ADS)

Thomas, D. Denee; Navab, Mahamad; Haake, David A.; Fogelman, Alan M.; Miller, James N.; Lovett, Michael A.

1988-05-01

The pathogenesis of syphilis reflects invasive properties of Treponema pallidum, but the actual mode of tissue invasion is unknown. We have found two in vitro parallels of treponemal invasiveness. We tested whether motile T. pallidum could invade host cells by determining the fate of radiolabeled motile organisms added to a HeLa cell monolayer; 26% of treponemes associated with the monolayer in a trypsin-resistant niche, presumably between the monolayer and the surface to which it adhered, but did not attain intracellularity. Attachment of T. pallidum to cultured human and rabbit aortic and human umbilical vein endothelial cells was 2-fold greater than to HeLa cells. We added T. pallidum to aortic endothelial cells grown on membrane filters under conditions in which tight intercellular junctions had formed. T. pallidum was able to pass through the endothelial cell monolayers without altering tight junctions, as measured by electrical resistance. In contrast, heat-killed T. pallidum and the nonpathogen Treponema phagedenis biotype Reiter failed to penetrate the monolayer. Transmission electron micrographs of sections of the monolayer showed T. pallidum in intercellular junctions. Our in vitro observations suggest that these highly motile spirochetes may leave the circulation by invading the junctions between endothelial cells.

Pharmaceutical Activation or Genetic Absence of ClC-2 Alters Tight Junctions During Experimental Colitis.

PubMed

Jin, Younggeon; Pridgen, Tiffany A; Blikslager, Anthony T

2015-12-01

We have previously reported that the ClC-2 chloride channel has an important role in regulation of tight junction barrier function during experimental colitis, and the pharmaceutical ClC-2 activator lubiprostone initiates intestinal barrier repair in ischemic-injured intestine. Thus, we hypothesized that pharmaceutical ClC-2 activation would have a protective and therapeutic effect in murine models of colitis, which would be absent in ClC-2 mice. We administered lubiprostone to wild-type or ClC-2 mice with dextran sulfate sodium (DSS) or 2, 4, 5-trinitrobenzene sulfonic acid-induced colitis. We determined the severity of colitis and assessed intestinal permeability. Selected tight junction proteins were analyzed by Western blotting and immunofluorescence/confocal microscopy, whereas proliferative and differentiated cells were examined with special staining and immunohistochemistry. Oral preventive or therapeutic administration of lubiprostone significantly reduced the severity of colitis and reduced intestinal permeability in both DSS and trinitrobenzene sulfonic acid-induced colitis. Preventive treatment with lubiprostone induced significant recovery of the expression and distribution of selected sealing tight junction proteins in mice with DSS-induced colitis. In addition, lubiprostone reduced crypt proliferation and increased the number of differentiated epithelial cells. Alternatively, when lubiprostone was administered to ClC-2 mice, the protective effect against DSS colitis was limited. This study suggests a central role for ClC-2 in restoration of barrier function and tight junction architecture in experimental murine colitis, which can be therapeutically targeted with lubiprostone.

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro.

PubMed

Tansriratanawong, Kallapat; Ishikawa, Hiroshi; Toyomura, Junko; Sato, Soh

2017-10-01

In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial-endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.

A mechanism enhancing macromolecule transport through paracellular spaces induced by Poly-L-Arginine: Poly-L-Arginine induces the internalization of tight junction proteins via clathrin-mediated endocytosis.

PubMed

Yamaki, Tsutomu; Kamiya, Yusuke; Ohtake, Kazuo; Uchida, Masaki; Seki, Toshinobu; Ueda, Hideo; Kobayashi, Jun; Morimoto, Yasunori; Natsume, Hideshi

2014-09-01

Poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules by disappearance of tight junction (TJ) proteins from cell-cell junctions. However, the mechanism of the disappearance of TJ proteins in response to PLA has been unclear. In this study, we investigated the mechanism of disappearance of TJ proteins from cell-cell junctions after the application of PLA to Caco-2 cell monolayers. The membrane conductance (Gt), FITC-dextran (FD-4) permeability, and localization of TJ proteins were examined after the treatment of Caco-2 cell monolayers with PLA in the presence of various endocytosis inhibitors. In addition, the localization of endosome marker proteins was also observed. Clathrin-mediated endocytosis inhibitors suppressed the increase in Gt and Papp of FD-4 induced by PLA, and also significantly suppressed the disappearance of TJ proteins induced by PLA. Furthermore, occludin, one of the TJ proteins, colocalized with early endosome and recycling endosomes after the internalization of occludin induced by PLA, and then was recycled to the cell-cell junctions. PLA induced the transient internalization of TJ proteins in cell-cell junctions via clathrin-mediated endocytosis, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

Fluorescence contrast-enhanced proliferative lesion imaging by enema administration of indocyanine green in a rat model of colon carcinogenesis

PubMed Central

Onda, Nobuhiko; Mizutani-Morita, Reiko; Yamashita, Susumu; Nagahara, Rei; Matsumoto, Shinya; Yoshida, Toshinori; Shibutani, Makoto

2017-01-01

The fluorescent contrast agent indocyanine green (ICG) is approved by the Food and Drug Administration for clinical applications. We previously reported that cultured human colon tumor cells preferentially take up ICG by endocytic activity in association with disruption of their tight junctions. The present study explored ICG availability in fluorescence imaging of the colon to identify proliferative lesions during colonoscopy. The cellular uptake of ICG in cultured rat colon tumor cells was examined using live-cell imaging. Colon lesions in rats administered an ICG-containing enema were further assessed in rats with azoxymethane-induced colon carcinogenesis, using in vivo endoscopy, ex vivo microscopy, and immunofluorescence microscopy. The uptake of ICG by the cultured cells was temperature-dependent. The intracellular retention of the dye in the membrane trafficking system suggested endocytosis as the uptake mechanism. ICG administered via enema accumulated in colon proliferative lesions ranging from tiny aberrant crypt foci to adenomas and localized in proliferating cells. Fluorescence endoscopy detected these ICG-positive colonic proliferative lesions in vivo. The immunoreactivity of the tight-junction molecule occludin was altered in the proliferative lesions, suggesting the disruption of the integrity of tight junctions. These results suggest that fluorescence contrast-enhanced imaging following the administration of an ICG-containing enema can enhance the detection of mucosal proliferative lesions of the colon during colonoscopy. The tissue preference of ICG in the rat model evaluated in this study can be attributed to the disruption of tight junctions, which in turn promotes endocytosis by proliferative cells and the cellular uptake of ICG. PMID:29163827

House dust mite allergen Der p 1 effects on sinonasal epithelial tight junctions.

PubMed

Henriquez, Oswaldo A; Den Beste, Kyle; Hoddeson, Elizabeth K; Parkos, Charles A; Nusrat, Asma; Wise, Sarah K

2013-08-01

Epithelial permeability is highly dependent upon the integrity of tight junctions, which are cell-cell adhesion complexes located at the apical aspect of the lateral membrane of polarized epithelial cells. We hypothesize that sinonasal epithelial exposure to Der p 1 house dust mite antigen decreases expression of tight junction proteins (TJPs), representing a potential mechanism for increased permeability and presentation of antigens across the sinonasal epithelial layer. Confluent cultured primary human sinonasal epithelial cells were exposed to recombinant Der p 1 antigen vs control, and transepithelial resistance measurements were performed over 24 hours. Antibody staining for a panel of TJPs was examined with immunofluorescence/confocal microscopy and Western blotting. Tissue for these experiments was obtained from 4 patients total. Der p 1 exposed sinonasal cells showed a marked decrease in transepithelial resistance when compared to control cells. In addition, results of Western immunoblot and immunofluorescent labeling demonstrated decreased expression of TJPs claudin-1 and junction adhesion molecule-A (JAM-A) in Der p 1-exposed cultured sinonasal cells vs controls. Der p 1 antigen exposure decreases sinonasal epithelium TJP expression, most notably seen in JAM-A and claudin-1 in these preliminary experiments. This decreased TJP expression likely contributes to increased epithelial permeability and represents a potential mechanism for transepithelial antigen exposure in allergic rhinitis. © 2013 ARS-AAOA, LLC.

[Effect of self-microemulsifying system on cell tight junctions].

PubMed

Sha, Xian-Yi; Fang, Xiao-Ling

2006-01-01

To study the effect of negatively charged and positively charged self-microemulsifying systems (SMES) on the cellular tight junction complex was to be investigated at molecular cell level. Human intestinal epithelial Caco-2 cell model was established. Effect of formulations on the transepithelial electrical resistance (TEER) and permeability of the paracellular transport marker mannitol were measured to evaluate the cell integrity. Changes in subcellular localization of the tight junction protein zona occludens 1 (ZO-1) and cytoskeleton protein actin by immunofluorescence were also assessed after treatment of two SMESs in different dilutions. The TEER of cell monolayers was not markedly affected by negatively charged SMES in different dilutions. The positively charged SMES could significantly decrease the TEER (P < 0.05) in three dilutions. The full recovery of TEER was found after the treatment of lower dilution for 2 h, then cultured for 48 h, while the recovery of TEER was 81.3% of control in 1 : 50 dilution. Two SMESs could enhance the apparent permeability coefficient of mannitol (2.9 - 64.6 folds), which depended on the dilution times. The immunofluorescent results indicated that the distribution of ZO-1 and actin were discrete in cell membrane after the treatment of formulation. Since the positively charged microemulsion could bind to the epithelial cell membrane by electrostatic interaction, the actin of the cells undergone some kind of stress stimulated by the higher concentration of microemulsion was more markedly affected than the negatively charged SMES. Effect of formulations on ZO-1 and actin relied on the dilution. SMES is able to enhance the paracellular transport marker mannitol. The mechanism of opening of tight junctions by SMES might be the change of distribution of ZO-1 and actin.

HIV-1 gp120 Glycoprotein Interacting with Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Non-integrin (DC-SIGN) Down-Regulates Tight Junction Proteins to Disrupt the Blood Retinal Barrier and Increase Its Permeability.

PubMed

Qian, Yi-Wen; Li, Chuan; Jiang, Ai-Ping; Ge, Shengfang; Gu, Ping; Fan, Xianqun; Li, Tai-Sheng; Jin, Xia; Wang, Jian-Hua; Wang, Zhi-Liang

2016-10-28

Approximately 70% of HIV-1 infected patients acquire ocular opportunistic infections and manifest eye disorders during the course of their illness. The mechanisms by which pathogens invade the ocular site, however, are unclear. Under normal circumstances, vascular endothelium and retinal pigment epithelium (RPE), which possess a well developed tight junction complex, form the blood-retinal barrier (BRB) to prevent pathogen invasion. We hypothesize that disruption of the BRB allows pathogen entry into ocular sites. The hypothesis was tested using in vitro models. We discovered that human RPE cells could bind to either HIV-1 gp120 glycoproteins or HIV-1 viral particles. Furthermore, the binding was mediated by dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expressed on RPE cells. Upon gp120 binding to DC-SIGN, cellular NF-κB signaling was triggered, leading to the induction of matrix metalloproteinases, which subsequently degraded tight junction proteins and disrupted the BRB integrity. DC-SIGN knockdown or prior blocking with a specific antibody abolished gp120-induced matrix metalloproteinase expression and reduced the degradation of tight junction proteins. This study elucidates a novel mechanism by which HIV, type 1 invades ocular tissues and provides additional insights into the translocation or invasion process of ocular complication-associated pathogens. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cooling treatment transiently increases the permeability of brain capillary endothelial cells through translocation of claudin-5.

PubMed

Inamura, Akinori; Adachi, Yasuhiro; Inoue, Takao; He, Yeting; Tokuda, Nobuko; Nawata, Takashi; Shirao, Satoshi; Nomura, Sadahiro; Fujii, Masami; Ikeda, Eiji; Owada, Yuji; Suzuki, Michiyasu

2013-08-01

The blood-brain-barrier (BBB) is formed by different cell types, of which brain microvascular endothelial cells are major structural constituents. The goal of this study was to examine the effects of cooling on the permeability of the BBB with reference to tight junction formation of brain microendothelial cells. The sensorimotor cortex above the dura mater in adult male Wistar rats was focally cooled to a temperature of 5 °C for 1 h, then immunostaining for immunoglobulin G (IgG) was performed to evaluate the permeability of the BBB. Permeability produced by cooling was also evaluated in cultured murine brain endothelial cells (bEnd3) based on measurement of trans-epithelial electric resistance (TEER). Immunocytochemistry and Western blotting of proteins associated with tight junctions in bEnd3 were performed to determine protein distribution before and after cooling. After focal cooling of the rat brain cortex, diffuse immunostaining for IgG was observed primarily around the small vasculature and in the extracellular spaces of parenchyma of the cortex. In cultured bEnd3, TEER significantly decreased during cooling (15 °C) and recovered to normal levels after rewarming to 37 °C. Immunocytochemistry and Western blotting showed that claudin-5, a critical regulatory protein for tight junctions, was translocated from the membrane to the cytoplasm after cooling in cultured bEnd3 cells. These results suggest that focal brain cooling may open the BBB transiently through an effect on tight junctions of brain microendothelial cells, and that therapeutically this approach may allow control of BBB function and drug delivery through the BBB.

Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse

PubMed Central

Wang, Hehai; Ding, Tianbing; Brown, Naoko; Yamamoto, Yasutoshi; Prince, Lawrence S.; Reese, Jeff; Paria, B. C.

2008-01-01

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells. PMID:18423437

Loss of tight junction barrier function and its role in cancer metastasis.

PubMed

Martin, Tracey A; Jiang, Wen G

2009-04-01

As the most apical structure between epithelial and endothelial cells, tight junctions (TJ) are well known as functioning as a control for the paracellular diffusion of ions and certain molecules. It has however, become increasingly apparent that the TJ has a vital role in maintaining cell to cell integrity and that the loss of cohesion of the structure can lead to invasion and thus metastasis of cancer cells. This article will present data showing how modulation of expression of TJ molecules results in key changes in TJ barrier function leading to the successful metastasis of a number of different cancer types.

ACF7 regulates inflammatory colitis and intestinal wound response by orchestrating tight junction dynamics

PubMed Central

Ma, Yanlei; Yue, Jiping; Zhang, Yao; Shi, Chenzhang; Odenwald, Matt; Liang, Wenguang G.; Wei, Qing; Goel, Ajay; Gou, Xuewen; Zhang, Jamie; Chen, Shao-Yu; Tang, Wei-Jen; Turner, Jerrold R.; Yang, Feng; Liang, Hong; Qin, Huanlong; Wu, Xiaoyang

2017-01-01

In the intestinal epithelium, the aberrant regulation of cell/cell junctions leads to intestinal barrier defects, which may promote the onset and enhance the severity of inflammatory bowel disease (IBD). However, it remains unclear how the coordinated behaviour of cytoskeletal network may contribute to cell junctional dynamics. In this report, we identified ACF7, a crosslinker of microtubules and F-actin, as an essential player in this process. Loss of ACF7 leads to aberrant microtubule organization, tight junction stabilization and impaired wound closure in vitro. With the mouse genetics approach, we show that ablation of ACF7 inhibits intestinal wound healing and greatly increases susceptibility to experimental colitis in mice. ACF7 level is also correlated with development and progression of ulcerative colitis (UC) in human patients. Together, our results reveal an important molecular mechanism whereby coordinated cytoskeletal dynamics contributes to cell adhesion regulation during intestinal wound repair and the development of IBD. PMID:28541346

IGF-1 decreases portal vein endotoxin via regulating intestinal tight junctions and plays a role in attenuating portal hypertension of cirrhotic rats.

PubMed

Zhao, Tian-Yu; Su, Li-Ping; Ma, Chun-Ye; Zhai, Xiao-Han; Duan, Zhi-Jun; Zhu, Ying; Zhao, Gang; Li, Chun-Yan; Wang, Li-Xia; Yang, Dong

2015-07-08

Intestinal barrier dysfunction is not only the consequence of liver cirrhosis, but also an active participant in the development of liver cirrhosis. Previous studies showed that external administration of insulin-like growth factor 1 (IGF-1) improved intestinal barrier function in liver cirrhosis. However, the mechanism of IGF-1 on intestinal barrier in liver cirrhosis is not fully elucidated. The present study aims to investigate the mechanisms of IGF-1 improving intestinal barrier function via regulating tight junctions in intestines. We used carbon tetrachloride induced liver cirrhotic rats to investigate the effect of IGF-1 on intestinal claudin-1 and occludin expressions, serum alanine transaminase (ALT) and aspartate transaminase (AST) levels, severity of liver fibrosis, portal pressures, enterocytic apoptosis and lipopolysaccharides (LPS) levels in portal vein. The changes of IGF-1 in serum during the development of rat liver cirrhosis were also evaluated. Additionally, we assessed the effect of IGF-1 on claudin-1 and occludin expressions, changes of transepithelial electrical resistance (TEER) and apoptosis in Caco-2 cells to confirm in vivo findings. Serum IGF-1 levels were decreased in the development of rat liver cirrhosis, and external administration of IGF-1 restored serum IGF-1 levels. External administration of IGF-1 reduced serum ALT and AST levels, severity of liver fibrosis, LPS levels in portal vein, enterocytic apoptosis and portal pressure in cirrhotic rats. External administration of IGF-1 increased the expressions of claudin-1 and occludin in enterocytes, and attenuated tight junction dysfunction in intestines of cirrhotic rats. LPS decreased TEER in Caco-2 cell monolayer. LPS also decreased claudin-1 and occludin expressions and increased apoptosis in Caco-2 cells. Furthermore, IGF-1 attenuated the effect of LPS on TEER, claudin-1 expression, occludin expression and apoptosis in Caco-2 cells. Tight junction dysfunction develops during the development of liver cirrhosis, and endotoxemia will develop subsequently. Correspondingly, increased endotoxin in portal system worsens tight junction dysfunction via decreasing intestinal occludin and claudin-1 expressions and increasing enterocytic apoptosis. Endotoxemia and intestinal barrier dysfunction form a vicious circle. External administration of IGF-1 breaks this vicious circle. Improvement of tight junctions might be one possible mechanism of the restoration of intestinal barrier function mediated by IGF-1.

Disruption of the epithelial barrier during intestinal inflammation: Quest for new molecules and mechanisms.

PubMed

Lechuga, Susana; Ivanov, Andrei I

2017-07-01

The intestinal epithelium forms a key protective barrier that separates internal organs from the harmful environment of the gut lumen. Increased permeability of the gut barrier is a common manifestation of different inflammatory disorders contributing to the severity of disease. Barrier permeability is controlled by epithelial adherens junctions and tight junctions. Junctional assembly and integrity depend on fundamental homeostatic processes such as cell differentiation, rearrangements of the cytoskeleton, and vesicle trafficking. Alterations of intestinal epithelial homeostasis during mucosal inflammation may impair structure and remodeling of apical junctions, resulting in increased permeability of the gut barrier. In this review, we summarize recent advances in our understanding of how altered epithelial homeostasis affects the structure and function of adherens junctions and tight junctions in the inflamed gut. Specifically, we focus on the transcription reprogramming of the cell, alterations in the actin cytoskeleton, and junctional endocytosis and exocytosis. We pay special attention to knockout mouse model studies and discuss the relevance of these mechanisms to human gastrointestinal disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Minimal Effects of VEGF and Anti-VEGF Drugs on the Permeability or Selectivity of RPE Tight Junctions

PubMed Central

Peng, Shaomin; Adelman, Ron A.

2010-01-01

Purpose. Bevacizumab and ranibizumab are currently used to treat age-related macular degeneration by neutralizing vascular endothelial growth factor (VEGF). In this study, the potential side effects on the outer blood–retinal barrier were examined. Methods. Human fetal RPE (hfRPE) cells were used because they are highly differentiated in culture. The claudin composition of RPE tight junctions was determined by RT-PCR, immunoblot analysis, and immunofluorescence. ELISA assays monitored the secretion and trafficking of VEGF and a fluid-phase marker, methylpolyethylene glycol (mPEG). Tight junction functions were assessed by the conductance of K+ and Na+ (derived from the transepithelial electrical resistance, TER) and the flux of NaCl and mPEG. Results. Claudin-3, claudin-10, and claudin-19 were detected in RPE tight junctions. VEGF was secreted in equal amounts across the apical and basolateral membranes, but the apical membrane was more active in endocytosing and degrading VEGF. Exogenous VEGF and mPEG crossed the RPE monolayer by transcytosis, predominantly in the apical-to-basal direction. RPE tight junctions were selective for K+, but did not discriminate between Na+ and Cl−. VEGF, bevacizumab, and ranibizumab had minimal effects on TER, permeation of mPEG, and selectivity for K+, Na+, and Cl−. They had minimal effects on the expression and distribution of the claudins. Conclusions. RPE has mechanisms for maintaining low concentrations of VEGF in the subretinal space that include endocytosis and degradation and fluid-phase transcytosis in the apical-to-basal direction. RPE tight junctions are selective for K+ over Na+ and Cl−. Permeability and selectivity of the junctions are not affected by VEGF, bevacizumab, or ranibizumab. PMID:20042644

Sodium Butyrate Attenuates Diarrhea in Weaned Piglets and Promotes Tight Junction Protein Expression in Colon in a GPR109A-Dependent Manner.

PubMed

Feng, Wenqian; Wu, Yancheng; Chen, Guangxin; Fu, Shoupeng; Li, Bai; Huang, Bingxu; Wang, Dali; Wang, Wei; Liu, Juxiong

2018-06-27

Butyric acid plays an important role in maintaining intestinal health. Butyric acid has received special attention as a short-chain fatty acid, but its role in protecting the intestinal barrier is poorly characterized. Butyric acid not only provides energy for epithelial cells but also acts as a histone deacetylase inhibitor; it is also a natural ligand for G protein-coupled receptor 109A (GPR109A). A GPR109A analog was expressed in Sus scrofa and mediated the anti-inflammatory effects of beta-hydroxybutyric acid. This study investigated the effects of butyrate on growth performance, diarrhea symptoms, and tight junction protein levels in 21-day-old weaned piglets. We also studied the mechanism by which butyric acid regulates intestinal permeability. Twenty-four piglets that had been weaned at an age of 21 days were divided randomly into 2 equal groups: basal diet group and sodium butyrate + basal diet group. Diarrhea rate, growth performance during 3 weeks of feeding on these diets were observed, the lactulose-mannitol ratio in urine were detected by High Performance Liquid Chromatography, the expression levels of tight junction proteins in the intestinal tract and related signaling molecules, such as GPR109A and Akt, in the colon were examined by quantitative real-time PCR or western blot analyses on day 21. Caco-2 cells were used as a colon cell model and cultured with or without sodium butyrate to assess the expression of tight junction proteins and the activation of related signaling molecules. GPR109A-short hairpin RNA (shRNA) and specific antagonists of Akt and ERK1/2 were used as signaling pathway inhibitors to elucidate the mechanism by which butyric acid regulates the expression of tight junction proteins and the colonic epithelial barrier. The sodium butyrate diet alleviated diarrhea symptoms and decreased intestinal permeability without affecting the growth of early weaned piglets. The expression levels of the tight junction proteins Claudin-3, Occludin, and zonula occludens 1 were up-regulated by sodium butyrate in the colon and Caco-2 cells. GPR109A knockdown using shRNA or blockade of the Akt signaling pathway in Caco-2 cells suppressed sodium butyrate-induced Claudin-3 expression. Sodium butyrate acts on the Akt signaling pathway to facilitate Claudin-3 expression in the colon in a GPR109A-dependent manner. © 2018 The Author(s). Published by S. Karger AG, Basel.

Time-dependent effects of low-temperature atmospheric-pressure argon plasma on epithelial cell attachment, viability and tight junction formation in vitro

NASA Astrophysics Data System (ADS)

Hoentsch, Maxi; von Woedtke, Thomas; Weltmann, Klaus-Dieter; Nebe, J. Barbara

2012-01-01

The application of physical plasma to living tissues is expected to promote wound healing by plasma disinfection and stimulation of tissue regeneration. However, the effects of plasma on healthy cells must be studied and understood. In our experiments we used an argon plasma jet (kINPen®09) to gain insights into time-dependent plasma effects on cell attachment, viability and tight junction formation in vitro. Murine epithelial cells mHepR1 were suspended in complete cell culture medium and were irradiated with argon plasma (direct approach) for 30, 60 and 120 s. Suspecting that physical plasma may exert its effect via the medium, cell culture medium alone was first treated with argon plasma (indirect approach) and immediately afterwards, cells were added and also cultured for 24 h. Cell morphology and vitality were verified using light microscopy and an enzyme-linked immunosorbent assay. Already after 30 s of treatment the mHepR1 cells lost their capability to adhere and the cell vitality decreased with increasing treatment time. Interestingly, the same inhibitory effect was observed in the indirect approach. Furthermore, the argon plasma-treated culture medium-induced large openings of the cell's tight junctions, were verified by the zonula occludens protein ZO-1, which we observed for the first time in confluently grown epithelial cells.

Effect of lactobacilli on paracellular permeability in the gut.

PubMed

Ahrne, Siv; Hagslatt, Marie-Louise Johansson

2011-01-01

Paracellular permeability is determined by the complex structures of junctions that are located between the epithelial cells. Already in 1996, it was shown that the human probiotic strain Lactobacillus plantarum 299v and the rat-originating strain Lactobacillus reuteri R2LC could reduce this permeability in a methotrexate-induced colitis model in the rat. Subsequently, many animal models and cell culture systems have shown indications that lactobacilli are able to counteract increased paracellular permeability evoked by cytokines, chemicals, infections, or stress. There have been few human studies focusing on the effect of lactobacilli on intestinal paracellular permeability but recently it has been shown that they could influence the tight junctions. More precisely, short-term administration of L. plantarum WCSF1 to healthy volunteers increased the relocation of occludin and ZO-1 into the tight junction area between duodenal epithelial cells.

Novel effects of edaravone on human brain microvascular endothelial cells revealed by a proteomic approach.

PubMed

Onodera, Hidetaka; Arito, Mitsumi; Sato, Toshiyuki; Ito, Hidemichi; Hashimoto, Takuo; Tanaka, Yuichiro; Kurokawa, Manae S; Okamoto, Kazuki; Suematsu, Naoya; Kato, Tomohiro

2013-10-09

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a free radical scavenger used for acute ischemic stroke. However, it is not known whether edaravone works only as a free radical scavenger or possess other pharmacological actions. Therefore, we elucidated the effects of edaravone on human brain microvascular endothelial cells (HBMECs) by 2 dimensional fluorescence difference gel electrophoresis (2D-DIGE). We found 38 protein spots the intensity of which was significantly altered 1.3 fold on average (p< 0.05) by the edaravone treatment and successfully identified 17 proteins of those. Four of those 17 proteins were cytoskeleton proteins or cytoskeleton-regulating proteins. Therefore, we subsequently investigated the change of size and shape of the cells, the actin network, and the tight junction of HBMEC by immunocytochemistry. As a result, most edaravone-treated HBMECs became larger and rounder compared with those that were not treated. Furthermore, edaravone-treated HBMECs formed gathering zona occludens (ZO)-1, a tight junction protein, along the junction of the cells. In addition, we found that edaravone suppressed interleukin (IL)-1β-induced secretion of monocyte chemoattractant protein-1 (MCP-1), which was reported to increase cell permeability. We found a novel function of edaravone is the promotion of tight junction formations of vascular endothelial cells partly via the down-regulation of MCP-1 secretion. These data provide fundamental and useful information in the clinical use of edaravone in patients with cerebral vascular diseases. © 2013 Elsevier B.V. All rights reserved.

Possible Involvement of Tight Junctions, Extracellular Matrix and Nuclear Receptors in Epithelial Differentiation

PubMed Central

Ichikawa-Tomikawa, Naoki; Sugimoto, Kotaro; Satohisa, Seiro; Nishiura, Keisuke; Chiba, Hideki

2011-01-01

Tight junctions are intercellular junctions localized at the most apical end of the lateral plasma membrane. They consist of four kinds of transmembrane proteins (occludin, claudins, junctional adhesion molecules, and tricellulin) and huge numbers of scaffolding proteins and contribute to the paracellular barrier and fence function. The mutation and deletion of these proteins impair the functions of tight junctions and cause various human diseases. In this paper, we provide an overview of recent studies on transmembrane proteins of tight junctions and highlight the functional significance of tight junctions, extracellular matrix, and nuclear receptors in epithelial differentiation. PMID:22162632

The stress polarity pathway: AMPK ‘GIV’-es protection against metabolic insults

PubMed Central

Ghosh, Pradipta

2017-01-01

Loss of cell polarity impairs organ development and function; it can also serve as one of the first triggers for oncogenesis. In 2006-2007 two groups simultaneously reported the existence of a special pathway for maintaining epithelial polarity in the face of environmental stressors. In this pathway, AMPK, a key sensor of metabolic stress stabilizes tight junctions, preserves cell polarity, and thereby, maintains epithelial barrier functions. Accumulating evidence since has shown that pharmacologic activation of AMPK by Metformin protects the epithelial barrier against multiple environmental and pathological stressful states and suppresses tumorigenesis. How AMPK protects the epithelium remained unknown until recently Aznar et al. identified GIV/Girdin as a novel effector of AMPK at the cell-cell junctions; phosphorylation of GIV at a single site by AMPK appears to be both necessary and sufficient for strengthening tight junctions and preserving cell polarity and epithelial barrier function in the face of energetic stress. Here we review the fundamentals of this specialized signaling pathway that buttresses cell-cell junctions against stress-induced collapse and discuss its pathophysiologic relevance in the context of a variety of diseases, including cancers, diabetes, aging, and the growing list of beneficial effects of the AMPK-activator, Metformin. PMID:28209925

Scribble is required for normal epithelial cell–cell contacts and lumen morphogenesis in the mammalian lung

PubMed Central

Yates, Laura L.; Schnatwinkel, Carsten; Hazelwood, Lee; Chessum, Lauren; Paudyal, Anju; Hilton, Helen; Romero, M. Rosario; Wilde, Jonathan; Bogani, Debora; Sanderson, Jeremy; Formstone, Caroline; Murdoch, Jennifer N.; Niswander, Lee A.; Greenfield, Andy; Dean, Charlotte H.

2013-01-01

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in ScribCrc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell–cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion. PMID:23195221

Estrogen decreases tight junction protein ZO-1 expression in human primary gut tissues.

PubMed

Zhou, Zejun; Zhang, Lumin; Ding, Miao; Luo, Zhenwu; Yuan, Shao; Bansal, Meena B; Gilkeson, Gary; Lang, Ren; Jiang, Wei

2017-10-01

Females have a higher prevalence of most autoimmune diseases; however, the mechanism is unknown. In this study, we examined the expression of tight junction protein zonula occludens 1 (ZO-1) and estrogen receptor (ER)-α/β in human primary gut tissues by immunohistochemistry, immunofluorescence and qPCR. The expression of ZO-1 and ER-β but not ER-α was present in both male and female gut tissues. There was no sex difference in ER-β expression, but ZO-1 expression was decreased in females compared to males. In vitro, estrogen treatment decreased ZO-1 mRNA and protein expression, ZO-1 promoter activity, IL-6 production, and NF-κB activation in human primary gut tissues or the Caco-2 cells, but increased the ER-β expression in Caco-2 cells. Consistently, plasma IL-6 levels in females were reduced relative to males in vivo. Our finding indicates that estrogen may play a role in gut tight junction expression and permeability. Copyright © 2017 Elsevier Inc. All rights reserved.

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

PubMed Central

Zihni, Ceniz; Munro, Peter M.G.; Elbediwy, Ahmed; Keep, Nicholas H.; Terry, Stephen J.; Harris, John

2014-01-01

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation. PMID:24379416

C-terminal Src kinase (Csk) regulates the tricellular junction protein Gliotactin independent of Src

PubMed Central

Samarasekera, G. D. N. Gayathri; Auld, Vanessa Jane

2018-01-01

Tricellular junctions (TCJs) are uniquely placed permeability barriers formed at the corners of polarized epithelia where tight junctions in vertebrates or septate junctions (SJ) in invertebrates from three cells converge. Gliotactin is a Drosophila TCJ protein, and loss of Gliotactin results in SJ and TCJ breakdown and permeability barrier loss. When overexpressed, Gliotactin spreads away from the TCJs, resulting in disrupted epithelial architecture, including overproliferation, cell delamination, and migration. Gliotactin levels are tightly controlled at the mRNA level and at the protein level through endocytosis and degradation triggered by tyrosine phosphorylation. We identified C-terminal Src kinase (Csk) as a tyrosine kinase responsible for regulating Gliotactin endocytosis. Increased Csk suppresses the Gliotactin overexpression phenotypes by increasing endocytosis. Loss of Csk causes Gliotactin to spread away from the TCJ. Although Csk is known as a negative regulator of Src kinases, the effects of Csk on Gliotactin are independent of Src and likely occur through an adherens junction associated complex. Overall, we identified a new Src-independent role for Csk in the control of Gliotactin, a key tricellular junction protein. PMID:29167383

Angubindin-1 opens the blood-brain barrier in vivo for delivery of antisense oligonucleotide to the central nervous system.

PubMed

Zeniya, Satoshi; Kuwahara, Hiroya; Daizo, Kaiichi; Watari, Akihiro; Kondoh, Masuo; Yoshida-Tanaka, Kie; Kaburagi, Hidetoshi; Asada, Ken; Nagata, Tetsuya; Nagahama, Masahiro; Yagi, Kiyohito; Yokota, Takanori

2018-05-17

Within the field of RNA therapeutics, antisense oligonucleotide-based therapeutics are a potentially powerful means of treating intractable diseases. However, if these therapeutics are used for the treatment of neurological disorders, safe yet efficient methods of delivering antisense oligonucleotides across the blood-brain barrier to the central nervous system must be developed. Here, we examined the use of angubindin-1, a binder to the tricellular tight junction, to modulate paracellular transport between brain microvascular endothelial cells in the blood-brain barrier for the delivery of antisense oligonucleotides to the central nervous system. This proof-of-concept study demonstrated that intravenously injected angubindin-1 increased the permeability of the blood-brain barrier and enabled transient delivery of subsequently administered antisense oligonucleotides into the mouse brain and spinal cord, leading to silencing of a target RNA without any overt adverse effects. We also found that two bicellular tight junction modulators did not produce such a silencing effect, suggesting that the tricellular tight junction is likely a better target for the delivery of antisense oligonucleotides than the bicellular tight junction. Our delivery strategy of modulating the tricellular tight junction in the blood-brain barrier via angubindin-1 provides a novel avenue of research for the development of antisense oligonucleotide-based therapeutics for the treatment of neurological disorders. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier

PubMed Central

Mei, Shenghui; Jin, Hong; Zhu, Bin; Tian, Yue; Huo, Jiping; Cui, Xu; Guo, Anchen; Zhao, Zhigang

2017-01-01

To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted. PMID:29059256

Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier.

PubMed

Yang, Shu; Mei, Shenghui; Jin, Hong; Zhu, Bin; Tian, Yue; Huo, Jiping; Cui, Xu; Guo, Anchen; Zhao, Zhigang

2017-01-01

To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted.

Intestinal epithelial barrier function and tight junction proteins with heat and exercise

PubMed Central

Zuhl, Micah N.; Moseley, Pope L.

2015-01-01

A single layer of enterocytes and tight junctions (intercellular multiprotein complexes) form the intestinal epithelial barrier that controls transport of molecules through transcellular and paracellular pathways. A dysfunctional or “leaky” intestinal tight junction barrier allows augmented permeation of luminal antigens, endotoxins, and bacteria into the blood stream. Various substances and conditions have been shown to affect the maintenance of the intestinal epithelial tight junction barrier. The primary focus of the present review is to analyze the effects of exertional or nonexertional (passive hyperthermia) heat stress on tight junction barrier function in in vitro and in vivo (animals and humans) models. Our secondary focus is to review changes in tight junction proteins in response to exercise or hyperthermic conditions. Finally, we discuss some pharmacological or nutritional interventions that may affect the cellular mechanisms involved in maintaining homeostasis of the intestinal epithelial tight junction barrier during heat stress or exercise. PMID:26359485

Intestinal epithelial barrier function and tight junction proteins with heat and exercise.

PubMed

Dokladny, Karol; Zuhl, Micah N; Moseley, Pope L

2016-03-15

A single layer of enterocytes and tight junctions (intercellular multiprotein complexes) form the intestinal epithelial barrier that controls transport of molecules through transcellular and paracellular pathways. A dysfunctional or "leaky" intestinal tight junction barrier allows augmented permeation of luminal antigens, endotoxins, and bacteria into the blood stream. Various substances and conditions have been shown to affect the maintenance of the intestinal epithelial tight junction barrier. The primary focus of the present review is to analyze the effects of exertional or nonexertional (passive hyperthermia) heat stress on tight junction barrier function in in vitro and in vivo (animals and humans) models. Our secondary focus is to review changes in tight junction proteins in response to exercise or hyperthermic conditions. Finally, we discuss some pharmacological or nutritional interventions that may affect the cellular mechanisms involved in maintaining homeostasis of the intestinal epithelial tight junction barrier during heat stress or exercise. Copyright © 2016 the American Physiological Society.

Effect of Lactobacilli on Paracellular Permeability in the Gut

PubMed Central

Ahrne, Siv; Hagslatt, Marie-Louise Johansson

2011-01-01

Paracellular permeability is determined by the complex structures of junctions that are located between the epithelial cells. Already in 1996, it was shown that the human probiotic strain Lactobacillus plantarum 299v and the rat-originating strain Lactobacillus reuteri R2LC could reduce this permeability in a methotrexate-induced colitis model in the rat. Subsequently, many animal models and cell culture systems have shown indications that lactobacilli are able to counteract increased paracellular permeability evoked by cytokines, chemicals, infections, or stress. There have been few human studies focusing on the effect of lactobacilli on intestinal paracellular permeability but recently it has been shown that they could influence the tight junctions. More precisely, short-term administration of L. plantarum WCSF1 to healthy volunteers increased the relocation of occludin and ZO-1 into the tight junction area between duodenal epithelial cells. PMID:22254077

Up-regulation of the tight-junction protein ZO-1 by substance P and IGF-1 in A431 cells.

PubMed

Ko, Ji-Ae; Murata, Shizuka; Nishida, Teruo

2009-08-01

The formation of a barrier by tight junctions is important in epithelia of various tissues. Substance P (SP) and insulin-like growth factor (IGF)-1 synergistically promote barrier function in the corneal epithelium. We have now examined the effects of SP and IGF-1 on expression of the tight-junction protein zonula occludens (ZO)-1 in A431 human epidermoid carcinoma cells. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot analyses revealed that SP and IGF-1 increased the amounts of ZO-1 mRNA and protein in these cells in a concentration-dependent manner, with neither SP nor IGF-1 alone having such an effect. The SP- and IGF-1-induced up-regulation of ZO-1 was accompanied by phosphorylation of extracellular signal-regulated kinase (ERK), and both of these effects were blocked by PD98059, an inhibitor of ERK activation. SP and IGF-1 also increased the transepithelial electrical resistance (TER) (an indicator of barrier function) of an A431 cell monolayer in a manner sensitive to PD98059. Our results thus suggest that the synergistic induction of ZO-1 expression by SP and IGF-1 may promote barrier function in skin epithelial cells. (c) 2009 John Wiley & Sons, Ltd.

A refined model of claudin-15 tight junction paracellular architecture by molecular dynamics simulations

PubMed Central

Alberini, Giulio; Benfenati, Fabio

2017-01-01

Tight-junctions between epithelial cells of biological barriers are specialized molecular structures that regulate the flux of solutes across the barrier, parallel to cell walls. The tight-junction backbone is made of strands of transmembrane proteins from the claudin family, but the molecular mechanism of its function is still not completely understood. Recently, the crystal structure of a mammalian claudin-15 was reported, displaying for the first time the detailed features of transmembrane and extracellular domains. Successively, a structural model of claudin-15-based paracellular channels has been proposed, suggesting a putative assembly that illustrates how claudins associate in the same cell (via cis interactions) and across adjacent cells (via trans interactions). Although very promising, the model offers only a static conformation, with residues missing in the most important extracellular regions and potential steric clashes. Here we present detailed atomic models of paracellular single and double pore architectures, obtained from the putative assembly and refined via structural modeling and all-atom molecular dynamics simulations in double membrane bilayer and water environment. Our results show an overall stable configuration of the complex with a fluctuating pore size. Extracellular residue loops in trans interaction are able to form stable contacts and regulate the size of the pore, which displays a stationary radius of 2.5–3.0 Å at the narrowest region. The side-by-side interactions of the cis configuration are preserved via stable hydrogen bonds, already predicted by cysteine crosslinking experiments. Overall, this work introduces an improved version of the claudin-15-based paracellular channel model that strengthens its validity and that can be used in further computational studies to understand the structural features of tight-junctions regulation. PMID:28863193

Altered Expression of ZO-1 and ZO-2 in Sertoli Cells and Loss of Blood-Testis Barrier Integrity in Testicular Carcinoma In Situ1

PubMed Central

Fink, Cornelia; Weigel, Roswitha; Hembes, Tanja; Lauke-Wettwer, Heidrun; Kliesch, Sabine; Bergmann, Martin; Brehm, Ralph H

2006-01-01

Abstract Carcinoma in situ (CIS) is the noninvasive precursor of most human testicular germ cell tumors. In normal seminiferous epithelium, specialized tight junctions between Sertoli cells constitute the major component of the blood-testis barrier. Sertoli cells associated with CIS exhibit impaired maturation status, but their functional significance remains unknown. The aim was to determine whether the blood-testis barrier is morphologically and/or functionally altered. We investigated the expression and distribution pattern of the tight junction proteins zonula occludens (ZO) 1 and 2 in normal seminiferous tubules compared to tubules showing CIS. In normal tubules, ZO-1 and ZO-2 immunostaining was observed at the blood-testis barrier region of adjacent Sertoli cells. Within CIS tubules, ZO-1 and ZO-2 immunoreactivity was reduced at the blood-testis barrier region, but spread to stain the Sertoli cell cytoplasm. Western blot analysis confirmed ZO-1 and ZO-2, and their respective mRNA were shown by RT-PCR. Additionally, we assessed the functional integrity of the blood-testis barrier by lanthanum tracer study. Lanthanum permeated tight junctions in CIS tubules, indicating disruption of the blood-testis barrier. In conclusion, Sertoli cells associated with CIS show an altered distribution of ZO-1 and ZO-2 and lose their blood-testis barrier function. PMID:17217619

Gastrointestinal cell lines form polarized epithelia with an adherent mucus layer when cultured in semi-wet interfaces with mechanical stimulation.

PubMed

Navabi, Nazanin; McGuckin, Michael A; Lindén, Sara K

2013-01-01

Mucin glycoproteins are secreted in large quantities by mucosal epithelia and cell surface mucins are a prominent feature of the glycocalyx of all mucosal epithelia. Currently, studies investigating the gastrointestinal mucosal barrier use either animal experiments or non-in vivo like cell cultures. Many pathogens cause different pathology in mice compared to humans and the in vitro cell cultures used are suboptimal because they are very different from an in vivo mucosal surface, are often not polarized, lack important components of the glycocalyx, and often lack the mucus layer. Although gastrointestinal cell lines exist that produce mucins or polarize, human cell line models that reproducibly create the combination of a polarized epithelial cell layer, functional tight junctions and an adherent mucus layer have been missing until now. We trialed a range of treatments to induce polarization, 3D-organization, tight junctions, mucin production, mucus secretion, and formation of an adherent mucus layer that can be carried out using standard equipment. These treatments were tested on cell lines of intestinal (Caco-2, LS513, HT29, T84, LS174T, HT29 MTX-P8 and HT29 MTX-E12) and gastric (MKN7, MKN45, AGS, NCI-N87 and its hTERT Clone5 and Clone6) origins using Ussing chamber methodology and (immuno)histology. Semi-wet interface culture in combination with mechanical stimulation and DAPT caused HT29 MTX-P8, HT29 MTX-E12 and LS513 cells to polarize, form functional tight junctions, a three-dimensional architecture resembling colonic crypts, and produce an adherent mucus layer. Caco-2 and T84 cells also polarized, formed functional tight junctions and produced a thin adherent mucus layer after this treatment, but with less consistency. In conclusion, culture methods affect cell lines differently, and testing a matrix of methods vs. cell lines may be important to develop better in vitro models. The methods developed herein create in vitro mucosal surfaces suitable for studies of host-pathogen interactions at the mucosal surface.

Gastrointestinal Cell Lines Form Polarized Epithelia with an Adherent Mucus Layer when Cultured in Semi-Wet Interfaces with Mechanical Stimulation

PubMed Central

Navabi, Nazanin; McGuckin, Michael A.; Lindén, Sara K.

2013-01-01

Mucin glycoproteins are secreted in large quantities by mucosal epithelia and cell surface mucins are a prominent feature of the glycocalyx of all mucosal epithelia. Currently, studies investigating the gastrointestinal mucosal barrier use either animal experiments or non-in vivo like cell cultures. Many pathogens cause different pathology in mice compared to humans and the in vitro cell cultures used are suboptimal because they are very different from an in vivo mucosal surface, are often not polarized, lack important components of the glycocalyx, and often lack the mucus layer. Although gastrointestinal cell lines exist that produce mucins or polarize, human cell line models that reproducibly create the combination of a polarized epithelial cell layer, functional tight junctions and an adherent mucus layer have been missing until now. We trialed a range of treatments to induce polarization, 3D-organization, tight junctions, mucin production, mucus secretion, and formation of an adherent mucus layer that can be carried out using standard equipment. These treatments were tested on cell lines of intestinal (Caco-2, LS513, HT29, T84, LS174T, HT29 MTX-P8 and HT29 MTX-E12) and gastric (MKN7, MKN45, AGS, NCI-N87 and its hTERT Clone5 and Clone6) origins using Ussing chamber methodology and (immuno)histology. Semi-wet interface culture in combination with mechanical stimulation and DAPT caused HT29 MTX-P8, HT29 MTX-E12 and LS513 cells to polarize, form functional tight junctions, a three-dimensional architecture resembling colonic crypts, and produce an adherent mucus layer. Caco-2 and T84 cells also polarized, formed functional tight junctions and produced a thin adherent mucus layer after this treatment, but with less consistency. In conclusion, culture methods affect cell lines differently, and testing a matrix of methods vs. cell lines may be important to develop better in vitro models. The methods developed herein create in vitro mucosal surfaces suitable for studies of host-pathogen interactions at the mucosal surface. PMID:23869232

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

PubMed

Yamada, Akio; Irie, Kenji; Fukuhara, Atsunori; Ooshio, Takako; Takai, Yoshimi

2004-09-01

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.

Systems Proteomics View of the Endogenous Human Claudin Protein Family

PubMed Central

Liu, Fei; Koval, Michael; Ranganathan, Shoba; Fanayan, Susan; Hancock, William S.; Lundberg, Emma K.; Beavis, Ronald C.; Lane, Lydie; Duek, Paula; McQuade, Leon; Kelleher, Neil L.; Baker, Mark S.

2016-01-01

Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junctions in cell permeability control but also vital in organizing cell contact signaling by protein–protein interactions. How this protein family is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity. The focus of this review is to first provide a brief overview of the functional context, on the basis of the extensive body of claudin biology research that has been thoroughly reviewed, for endogenous human claudin members and then ascertain existing and future proteomics techniques that may be applicable to systematically characterizing the chemical forms and interacting protein partners of this protein family in human. The ability to elucidate claudin-based signaling networks may provide new insight into cell development and differentiation programs that are crucial to tissue stability and manipulation. PMID:26680015

A novel immunotoxin reveals a new role for CD321 in endothelial cells

PubMed Central

Kim, Jia; Hokaiwado, Shintaro; Nawa, Makiko; Okamoto, Hayato; Kogiso, Tomohiko; Watabe, Tetsuro; Hattori, Nobutaka

2017-01-01

There are currently several antibody therapies that directly target tumors, and antibody-drug conjugates represent a novel moiety as next generation therapeutics. Here, we used a unique screening probe, DT3C, to identify functional antibodies that recognized surface molecules and functional epitopes, and which provided toxin delivery capability. Accordingly, we generated the 90G4 antibody, which induced DT3C-dependent cytotoxicity in endothelial cells. Molecular analysis revealed that 90G4 recognized CD321, a protein localized at tight junctions. Although CD321 plays a pivotal role in inflammation and lymphocyte trans-endothelial migration, little is known about its mechanism of action in endothelial cells. Targeting of CD321 by the 90G4 immunotoxin induced cell death. Moreover, 90G4 immunotoxin caused cytotoxicity primarily in migratory endothelial cells, but not in those forming sheets, suggesting a critical role for CD321 in tumor angiogenesis. We also found that hypoxia triggered redistribution of CD321 to a punctate localization on the basal side of cells, resulting in functional impairment of tight junctions and increased motility. Thus, our findings raise the intriguing possibility that endothelial CD321 presented cellular localization in tight junction as well as multifunctional dynamics in several conditions, leading to illuminate the importance of widely-expressed CD321 as a potential target for antitumor therapy. PMID:29028806

Manufactured aluminum oxide nanoparticles decrease expression of tight junction proteins in brain vasculature.

PubMed

Chen, Lei; Yokel, Robert A; Hennig, Bernhard; Toborek, Michal

2008-12-01

Manufactured nanoparticles of aluminum oxide (nano-alumina) have been widely used in the environment; however, their potential toxicity provides a growing concern for human health. The present study focuses on the hypothesis that nano-alumina can affect the blood-brain barrier and induce endothelial toxicity. In the first series of experiments, human brain microvascular endothelial cells (HBMEC) were exposed to alumina and control nanoparticles in dose- and time-responsive manners. Treatment with nano-alumina markedly reduced HBMEC viability, altered mitochondrial potential, increased cellular oxidation, and decreased tight junction protein expression as compared to control nanoparticles. Alterations of tight junction protein levels were prevented by cellular enrichment with glutathione. In the second series of experiments, rats were infused with nano-alumina at the dose of 29 mg/kg and the brains were stained for expression of tight junction proteins. Treatment with nano-alumina resulted in a marked fragmentation and disruption of integrity of claudin-5 and occludin. These results indicate that cerebral vasculature can be affected by nano-alumina. In addition, our data indicate that alterations of mitochondrial functions may be the underlying mechanism of nano-alumina toxicity.

Occludin confers adhesiveness when expressed in fibroblasts.

PubMed

Van Itallie, C M; Anderson, J M

1997-05-01

Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cell-cell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.

Downregulation of tight junction-associated MARVEL protein marvelD3 during epithelial-mesenchymal transition in human pancreatic cancer cells.

PubMed

Kojima, Takashi; Takasawa, Akira; Kyuno, Daisuke; Ito, Tatsuya; Yamaguchi, Hiroshi; Hirata, Koichi; Tsujiwaki, Mitsuhiro; Murata, Masaki; Tanaka, Satoshi; Sawada, Norimasa

2011-10-01

The novel tight junction protein marvelD3 contains a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain like occludin and tricellulin. However, little is yet known about the detailed role and regulation of marvelD3 in normal epithelial cells and cancer cells, including pancreatic cancer. In the present study, we investigated marvelD3 expression in well and poorly differentiated human pancreatic cancer cell lines and normal pancreatic duct epithelial cells in which the hTERT gene was introduced into human pancreatic duct epithelial cells in primary culture, and the changes of marvelD3 during Snail-induced epithelial-mesenchymal transition (EMT) under hypoxia, TGF-β treatment and knockdown of FOXA2 in well differentiated pancreatic cancer HPAC cells. MarvelD3 was transcriptionally downregulated in poorly differentiated pancreatic cancer cells and during Snail-induced EMT of pancreatic cancer cells in which Snail was highly expressed and the fence function downregulated, whereas it was maintained in well differentiated human pancreatic cancer cells and normal pancreatic duct epithelial cells. Depletion of marvelD3 by siRNAs in HPAC cells resulted in downregulation of barrier functions indicated as a decrease in transepithelial electric resistance and an increase of permeability to fluorescent dextran tracers, whereas it did not affect fence function of tight junctions. In conclusion, marvelD3 is transcriptionally downregulated in Snail-induced EMT during the progression for the pancreatic cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

Effects of Human Parvovirus B19 and Bocavirus VP1 Unique Region on Tight Junction of Human Airway Epithelial A549 Cells

PubMed Central

Chiu, Chun-Ching; Shi, Ya-Fang; Yang, Jiann-Jou; Hsiao, Yuan-Chao; Tzang, Bor-Show; Hsu, Tsai-Ching

2014-01-01

As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity. PMID:25268969

Bovine dairy complex lipids improve in vitro measures of small intestinal epithelial barrier integrity.

PubMed

Anderson, Rachel C; MacGibbon, Alastair K H; Haggarty, Neill; Armstrong, Kelly M; Roy, Nicole C

2018-01-01

Appropriate intestinal barrier maturation is essential for absorbing nutrients and preventing pathogens and toxins from entering the body. Compared to breast-fed infants, formula-fed infants are more susceptible to barrier dysfunction-associated illnesses. In infant formula dairy lipids are usually replaced with plant lipids. We hypothesised that dairy complex lipids improve in vitro intestinal epithelial barrier integrity. We tested milkfat high in conjugated linoleic acid, beta serum (SureStart™Lipid100), beta serum concentrate (BSC) and a ganglioside-rich fraction (G600). Using Caco-2 cells as a model of the human small intestinal epithelium, we analysed the effects of the ingredients on trans-epithelial electrical resistance (TEER), mannitol flux, and tight junction protein co-localisation. BSC induced a dose-dependent improvement in TEER across unchallenged cell layers, maintained the co-localisation of tight junction proteins in TNFα-challenged cells with increased permeability, and mitigated the TEER-reducing effects of lipopolysaccharide (LPS). G600 also increased TEER across healthy and LPS-challenged cells, but it did not alter the co-location of tight junction proteins in TNFα-challenged cells. SureStart™Lipid100 had similar TEER-increasing effects to BSC when added at twice the concentration (similar lipid concentration). Ultimately, this research aims to contribute to the development of infant formulas supplemented with dairy complex lipids that support infant intestinal barrier maturation.

Oxidative stress induced by potassium bromate exposure results in altered tight junction protein expression in renal proximal tubule cells.

PubMed

Limonciel, Alice; Wilmes, Anja; Aschauer, Lydia; Radford, Robert; Bloch, Katarzyna M; McMorrow, Tara; Pfaller, Walter; van Delft, Joost H; Slattery, Craig; Ryan, Michael P; Lock, Edward A; Jennings, Paul

2012-11-01

Potassium bromate (KBrO(3)) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO(3) in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E. A genome-wide transcriptomic screen was carried out from cells exposed to a sub-lethal concentration of KBrO(3) for 6, 24 and 72 h. Pathway analysis identified "glutathione metabolism", "Nrf2-mediated oxidative stress" and "tight junction (TJ) signalling" as the most enriched pathways. TJ signalling was less impacted in the rat model, and further studies revealed low transepithelial electrical resistance (TEER) and an absence of several TJ proteins in NRK-52E cells. In RPTEC/TERT1 cells, KBrO(3) exposure caused a decrease in TEER and resulted in altered expression of several TJ proteins. N-Acetylcysteine co-incubation prevented these effects. These results demonstrate that oxidative stress has, in conjunction with the activation of the cytoprotective Nrf2 pathway, a dramatic effect on the expression of tight junction proteins. The further understanding of the cross-talk between these two pathways could have major implications for epithelial repair, carcinogenesis and metastasis.

Bovine dairy complex lipids improve in vitro measures of small intestinal epithelial barrier integrity

PubMed Central

MacGibbon, Alastair K. H.; Haggarty, Neill; Armstrong, Kelly M.; Roy, Nicole C.

2018-01-01

Appropriate intestinal barrier maturation is essential for absorbing nutrients and preventing pathogens and toxins from entering the body. Compared to breast-fed infants, formula-fed infants are more susceptible to barrier dysfunction-associated illnesses. In infant formula dairy lipids are usually replaced with plant lipids. We hypothesised that dairy complex lipids improve in vitro intestinal epithelial barrier integrity. We tested milkfat high in conjugated linoleic acid, beta serum (SureStart™Lipid100), beta serum concentrate (BSC) and a ganglioside-rich fraction (G600). Using Caco-2 cells as a model of the human small intestinal epithelium, we analysed the effects of the ingredients on trans-epithelial electrical resistance (TEER), mannitol flux, and tight junction protein co-localisation. BSC induced a dose-dependent improvement in TEER across unchallenged cell layers, maintained the co-localisation of tight junction proteins in TNFα-challenged cells with increased permeability, and mitigated the TEER-reducing effects of lipopolysaccharide (LPS). G600 also increased TEER across healthy and LPS-challenged cells, but it did not alter the co-location of tight junction proteins in TNFα-challenged cells. SureStart™Lipid100 had similar TEER-increasing effects to BSC when added at twice the concentration (similar lipid concentration). Ultimately, this research aims to contribute to the development of infant formulas supplemented with dairy complex lipids that support infant intestinal barrier maturation. PMID:29304106

Physiological roles of claudins in kidney tubule paracellular transport.

PubMed

Muto, Shigeaki

2017-01-01

The paracellular pathways in renal tubular epithelia such as the proximal tubules, which reabsorb the largest fraction of filtered solutes and water and are leaky epithelia, are important routes for transepithelial transport of solutes and water. Movement occurs passively via an extracellular route through the tight junction between cells. The characteristics of paracellular transport vary among different nephron segments with leaky or tighter epithelia. Claudins expressed at tight junctions form pores and barriers for paracellular transport. Claudins are from a multigene family, comprising at least 27 members in mammals. Multiple claudins are expressed at tight junctions of individual nephron segments in a nephron segment-specific manner. Over the last decade, there have been advances in our understanding of the structure and functions of claudins. This paper is a review of our current knowledge of claudins, with special emphasis on their physiological roles in proximal tubule paracellular solute and water transport. Copyright © 2017 the American Physiological Society.

In Vitro Evaluation of the Impact of the Probiotic E. coli Nissle 1917 on Campylobacter jejuni's Invasion and Intracellular Survival in Human Colonic Cells.

PubMed

Helmy, Yosra A; Kassem, Issmat I; Kumar, Anand; Rajashekara, Gireesh

2017-01-01

Campylobacter jejuni is a leading cause of bacterial food poisoning in humans. Due to the rise in antibiotic-resistant Campylobacter , there exists a need to develop antibiotic-independent interventions to control infections in humans. Here, we evaluated the impact of Escherichia coli Nissle 1917 (EcN), a probiotic strain, on C. jejuni's invasion and intracellular survival in polarized human colonic cells (HT-29). To further understand how EcN mediates its impact, the expression of 84 genes associated with tight junctions and cell adhesion was profiled in HT-29 cells after treatment with EcN and challenge with C. jejuni . The pre-treatment of polarized HT-29 cells with EcN for 4 h showed a significant effect on C. jejuni 's invasion (∼2 log reduction) of the colonic cells. Furthermore, no intracellular C. jejuni were recovered from EcN pre-treated HT-29 cells at 24 h post-infection. Other probiotic strains tested had no significant impact on C. jejuni invasion and intracellular survival. C. jejuni decreased the expression of genes associated with epithelial cells permeability and barrier function in untreated HT-29 cells. However, EcN positively affected the expression of genes that are involved in enhanced intestinal barrier function, decreased cell permeability, and increased tight junction integrity. The results suggest that EcN impedes C. jejuni invasion and subsequent intracellular survival by affecting HT-29 cells barrier function and tight junction integrity. We conclude that EcN might be a viable alternative for controlling C. jejuni infections.

Angelica archengelica extract induced perturbation of rat skin and tight junctional protein (ZO-1) of HaCaT cells

PubMed Central

Kaushal, N.; Naz, S.; Tiwary, AK.

2011-01-01

Background and purpose of the study Herbal enhancers compared to the synthetic ones have shown less toxis effects. Coumarins have been shown at concentrations inhibiting phospoliphase C-Y (Phc-Y) are able to enhance tight junction (TJ) permeability due to hyperpoalation of Zonolous Occludense-1 (ZO-1) proteins. The purpose of this study was to evaluate the influence of ethanolic extract of Angelica archengelica (AA-E) which contain coumarin on permeation of repaglinide across rat epidermis and on the tight junction plaque protein ZO-1 in HaCaT cells. Methods Transepidermal water loss (TEWL) from the rat skin treated with different concentrations of AA-E was assessed by Tewameter. Scanning and Transmission Electron Microscopy (TEM) on were performed on AA-E treated rat skin portions. The possibility of AA-E influence on the architecture of tight junctions by adverse effect on the cytoplasmic ZO-1 in HaCaT cells was investigated. Finally, the systemic delivery of repaglinide from the optimized transdermal formulation was investigated in rats. Results The permeation of repaglinide across excised rat epidermis was 7-fold higher in the presence of AA-E (5% w/v) as compared to propylene glycol:ethanol (7:3) mixture. The extract was found to perturb the lipid microconstituents in both excised and viable rat skin, although, the effect was less intense in the later. The enhanced permeation of repaglinide across rat epidermis excised after treatment with AA-E (5% w/v) for different periods was in concordance with the high TEWL values of similarly treated viable rat skin. Further, the observed increase in intercellular space, disordering of lipid structure and corneocyte detachment indicated considerable effect on the ultrastructure of rat epidermis. Treatment of HaCaT cell line with AA-E (0.16% w/v) for 6 hrs influenced ZO-1 as evidenced by reduced immunofluorescence of anti-TJP1 (ZO-1) antibody in Confocal Laser Scanning Microscopy studies (CLSM) studies. The plasma concentration of repaglinide from transdermal formulation was maintained higher and for longer time as compared to oral administration of repaglinide. Major conclusion Results suggest the overwhelming influence of Angelica archengelica in enhancing the percutaneous permeation of repaglinide to be mediated through perturbation of skin lipids and tight junction protein (ZO-1). PMID:22615634

Tight Junction Protein 1a regulates pigment cell organisation during zebrafish colour patterning.

PubMed

Fadeev, Andrey; Krauss, Jana; Frohnhöfer, Hans Georg; Irion, Uwe; Nüsslein-Volhard, Christiane

2015-04-27

Zebrafish display a prominent pattern of alternating dark and light stripes generated by the precise positioning of pigment cells in the skin. This arrangement is the result of coordinated cell movements, cell shape changes, and the organisation of pigment cells during metamorphosis. Iridophores play a crucial part in this process by switching between the dense form of the light stripes and the loose form of the dark stripes. Adult schachbrett (sbr) mutants exhibit delayed changes in iridophore shape and organisation caused by truncations in Tight Junction Protein 1a (ZO-1a). In sbr mutants, the dark stripes are interrupted by dense iridophores invading as coherent sheets. Immuno-labelling and chimeric analyses indicate that Tjp1a is expressed in dense iridophores but down-regulated in the loose form. Tjp1a is a novel regulator of cell shape changes during colour pattern formation and the first cytoplasmic protein implicated in this process.

How hormones influence composition and physiological function of the brain-blood barrier.

PubMed

Hampl, R; Bičíková, M; Sosvorová, L

2015-01-01

Hormones exert many actions in the brain. Their access and effects in the brain are regulated by the blood-brain barrier (BBB). Hormones as other substances may enter the brain and vice versa either by paracellular way requiring breaching tight junctions stitching the endothelial cells composing the BBB, or by passage through the cells (transcellular way). Hormones influence both ways through their receptors, both membrane and intracellular, present on/in the BBB. In the review the main examples are outlined how hormones influence the expression and function of proteins forming the tight junctions, as well as how they regulate expression and function of major protein transporters mediating transport of various substances including hormone themselves.

Polarity Proteins as Regulators of Cell Junction Complexes: Implications for Breast Cancer

PubMed Central

Bazzoun, Dana; Lelièvre, Sophie; Talhouk, Rabih

2013-01-01

The epithelium of multicellular organisms possesses a well-defined architecture, referred to as polarity that coordinates the regulation of essential cell features. Polarity proteins are intimately linked to the protein complexes that make the tight, adherens and gap junctions; they contribute to the proper localization and assembly of these cell-cell junctions within cells and consequently to functional tissue organization. The establishment of cell-cell junctions and polarity are both implicated in the regulation of epithelial modifications in normal and cancer situations. Uncovering the mechanisms through which cell-cell junctions and epithelial polarization are established and how their interaction with the microenvironment direct cell and tissue organization has opened new venues for the development of cancer therapies. In this review, we focus on the breast epithelium to highlight how polarity and cell-cell junction proteins interact together in normal and cancerous contexts to regulate major cellular mechanisms such as migration. The impact of these proteins on epigenetic mechanisms responsible for resetting cells towards oncogenesis is discussed in light of increasing evidence that tissue polarity modulates chromatin function. Finally, we give an overview of recent breast cancer therapies that target proteins involved in cell-cell junctions. PMID:23458609

A permeability barrier surrounds taste buds in lingual epithelia

PubMed Central

Dando, Robin; Pereira, Elizabeth; Kurian, Mani; Barro-Soria, Rene; Chaudhari, Nirupa

2014-01-01

Epithelial tissues are characterized by specialized cell-cell junctions, typically localized to the apical regions of cells. These junctions are formed by interacting membrane proteins and by cytoskeletal and extracellular matrix components. Within the lingual epithelium, tight junctions join the apical tips of the gustatory sensory cells in taste buds. These junctions constitute a selective barrier that limits penetration of chemosensory stimuli into taste buds (Michlig et al. J Comp Neurol 502: 1003–1011, 2007). We tested the ability of chemical compounds to permeate into sensory end organs in the lingual epithelium. Our findings reveal a robust barrier that surrounds the entire body of taste buds, not limited to the apical tight junctions. This barrier prevents penetration of many, but not all, compounds, whether they are applied topically, injected into the parenchyma of the tongue, or circulating in the blood supply, into taste buds. Enzymatic treatments indicate that this barrier likely includes glycosaminoglycans, as it was disrupted by chondroitinase but, less effectively, by proteases. The barrier surrounding taste buds could also be disrupted by brief treatment of lingual tissue samples with DMSO. Brief exposure of lingual slices to DMSO did not affect the ability of taste buds within the slice to respond to chemical stimulation. The existence of a highly impermeable barrier surrounding taste buds and methods to break through this barrier may be relevant to basic research and to clinical treatments of taste. PMID:25209263

Comparison of tight junction protein expression in the ciliary epithelia of mouse, rabbit, cat and human eyes.

PubMed

Karim, M J; Biswas, S; Bhattacherjee, P; Paterson, C A

2011-06-01

Tight junctions in the nonpigmented epithelium (NPE) of the ciliary processes and the iris vascular endothelium form the ocular blood aqueous barrier that prevents leakage of proteins, immune cells and non-immune cells of blood into the anterior chamber. We attempted to determine whether ultrastructural differences in tight junctions reported in earlier studies are reflected in the expression pattern of tight junction proteins (TJP) and whether the TJP in mice, rabbits and cats resemble those of humans. For immunohistochemistry, 10 μm thick cryosections were rehydrated in PBS and fixed in 50 mM ammonium chloride at room temperature. After rinses in PBS, the sections were incubated twice in 0.1% Triton X-100, 10% goat serum, specific primary antibody or in PBS. After rinses in PBS, the sections were incubated in FITC-conjugated secondary antibody. After rinses in PBS, the sections were mounted with Vectashield mounting medium with propidium iodide, examined and photographed using a confocal microscope. The expression patterns of TJP in ocular ciliary epithelium of human, rabbit, cat and mouse were similar. Occludin immunoreactivity was observed as a sharp line along the junction between pigmented epithelium (PE) and NPE, and along the apico-lateral surfaces of NPE. Very light staining of the ciliary stroma was observed in cat and mouse. Claudin-1 was expressed along the entire boundaries of NPE and was more distinct between PE and NPE in rabbit. The ciliary stroma showed faint staining in cat and mouse. ZO-1 showed staining between PE and NPE, and at the adjacent membrane. Moderate staining was seen in PE in cat and mouse, which suggests that claudin-1, occludin and ZO-1 are expressed along the junction between PE and NPE, and the apico-lateral border of NPE. Lack of major difference in the expression patterns among the different species is important for validating the use of rabbit, mouse and cat in studies of intraocular inflammation in humans.

Macrophages induce "budding" in aggressive human colon cancer subtypes by protease-mediated disruption of tight junctions.

PubMed

Trumpi, Kari; Frenkel, Nicola; Peters, Timo; Korthagen, Nicoline M; Jongen, Jennifer M J; Raats, Daniëlle; van Grevenstein, Helma; Backes, Yara; Moons, Leon M; Lacle, Miangela M; Koster, Jan; Zwijnenburg, Danny; Borel Rinkes, Inne H M; Kranenburg, Onno

2018-04-13

Primary human colorectal tumors with a high stromal content have an increased capacity to metastasize. Cancer-associated fibroblasts (CAFs) promote metastasis, but the contribution of other stromal cell types is unclear. Here we searched for additional stromal cell types that contribute to aggressive tumor cell behavior. By making use of the 'immunome compendium'-a collection of gene signatures reflecting the presence of specific immune cell-types-we show that macrophage signatures are most strongly associated with a high CAF content and with poor prognosis in multiple large cohorts of primary tumors and liver metastases. Co-culturing macrophages with patient-derived colonospheres promoted 'budding' of small clusters of tumor cells from the bulk. Immunohistochemistry showed that budding tumor clusters in stroma-rich areas of T1 colorectal carcinomas were surrounded by macrophages. In vitro budding was accompanied by reduced levels of the tight junction protein occludin, but OCLN mRNA levels did not change, nor did markers of epithelial mesenchymal transition. Budding was accompanied by nuclear accumulation of β-catenin, which was also observed in budding tumor cell clusters in situ . The NFκB inhibitor Sanguinarine resulted in a decrease in MMP7 protein expression and both NFκB inhibitor Sanguinarine and MMP inhibitor Batimastat prevented occludin degradation and budding. We conclude that macrophages contribute to the aggressive nature of stroma-rich colon tumors by promoting an MMP-dependent pathway that operates in parallel to classical EMT and leads to tight junction disruption.

Changes in intestinal tight junction permeability associated with industrial food additives explain the rising incidence of autoimmune disease.

PubMed

Lerner, Aaron; Matthias, Torsten

2015-06-01

The incidence of autoimmune diseases is increasing along with the expansion of industrial food processing and food additive consumption. The intestinal epithelial barrier, with its intercellular tight junction, controls the equilibrium between tolerance and immunity to non-self-antigens. As a result, particular attention is being placed on the role of tight junction dysfunction in the pathogenesis of AD. Tight junction leakage is enhanced by many luminal components, commonly used industrial food additives being some of them. Glucose, salt, emulsifiers, organic solvents, gluten, microbial transglutaminase, and nanoparticles are extensively and increasingly used by the food industry, claim the manufacturers, to improve the qualities of food. However, all of the aforementioned additives increase intestinal permeability by breaching the integrity of tight junction paracellular transfer. In fact, tight junction dysfunction is common in multiple autoimmune diseases and the central part played by the tight junction in autoimmune diseases pathogenesis is extensively described. It is hypothesized that commonly used industrial food additives abrogate human epithelial barrier function, thus, increasing intestinal permeability through the opened tight junction, resulting in entry of foreign immunogenic antigens and activation of the autoimmune cascade. Future research on food additives exposure-intestinal permeability-autoimmunity interplay will enhance our knowledge of the common mechanisms associated with autoimmune progression. Copyright © 2015. Published by Elsevier B.V.

Spatiotemporal loss of K+ transport proteins in the developing cochlear lateral wall of guinea pigs with hereditary deafness.

PubMed

Jin, Zhe; Ulfendahl, Mats; Järlebark, Leif

2008-01-01

Genetic deafness is one of the most common human genetic birth defects. To understand the molecular mechanisms underlying human hereditary deafness, deaf animal strains have proved to be invaluable models. The German waltzing guinea pig is a new strain of animals with unidentified gene mutation(s), displaying recessively inherited cochleovestibular impairment. Histological investigations of the homozygous animals (gw/gw) revealed a collapse of the endolymphatic compartment and malformation of stria vascularis. RT-PCR showed a significant reduction in expression of the strial intermediate cell-specific gene Dct and the tight-junction gene Cldn11 in the embryonic day (E)40 and adult gw/gw cochlear lateral wall. Immunohistochemical analysis of the gw/gw cochlea showed loss of the tight junction protein CLDN11 in strial basal cells from E40, loss of the potassium channel subunit KCNJ10 in strial intermediate cells from E50, and loss of the Na-K-Cl cotransporter SLC12A2 in strial marginal cells from E50. In addition, a temporary loss of the gap junction protein GJB2 (connexin 26) between fibrocytes in the spiral ligament of the E50 gw/gw cochlea was observed. The barrier composed of tight junctions between strial basal cells was disrupted in the gw/gw cochlea as indicated by a biotin tracer permeability assay. In conclusion, spatiotemporal loss of K+ transport proteins in the cochlear lateral wall is caused by malformation of the stria vascularis in the developing German waltzing guinea pig inner ear. This new animal strain may serve as a good model for studying human genetic deafness due to disruption of inner ear ion homeostasis.

Garlic (Allium sativum) feeding impairs Sertoli cell junctional proteins in male Wistar rat testis: microscopy study.

PubMed

Hammami, I; Nahdi, A; Atig, F; El May, A; El May, M V

2016-12-01

Sertoli cell junctions, such as adhesion junction (AJ), gap junction (GJ) and tight junction (TJ), are important for maintaining spermatogenesis. In previous studies, we showed the inhibitory effect of crude garlic (Allium sativum, As) on spermatogenesis and steroidogenesis. The aim of this work was to complete our investigation on the impact of this plant, especially on Sertoli cell junctional proteins (SCJPs). During 1 month, 24 male rats were divided into groups: group control (0% of As) and treated groups fed 5%, 10% and 15% of As. Light and electron microscopy observations were performed to localise junctional proteins: connexin-43, Zona Occluding-1 and N-cadherin (immunohistochemistry) and to describe junctions. We showed that the specific cells involved in the localisation of the SCJP were similar in both control and treated groups, but with different immunoreactivity intensity between them. The electron microscopy observation focused on TJs between Sertoli cells, constituting the blood-testis barrier, showed ultrastructural changes such as fragmentation of TJs between adjacent Sertoli cell membranes and dilatation of rough endoplasmic reticulum saccules giving an aspect of scale to these junctions. We concluded that crude garlic consumption during 1 month induces perturbations on Sertoli cell junctions. These alterations can explain apoptosis in testicular germ cells previously showed. © 2016 Blackwell Verlag GmbH.

Epidermal cell turnover across tight junctions based on Kelvin's tetrakaidecahedron cell shape

PubMed Central

Yokouchi, Mariko; Atsugi, Toru; van Logtestijn, Mark; Tanaka, Reiko J; Kajimura, Mayumi; Suematsu, Makoto; Furuse, Mikio; Amagai, Masayuki; Kubo, Akiharu

2016-01-01

In multicellular organisms, cells adopt various shapes, from flattened sheets of endothelium to dendritic neurons, that allow the cells to function effectively. Here, we elucidated the unique shape of cells in the cornified stratified epithelia of the mammalian epidermis that allows them to achieve homeostasis of the tight junction (TJ) barrier. Using intimate in vivo 3D imaging, we found that the basic shape of TJ-bearing cells is a flattened Kelvin's tetrakaidecahedron (f-TKD), an optimal shape for filling space. In vivo live imaging further elucidated the dynamic replacement of TJs on the edges of f-TKD cells that enables the TJ-bearing cells to translocate across the TJ barrier. We propose a spatiotemporal orchestration model of f-TKD cell turnover, where in the classic context of 'form follows function', cell shape provides a fundamental basis for the barrier homeostasis and physical strength of cornified stratified epithelia. DOI: http://dx.doi.org/10.7554/eLife.19593.001 PMID:27894419

Occludin Independently Regulates Permeability under Hydrostatic Pressure and Cell Division in Retinal Pigment Epithelial Cells

PubMed Central

Phillips, Brett E.; Cancel, Limary; Tarbell, John M.; Antonetti, David A.

2008-01-01

Purpose The aim of this study was to determine the function of the tight junction protein occludin in the control of permeability, under diffusive and hydrostatic pressures, and its contribution to the control of cell division in retinal pigment epithelium. Methods Occludin expression was inhibited in the human retinal pigment epithelial cell line ARPE-19 by siRNA. Depletion of occludin was confirmed by Western blot, confocal microscopy, and RT-PCR. Paracellular permeability of cell monolayers to fluorescently labeled 70 kDa dextran, 10 kDa dextran, and 467 Da tetramethylrhodamine (TAMRA) was examined under diffusive conditions or after the application of 10 cm H2O transmural pressure. Cell division rates were determined by tritiated thymidine incorporation and Ki67 immunoreactivity. Cell cycle inhibitors were used to determine whether changes in cell division affected permeability. Results Occludin depletion increased diffusive paracellular permeability to 467 Da TAMRA by 15%, and permeability under hydrostatic pressure was increased 50% compared with control. Conversely, depletion of occludin protein with siRNA did not alter diffusive permeability to 70 kDa and 10 kDa RITC-dextran, and permeability to 70 kDa dextran was twofold lower in occludin-depleted cells under hydrostatic pressure conditions. Occludin depletion also increased thymidine incorporation by 90% and Ki67-positive cells by 50%. Finally, cell cycle inhibitors did not alter the effect of occludin siRNA on paracellular permeability. Conclusions The data suggest that occludin regulates tight junction permeability in response to changes in hydrostatic pressure. Furthermore, these data suggest that occludin also contributes to the control of cell division, demonstrating a novel function for this tight junction protein. PMID:18263810

Eye lens membrane junctional microdomains: a comparison between healthy and pathological cases

NASA Astrophysics Data System (ADS)

Buzhynskyy, Nikolay; Sens, Pierre; Behar-Cohen, Francine; Scheuring, Simon

2011-08-01

The eye lens is a transparent tissue constituted of tightly packed fiber cells. To maintain homeostasis and transparency of the lens, the circulation of water, ions and metabolites is required. Junctional microdomains connect the lens cells and ensure both tight cell-to-cell adhesion and intercellular flow of fluids through a microcirculation system. Here, we overview membrane morphology and tissue functional requirements of the mammalian lens. Atomic force microscopy (AFM) has opened up the possibility of visualizing the junctional microdomains at unprecedented submolecular resolution, revealing the supramolecular assembly of lens-specific aquaporin-0 (AQP0) and connexins (Cx). We compare the membrane protein assembly in healthy lenses with senile and diabetes-II cataract cases and novel data of the lens membranes from a congenital cataract. In the healthy case, AQP0s form characteristic square arrays confined by connexons. In the cases of senile and diabetes-II cataract patients, connexons were degraded, leading to malformation of AQP0 arrays and breakdown of the microcirculation system. In the congenital cataract, connexons are present, indicating probable non-membranous grounds for lens opacification. Further, we discuss the energetic aspects of the membrane organization in junctional microdomains. The AFM hence becomes a biomedical nano-imaging tool for the analysis of single-membrane protein supramolecular association in healthy and pathological membranes.

The Nuclear and Adherent Junction Complex Component Protein Ubinuclein Negatively Regulates the Productive Cycle of Epstein-Barr Virus in Epithelial Cells▿

PubMed Central

Gruffat, Henri; Lupo, Julien; Morand, Patrice; Boyer, Véronique; Manet, Evelyne

2011-01-01

The Epstein-Barr Virus (EBV) productive cycle is initiated by the expression of the viral trans-activator EB1 (also called Zebra, Zta, or BZLF1), which belongs to the basic leucine zipper transcription factor family. We have previously identified the cellular NACos (nuclear and adherent junction complex components) protein ubinuclein (Ubn-1) as a partner for EB1, but the function of this complex has never been studied. Here, we have evaluated the consequences of this interaction on the EBV productive cycle and find that Ubn-1 overexpression represses the EBV productive cycle whereas Ubn-1 downregulation by short hairpin RNA (shRNA) increases virus production. By a chromatin immunoprecipitation (ChIP) assay, we show that Ubn-1 blocks EB1-DNA interaction. We also show that in epithelial cells, relocalization and sequestration of Ubn-1 to the tight junctions of nondividing cells allow increased activation of the productive cycle. We propose a model in which Ubn-1 is a modulator of the EBV productive cycle: in proliferating epithelial cells, Ubn-1 is nuclear and inhibits activation of the productive cycle, whereas in differentiated cells, Ubn-1 is sequestrated to tight junctions, thereby allowing EB1 to fully function in the nucleus. PMID:21084479

Delocalized Claudin-1 promotes metastasis of human osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jian, Yuekui; Chen, Changqiong; Li, Bo

2015-10-23

Tight junction proteins (TJPs) including Claudins, Occludin and tight junction associated protein Zonula occludens-1 (ZO-1), are the most apical component of junctional complex that mediates cell–cell adhesion in epithelial and endothelial cells. In human malignancies, TJPs are often deregulated and affect cellular behaviors of tumor cells. In this study, we investigated alternations of TJPs and related biological characteristics in human osteosarcoma (OS). Claudin1 was increased in the metastatic OS cells (KRIB and KHOS) compared with the normal osteoblast cells (hFOB1.19) or primary tumor cells (HOS and U2OS), whereas no significant difference was found in Occludin and ZO-1. Immunohistochemistry, immunofluorescence andmore » Western blotting revealed that Claudin1 was initially localized at cell junctions of normal osteoblasts, but substantially delocalized to the nucleus of metastatic OS cells. Phenotypically, inhibition of the nucleus Claudin1 expression compromised the metastatic potential of KRIB and KHOS cells. Moreover, we found that protein kinase C (PKC) but not PKA phosphorylation influenced Claudin1 expression and cellular functions, as PKC inhibitor (Go 6983 and Staurosporine) or genetic silencing of PKC reduced Claudin1 expression and decreased the motility of KRIB and KHOS cells. Taken together, our study implied that delocalization of claudin-1 induced by PKC phosphorylation contributes to metastatic capacity of OS cells. - Highlights: • Claudin1 is increased during the malignant transformation of human OS. • Delocalization of Claudin1 in metastatic OS cells. • Silencing nuclear Claudin1 expression inhibits cell invasion of OS. • Deregulated Claudin1 is regulated by PKC.« less

Involvement of the interaction of afadin with ZO-1 in the formation of tight junctions in Madin-Darby canine kidney cells.

PubMed

Ooshio, Takako; Kobayashi, Reiko; Ikeda, Wataru; Miyata, Muneaki; Fukumoto, Yuri; Matsuzawa, Naomi; Ogita, Hisakazu; Takai, Yoshimi

2010-02-12

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with beta- and alpha-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-DeltaPR1-2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.

Involvement of the Interaction of Afadin with ZO-1 in the Formation of Tight Junctions in Madin-Darby Canine Kidney Cells*

PubMed Central

Ooshio, Takako; Kobayashi, Reiko; Ikeda, Wataru; Miyata, Muneaki; Fukumoto, Yuri; Matsuzawa, Naomi; Ogita, Hisakazu; Takai, Yoshimi

2010-01-01

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with β- and α-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-ΔPR1–2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells. PMID:20008323

Claudins and the Modulation of Tight Junction Permeability

PubMed Central

Günzel, Dorothee

2013-01-01

Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. This review summarizes our current knowledge of this large protein family and discusses recent advances in our understanding of their structure and physiological functions. PMID:23589827

Characterization of the column and autocellular junctions that define the vasculature of gill lamellae.

PubMed

Kato, Akira; Nakamura, Korefumi; Kudo, Hisayuki; Tran, Yen Ha; Yamamoto, Yoko; Doi, Hiroyuki; Hirose, Shigehisa

2007-09-01

Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the "column junction" and "autocellular junction" and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the "column junction." In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae.

Tight junctions of the proximal tubule and their channel proteins.

PubMed

Fromm, Michael; Piontek, Jörg; Rosenthal, Rita; Günzel, Dorothee; Krug, Susanne M

2017-08-01

The renal proximal tubule achieves the majority of renal water and solute reabsorption with the help of paracellular channels which lead through the tight junction. The proteins forming such channels in the proximal tubule are claudin-2, claudin-10a, and possibly claudin-17. Claudin-2 forms paracellular channels selective for small cations like Na + and K + . Independently of each other, claudin-10a and claudin-17 form anion-selective channels. The claudins form the paracellular "pore pathway" and are integrated, together with purely sealing claudins and other tight junction proteins, in the belt of tight junction strands surrounding the tubular epithelial cells. In most species, the proximal tubular tight junction consists of only 1-2 (pars convoluta) to 3-5 (pars recta) horizontal strands. Even so, they seal the tubule very effectively against leak passage of nutrients and larger molecules. Remarkably, claudin-2 channels are also permeable to water so that 20-25% of proximal water absorption may occur paracellularly. Although the exact structure of the claudin-2 channel is still unknown, it is clear that Na + and water share the same pore. Already solved claudin crystal structures reveal a characteristic β-sheet, comprising β-strands from both extracellular loops, which is anchored to a left-handed four-transmembrane helix bundle. This allowed homology modeling of channel-forming claudins present in the proximal tubule. The surface of cation- and anion-selective claudins differ in electrostatic potentials in the area of the proposed ion channel, resulting in the opposite charge selectivity of these claudins. Presently, while models of the molecular structure of the claudin-based oligomeric channels have been proposed, its full understanding has only started.

Synergistic effects of arsenic trioxide combined with ascorbic acid in human osteosarcoma MG-63 cells: a systems biology analysis.

PubMed

Huang, X C; Maimaiti, X Y M; Huang, C W; Zhang, L; Li, Z B; Chen, Z G; Gao, X; Chen, T Y

2014-01-01

To further understand the synergistic mechanism of As2O3 and asscorbic acid (AA) in human osteosarcoma MG-63 cells by systems biology analysis. Human osteosarcoma MG-63 cells were treated by As2O3 (1 µmol/L), AA (62.5 µmol/L) and combined drugs (1 µmol/L As2O3 plus 62.5 µmol/L AA). Dynamic morphological characteristics were recorded by Cell-IQ system, and growth rate was calculated. Illumina beadchip assay was used to analyze the differential expression genes in different groups. Synergic effects on differential expression genes (DEGs) were analyzed by mixture linear model and singular value decomposition model. KEGG pathway annotations and GO enrichment analysis were performed to figure out the pathways involved in the synergic effects. We captured 1987 differential expression genes in combined therapy MG-63 cells. FAT1 gene was significantly upregulated in all three groups, which is a promising drug target as an important tumor suppressor analogue; meanwhile, HIST1H2BD gene was markedly downregulated in the As2O3 monotherapy group and the combined therapy group, which was found to be upregulated in prostatic cancer. These two genes might play critical roles in synergetic effects of AA and As2O3, although the exact mechanism needs further investigation. KEGG pathway analysis showed many DEGs were related with tight junction, and GO analysis also indicated that DEGs in the combined therapy cells gathered in occluding junction, apical junction complex, cell junction, and tight junction. AA potentiates the efficacy of As2O3 in MG-63 cells. Systems biology analysis showed the synergic effect on the DEGs.

Epithelial Permeability Alterations in an In Vitro Air-Liquid Interface Model of Allergic Fungal Rhinosinusitis

PubMed Central

Den Beste, Kyle A.; Hoddeson, Elizabeth K.; Parkos, Charles A.; Nusrat, Asma; Wise, Sarah K.

2012-01-01

Background Chronic rhinosinusitis (CRS) is an inflammatory upper-airway disease with numerous etiologies. Patients with a characteristic subtype of CRS, allergic fungal rhinosinusitis (AFRS), display increased expression of Th2 cytokines and antigen-specific IgE. Various sinonasal inflammatory conditions are associated with alterations in epithelial barrier function. The aim of this study was to compare epithelial permeability and intercellular junctional protein expression amongst cultured primary sinonasal cells from AFRS patients versus non-inflammatory controls. Methods Epithelial cells isolated from paranasal sinus mucosa of AFRS and non-inflammatory control patients were grown to confluence on permeable supports and transitioned to air-liquid interface (ALI). Trans-epithelial resistance (TER) was measured with a horizontal Ussing chamber to characterize the functional permeability of each cell type. After TER recordings were complete, a panel of intercellular junctional proteins was assessed by Western blot and immunofluorescence labeling followed by confocal microscopy. Results After 12 samples were measured from each group, we observed a 41% mean decrease in TER in AFRS cells (296±89 ohms × cm2) compared to control (503±134 ohms × cm2, P=0.006). TER deficits observed in AFRS were associated with decreased expression of the tight junction proteins occludin and Junctional Adhesion Molecule-A (JAM-A), and increased expression of a leaky tight junction protein claudin-2. Conclusions Cultured sinonasal epithelium from AFRS patients displayed increased epithelial permeability and altered expression of intercellular junctional proteins. Given that these cells were not incubated with inflammatory cytokines in vitro, the cultured AFRS epithelial alterations may represent a retained modification in protein expression from the in vivo phenotype. PMID:22927233

Non-nucleoside reverse transcriptase inhibitor efavirenz increases monolayer permeability of human coronary artery endothelial cells

PubMed Central

Jamaluddin, Md Saha; Lin, Peter H.; Yao, Qizhi; Chen, Changyi

2009-01-01

Highly active antiretroviral therapy (HAART) is often associated with endothelial dysfunction and cardiovascular complications. In this study, we determined whether HIV non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) could increase endothelial permeability. Human coronary artery endothelial cells (HCAECs) were treated with EFV (1, 5 and 10 µg/ml) and endothelial permeability was determined by a transwell system with a fluorescence-labeled dextran tracer. HCAECs treated with EFV showed a significant increase of endothelial permeability in a concentration-dependent manner. With real time PCR analysis, EFV significantly reduced the mRNA levels of tight junction proteins claudin-1, occludin, zonula occluden-1 and junctional adhesion molecule-1 compared with controls (P < 0.05). Protein levels of these tight junction molecules were also reduced substantially in the EFV-treated cells by western blot and flow cytometry analyses. In addition, EFV also increased superoxide anion production with dihydroethidium and cellular glutathione assays, while it decreased mitochondrial membrane potential with JC-staining. Antioxidants (ginkgolide B and MnTBAP) effectively blocked EFV-induced endothelial permeability and mitochondrial dysfunction. Furthermore, EFV increased the phosphorylation of MAPK JNK and IκBα, thereby increasing NFκB translocation to the nucleus. Chemical JNK inhibitor and dominant negative mutant JNK and IkBa adenoviruses effectively blocked the effects of EFV on HCAECs. Thus, EFV increases endothelial permeability which may be due to the decrease of tight junction proteins and the increase of superoxide anion. JNK and NFκB activation may be directly involved in the signal transduction pathway of EFV action in HCAECs. PMID:19674747

A permeability barrier surrounds taste buds in lingual epithelia.

PubMed

Dando, Robin; Pereira, Elizabeth; Kurian, Mani; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D

2015-01-01

Epithelial tissues are characterized by specialized cell-cell junctions, typically localized to the apical regions of cells. These junctions are formed by interacting membrane proteins and by cytoskeletal and extracellular matrix components. Within the lingual epithelium, tight junctions join the apical tips of the gustatory sensory cells in taste buds. These junctions constitute a selective barrier that limits penetration of chemosensory stimuli into taste buds (Michlig et al. J Comp Neurol 502: 1003-1011, 2007). We tested the ability of chemical compounds to permeate into sensory end organs in the lingual epithelium. Our findings reveal a robust barrier that surrounds the entire body of taste buds, not limited to the apical tight junctions. This barrier prevents penetration of many, but not all, compounds, whether they are applied topically, injected into the parenchyma of the tongue, or circulating in the blood supply, into taste buds. Enzymatic treatments indicate that this barrier likely includes glycosaminoglycans, as it was disrupted by chondroitinase but, less effectively, by proteases. The barrier surrounding taste buds could also be disrupted by brief treatment of lingual tissue samples with DMSO. Brief exposure of lingual slices to DMSO did not affect the ability of taste buds within the slice to respond to chemical stimulation. The existence of a highly impermeable barrier surrounding taste buds and methods to break through this barrier may be relevant to basic research and to clinical treatments of taste. Copyright © 2015 the American Physiological Society.

Mode of action of claudin peptidomimetics in the transient opening of cellular tight junction barriers.

PubMed

Staat, Christian; Coisne, Caroline; Dabrowski, Sebastian; Stamatovic, Svetlana M; Andjelkovic, Anuska V; Wolburg, Hartwig; Engelhardt, Britta; Blasig, Ingolf E

2015-06-01

In epithelial/endothelial barriers, claudins form tight junctions, seal the paracellular cleft, and limit the uptake of solutes and drugs. The peptidomimetic C1C2 from the C-terminal half of claudin-1's first extracellular loop increases drug delivery through epithelial claudin-1 barriers. However, its molecular and structural mode of action remains unknown. In the present study, >100 μM C1C2 caused paracellular opening of various barriers with different claudin compositions, ranging from epithelial to endothelial cells, preferentially modulating claudin-1 and claudin-5. After 6 h incubation, C1C2 reversibly increased the permeability to molecules of different sizes; this was accompanied by redistribution of claudins and occludin from junctions to cytosol. Internalization of C1C2 in epithelial cells depended on claudin-1 expression and clathrin pathway, whereby most C1C2 was retained in recyclosomes >2 h. In freeze-fracture electron microscopy, C1C2 changed claudin-1 tight junction strands to a more parallel arrangement and claudin-5 strands from E-face to P-face association - drastic and novel effects. In conclusion, C1C2 is largely recycled in the presence of a claudin, which explains the delayed onset of barrier and junction loss, the high peptide concentration required and the long-lasting effect. Epithelial/endothelial barriers are specifically modulated via claudin-1/claudin-5, which can be targeted to improve drug delivery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Lactic Acid Bacteria Improves Peyer's Patch Cell-Mediated Immunoglobulin A and Tight-Junction Expression in a Destructed Gut Microbial Environment.

PubMed

Kim, Sung Hwan; Jeung, Woonhee; Choi, Il-Dong; Jeong, Ji-Woong; Lee, Dong Eun; Huh, Chul-Sung; Kim, Geun-Bae; Hong, Seong Soo; Shim, Jae-Jung; Lee, Jung Lyoul; Sim, Jae-Hun; Ahn, Young-Tae

2016-06-28

To evaluate the effects of lactic acid bacteria (LAB) on Peyer's patch cells, mice were treated with a high dose of kanamycin to disturb the gut microbial environment. The overarching goal was to explore the potential of LAB for use as a dietary probiotic that buffers the negative consequences of antibiotic treatment. In vitro, LAB stimulated the production of immunoglobulin A (IgA) from isolated Peyer's patch cells. Inflammation-related genes (TNF-α, IL-1β, and IL-8) were up-regulated in Caco-2 cells stimulated with lipopolysaccharide (LPS), while tight-junction-related genes (ZO-1 and occludin) were down-regulated; the effects of LPS on inflammatory gene and tight-junction gene expression were reversed by treatment with LAB. Mice treated with a high dose of kanamycin showed increased serum IgE levels and decreases in serum IgA and fecal IgA levels; the number of Peyer's patch cells decreased with kanamycin treatment. However, subsequent LAB treatment was effective in reducing the serum IgE level and recovering the serum IgA and fecal IgA levels, as well as the number of Peyer's patch cells. In addition, ZO-1 and occludin mRNA levels were up-regulated in the ileum tissues of mice receiving LAB treatment. Lactic acid bacteria can enhance the intestinal immune system by improving the integrity of the intestinal barrier and increasing the production of IgA in Peyer's patches. Lactic acid bacteria should be considered a potential probiotic candidate for improving intestinal immunity, particularly in mitigating the negative consequences of antibiotic use.

Ultrastructural study of the semicircular canal cells of the frog Rana esculenta.

PubMed

Oudar, O; Ferrary, E; Feldmann, G

1988-03-01

The ultrastructure of the nonsensory cells (dark cells, transitional cells, and undifferentiated cells) of the frog semicircular canal was studied by using transmission electron microscopy in an attempt to correlate the structure with the functions of these epithelial cells. All the nonsensory cells were linked by tight junctions and desmosomes; this suggested that there is little paracellular ionic transport from perilymph to endolymph. In the dark cell epithelium, the apical intercellular spaces were dilated; in the basal part, numerous basolateral plasma membrane infoldings, containing mitochondria, delimited electron-lucent spaces. The undifferentiated cells and the transitional cells were devoid of any basal membrane infolding. Surrounding the semicircular canal, very flattened and interdigitated mesothelial cells constituted a thin multilayer tissue which limited the perilymphatic space. The morphological aspect of the dark cells suggests that they may play a role in the secretion and/or in the reabsorption of endolymph, which bathes the apical pole of these cells. The undifferentiated and transitional cells can play a role in the maintenance of the endolymphatic ionic composition because of their apical tight junctions and desmosomes.

Ablation of CD11c(hi) dendritic cells exacerbates Japanese encephalitis by regulating blood-brain barrier permeability and altering tight junction/adhesion molecules.

PubMed

Kim, Jin Hyoung; Hossain, Ferdaus Mohd Altaf; Patil, Ajit Mahadev; Choi, Jin Young; Kim, Seong Bum; Uyangaa, Erdenebelig; Park, Sang-Youel; Lee, John-Hwa; Kim, Bumseok; Kim, Koanhoi; Eo, Seong Kug

2016-10-01

Japanese encephalitis (JE), characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV), is becoming a leading cause of viral encephalitis due to rapid changes in climate and demography. The blood-brain barrier (BBB) plays an important role in restricting neuroinvasion of peripheral leukocytes and virus, thereby regulating the progression of viral encephalitis. In this study, we explored the role of CD11c(hi) dendritic cells (DCs) in regulating BBB integrity and JE progression using a conditional depletion model of CD11c(hi) DCs. Transient ablation of CD11c(hi) DCs resulted in markedly increased susceptibility to JE progression along with highly increased neuro-invasion of JEV. In addition, exacerbated JE progression in CD11c(hi) DC-ablated hosts was closely associated with increased expression of proinflammatory cytokines (IFN-β, IL-6, and TNF-α) and CC chemokines (CCL2, CCL3, CXCL2) in the brain. Moreover, our results revealed that the exacerbation of JE progression in CD11c(hi) DC-ablated hosts was correlated with enhanced BBB permeability and reduced expression of tight junction and adhesion molecules (claudin-5, ZO-1, occluding, JAMs). Ultimately, our data conclude that the ablation of CD11c(hi) DCs provided a subsidiary impact on BBB integrity and the expression of tight junction/adhesion molecules, thereby leading to exacerbated JE progression. These findings provide insight into the secondary role of CD11c(hi) DCs in JE progression through regulation of BBB integrity and the expression of tight junction/adhesion molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ischemic preconditioning enhances integrity of coronary endothelial tight junctions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhao; Jin, Zhu-Qiu, E-mail: zhu-qiu.jin@sdstate.edu

2012-08-31

Highlights: Black-Right-Pointing-Pointer Cardiac tight junctions are present between coronary endothelial cells. Black-Right-Pointing-Pointer Ischemic preconditioning preserves the structural and functional integrity of tight junctions. Black-Right-Pointing-Pointer Myocardial edema is prevented in hearts subjected to ischemic preconditioning. Black-Right-Pointing-Pointer Ischemic preconditioning enhances translocation of ZO-2 from cytosol to cytoskeleton. -- Abstract: Ischemic preconditioning (IPC) is one of the most effective procedures known to protect hearts against ischemia/reperfusion (IR) injury. Tight junction (TJ) barriers occur between coronary endothelial cells. TJs provide barrier function to maintain the homeostasis of the inner environment of tissues. However, the effect of IPC on the structure and function of cardiacmore » TJs remains unknown. We tested the hypothesis that myocardial IR injury ruptures the structure of TJs and impairs endothelial permeability whereas IPC preserves the structural and functional integrity of TJs in the blood-heart barrier. Langendorff hearts from C57BL/6J mice were prepared and perfused with Krebs-Henseleit buffer. Cardiac function, creatine kinase release, and myocardial edema were measured. Cardiac TJ function was evaluated by measuring Evans blue-conjugated albumin (EBA) content in the extravascular compartment of hearts. Expression and translocation of zonula occludens (ZO)-2 in IR and IPC hearts were detected with Western blot. A subset of hearts was processed for the observation of ultra-structure of cardiac TJs with transmission electron microscopy. There were clear TJs between coronary endothelial cells of mouse hearts. IR caused the collapse of TJs whereas IPC sustained the structure of TJs. IR increased extravascular EBA content in the heart and myocardial edema but decreased the expression of ZO-2 in the cytoskeleton. IPC maintained the structure of TJs. Cardiac EBA content and edema were reduced in IPC hearts. IPC enhanced the translocation of ZO-2 from cytosol to cytoskeleton. In conclusion, TJs occur in normal mouse heart. IPC preserves the integrity of TJ structure and function that are vulnerable to IR injury.« less

Glutamine Supplementation Attenuates Ethanol-Induced Disruption of Apical Junctional Complexes in Colonic Epithelium and Ameliorates Gut Barrier Dysfunction and Fatty Liver in Mice

PubMed Central

Chaudhry, Kamaljit K.; Shukla, Pradeep K.; Mir, Hina; Manda, Bhargavi; Gangwar, Ruchika; Yadav, Nikki; McMullen, Megan; Nagy, Laura E.; Rao, RadhaKrishna

2015-01-01

Previous in vitro studies showed that glutamine (Gln) prevents acetaldehyde-induced disruption of tight junctions and adherens junctions in Caco-2 cell monolayers and human colonic mucosa. In the present study, we evaluated the effect of Gln supplementation on ethanol-induced gut barrier dysfunction and liver injury in mice in vivo. Ethanol feeding caused a significant increase in inulin permeability in distal colon. Elevated permeability was associated with a redistribution of tight junction and adherens junction proteins and depletion of detergent-insoluble fractions of these proteins, suggesting that ethanol disrupts apical junctional complexes in colonic epithelium and increases paracellular permeability. Ethanol-induced increase in colonic mucosal permeability and disruption of junctional complexes were most severe in mice fed Gln-free diet. Gln supplementation attenuated ethanol-induced mucosal permeability and disruption of tight junctions and adherens junctions in a dose-dependent manner, indicating the potential role of glutamine in nutritional intervention to alcoholic tissue injury. Gln supplementation dose-dependently elevated reduced-protein thiols in colon without affecting the level of oxidized-protein thiols. Ethanol feeding depleted reduced protein thiols and elevated oxidized protein thiols. Ethanol-induced protein thiol oxidation was most severe in mice fed Gln-free diet and absent in mice fed Gln-supplemented diet, suggesting that antioxidant effect is one of the likely mechanisms involved in Gln-mediated amelioration of ethanol-induced gut barrier dysfunction. Ethanol feeding elevated plasma transaminase and liver triglyceride, which was accompanied by histopathologic lesions in the liver; ethanol-induced liver damage was attenuated by Gln supplementation. These results indicate that Gln supplementation ameliorates alcohol-induced gut and liver injury. PMID:26365579

The status of intercellular junctions in established lens epithelial cell lines

PubMed Central

Dave, Alpana; Craig, Jamie E.

2012-01-01

Purpose Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. Methods The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT–PCR), and localization was determined by immunofluorescence labeling. Results Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. Conclusions The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that these cell lines form tight junctions but do not form E-cadherin-based adherence junctions. These data further indicate that the regulatory role of NHS in actin remodeling, suggested in another study, is cell type dependent. In conclusion, the SRA 01/04 and αTN4 lens epithelial cell lines model some characteristics of an epithelium. PMID:23288986

The status of intercellular junctions in established lens epithelial cell lines.

PubMed

Dave, Alpana; Craig, Jamie E; Sharma, Shiwani

2012-01-01

Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT-PCR), and localization was determined by immunofluorescence labeling. Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that these cell lines form tight junctions but do not form E-cadherin-based adherence junctions. These data further indicate that the regulatory role of NHS in actin remodeling, suggested in another study, is cell type dependent. In conclusion, the SRA 01/04 and αTN4 lens epithelial cell lines model some characteristics of an epithelium.

Redox Regulation of Cell Contacts by Tricellulin and Occludin: Redox-Sensitive Cysteine Sites in Tricellulin Regulate Both Tri- and Bicellular Junctions in Tissue Barriers as Shown in Hypoxia and Ischemia.

PubMed

Cording, Jimmi; Günther, Ramona; Vigolo, Emilia; Tscheik, Christian; Winkler, Lars; Schlattner, Isabella; Lorenz, Dorothea; Haseloff, Reiner F; Schmidt-Ott, Kai M; Wolburg, Hartwig; Blasig, Ingolf E

2015-11-01

Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs; tricellulin, occludin, marvelD3) are thought to be relevant to regulation. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts. Ischemia, hypoxia, and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along the cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules. Tricellulin forms TJs via trans- and cis-association in 3-cell contacts, as demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox dependently; a structural model is proposed. TAMP ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox sensor and sealing element at 3-cell contacts and may compensate as a redox mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox regulator for ischemia-related alterations.

Managing the complexity of communication: regulation of gap junctions by post-translational modification

PubMed Central

Axelsen, Lene N.; Calloe, Kirstine; Holstein-Rathlou, Niels-Henrik; Nielsen, Morten S.

2013-01-01

Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins. PMID:24155720

The ZO-1–associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density

PubMed Central

Balda, Maria S.; Garrett, Michelle D.; Matter, Karl

2003-01-01

Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1–associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction–associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1–based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4. PMID:12566432

The atypical mitogen-activated protein kinase ERK3 is essential for establishment of epithelial architecture.

PubMed

Takahashi, Chika; Miyatake, Koichi; Kusakabe, Morioh; Nishida, Eisuke

2018-06-01

Epithelia contribute to physical barriers that protect internal tissues from the external environment and also support organ structure. Accordingly, establishment and maintenance of epithelial architecture are essential for both embryonic development and adult physiology. Here, using gene knockout and knockdown techniques along with gene profiling, we show that extracellular signal-regulated kinase 3 (ERK3), a poorly characterized atypical mitogen-activated protein kinase (MAPK), regulates the epithelial architecture in vertebrates. We found that in Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight-junction protein distribution, as well as tight-junction barrier function, resulting in epidermal breakdown. Moreover, in human epithelial breast cancer cells, inhibition of ERK3 expression induced thickened epithelia with aberrant adherens and tight junctions. Results from microarray analyses suggested that transcription factor AP-2α (TFAP2A), a transcriptional regulator important for epithelial gene expression, is involved in ERK3-dependent changes in gene expression. Of note, TFAP2A knockdown phenocopied ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 was required for full activation of TFAP2A-dependent transcription. Our findings reveal that ERK3 regulates epithelial architecture, possibly together with TFAP2A. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Poly(I:C) Induces Human Lung Endothelial Barrier Dysfunction by Disrupting Tight Junction Expression of Claudin-5

DOE PAGES

Huang, Li -Yun; Stuart, Christine; Takeda, Kazuyo; ...

2016-08-09

Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNβ production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelialmonolayer permeability with a corresponding dose- and time-dependent decrease in themore » expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Altogether, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.« less

Evaluation of Differentiated Human Bronchial Epithelial Cell Culture Systems for Asthma Research

PubMed Central

Stewart, Ceri E.; Torr, Elizabeth E.; Mohd Jamili, Nur H.; Bosquillon, Cynthia; Sayers, Ian

2012-01-01

The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC) and non-primary (Calu-3, BEAS-2B, BEAS-2B R1) bronchial epithelial cell culture systems as air-liquid interface- (ALI-) differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+) and ciliated (β-Tubulin IV+) cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin) and development of transepithelial electrical resistance (TEER) were assessed. Primary cells showed localised MUC5AC, β-Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse β-Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions. PMID:22287976

Expression of ZO-1 and claudin-1 in a 3D epidermal equivalent using canine progenitor epidermal keratinocytes.

PubMed

Teramoto, Keiji; Asahina, Ryota; Nishida, Hidetaka; Kamishina, Hiroaki; Maeda, Sadatoshi

2018-05-21

Previous studies indicate that tight junctions are involved in the pathogenesis of canine atopic dermatitis (cAD). An in vitro skin model is needed to elucidate the specific role of tight junctions in cAD. A 3D epidermal equivalent model using canine progenitor epidermal keratinocytes (CPEK) has been established; the expression of tight junctions within this model is uncharacterized. To investigate the expression of tight junctions in the 3D epidermal equivalent. Two normal laboratory beagle dogs served as donors of full-thickness skin biopsy samples for comparison to the in vitro model. Immunohistochemical techniques were employed to investigate the expression of tight junctions including zonula occludens (ZO)-1 and claudin-1 in normal canine skin, and in the CPEK 3D epidermal equivalent. Results demonstrated the expression of ZO-1 and claudin-1 in the CPEK 3D epidermal equivalent, with staining patterns that were similar to those in normal canine skin. The CPEK 3D epidermal equivalent has the potential to be a suitable in vitro research tool for clarifying the specific role of tight junctions in cAD. © 2018 ESVD and ACVD.

Tight junction-associated MARVEL proteins marveld3, tricellulin, and occludin have distinct but overlapping functions.

PubMed

Raleigh, David R; Marchiando, Amanda M; Zhang, Yong; Shen, Le; Sasaki, Hiroyuki; Wang, Yingmin; Long, Manyuan; Turner, Jerrold R

2010-04-01

In vitro studies have demonstrated that occludin and tricellulin are important for tight junction barrier function, but in vivo data suggest that loss of these proteins can be overcome. The presence of a heretofore unknown, yet related, protein could explain these observations. Here, we report marvelD3, a novel tight junction protein that, like occludin and tricellulin, contains a conserved four-transmembrane MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain. Phylogenetic tree reconstruction; analysis of RNA and protein tissue distribution; immunofluorescent and electron microscopic examination of subcellular localization; characterization of intracellular trafficking, protein interactions, dynamic behavior, and siRNA knockdown effects; and description of remodeling after in vivo immune activation show that marvelD3, occludin, and tricellulin have distinct but overlapping functions at the tight junction. Although marvelD3 is able to partially compensate for occludin or tricellulin loss, it cannot fully restore function. We conclude that marvelD3, occludin, and tricellulin define the tight junction-associated MARVEL protein family. The data further suggest that these proteins are best considered as a group with both redundant and unique contributions to epithelial function and tight junction regulation.

Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting.

PubMed

Ban, Yuriko; Cooper, Leanne J; Fullwood, Nigel J; Nakamura, Takahiro; Tsuzuki, Masakatsu; Koizumi, Noriko; Dota, Atsuyoshi; Mochida, Chikako; Kinoshita, Shigeru

2003-06-01

To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.

Small synthetic peptides homologous to segments of the first external loop of occludin impair tight junction resealing.

PubMed

Lacaz-Vieira, F; Jaeger, M M; Farshori, P; Kachar, B

1999-04-01

This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca2+ removal and was characterized by a marked drop of TER. The reintroduction of Ca2+ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca2+ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions.

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

PubMed Central

Anderson, J. M.; Glade, J. L.; Stevenson, B. R.; Boyer, J. L.; Mooseker, M. S.

1989-01-01

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. Images Figure 1 Figure 2 PMID:2719075

Molecular cell biology and physiology of solute transport

PubMed Central

Caplan, Michael J.; Seo-Mayer, Patricia; Zhang, Li

2010-01-01

Purpose of review An enormous body of research has been focused on exploring the mechanisms through which epithelial cells establish their characteristic polarity. It is clear that under normal circumstances cell–cell contacts mediated by the calcium-dependent adhesion proteins of the intercellular adhesion junctions are required to initiate complete polarization. Furthermore, formation of the tight, or occluding, junctions that limit paracellular permeability has long been thought to help to establish polarity by preventing the diffusion of membrane proteins between the two plasmalemmal domains. This review will discuss several selected kinases and protein complexes and highlight their relevance to transporting epithelial cell polarization. Recent findings Recent work has shed new light on the roles of junctional complexes in establishing and maintaining epithelial cell polarity. In addition, work from several laboratories, suggests that the formation of these junctions is tied to processes that regulate cellular energy metabolism. Summary Junctional complexes and energy sensing kinases constitute a novel class of machinery whose capacity to generate and modulate epithelial cell polarity is likely to have wide ranging and important physiological ramifications. PMID:18695392

Scaffolding proteins in the development and maintenance of the epidermal permeability barrier.

PubMed

Crawford, Melissa; Dagnino, Lina

2017-10-02

The skin of mammals and other terrestrial vertebrates protects the organism against the external environment, preventing heat, water and electrolyte loss, as well as entry of chemicals and pathogens. Impairments in the epidermal permeability barrier function are associated with the genesis and/or progression of a variety of pathological conditions, including genetic inflammatory diseases, microbial and viral infections, and photodamage induced by UV radiation. In mammals, the outside-in epidermal permeability barrier is provided by the joint action of the outermost cornified layer, together with assembled tight junctions in granular keratinocytes found in the layers underneath. Tight junctions serve as both outside-in and inside-out barriers, and impede paracellular movements of ions, water, macromolecules and microorganisms. At the molecular level, tight junctions consist of integral membrane proteins that form an extracellular seal between adjacent cells, and associate with cytoplasmic scaffold proteins that serve as links with the actin cytoskeleton. In this review, we address the roles that scaffold proteins play specifically in the establishment and maintenance of the epidermal permeability barrier, and how various pathologies alter or impair their functions.

Toxicants target cell junctions in the testis: Insights from the indazole-carboxylic acid model

PubMed Central

Cheng, C Yan

2014-01-01

There are numerous types of junctions in the seminiferous epithelium which are integrated with, and critically dependent on the Sertoli cell cytoskeleton. These include the basal tight junctions between Sertoli cells that form the main component of the blood–testis barrier, the basal ectoplasmic specializations (basal ES) and basal tubulobulbar complexes (basal TBC) between Sertoli cells; as well as apical ES and apical TBC between Sertoli cells and the developing spermatids that orchestrate spermiogenesis and spermiation. These junctions, namely TJ, ES, and TBC interact with actin microfilament-based cytoskeleton, which together with the desmosomal junctions that interact with the intermediate filament-based cytoskeleton plus the highly polarized microtubule-based cytoskeleton are working in concert to move spermatocytes and spermatids between the basal and luminal aspect of the seminiferous epithelium. In short, these various junctions are structurally complexed with the actin- and microtubule-based cytoskeleton or intermediate filaments of the Sertoli cell. Studies have shown toxicants (e.g., cadmium, bisphenol A (BPA), perfluorooctanesulfonate (PFOS), phthalates, and glycerol), and some male contraceptives under development (e.g., adjudin, gamendazole), exert their effects, at least in part, by targeting cell junctions in the testis. The disruption of Sertoli–Sertoli cell and Sertoli–germ cell junctions, results in the loss of germ cells from the seminiferous epithelium. Adjudin, a potential male contraceptive under investigation in our laboratory, produces loss of spermatids from the seminiferous tubules through disruption of the Sertoli cell spermatid junctions and disruption of the Sertoli cell cytoskeleton. The molecular and structural changes associated with adjudin administration are described, to provide an example of the profile of changes caused by disturbance of Sertoli-germ cell and also Sertoli cell-cell junctions. PMID:26413399

Baicalin Protects against TNF-α-Induced Injury by Down-Regulating miR-191a That Targets the Tight Junction Protein ZO-1 in IEC-6 Cells.

PubMed

Wang, Li; Zhang, Ren; Chen, Jian; Wu, Qihui; Kuang, Zaoyuan

2017-04-01

Tumor necrosis factor-alpha (TNF-α) plays an important role in the developing process of inflammatory bowel disease. Tight junction protein zonula occludens-1 (ZO-1), one of epithelial junctional proteins, maintains the permeability of intestinal barrier. The objective of this study was to investigate the mechanism of the protective effect of baicalin on TNF-α-induced injury and ZO-1 expression in intestinal epithelial cells (IECs). We found that baicalin pretreatment significantly improved cell viability and cell migration following TNF-α stimulation. miR-191a inhibitor increased the protective effect of baicalin on cell motility injured by TNF-α. In addition, miR-191a down-regulated the mRNA and protein level of its target gene ZO-1. TNF-α stimulation increased miR-191a expression, leading to the decline of ZO-1 mRNA and protein. Moreover, pretreatment with baicalin reversed TNF-α induced decrease of ZO-1 and increase of miR-191a, miR-191a inhibitor significantly enhanced ZO-1 protein expression restored by baicalin. These results indicate that baicalin exerts a protective effect on IEC-6 (rat small intestinal epithelial cells) cells against TNF-α-induced injury, which is at least partly via inhibiting the expression of miR-191a, thus increasing ZO-1 mRNA and protein levels.

Hormones and the blood-brain barrier.

PubMed

Hampl, Richard; Bičíková, Marie; Sosvorová, Lucie

2015-03-01

Hormones exert many actions in the brain, and brain cells are also hormonally active. To reach their targets in brain structures, hormones must overcome the blood-brain barrier (BBB). The BBB is a unique device selecting desired/undesired molecules to reach or leave the brain, and it is composed of endothelial cells forming the brain vasculature. These cells differ from other endothelial cells in their almost impermeable tight junctions and in possessing several membrane structures such as receptors, transporters, and metabolically active molecules, ensuring their selection function. The main ways how compounds pass through the BBB are briefly outlined in this review. The main part concerns the transport of major classes of hormones: steroids, including neurosteroids, thyroid hormones, insulin, and other peptide hormones regulating energy homeostasis, growth hormone, and also various cytokines. Peptide transporters mediating the saturable transport of individual classes of hormones are reviewed. The last paragraph provides examples of how hormones affect the permeability and function of the BBB either at the level of tight junctions or by various transporters.

MarvelD3 regulates the c-Jun N-terminal kinase pathway during eye development in Xenopus

PubMed Central

Vacca, Barbara; Sanchez-Heras, Elena; Steed, Emily; Balda, Maria S.; Ohnuma, Shin-Ichi; Sasai, Noriaki; Mayor, Roberto

2016-01-01

ABSTRACT Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus. MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis. PMID:27870636

Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van De Walle, Jacqueline; Sergent, Therese; Piront, Neil

Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation ofmore » [{sup 3}H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [{sup 3}H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-{kappa}B, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.« less

Integrin-Linked Kinase Is Indispensable for Keratinocyte Differentiation and Epidermal Barrier Function.

PubMed

Sayedyahossein, Samar; Rudkouskaya, Alena; Leclerc, Valerie; Dagnino, Lina

2016-02-01

A functional permeability barrier is essential to prevent the passage of water and electrolytes, macromolecules, and pathogens through the epidermis. This is accomplished in terminally differentiated keratinocytes through formation of a cornified envelope and the assembly of tight intercellular junctions. Integrin-linked kinase (ILK) is a scaffold protein essential for hair follicle morphogenesis and epidermal attachment to the basement membrane. However, the biological functions of ILK in differentiated keratinocytes remain poorly understood. Furthermore, whether ILK is implicated in keratinocyte differentiation and intercellular junction formation has remained an unresolved issue. Here we describe a pivotal role for ILK in keratinocyte differentiation responses to increased extracellular Ca(2+), regulation of adherens and tight junction assembly, and the formation of an outside-in permeability barrier toward macromolecules. In the absence of ILK, the calcium sensing receptor, E-cadherin, and ZO-1 fail to translocate to the cell membrane, through mechanisms that involve abnormalities in microtubules and in RhoA activation. In situ, ILK-deficient epidermis exhibits reduced tight junction formation and increased outside-in permeability to a dextran tracer, indicating reduced barrier properties toward macromolecules. Therefore, ILK is an essential component of keratinocyte differentiation programs that contribute to epidermal integrity and the establishment of its barrier properties. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The role of apoptosis in LDL transport through cultured endothelial cell monolayers

PubMed Central

Cancel, Limary M.; Tarbell, John M.

2009-01-01

We have previously shown that leaky junctions associated with dying or dividing cells are the dominant pathway for LDL transport under convective conditions, accounting for more than 90% of the transport [1]. To explore the role of apoptosis in the leaky junction pathway, TNFα and cycloheximide (TNFα/CHX) were used to induce an elevated rate of apoptosis in cultured bovine aortic endothelial cell (BAEC) monolayers and the convective fluxes of LDL and water were measured. Treatment with TNFα/CHX induced a 18.3-fold increase in apoptosis and a 4.4-fold increase in LDL permeability. Increases in apoptosis and permeability were attenuated by treatment with the caspase inhibitor Z-VAD-FMK. Water flux increased by 2.7-fold after treatment with TNFα/CHX, and this increase was not attenuated by treatment with Z-VAD-FMK. Immunostaining of the tight junction protein ZO-1 showed that TNFα/CHX treatment disrupts the tight junction in addition to inducing apoptosis. This disruption is present even when Z-VAD-FMK is used to inhibit apoptosis, and likely accounts for the increase in water flux. We found a strong correlation between the rate of apoptosis and the permeability of BAEC monolayers to LDL. These results demonstrate the potential of manipulating endothelial monolayer permeability by altering the rate of apoptosis pharmacollogicaly. This has implications for the treatment of atherosclerosis. PMID:19709659

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Qingqiao; Xia, Yuanyu, E-mail: xiayuanyu.wh@gmail.com; Wang, Guan

As an early sign of diabetic cardiovascular disease, endothelial dysfunction may contribute to progressive diabetic nephropathy (DN). Endothelial hyperpermeability induced by hyperglycemia (HG) is a central pathogenesis for DN. Sinomenine (SIN) has strong anti-inflammatory and renal protective effects, following an unknown protective mechanism against HG-induced hyperpermeability. We herein explored the role of SIN in vitro in an HG-induced barrier dysfunction model in human renal glomerular endothelial cells (HRGECs). The cells were exposed to SIN and/or HG for 24 h, the permeability of which was significantly increased by HG. Moreover, junction protein occludin in the cell-cell junction area and its total expression inmore » HRGECs were significantly decreased by HG. However, the dysfunction of tight junction and hyperpermeability of HRGECs were significantly reversed by SIN. Furthermore, SIN prevented HG-increased reactive oxygen species (ROS) by activating nuclear factor-E2-related factor 2 (Nrf2). Interestingly, activation of RhoA/ROCK induced by HG was reversed by SIN or ROCK inhibitor. HG-induced hyperpermeability was prevented by SIN. High ROS level, tight junction dysfunction and RhoA/ROCK activation were significantly attenuated with knockdown of Nrf2. Mediated by activation of Nrf2, SIN managed to significantly prevent HG-disrupted renal endothelial barrier function by suppressing the RhoA/ROCK signaling pathway through reducing ROS. We successfully identified a novel pathway via which SIN exerted antioxidative and renal protective functions, and provided a molecular basis for potential SIN applications in treating DN vascular disorders.« less

In vitro study of LDL transport under pressurized (convective) conditions.

PubMed

Cancel, Limary M; Fitting, Andrew; Tarbell, John M

2007-07-01

It is difficult to assess the transport pathways that carry low-density lipoprotein (LDL) into the artery wall in vivo, and there has been no previous in vitro study that has examined transendothelial transport under physiologically relevant pressurized (convective) conditions. Therefore, we measured water, albumin, and LDL fluxes across bovine aortic endothelial cell (BAEC) monolayers in vitro and determined the relative contributions of vesicles, paracellular transport through "breaks" in the tight junction, and "leaky" junctions associated with dying or dividing cells. Our results show that leaky junctions are the dominant pathway for LDL transport (>90%) under convective conditions and that albumin also has a significant component of transport through leaky junctions (44%). Transcellular transport of LDL by receptor-mediated processes makes a minor contribution (<10%) to overall transport under convective conditions.

The Human “Cochlear Battery” – Claudin-11 Barrier and Ion Transport Proteins in the Lateral Wall of the Cochlea

PubMed Central

Liu, Wei; Schrott-Fischer, Annelies; Glueckert, Rudolf; Benav, Heval; Rask-Andersen, Helge

2017-01-01

Background: The cochlea produces an electric field potential essential for hair cell transduction and hearing. This biological “battery” is situated in the lateral wall of the cochlea and contains molecular machinery that secretes and recycles K+ ions. Its functioning depends on junctional proteins that restrict the para-cellular escape of ions. The tight junction protein Claudin-11 has been found to be one of the major constituents of this barrier that maintains ion gradients (Gow et al., 2004; Kitajiri et al., 2004a). We are the first to elucidate the human Claudin-11 framework and the associated ion transport machinery using super-resolution fluorescence illumination microscopy (SR-SIM). Methods: Archival cochleae obtained during meningioma surgery were used for SR-SIM together with transmission electron microscopy after ethical consent. Results: Claudin-11-expressing cells formed parallel tight junction lamellae that insulated the epithelial syncytium of the stria vascularis and extended to the suprastrial region. Intercellular gap junctions were found between the barrier cells and fibrocytes. Conclusion: Transmission electron microscopy, confocal microscopy and SR-SIM revealed exclusive cell specialization in the various subdomains of the lateral wall of the human cochlea. The Claudin-11-expressing cells exhibited both conductor and isolator characteristics, and these micro-porous separators may selectively mediate the movement of charged units to the intrastrial space in a manner that is analogous to a conventional electrochemical “battery.” The function and relevance of this battery for the development of inner ear disease are discussed. PMID:28848383

Sertoli Cells Are Susceptible to ZIKV Infection in Mouse Testis.

PubMed

Sheng, Zi-Yang; Gao, Na; Wang, Zhao-Yang; Cui, Xiao-Yun; Zhou, De-Shan; Fan, Dong-Ying; Chen, Hui; Wang, Pei-Gang; An, Jing

2017-01-01

Flaviviruses including Dengue virus (DENV), Yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) are global health problems that caused several serious diseases such as fever, hemorrhagic fever, and encephalitis in the past century. Recently, Zika virus (ZIKV) which spreads from Asia to American and causes millions of infections emerges as a new dangerous member of the genus of Flavivirus . Unlike other well-known flaviviruses, ZIKV can be transmitted sexually and infect testes in murine models. Its impacts on sperm functions, and the exact susceptible cells, however, are not entirely clear. To investigate these issues, we infected interferon α/β and γ receptors deficient AG6 mice with ZIKV and examined the outcomes of infection using an assortment of physiological, histopathological, immunological, and virological techniques. We found that infected mice displayed signs of reproductive system disorder, altered androgen levels in serum, and high viral load in semen and testes. Additionally, histopathological examinations revealed marked atrophy of seminiferous tubules and significant reduction in lumen size. Notably, these were accompanied by positive staining of ZIKV antigens on sertoli cells, detection of viral particles and vacuole changes within cytoplasm of sertoli cells. The susceptibility of sertoli cells to ZIKV was further validated in vitro study using cell lines. Importantly, the disruption of tight junctions within testis and altered sperm morphology were also observed in ZIKV infected mice. It is well-known that tight junctions formed by adjacent sertoli cells are major component of blood testis barrier, which plays important roles in maintenance of microenvironment for spermagenesis in testis. Taken together, these results demonstrate that sertoli cells are susceptible to ZIKV infection, which results in the disruption of tight junctions in testis and causes abnormal spermatogenesis in mice. These results also imply that long-term impact of ZIKV infection on human male reproductive system requires close monitoring.

Ischemia-reperfusion impairs blood-brain barrier function and alters tight junction protein expression in the ovine fetus

PubMed Central

Chen, Xiaodi; Threlkeld, Steven W.; Cummings, Erin E.; Juan, Ilona; Makeyev, Oleksandr; Besio, Walter G.; Gaitanis, John; Banks, William A.; Sadowska, Grazyna B.; Stonestreet, Barbara S.

2012-01-01

The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (Ki) and tight junction proteins by Western immunoblot in fetal sheep at 127 days-of-gestation without ischemia, and 4-, 24-, or 48-h after ischemia. The largest increase in Ki (P<0.05) was 4-h after ischemia. Occludin and claudin-5 expressions decreased at 4-h, but returned toward control levels 24- and 48-h after ischemia. Zonula occludens-1 and -2 decreased after ischemia. Inverse correlations between Ki and tight junction proteins suggest that the decreases in tight junction proteins contribute to impaired blood-brain barrier function after ischemia. We conclude that impaired blood-brain barrier function is an important component of hypoxic-ischemic brain injury in the fetus, and that increases in quantitatively measured barrier permeability (Ki) change as a function of the duration of reperfusion after ischemia. The largest increase in permeability occurs 4-h after ischemia and blood-brain barrier function improves early after injury because the blood-brain barrier is less permeable 24- and 48- than 4-h after ischemia. Changes in the tight junction molecular composition are associated with increases in blood-brain barrier permeability after ischemia. PMID:22986172

Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans

PubMed Central

Farnsworth, Nikki L; Hemmati, Alireza; Pozzoli, Marina; Benninger, Richard K P

2014-01-01

The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose-stimulated insulin secretion is tightly linked to electrical activity in β cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre-diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose- and cAMP-dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between α cells and β cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies. PMID:25172942

Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans.

PubMed

Farnsworth, Nikki L; Hemmati, Alireza; Pozzoli, Marina; Benninger, Richard K P

2014-10-15

The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose‐stimulated insulin secretion is tightly linked to electrical activity in β cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre‐diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose‐ and cAMP‐dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between α cells and β cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies.

Claudin gene expression patterns do not associate with interspecific differences in paracellular nutrient absorption.

PubMed

Price, Edwin R; Rott, Katherine H; Caviedes-Vidal, Enrique; Karasov, William H

2016-01-01

Bats exhibit higher paracellular absorption of glucose-sized molecules than non-flying mammals, a phenomenon that may be driven by higher permeability of the intestinal tight junctions. The various claudins, occludin, and other proteins making up the tight junctions are thought to determine their permeability properties. Here we show that absorption of the paracellular probe l-arabinose is higher in a bat (Eptesicus fuscus) than in a vole (Microtus pennsylvanicus) or a hedgehog (Atelerix albiventris). Furthermore, histological measurements demonstrated that hedgehogs have many more enterocytes in their intestines, suggesting that bats cannot have higher absorption of arabinose simply by having more tight junctions. We therefore investigated the mRNA levels of several claudins and occludin, because these proteins may affect permeability of tight junctions to macronutrients. To assess the expression levels of claudins per tight junction, we normalized the mRNA levels of the claudins to the constitutively expressed tight junction protein ZO-1, and combined these with measurements previously made in a bat and a rodent to determine if there were among-species differences. Although expression ratios of several genes varied among species, there was not a consistent difference between bats and non-flyers in the expression ratio of any particular gene. Protein expression patterns may differ from mRNA expression patterns, and might better explain differences among species in arabinose absorption. Copyright © 2015 Elsevier Inc. All rights reserved.

Tight junctions in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer

PubMed Central

Landy, Jonathan; Ronde, Emma; English, Nick; Clark, Sue K; Hart, Ailsa L; Knight, Stella C; Ciclitira, Paul J; Al-Hassi, Hafid Omar

2016-01-01

Inflammatory bowel diseases are characterised by inflammation that compromises the integrity of the epithelial barrier. The intestinal epithelium is not only a static barrier but has evolved complex mechanisms to control and regulate bacterial interactions with the mucosal surface. Apical tight junction proteins are critical in the maintenance of epithelial barrier function and control of paracellular permeability. The characterisation of alterations in tight junction proteins as key players in epithelial barrier function in inflammatory bowel diseases is rapidly enhancing our understanding of critical mechanisms in disease pathogenesis as well as novel therapeutic opportunities. Here we give an overview of recent literature focusing on the role of tight junction proteins, in particular claudins, in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer. PMID:27003989

Effects of metformin treatment on glioma-induced brain edema

PubMed Central

Zhao, Bin; Wang, Xiaoke; Zheng, Jun; Wang, Hailiang; Liu, Jun

2016-01-01

Considerable evidence has demonstrated that metformin can activate 5’-AMP-activated protein kinase (AMPK) signaling pathway, which plays a critical role in protection of endothelial cell permeability. Hence, the present study evaluated the effects of metformin on blood brain barrier permeability and AQP4 expression in vitro, and assessed the effects of metformin treatment on tumor-induced brain edema in vivo. Hypoxia or VEGF exposure enhanced bEnd3 endothelial cell monolayer permeability and attenuated the expression of tight junction proteins including Occludin, Claudin-5, ZO-1, and ZO-2. However, 0.5 mM metformin treatment protected bEnd3 endothelial cell monolayer from hypoxia or VEGF-induced permeability, which was correlated with increased expression of tight junction proteins. Furthermore, metformin treatment attenuated AQP4 protein expression in cultured astrocytes. Such an effect involved the activation of AMPK and inhibition of NF-κB. Finally, metformin treatment dose-dependently reduced glioma induced vascular permeability and cerebral edema in vivo in rats. Thus, our results suggested that metformin may protect endothelial cell tight junction, prevent damage to the blood brain barrier induced by brain tumor growth, and alleviate the formation of cerebral edema. Furthermore, since the formation of cytotoxic edema and AQP4 expression was positively correlated, our results indicated that metformin may reduce the formation of cytotoxic edema. However, given that AQP4 plays a key role in the elimination of cerebral edema, attenuation of AQP4 expression by metformin may reduce the elimination of cerebral edema. Hence, future studies will be necessary to dissect the specific mechanisms of metformin underlying the dynamics of tumor-induced brain edema in vivo. PMID:27648126

Effects of metformin treatment on glioma-induced brain edema.

PubMed

Zhao, Bin; Wang, Xiaoke; Zheng, Jun; Wang, Hailiang; Liu, Jun

2016-01-01

Considerable evidence has demonstrated that metformin can activate 5'-AMP-activated protein kinase (AMPK) signaling pathway, which plays a critical role in protection of endothelial cell permeability. Hence, the present study evaluated the effects of metformin on blood brain barrier permeability and AQP4 expression in vitro, and assessed the effects of metformin treatment on tumor-induced brain edema in vivo. Hypoxia or VEGF exposure enhanced bEnd3 endothelial cell monolayer permeability and attenuated the expression of tight junction proteins including Occludin, Claudin-5, ZO-1, and ZO-2. However, 0.5 mM metformin treatment protected bEnd3 endothelial cell monolayer from hypoxia or VEGF-induced permeability, which was correlated with increased expression of tight junction proteins. Furthermore, metformin treatment attenuated AQP4 protein expression in cultured astrocytes. Such an effect involved the activation of AMPK and inhibition of NF-κB. Finally, metformin treatment dose-dependently reduced glioma induced vascular permeability and cerebral edema in vivo in rats. Thus, our results suggested that metformin may protect endothelial cell tight junction, prevent damage to the blood brain barrier induced by brain tumor growth, and alleviate the formation of cerebral edema. Furthermore, since the formation of cytotoxic edema and AQP4 expression was positively correlated, our results indicated that metformin may reduce the formation of cytotoxic edema. However, given that AQP4 plays a key role in the elimination of cerebral edema, attenuation of AQP4 expression by metformin may reduce the elimination of cerebral edema. Hence, future studies will be necessary to dissect the specific mechanisms of metformin underlying the dynamics of tumor-induced brain edema in vivo.

A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

PubMed

Thomsen, Louiza Bohn; Burkhart, Annette; Moos, Torben

2015-01-01

In vitro blood-brain barrier (BBB) models based on primary brain endothelial cells (BECs) cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER) and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs) in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP) and breast cancer related protein (BCRP), and the transferrin receptor).

Uropathogenic E. coli Promote a Paracellular Urothelial Barrier Defect Characterized by Altered Tight Junction Integrity, Epithelial Cell Sloughing and Cytokine Release

PubMed Central

Wood, M. W.; Breitschwerdt, E. B.; Nordone, S. K.; Linder, K. E.; Gookin, J. L.

2013-01-01

Summary The urinary bladder is a common site of bacterial infection with a majority of cases attributed to uropathogenic Escherichia coli. Sequels of urinary tract infections (UTIs) include the loss of urothelial barrier function and subsequent clinical morbidity secondary to the permeation of urine potassium, urea and ammonia into the subepithelium. To date there has been limited research describing the mechanism by which this urothelial permeability defect develops. The present study models acute uropathogenic E. coli infection in vitro using intact canine bladder mucosa mounted in Ussing chambers to determine whether infection induces primarily a transcellular or paracellular permeability defect. The Ussing chamber sustains tissue viability while physically separating submucosal and lumen influences, so this model is ideal for quantitative measurement of transepithelial electrical resistance (TER) to assess alterations of urothelial barrier function. Using this model, changes in both tissue ultrastructure and TER indicated that uropathogenic E. coli infection promotes a paracellular permeability defect associated with the failure of umbrella cell tight junction formation and umbrella cell sloughing. In addition, bacterial interaction with the urothelium promoted secretion of cytokines from the urinary bladder with bioactivity capable of modulating epithelial barrier function including tumour necrosis factor-α, interleukin (IL)-6 and IL-15. IL-15 secretion by the infected bladder mucosa is a novel finding and, because IL-15 plays key roles in reconstitution of tight junction function in damaged intestine, this study points to a potential role for IL-15 in UTI-induced urothelial injury. PMID:22014415

Celiac Disease: Role of the Epithelial Barrier.

PubMed

Schumann, Michael; Siegmund, Britta; Schulzke, Jörg D; Fromm, Michael

2017-03-01

In celiac disease (CD) a T-cell-mediated response to gluten is mounted in genetically predisposed individuals, resulting in a malabsorptive enteropathy histologically highlighted by villous atrophy and crypt hyperplasia. Recent data point to the epithelial layer as an under-rated hot spot in celiac pathophysiology to date. This overview summarizes current functional and genetic evidence on the role of the epithelial barrier in CD, consisting of the cell membranes and the apical junctional complex comprising sealing as well as ion and water channel-forming tight junction proteins and the adherens junction. Moreover, the underlying mechanisms are discussed, including apoptosis of intestinal epithelial cells, biology of intestinal stem cells, alterations in the apical junctional complex, transcytotic uptake of gluten peptides, and possible implications of a defective epithelial polarity. Current research is directed toward new treatment options for CD that are alternatives or complementary therapeutics to a gluten-free diet. Thus, strategies to target an altered epithelial barrier therapeutically also are discussed.

Changes of Tight Junction Protein Claudins in Small Intestine and Kidney Tissues of Mice Fed a DDC Diet.

PubMed

Abiko, Yukie; Kojima, Takashi; Murata, Masaki; Tsujiwaki, Mitsuhiro; Takeuchi, Masaya; Sawada, Norimasa; Mori, Michio

2013-12-01

DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine)-fed mice are widely used as a model for cholestatic liver disease. We examined the expression of tight junction protein claudin subspecies by immunofluorescent histochemistry in small intestine and kidney tissues of mice fed a DDC diet for 12 weeks. In the small intestine, decreases in claudin-3, claudin-7 and claudin-15 were observed in villous epithelial cells corresponding to the severity of histological changes while leaving the abundance of these claudin subspecies unchanged in crypt cells. Nevertheless, the proliferative activity of intestinal crypt cells measured by immunohistochemistry for Ki-67 decreased in the mice fed the DDC diet compared with that of control mice. These results suggest the possibility that DDC feeding affects the barrier function of villous epithelial cells and thus inhibits the proliferative activity of crypt epithelial cells. On the other hand, in the kidney, remarkable changes were found in the subcellular localization of claudin subspecies in a segment-specific manner, although histological changes of renal epithelial cells were quite minimal. These results indicate that immunohistochemistry for claudin subspecies can serve as a useful tool for detecting minute functional alterations of intestinal and renal epithelial cells.

Epithelial cell specific properties and genetic complementation in a delta F508 cystic fibrosis nasal polyp cell line.

PubMed

Kunzelmann, K; Lei, D C; Eng, K; Escobar, L C; Koslowsky, T; Gruenert, D C

1995-09-01

Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to lose their tight junctions with multiple subcultures. To elucidate ion transport and protein trafficking in CF epithelial cells, a polar cell line with apical and basolateral compartments will facilitate analysis of the efficacy of different gene therapy strategies in a "tight epithelium" in vitro. This study investigates the genotypic and phenotypic properties of a CF nasal polyp epithelial, delta F508 homozygote, cell line that has tight junctions pre-crisis. The cells (sigma CFNPE14o-) were transformed with an origin-of-replication defective SV40 plasmid. They develop transepithelial resistance in Ussing chambers and are defective in cAMP-dependent Cl- transport as measured by efflux of radioactive Cl-, short circuit current (Isc), or whole-cell patch clamp. Stimulation of the cells by bradykinin, histamine, or ATP seems to activate both K(+)- and Ca(+2)-dependent Cl- transport. Measurement of 36Cl- efflux following stimulation with A23187 and ionomycin indicate a Ca(+2)-dependent Cl- transport. Volume regulatory capacity of the cells is indicated by cell swelling conductance. Expression of the CF transmembrane conductance regulator mRNA was indicated by RT-PCR amplification. When cells are grown at 26 degrees C for 48 h there is no indication of cAMP-dependent Cl- as has been previously indicated in heterologous expression systems. Antibodies specific for secretory cell antigens indicate the presence of antigens found in goblet, serous, and mucous cells; in goblet and serous cells; or in goblet and mucous cells; but not antigens found exclusively in mucous or serous cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostate cancer cells specifically reorganize epithelial cell-fibroblast communication through proteoglycan and junction pathways.

PubMed

Suhovskih, Anastasia V; Kashuba, Vladimir I; Klein, George; Grigorieva, Elvira V

2017-01-02

Microenvironment and stromal fibroblasts are able to inhibit tumor cell proliferation both through secreted signaling molecules and direct cell-cell interactions but molecular mechanisms of these effects remain unclear. In this study, we investigated a role of cell-cell contact-related molecules (protein ECM components, proteoglycans (PGs) and junction-related molecules) in intercellular communications between the human TERT immortalized fibroblasts (BjTERT fibroblasts) and normal (PNT2) or cancer (LNCaP, PC3, DU145) prostate epithelial cells. It was shown that BjTERT-PNT2 cell coculture resulted in significant decrease of both BjTERT and PNT2 proliferation rates and reorganization of transcriptional activity of cell-cell contact-related genes in both cell types. Immunocytochemical staining revealed redistribution of DCN and LUM in PNT2 cells and significant increase of SDC1 at the intercellular contact zones between BjTERT and PNT2 cells, suggesting active involvement of the PGs in cell-cell contacts and contact inhibition of cell proliferation. Unlike to PNT2 cells, PC3 cells did not respond to BjTERT in terms of PGs expression, moderately increased transcriptional activity of junctions-related genes (especially tight junction) and failed to establish PC3-BjTERT contacts. At the same time, PC3 cells significantly down-regulated junctions-related genes (especially focal adhesions and adherens junctions) in BjTERT fibroblasts resulting in visible preference for homotypic PC3-PC3 over heterotypic PC3-BjTERT contacts and autonomous growth of PC3 clones. Taken together, the results demonstrate that an instructing role of fibroblasts to normal prostate epithelial cells is revoked by cancer cells through deregulation of proteoglycans and junction molecules expression and overall disorganization of fibroblast-cancer cell communication.

Long-term forskolin stimulation induces AMPK activation and thereby enhances tight junction formation in human placental trophoblast BeWo cells.

PubMed

Egawa, M; Kamata, H; Kushiyama, A; Sakoda, H; Fujishiro, M; Horike, N; Yoneda, M; Nakatsu, Y; Ying, Guo; Jun, Zhang; Tsuchiya, Y; Takata, K; Kurihara, H; Asano, T

2008-12-01

BeWo cells, derived from human choriocarcinoma, have been known to respond to forskolin or cAMP analogues by differentiating into multinucleated cells- like syncytiotrophoblasts on the surfaces of chorionic villi of the human placenta. In this study, we demonstrated that long-term treatment with forskolin enhances the tight junction (TJ) formation in human placental BeWo cells. Interestingly, AMPK activation and phosphorylation of acetyl-CoA carboxylase (ACC), a molecule downstream from AMPK, were induced by long-term incubation (>12h) with forskolin, despite not being induced by acute stimulation with forskolin. In addition, co-incubation with an AMPK inhibitor, compound C, as well as overexpression of an AMPK dominant negative mutant inhibited forskolin-induced TJ formation. Thus, although the molecular mechanism underlying AMPK activation via the forskolin stimulation is unclear, the TJ formation induced by forskolin is likely to be mediated by the AMPK pathway. Taking into consideration that TJs are present in the normal human placenta, this mechanism may be important for forming the placental barrier system between the fetal and maternal circulations.

The Saccharomyces boulardii CNCM I-745 strain shows protective effects against the B. anthracis LT toxin.

PubMed

Pontier-Bres, Rodolphe; Rampal, Patrick; Peyron, Jean-François; Munro, Patrick; Lemichez, Emmanuel; Czerucka, Dorota

2015-10-30

The probiotic yeast Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.

The Saccharomyces boulardii CNCM I-745 Strain Shows Protective Effects against the B. anthracis LT Toxin

PubMed Central

Pontier-Bres, Rodolphe; Rampal, Patrick; Peyron, Jean-François; Munro, Patrick; Lemichez, Emmanuel; Czerucka, Dorota

2015-01-01

The probiotic yeast Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax. PMID:26529015

Sinomenine alleviates high glucose-induced renal glomerular endothelial hyperpermeability by inhibiting the activation of RhoA/ROCK signaling pathway.

PubMed

Yin, Qingqiao; Xia, Yuanyu; Wang, Guan

2016-09-02

As an early sign of diabetic cardiovascular disease, endothelial dysfunction may contribute to progressive diabetic nephropathy (DN). Endothelial hyperpermeability induced by hyperglycemia (HG) is a central pathogenesis for DN. Sinomenine (SIN) has strong anti-inflammatory and renal protective effects, following an unknown protective mechanism against HG-induced hyperpermeability. We herein explored the role of SIN in vitro in an HG-induced barrier dysfunction model in human renal glomerular endothelial cells (HRGECs). The cells were exposed to SIN and/or HG for 24 h, the permeability of which was significantly increased by HG. Moreover, junction protein occludin in the cell-cell junction area and its total expression in HRGECs were significantly decreased by HG. However, the dysfunction of tight junction and hyperpermeability of HRGECs were significantly reversed by SIN. Furthermore, SIN prevented HG-increased reactive oxygen species (ROS) by activating nuclear factor-E2-related factor 2 (Nrf2). Interestingly, activation of RhoA/ROCK induced by HG was reversed by SIN or ROCK inhibitor. HG-induced hyperpermeability was prevented by SIN. High ROS level, tight junction dysfunction and RhoA/ROCK activation were significantly attenuated with knockdown of Nrf2. Mediated by activation of Nrf2, SIN managed to significantly prevent HG-disrupted renal endothelial barrier function by suppressing the RhoA/ROCK signaling pathway through reducing ROS. We successfully identified a novel pathway via which SIN exerted antioxidative and renal protective functions, and provided a molecular basis for potential SIN applications in treating DN vascular disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

The heat-shock protein Apg-2 binds to the tight junction protein ZO-1 and regulates transcriptional activity of ZONAB.

PubMed

Tsapara, Anna; Matter, Karl; Balda, Maria S

2006-03-01

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1-ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G(1)/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1-ZONAB signaling in epithelial cells in response to cellular stress.

The Heat-Shock Protein Apg-2 Binds to the Tight Junction Protein ZO-1 and Regulates Transcriptional Activity of ZONAB

PubMed Central

Tsapara, Anna; Matter, Karl; Balda, Maria S.

2006-01-01

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1–ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G1/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1–ZONAB signaling in epithelial cells in response to cellular stress. PMID:16407410

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

PubMed Central

Nunbhakdi-Craig, Viyada; Machleidt, Thomas; Ogris, Egon; Bellotto, Dennis; White, Charles L.; Sontag, Estelle

2002-01-01

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABαC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function. PMID:12196510

Expression pattern of adhesion molecules in junctional epithelium differs from that in other gingival epithelia.

PubMed

Hatakeyama, S; Yaegashi, T; Oikawa, Y; Fujiwara, H; Mikami, T; Takeda, Y; Satoh, M

2006-08-01

The gingival epithelium is the physiologically important interface between the bacterially colonized gingival sulcus and periodontal soft and mineralized connective tissues, requiring protection from exposure to bacteria and their products. However, of the three epithelia comprising the gingival epithelium, the junctional epithelium has much wider intercellular spaces than the sulcular epithelium and oral gingival epithelium. Hence, the aim of the present study was to characterize the cell adhesion structure in the junctional epithelium compared with the other two epithelia. Gingival epithelia excised at therapeutic flap surgery from patients with periodontitis were examined for expression of adhesion molecules by immunofluorescence. In the oral gingival epithelium and sulcular epithelium, but not in the junctional epithelium, desmoglein 1 and 2 in cell-cell contact sites were more abundant in the upper than the suprabasal layers. E-cadherin, the main transmembranous molecule of adherens junctions, was present in spinous layers of the oral gingival epithelium and sulcular epithelium, but was scarce in the junctional epithelium. In contrast, desmoglein 3 and P-cadherin were present in all layers of the junctional epithelium as well as the oral gingival epithelium and sulcular epithelium. Connexin 43 was clearly localized to spinous layers of the oral gingival epithelium, sulcular epithelium and parts of the junctional epithelium. Claudin-1 and occludin were expressed in the cell membranes of a few superficial layers of the oral gingival epithelium. These findings indicated that the junctional epithelium contains only a few desmosomes, composed of only desmoglein 3; adherens junctions are probably absent because of defective E-cadherin. Thus, the anchoring junctions connecting junctional epithelium cells are lax, causing widened intercellular spaces. In contrast, the oral gingival epithelium, which has a few tight junctions, functions as a barrier.

Claudin-4 Overexpression in Epithelial Ovarian Cancer Is Associated with Hypomethylation and Is a Potential Target for Modulation of Tight Junction Barrier Function Using a C-Terminal Fragment of Clostridium perfringens Enterotoxin1

PubMed Central

Litkouhi, Babak; Kwong, Joseph; Lo, Chun-Min; Smedley, James G; McClane, Bruce A; Aponte, Margarita; Gao, Zhijian; Sarno, Jennifer L; Hinners, Jennifer; Welch, William R; Berkowitz, Ross S; Mok, Samuel C; Garner, Elizabeth I O

2007-01-01

Background Claudin-4, a tight junction (TJ) protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is overexpressed in epithelial ovarian cancer (EOC). Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4-expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. Methods Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular) resistance (Rb) in EOC cells after claudin-4 silencing and after C-CPE treatment. Results Claudin-4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. Conclusions C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies. PMID:17460774

An In Vitro Model of the Blood-Brain Barrier: Naegleria fowleri Affects the Tight Junction Proteins and Activates the Microvascular Endothelial Cells.

PubMed

Coronado-Velázquez, Daniel; Betanzos, Abigail; Serrano-Luna, Jesús; Shibayama, Mineko

2018-04-14

Naegleria fowleri causes a fatal disease known as primary amoebic meningoencephalitis. This condition is characterized by an acute inflammation that originates from the free passage of peripheral blood cells to the central nervous system through the alteration of the blood-brain barrier. In this work, we established models of the infection in rats and in a primary culture of endothelial cells from rat brains with the aim of evaluating the activation and the alterations of these cells by N. fowleri. We proved that the rat develops the infection similar to the mouse model. We also found that amoebic cysteine proteases produced by the trophozoites and the conditioned medium induced cytopathic effect in the endothelial cells. In addition, N. fowleri can decrease the transendothelial electrical resistance by triggering the destabilization of the tight junction proteins claudin-5, occludin, and ZO-1 in a time-dependent manner. Furthermore, N. fowleri induced the expression of VCAM-1 and ICAM-1 and the production of IL-8, IL-1β, TNF-α, and IL-6 as well as nitric oxide. We conclude that N. fowleri damaged the blood-brain barrier model by disrupting the intercellular junctions and induced the presence of inflammatory mediators by allowing the access of inflammatory cells to the olfactory bulbs. © 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.

Tight junction-based epithelial microenvironment and cell proliferation.

PubMed

Tsukita, S; Yamazaki, Y; Katsuno, T; Tamura, A; Tsukita, S

2008-11-24

Belt-like tight junctions (TJs), referred to as zonula occludens, have long been regarded as a specialized differentiation of epithelial cell membranes. They are required for cell adhesion and paracellular barrier functions, and are now thought to be partly involved in fence functions and in cell polarization. Recently, the molecular bases of TJs have gradually been unveiled. TJs are constructed by TJ strands, whose basic frameworks are composed of integral membrane proteins with four transmembrane domains, designated claudins. The claudin family is supposedly composed of at least 24 members in mice and humans. Other types of integral membrane proteins with four transmembrane domains, namely occludin and tricellulin, as well as the single transmembrane proteins, JAMs (junctional adhesion molecules) and CAR (coxsackie and adenovirus receptor), are associated with TJ strands, and the high-level organization of TJ strands is likely to be established by membrane-anchored scaffolding proteins, such as ZO-1/2. Recent functional analyses of claudins in cell cultures and in mice have suggested that claudin-based TJs may have pivotal functions in the regulation of the epithelial microenvironment, which is critical for various biological functions such as control of cell proliferation. These represent the dawn of 'Barriology' (defined by Shoichiro Tsukita as the science of barriers in multicellular organisms). Taken together with recent reports regarding changes in claudin expression levels, understanding the regulation of the TJ-based microenvironment system will provide new insights into the regulation of polarization in the respect of epithelial microenvironment system and new viewpoints for developing anticancer strategies.

Intracellular Ca2+ release mediates cationic but not anionic poly(amidoamine) (PAMAM) dendrimer-induced tight junction modulation.

PubMed

Avaritt, Brittany R; Swaan, Peter W

2014-09-01

Poly(amidoamine) (PAMAM) dendrimers show great promise for utilization as oral drug delivery vehicles. These polymers are capable of traversing epithelial barriers, and have been shown to translocate by both transcellular and paracellular routes. While many proof-of-concept studies have shown that PAMAM dendrimers improve intestinal transport, little information exists on the mechanisms of paracellular transport, specifically dendrimer-induced tight junction modulation. Using anionic G3.5 and cationic G4 PAMAM dendrimers with known absorption enhancers, we investigated tight junction modulation in Caco-2 monolayers by visualization and mannitol permeability and compared dendrimer-mediated tight junction modulation to that of established permeation enhancers. [(14)C]-Mannitol permeability in the presence and absence of phospholipase C-dependent signaling pathway inhibitors was also examined and indicated that this pathway may mediate dendrimer-induced changes in permeability. Differences between G3.5 and G4 in tight junction protein staining and permeability with inhibitors were evident, suggesting divergent mechanisms were responsible for tight junction modulation. These dissimilarities are further intimated by the intracellular calcium release caused by G4 but not G3.5. Based on our results, it is apparent that the underlying mechanisms of dendrimer permeability are complex, and the complexities are likely a result of the density and sign of the surface charges of PAMAM dendrimers. The results of this study will have implications on the future use of PAMAM dendrimers for oral drug delivery.

Nerve cables formed in silicone chambers reconstitute a perineurial but not a vascular endoneurial permeability barrier.

PubMed

Azzam, N A; Zalewski, A A; Williams, L R; Azzam, R N

1991-12-22

The passage of molecules into the endoneurial environment of the axons of normal peripheral nerve is regulated by two permeability barriers, the perineurial-nerve barrier and the endoneurial blood-nerve barrier. These barriers exist because of the presence of tight junctions between adjacent perineurial cells and adjacent endothelial cells. In the present study we investigated whether permeability barriers form in nerve cables, which develop inside silicone chambers. The sciatic nerves of adult rats were cut, and the proximal and distal ends sutured into opposite ends of silicone chambers that were filled with dialyzed plasma. The presence of barriers was determined with the tracer horseradish peroxidase (HRP), which was injected intravenously and detected histochemically in tissues by light and electron microscopy. At four weeks, a regenerated nerve cable extended across the 10 mm length of each chamber. However, no permeability barriers were present since the reaction product for HRP was visible throughout the cable. At twenty-six weeks, all the axons in cables were gathered into minifascicles. Each minifascicle of axons was surrounded by perineurial cells. Blood vessels were excluded from the minifascicles by the perineurial cells and the vessels were permeable to HRP, thus indicating that their endothelial cells had not formed tight junctions. Despite the leakage of HRP from the excluded vessels, the tracer did not reach the axons because the perineurial cells encircling the minifascicles developed tight junctions. In some animals, the chambers were removed at four weeks to determine whether the chamber influenced barrier development. This manipulation had no effect since cables, with or without chambers, exhibited similar findings at twenty-six weeks. Our results indicate that nerve cables regenerate a perineurial but not an endoneurial permeability barrier. We conclude that axons in long-term cables are protected by only a perineurial permeability barrier.

The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks*

PubMed Central

Van Itallie, Christina M.; Aponte, Angel; Tietgens, Amber Jean; Gucek, Marjan; Fredriksson, Karin; Anderson, James Melvin

2013-01-01

The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization. PMID:23553632

Eicosapentaenoic Acid Enhances Heat Stress-Impaired Intestinal Epithelial Barrier Function in Caco-2 Cells

PubMed Central

Xiao, Guizhen; Tang, Liqun; Yuan, Fangfang; Zhu, Wei; Zhang, Shaoheng; Liu, Zhifeng; Geng, Yan; Qiu, Xiaowen

2013-01-01

Objective Dysfunction of the intestinal epithelial tight junction (TJ) barrier is known to have an important etiologic role in the pathophysiology of heat stroke. N-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), play a role in maintaining and protecting the TJ structure and function. This study is aimed at investigating whether n-3 PUFAs could alleviate heat stress-induced dysfunction of intestinal tight junction. Methods Human intestinal epithelial Caco-2 cells were pre-incubated with EPA, DHA or arachidonic acid (AA) and then exposed to heat stress. Transepithelial electrical resistance (TEER) and Horseradish Peroxidase (HRP) permeability were measured to analyze barrier integrity. Levels of TJ proteins, including occludin, ZO-1 and claudin-2, were analyzed by Western blot and localized by immunofluorescence microscopy. Messenger RNA levels were determined by quantitative real time polymerase chain reaction (Q-PCR). TJ morphology was observed by transmission electron microscopy. Results EPA effectively attenuated the decrease in TEER and impairment of intestinal permeability in HRP flux induced by heat exposure. EPA significantly elevated the expression of occludin and ZO-1, while DHA was less effective and AA was not at all effective. The distortion and redistribution of TJ proteins, and disruption of morphology were also effectively prevented by pretreatment with EPA. Conclusion This study indicates for the first time that EPA is more potent than DHA in protecting against heat-induced permeability dysfunction and epithelial barrier damage of tight junction. PMID:24066055

Bifidobacteria isolated from infants and cultured on human milk oligosaccharides affect intestinal epithelial function

PubMed Central

Chichlowski, Maciej; De Lartigue, Guillaume; German, J. Bruce; Raybould, Helen E.; Mills, David A.

2012-01-01

Objectives Human milk oligosaccharides (HMO) are the third most abundant component of breast milk. Our laboratory has previously revealed gene clusters specifically linked to HMO metabolism in select bifidobacteria isolated from fecal samples of infants. Our objective was to test the hypothesis that growth of select bifidobacteria on HMO stimulates the intestinal epithelium. Methods Caco-2 and HT-29 cells were incubated with lactose (LAC) or HMO-grown Bifidobacterium longum subsp. infantis (B. infantis) or B. bifidum. Bacterial adhesion and translocation was measured by real-time quantitative PCR. Expression of pro- and anti-inflammatory cytokines and tight junction proteins was analyzed by real time reverse transcriptase. Distribution of tight junction proteins was measured using immunofluorescent microscopy. Results We showed that HMO-grown B. infantis had significantly higher rate of adhesion to HT-29 cells compared to B. bifidum. B. infantis also induced expression of a cell membrane glycoprotein, P-selectin glycoprotein ligand -1. Both B. infantis and B. bifidum grown on HMO caused less occludin relocalization and higher expression of anti-inflammatory cytokine, interleukin (IL)-10 compared to LAC-grown bacteria in Caco-2 cells. B. bifidum grown on HMO showed higher expression of junctional adhesion molecule and occludin in Caco-2 cell and HT-29 cells. There were no significant differences between LAC or HMO treatments in bacterial translocation. Conclusions This study provides evidence for the specific relationship between HMO-grown bifidobacteria and intestinal epithelial cells. To our knowledge, this is the first study describing HMO-induced changes in the bifidobacteria-intestinal cells interaction. PMID:22383026

Multiple protocadherins are expressed in brain microvascular endothelial cells and might play a role in tight junction protein regulation.

PubMed

Dilling, Christina; Roewer, Norbert; Förster, Carola Y; Burek, Malgorzata

2017-10-01

Protocadherins (Pcdhs) are a large family of cadherin-related molecules. They play a role in cell adhesion, cellular interactions, and development of the central nervous system. However, their expression and role in endothelial cells has not yet been characterized. Here, we examined the expression of selected clustered Pcdhs in endothelial cells from several vascular beds. We analyzed human and mouse brain microvascular endothelial cell (BMEC) lines and primary cells, mouse myocardial microvascular endothelial cell line, and human umbilical vein endothelial cells. We examined the mRNA and protein expression of selected Pcdhs using RT-PCR, Western blot, and immunostaining. A strong mRNA expression of Pcdhs was observed in all endothelial cells tested. At the protein level, Pcdhs-gamma were detected using an antibody against the conserved C-terminal domain of Pcdhs-gamma or an antibody against PcdhgC3. Deletion of highly expressed PcdhgC3 led to differences in the tight junction protein expression and mRNA expression of Wnt/mTOR (mechanistic target of rapamycin) pathway genes as well as lower transendothelial electrical resistance. Staining of PcdhgC3 showed diffused cytoplasmic localization in mouse BMEC. Our results suggest that Pcdhs may play a critical role in the barrier-stabilizing pathways at the blood-brain barrier.

Ischemia-reperfusion impairs blood-brain barrier function and alters tight junction protein expression in the ovine fetus.

PubMed

Chen, X; Threlkeld, S W; Cummings, E E; Juan, I; Makeyev, O; Besio, W G; Gaitanis, J; Banks, W A; Sadowska, G B; Stonestreet, B S

2012-12-13

The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) and tight junction proteins by Western immunoblot in fetal sheep at 127 days of gestation without ischemia, and 4, 24, or 48 h after ischemia. The largest increase in K(i) (P<0.05) was 4 h after ischemia. Occludin and claudin-5 expressions decreased at 4 h, but returned toward control levels 24 and 48 h after ischemia. Zonula occludens-1 and -2 decreased after ischemia. Inverse correlations between K(i) and tight junction proteins suggest that the decreases in tight junction proteins contribute to impaired blood-brain barrier function after ischemia. We conclude that impaired blood-brain barrier function is an important component of hypoxic-ischemic brain injury in the fetus, and that increases in quantitatively measured barrier permeability (K(i)) change as a function of the duration of reperfusion after ischemia. The largest increase in permeability occurs 4 h after ischemia and blood-brain barrier function improves early after injury because the blood-brain barrier is less permeable 24 and 48 than 4 h after ischemia. Changes in the tight junction molecular composition are associated with increases in blood-brain barrier permeability after ischemia. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Adherens Junctions Modulate Diffusion between Epithelial Cells in Trichoplax adhaerens.

PubMed

Smith, Carolyn L; Reese, Thomas S

2016-12-01

Trichoplax adhaerens is the sole named member of Placozoa, an ancient metazoan phylum. This coin-shaped animal glides on ventral cilia to find and digest algae on the substrate. It has only six cell types, all but two of which are incorporated into the epithelium that encloses it. The upper epithelium is thin, composed of a pavement of relatively large polygonal disks, each bearing a cilium. The lower epithelium is thick and composed primarily of narrow ciliated cells that power locomotion. Interspersed among these cells are two different secretory cells: one containing large lipophilic granules that, when released, lyse algae under the animal; the other, less abundant, is replete with smaller secretory granules containing neuropeptides. All cells within both epithelia are joined by adherens junctions that are stabilized by apical actin networks. Cells are held in place during shape changes or under osmotic stress, but dissociate in low calcium. Neither tight, septate, nor gap junctions are evident, leaving only the adherens junction to control the permeability of the epithelium. Small (<4 kDa) fluorescent dextrans introduced into artificial seawater readily penetrate into the animal between the cells. Larger dextrans enter slowly, except in animals treated with reduced calcium, indicating that the adherens junctions form a circumferential belt around each cell that impedes diffusion into the animal. During feeding, the limited permeability of the adherens junctions helps to confine material released from lysed algae within the narrow space under the animal, where it is absorbed by endocytosis.

Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation.

PubMed

Zenker, Jennifer; White, Melanie D; Gasnier, Maxime; Alvarez, Yanina D; Lim, Hui Yi Grace; Bissiere, Stephanie; Biro, Maté; Plachta, Nicolas

2018-04-19

Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation. Copyright © 2018 Elsevier Inc. All rights reserved.

Interactions of Entamoeba Histolytica with Host Cells in the Gut Mucosa,

DTIC Science & Technology

1977-01-01

amebae were observed close to the cecal epithelium, the apposing epithelial cell was found to have protruded in the lumen and making contact with the... amebae . Such cells often had become detached from their basal lamina and intercellular tight junctions, affording spaces through which amebae invaded the...mucosa. Other epithelial cells in close proximity to amebae showed degenerative changes characterized by swelling of mitochondria and endoplasmic

Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

NASA Astrophysics Data System (ADS)

Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

2016-07-01

Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

Intestinal Alkaline Phosphatase Regulates Tight Junction Protein Levels

PubMed Central

Liu, Wei; Hu, Dong; Huo, Haizhong; Zhang, Weifeng; Adiliaghdam, Fatemeh; Morrison, Sarah; Ramirez, Juan M; Gul, Sarah S; Hamarneh, Sulaiman R; Hodin, Richard A

2017-01-01

BACKGROUND Intestinal alkaline phosphatase (IAP) plays a pivotal role in maintaining gut health and well-being. Oral supplementation with IAP in mice improves gut barrier function and prevents luminal proinflammatory factors from gaining access to the circulation. In this study, we sought to explore the relationship between IAP and tight junction protein (TJP) expression and function. STUDY DESIGN The effect of IAP deletion on TJP levels was studied in mouse embryonic fibroblasts (MEFs) generated from IAP-knockout and wild type mice. Regulation of TJPs by IAP was assayed in the human colon cancer Caco-2 and T84 cells by overexpressing the human IAP gene. Tight junction protein levels and localization were measured by using RT q-PCR and antibodies targeting the specific TJPs. Finally, the effect of IAP on inflammation-induced intestinal permeability was measured by in vitro trans-well epithelial electrical resistance (TEER). RESULTS Intestinal alkaline phosphatase gene deletion in MEFs resulted in significantly lower levels of ZO-1, ZO-2, and Occludin compared with levels in wild-type control cells; IAP over-expression in Caco-2 and T84 cells resulted in approximate 2-fold increases in the mRNA levels of ZO-1 and ZO-2. The IAP treatment ameliorated lipopolysaccharide-induced increased permeability in the Caco-2 trans-well system. Furthermore, IAP treatment preserved the localization of the ZO-1 and Occludin proteins during inflammation and was also associated with improved epithelial barrier function. CONCLUSIONS Intestinal alkaline phosphatase is a major regulator of gut mucosal permeability and appears to work at least partly through improving TJP levels and localization. These data provide a strong foundation to develop IAP as a novel therapy to maintain gut barrier function. PMID:27106638

Intestinal Alkaline Phosphatase Regulates Tight Junction Protein Levels.

PubMed

Liu, Wei; Hu, Dong; Huo, Haizhong; Zhang, Weifeng; Adiliaghdam, Fatemeh; Morrison, Sarah; Ramirez, Juan M; Gul, Sarah S; Hamarneh, Sulaiman R; Hodin, Richard A

2016-06-01

Intestinal alkaline phosphatase (IAP) plays a pivotal role in maintaining gut health and well-being. Oral supplementation with IAP in mice improves gut barrier function and prevents luminal proinflammatory factors from gaining access to the circulation. In this study, we sought to explore the relationship between IAP and tight junction protein (TJP) expression and function. The effect of IAP deletion on TJP levels was studied in mouse embryonic fibroblasts (MEFs) generated from IAP-knockout and wild type mice. Regulation of TJPs by IAP was assayed in the human colon cancer Caco-2 and T84 cells by overexpressing the human IAP gene. Tight junction protein levels and localization were measured by using RT q-PCR and antibodies targeting the specific TJPs. Finally, the effect of IAP on inflammation-induced intestinal permeability was measured by in vitro trans-well epithelial electrical resistance (TEER). Intestinal alkaline phosphatase gene deletion in MEFs resulted in significantly lower levels of ZO-1, ZO-2, and Occludin compared with levels in wild-type control cells; IAP overexpression in Caco-2 and T84 cells resulted in approximate 2-fold increases in the mRNA levels of ZO-1 and ZO-2. The IAP treatment ameliorated lipopolysaccharide-induced increased permeability in the Caco-2 trans-well system. Furthermore, IAP treatment preserved the localization of the ZO-1 and Occludin proteins during inflammation and was also associated with improved epithelial barrier function. Intestinal alkaline phosphatase is a major regulator of gut mucosal permeability and appears to work at least partly through improving TJP levels and localization. These data provide a strong foundation to develop IAP as a novel therapy to maintain gut barrier function. Copyright © 2016. Published by Elsevier Inc.

Claudin 5 Expression in Mouse Seminiferous Epithelium Is Dependent upon the Transcription Factor Ets Variant 5 and Contributes to Blood-Testis Barrier Function1

PubMed Central

Morrow, Carla M.K.; Tyagi, Gaurav; Simon, Liz; Carnes, Kay; Murphy, Kenneth M.; Cooke, Paul S.; Hofmann, Marie-Claude C.; Hess, Rex A.

2009-01-01

The blood-testis barrier (BTB) is formed by tight junctions between Sertoli cells. Results of previous studies suggested that the barrier is deficient in ets variant 5 (ETV5) gene-deleted mice; therefore, microarray data were examined for changes in tight junction-associated genes. The tight junctional protein claudin 5 (CLDN5) was decreased in testes of 8-day-old Etv5−/− pups. The study reported herein examined the expression of CLDN5 in wild-type (WT) and Etv5−/− mice and evaluated its contribution to BTB function. CLDN5 protein expression was evaluated in 8-day-old WT and Etv5−/− and adult WT, Etv5−/−, and W/Wv testes by immunohistochemistry and in 8-day-old WT Sertoli cell-enriched and germ cell-enriched fractions by immunocytochemistry. Cldn5 mRNA expression was evaluated in 0- to 20-day-old and adult WT mice and in 8-day-old and adult Etv5−/− mice via quantitative PCR. Tracer studies were performed in adult WT, Etv5−/−, and W/Wv mice. The results indicate the following: 1) CLDN5 was expressed in Sertoli cells, spermatogonia, and preleptotene spermatocytes. 2) Seminiferous epithelial CLDN5 expression depended upon both the presence of germ cells and ETV5. 3) CLDN5 expression in testicular vascular endothelium and rete testis epithelium was ETV5 independent. 4) Cldn5 mRNA expression increased in the testes of juvenile mice at the time of BTB formation. 5) Testes of Etv5−/− and W/Wv mice, which are both deficient in seminiferous epithelial CLDN5 expression, had biotin tracer leakage from the interstitial space into the seminiferous tubule lumen. In conclusion, CLDN5 is expressed in the seminiferous epithelium, appears to be regulated by multiple influences, and contributes to BTB function. PMID:19571261

Methyl-beta-cyclodextrin increases permeability of Caco-2 cell monolayers by displacing specific claudins from cholesterol rich domains associated with tight junctions.

PubMed

Lambert, Daniel; O'Neill, Catherine A; Padfield, Philip J

2007-01-01

In a previous study we demonstrated that depletion of Caco-2 cell cholesterol results in the loss of tight junction (TJ) integrity through the movement of claudins 3 and 4 and occludin, but not claudin 1, out of the TJs [1]. The aims of this study were to determine whether the major tight junction (TJ) proteins in Caco-2 cells are associated with cholesterol rich, membrane raft-like domains and if the loss of TJ integrity produced by the extraction of cholesterol reflects the dissolution of these domains resulting in the loss of TJ organisation. We have demonstrated that in Caco-2 cells claudins 1, 3, 4 and 7, JAM-A and occludin, are associated with cholesterol rich membrane domains that are insoluble in Lubrol WX. Co-immunoprecipitation studies demonstrated that there is no apparent restriction on the combination of claudins present in the rafts and that interaction between the proteins is dependent on cholesterol. JAM-A was not co-immunoprecipitated with the other TJ proteins indicating that it is resident within in a distinct population of rafts and therefore is likely not directly associated with the claudins/occludin present in the TJ complexes. Depletion of Caco-2 cell cholesterol with methyl-beta-cyclodextrin resulted in the displacement of claudins 3, 4 and 7, JAM-A and occludin, but not claudin 1, out of the cholesterol rich domains. Our data indicate that depletion of cholesterol does not result in the loss of the TJ-associated membrane rafts. However, the sterol is required to maintain the association of key proteins with the TJ associated membrane rafts and therefore the TJs. Furthermore, the data suggest that cholesterol may actually directly stabilise the multi-protein complexes that form the TJ strands. Copyright (c) 2007 S. Karger AG, Basel.

Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development.

PubMed

Cheng, C Yan; Mruk, Dolores D

2002-10-01

Spermatogenesis is an intriguing but complicated biological process. However, many studies since the 1960s have focused either on the hormonal events of the hypothalamus-pituitary-testicular axis or morphological events that take place in the seminiferous epithelium. Recent advances in biochemistry, cell biology, and molecular biology have shifted attention to understanding some of the key events that regulate spermatogenesis, such as germ cell apoptosis, cell cycle regulation, Sertoli-germ cell communication, and junction dynamics. In this review, we discuss the physiology and biology of junction dynamics in the testis, in particular how these events affect interactions of Sertoli and germ cells in the seminiferous epithelium behind the blood-testis barrier. We also discuss how these events regulate the opening and closing of the blood-testis barrier to permit the timely passage of preleptotene and leptotene spermatocytes across the blood-testis barrier. This is physiologically important since developing germ cells must translocate across the blood-testis barrier as well as traverse the seminiferous epithelium during their development. We also discuss several available in vitro and in vivo models that can be used to study Sertoli-germ cell anchoring junctions and Sertoli-Sertoli tight junctions. An in-depth survey in this subject has also identified several potential targets to be tackled to perturb spermatogenesis, which will likely lead to the development of novel male contraceptives.

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imamura, Masafumi; Department of Pathology, Sapporo Medical University School of Medicine, S1. W17. Sapporo 060-8556; Kojima, Takashi

2007-05-15

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions inmore » fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.« less

Claudins and the Kidney Volume 75: Annual Review of Physiology

PubMed Central

Hou, Jianghui; Rajagopal, Madhumitha; Yu, Alan S. L.

2013-01-01

Claudins are tight junction membrane proteins that regulate paracellular permeability of renal epithelia to small ions, solutes and water. Claudins interact within the cell membrane and between neighboring cells to form tight junction strands and constitute both the paracellular barrier and pore. The first extracellular domain of claudins is thought to be the pore-lining domain and contains the determinants of charge selectivity. Multiple claudins are expressed in different nephron segments and this likely determines the permeability properties of each segment. Recent evidence has identified claudin-2 as constituting the cation-reabsorptive pathway in the proximal tubule, claudin-14, -16 and -19 as forming a complex that regulates calcium transport in the thick ascending limb of the loop of Henle, and claudin-4, -7 and -8 as determinants of collecting duct choride permeability. Mutations in claudin-16 and -19 cause familial hypercalciuric hypomagnesemia. The roles of other claudins in kidney diseases remain to be fully elucidated. PMID:23140368

TNFalpha-induced and berberine-antagonized tight junction barrier impairment via tyrosine kinase, Akt and NFkappaB signaling.

PubMed

Amasheh, Maren; Fromm, Anja; Krug, Susanne M; Amasheh, Salah; Andres, Susanne; Zeitz, Martin; Fromm, Michael; Schulzke, Jörg-Dieter

2010-12-01

TNFα-mediated tight junction defects contribute to diarrhea in inflammatory bowel diseases (IBDs). In our study, the signaling pathways of the TNFα effect on barrier- or pore-forming claudins were analyzed in HT-29/B6 human colon monolayers. Berberine, a herbal therapeutic agent that has been recently established as a therapy for diabetes and hypercholesterinemia, was able to completely antagonize the TNFα-mediated barrier defects in the cell model and in rat colon. Ussing chamber experiments and two-path impedance spectroscopy revealed a decrease of paracellular resistance after TNFα to 11±4%, whereas transcellular resistance was unchanged. The permeability of the paracellular marker fluorescein was increased fourfold. Berberine alone had no effect while it fully prevented the TNFα-induced barrier defects. This effect on resistance was confirmed in rat colon. TNFα removed claudin-1 from the tight junction and increased claudin-2 expression. Berberine prevented TNFα-induced claudin-1 disassembly and upregulation of claudin-2. The effects of berberine were mimicked by genistein plus BAY11-7082, indicating that they are mediated via tyrosine kinase, pAkt and NFκB pathways. In conclusion, the anti-diarrheal effect of berberine is explained by a novel mechanism, suggesting a therapeutic approach against barrier breakdown in intestinal inflammation.

Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs).

PubMed

DeStefano, Jackson G; Xu, Zinnia S; Williams, Ashley J; Yimam, Nahom; Searson, Peter C

2017-08-04

The endothelial cells that form the lumen of capillaries and microvessels are an important component of the blood-brain barrier. Cell phenotype is regulated by transducing a range of biomechanical and biochemical signals in the local microenvironment. Here we report on the role of shear stress in modulating the morphology, motility, proliferation, apoptosis, and protein and gene expression, of confluent monolayers of human brain microvascular endothelial cells derived from induced pluripotent stem cells. To assess the response of derived human brain microvascular endothelial cells (dhBMECs) to shear stress, confluent monolayers were formed in a microfluidic device. Monolayers were subjected to a shear stress of 4 or 12 dyne cm -2 for 40 h. Static conditions were used as the control. Live cell imaging was used to assess cell morphology, cell speed, persistence, and the rates of proliferation and apoptosis as a function of time. In addition, immunofluorescence imaging and protein and gene expression analysis of key markers of the blood-brain barrier were performed. Human brain microvascular endothelial cells exhibit a unique phenotype in response to shear stress compared to static conditions: (1) they do not elongate and align, (2) the rates of proliferation and apoptosis decrease significantly, (3) the mean displacement of individual cells within the monolayer over time is significantly decreased, (4) there is no cytoskeletal reorganization or formation of stress fibers within the cell, and (5) there is no change in expression levels of key blood-brain barrier markers. The characteristic response of dhBMECs to shear stress is significantly different from human and animal-derived endothelial cells from other tissues, suggesting that this unique phenotype that may be important in maintenance of the blood-brain barrier. The implications of this work are that: (1) in confluent monolayers of dhBMECs, tight junctions are formed under static conditions, (2) the formation of tight junctions decreases cell motility and prevents any morphological transitions, (3) flow serves to increase the contact area between cells, resulting in very low cell displacement in the monolayer, (4) since tight junctions are already formed under static conditions, increasing the contact area between cells does not cause upregulation in protein and gene expression of BBB markers, and (5) the increase in contact area induced by flow makes barrier function more robust.

[Research progress of relationship between diabetes and intestinal epithelial tight junction barrier and intervetion of berberine].

PubMed

Qin, Xin; Dong, Hui; Lu, Fu-Er

2016-06-01

Intestinal tight junction is an important part of the small intestinal mucosa barrier. It plays a very significant role in maintaining the intestinal mucosal permeability and integrity, preventing the bacterial endotoxin and toxic macromolecular substances into the body so as to keep a stable internal environment. Numerous studies have shown that intestinal mucosal barrier dysfunction is closely related to the development of diabetes. Therefore, protecting intestinal tight junction and maintaining the mucosal barrier have great significance in the prevention and treatment of diabetes. The effect of berberine in diabetes treatment is obvious. However, the pharmacological study found that the bioavailability of berberine is extremely low. Some scholars put forward that the major site of pharmaceutical action of berberine might be in the gut. Studies have shown that berberine could regulate the intestinal flora and intestinal hormone secretion, protect the intestinal barrier, inhibit the absorption of glucose, eliminate the intestinal inflammation and so on. Recently studies have found that the hypoglycemic effect of berberine is likely to relate with the influence on intestinal tight junction and the protection of mucosal barrier. Here is the review about the association between intestinal tight junction barrier dysfunction and diabetes, and the related hypoglycemic mechanism of berberine. Copyright© by the Chinese Pharmaceutical Association.

Estrogen Modulates Expression of Tight Junction Proteins in Rat Vagina

PubMed Central

Oh, Kyung-Jin; Ahn, Kyuyoun

2016-01-01

Background. The objectives of this study were to investigate the localization of tight junctions and the modulation of zonula occludens- (ZO-) 1, occludin and claudin-1 expression by estrogen in castrated female rat vagina. Female Sprague-Dawley rats (230–240 g, n = 45) were divided into three groups and subjected to a sham operation (control group, n = 15), bilateral ovariectomy (Ovx group, n = 15), or bilateral ovariectomy followed by daily subcutaneous injection of 17β-estradiol (50 μg/kg/day, Ovx + Est group, n = 15). The cellular localization and expression of ZO-1, occludin, and claudin-1 were determined in each group by immunohistochemistry and western blot. Results. Expression of ZO-1 was diffuse in all groups, with the highest intensity in the superficial epithelium in the control group. Occludin was localized in the intermediate and basal epithelium. Claudin-1 was most intense in the superficial layer of the vaginal epithelium in the control group. Expression of ZO-1, occludin, and claudin-1 was significantly decreased after ovariectomy and was restored to the level of the control after estrogen replacement. Conclusions. Tight junctions are distinctly localized in rat vagina, and estrogen modulates the expression of tight junctions. Further researches are needed to clarify the functional role of tight junctions in vaginal lubrication. PMID:27127786

Estrogen Modulates Expression of Tight Junction Proteins in Rat Vagina.

PubMed

Oh, Kyung-Jin; Lee, Hyun-Suk; Ahn, Kyuyoun; Park, Kwangsung

2016-01-01

Background. The objectives of this study were to investigate the localization of tight junctions and the modulation of zonula occludens- (ZO-) 1, occludin and claudin-1 expression by estrogen in castrated female rat vagina. Female Sprague-Dawley rats (230-240 g, n = 45) were divided into three groups and subjected to a sham operation (control group, n = 15), bilateral ovariectomy (Ovx group, n = 15), or bilateral ovariectomy followed by daily subcutaneous injection of 17β-estradiol (50 μg/kg/day, Ovx + Est group, n = 15). The cellular localization and expression of ZO-1, occludin, and claudin-1 were determined in each group by immunohistochemistry and western blot. Results. Expression of ZO-1 was diffuse in all groups, with the highest intensity in the superficial epithelium in the control group. Occludin was localized in the intermediate and basal epithelium. Claudin-1 was most intense in the superficial layer of the vaginal epithelium in the control group. Expression of ZO-1, occludin, and claudin-1 was significantly decreased after ovariectomy and was restored to the level of the control after estrogen replacement. Conclusions. Tight junctions are distinctly localized in rat vagina, and estrogen modulates the expression of tight junctions. Further researches are needed to clarify the functional role of tight junctions in vaginal lubrication.

The role of apoptosis in LDL transport through cultured endothelial cell monolayers.

PubMed

Cancel, Limary M; Tarbell, John M

2010-02-01

We have previously shown that leaky junctions associated with dying or dividing cells are the dominant pathway for low density lipoprotein (LDL) transport under convective conditions, accounting for more than 90% of the transport [Cancel LM, Fitting A, Tarbell JM. In vitro study of LDL transport under pressurized (convective) conditions. Am J Physiol Heart Circ Physiol 2007;293:H126-32]. To explore the role of apoptosis in the leaky junction pathway, TNFalpha and cycloheximide (TNFalpha/CHX) were used to induce an elevated rate of apoptosis in cultured bovine aortic endothelial cell (BAEC) monolayers and the convective fluxes of LDL and water were measured. Treatment with TNFalpha/CHX induced a 18.3-fold increase in apoptosis and a 4.4-fold increase in LDL permeability. Increases in apoptosis and permeability were attenuated by treatment with the caspase inhibitor Z-VAD-FMK. Water flux increased by 2.7-fold after treatment with TNFalpha/CHX, and this increase was not attenuated by treatment with Z-VAD-FMK. Immunostaining of the tight junction protein ZO-1 showed that TNFalpha/CHX treatment disrupts the tight junction in addition to inducing apoptosis. This disruption is present even when Z-VAD-FMK is used to inhibit apoptosis, and likely accounts for the increase in water flux. We found a strong correlation between the rate of apoptosis and the permeability of BAEC monolayers to LDL. These results demonstrate the potential of manipulating endothelial monolayer permeability by altering the rate of apoptosis pharmacollogicaly. This has implications for the treatment of atherosclerosis. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Cobalt chloride compromises transepithelial barrier properties of CaCo-2 BBe human gastrointestinal epithelial cell layers.

PubMed

DiGuilio, K M; Valenzano, M C; Rybakovsky, E; Mullin, J M

2018-01-05

Elevation of the transcription factor HIF-1 is a prominent mediator of not only processes that accompany hypoxia, but also the tumor microenvironment and tissue regeneration. This study uses mediators of "chemical hypoxia" to ask the question whether HIF-1α elevation in a healthy epithelial cell layer leads to leakiness in its tight junctional seals. Transepithelial electrical resistance and transepithelial diffusion of 14 C-D-mannitol and other radiolabeled probes are used as indicators of transepithelial barrier function of CaCo-2 BBe human gastrointestinal epithelial cell layers cultured on permeable supports. Western immunoblot analyses of integral tight junctional proteins (occludin and claudins) are used as further indicators of barrier function change. Cobalt, an inhibitor of the prolyl hydroxylase enzymes governing HIF-1α breakdown in the cell, induces transepithelial leakiness in CaCo-2 BBe cell layers in a time and concentration-dependent manner. This increased leakiness is accompanied by significant changes in certain specific integral tight junctional (TJ) proteins such as a decreased level of occludin and increased level of claudin-5. Similar results regarding barrier function compromise also occur with other chemical inhibitors of HIF-1α breakdown, namely ciclopiroxolamine (CPX) and dimethyloxalylglycine (DMOG). The increased leak is manifested by both decreased transepithelial electrical resistance (R t ) and increased paracellular diffusion of D-mannitol (J m ). The induced transepithelial leak shows significant size selectivity, consistent with induced effects on TJ permeability. Less-differentiated cell layers were significantly more affected than well-differentiated cell layers regarding induced transepithelial leak. A genetically modified CaCo-2 variant with reduced levels of HIF-1β, showed reduced transepithelial leak in response to cobalt exposure, further indicating that elevation of HIF-1α levels induced by agents of "chemical hypoxia" is responsible for the compromised barrier function of the CaCo-2 BBe cell layers. Exposure to inducers of chemical hypoxia elevated HIF-1α levels and increased transepithelial leak. The degree of epithelial differentiation has significant effects on this action, possibly explaining the varying effects of HIF-1 modulation in epithelial and endothelial barrier function in different physiological and pathophysiological conditions.

Environmental toxicants and male reproductive function

PubMed Central

Wong, Elissa W.P; Lie, Pearl P.Y; Li, Michelle W.M; Su, Linlin; Siu, Erica R; Yan, Helen H.N; Mannu, Jayakanthan; Mathur, Premendu P; Bonanomi, Michele; Silvestrini, Bruno; Mruk, Dolores D

2011-01-01

Environmental toxicants, such as cadmium and bisphenol A (BPA) are endocrine disruptors. In utero, perinatal or neonatal exposure of BPA to rats affect the male reproductive function, such as the blood-testis barrier (BTB) integrity. This effect of BPA on BTB integrity in immature rats is likely mediated via a loss of gap junction function at the BTB, failing to coordinate tight junction and anchoring junction function at the site to maintain the immunological barrier integrity. This in turn activates the extracellular signal-regulated kinases 1/2 (Erk1/2) downstream and an increase in protein endocytosis, destabilizing the BTB. The cadmium-induced disruption of testicular dysfunction is mediated initially via its effects on the occludin/ZO-1/focal adhesion kinase (FAK) complex at the BTB, causing redistribution of proteins at the Sertoli-Sertoli cell interface, leading to the BTB disruption. The damaging effects of these toxicants to testicular function are mediated by mitogen-activated protein kinases (MAPK) downstream, which in turn perturbs the actin bundling and accelerates the actin-branching activity, causing disruption of the Sertoli cell tight junction (TJ)-barrier function at the BTB and perturbing spermatid adhesion at the apical ectoplasmic specialization (apical ES, a testis-specific anchoring junction type) that leads to premature release of germ cells from the testis. However, the use of specific inhibitors against MAPK was shown to block or delay the cadmium-induced testicular injury, such as BTB disruption and germ cell loss. These findings suggest that there may be a common downstream p38 and/or Erk1/2 MAPK-based signaling pathway involving polarity proteins and actin regulators that is shared between different toxicants that induce male reproductive dysfunction. As such, the use of inhibitors and/or antagonists against specific MAPKs can possibly be used to “manage” the illnesses caused by these toxicants and/or “protect” industrial workers being exposed to high levels of these toxicants in their work environment. PMID:21866273

HIV Exposure to the Epithelia in Ectocervical and Colon Tissues Induces Inflammatory Cytokines Without Tight Junction Disruption

PubMed Central

Sankapal, Soni; Gupta, Phalguni; Ratner, Deena; Ding, Ming; Shen, Chengli; Sanyal, Anwesha; Stolz, Donna; Cu-Uvin, Susan; Ramratnam, Bharat

2016-01-01

Abstract Epithelial cells in human cervical and colonic mucosa do not express HIV receptor. However, HIV transmission occurs across the unbreached epithelia by an unknown mechanism. In this study, the effect of HIV exposure on tight junction (TJ) and cytokine production in ectocervical and colon mucosal epithelia in tissue biopsies was investigated in an organ culture model. After HIV exposure, the distribution patterns and quantities of epithelial TJ and adherens proteins were evaluated by immunofluorescence staining followed by confocal microscopy. Cytokine mRNA in the mucosal epithelia was also evaluated by real-time reverse transcription–polymerase chain reaction (RT-PCR). HIV transmission was evaluated by measuring p24 production in culture supernatant. Our results showed there were no significant changes in the distribution and quantities of epithelial TJ/adherens junction (AJ) proteins after exposure to HIV. However, higher levels of CXCL10 and CXCL11 mRNA expression were detected in HIV-exposed ectocervical epithelia. In case of colon mucosa, higher levels of CXCL10 and IL-6 mRNA expression were detected in HIV-exposed colon mucosa. Our study suggests that HIV induces cytokine production in epithelial cells, which may facilitate HIV transmission by recruiting HIV target cells in the submucosal region. Furthermore, HIV transmission may not occur through epithelial TJ/AJ disruption. PMID:27153934

Transmembrane protein PERP is a component of tessellate junctions and of other junctional and non-junctional plasma membrane regions in diverse epithelial and epithelium-derived cells.

PubMed

Franke, Werner W; Heid, Hans; Zimbelmann, Ralf; Kuhn, Caecilia; Winter-Simanowski, Stefanie; Dörflinger, Yvette; Grund, Christine; Rickelt, Steffen

2013-07-01

Protein PERP (p53 apoptosis effector related to PMP-22) is a small (21.4 kDa) transmembrane polypeptide with an amino acid sequence indicative of a tetraspanin character. It is enriched in the plasma membrane and apparently contributes to cell-cell contacts. Hitherto, it has been reported to be exclusively a component of desmosomes of some stratified epithelia. However, by using a series of newly generated mono- and polyclonal antibodies, we show that protein PERP is not only present in all kinds of stratified epithelia but also occurs in simple, columnar, complex and transitional epithelia, in various types of squamous metaplasia and epithelium-derived tumors, in diverse epithelium-derived cell cultures and in myocardial tissue. Immunofluorescence and immunoelectron microscopy allow us to localize PERP predominantly in small intradesmosomal locations and in variously sized, junction-like peri- and interdesmosomal regions ("tessellate junctions"), mostly in mosaic or amalgamated combinations with other molecules believed, to date, to be exclusive components of tight and adherens junctions. In the heart, PERP is a major component of the composite junctions of the intercalated disks connecting cardiomyocytes. Finally, protein PERP is a cobblestone-like general component of special plasma membrane regions such as the bile canaliculi of liver and subapical-to-lateral zones of diverse columnar epithelia and upper urothelial cell layers. We discuss possible organizational and architectonic functions of protein PERP and its potential value as an immunohistochemical diagnostic marker.

Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells.

PubMed

Brown, Rachel C; Morris, Andrew P; O'Neil, Roger G

2007-01-26

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions.

TIGHT JUNCTION PROTEIN EXPRESSION AND BARRIER PROPERTIES OF IMMORTALIZED MOUSE BRAIN MICROVESSEL ENDOTHELIAL CELLS

PubMed Central

Brown, Rachel C.; Morris, Andrew P.; O’Neil, Roger G.

2007-01-01

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions. PMID:17169347

Assessment of the Activation of Rho Family GTP-Binding Proteins in Breast Cancer Cells and Specimens

DTIC Science & Technology

2001-08-01

lymphopenia Vav2 GEF for Rho, Rac, and Cdc42 proto-oncogene product; NM009500 Vav3 GEF for Rho and Rac proto-oncogene product; NM020505 hPEM -2 GEF for...junctions, and desmosomes play a fundamental role in maintaining the polarized phenotype and vectorial transport functions of epithelial cells. The tight

Hypoxia/Aglycemia-Induced Endothelial Barrier Dysfunction and Tight Junction Protein Downregulation Can Be Ameliorated by Citicoline

PubMed Central

Pan, Qunwen; Zhao, Yuhui; Chen, Ji; Zhao, Bin; Chen, Yanfang

2013-01-01

This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs. PMID:24358213

MicroRNAs as regulators of drug transporters, drug-metabolizing enzymes, and tight junctions: implication for intestinal barrier function.

PubMed

Ikemura, Kenji; Iwamoto, Takuya; Okuda, Masahiro

2014-08-01

Drug transporters, drug-metabolizing enzymes, and tight junctions in the small intestine function as an absorption barrier and sometimes as a facilitator of orally administered drugs. The expression of these proteins often fluctuates and thereby causes individual pharmacokinetic variability. MicroRNAs (miRNAs), which are small non-coding RNAs, have recently emerged as a new class of gene regulator. MiRNAs post-transcriptionally regulate gene expression by binding to target mRNA to suppress its translation or regulate its degradation. They have been shown to be key regulators of proteins associated with pharmacokinetics. Moreover, the role of miRNAs on the expression of some proteins expressed in the small intestine has recently been clarified. In this review, we summarize current knowledge regarding the role of miRNAs in the regulation of drug transporters, drug-metabolizing enzymes, and tight junctions as well as its implication for intestinal barrier function. MiRNAs play vital roles in the differentiation, architecture, and barrier function of intestinal epithelial cells, and directly and/or indirectly regulate the expression and function of proteins associated with drug absorption in intestinal epithelial cells. Moreover, the variation of miRNA expression caused by pathological and physiological conditions as well as genetic factors should affect the expression of these proteins. Therefore, miRNAs could be significant factors affecting inter- and intra-individual variations in the pharmacokinetics and intestinal absorption of drugs. Overall, miRNAs could be promising targets for personalized pharmacotherapy or other attractive therapies through intestinal absorption of drugs. Copyright © 2014 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohkuni, Tsuyoshi; Department of Pathology, Sapporo Medical University School of Medicine, Sapporo; Kojima, Takashi, E-mail: ktakashi@sapmed.ac.jp

Human nasal epithelium is an important physical barrier and innate immune defense protecting against inhaled substances and pathogens. Toll-like receptor (TLR) signaling, which plays a key role in the innate immune response, has not been well characterized in human nasal epithelial cells (HNECs), including the epithelial tight junctional barrier. In the present study, mRNAs of TLR1-10 were detected in hTERT-transfected HNECs, which can be used as an indispensable and stable model of normal HNECs, similar to primary cultured HNECs. To investigate the changes of tight junction proteins and the signal transduction pathways via TLRs in HNECs in vitro, hTERT-transfected HNECsmore » were treated with TLR2 ligand P{sub 3}CSK{sub 4}, TLR3 ligand poly(I:C), TLR4 ligand LPS, TLR7/8 ligand CL097, TLR8 ligand ssRNA40/LyoVec, and TLR9 ligand ODN2006. In hTERT-transfected HNECs, treatment with poly(I:C) significantly reduced expression of the tight junction protein JAM-A and induced secretion of proinflammatory cytokines IL-8 and TNF-{alpha}. Both the reduction of JAM-A expression and the induction of secretion of IL-8 and TNF-{alpha} after treatment with poly(I:C) were modulated by distinct signal transduction pathways via EGFR, PI3K, and p38 MAPK and finally regulated by a TLR3-mediated NF-{kappa}B pathway. The control of TLR3-mediated signaling pathways in HNECs may be important not only in infection by viral dsRNA but also in autoimmune diseases caused by endogenous dsRNA released from necrotic cells.« less

Effects of Mesalamine Treatment on Gut Barrier Integrity Following Burn Injury

PubMed Central

Cannon, Abigail R.; Akhtar, Suhail; Hammer, Adam M.; Morris, Niya L.; Javorski, Mike J.; Li, Xiaoling; Kennedy, Richard H.; Gamelli, Richard L.; Choudhry, Mashkoor A.

2016-01-01

Gut barrier disruption is often implicated in pathogenesis associated with burn and other traumatic injuries. In this study, we examined whether therapeutic intervention with mesalamine (5-ASA), a common anti-inflammatory treatment for patients with inflammatory bowel disease, reduces intestinal inflammation and maintains normal barrier integrity after burn injury. Male C57BL/6 mice were administered an ~20% total body surface area dorsal scald burn and resuscitated with either 1mL normal saline or 100mg/kg of 5-ASA dissolved in saline. We examined intestinal transit and permeability along with levels of small intestine epithelial cell pro-inflammatory cytokines and tight junction protein expression one day after burn injury in the presence or absence of 5-ASA. A significant decrease in intestinal transit was observed one day after burn injury, which accompanied a significant increase in gut permeability. We found a substantial increase in the levels of IL-6 (by ~1.5 fold) and IL-18 (by ~2.5 fold) in small intestine epithelial cells one day after injury. Furthermore, burn injury decreases expression of the tight junction proteins claudin-4, claudin-8, and occludin. Treatment with 5-ASA after burn injury prevented the burn induced increase in permeability, partially restored normal intestinal transit, normalized levels of the pro-inflammatory cytokines IL-6 and IL-18, and restored tight junction protein expression of claudin-4 and occludin to that of sham levels. Together these findings suggest that 5-ASA can potentially be used as treatment to decrease intestinal inflammation and normalize intestinal function after burn injury. PMID:27388883

Effects of Mesalamine Treatment on Gut Barrier Integrity After Burn Injury.

PubMed

Cannon, Abigail R; Akhtar, Suhail; Hammer, Adam M; Morris, Niya L; Javorski, Michael J; Li, Xiaoling; Kennedy, Richard H; Gamelli, Richard L; Choudhry, Mashkoor A

2016-01-01

Gut barrier disruption is often implicated in pathogenesis associated with burn and other traumatic injuries. In this study, the authors examined whether therapeutic intervention with mesalamine (5-aminosalicylic acid [5-ASA]), a common anti-inflammatory treatment for patients with inflammatory bowel disease, reduces intestinal inflammation and maintains normal barrier integrity after burn injury. Male C57BL/6 mice were administered an approximately 20% TBSA dorsal scald burn and resuscitated with either 1 ml normal saline or 100 mg/kg of 5-ASA dissolved in saline. The authors examined intestinal transit and permeability along with the levels of small intestine epithelial cell proinflammatory cytokines and tight junction protein expression 1 day after burn injury in the presence or absence of 5-ASA. A significant decrease in intestinal transit was observed 1 day after burn injury, which accompanied a significant increase in gut permeability. The authors found a substantial increase in the levels of interleukin (IL)-6 (by ~1.5-fold) and IL-18 (by ~2.5-fold) in the small intestine epithelial cells 1 day after injury. Furthermore, burn injury decreases the expression of the tight junction proteins claudin-4, claudin-8, and occludin. Treatment with 5-ASA after burn injury prevented the burn-induced increase in permeability, partially restored normal intestinal transit, normalized the levels of the proinflammatory cytokines IL-6 and IL-18, and restored tight junction protein expression of claudin-4 and occludin compared with that of sham levels. Together these findings suggest that 5-ASA can potentially be used as treatment to decrease intestinal inflammation and normalize intestinal function after burn injury.

A 3-D well-differentiated model of pediatric bronchial epithelium demonstrates unstimulated morphological differences between asthmatic and nonasthmatic cells.

PubMed

Parker, Jeremy; Sarlang, Severine; Thavagnanam, Surendran; Williamson, Grace; O'donoghue, Dara; Villenave, Remi; Power, Ultan; Shields, Michael; Heaney, Liam; Skibinski, Grzegorz

2010-01-01

There is a need for reproducible and effective models of pediatric bronchial epithelium to study disease states such as asthma. We aimed to develop, characterize, and differentiate an effective, an efficient, and a reliable three-dimensional model of pediatric bronchial epithelium to test the hypothesis that children with asthma differ in their epithelial morphologic phenotype when compared with nonasthmatic children. Primary cell cultures from both asthmatic and nonasthmatic children were grown and differentiated at the air-liquid interface for 28 d. Tight junction formation, MUC5AC secretion, IL-8, IL-6, prostaglandin E2 production, and the percentage of goblet and ciliated cells in culture were assessed. Well-differentiated, multilayered, columnar epithelium containing both ciliated and goblet cells from asthmatic and nonasthmatic subjects were generated. All cultures demonstrated tight junction formation at the apical surface and exhibited mucus production and secretion. Asthmatic and nonasthmatic cultures secreted similar quantities of IL-8, IL-6, and prostaglandin E2. Cultures developed from asthmatic children contained considerably more goblet cells and fewer ciliated cells compared with those from nonasthmatic children. A well-differentiated model of pediatric epithelium has been developed that will be useful for more in vivo like study of the mechanisms at play during asthma.

Tenofovir Inhibits Wound Healing of Epithelial Cells and Fibroblasts from the Upper and Lower Human Female Reproductive Tract

PubMed Central

Rodriguez-Garcia, Marta; Patel, Mickey V.; Shen, Zheng; Bodwell, Jack; Rossoll, Richard M.; Wira, Charles R.

2017-01-01

Disruption of the epithelium in the female reproductive tract (FRT) is hypothesized to increase HIV infection risk by interfering with barrier protection and facilitating HIV-target cell recruitment. Here we determined whether Tenofovir (TFV), used vaginally in HIV prevention trials, and Tenofovir alafenamide (TAF), an improved prodrug of TFV, interfere with wound healing in the human FRT. TFV treatment of primary epithelial cells and fibroblasts from the endometrium (EM), endocervix (CX) and ectocervix (ECX) significantly delayed wound closure. Reestablishment of tight junctions was compromised in EM and CX epithelial cells even after wound closure occurred. In contrast, TAF had no inhibitory effect on wound closure or tight junction formation following injury. TAF accumulated inside genital epithelial cells as TFV-DP, the active drug form. At elevated levels of TAF treatment to match TFV intracellular TFV-DP concentrations, both equally impaired barrier function, while wound closure was more sensitive to TFV. Furthermore, TFV but not TAF increased elafin and MIP3a secretion following injury, molecules known to be chemotactic for HIV-target cells. Our results highlight the need of evaluating antiretroviral effects on genital wound healing in future clinical trials. A possible link between delayed wound healing and increased risk of HIV acquisition deserves further investigation. PMID:28368028

Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures - a new donor for cell therapy.

PubMed

Wu, Wei; Zeng, Yuxiao; Li, Zhengya; Li, Qiyou; Xu, Haiwei; Yin, Zheng Qin

2016-04-19

Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases.

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells

PubMed Central

Cheng, Catherine; Nowak, Roberta B.; Gao, Junyuan; Sun, Xiurong; Biswas, Sondip K.; Lo, Woo-Kuen; Mathias, Richard T.

2015-01-01

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1−/−;CP49−/− double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance. PMID:25740157

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells.

PubMed

Cheng, Catherine; Nowak, Roberta B; Gao, Junyuan; Sun, Xiurong; Biswas, Sondip K; Lo, Woo-Kuen; Mathias, Richard T; Fowler, Velia M

2015-05-15

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance. Copyright © 2015 the American Physiological Society.

Characterization of tight junction proteins in cultured human urothelial cells.

PubMed

Rickard, Alice; Dorokhov, Nikolay; Ryerse, Jan; Klumpp, David J; McHowat, Jane

2008-01-01

Tight junctions (TJs) are essential for normal function of epithelia, restricting paracellular diffusion and contributing to the maintenance of cell surface polarity. Superficial cells of the urothelium develop TJs, the basis for the paracellular permeability barrier of the bladder against diffusion of urinary solutes. Focusing on the superficial cell layer of stratified cell cultures of an immortalized human ureteral cell line, TEU-2 cells, we have examined the presence of TJ and TJ-associated proteins. TEU-2 cells were treated with calcium chloride and fetal bovine serum culture conditions used to induce stratification that resembles the normal transitional epithelial phenotype. Cultures were examined for TJ and TJ-associated proteins by confocal immunofluorescence microscopy and evaluated for TJ mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). TEU-2 cultures exhibited immunoreactivity at intercellular margins for claudins 1, 4, 5, 7, 14, and 16 whereas claudins 2, 8, and 12 were intracellular. RT-PCR corroborated the presence of these claudins at the mRNA level. The TJ-associated proteins occludin, JAM-1, and zonula occludens (ZO-1, ZO-2, and ZO-3) were localized at cell margins. We have found that numerous TJs and TJ-associated proteins are expressed in stratified TEU-2 cultures. Further, we propose TEU-2s provide a useful ureteral model for future studies on the involvement of TJs proteins in the normal and pathological physiology of the human urinary system.

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

PubMed Central

Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

2018-01-01

Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488

Possible role of HIWI2 in modulating tight junction proteins in retinal pigment epithelial cells through Akt signaling pathway.

PubMed

Sivagurunathan, Suganya; Palanisamy, Karthikka; Arunachalam, Jayamuruga Pandian; Chidambaram, Subbulakshmi

2017-03-01

PIWI subfamily of proteins is shown to be primarily expressed in germline cells. They maintain the genomic integrity by silencing the transposable elements. Although the role of PIWI proteins in germ cells has been documented, their presence and function in somatic cells remains unclear. Intriguingly, we detected all four members of PIWI-like proteins in human ocular tissues and somatic cell lines. When HIWI2 was knocked down in retinal pigment epithelial cells, the typical honeycomb morphology was affected. Further analysis showed that the expression of tight junction (TJ) proteins, CLDN1, and TJP1 were altered in HIWI2 knockdown. Moreover, confocal imaging revealed disrupted TJP1 assembly at the TJ. Previous studies report the role of GSK3β in regulating TJ proteins. Accordingly, phospho-kinase proteome profiler array indicated increased phosphorylation of Akt and GSK3α/β in HIWI2 knockdown, suggesting that HIWI2 might affect TJ proteins through Akt-GSK3α/β signaling axis. Moreover, treating the HIWI2 knockdown cells with wortmannin increased the levels of TJP1 and CLDN1. Taken together, our study demonstrates the presence of PIWI-like proteins in somatic cells and the possible role of HIWI2 in preserving the functional integrity of epithelial cells probably by modulating the phosphorylation status of Akt.

Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

PubMed Central

Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

2016-01-01

Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction. PMID:27436542

Myosin-X functions in polarized epithelial cells

PubMed Central

Liu, Katy C.; Jacobs, Damon T.; Dunn, Brian D.; Fanning, Alan S.; Cheney, Richard E.

2012-01-01

Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein–Myo10 localizes to lateral membrane cell–cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis. PMID:22419816

Simultaneous cell death and desquamation of the embryonic diffusion barrier during epidermal development.

PubMed

Saathoff, Manuela; Blum, Barbara; Quast, Thomas; Kirfel, Gregor; Herzog, Volker

2004-10-01

The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier. Copyright 2004 Elsevier Inc.

Blood-brain Barrier Disruption Leads to Postoperative Cognitive Dysfunction.

PubMed

Wang, Bin; Li, Siyuan; Cao, Xipeng; Dou, Xinghui; Li, Jingzhu; Wang, Ling; Wang, Mingshan; Bi, Yanlin

2017-01-01

Postoperative Cognitive Dysfunction (POCD) has received considerable attention as one of the main postoperative complications. The underlying mechanism of POCD in elderly subjects has not been fully elucidated to date. The Central Nervous System (CNS) is isolated from the bloodstream by the Blood Brain Barrier (BBB) that consists of endothelial cells, capillary blood vessels and tight junctions. The tight junctions carry out significant biological functions that are associated with the CNS and blood circulation. In this review, I present a hypothesis that blood-brain barrier disruption leads to postoperative cognitive dysfunction. A total of 81 healthy male Wistar rats were used for the present study. All the experimental animals were randomly divided into 3 groups: normal control group, isoflurane group and splenectomy group. The control group was not subjected to any form of treatment. The rats in isoflurane group were given 1.5-2% isoflurane under intubation and mechanical ventilation. The rats in splenectomy group underwent splenectomy under the same anesthesia as the isoflurane group. The Morris water maze was used to examine the learning and memory ability of the animals. The expression of the Tight Junctions Proteins (TJPs) in the hippocampus was analyzed using Western blotting. The concentration of Evans Blue (EB) in the supernatant was analyzed using UV spectroscopy. Ultrastructure changes in the basal laminas, the Tight Junctions (TJs), mitochondria and the endoplasmic reticulum surrounding the capillaries were assessed by Transmission Electron Microscopy (TEM). Following splenectomy, the rats displayed concomitant significant cognitive deficits in the Morris water maze test. Taken together, the results indicate that the expression levels of occludin (65KD) following splenectomy were reduced on days one and three in aged rats. No significant difference was noted in the expression levels of claudin-5, except for a reduction after surgery on day one. The leakage of EB was higher following splenectomy than control group and isoflurane group. The ultrastructure of the neurovascular unit was monitored on the day prior to surgery and on the 1st, 3rd and 7th day following surgery using a transmission electronmicroscope. The alterations in the levels of tight junction proteins following splenectomy may contribute to the BBB permeability increase, which in turn will induce postoperative cognitive dysfunction. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood-brain barrier primary triple coculture model.

PubMed

Xu, Liming; Dan, Mo; Shao, Anliang; Cheng, Xiang; Zhang, Cuiping; Yokel, Robert A; Takemura, Taro; Hanagata, Nobutaka; Niwa, Masami; Watanabe, Daisuke

2015-01-01

Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm(2). After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes.

Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer

PubMed Central

de Semir, D.; Maurisse, R.; Du, F.; Xu, J.; Yang, X.; Illek, B.; Gruenert, D. C.

2013-01-01

The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl− ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology. PMID:22234514

Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions

PubMed Central

Baranwal, Somesh

2015-01-01

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. PMID:25792565

Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions.

PubMed

Lechuga, Susana; Baranwal, Somesh; Ivanov, Andrei I

2015-05-01

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. Copyright © 2015 the American Physiological Society.

bFGF Protects Against Blood-Brain Barrier Damage Through Junction Protein Regulation via PI3K-Akt-Rac1 Pathway Following Traumatic Brain Injury.

PubMed

Wang, Zhou-Guang; Cheng, Yi; Yu, Xi-Chong; Ye, Li-Bing; Xia, Qing-Hai; Johnson, Noah R; Wei, Xiaojie; Chen, Da-Qing; Cao, Guodong; Fu, Xiao-Bing; Li, Xiao-Kun; Zhang, Hong-Yu; Xiao, Jian

2016-12-01

Many traumatic brain injury (TBI) survivors sustain neurological disability and cognitive impairments due to the lack of defined therapies to reduce TBI-induced blood-brain barrier (BBB) breakdown. Exogenous basic fibroblast growth factor (bFGF) has been shown to have neuroprotective function in brain injury. The present study therefore investigates the beneficial effects of bFGF on the BBB after TBI and the underlying mechanisms. In this study, we demonstrate that bFGF reduces neurofunctional deficits and preserves BBB integrity in a mouse model of TBI. bFGF suppresses RhoA and upregulates tight junction proteins, thereby mitigating BBB breakdown. In vitro, bFGF exerts a protective effect on BBB by upregulating tight junction proteins claudin-5, occludin, zonula occludens-1, p120-catenin, and β-catenin under oxygen glucose deprivation/reoxygenation (OGD) in human brain microvascular endothelial cells (HBMECs). Both the in vivo and in vitro effects are related to the activation of the downstream signaling pathway, PI3K/Akt/Rac-1. Inhibition of the PI3K/Akt or Rac-1 by specific inhibitors LY294002 or si-Rac-1, respectively, partially reduces the protective effect of bFGF on BBB integrity. Overall, our results indicate that the protective role of bFGF on BBB involves the regulation of tight junction proteins and RhoA in the TBI model and OGD-induced HBMECs injury, and that activation of the PI3K/Akt /Rac-1 signaling pathway underlies these effects.

Tight Junction–Associated Signaling Pathways Modulate Cell Proliferation in Uveal Melanoma

PubMed Central

Jayagopal, Ashwath; Yang, Jin-Long; Haselton, Frederick R.; Chang, Min S.

2011-01-01

Purpose. To investigate the role of tight junction (TJ)–associated signaling pathways in the proliferation of uveal melanoma. Methods. Human uveal melanoma cell lines overexpressing the TJ molecule blood vessel epicardial substance (Bves) were generated. The effects of Bves overexpression on TJ protein expression, cell proliferation, and cell cycle distribution were quantified. In addition, localization and transcription activity of the TJ-associated protein ZO-1–associated nucleic acid binding protein (ZONAB) were evaluated using immunofluorescence and bioluminescence reporter assays to study the involvement of Bves signaling in cell proliferation-associated pathways. Results. Bves overexpression in uveal melanoma cell lines resulted in increased expression of the TJ proteins occludin and ZO-1, reduced cell proliferation, and increased sequestration of ZONAB at TJs and reduced ZONAB transcriptional activity. Conclusions. TJ proteins are present in uveal melanoma, and TJ-associated signaling pathways modulate cell signaling pathways relevant to proliferation in uveal melanoma. PMID:20861479

The Drosophila blood-brain barrier: development and function of a glial endothelium.

PubMed

Limmer, Stefanie; Weiler, Astrid; Volkenhoff, Anne; Babatz, Felix; Klämbt, Christian

2014-01-01

The efficacy of neuronal function requires a well-balanced extracellular ion homeostasis and a steady supply with nutrients and metabolites. Therefore, all organisms equipped with a complex nervous system developed a so-called blood-brain barrier, protecting it from an uncontrolled entry of solutes, metabolites or pathogens. In higher vertebrates, this diffusion barrier is established by polarized endothelial cells that form extensive tight junctions, whereas in lower vertebrates and invertebrates the blood-brain barrier is exclusively formed by glial cells. Here, we review the development and function of the glial blood-brain barrier of Drosophila melanogaster. In the Drosophila nervous system, at least seven morphologically distinct glial cell classes can be distinguished. Two of these glial classes form the blood-brain barrier. Perineurial glial cells participate in nutrient uptake and establish a first diffusion barrier. The subperineurial glial (SPG) cells form septate junctions, which block paracellular diffusion and thus seal the nervous system from the hemolymph. We summarize the molecular basis of septate junction formation and address the different transport systems expressed by the blood-brain barrier forming glial cells.

Aberrant expression of the tight junction molecules claudin-1 and zonula occludens-1 mediates cell growth and invasion in oral squamous cell carcinoma.

PubMed

Babkair, Hamzah; Yamazaki, Manabu; Uddin, Md Shihab; Maruyama, Satoshi; Abé, Tatsuya; Essa, Ahmed; Sumita, Yoshimasa; Ahsan, Md Shahidul; Swelam, Wael; Cheng, Jun; Saku, Takashi

2016-11-01

We reported that altered cell contact mediated by E-cadherin is an initial event in the pathogenesis of oral epithelial malignancies. To assess other effects of cell adhesion, we examined the expression levels of tight junction (TJ) molecules in oral carcinoma in situ (CIS) and squamous cell carcinoma (SCC). To identify changes in the expression of TJ molecules, we conducted an analysis of the immunohistochemical profiles of claudin-1 (CLDN-1) and zonula occludens-1 (ZO-1) in surgical specimens acquired from patients with oral SCC containing foci of epithelial dysplasia or from patients with CIS. We used immunofluorescence, Western blotting, reverse-transcription polymerase chain reaction, and RNA interference to evaluate the functions of CLDN-1 and ZO-1 in cultured oral SCC cells. TJ molecules were not detected in normal oral epithelial tissues but were expressed in SCC/CIS cells. ZO-1 was localized within the nucleus of proliferating cells. When CLDN-1 expression was inhibited by transfecting cells with specific small interference RNAs, SCC cells dissociated, and their ability to proliferate and invade Matrigel was inhibited. In contrast, although RNA interference-mediated inhibition of ZO-1 expression did not affect cell morphology, it inhibited cell proliferation and invasiveness. Our findings indicated that the detection of TJ molecules in the oral epithelia may serve as a marker for the malignant phenotype of cells in which CLDN-1 regulates proliferation and invasion. Copyright © 2016 Elsevier Inc. All rights reserved.

The adherens junction is lost during normal pregnancy but not during ovarian hyperstimulated pregnancy.

PubMed

Dowland, Samson N; Madawala, Romanthi J; Lindsay, Laura A; Murphy, Christopher R

2016-03-01

During early pregnancy in the rat, the luminal uterine epithelial cells (UECs) must transform to a receptive state to permit blastocyst attachment and implantation. The implantation process involves penetration of the epithelial barrier, so it is expected that the transformation of UECs includes alterations in the lateral junctional complex. Previous studies have demonstrated a deepening of the tight junction (zonula occludens) and a reduction in the number of desmosomes (macula adherens) in UECs at the time of implantation. However, the adherens junction (zonula adherens), which is primarily responsible for cell-cell adhesion, has been little studied during early pregnancy. This study investigated the adherens junction in rat UECs during the early stages of normal pregnancy and ovarian hyperstimulated (OH) pregnancy using transmission electron microscopy. The adherens junction is present in UECs at the time of fertilisation, but is lost at the time of blastocyst implantation during normal pregnancy. Interestingly, at the time of implantation after OH, adherens junctions are retained and may impede blastocyst penetration of the epithelium. The adherens junction anchors the actin-based terminal web, which is known to be disrupted in UECs during early pregnancy. However, artificial disruption of the terminal web, using cytochalasin D, did not cause removal of the adherens junction in UECs. This study revealed that adherens junction disassembly occurs during early pregnancy, but that this process does not occur during OH pregnancy. Such disassembly does not appear to depend on the disruption of the terminal web. Copyright © 2015 Elsevier GmbH. All rights reserved.

Effect of High Dietary Tryptophan on Intestinal Morphology and Tight Junction Protein of Weaned Pig.

PubMed

Tossou, Myrlene Carine B; Liu, Hongnan; Bai, Miaomiao; Chen, Shuai; Cai, Yinghua; Duraipandiyan, Veeramuthu; Liu, Hongbin; Adebowale, Tolulope O; Al-Dhabi, Naif Abdullah; Long, Lina; Tarique, Hussain; Oso, Abimbola O; Liu, Gang; Yin, Yulong

2016-01-01

Tryptophan (Trp) plays an essential role in pig behavior and growth performances. However, little is known about Trp's effects on tight junction barrier and intestinal health in weaned pigs. In the present study, twenty-four (24) weaned pigs were randomly assigned to one of the three treatments with 8 piglets/treatments. The piglets were fed different amounts of L-tryptophan (L-Trp) as follows: 0.0%, 0.15, and 0.75%, respectively, named zero Trp (ZTS), low Trp (LTS), and high Trp (HTS), respectively. No significant differences were observed in average daily gain (ADG), average daily feed intake (ADFI), and gain: feed (G/F) ratio between the groups. After 21 days of the feeding trial, results showed that dietary Trp significantly increased (P < 0.05) crypt depth and significantly decreased (P < 0.05) villus height to crypt depth ratio (VH/CD) in the jejunum of pig fed HTS. In addition, pig fed HTS had higher (P < 0.05) serum diamine oxidase (DAO) and D-lactate. Furthermore, pig fed HTS significantly decreased mRNA expression of tight junction proteins occludin and ZO-1 but not claudin-1 in the jejunum. The number of intraepithelial lymphocytes and goblet cells were not significantly different (P > 0.05) between the groups. Collectively, these data suggest that dietary Trp supplementation at a certain level (0.75%) may negatively affect the small intestinal structure in weaned pig.

Lecithin in mixed micelles attenuates the cytotoxicity of bile salts in Caco-2 cells.

PubMed

Tan, Ya'nan; Qi, Jianping; Lu, Yi; Hu, Fuqiang; Yin, Zongning; Wu, Wei

2013-03-01

This study was designed to investigate the cytotoxicity of bile salt-lecithin mixed micelles on the Caco-2 cell model. Cell viability and proliferation after mixed micelles treatments were evaluated with the MTT assay, and the integrity of Caco-2 cell monolayer was determined by quantitating the transepithelial electrical resistance and the flux of tracer, FITC-dextran 4400. The apoptosis induced by mixed micelles treatments was investigated with the annexin V/PI protocol. The particle size of mixed micelles was all smaller than 100 nm. The mixed micelles with lower than 0.2mM sodium deoxycholate (SDC) had no significant effects on cell viability and proliferation. When the level of SDC was higher than 0.4mM and the lecithin/SDC ratio was lower than 2:1, the mixed micelles caused significant changes in cell viability and proliferation. Furthermore, the mixed micelles affected tight junctions in a composition-dependent manner. Specifically, the tight junctions were transiently opened rather than damaged by the mixed micelles with SDC of between 0.2 and 0.6mM. The mixed micelles with more lecithin also induced less apoptosis. These results demonstrate that relatively higher concentrations of mixed micelles are toxic to Caco-2 cells, while phospholipids can attenuate the toxicity of the bile salts. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Artificial sweetener saccharin disrupts intestinal epithelial cells' barrier function in vitro.

PubMed

Santos, P S; Caria, C R P; Gotardo, E M F; Ribeiro, M L; Pedrazzoli, J; Gambero, A

2018-06-25

Consumption of non-nutritive sweeteners (NNS) is a dietary practice used by those who wish to lose weight or by patients on a sugar-restricted diet such as those with DM2. Although these substances are safe, possible biological interactions with the digestive tract, particularly in relation to intestinal permeability, have not been studied. Thus, the current work sought to investigate the action of different NNS on intestinal permeability using an in vitro Caco-2 cell model. Caco-2 cells were incubated with acesulfame K, aspartame, saccharin, or sucralose at equimolar concentrations. Acesulfame K, aspartame, and sucralose did not disrupt monolayer integrity in the cells. However, saccharin increased paracellular permeability and decreased transepithelial electrical resistance (TEER) via a non-cytotoxic mechanism. The levels of the tight junction protein claudin-1 were reduced in Caco-2 cells that had previously been exposed to saccharin. The inhibition of nuclear factor-κB (NF-κB) was able to prevent the reduction in TEER induced by saccharin treatment. Thalidomide, as an inhibitor of ubiquitin ligase, was able to prevent the decrease in claudin-1 protein expression and the TEER reduction in Caco-2 cells. Saccharin disrupts monolayer integrity and alters paracellular permeability in a Caco-2 cell monolayer model, via a mechanism involving NF-κB activation, resulting in the ubiquitination of the tight junction protein claudin-1. Saccharin consumption may potentially alter the intestinal integrity in humans.

Integrin-linked kinase and ELMO2 modulate recycling endosomes in keratinocytes.

PubMed

Ho, Ernest; Ivanova, Iordanka A; Dagnino, Lina

2016-12-01

The formation of tight cell-cell junctions is essential in the epidermis for its barrier properties. In this tissue, keratinocytes follow a differentiation program tightly associated with their movement from the innermost basal to the outer suprabasal layers, and with changes in their cell-cell adhesion profile. Intercellular adhesion in keratinocytes is mediated through cell-cell contacts, including E-cadherin-based adherens junctions. Although the mechanisms that mediate E-cadherin delivery to the plasma membrane have been widely studied in simple epithelia, this process is less well understood in the stratified epidermis. In this study, we have investigated the role of Engulfment and Cell Motility 2 (ELMO2) and integrin-linked kinase (ILK) in the positioning of E-cadherin-containing recycling endosomes during establishment of cell-cell contacts in differentiating keratinocytes. We now show that induction of keratinocyte differentiation by Ca 2+ is accompanied by localization of ELMO2 and ILK to Rab4- and Rab11a-containing recycling endosomes. The positioning of long-loop Rab11a-positive endosomes at areas adjacent to cell-cell contacts is disrupted in ELMO2- or ILK-deficient keratinocytes, and is associated with impaired localization of E-cadherin to cell borders. Our studies show a previously unrecognized role for ELMO2 and ILK in modulation of endosomal positioning, which may play key roles in epidermal sheet maintenance and permeability barrier function. Copyright © 2016 Elsevier B.V. All rights reserved.

Correlative study of functional and structural regeneration of urothelium after chitosan-induced injury.

PubMed

Erman, Andreja; Kerec Kos, Mojca; Žakelj, Simon; Resnik, Nataša; Romih, Rok; Veranič, Peter

2013-11-01

High transepithelial electrical resistance (TEER) demonstrates a functional permeability barrier of the normal urothelium, which is maintained by a layer of highly differentiated superficial cells. When the barrier is challenged, a quick regeneration is induced. We used side-by-side diffusion chambers as an ex vivo system to determine the time course of functional and structural urothelial regeneration after chitosan-induced injury. The exposure of the urothelium to chitosan caused a 60 % decrease in TEER, the exposure of undifferentiated urothelial cells to the luminal surface and leaky tight junctions. During the regeneration period (350 min), TEER recovered to control values after approximately 200 min, while structural regeneration continued until 350 min after injury. The tight junctions are the earliest and predominant component of the barrier to appear, while complete barrier regeneration is achieved by delayed superficial cell terminal differentiation. The barrier function and the structure of untreated urothelium were unaffected in side-by-side diffusion chambers for at least 6 h. The urinary bladder tissue excised from an animal thus retains the ability to maintain and restore the transepithelial barrier and cellular ultrastructure for a sufficient period to allow for studies of regeneration in ex vivo conditions.

Male reproductive toxicity of CrVI: In-utero exposure to CrVI at the critical window of testis differentiation represses the expression of Sertoli cell tight junction proteins and hormone receptors in adult F1 progeny rats.

PubMed

Kumar, Kathiresh M; Aruldhas, Mariajoseph Michael; Banu, Sheerin L; Sadasivam, Balaji; Vengatesh, Ganapathy; Ganesh, Karthik M; Navaneethabalakrishnan, Shobana; Navin, Ajith Kumar; Michael, Felicia Mary; Venkatachalam, Sankar; Stanley, Jone A; Ramachandran, Ilangovan; Banu, Sakhila K; Akbarsha, Mohammad Abdulkader

2017-04-01

The effect of gestational exposure to CrVI (occupational/environmental pollutant and target to Sertoli cells(SC)) was tested in a rat model during the testicular differentiation from the bipotential gonad may interrupt spermatogenesis by disrupting SC tight junctions(TJ) and it's proteins and hormone receptors. Pregnant Wistar rats were exposed to 50/100/200ppm CrVI through drinking water during embryonic days 9-14. On Postnatal day 120, testes were subjected to ion exchange chromatographic analysis and revealed increased level of CrIII in SCs and germ cells, serum and testicular interstitial fluid(TIF). Microscopic analyses showed seminiferous tubules atrophy and disruption of SC TJ, which also recorded decreased testosterone in TIF. mRNA and Protein expression analyses attested decreased level of Fshr, Ar, occludin and claudin-11 in SCs. Immunofluorescent detection revealed weak signal of TJ proteins. Taken together, we concluded that gestational exposure to CrVI interferes with the expression of SC TJ proteins due to attenuated expression of hormone receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystallization and Preliminary X-ray Analysis of the Human Long Myosin Light-Chain Kinase 1-Specific Domain IgCAM3

DOE Office of Scientific and Technical Information (OSTI.GOV)

W Vallen Graham; A Magis; K Bailey

2011-12-31

Myosin light-chain kinase-dependent tight junction regulation is a critical event in inflammatory cytokine-induced increases in epithelial paracellular permeability. MLCK is expressed in human intestinal epithelium as two isoforms, long MLCK1 and long MLCK2, and MLCK1 is specifically localized to the tight junction, where it regulates paracellular permeability. The sole difference between these long MLCK splice variants is the presence of an immunoglobulin-like cell-adhesion molecule domain, IgCAM3, in MLCK1. To gain insight into the structure of the IgCAM3 domain, the IgCAM3 domain of MLCK1 has been expressed, purified and crystallized. Preliminary X-ray diffraction data were collected to 2.0 {angstrom} resolution andmore » were consistent with the primitive trigonal space group P2{sub 1}2{sub 1}2{sub 1}.« less

Tight junction gene expression in gastrointestinal tract of dairy calves with coccidiosis and treated with glucagon-like peptide-2

USDA-ARS?s Scientific Manuscript database

Selective permeability of the intestinal epithelium and efficient nutrient absorption are important functions for proper growth and development of calves. Damage to the intestinal mucosa can give rise to harmful long-term health effects and reduce productivity of the mature animal. Tight junction pr...

The ultrastructure of cerebral blood capillaries in the ratfish, Chimaera monstrosa.

PubMed

Bundgaard, M

1982-01-01

Sharks and skates (Chondrichthyes: Elasmobranchii) have a glial blood-brain barrier, while all other vertebrates examined so far have an endothelial barrier. For comparative reasons it is desirable to examine the blood-brain barrier in species from the other subclass of cartilaginous fish, the holocephalans. The ultrastructure of cerebral capillaries in the chimaera (Chondrichthyes: Holocephali) is described in the present study. The endothelial cells are remarkably thick. Fenestrae and transendothelial channels were not observed. The endothelial cells are joined by elaborate tight junctions. The perivascular glial processes are separated by wide spaces (15-60 nm) without obliterating junctional complexes. These findings indicate that the chimaera has an endothelial blood-brain barrier.

Escherichia coli K1 invasion increases human brain microvascular endothelial cell monolayer permeability by disassembling vascular-endothelial cadherins at tight junctions.

PubMed

Sukumaran, Sunil K; Prasadarao, Nemani V

2003-11-01

We investigated the permeability changes that occur in the human brain microvascular endothelial cell (HBMEC) monolayer, an in vitro model of the blood-brain barrier, during Escherichia coli K1 infection. An increase in permeability of HBMECs and a decrease in transendothelial electrical resistance were observed. These permeability changes occurred only when HBMECs were infected with E. coli expressing outer membrane protein A (OmpA) and preceded the traversal of bacteria across the monolayer. Activated protein kinase C (PKC)-alpha interacts with vascular-endothelial cadherins (VECs) at the tight junctions of HBMECs, resulting in the dissociation of beta-catenins from VECs and leading to the increased permeability of the HBMEC monolayer. Overexpression of a dominant negative form of PKC-alpha in HBMECs blocked the E. coli-induced increase in permeability of HBMECs. Anti-OmpA and anti-OmpA receptor antibodies exerted inhibition of E. coli-induced permeability of HBMEC monolayers. This inhibition was the result of the absence of PKC-alpha activation in HBMECs treated with the antibodies.

Effects of Intercellular Junction Protein Expression on Intracellular Ice Formation in Mouse Insulinoma Cells

PubMed Central

Higgins, Adam Z.; Karlsson, Jens O.M.

2013-01-01

The development of cryopreservation procedures for tissues has proven to be difficult in part because cells within tissue are more susceptible to intracellular ice formation (IIF) than are isolated cells. In particular, previous studies suggest that cell-cell interactions increase the likelihood of IIF by enabling propagation of ice between neighboring cells, a process thought to be mediated by gap junction channels. In this study, we investigated the effects of cell-cell interactions on IIF using three genetically modified strains of the mouse insulinoma cell line MIN6, each of which expressed key intercellular junction proteins (connexin-36, E-cadherin, and occludin) at different levels. High-speed video cryomicroscopy was used to visualize the freezing process in pairs of adherent cells, revealing that the initial IIF event in a given cell pair was correlated with a hitherto unrecognized precursor phenomenon: penetration of extracellular ice into paracellular spaces at the cell-cell interface. Such paracellular ice penetration occurred in the majority of cell pairs observed, and typically preceded and colocalized with the IIF initiation events. Paracellular ice penetration was generally not observed at temperatures >−5.65°C, which is consistent with a penetration mechanism via defects in tight-junction barriers at the cell-cell interface. Although the maximum temperature of paracellular penetration was similar for all four cell strains, genetically modified cells exhibited a significantly higher frequency of ice penetration and a higher mean IIF temperature than did wild-type cells. A four-state Markov chain model was used to quantify the rate constants of the paracellular ice penetration process, the penetration-associated IIF initiation process, and the intercellular ice propagation process. In the initial stages of freezing (>−15°C), junction protein expression appeared to only have a modest effect on the kinetics of propagative IIF, and even cell strains lacking the gap junction protein connexin-36 exhibited nonnegligible ice propagation rates. PMID:24209845

Decrease of tight junction integrity in the ipsilateral thalamus during the acute stage after focal infarction and ablation of the cerebral cortex in rats.

PubMed

Li, Jing-Jing; Xing, Shi-Hui; Zhang, Jian; Hong, Hua; Li, Yi-Liang; Dang, Chao; Zhang, Yu-Sheng; Li, Chuo; Fan, Yu-Hua; Yu, Jian; Pei, Zhong; Zeng, Jin-Sheng

2011-11-01

1. Whether damage to the blood-brain barrier (BBB) occurs in remote areas after a focal cortical lesion remains unknown. The present study investigated tight junction-related proteins and tight junction microstructure in the ipsilateral thalamus during the acute stage after middle cerebral artery occlusion (MCAO) and cortical aspiration lesion (CAL) in rats. 2. Thirty-six hypertensive and normotensive rats were subjected to MCAO or CAL; another 18 rats in each group were submitted to sham operation. Zonula Occluden (ZO)-1, occludin and albumin were detected by western blotting 12 and 24 h after surgery. Tight junction microstructure was evaluated using electron microscopy, whereas albumin location in the ipsilateral thalamus was determined using double immunostaining for albumin and occludin or albumin and neuronal nuclei (NeuN) 24 h after surgery. 3. Twenty-four hours after MCAO or CAL, occludin expression was reduced to 78.4% and 81.3%, respectively, compared with control. A reduction in ZO-1 expression in the ipsilateral thalamus (to 79%) was seen only after CAL (P < 0.05). Membrane contact at the tight junction was discontinuous in the ipsilateral thalamus in both MCAO and CAL rats. Albumin levels were 23.2% and 82.5% higher in the ipsilateral thalamus after MCAO and CAL, respectively (P < 0.05). The percentage of the albumin-positive area that coincided with the occludin-positive area in the MCAO and CAL groups was 76.8% and 64.6%, respectively, indicating that albumin was mainly localized around the microvessels. 4. The results of the present study suggest that tight junction integrity decreases during the acute stage in the ipsilateral thalamus after MCAO and CAL in rats. © 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.

Functional Genomics to Identify Therapeutic Targets in Cancer Stem Cells Using a Novel Murine CRPC Model

DTIC Science & Technology

2014-09-01

previous report [6, 7], Ptenpc-/- tumors are sensitive to ADT (Fig 1A). A significant amount of normal epithelium was identified in castrated...histopathological analysis by H & E staining (Fig 1A) and MRI analysis (data not shown). There is no noticeable normal epithelium in the castrated Ptenpc...indicated that while tumor cells are enriched for pathways involving cell adhesion molecules and tight junction (consistent with their epithelial

Astrocytes increase barrier properties and ZO-1 expression in retinal vascular endothelial cells.

PubMed

Gardner, T W; Lieth, E; Khin, S A; Barber, A J; Bonsall, D J; Lesher, T; Rice, K; Brennan, W A

1997-10-01

Diabetic retinopathy and other diseases associated with retinal edema are characterized by increased microvascular leakage. Astrocytes have been proposed to maintain endothelial function in the brain, suggesting that glial impairment may underlie the development of retinal edema. The purpose of this study was to test the effects of astrocytes on barrier properties in retinal microvascular endothelial cells. Bovine retinal microvascular endothelial cells were exposed to conditioned media from rat brain astrocytes. Transendothelial electrical resistance (TER) was determined on 24-mm Transwell (Cambridge, MA) polycarbonate filters with the End-Ohm device (World Precision Instruments, Sarasota, FL). ZO-1 protein content was quantified by microtiter enzyme-linked immunosorbent assay. Astrocyte-conditioned medium (ACM) significantly increased TER (P < 0.0001) and ZO-1 content (P < 0.01). Both serum-containing and serum-free N1B defined ACM increased ZO-1 expression, but heating abolished the effect. Serum-free ACM decreased cell proliferation by 16%. Astrocytes release soluble, heat-labile factors that increase barrier properties and tight junction protein content. These results suggest that astrocytes enhance blood-retinal barrier properties, at least in part by increasing tight junction protein expression. Our findings suggest that glial malfunction plays an important role in the pathogenesis of vasogenic retinal edema.

A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

PubMed

Vespa, Alisa; D'Souza, Sudhir J A; Dagnino, Lina

2005-09-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

Alterations of intercellular junctions in peritoneal mesothelial cells from patients undergoing dialysis: effect of retinoic Acid.

PubMed

Retana, Carmen; Sanchez, Elsa; Perez-Lopez, Alejandro; Cruz, Armando; Lagunas, Jesus; Cruz, Carmen; Vital, Socorro; Reyes, Jose L

2015-01-01

Dialysis patients are classified according to their peritoneal permeability as low transporter (LT, low solute permeability) or high transporter (HT, high solute permeability). Tight junction (TJ) proteins are critical to maintain ions, molecules and water paracellular transport through peritoneum. Exposure to peritoneal dialysis solutions causes damage to TJ in human peritoneal mesothelial cells (HPMCs). We analyzed the quantity, distribution and function of TJ proteins: claudin-1, -2 and -8, ZO-1 and occludin, in HPMC cultures from LT and HT patients. Since all-trans retinoic acid (ATRA) might modify the expression of TJ proteins, we studied its effect on HPMCs. Control HPMCs were isolated from human omentum, while HT or LT cells were obtained from dialysis effluents. Cells were cultured in presence of ATRA 0, 50 or 100 nM. Transepithelial electrical resistance (TER) measurement, immunostaining and Western blot analyses were performed. HT exhibited lower TER than control and LT monolayers. Immunofluorescence for TJ was weak and discontinuous along the cell contour, in LT and HT. Furthermore, claudin-1, occludin and ZO-1 expressions were decreased. In all groups, claudin-2 was localized at nuclei. We observed that ATRA improved TJ distribution and increased TJ expression in HT. This retinoid did not modify claudin-2 and -8 expressions. All-trans retinoic acid decreased TER in HT, but had no effect in LT. Tight junctions were altered in HPMCs from dialyzed patients. The HT monolayer has lower TER than LT, which might be associated with the peritoneal permeability in these patients. ATRA might be a therapeutic alternative to maintain mesothelial integrity, since it improved TJ localization and expression. Copyright © 2015 International Society for Peritoneal Dialysis.

Alterations of Intercellular Junctions in Peritoneal Mesothelial Cells from Patients Undergoing Dialysis: Effect of Retinoic Acid

PubMed Central

Retana, Carmen; Sanchez, Elsa; Perez-Lopez, Alejandro; Cruz, Armando; Lagunas, Jesus; Cruz, Carmen; Vital, Socorro; Reyes, Jose L.

2015-01-01

♦ Background: Dialysis patients are classified according to their peritoneal permeability as low transporter (LT, low solute permeability) or high transporter (HT, high solute permeability). Tight junction (TJ) proteins are critical to maintain ions, molecules and water paracellular transport through peritoneum. Exposure to peritoneal dialysis solutions causes damage to TJ in human peritoneal mesothelial cells (HPMCs). We analyzed the quantity, distribution and function of TJ proteins: claudin-1, -2 and -8, ZO-1 and occludin, in HPMC cultures from LT and HT patients. Since all-trans retinoic acid (ATRA) might modify the expression of TJ proteins, we studied its effect on HPMCs. ♦ Methods: Control HPMCs were isolated from human omentum, while HT or LT cells were obtained from dialysis effluents. Cells were cultured in presence of ATRA 0, 50 or 100 nM. Transepithelial electrical resistance (TER) measurement, immunostaining and Western blot analyses were performed. ♦ Results: HT exhibited lower TER than control and LT monolayers. Immunofluorescence for TJ was weak and discontinuous along the cell contour, in LT and HT. Furthermore, claudin-1, occludin and ZO-1 expressions were decreased. In all groups, claudin-2 was localized at nuclei. We observed that ATRA improved TJ distribution and increased TJ expression in HT. This retinoid did not modify claudin-2 and -8 expressions. All-trans retinoic acid decreased TER in HT, but had no effect in LT. ♦ Conclusions: Tight junctions were altered in HPMCs from dialyzed patients. The HT monolayer has lower TER than LT, which might be associated with the peritoneal permeability in these patients. ATRA might be a therapeutic alternative to maintain mesothelial integrity, since it improved TJ localization and expression. PMID:24584604

Virus interaction with the apical junctional complex.

PubMed

Gonzalez-Mariscal, Lorenza; Garay, Erika; Lechuga, Susana

2009-01-01

In order to infect pathogens must breach the epithelial barriers that separate the organism from the external environment or that cover the internal cavities and ducts of the body. Epithelia seal the passage through the paracellular pathway with the apical junctional complex integrated by tight and adherens junctions. In this review we describe how viruses like coxsackie, swine vesicular disease virus, adenovirus, reovirus, feline calcivirus, herpes viruses 1 and 2, pseudorabies, bovine herpes virus 1, poliovirus and hepatitis C use as cellular receptors integral proteins present at the AJC of epithelial cells. Interaction with these proteins contributes in a significant manner in defining the particular tropism of each virus. Besides these proteins, viruses exhibit a wide range of cellular co-receptors among which proteins present in the basolateral cell surface like integrins are often found. Therefore targeting proteins of the AJC constitutes a strategy that might allow viruses to bypass the physical barrier that blocks their access to receptors expressed on the basolateral surface of epithelial cells.

MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

PubMed

Van Itallie, Christina M; Tietgens, Amber Jean; Aponte, Angel; Gucek, Marjan; Cartagena-Rivera, Alexander X; Chadwick, Richard S; Anderson, James M

2018-02-02

Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells. © 2018. Published by The Company of Biologists Ltd.

Perfluorooctanesulfonate (PFOS) Perturbs Male Rat Sertoli Cell Blood-Testis Barrier Function by Affecting F-Actin Organization via p-FAK-Tyr407: An in Vitro Study

PubMed Central

Wan, Hin-Ting; Mruk, Dolores D.; Wong, Chris K. C.

2014-01-01

Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10–20 μM (∼5–10 μg/mL) was found to be more potent than bisphenol A (100 μM) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr407 was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr407 was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr407 and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB. PMID:24169556

A Novel Role for Integrin-linked Kinase in Epithelial Sheet Morphogenesis

PubMed Central

Vespa, Alisa; D'Souza, Sudhir J.A.; Dagnino, Lina

2005-01-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca2+ triggers formation of cell–cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca2+ treatment of keratinocytes induces rapid (≤1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and β-catenin. This is followed by slower (>6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell–cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/β-catenin to cell borders, precluding Ca2+-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell–cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell–cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration. PMID:15975904

In vitro studies of the blood-brain barrier using isolated brain capillaries and cultured endothelial cells.

PubMed

Goldstein, G W; Betz, A L; Bowman, P D; Dorovini-Zis, K

1986-01-01

The endothelial cells in brain capillaries are the anatomic site of the blood-brain barrier. To learn more about the biology of these specialized cells, we developed methods to prepare suspensions of purified brain microvessels as well as primary cultures of endothelial cells in monolayer. These two preparations allow for direct investigation of the metabolism, transport properties, and receptor content of the brain capillary. We used isolated brain microvessels to study distribution of membrane carriers between the luminal and the abluminal plasma membrane of endothelial cells. We found that Na+K+-ATPase and the A-system amino-acid transport system are located predominantly on the abluminal surface of brain capillary endothelial cells. This distribution of transport carriers is consistent with the low permeability of potassium and small neutral amino acids in the blood-to-brain direction. It suggests, however, that both solutes can be actively transported across brain capillaries from the brain interstitial fluid to the blood. In tissue culture, the endothelial cells form continuous tight junctions with their neighbors. This results in a cellular layer impermeable to protein tracers. When exposed to hyperosmolar solutions, in an attempt to mimic the conditions that open the blood-brain barrier in vivo, we found a reversible separation of the tight junctions between contiguous endothelial cells. No indication of activation of pinocytosis was observed. In vitro systems provide a novel approach for studying the function of the blood-brain barrier and allow for observations not possible with intact animals.

Primary adenocarcinomas of the human urinary bladder: histochemical, immunological and ultrastructural studies.

PubMed

Alroy, J; Roganovic, D; Banner, B F; Jacobs, J B; Merk, F B; Ucci, A A; Kwan, P W; Coon, J S; Miller, A W

1981-01-01

Neoplastic and non-neoplastic tissue specimens from ten patients with primary adenocarcinoma of the urinary bladder were examined. Most of these tumors were associated with either foci of transitional cell carcinoma and/or with glandular metaplasia of the bladder epithelium. The mucin produced by the neoplastic cells was PAS, alcian blue, mucicarmine, PB/KOH/PAS, and RPB/KOH/PAS-positive. ABH isoantigens of these tumors were not always deleted. Ultrastructurally, the neoplastic cells resembled goblet cells. Their plasma membrane had numerous microvilli with prominent glycocalyx. Proliferation and attenuation of tight junctions were noted. The gap junctions were few and small. Two types of desmosomes were found. The ultrastructural features of the neoplastic cells were attributed in part to the malignant transformation and in part to the direction of their differentiation. We have not observed any distinctive morphologic, histochemical, immunologic or ultrastructural features that might be diagnostic for these adenocarcinomas.

NHS-A isoform of the NHS gene is a novel interactor of ZO-1.

PubMed

Sharma, Shiwani; Koh, Katrina S Y; Collin, Caitlin; Dave, Alpana; McMellon, Amy; Sugiyama, Yuki; McAvoy, John W; Voss, Anne K; Gécz, Jozef; Craig, Jamie E

2009-08-15

Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.

The Effect of Capsaicin Derivatives on Tight-Junction Integrity and Permeability of Madin-Darby Canine Kidney Cells.

PubMed

Kaiser, Mathias; Chalapala, Sudharani; Gorzelanny, Christian; Perali, Ramu Sridhar; Goycoolea, Francisco Martin

2016-02-01

Capsaicin is known to interfere with tight junctions (TJs) of epithelial cells and therefore to enhance paracellular permeability of poorly absorbable drugs. However, due to its low water solubility, pungency, and cytotoxicity, its pharmacologic use is limited. In this study, we investigated the effect of capsaicin derivatives of synthetic (e.g., 10-hydroxy-N-(4-hydroxy-3-methoxybenzyl)decanamide, etc.) and natural (olvanil and dihydrocapsaicin) origin on Madin-Darby Canine Kidney-C7 cells. Impedance spectroscopy was used to determine the transepithelial electrical resistance and the capacitance. Permeability assays with fluorescein isothiocyanate-dextran were carried out to evaluate the impact on cell permeability. The results show that lipophilicity could play an important role for the interference with TJ and that the mechanism is independent from the ion channel TRPV-1 and hence on the flux of calcium into the cells. In summary, we synthesized 4 derivatives of capsaicin of lower lipophilicity and compared their properties with other well-known vanilloids. We show that these compounds are able to enhance the permeability of a hydrophilic macromolecule, by opening the TJ for a shorter time than capsaicin. This behavior is dependent on the lipophilicity of the molecule. Understanding of these phenomena may lead to better control of administration of therapeutic molecules. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Segmentation and Quantitative Analysis of Epithelial Tissues.

PubMed

Aigouy, Benoit; Umetsu, Daiki; Eaton, Suzanne

2016-01-01

Epithelia are tissues that regulate exchanges with the environment. They are very dynamic and can acquire virtually any shape; at the cellular level, they are composed of cells tightly connected by junctions. Most often epithelia are amenable to live imaging; however, the large number of cells composing an epithelium and the absence of informatics tools dedicated to epithelial analysis largely prevented tissue scale studies. Here we present Tissue Analyzer, a free tool that can be used to segment and analyze epithelial cells and monitor tissue dynamics.

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies

PubMed Central

2013-01-01

Background Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Methods Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. Results The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Conclusions Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products. PMID:24262108

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.

PubMed

Eigenmann, Daniela E; Xue, Gongda; Kim, Kwang S; Moses, Ashlee V; Hamburger, Matthias; Oufir, Mouhssin

2013-11-22

Reliable human in vitro blood-brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time.Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products.

Effects of soybean agglutinin on intestinal barrier permeability and tight junction protein expression in weaned piglets.

PubMed

Zhao, Yuan; Qin, Guixin; Sun, Zewei; Che, Dongsheng; Bao, Nan; Zhang, Xiaodong

2011-01-01

This study was developed to provide further information on the intestinal barrier permeability and the tight junction protein expression in weaned piglets fed with different levels of soybean agglutinin (SBA). Twenty-five weaned crossbred barrows (Duroc × Landrace × Yorkshire) were selected and randomly allotted to five groups, each group with five replicates. The piglets in the control group were not fed with leguminous products. 0.05, 0.1, 0.15 and 0.2% SBA was added to the control diet to form four experimental diets, respectively. After the experimental period of 7 days (for each group), all the piglets were anesthetized with excess procaine and slaughtered. The d-lactic acid in plasma and the Ileal mucosa diamine oxidase (DAO) was analyzed to observe the change in the intestinal permeability. The tight junction proteins occludin and ZO-1 in the jejunum tissue distribution and relative expression were detected by immunohistochemistry and Western Blot. The results illustrated that a high dose of SBA (0.1-0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no significant affects. The contents of DAO, d-lactic acid, occludin or ZO-1, had a linear relationship with the SBA levels (0-0.2%) in diets. The high dose SBA (0.1-0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no affects.

Role of Integrin-Beta 1 in Polycystic Kidney Disease

DTIC Science & Technology

2011-04-01

characterized a novel cell line from human loop of Henle epithelium that can serve as a unique model to study medullary cystic kidney disease-2 (MCKD2) and...Therefore, we further characterized the TIRE131 clone to confirm their loop of Henle origin. Similarly to the loop of Henle epithelium , the...TIRE131 cells: 1) possessed a significant resistance to hyperosmotic growth conditions; 2) formed a functional epithelium with tight junction and

Transmigration characteristics of breast cancer and melanoma cells through the brain endothelium: Role of Rac and PI3K.

PubMed

Molnár, Judit; Fazakas, Csilla; Haskó, János; Sipos, Orsolya; Nagy, Krisztina; Nyúl-Tóth, Ádám; Farkas, Attila E; Végh, Attila G; Váró, György; Galajda, Péter; Krizbai, István A; Wilhelm, Imola

2016-05-03

Brain metastases are common and devastating complications of both breast cancer and melanoma. Although mammary carcinoma brain metastases are more frequent than those originating from melanoma, this latter has the highest tropism to the brain. Using static and dynamic in vitro approaches, here we show that melanoma cells have increased adhesion to the brain endothelium in comparison to breast cancer cells. Moreover, melanoma cells can transmigrate more rapidly and in a higher number through brain endothelial monolayers than breast cancer cells. In addition, melanoma cells have increased ability to impair tight junctions of cerebral endothelial cells. We also show that inhibition of Rac or PI3K impedes adhesion of breast cancer cells and melanoma cells to the brain endothelium. In addition, inhibition of Rac or PI3K inhibits the late phase of transmigration of breast cancer cells and the early phase of transmigration of melanoma cells. On the other hand, the Rac inhibitor EHT1864 impairs the junctional integrity of the brain endothelium, while the PI3K inhibitor LY294002 has no damaging effect on interendothelial junctions. We suggest that targeting the PI3K/Akt pathway may represent a novel opportunity in preventing the formation of brain metastases of melanoma and breast cancer.

Protein myozap--a late addition to the molecular ensembles of various kinds of adherens junctions.

PubMed

Rickelt, Steffen; Kuhn, Caecilia; Winter-Simanowski, Stefanie; Zimbelmann, Ralf; Frey, Norbert; Franke, Werner Wilhelm

2011-12-01

The protein myozap, a polypeptide of 54 kDa, has recently been identified as a component of the cytoplasmic plaques of the composite junctions (areae compositae) in the myocardiac intercalated disks and of the adherens junctions (AJs) in vascular endothelia. Now we report that using very sensitive new antibodies and drastic localization methods, we have also identified this protein as a component of the AJ plaques in simple and complex epithelia, in the adluminal cell layer of the transitional epithelium of the urinary tract and in certain cell layers of diverse stratified epithelia, including gingiva, tongue, pharynx and esophagus, cervix, vagina and epidermis. Myozap has not been identified in desmosomal and tight junction plaques. We have also detected protein myozap in AJ structures of carcinomas. The discovery of a novel major protein in AJ plaques now calls for re-examinations of molecular interactions in AJ formation and maintenance and also offers a new marker for diagnostic immunocytochemistry. We also discuss the need for progressive unravelling, extractive treatments and buffer rinses of sections and cultured cells to reveal obscured or masked antigens, before definitive negative conclusions in immunohistochemistry can be made.

Effect of High Dietary Tryptophan on Intestinal Morphology and Tight Junction Protein of Weaned Pig

PubMed Central

Tossou, Myrlene Carine B.; Bai, Miaomiao; Chen, Shuai; Cai, Yinghua; Duraipandiyan, Veeramuthu; Liu, Hongbin; Adebowale, Tolulope O.; Al-Dhabi, Naif Abdullah; Long, Lina; Tarique, Hussain; Oso, Abimbola O.; Liu, Gang; Yin, Yulong

2016-01-01

Tryptophan (Trp) plays an essential role in pig behavior and growth performances. However, little is known about Trp's effects on tight junction barrier and intestinal health in weaned pigs. In the present study, twenty-four (24) weaned pigs were randomly assigned to one of the three treatments with 8 piglets/treatments. The piglets were fed different amounts of L-tryptophan (L-Trp) as follows: 0.0%, 0.15, and 0.75%, respectively, named zero Trp (ZTS), low Trp (LTS), and high Trp (HTS), respectively. No significant differences were observed in average daily gain (ADG), average daily feed intake (ADFI), and gain: feed (G/F) ratio between the groups. After 21 days of the feeding trial, results showed that dietary Trp significantly increased (P < 0.05) crypt depth and significantly decreased (P < 0.05) villus height to crypt depth ratio (VH/CD) in the jejunum of pig fed HTS. In addition, pig fed HTS had higher (P < 0.05) serum diamine oxidase (DAO) and D-lactate. Furthermore, pig fed HTS significantly decreased mRNA expression of tight junction proteins occludin and ZO-1 but not claudin-1 in the jejunum. The number of intraepithelial lymphocytes and goblet cells were not significantly different (P > 0.05) between the groups. Collectively, these data suggest that dietary Trp supplementation at a certain level (0.75%) may negatively affect the small intestinal structure in weaned pig. PMID:27366740

Intestinal barrier: A gentlemen’s agreement between microbiota and immunity

PubMed Central

Caricilli, Andrea Moro; Castoldi, Angela; Câmara, Niels Olsen Saraiva

2014-01-01

Our body is colonized by more than a hundred trillion commensals, represented by viruses, bacteria and fungi. This complex interaction has shown that the microbiome system contributes to the host’s adaptation to its environment, providing genes and functionality that give flexibility of diet and modulate the immune system in order not to reject these symbionts. In the intestine, specifically, the microbiota helps developing organ structures, participates of the metabolism of nutrients and induces immunity. Certain components of the microbiota have been shown to trigger inflammatory responses, whereas others, anti-inflammatory responses. The diversity and the composition of the microbiota, thus, play a key role in the maintenance of intestinal homeostasis and explain partially the link between intestinal microbiota changes and gut-related disorders in humans. Tight junction proteins are key molecules for determination of the paracellular permeability. In the context of intestinal inflammatory diseases, the intestinal barrier is compromised, and decreased expression and differential distribution of tight junction proteins is observed. It is still unclear what is the nature of the luminal or mucosal factors that affect the tight junction proteins function, but the modulation of the immune cells found in the intestinal lamina propria is hypothesized as having a role in this modulation. In this review, we provide an overview of the current understanding of the interaction of the gut microbiota with the immune system in the development and maintenance of the intestinal barrier. PMID:24891972

Anti-IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus.

PubMed

Zhang, Jiyong; Sadowska, Grazyna B; Chen, Xiaodi; Park, Seon Yeong; Kim, Jeong-Eun; Bodge, Courtney A; Cummings, Erin; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G; Gaitanis, John; Banks, William A; Stonestreet, Barbara S

2015-05-01

Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti-IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti-IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti-IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia. © FASEB.

Transforming growth factor β-induced epithelial to mesenchymal transition requires the Ste20-like kinase SLK independently of its catalytic activity

PubMed Central

Conway, Jillian; Al-Zahrani, Khalid N.; Pryce, Benjamin R.; Abou-Hamad, John; Sabourin, Luc A.

2017-01-01

Invasion can be stimulated in vitro using the soluble ligand transforming growth factor-β (TGFβ) to induce a process called epithelial-to-mesenchymal transition (EMT) characterized by cell-cell junction breakdown and an invasive phenotype. We have previously demonstrated a role for Ste20-like kinase SLK cell migration and invasion. Here we show that SLK depletion in NMuMG mammary epithelial cells significantly impairs their TGFβ-induced migration and invasion. Immunofluorescence studies show that a fraction of SLK localizes to E-cadherin-positive adherens junction and that SLK impairs the breakdown of cell-cell contacts. We find that SLK-depleted cultures express significantly lower levels of vimentin protein as well as Snai1 and E-cadherin mRNA levels following TGF-β treatment. Surprisingly, our data show that SLK depletion does not affect the activation and nuclear translocation of Smad3. Furthermore, we show that expression of a dominant negative kinase does not impair tight junction breakdown and rescues Snai1 mRNA expression levels. Together these data suggest that SLK plays a novel role in TGFβ-induced EMT, independent of Smads, in a kinase activity-independent manner. PMID:29228724

Junction restructuring and spermatogenesis: the biology, regulation, and implication in male contraceptive development.

PubMed

Yan, Helen H N; Mruk, Dolores D; Cheng, C Yan

2008-01-01

Spermatogenesis that occurs in the seminiferous epithelium of adult mammalian testes is associated with extensive junction restructuring at the Sertoli-Sertoli cell, Sertoli-germ cell, and Sertoli-basement membrane interface. While this morphological phenomenon is known and has been described in great details for decades, the biochemical and molecular changes as well as the mechanisms/signaling pathways that define changes at the cell-cell and cell-matrix interface remain largely unknown until recently. In this chapter, we summarize and discuss findings in the field regarding the coordinated efforts of the anchoring [e.g., adherens junction (AJ), such as basal ectoplasmic specialization (basal ES)] and tight junctions (TJs) that are present in the same microenvironment, such as at the blood-testis barrier (BTB), or at distinctly opposite ends of the Sertoli cell epithelium, such as between apical ectoplasmic specialization (apical ES) in the apical compartment, and the BTB adjacent to the basal compartment of the epithelium. These efforts, in turn, regulate and coordinate different cellular events that occur during the seminiferous epithelial cycle. For instance, the events of spermiation and of preleptotene spermatocyte migration across the BTB both take place concurrently at stage VIII of the epithelial cycle of spermatogenesis. Recent findings suggest that these events are coordinated by protein complexes found at the apical and basal ES and TJ, which are located at different ends of the Sertoli cell epithelium. Besides, we highlight important areas of research that can now be undertaken, and functional studies that can be designed to tackle different issues pertinent to junction restructuring during spermatogenesis.

[Peripheral neuropathy and blood-nerve barrier].

PubMed

Kanda, Takashi

2009-11-01

It is important to know the cellular properties of endoneurial microvascular endothelial cells (PnMECs) and microvascular pericytes which constitute blood-nerve barrier (BNB), since this barrier structure in the peripheral nervous system (PNS) may play pivotal pathophysiological roles in various disorders of the PNS including inflammatory neuropathies (i.e. Guillain-Barré syndrome), vasculitic neuropathies, hereditary neuropathies and diabetic neuropathy. However, in contrast to blood-brain barrier (BBB), very few studies have been directed to BNB and no adequate cell lines originating from BNB had been launched. In our laboratory, we successfully established human immortalized cell lines originating from BNB using temperature-sensitive SV40 large T antigen and the cellular properties of human cell lines are presented in this paper. Human PnMEC cell line showed high transendothelial electrical resistance and expressed tight junction components and various types of influx as well as efflux transporters that have been reported to function at BBB. Human pericyte cell line also possessed tight junction proteins except claudin-5 and secrete various cytokines and growth factors including bFGF, VEGF, GDNF, NGF, BDNF and angiopoietin-1. Co-culture with pericytes or pericyte-conditioned media strengthend barrier properties of PnMEC, suggesting that in the PNS, peripheral nerve pericytes support the BNB function and play the same role of astrocytes in the BBB. Future accumulation of the knowledge concerning the cellular properties of BNB-forming cells will open the door to novel therapeutic strategies for intractable peripheral neuropathies.

Evidence for a Role of Connexin 43 in Trigeminal Pain Using RNA Interference In Vivo

PubMed Central

Ohara, Peter T.; Vit, Jean-Philippe; Bhargava, Aditi; Jasmin, Luc

2008-01-01

The importance of glial cells in the generation and maintenance of neuropathic pain is becoming widely accepted. We examined the role of glial-specific gap junctions in nociception in the rat trigeminal ganglion in nerve-injured and -uninjured states. The connexin 43 (Cx43) gap-junction subunit was found to be confined to the satellite glial cells (SGCs) that tightly envelop primary sensory neurons in the trigeminal ganglion and we therefore used Cx43 RNA interference (RNAi) to alter gap-junction function in SGCs. Using behavioral evaluation, together with immunocytochemical and Western blot monitoring, we show that Cx43 increased in the trigeminal ganglion in rats with a chronic constriction injury (CCI) of the infraorbital nerve. Reducing Cx43 expression using RNAi in CCI rats reduced painlike behavior, whereas in non-CCI rats, reducing Cx43 expression increased painlike behavior. The degree of painlike behavior in CCI rats and intact, Cx43-silenced rats was similar. Our results support previous suggestions that increases in glial gap junctions after nerve injury increases nociceptive behavior but paradoxically the reduction of gap junctions in normal ganglia also increases nociceptive behavior, possibly a reflection of the multiple functions performed by glia. PMID:18715894

Novel In Vitro/Ex Vivo Animal Modeling for Filovirus Aerosol Infection

DTIC Science & Technology

2014-09-01

junction cell‐cell, cell‐substrate  contact  was measured by  the  addition  of  10KDa,  70KDa  and  500KDa  FITC‐dextran molecules,  to  better...or  “layering  effect”.  Thus,  a  stable  TEER  reading  is  reflective  cellular  growth  plateau  or  contact   inhibition  leading  to  a  stable...relatively wider range of TEER readouts. This suggests a less  stable  tissue barrier  function or reduced cell‐cell  tight  junction  contact  by  the

[Effect of eicosapentaenoic acid on mRNA expression of tight junction protein ZO-1 in intestinal epithelial cells after Escherichia coli LF82 infection].

PubMed

Hao, Li-Jun; Lin, Yan; Zhang, Wei; Tian, Jiao; Wang, Ya; Chen, Peng-De; Hu, Chong-Kang; Zeng, Ling-Chao; Yang, Jie; Wang, Bao-Xi; Jiang, Xun

2017-06-01

To investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection. The Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 μmol/L EPA) and EPA (0, 25, 50, 100, and 200 μmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant. After EPA treatment (25 and 50 μmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant inhibitory effect on the proliferation of Caco-2 cells in a dose-dependent manner. The survival rates of the cells were significantly lower than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant increase in cell apoptosis rate compared with the control group (P<0.05). The 6- and 12-hour E.coli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). The Caco-2 cells treated with E.coli LF82 alone for 6 or 12 hours had increasing secretion of TNF-α over the time of treatment and had significantly higher secretion than the untreated Caco-2 cells (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed a reduction in the secretion of TNF-α with the increasing concentration of EPA and had significantly lower secretion than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). EPA can effectively prevent the destruction of tight junction of intestinal epithelial cells induced by E.coli LF82 infection and inhibit the secretion of inflammatory factors. Therefore, it has a certain protective effect on intestinal mucosal barrier.

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis.

PubMed

Pfeiffer, Friederike; Schäfer, Julia; Lyck, Ruth; Makrides, Victoria; Brunner, Sarah; Schaeren-Wiemers, Nicole; Deutsch, Urban; Engelhardt, Britta

2011-11-01

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

Multifunctional Role of 35 Kilodalton Hyaluronan in Promoting Defense of the Intestinal Epithelium.

PubMed

Kessler, Sean P; Obery, Dana R; Nickerson, Kourtney P; Petrey, Aaron C; McDonald, Christine; de la Motte, Carol A

2018-04-01

Intestinal epithelium plays a critical role in host defense against orally acquired pathogens. Dysregulation of this protective barrier is a primary driver of inflammatory bowel diseases (Crohn's and ulcerative colitis) and also infant gastrointestinal infections. Previously, our lab reported that hyaluronan (HA) isolated from human milk induces the expression of the antimicrobial peptide β-defensin in vivo and protects against Salmonella Typhimurium infection of epithelial cells in vitro. In addition, we demonstrated that commercially available 35 kDa size HA induces the expression of β-defensin, upregulates the expression of tight junction protein zonula occludens-1 (ZO-1), and attenuates murine Citrobacter rodentium infection in vivo. In this current study, we report that HA35 remains largely intact and biologically active during transit through the digestive tract where it directly induces β-defensin expression upon epithelial cell contact. We also demonstrate HA35 abrogation of murine Salmonella Typhimurium infection as well as downregulation of leaky tight junction protein claudin-2 expression. Taken together, we propose a dual role for HA in host innate immune defense at the epithelial cell surface, acting to induce antimicrobial peptide production and also block pathogen-induced leaky gut. HA35 is therefore a promising therapeutic in the defense against bacterially induced colitis in compromised adults and vulnerable newborns.

Direct effects of fermented cow's milk product with Lactobacillus paracasei CBA L74 on human enterocytes.

PubMed

Paparo, L; Aitoro, R; Nocerino, R; Fierro, C; Bruno, C; Canani, R Berni

2018-01-29

Cow's milk fermented with Lactobacillus paracasei CBA L74 (FM-CBAL74) exerts a preventive effect against infectious diseases in children. We evaluated if this effect is at least in part related to a direct modulation of non-immune and immune defence mechanisms in human enterocytes. Human enterocytes (Caco-2) were stimulated for 48 h with FM-CBAL74 at different concentrations. Cell growth was assessed by colorimetric assay; cell differentiation (assessed by lactase expression), tight junction proteins (zonula occludens1 and occludin), mucin 2, and toll-like receptor (TRL) pathways were analysed by real-time PCR; innate immunity peptide synthesis, beta-defensin-2 (HBD-2) and cathelicidin (LL-37) were evaluated by ELISA. Mucus layer thickness was analysed by histochemistry. FMCBA L74 stimulated cell growth and differentiation, tight junction proteins and mucin 2 expression, and mucus layer thickness in a dose-dependent fashion. A significant stimulation of HBD-2 and LL-37 synthesis, associated with a modulation of TLR pathway, was also observed. FM-CBAL74 regulates non-immune and immune defence mechanisms through a direct interaction with the enterocytes. These effects could be involved in the preventive action against infectious diseases demonstrated by this fermented product in children.

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood–brain barrier primary triple coculture model

PubMed Central

Xu, Liming; Dan, Mo; Shao, Anliang; Cheng, Xiang; Zhang, Cuiping; Yokel, Robert A; Takemura, Taro; Hanagata, Nobutaka; Niwa, Masami; Watanabe, Daisuke

2015-01-01

Background Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood–brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. Method To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. Results A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm2. After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. Conclusion These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes. PMID:26491287

Krüppel-like factor 5 is essential for maintenance of barrier function in mouse colon.

PubMed

Liu, Yang; Chidgey, Martyn; Yang, Vincent W; Bialkowska, Agnieszka B

2017-11-01

Krüppel-like factor 5 (KLF5) is a member of the zinc finger family of transcription factors that regulates homeostasis of the intestinal epithelium. Previous studies suggested an indispensable role of KLF5 in maintaining intestinal barrier function. In the current study, we investigated the mechanisms by which KLF5 regulates colonic barrier function in vivo and in vitro. We used an inducible and a constitutive intestine-specific Klf5 knockout mouse models ( Villin-CreER T2 ;Klf5 fl/fl designated as Klf5 ΔIND and Villin-Cre;Klf5 fl/fl as Klf5 ΔIS ) and studied an inducible KLF5 knockdown in Caco-2 BBe cells using a lentiviral Tet-on system (Caco-2 BBe KLF5ΔIND ). Specific knockout of Klf5 in colonic tissues, either inducible or constitutive, resulted in increased intestinal permeability. The phenotype was accompanied by a significant reduction in Dsg2 , which encodes desmoglein-2, a desmosomal cadherin, at both mRNA and protein levels. Transmission electron microscopy showed alterations of desmosomal morphology in both KLF5 knockdown Caco-2 BBe cells and Klf5 knockout mouse colonic tissues. Inducible knockdown of KLF5 in Caco-2BBe cells grown on Transwell plates led to impaired barrier function as evidenced by decreased transepithelial electrical resistance and increased paracellular permeability to fluorescein isothiocyanate-4 kDa dextran. Furthermore, DSG2 was significantly decreased in KLF5 knockdown cells, and DSG2 overexpression partially rescued the impaired barrier function caused by KLF5 knockdown. Electron microscopy studies demonstrated altered desmosomal morphology after KLF5 knockdown. In combination with chromatin immunoprecipitation analysis and promoter study, our data show that KLF5 regulates intestinal barrier function by mediating the transcription of DSG2 , a gene encoding a major component of desmosome structures. NEW & NOTEWORTHY The study is original research on the direct function of a Krüppel-like factor on intestinal barrier function, which is commonly exerted by cell junctions, including tight junctions, adherens junctions, and desmosomes. Numerous previous studies were focused on tight junctions and adherens junctions. However, this study provided a new perspective on how the intestinal barrier function is regulated by KLF5 through DSG2, a component of desmosome complexes. Copyright © 2017 the American Physiological Society.

Phosphorylation of the Tight Junction Protein Occludin Regulates Epithelial Monolayer Proliferation and Maturation

NASA Astrophysics Data System (ADS)

Bolinger, Mark Thomas

Barriers against the external environment are crucial for sustaining life in multicellular organisms, and form following convergent growth and development of cell-cell junctions. At least four types of epithelial cell-cell junctions exist, the most apical of which is known as the tight junction (TJ). A specific transmembrane protein known as occludin is highly phosphorylated on its C-terminal coiled-coil, and certain sites have been found to regulate specific aspects of TJ function, including the response to certain cytokines. Previously, our lab discovered a novel phosphosite at serine 471 that is located at a contact site with an important central organizer of the TJ, zonula occludens-1. Phosphoinhibitory, serine to alanine (S471A) occludin point mutant MDCK cell lines demonstrate that S471A monolayers are poorly organized compared to WT occludin (WT Occ) or phosphomimetic, serine to aspartic acid (S471D) lines. Additionally, S471A monolayers are composed of fewer, larger cells than controls, and exhibit proliferative arrest almost immediately following confluency, in contrast to control lines, which go through at least one additional round of proliferation. This phenotype can be recapitulated with a cell cycle inhibitor, demonstrating that confluent proliferation or cell packing is necessary for barrier maturation. G-protein coupled receptor kinase (GRK) was confirmed to be an S471 kinase by inhibitor experiments from a bioinformatically compiled candidate kinase list, and GRK inhibitors were able to recapitulate the phenotype of S471A lines. Finally, S471A expression perturbed purified coiled-coil stability as determined by NMR. Modeling of inter-coil interactions identified several possible hydrogen bonds that differ between the phosphorylated and non-phosphorylated forms. Expression of S471N (asparagine) transgenic occludin in vitro demonstrated highly organized border organization despite the lack of a negative charge at the S471 position. This result suggests that the border organization of p-S471 is not due to the negative charge at S471, and may be the result of differential intra-coil hydrogen bonding. In conclusion, cell packing is necessary for barrier maturation, and is regulated by the novel phosphosite, occludin S471. S471 is an important contributor to confluent proliferation, monolayer maturation, and barrier resistance, and plays a role in the barrier regulatory function of occludin.

Effect of tributyltin on mammalian endothelial cell integrity.

PubMed

Botelho, G; Bernardini, C; Zannoni, A; Ventrella, V; Bacci, M L; Forni, M

2015-01-01

Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Deciliation Is Associated with Dramatic Remodeling of Epithelial Cell Junctions and Surface Domains

PubMed Central

Overgaard, Christian E.; Sanzone, Kaitlin M.; Spiczka, Krystle S.; Sheff, David R.; Sandra, Alexander

2009-01-01

Stress-induced shedding of motile cilia (autotomy) has been documented in diverse organisms and likely represents a conserved cellular reaction. However, little is known about whether primary cilia are shed from mammalian epithelial cells and what impact deciliation has on polarized cellular organization. We show that several chemically distinct agents trigger autotomy in epithelial cells. Surprisingly, deciliation is associated with a significant, but reversible increase in transepithelial resistance. This reflects substantial reductions in tight junction proteins associated with “leaky” nephron segments (e.g., claudin-2). At the same time, apical trafficking of gp80/clusterin and gp114/CEACAM becomes randomized, basal-lateral delivery of Na,K-ATPase is reduced, and expression of the nonciliary apical protein gp135/podocalyxin is greatly decreased. However, ciliogenesis-impaired MDCK cells do not undergo continual junction remodeling, and mature cilia are not required for autotomy-associated remodeling events. Deciliation and epithelial remodeling may be mechanistically linked processes, because RNAi-mediated reduction of Exocyst subunit Sec6 inhibits ciliary shedding and specifically blocks deciliation-associated down-regulation of claudin-2 and gp135. We propose that ciliary autotomy represents a signaling pathway that impacts the organization and function of polarized epithelial cells. PMID:19005211

Endothelial β-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System Homeostasis.

PubMed

Tran, Khiem A; Zhang, Xianming; Predescu, Dan; Huang, Xiaojia; Machado, Roberto F; Göthert, Joachim R; Malik, Asrar B; Valyi-Nagy, Tibor; Zhao, You-Yang

2016-01-12

The blood-brain barrier (BBB) formed by brain endothelial cells interconnected by tight junctions is essential for the homeostasis of the central nervous system. Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. Using a mouse model with tamoxifen-inducible endothelial cell-restricted disruption of ctnnb1 (iCKO), we show here that endothelial β-catenin signaling is essential for maintaining BBB integrity and central nervous system homeostasis in adult mice. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and central nervous system inflammation, and all had postictal death. Disruption of endothelial β-catenin induced BBB breakdown and downregulation of the specific tight junction proteins claudin-1 and -3 in adult brain endothelial cells. The clinical relevance of the data is indicated by the observation of decreased expression of claudin-1 and nuclear β-catenin in brain endothelial cells of hemorrhagic lesions of hemorrhagic stroke patients. These results demonstrate the prerequisite role of endothelial β-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective β-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity, and central nervous system inflammation. © 2015 American Heart Association, Inc.

Live Faecalibacterium prausnitzii Does Not Enhance Epithelial Barrier Integrity in an Apical Anaerobic Co-Culture Model of the Large Intestine.

PubMed

Maier, Eva; Anderson, Rachel C; Roy, Nicole C

2017-12-12

Appropriate intestinal barrier maturation during infancy largely depends on colonization with commensal bacteria. Faecalibacterium prausnitzii is an abundant obligate anaerobe that colonizes during weaning and is thought to maintain colonic health throughout life. We previously showed that F. prausnitzii induced Toll-like receptor 2 (TLR2) activation, which is linked to enhanced tight junction formation. Therefore, we hypothesized that F. prausnitzii enhances barrier integrity, an important factor in appropriate intestinal barrier maturation. In order to test metabolically active bacteria, we used a novel apical anaerobic co-culture system that allows the survival of both obligate anaerobic bacteria and oxygen-requiring intestinal epithelial cells (Caco-2). The first aim was to optimize the culture medium to enable growth and active metabolism of F. prausnitzii while maintaining the viability and barrier integrity, as measured by trans-epithelial electrical resistance (TEER), of the Caco-2 cells. This was achieved by supplementing the apical cell culture medium with bacterial culture medium. The second aim was to test the effect of F. prausnitzii on TEER across Caco-2 cell layers. Live F. prausnitzii did not improve TEER, which indicates that its benefits are not via altering tight junction integrity. The optimization of the novel dual-environment co-culturing system performed in this research will enable the investigation of new probiotics originating from indigenous beneficial bacteria.

Live Faecalibacterium prausnitzii Does Not Enhance Epithelial Barrier Integrity in an Apical Anaerobic Co-Culture Model of the Large Intestine

PubMed Central

Maier, Eva; Anderson, Rachel C.; Roy, Nicole C.

2017-01-01

Appropriate intestinal barrier maturation during infancy largely depends on colonization with commensal bacteria. Faecalibacterium prausnitzii is an abundant obligate anaerobe that colonizes during weaning and is thought to maintain colonic health throughout life. We previously showed that F. prausnitzii induced Toll-like receptor 2 (TLR2) activation, which is linked to enhanced tight junction formation. Therefore, we hypothesized that F. prausnitzii enhances barrier integrity, an important factor in appropriate intestinal barrier maturation. In order to test metabolically active bacteria, we used a novel apical anaerobic co-culture system that allows the survival of both obligate anaerobic bacteria and oxygen-requiring intestinal epithelial cells (Caco-2). The first aim was to optimize the culture medium to enable growth and active metabolism of F. prausnitzii while maintaining the viability and barrier integrity, as measured by trans-epithelial electrical resistance (TEER), of the Caco-2 cells. This was achieved by supplementing the apical cell culture medium with bacterial culture medium. The second aim was to test the effect of F. prausnitzii on TEER across Caco-2 cell layers. Live F. prausnitzii did not improve TEER, which indicates that its benefits are not via altering tight junction integrity. The optimization of the novel dual-environment co-culturing system performed in this research will enable the investigation of new probiotics originating from indigenous beneficial bacteria. PMID:29231875

Human serum albumin nanoparticles modified with apolipoprotein A-I cross the blood-brain barrier and enter the rodent brain.

PubMed

Zensi, Anja; Begley, David; Pontikis, Charles; Legros, Celine; Mihoreanu, Larisa; Büchel, Claudia; Kreuter, Jörg

2010-12-01

Nanoparticles made of human serum albumin (HSA) and modified with apolipoproteins have previously been shown to transport drugs, which normally do not enter the brain, across the blood-brain barrier (BBB). However the precise mechanism by which nanoparticles with different apolipoproteins on their surface can target to the brain, as yet, has not been totally elucidated. In the present study, HSA nanoparticles with covalently bound apolipoprotein A-I (Apo A-I) as a targetor for brain capillary endothelial cells were injected intravenously into SV 129 mice and Wistar rats. The rodents were sacrificed after 15 or 30 min, and their brains were examined by transmission electron microscopy. Apo A-I nanoparticles could be found inside the endothelial cells of brain capillaries as well as within parenchymal brain tissue of both, mice and rats, whereas control particles without Apo A-I on their surface did not cross the BBB during our experiments. The maintenance of tight junction integrity and barrier function during treatment with nanoparticles was demonstrated by perfusion with a fixative containing lanthanum nitrate as an electron dense marker for the permeability of tight junctions.

TRPA1-dependent reversible opening of tight junction by natural compounds with an α,β-unsaturated moiety and capsaicin.

PubMed

Kanda, Yusuke; Yamasaki, Youhei; Sasaki-Yamaguchi, Yoshie; Ida-Koga, Noriko; Kamisuki, Shinji; Sugawara, Fumio; Nagumo, Yoko; Usui, Takeo

2018-02-02

The delivery of hydrophilic macromolecules runs into difficulties such as penetration of the cell membrane lipid bilayer. Our prior experiment demonstrated that capsaicin induces the reversible opening of tight junctions (TJs) and enhances the delivery of hydrophilic macromolecules through a paracellular route. Herein, we screened paracellular permeability enhancers other than capsaicin. As TJ opening by capsaicin is associated with Ca 2+ influx, we first screened the compounds that induce Ca 2+ influx in layered MDCK II cells, and then we determined the compounds' abilities to open TJs. Our results identified several natural compounds with α,β-unsaturated moiety. A structure-activity relationship (SAR) analysis and the results of pretreatment with reducing reagent DTT suggested the importance of α,β-unsaturated moiety. We also examined the underlying mechanisms, and our findings suggest that the actin reorganization seen in capsaicin treatment is important for the reversibility of TJ opening. Furthermore, our analyses revealed that TRPA1 is involved in the Ca 2+ influx and TJ permeability increase not only by an α,β-unsaturated compound but also by capsaicin. Our results indicate that the α,β-unsaturated moiety can be a potent pharmacophore for TJ opening.

Effects of Soybean Agglutinin on Intestinal Barrier Permeability and Tight Junction Protein Expression in Weaned Piglets

PubMed Central

Zhao, Yuan; Qin, Guixin; Sun, Zewei; Che, Dongsheng; Bao, Nan; Zhang, Xiaodong

2011-01-01

This study was developed to provide further information on the intestinal barrier permeability and the tight junction protein expression in weaned piglets fed with different levels of soybean agglutinin (SBA). Twenty-five weaned crossbred barrows (Duroc × Landrace × Yorkshire) were selected and randomly allotted to five groups, each group with five replicates. The piglets in the control group were not fed with leguminous products. 0.05, 0.1, 0.15 and 0.2% SBA was added to the control diet to form four experimental diets, respectively. After the experimental period of 7 days (for each group), all the piglets were anesthetized with excess procaine and slaughtered. The d-lactic acid in plasma and the Ileal mucosa diamine oxidase (DAO) was analyzed to observe the change in the intestinal permeability. The tight junction proteins occludin and ZO-1 in the jejunum tissue distribution and relative expression were detected by immunohistochemistry and Western Blot. The results illustrated that a high dose of SBA (0.1–0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no significant affects. The contents of DAO, d-lactic acid, occludin or ZO-1, had a linear relationship with the SBA levels (0–0.2%) in diets. The high dose SBA (0.1–0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no affects. PMID:22272087

Social stress induces neurovascular pathology promoting depression

PubMed Central

Menard, Caroline; Pfau, Madeline L.; Hodes, Georgia E.; Kana, Veronika; Wang, Victoria X.; Bouchard, Sylvain; Takahashi, Aki; Flanigan, Meghan E.; Aleyasin, Hossein; LeClair, Katherine B.; Janssen, William G.; Labonté, Benoit; Parise, Eric M.; Lorsch, Zachary S.; Golden, Sam A.; Heshmati, Mitra; Tamminga, Carol; Turecki, Gustavo; Campbell, Matthew; Fayad, Zahi; Tang, Cheuk Ying; Merad, Miriam; Russo, Scott J.

2017-01-01

Studies suggest that heightened peripheral inflammation contributes to the pathogenesis of major depressive disorder. We investigated the effect of chronic social defeat stress, a mouse model of depression, on blood-brain barrier (BBB) permeability and infiltration of peripheral immune signals. We found reduced expression of endothelial cell tight junction protein claudin-5 (cldn5) and abnormal blood vessel morphology in nucleus accumbens (NAc) of stress-susceptible but not resilient mice. CLDN5 expression was also decreased in NAc of depressed patients. Cldn5 down-regulation was sufficient to induce depression-like behaviors following subthreshold social stress while chronic antidepressant treatment rescued cldn5 loss and promoted resilience. Reduced BBB integrity in NAc of stress-susceptible or AAV-shRNA-cldn5-injected mice caused infiltration of peripheral cytokine interleukin-6 (IL-6) into brain parenchyma and subsequent expression of depression-like behaviors. These findings suggest that chronic social stress alters BBB integrity through loss of tight junction protein cldn5, promoting peripheral IL-6 passage across the BBB and depression. PMID:29184215

ZO proteins redundantly regulate the transcription factor DbpA/ZONAB.

PubMed

Spadaro, Domenica; Tapia, Rocio; Jond, Lionel; Sudol, Marius; Fanning, Alan S; Citi, Sandra

2014-08-08

The localization and activities of DbpA/ZONAB and YAP transcription factors are in part regulated by the density-dependent assembly of epithelial junctions. DbpA activity and cell proliferation are inhibited by exogenous overexpression of the tight junction (TJ) protein ZO-1, leading to a model whereby ZO-1 acts by sequestering DbpA at the TJ. However, mammary epithelial cells and mouse tissues knock-out for ZO-1 do not show increased proliferation, as predicted by this model. To address this discrepancy, we examined the localization and activity of DbpA and YAP in Madin-Darby canine kidney cells depleted either of ZO-1, or one of the related proteins ZO-2 and ZO-3 (ZO proteins), or all three together. Depletion of only one ZO protein had no effect on DbpA localization and activity, whereas depletion of ZO-1 and ZO-2, which is associated with reduced ZO-3 expression, resulted in increased DbpA localization in the cytoplasm. Only depletion of ZO-2 reduced the nuclear import of YAP. Mammary epithelial (Eph4) cells KO for ZO-1 showed junctional DbpA, demonstrating that ZO-1 is not required to sequester DbpA at junctions. However, further depletion of ZO-2 in Eph4 ZO-1KO cells, which do not express ZO-3, caused decreased junctional localization and expression of DbpA, which were rescued by the proteasome inhibitor MG132. In vitro binding assays showed that full-length ZO-1 does not interact with DbpA. These results show that ZO-2 is implicated in regulating the nuclear shuttling of YAP, whereas ZO proteins redundantly control the junctional retention and stability of DbpA, without affecting its shuttling to the nucleus. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon

PubMed Central

Cohen, L; Sekler, I; Hershfinkel, M

2014-01-01

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn2+ are not well understood. Here, we determined a role of the colonocytic Zn2+ sensing receptor, ZnR/GPR39, in mediating Zn2+-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn2+-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn2+ by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases. PMID:24967969

The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon.

PubMed

Cohen, L; Sekler, I; Hershfinkel, M

2014-06-26

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn(2+) are not well understood. Here, we determined a role of the colonocytic Zn(2+) sensing receptor, ZnR/GPR39, in mediating Zn(2+)-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn(2+)-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn(2+) by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases.

Ephrin-Bs Drive Junctional Downregulation and Actin Stress Fiber Disassembly to Enable Wound Re-epithelialization

PubMed Central

Nunan, Robert; Campbell, Jessica; Mori, Ryoichi; Pitulescu, Mara E.; Jiang, Wen G.; Harding, Keith G.; Adams, Ralf H.; Nobes, Catherine D.; Martin, Paul

2015-01-01

Summary For a skin wound to successfully heal, the cut epidermal-edge cells have to migrate forward at the interface between scab and healthy granulation tissue. Much is known about how lead-edge cells migrate, but very little is known about the mechanisms that enable active participation by cells further back. Here we show that ephrin-B1 and its receptor EphB2 are both upregulated in vivo, just for the duration of repair, in the first 70 or so rows of epidermal cells, and this signal leads to downregulation of the molecular components of adherens and tight (but not desmosomal) junctions, leading to loosening between neighbors and enabling shuffle room among epidermal cells. Additionally, this signaling leads to the shutdown of actomyosin stress fibers in these same epidermal cells, which may act to release tension within the wound monolayer. If this signaling axis is perturbed, then disrupted healing is a consequence in mouse and man. PMID:26549443

Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats.

PubMed

Arnhold, Stefan; Heiduschka, Peter; Klein, Helmut; Absenger, Yvonne; Basnaoglu, Serkan; Kreppel, Florian; Henke-Fahle, Sylvia; Kochanek, Stefan; Bartz-Schmidt, Karl-Ulrich; Addicks, Klaus; Schraermeyer, Ulrich

2006-09-01

To determine the potential of adenovirally transduced bone marrow stromal cells (BMSCs) to differentiate into retinal pigment epithelial-like cells and to evaluabe possible rescue effects after transplantation into the retinas of Royal College of Surgeons (RCS) rats. Through a high-capacity adenoviral vector expressing either green fluorescent protein (GFP) or pigment epithelial-derived factor (PEDF), rat MSCs were transduced in vitro before subretinal transplantation into Wistar rats or, alternatively, RCS rats. Two months after cell injection, the rats were killed and the eyes enucleated. The eyes were then investigated light microscopically or processed for electron microscopic investigations. Cell differentiation and integration were analyzed immunocytochemically using antibodies against cytokeratin and the tight junction protein ZO-1. Electroretinography was performed 16 days after injection of cells, to check whether a functional rescue could be detected. In vitro experiments in cocultured human MSCs and human RPE cells showed that MSCs adopted RPE-like characteristics. In grafting experiments, some rat MSCs integrate into the host RPE cell layer of Wistar and RCS rats, indicated by their hexagonal morphology. Subretinally transplanted cells express the epithelial marker cytokeratin and establish tight junctions with the host RPE cells. Furthermore, rescue effects can be demonstrated after grafting of vector-transduced and nontransduced MSCs in semithin sections of dystrophic retinas. Ultrastructurally, MSCs can be detected on top of host RPE and in close contact with photoreceptor outer segments phagocytosing rod outer segments. Taken together, these results raise the possibility that MSCs have the potency to replace diseased RPE cells and deliver therapeutic proteins into the subretinal space to protect photoreceptor cells from degeneration.

Endocochlear potential generation is associated with intercellular communication in the stria vascularis: structural analysis in the viable dominant spotting mouse mutant.

PubMed

Carlisle, L; Steel, K; Forge, A

1990-11-01

Deafness in the viable dominant spotting mouse mutant is due to a primary defect of the stria vascularis which results in absence of the positive endocochlear potential in scala media. Endocochlear potentials were measured and the structure of stria vascularis of mutants with potentials close to zero was compared with that in normal littermate controls by use of morphometric methods. The stria vascularis was significantly thinner in mutants. Marginal cells were not significantly different from controls in terms of volume density or intramembrane particle density but the network density of tight junctions was significantly reduced in the mutants. A virtual absence of gap junctions between basal cells and marginal or intermediate cells was observed, but intramembrane particle density and junctional complexes between adjacent basal cells were not different from controls. The volume density of basal cells was significantly greater in mutants. Intermediate cells accounted for a significantly smaller volume density of the stria vascularis in mutants and had a lower density of intramembrane particles than controls. Melanocytes were not identified in the stria vascularis of mutants. These results suggest that communication between marginal, intermediate and basal cells might be important to the normal function of the stria vascularis.

Intracellular cytoskeleton and junction proteins of endothelial cells in the porcine iris microvasculature.

PubMed

Yang, Hongfang; Yu, Paula K; Cringle, Stephen J; Sun, Xinghuai; Yu, Dao-Yi

2015-11-01

Recently we reported studies of the iris microvasculature and its endothelial cells using intra-luminal micro-perfusion, fixation, and silver staining, suggesting that the iris vascular endothelium may be crucial for maintaining homeostasis in the ocular anterior segment. Here we present information regarding the intracellular structure and cell junctions of the iris endothelium. Thirty-seven porcine eyes were used for this study. The temporal long posterior ciliary artery was cannulated to assess the iris microvascular network and its endothelium using intra-luminal micro-perfusion, fixation, and staining with phalloidin for intracellular cytoskeleton f-actin, and with antibodies against claudin-5 and VE-cadherin for junction proteins. Nuclei were counterstained with Hoechst. The iris was flat-mounted for confocal imaging. The iris microvasculature was studied for its distribution, branch orders and endothelial morphometrics with endothelial cell length measured for each vessel order. Our results showed that morphometrics of the iris microvasculature was comparable with our previous silver staining. Abundant stress fibres and peripheral border staining were seen within the endothelial cells in larger arteries. An obvious decrease in cytoplasmic stress fibres was evident further downstream in the smaller arterioles, and they tended to be absent from capillaries and veins. Endothelial intercellular junctions throughout the iris vasculature were VE-cadherin and claudin-5 immuno-positive, indicating the presence of both adherent junctions and tight junctions between vascular endothelial cells throughout the iris microvasculature. Unevenness of claudin-5 staining was noted along the endothelial cell borders in almost every order of vessels, especially in veins and small arterioles. Our results suggest that significant heterogeneity of intracellular structure and junction proteins is present in different orders of the iris vasculature in addition to vascular diameter and shape of the endothelia. Detailed information of the topography and intracellular structure and junction proteins of the endothelium of the iris microvasculature combined with unique structural features of the iris may help us to further understand the physiological and pathogenic roles of the iris vasculature in relevant ocular diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Changes in the blood-nerve barrier after sciatic nerve cold injury: indications supporting early treatment

PubMed Central

Li, Hao; Jia, Jian-ping; Xu, Min; Zhang, Lei

2015-01-01

Severe edema in the endoneurium can occur after non-freezing cold injury to the peripheral nerve, which suggests damage to the blood-nerve barrier. To determine the effects of cold injury on the blood-nerve barrier, the sciatic nerve on one side of Wistar rats was treated with low temperatures (3–5°C) for 2 hours. The contralateral sciatic nerve was used as a control. We assessed changes in the nerves using Evans blue as a fluid tracer and morphological methods. Excess fluid was found in the endoneurium 1 day after cold injury, though the tight junctions between cells remained closed. From 3 to 5 days after the cold injury, the fluid was still present, but the tight junctions were open. Less tracer leakage was found from 3 to 5 days after the cold injury compared with 1 day after injury. The cold injury resulted in a breakdown of the blood-nerve barrier function, which caused endoneurial edema. However, during the early period, the breakdown of the blood-nerve barrier did not include the opening of tight junctions, but was due to other factors. Excessive fluid volume produced a large increase in the endoneurial fluid pressure, prevented liquid penetration into the endoneurium from the microvasculature. These results suggest that drug treatment to patients with cold injuries should be administered during the early period after injury because it may be more difficult for the drug to reach the injury site through the microcirculation after the tissue fluid pressure becomes elevated. PMID:25878590

Dapsone protects brain microvascular integrity from high-fat diet induced LDL oxidation.

PubMed

Zhan, Rui; Zhao, Mingming; Zhou, Ting; Chen, Yue; Yu, Weiwei; Zhao, Lei; Zhang, Tao; Wang, Hecheng; Yang, Huan; Jin, Yinglan; He, Qihua; Yang, Xiaoda; Guo, Xiangyang; Willard, Belinda; Pan, Bing; Huang, Yining; Chen, Yingyu; Chui, Dehua; Zheng, Lemin

2018-06-07

Atherosclerosis was considered to induce many vascular-related complications, such as acute myocardial infarction and stroke. Abnormal lipid metabolism and its peroxidation inducing blood-brain barrier (BBB) leakage were associated with the pre-clinical stage of stroke. Dapsone (DDS), an anti-inflammation and anti-oxidation drug, has been found to have protective effects on vascular. However, whether DDS has a protective role on brain microvessels during lipid oxidation had yet to be elucidated. We investigated brain microvascular integrity in a high-fat diet (HFD) mouse model. We designed this study to explore whether DDS had protective effects on brain microvessels under lipid oxidation and tried to explain the underlying mechanism. In our live optical study, we found that DDS significantly attenuated brain microvascular leakage through reducing serum oxidized low-density lipoprotein (oxLDL) in HFD mice (p < 0.001), and DDS significantly inhibited LDL oxidation in vitro (p < 0.001). Our study showed that DDS protected tight junction proteins: ZO-1 (p < 0.001), occludin (p < 0.01), claudin-5 (p < 0.05) of microvascular endothelial cells in vivo and in vitro. DDS reversed LAMP1 aggregation in cytoplasm, and decreased the destruction of tight junction protein: ZO-1 in vitro. We first revealed that DDS had a protective role on cerebral microvessels through preventing tight junction ZO-1 from abnormal degradation by autophagy and reducing lysosome accumulation. Our findings suggested the significance of DDS in protecting brain microvessels under lipid metabolic disorders, which revealed a novel potential therapeutic strategy in brain microvascular-related diseases.

Preferential tumor cellular uptake and retention of indocyanine green for in vivo tumor imaging.

PubMed

Onda, Nobuhiko; Kimura, Masayuki; Yoshida, Toshinori; Shibutani, Makoto

2016-08-01

Indocyanine green (ICG) is a fluorescent agent approved for clinical applications by the Food and Drug Administration and European Medicines Agency. This study examined the mechanism of tumor imaging using intravenously administered ICG. The in vivo kinetics of intravenously administered ICG were determined in tumor xenografts using microscopic approaches that enabled both spatio-temporal and high-magnification analyses. The mechanism of ICG-based tumor imaging was examined at the cellular level in six phenotypically different human colon cancer cell lines exhibiting different grades of epithelioid organization. ICG fluorescence imaging detected xenograft tumors, even those < 1 mm in size, based on their preferential cellular uptake and retention of the dye following its rapid tissue-non-specific delivery, in contrast to its rapid clearance by normal tissue. Live-cell imaging revealed that cellular ICG uptake is temperature-dependent and occurs after ICG binding to the cellular membrane, a pattern suggesting endocytic uptake as the mechanism. Cellular ICG uptake correlated inversely with the formation of tight junctions. Intracellular ICG was entrapped in the membrane traffic system, resulting in its slow turnover and prolonged retention by tumor cells. Our results suggest that tumor-specific imaging by ICG involves non-specific delivery of the dye to tissues followed by preferential tumor cellular uptake and retention. The tumor cell-preference of ICG is driven by passive tumor cell-targeting, the inherent ability of ICG to bind to cell membranes, and the high endocytic activity of tumor cells in association with the disruption of their tight junctions. © 2016 UICC.

Claudin-1 has tumor suppressive activity and is a direct target of RUNX3 in gastric epithelial cells.

PubMed

Chang, Ti Ling; Ito, Kosei; Ko, Tun Kiat; Liu, Qiang; Salto-Tellez, Manuel; Yeoh, Khay Guan; Fukamachi, Hiroshi; Ito, Yoshiaki

2010-01-01

The transcription factor RUNX3 is a gastric tumor suppressor. Tumorigenic Runx3(-/-) gastric epithelial cells attach weakly to each other, compared with nontumorigenic Runx3(+/+) cells. We aimed to identify RUNX3 target genes that promote cell-cell contact to improve our understanding of RUNX3's role in suppressing gastric carcinogenesis. We compared gene expression profiles of Runx3(+/+) and Runx3(-/-) cells and observed down-regulation of genes associated with cell-cell adhesion in Runx3(-/-) cells. Reporter, mobility shift, and chromatin immunoprecipitation assays were used to examine the regulation of these genes by RUNX3. Tumorigenesis assays and immunohistological analyses of human gastric tumors were performed to confirm the role of the candidate genes in gastric tumor development. Mobility shift and chromatin immunoprecipitation assays revealed that the promoter activity of the gene that encodes the tight junction protein claudin-1 was up-regulated via the binding of RUNX3 to the RUNX consensus sites. The tumorigenicity of gastric epithelial cells from Runx3(-/-) mice was significantly reduced by restoration of claudin-1 expression, whereas knockdown of claudin-1 increased the tumorigenicity of human gastric cancer cells. Concomitant expression of RUNX3 and claudin-1 was observed in human normal gastric epithelium and cancers. The tight junction protein claudin-1 has gastric tumor suppressive activity and is a direct transcriptional target of RUNX3. Claudin-1 is down-regulated during the epithelial-mesenchymal transition; RUNX3 might therefore act as a tumor suppressor to antagonize the epithelial-mesenchymal transition. Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

Calcium Dobesilate Inhibits the Alterations in Tight Junction Proteins and Leukocyte Adhesion to Retinal Endothelial Cells Induced by Diabetes

PubMed Central

Leal, Ermelindo C.; Martins, João; Voabil, Paula; Liberal, Joana; Chiavaroli, Carlo; Bauer, Jacques; Cunha-Vaz, José; Ambrósio, António F.

2010-01-01

OBJECTIVE Calcium dobesilate (CaD) has been used in the treatment of diabetic retinopathy in the last decades, but its mechanisms of action are not elucidated. CaD is able to correct the excessive vascular permeability in the retina of diabetic patients and in experimental diabetes. We investigated the molecular and cellular mechanisms underlying the protective effects of CaD against the increase in blood–retinal barrier (BRB) permeability induced by diabetes. RESEARCH DESIGN AND METHODS Wistar rats were divided into three groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with CaD. The BRB breakdown was evaluated using Evans blue. The content or distribution of tight junction proteins (occludin, claudin-5, and zonula occluden-1 [ZO-1]), intercellular adhesion molecule-1 (ICAM-1), and p38 mitogen-activated protein kinase (p38 MAPK) was evaluated by Western blotting and immunohistochemistry. Leukocyte adhesion was evaluated in retinal vessels and in vitro. Oxidative stress was evaluated by the detection of oxidized carbonyls and tyrosine nitration. NF-κB activation was measured by enzyme-linked immunosorbent assay. RESULTS Diabetes increased the BRB permeability and retinal thickness. Diabetes also decreased occludin and claudin-5 levels and altered the distribution of ZO-1 and occludin in retinal vessels. These changes were inhibited by CaD treatment. CaD also inhibited the increase in leukocyte adhesion to retinal vessels or endothelial cells and in ICAM-1 levels, induced by diabetes or elevated glucose. Moreover, CaD decreased oxidative stress and p38 MAPK and NF-κB activation caused by diabetes. CONCLUSIONS CaD prevents the BRB breakdown induced by diabetes, by restoring tight junction protein levels and organization and decreasing leukocyte adhesion to retinal vessels. The protective effects of CaD are likely to involve the inhibition of p38 MAPK and NF-κB activation, possibly through the inhibition of oxidative/nitrosative stress. PMID:20627932

Molecular mechanisms of cell-cell spread of intracellular bacterial pathogens.

PubMed

Ireton, Keith

2013-07-17

Several bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and Rickettsia spp., have evolved mechanisms to actively spread within human tissues. Spreading is initiated by the pathogen-induced recruitment of host filamentous (F)-actin. F-actin forms a tail behind the microbe, propelling it through the cytoplasm. The motile pathogen then encounters the host plasma membrane, forming a bacterium-containing protrusion that is engulfed by an adjacent cell. Over the past two decades, much progress has been made in elucidating mechanisms of F-actin tail formation. Listeria and Shigella produce tails of branched actin filaments by subverting the host Arp2/3 complex. By contrast, Rickettsia forms tails with linear actin filaments through a bacterial mimic of eukaryotic formins. Compared with F-actin tail formation, mechanisms controlling bacterial protrusions are less well understood. However, recent findings have highlighted the importance of pathogen manipulation of host cell-cell junctions in spread. Listeria produces a soluble protein that enhances bacterial protrusions by perturbing tight junctions. Shigella protrusions are engulfed through a clathrin-mediated pathway at 'tricellular junctions'--specialized membrane regions at the intersection of three epithelial cells. This review summarizes key past findings in pathogen spread, and focuses on recent developments in actin-based motility and the formation and internalization of bacterial protrusions.

Actomyosin tension is required for correct recruitment of adherens junction components and zonula occludens formation.

PubMed

Miyake, Yuka; Inoue, Naoko; Nishimura, Koji; Kinoshita, Nagatoki; Hosoya, Hiroshi; Yonemura, Shigenobu

2006-05-15

The adherens junction (AJ) densely associated with actin filaments is a major cell-cell adhesion structure. To understand the importance of actin filament association in AJ formation, we first analyzed punctate AJs in NRK fibroblasts where one actin cable binds to one AJ structure unit. The accumulation of AJ components such as the cadherin/catenin complex and vinculin, as well as the formation of AJ-associated actin cables depended on Rho activity. Inhibitors for the Rho target, ROCK, which regulates myosin II activity, and for myosin II ATPase prevented the accumulation of AJ components, indicating that myosin II activity is more directly involved than Rho activity. Depletion of myosin II by RNAi showed similar results. The inhibition of myosin II activity in polarized epithelial MTD-1A cells affected the accumulation of vinculin to circumferential AJ (zonula adherens). Furthermore, correct zonula occludens (tight junction) formation along the apicobasal axis that requires cadherin activity was also impaired. Although MDCK cells which are often used as typical epithelial cells do not have a typical zonula adherens, punctate AJs formed dependently on myosin II activity by inducing wound closure in a MDCK cell sheet. These findings suggest that tension generated by actomyosin is essential for correct AJ assembly.

Dietary glucosylceramide enhances tight junction function in skin epidermis via induction of claudin-1.

PubMed

Kawada, Chinatsu; Hasegawa, Tatsuya; Watanabe, Mutsuto; Nomura, Yoshihiro

2013-01-01

Dietary glucosylceramide increased the expression of claudin-1 in UVB-irradiated mouse epidermis. Sphingosine and phytosphingosine, metabolites of glucosylceramide, increased trans-epithelial electrical resistance, and phytosphingosine increased claudin-1 mRNA expression in cultured keratinocytes. Our results indicate that the skin barrier improvement induced by dietary glucosylceramide might be due to enhancement of tight junction function, mediated by increased expression of claudin-1 induced by sphingoid metabolites.

Perfect transmission at oblique incidence by trigonal warping in graphene P-N junctions

NASA Astrophysics Data System (ADS)

Zhang, Shu-Hui; Yang, Wen

2018-01-01

We develop an analytical mode-matching technique for the tight-binding model to describe electron transport across graphene P-N junctions. This method shares the simplicity of the conventional mode-matching technique for the low-energy continuum model and the accuracy of the tight-binding model over a wide range of energies. It further reveals an interesting phenomenon on a sharp P-N junction: the disappearance of the well-known Klein tunneling (i.e., perfect transmission) at normal incidence and the appearance of perfect transmission at oblique incidence due to trigonal warping at energies beyond the linear Dirac regime. We show that this phenomenon arises from the conservation of a generalized pseudospin in the tight-binding model. We expect this effect to be experimentally observable in graphene and other Dirac fermions systems, such as the surface of three-dimensional topological insulators.

Assessment of the Activation State of Rho Family GTP-Binding Proteins in Breast Cancer Cells and Specimens

DTIC Science & Technology

2004-08-01

AF038388 Vav3 GEF for Rho and Rac Proto-oncogene product NM020505 hPEM -2 GEF for Cdc42 Predominantly expressed in brain AB007884 GEF-H1 GEF for Rac and Rho...fence tions, and desmosomes play a fundamental role in maintain- function). A functional tight junction is crucial to maintain ing the polarized phenotype

Regulation of immune cell infiltration into the CNS by regional neural inputs explained by the gate theory.

PubMed

Arima, Yasunobu; Kamimura, Daisuke; Sabharwal, Lavannya; Yamada, Moe; Bando, Hidenori; Ogura, Hideki; Atsumi, Toru; Murakami, Masaaki

2013-01-01

The central nervous system (CNS) is an immune-privileged environment protected by the blood-brain barrier (BBB), which consists of specific endothelial cells that are brought together by tight junctions and tight liner sheets formed by pericytes and astrocytic end-feet. Despite the BBB, various immune and tumor cells can infiltrate the CNS parenchyma, as seen in several autoimmune diseases like multiple sclerosis (MS), cancer metastasis, and virus infections. Aside from a mechanical disruption of the BBB like trauma, how and where these cells enter and accumulate in the CNS from the blood is a matter of debate. Recently, using experimental autoimmune encephalomyelitis (EAE), an animal model of MS, we found a "gateway" at the fifth lumber cord where pathogenic autoreactive CD4+ T cells can cross the BBB. Interestingly, this gateway is regulated by regional neural stimulations that can be mechanistically explained by the gate theory. In this review, we also discuss this theory and its potential for treating human diseases.

Epithelial-stromal interface in normal and neoplastic human bladder epithelium.

PubMed

Alroy, J; Gould, V E

1980-01-01

The ultrastructure of the epithelial-stromal interface of the human urinary bladder was studied in biopsy specimens that included 7 normal controls, 1 inverted papilloma, 18 noninvasive papillary carcinomas, and 19 invasive transitional cell carcinomas. In the invasive foci of the transitional cell carcinomas, the underlying basal lamina was attenuated or absent and the number of hemidesmosomes was decreased. These neoplastic cells displayed notably increased numbers of lysosomes, some of which appeared to be in the process of exocytosis. Increased numbers of cytoplasmic filaments adjacent to the plasma membranes at the invading pole of these cells were also observed. Tight junctions and junctional complexes were noticed adjacent to the tumor-stromal interface. None of the aforementioned features was observed in normal transitional epithelium, in inverted papilloma, in noninvasive papillary carcinomas, or in the noninvasive portions of invasive transitional cell carcinomas. Alterations of the epithelial-stromal interface deserve additional studies for they may constitute important parameters in the evaluation of actual or potential invasiveness in the various types of carcinoma of the bladder.

Tick-borne encephalitis virus infects human brain microvascular endothelial cells without compromising blood-brain barrier integrity.

PubMed

Palus, Martin; Vancova, Marie; Sirmarova, Jana; Elsterova, Jana; Perner, Jan; Ruzek, Daniel

2017-07-01

Alteration of the blood-brain barrier (BBB) is a hallmark of tick-borne encephalitis (TBE), a life-threating human viral neuroinfection. However, the mechanism of BBB breakdown during TBE, as well as TBE virus (TBEV) entry into the brain is unclear. Here, primary human microvascular endothelial cells (HBMECs) were infected with TBEV to study interactions with the BBB. Although the number of infected cells was relatively low in culture (<5%), the infection was persistent with high TBEV yields (>10 6 pfu/ml). Infection did not induce any significant changes in the expression of key tight junction proteins or upregulate the expression of cell adhesion molecules, and did not alter the highly organized intercellular junctions between HBMECs. In an in vitro BBB model, the virus crossed the BBB via a transcellular pathway without compromising the integrity of the cell monolayer. The results indicate that HBMECs may support TBEV entry into the brain without altering BBB integrity. Copyright © 2017 Elsevier Inc. All rights reserved.

Establishment and characterisation of a novel bovine SV40 large T-antigen-transduced foetal hepatocyte-derived cell line.

PubMed

Gleich, Alexander; Kaiser, Bastian; Schumann, Julia; Fuhrmann, Herbert

2016-06-01

Due to lack of in vitro models for bovine hepatocytes apart from primary cells, there is demand for a bovine hepatocyte-derived cell line. Transduction of bovine foetal hepatocytes with SV40 large T-antigen was performed using the vector pRetro-E2 SV40. Phase contrast microscopy was carried out to evaluate morphology. Immunofluorescence staining was conducted to study expression of keratins, tight junction proteins zona occludens-1 and claudin-1, glucose transporter-2 and P-glycoprotein as well as phosphoenolpyruvate carboxykinase. Urea and triglyceride production was quantified photometrically. Histochemical staining of glycogen by Periodic acid-Schiff stain and of lipids with Oil red O was performed after 24 h incubation with 20 mM glucose and 85 μM palmitic acid, respectively. Gene expression analysis of hepatocyte-typical genes was conducted by reverse transcription PCR. We obtained a SV40LTAg-transduced extended passage cell line, referred to as BFH12. Polygonal growth, keratins, tight junction proteins zona occludens-1 and claudin-1 and glucose transporter-2 as well as P-glycoprotein and phosphoenolpyruvate carboxykinase were attested positively. Urea production calculated as cell-specific rate was 14.2 ± 2.0 fmol/h (early passage) and 17.6 ± 3.7 fmol/h (late passage). Cell-specific triglyceride production was 1.6 ± 0.5 fmol/h (early passage) and 2.1 ± 0.3 fmol/h (late passage). Additionally, cells were positive for glycogen and lipid storage and showed a gene expression pattern resembling foetal hepatocytes. With the properties described here, the novel cell line BFH12 is a hepatocyte-derived cell line which can be used as an in vitro whole cell model.

Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

PubMed

Katt, Moriah E; Xu, Zinnia S; Gerecht, Sharon; Searson, Peter C

2016-01-01

The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2-4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.

[Study of the effect of surface-active agents on living cells, used as component parts in microemulsions, based on their chemical structures and critical micelle-formative concentration (CMC)].

PubMed

Ujhelyi, Zoltán; Vecsernyés, Miklós; Bácskay, Ildikó

2013-01-01

The aim of this study was to examine the cellular effects of two nonionic amphiphilic tenside groups and their mixtures on human Caco-2 cell monolayers as dependent upon their chemical structures and physicochemical properties. The first group of polyethylene glycol esters is represented by Polysorbates and Labrasol alone and in blends, while the members of the second group:Capryol 90, Capryol PGMC, Lauroglycol 90 and Lauroglycol FCC were used as propylene glycol esters. They are increasingly used in SMEDDS as recent tensides or co-tensides to increase the solubility of hydrophobic drug. Critical micelle concentration was measured by determination of surface tension. CMC refers to the ability of solubilization of surfactants. Cytotoxicity tests were performed on Caco-2 cell monolayers by MTT and LDH methods. Caco-2 cell monolayers are convenient and reliable in vitro models of the gastrointestinal tract. Paracellular permeability was examined with Lucifer yellow assays. The integrity of cell monolayers was observed by TransEpithelial Electrical Resistance (TEER) measurements. Tight junction alterations effected by the surfactants were also characterized as evidence for paracellular pathway. Changes in sub cellular localization of the tight junction proteins: ZO-1, Claudin-land beta-cathenin, were examined by confocal laser scanning microscopy.The results of cytotoxicity assays were in agreement and showed significant differences among the cytotoxic properties of surfactants in a concentration-dependent manner. Polysorbates 20, 60, 80 are the most toxic compounds. In the case of Labrasol, the degree of esterification and lack of sorbit component decreased cytotoxicity. If the hydrophyl head was changed from polyethylene glycol to propylene glycol, the main determined factor of cytotoxicity was the monoester content and the length of carbon chain. In our CMC experiments, we found that only Labrasol showed expressed cytotoxicity above the CMC. It refers to good ability of micelle solubilization of Labrasol. In our paracellular transport experiments each of polyethylene glycol surfactants (Polysorbates and Labrasol) altered TEER values but propylene glycol esters did not modify the monolayer integrity. Polyethylene glycol esters alone and in blends (0.05% Labrasol--0.001% Polysorbates 20, 60, 80) were able to increase Lucifer yellow permeability significantly below the IC50 concentration. On the other hand Labrasol and Polysorbates 20 have expressed effect on tight junctions of Caco-2 monolayer. It could be concluded that polyethylene glycol ester-type tensides were able to enhance the paracellular permeability by the redistribution of junctional proteins. Our results might ensure useful data for selection of suitable tensides, co-tensides and tenside mixtures for SMEDDS formulations.

The effector and scaffolding proteins AF6 and MUPP1 interact with connexin36 and localize at gap junctions that form electrical synapses in rodent brain.

PubMed

Li, X; Lynn, B D; Nagy, J I

2012-01-01

Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) occur in most major structures in the mammalian central nervous system. These synapses link ensembles of neurons and influence their network properties. Little is known about the macromolecular constituents of neuronal gap junctions or how transmission through electrical synapses is regulated at the level of channel conductance or gap junction assembly/disassembly. Such knowledge is a prerequisite to understanding the roles of gap junctions in neuronal circuitry. Gap junctions share similarities with tight and adhesion junctions in that all three reside at close plasma membrane appositions, and therefore may associate with similar structural and regulatory proteins. Previously, we reported that the tight junction-associated protein zonula occludens-1 (ZO-1) interacts with Cx36 and is localized at gap junctions. Here, we demonstrate that two proteins known to be associated with tight and adherens junctions, namely AF6 and MUPP1, are components of neuronal gap junctions in rodent brain. By immunofluorescence, AF6 and MUPP1 were co-localized with Cx36 in many brain areas. Co-immunoprecipitation and pull-down approaches revealed an association of Cx36 with AF6 and MUPP1, which required the C-terminus PDZ domain interaction motif of Cx36 for interaction with the single PDZ domain of AF6 and with the 10th PDZ domain of MUPP1. As AF6 is a target of the cAMP/Epac/Rap1 signalling pathway and MUPP1 is a scaffolding protein that interacts with CaMKII, the present results suggest that AF6 may be a target for cAMP/Epac/Rap1 signalling at electrical synapses, and that MUPP1 may contribute to anchoring CaMKII at these synapses. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Effects of topical steroids on tight junction proteins and spongiosis in esophageal epithelia of patients with eosinophilic esophagitis.

PubMed

Katzka, David A; Tadi, Ravikanth; Smyrk, Thomas C; Katarya, Eesha; Sharma, Anamay; Geno, Deborah M; Camilleri, Michael; Iyer, Prasad G; Alexander, Jeffrey A; Buttar, Navtej S

2014-11-01

The allergic response associated with eosinophilic esophagitis (EoE) occurs when food antigens permeate tight junction-mediated epithelial dilated intercellular spaces. We assessed whether levels of tight junction proteins correlate with the dilation of intercellular spaces (spongiosis) and the effects of topical steroids on these parameters. We assessed esophageal biopsy samples from 10 patients with active EoE treated with topical fluticasone, 10 untreated patients, and 10 patients without esophageal disease (controls) for degree of spongiosis. Immunohistochemical assays were used to determine the levels of the tight junction proteins filaggrin, zonula occludens (ZO)-1, ZO-2, ZO-3, and claudin-1. Histology and immunohistochemistry results were assessed blindly, with levels of tight junction proteins and degree of spongiosis rated on scales of 0 to 3. The mean degrees of spongiosis in untreated and treated patients with EoE were 1.3 and 0.4, respectively (P = .016). Esophageal epithelia did not stain significantly for ZO-1 or ZO-2. Filaggrin was observed in a predominant cytoplasmic pattern, compared with the cytoplasmic and membranous patterns of ZO-3 and claudin-1. In biopsy specimens from patients with active EoE, the mean staining intensities for filaggrin, ZO-3, and claudin-1 were 1.6, 1.4, and 0.7, respectively. In biopsy specimens from patients treated with fluticasone, levels of filaggrin, ZO-3, and claudin-1 were 2.8 (P = .002 compared with untreated patients), 1.7 (P = .46 compared with untreated patients), and 1.3 (P = .25 compared with untreated patients), respectively. The correlation between the level of filaggrin and the degree of spongiosis was r = 0.23, and between ZO-3 staining and the degree of spongiosis was r = .016 (P = .001 for filaggrin vs ZO-3 staining). Filaggrin, ZO-3, and claudin-1 (but not ZO-1 or ZO-2) are detected in the esophageal mucosa of patients with EoE treated with steroids and individuals without esophageal disease. Without treatment, spongiosis increases, corresponding with reduced levels of filaggrin, ZO-3, and claudin-1. Loss of tight junction regulators and dilation of intercellular spaces appear to be involved in the pathophysiology of EoE and could be targets for treatment. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Shear stress and the endothelial transport barrier.

PubMed

Tarbell, John M

2010-07-15

The shear stress of flowing blood on the surfaces of endothelial cells that provide the barrier to transport of solutes and water between blood and the underlying tissue modulates the permeability to solutes and the hydraulic conductivity. This review begins with a discussion of transport pathways across the endothelium and then considers the experimental evidence from both in vivo and in vitro studies that shows an influence of shear stress on endothelial transport properties after both acute (minutes to hours) and chronic (hours to days) changes in shear stress. Next, the effects of shear stress on individual transport pathways (tight junctions, adherens junctions, vesicles and leaky junctions) are described, and this information is integrated with the transport experiments to suggest mechanisms controlling both acute and chronic responses of transport properties to shear stress. The review ends with a summary of future research challenges.

Role of the Polarity Determinant Crumbs in Suppressing Mammalian Epithelial Tumor Progression

PubMed Central

Karp, Cristina M.; Tan, Ting Ting; Mathew, Robin; Nelson, Deidre; Mukherjee, Chandreyee; Degenhardt, Kurt; Karantza-Wadsworth, Vassiliki; White, Eileen

2009-01-01

Most tumors are epithelial-derived, and although disruption of polarity and aberrant cellular junction formation is a poor prognosticator in human cancer, the role of polarity determinants in oncogenesis is poorly understood. Using in vivo selection, we identified a mammalian orthologue of the Drosophila polarity regulator crumbs as a gene whose loss of expression promotes tumor progression. Immortal baby mouse kidney epithelial (iBMK) cells selected in vivo to acquire tumorigenicity displayed dramatic repression of crumbs3 (crb3) expression associated with disruption of tight junction formation, apicobasal polarity, and contact-inhibited growth. Restoration of crb3 expression restored junctions, polarity and contact inhibition, while suppressing migration and metastasis. These findings suggest a role for mammalian polarity determinants in suppressing tumorigenesis that may be analogous to the well-studied polarity tumor suppressor mechanisms in Drosophila. PMID:18519669

Cardiotonic steroid ouabain stimulates expression of blood-testis barrier proteins claudin-1 and -11 and formation of tight junctions in Sertoli cells.

PubMed

Dietze, Raimund; Shihan, Mazen; Stammler, Angelika; Konrad, Lutz; Scheiner-Bobis, Georgios

2015-04-15

The interaction of ouabain with the sodium pump induces signalling cascades resembling those triggered by hormone/receptor interactions. In the rat Sertoli cell line 93RS2, ouabain at low concentrations stimulates the c-Src/c-Raf/Erk1/2 signalling cascade via its interaction with the α4 isoform of the sodium pump expressed in these cells, leading to the activation of the transcription factor CREB. As a result of this signalling sequence, ouabain stimulates expression of claudin-1 and claudin-11, which are also controlled by a CRE promoter. Both of these proteins are known to be essential constituents of tight junctions (TJ) between Sertoli cells, and as a result of the ouabain-induced signalling TJ formation between neighbouring Sertoli cells is significantly enhanced by the steroid. Thus, ouabain-treated cell monolayers display higher transepithelial resistance and reduced free diffusion of FITC-coupled dextran in tracer diffusion assays. Taking into consideration that the formation of TJ is indispensable for the maintenance of the blood-testis barrier (BTB) and therefore for male fertility, the actions of ouabain described here and the fact that this and other related cardiotonic steroids (CTS) are produced endogenously suggest a direct influence of ouabain/sodium pump interactions on the maintenance of the BTB and thereby an effect on male fertility. Since claudin-1 and claudin-11 are also present in other blood-tissue barriers, one can speculate that ouabain and perhaps other CTS influence the dynamics of these barriers as well. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Immune Consequences of Decreasing Tumor Vasculature with Antiangiogenic Tyrosine Kinase Inhibitors in Combination with Therapeutic Vaccines

PubMed Central

Farsaci, Benedetto; Donahue, Renee N.; Coplin, Michael A.; Grenga, Italia; Lepone, Lauren M.; Molinolo, Alfredo A.; Hodge, James W.

2014-01-01

This study investigated the effects on the tumor microenvironment of combining antiangiogenic tyrosine kinase inhibitors (TKI) with therapeutic vaccines, and in particular, how vascular changes affect tumor-infiltrating immune cells. We conducted studies using a TKI (sunitinib or sorafenib) in combination with recombinant vaccines in 2 murine tumor models: colon carcinoma (MC38-CEA) and breast cancer (4T1). Tumor vasculature was measured by immunohistochemistry using 3 endothelial cell markers: CD31 (mature), CD105 (immature/proliferating), and CD11b (monocytic). We assessed oxygenation, tight junctions, compactness, and pressure within tumors, along with the frequency and phenotype of tumor-infiltrating T lymphocytes (TIL), myeloid-derived suppressor cells (MDSC), and tumor-associated macrophages (TAM) following treatment with antiangiogenic TKIs alone, vaccine alone, or the combination of a TKI with vaccine. The combined regimen decreased tumor vasculature, compactness, tight junctions, and pressure, leading to vascular normalization and increased tumor oxygenation. This combination therapy also increased TILs, including tumor antigen-specific CD8 T cells, and elevated the expression of activation markers FAS-L, CXCL-9, CD31, and CD105 in MDSCs and TAMs, leading to reduced tumor volumes and an increase in the number of tumor-free animals. The improved antitumor activity induced by combining antiangiogenic TKIs with vaccine may be the result of activated lymphoid and myeloid cells in the tumor microenvironment, resulting from vascular normalization, decreased tumor-cell density, and the consequent improvement in vascular perfusion and oxygenation. Therapies that alter tumor architecture can thus have a dramatic impact on the effectiveness of cancer immunotherapy. PMID:25092771

Prophylactic effect of rebamipide on aspirin-induced gastric lesions and disruption of tight junctional protein zonula occludens-1 distribution.

PubMed

Suzuki, Takahiro; Yoshida, Norimasa; Nakabe, Nami; Isozaki, Yutaka; Kajikawa, Hirokazu; Takagi, Tomohisa; Handa, Osamu; Kokura, Satoshi; Ichikawa, Hiroshi; Naito, Yuji; Matsui, Hirofumi; Yoshikawa, Toshikazu

2008-03-01

Aspirin and nonsteroidal anti-inflammatory agents are known to induce gastroduodenal complications such as ulcer, bleeding, and dyspepsia. In this study, we examined the prophylactic effect of rebamipide, an anti-ulcer agent with free-radical scavenging and anti-inflammatory effect, on acidified aspirin-induced gastric mucosal injury in rats. In addition, we investigated the mucosal barrier functions disrupted by aspirin. Oral administration of acidified aspirin resulted in linear hemorrhagic erosions with increasing myeloperoxidase activity and thiobarbituric acid-reactive substance concentrations in the gastric mucosa. Rebamipide suppressed these acidified aspirin-induced gastric lesions and inflammatory changes significantly, and its protective effect was more potent in the case of repeated (twice daily for 3 days) treatment than single treatment before aspirin administration. Immunostaining of zonula occludens (ZO)-1, one of the tight junctional proteins, was strengthened in rat gastric mucosa after repeated administration of rebamipide. In addition, aspirin induced the increasing transport of fluorescine isothiocyanate-labeled dextrans with localized disruption and decreased expression of ZO-1 protein on rat gastric mucosal cell line RGM-1. Rebamipide effectively prevented aspirin-induced permeability changes and disruption of ZO-1 distribution. These results suggest that rebamipide protects against aspirin-induced gastric mucosal lesions by preserving gastric epithelial cell-to cell integrity in addition to the anti-inflammatory effects.

Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion.

PubMed

Scott, Kevin G-E; Meddings, Jonathon B; Kirk, David R; Lees-Miller, Susan P; Buret, André G

2002-10-01

Giardiasis causes malabsorptive diarrhea, and symptoms can be present in the absence of any significant morphologic injury to the intestinal mucosa. The effects of giardiasis on epithelial permeability in vivo remain unknown, and the role of T cells and myosin light chain kinase (MLCK) in altered intestinal barrier function is unclear. This study was conducted to determine whether Giardia spp. alters intestinal permeability in vivo, to assess whether these abnormalities are dependent on T cells, and to assess the role of MLCK in altered epithelial barrier function. Immunocompetent and isogenic athymic mice were inoculated with axenic Giardia muris trophozoites or sterile vehicle (control), then assessed for trophozoite colonization and gastrointestinal permeability. Mechanistic studies using nontransformed human duodenal epithelial monolayers (SCBN) determined the effects of Giardia on myosin light chain (MLC) phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, cytoskeletal F-actin, tight junctional zonula occludens-1 (ZO-1), and MLCK. Giardia infection caused a significant increase in small intestinal, but not gastric or colonic, permeability that correlated with trophozoite colonization in both immunocompetent and athymic mice. In vitro, Giardia increased permeability and phosphorylation of MLC and reorganized F-actin and ZO-1. These alterations were abolished with an MLCK inhibitor. Disruption of small intestinal barrier function is T cell independent, disappears on parasite clearance, and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1 in an MLCK-dependent fashion.

Mesenchymal stem cell therapy ameliorates diabetic nephropathy via the paracrine effect of renal trophic factors including exosomes

PubMed Central

Nagaishi, Kanna; Mizue, Yuka; Chikenji, Takako; Otani, Miho; Nakano, Masako; Konari, Naoto; Fujimiya, Mineko

2016-01-01

Bone marrow-derived mesenchymal stem cells (MSCs) have contributed to the improvement of diabetic nephropathy (DN); however, the actual mediator of this effect and its role has not been characterized thoroughly. We investigated the effects of MSC therapy on DN, focusing on the paracrine effect of renal trophic factors, including exosomes secreted by MSCs. MSCs and MSC-conditioned medium (MSC-CM) as renal trophic factors were administered in parallel to high-fat diet (HFD)-induced type 2 diabetic mice and streptozotocin (STZ)-induced insulin-deficient diabetic mice. Both therapies showed approximately equivalent curative effects, as each inhibited the exacerbation of albuminuria. They also suppressed the excessive infiltration of BMDCs into the kidney by regulating the expression of the adhesion molecule ICAM-1. Proinflammatory cytokine expression (e.g., TNF-α) and fibrosis in tubular interstitium were inhibited. TGF-β1 expression was down-regulated and tight junction protein expression (e.g., ZO-1) was maintained, which sequentially suppressed the epithelial-to-mesenchymal transition of tubular epithelial cells (TECs). Exosomes purified from MSC-CM exerted an anti-apoptotic effect and protected tight junction structure in TECs. The increase of glomerular mesangium substrate was inhibited in HFD-diabetic mice. MSC therapy is a promising tool to prevent DN via the paracrine effect of renal trophic factors including exosomes due to its multifactorial action. PMID:27721418

The angiopoietin1-Akt pathway regulates barrier function of the cultured spinal cord microvascular endothelial cells through Eps8.

PubMed

Liu, Xinchun; Zhou, Xiaoshu; Yuan, Wei

2014-10-15

In mammalian central nervous system (CNS), the integrity of the blood-spinal cord barrier (BSCB), formed by tight junctions (TJs) between adjacent microvascular endothelial cells near the basement membrane of capillaries and the accessory structures, is important for relatively independent activities of the cellular constituents inside the spinal cord. The barrier function of the BSCB are tightly regulated and coordinated by a variety of physiological or pathological factors, similar with but not quite the same as its counterpart, the blood-brain barrier (BBB). Herein, angiopoietin 1 (Ang1), an identified ligand of the endothelium-specific tyrosine kinase receptor Tie-2, was verified to regulate barrier functions, including permeability, junction protein interactions and F-actin organization, in cultured spinal cord microvascular endothelial cells (SCMEC) of rat through the activity of Akt. Besides, these roles of Ang1 in the BSCB in vitro were found to be accompanied with an increasing expression of epidermal growth factor receptor pathway substrate 8 (Eps8), an F-actin bundling protein. Furthermore, the silencing of Eps8 by lentiviral shRNA resulted in an antagonistic effect vs. Ang1 on the endothelial barrier function of SCMEC. In summary, the Ang1-Akt pathway serves as a regulator in the barrier function modulation of SCMEC via the actin-binding protein Eps8. Copyright © 2014 Elsevier Inc. All rights reserved.

Expression of drebrin, an actin binding protein, in basal cell carcinoma, trichoblastoma and trichoepithelioma.

PubMed

Mizutani, Yoko; Iwamoto, Ikuko; Kanoh, Hiroyuki; Seishima, Mariko; Nagata, Koh-ichi

2014-06-01

Drebrin, an F-actin binding protein, is known to play important roles in cell migration, synaptogenesis and neural plasticity. Although drebrin was long thought to be specific for neuronal cells, its expression has recently been reported in non-neuronal cells. As for skin-derived cells, drebrin was shown to be enriched at adhering junctions (AJs) in cultured primary keratinocytes and also be highly expressed in basal cell carcinoma (BCC) cells. Since BCC and two types of benign neoplasm, trichoblastoma and trichoepithelioma, are considered to derive from the same origin, follicular germinative cells, it is sometimes difficult to morphologically distinguish BCC from trichoblastoma and trichoepithelioma. In this study, we performed immunohistochemical staining of drebrin in BCC, trichoblastoma and trichoepithelioma, to examine whether drebrin could serve as a biomarker for BCC diagnosis. In western blotting, drebrin was detected highly and moderately in the lysates from a squamous cell carcinoma cell line, DJM-1, and normal human epidermis, respectively. In immunofluorescence analyses, drebrin was colocalized with markers of AJs and tight junctions in DJM-1 cells and detected at cell-cell junction areas of human normal epidermis tissue. We then examined the distribution patterns of drebrin in BCC, trichoblastoma and trichoepithelioma. In BCC tissues, intense and homogeneous drebrin expression was observed mainly at tumor cell-cell boundaries. In contrast, drebrin was stained only weakly and non-homogeneously in trichoblastoma and trichoepthelioma tissue samples. For differential diagnosis of BCC, drebrin may be a novel and useful marker.

Cultures of human liver cells in simulated microgravity environment

NASA Astrophysics Data System (ADS)

Yoffe, B.; Darlington, G. J.; Soriano, H. E.; Krishnan, B.; Risin, D.; Pellis, N. R.; Khaoustov, V. I.

1999-01-01

We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.

Single and collective cell migration: the mechanics of adhesions

PubMed Central

De Pascalis, Chiara; Etienne-Manneville, Sandrine

2017-01-01

Chemical and physical properties of the environment control cell proliferation, differentiation, or apoptosis in the long term. However, to be able to move and migrate through a complex three-dimensional environment, cells must quickly adapt in the short term to the physical properties of their surroundings. Interactions with the extracellular matrix (ECM) occur through focal adhesions or hemidesmosomes via the engagement of integrins with fibrillar ECM proteins. Cells also interact with their neighbors, and this involves various types of intercellular adhesive structures such as tight junctions, cadherin-based adherens junctions, and desmosomes. Mechanobiology studies have shown that cell–ECM and cell–cell adhesions participate in mechanosensing to transduce mechanical cues into biochemical signals and conversely are responsible for the transmission of intracellular forces to the extracellular environment. As they migrate, cells use these adhesive structures to probe their surroundings, adapt their mechanical properties, and exert the appropriate forces required for their movements. The focus of this review is to give an overview of recent developments showing the bidirectional relationship between the physical properties of the environment and the cell mechanical responses during single and collective cell migration. PMID:28684609

Effect of ceramide-1-phosphate transfer protein on intestinal bacterial translocation in severe acute pancreatitis.

PubMed

Wang, Jiang; Li, Chang; Jiang, Yingjian; Zheng, Hongmei; Li, Dehui; Liang, Yibo; Deng, Wensheng; Zhang, Dianliang

2017-02-01

The aim of the study was to investigate the effects of ceramide-1-phosphate transfer protein (CPTP) on the intestinal epithelial tight junction proteins in patients with severe acute pancreatitis (SAP). Fifty patients with SAP were classified into two groups according to the presence of bacterial translocation (BT) in the blood. Thirty healthy individuals were included in the control group. The presence of BT was analyzed by polymerase chain reaction. The expression of tight junction proteins and CPTP was determined using immunohistochemistry and western blotting. Bacterial DNA was detected in the peripheral blood of 62.0% of the patients with SAP. The expression of CPTP and tight junction proteins in SAP patients was lower than that in healthy controls. Among the patients with SAP, those positive for BT(+) showed a lower level of CPTP and occluding (OC) and zonula occludens-1 (ZO-1) expression and a higher level of IVA cPLA2 expression than BT(-) patients. Moreover, the expression of CPTP was significantly associated with ZO-1 and showed a negative correlation with expression of IVA cPLA2 in SAP-BT(+) patients. CPTP affects the expression of tight junction proteins and may protects the intestinal epithelial barrier by downregulating the expression of IVA cPLA2. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Tricellulin, occludin and claudin-3 expression in salmon intestine and kidney during salinity adaptation.

PubMed

Tipsmark, C K; Madsen, S S

2012-08-01

Molecular regulation of tight junctions in osmoregulatory epithelia of euryhaline fishes must be extensive during ontogeny and acclimation to salinity changes. In this study, five tight junction proteins were examined in Atlantic salmon (Salmo salar): tight junction associated tricellulin, occludin and claudin-3 isoforms (a, b, c). A survey of tissue distribution in freshwater (FW) salmon showed that tricellulin expression was highest in the intestine. Occludin was detected in tissues with importance for epithelial transport and the order of expression was gill>intestine>kidney. The three claudin-3 isoforms were expressed at highest level in kidney tissue. Transfer of juvenile FW salmon to seawater (SW) elevated intestinal tricellulin and occludin mRNA, and these transcripts were also elevated at the time of best SW-tolerance during the course of smoltification. In the kidney, expression of tricellulin and claudin-3 isoforms was elevated after SW-transfer and tricellulin, occludin, claudin-3a and -3b increased in March before the peak smolt stage. In the gill, none of the examined tight junction proteins were impacted by SW-transfer. The data suggest that expression of tricellulin and occludin is dynamically involved in reorganization of intestinal epithelium and possibly changed paracellular permeability during SW-acclimation. The increased renal tricellulin and claudin-3 expression in SW suggests a role in remodeling of the kidney during SW-acclimation. Copyright © 2012 Elsevier Inc. All rights reserved.

Immunohistochemical quantification of expression of a tight junction protein, claudin-7, in human lung cancer samples using digital image analysis method.

PubMed

Lu, Zhe; Liu, Yi; Xu, Junfeng; Yin, Hongping; Yuan, Haiying; Gu, Jinjing; Chen, Yan-Hua; Shi, Liyun; Chen, Dan; Xie, Bin

2018-03-01

Tight junction proteins are correlated with cancer development. As the pivotal proteins in epithelial cells, altered expression and distribution of different claudins have been reported in a wide variety of human malignancies. We have previously reported that claudin-7 was strongly expressed in benign bronchial epithelial cells at the cell-cell junction while expression of claudin-7 was either altered with discontinued weak expression or completely absent in lung cancers. Based on these results, we continued working on the expression pattern of claudin-7 and its relationship with lung cancer development. We herein proposed a new Digital Image Classification, Fragmentation index, Morphological analysis (DICFM) method for differentiating the normal lung tissues and lung cancer tissues based on the claudin-7 immunohistochemical staining. Seventy-seven lung cancer samples were obtained from the Second Affiliated Hospital of Zhejiang University and claudin-7 immunohistochemical staining was performed. Based on C++ and Open Source Computer Vision Library (OpenCV, version 2.4.4), the DICFM processing module was developed. Intensity and fragmentation of claudin-7 expression, as well as the morphological parameters of nuclei were calculated. Evaluation of results was performed using Receiver Operator Characteristic (ROC) analysis. Agreement between these computational results and the results obtained by two pathologists was demonstrated. The intensity of claudin-7 expression was significantly decreased while the fragmentation was significantly increased in the lung cancer tissues compared to the normal lung tissues and the intensity was strongly positively associated with the differentiation of lung cancer cells. Moreover, the perimeters of the nuclei of lung cancer cells were significantly greater than that of the normal lung cells, while the parameters of area and circularity revealed no statistical significance. Taken together, our DICFM approach may be applied as an appropriate approach to quantify the immunohistochemical staining of claudin-7 on the cell membrane and claudin-7 may serve as a marker for identification of lung cancer. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Poly(ADP-ribose) polymerase-1 regulates microglia mediated decrease of endothelial tight junction integrity.

PubMed

Mehrabadi, Abbas Rezaeian; Korolainen, Minna A; Odero, Gary; Miller, Donald W; Kauppinen, Tiina M

2017-09-01

Alzheimer's disease pathology includes, beside neuronal damage, reactive gliosis and reduced blood-brain barrier (BBB) integrity. Microglia are intimately associated with the BBB and upon AD pathology, pro-inflammatory responses of microglia could contribute to BBB damage. To study whether microglia can directly affect BBB integrity, the effects of amyloid beta (Aβ) -stimulated primary murine microglia on co-cultured mouse brain endothelial cells (bEnd3) and murine astrocyte cultures were assessed. We also assessed whether microglial phenotype modulation via poly(ADP-ribose) polymerase-1 (PARP-1) inhibition/ablation can reverse microglial impact on these BBB forming cells. Unstimulated microglia promoted expression of tight junction proteins (TJPs), zonula ocluden-1 (ZO-1) and occludin in co-cultured endothelia cells, whereas Aβ-stimulated microglia reduced endothelial expression of ZO-1 and occludin. Astrocytes co-cultured with microglia showed elevated glial fibrillary acidic protein (GFAP) expression, which was further increased if microglia had been stimulated with Aβ. Aβ induced microglial release of nitric oxide (NO) and tumour necrosis factor alpha (TNFα), which resulted in reduced endothelial expression of TJPs and increased paracellular permeability. Microglial PARP-1 inhibition attenuated these Aβ-induced events. These findings demonstrate that PARP-1 mediated microglial responses (NO and TNFα) can directly reduce BBB integrity by promoting TJP degradation, increasing endothelial cell permeability and inducing astrogliosis. PARP-1 as a modulator of microglial phenotype can prevent microglial BBB damaging events, and thus is a potential therapeutic target. Copyright © 2017 Elsevier Ltd. All rights reserved.

Salmonella enteritidis Effector AvrA Stabilizes Intestinal Tight Junctions via the JNK Pathway.

PubMed

Lin, Zhijie; Zhang, Yong-Guo; Xia, Yinglin; Xu, Xiulong; Jiao, Xinan; Sun, Jun

2016-12-23

Salmonella pathogenesis studies to date have focused on Salmonella typhimurium, and the pathogenesis of a second major serotype, Salmonella enteritidis, is poorly understood. Salmonella spp. possess effector proteins that display biochemical activities and modulate host functions. Here, we generated a deletion mutant of the effector AvrA, S.E-AvrA - , and a plasmid-mediated complementary strain, S.E-AvrA - /pAvrA + (S.E-AvrA + ), in S. Enteritidis. Using in vitro and in vivo infection models, we showed that AvrA stabilizes epithelial tight junction (TJ) proteins, such as ZO-1, in human intestinal epithelial cells. Transepithelial electrical resistance was significantly higher in cells infected with S.E-AvrA + than in cells infected with S.E-AvrA - Inhibition of the JNK pathway suppresses the disassembly of TJ proteins; we found that enteritidis AvrA inhibited JNK activity in cells infected with wild type or S.E-AvrA + strains. Therefore, Enteritidis AvrA-induced ZO-1 stability is achieved via suppression of the JNK pathway. Furthermore, the S.E-AvrA - strain led to enhanced bacterial invasion, both in vitro and in vivo Taken together, our data reveal a novel role for AvrA in S. Enteritidis: Enteritidis AvrA stabilizes intestinal TJs and attenuates bacterial invasion. The manipulation of JNK activity and TJs in microbial-epithelial interactions may be a novel therapeutic approach for the treatment of infectious diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Salmonella enteritidis Effector AvrA Stabilizes Intestinal Tight Junctions via the JNK Pathway*

PubMed Central

Lin, Zhijie; Zhang, Yong-Guo; Xia, Yinglin; Xu, Xiulong; Jiao, Xinan

2016-01-01

Salmonella pathogenesis studies to date have focused on Salmonella typhimurium, and the pathogenesis of a second major serotype, Salmonella enteritidis, is poorly understood. Salmonella spp. possess effector proteins that display biochemical activities and modulate host functions. Here, we generated a deletion mutant of the effector AvrA, S.E-AvrA−, and a plasmid-mediated complementary strain, S.E-AvrA−/pAvrA+ (S.E-AvrA+), in S. Enteritidis. Using in vitro and in vivo infection models, we showed that AvrA stabilizes epithelial tight junction (TJ) proteins, such as ZO-1, in human intestinal epithelial cells. Transepithelial electrical resistance was significantly higher in cells infected with S.E-AvrA+ than in cells infected with S.E-AvrA−. Inhibition of the JNK pathway suppresses the disassembly of TJ proteins; we found that enteritidis AvrA inhibited JNK activity in cells infected with wild type or S.E-AvrA+ strains. Therefore, Enteritidis AvrA-induced ZO-1 stability is achieved via suppression of the JNK pathway. Furthermore, the S.E-AvrA− strain led to enhanced bacterial invasion, both in vitro and in vivo. Taken together, our data reveal a novel role for AvrA in S. Enteritidis: Enteritidis AvrA stabilizes intestinal TJs and attenuates bacterial invasion. The manipulation of JNK activity and TJs in microbial-epithelial interactions may be a novel therapeutic approach for the treatment of infectious diseases. PMID:27875307

IL-4 and IL-13 Compromise the Sinonasal Epithelial Barrier and Perturb Intercellular Junction Protein Expression

PubMed Central

Wise, Sarah K.; Laury, Adrienne M.; Katz, Elizabeth H.; Den Beste, Kyle A.; Parkos, Charles A.; Nusrat, Asma

2014-01-01

Introduction Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a “leaky” epithelial barrier phenotype. We hypothesize that Th2 cytokines IL-4 and IL-13 modulate epithelial junction proteins thereby contributing to the leaky epithelial barrier. Methods Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. Results IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n=6) and 68.6% (n=8) of baseline, respectively. Tight junction protein JAM-A expression decreased 42.2% with IL-4 exposure (n=9) and 37.5% with IL-13 exposure (n=9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n=9) and 32.9% with IL-13 exposure (n=9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or ZO-1 with IL-4 or IL-13 exposure. Conclusion Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity. PMID:24510479

Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition.

PubMed

Li, Qisheng; Sodroski, Catherine; Lowey, Brianna; Schweitzer, Cameron J; Cha, Helen; Zhang, Fang; Liang, T Jake

2016-07-05

Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.

Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

PubMed Central

Sherwood, Cara L.; Liguori, Andrew E.; Olsen, Colin E.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

2013-01-01

Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway. PMID:24349408

Rhinovirus disrupts the barrier function of polarized airway epithelial cells.

PubMed

Sajjan, Umadevi; Wang, Qiong; Zhao, Ying; Gruenert, Dieter C; Hershenson, Marc B

2008-12-15

Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease. We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection. Primary human airway epithelial cells grown at air-liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (R(T)) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting. Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in R(T) without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease R(T), suggesting a requirement for viral replication. Reduced R(T) was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-alpha, IFN-gamma and IL-1beta reversed corresponding cytokine-induced reductions in R(T) but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo. RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function.

The effect of L-ascorbic acid and/or tocopherol supplementation on electrophysiological parameters of the colon of rats chronically exposed to lead

PubMed Central

Kosik-Bogacka, Danuta I.; Baranowska-Bosiacka, Irena; Marchlewicz, Mariola; Kolasa, Agnieszka; Jakubowska, Katarzyna; Olszewska, Maria; Łanocha, Natalia; Wiernicki, Ireneusz; Millo, Barbara; Wiszniewska, Barbara; Chlubek, Dariusz

2011-01-01

Summary Background The aim of this study was to assess the effect of diet supplementation with L-ascorbic acid (500 mg/L), tocopherol (3 mg/kg b.w.), and/or a water soluble analog of tocopherol (Trolox) (48 mg/L) on ion transport in the colon of rats subjected to a chronic exposure (9 months) to 0.1% lead acetate in drinking water. Material/Methods The electrophysiological parameters of the colon wall were measured with Ussing methods. Lead content in the whole blood was analyzed by graphite furnace atomic absorption spectrometry (GFAAS) using Zeeman correction. L-ascorbic acid and tocopherol in plasma was measured by high performance liquid chromatography. Immunohistochemical reaction was carried out for visualization of occludin, the intracellular tight junction protein. Results We showed a strong inhibitory effect of lead on the electrophysiological parameters, changes in intestinal permeability, disappearance of junctional occludin, decreased amount of mucus covering the colon surface, and the accumulation of PAS-positive substance in the apical region of the cytoplasm in the absorptive cells. Conclusions Supplementation with tocopherol or Trolox did not exert a beneficial influence on the studied parameters. L-ascorbic acid positively influenced the examined electrophysiological parameters, as it cancelled the inhibitory influence of lead on ion transport in the rat colon. L-ascorbic acid also protected against tight junction disruption of epithelial cells in the colon of the lead-treated rats. A similar effect was observed in the group of rats receiving lead and supplemented with L-ascorbic acid plus Trolox. PMID:21169903

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

PubMed Central

Salomon, Julie; Gaston, Cécile; Magescas, Jérémy; Duvauchelle, Boris; Canioni, Danielle; Sengmanivong, Lucie; Mayeux, Adeline; Michaux, Grégoire; Campeotto, Florence; Lemale, Julie; Viala, Jérôme; Poirier, Françoise; Minc, Nicolas; Schmitz, Jacques; Brousse, Nicole; Ladoux, Benoit; Goulet, Olivier; Delacour, Delphine

2017-01-01

Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture. PMID:28084299

JAM related proteins in mucosal homeostasis and inflammation

PubMed Central

Luissint, Anny-Claude; Nusrat, Asma; Parkos, Charles A.

2014-01-01

Mucosal surfaces are lined by epithelial cells that form a physical barrier protecting the body against external noxious substances and pathogens. At a molecular level, the mucosal barrier is regulated by tight junctions (TJs) that seal the paracellular space between adjacent epithelial cells. Transmembrane proteins within TJs include Junctional Adhesion Molecules (JAMs) that belong to the CTX (Cortical Thymocyte marker for Xenopus) family of proteins. JAM family encompasses three classical members (JAM-A, -B and –C) and related molecules including JAM4, JAM-Like protein (JAM-L), Coxsackie and Adenovirus Receptor (CAR), CAR-Like Membrane Protein (CLMP) and Endothelial cell-Selective Adhesion Molecule (ESAM). JAMs have multiple functions that include regulation of endothelial and epithelial paracellular permeability, leukocyte recruitment during inflammation, angiogenesis, cell migration and proliferation. In this review, we summarize the current knowledge regarding the roles of the JAM family members in the regulation of mucosal homeostasis and leukocyte trafficking with a particular emphasis on barrier function and its perturbation during pathological inflammation. PMID:24667924

Formononetin Administration Ameliorates Dextran Sulfate Sodium-Induced Acute Colitis by Inhibiting NLRP3 Inflammasome Signaling Pathway.

PubMed

Wu, Dacheng; Wu, Keyan; Zhu, Qingtian; Xiao, Weiming; Shan, Qing; Yan, Zhigang; Wu, Jian; Deng, Bin; Xue, Yan; Gong, Weijuan; Lu, Guotao; Ding, Yanbing

2018-01-01

Formononetin is a kind of isoflavone compound and has been reported to possess anti-inflammatory properties. In this present study, we aimed to explore the protective effects of formononetin on dextran sulfate sodium- (DSS-) induced acute colitis. By intraperitoneal injection of formononetin in mice, the disease severity of colitis was attenuated in a dose-dependent manner, mainly manifesting as relieved clinical symptoms of colitis, mitigated colonic epithelial cell injury, and upregulations of colonic tight junction proteins levels (ZO-1, claudin-1, and occludin). Meanwhile, our study found that formononetin significantly prevented acute injury of colonic cells induced by TNF- α in vitro, specifically manifesting as the increased expressions of colonic tight junction proteins (ZO-1, claudin-1, and occludin). In addition, the result showed that formononetin could reduce the NLRP3 pathway protein levels (NLRP3, ASC, IL-1 β ) in vivo and vitro, and MCC950, the NLRP3 specific inhibitor, could alleviate the DSS-induced mice acute colitis. Furthermore, in the foundation of administrating MCC950 to inhibit activation of NLRP3 inflammasome, we failed to observe the protective effects of formononetin on acute colitis in mice. Collectively, our study for the first time confirmed the protective effects of formononetin on DSS-induced acute colitis via inhibiting the NLRP3 inflammasome pathway activation.

Visceral adipose tissue and leptin increase colonic epithelial tight junction permeability via a RhoA-ROCK-dependent pathway.

PubMed

Le Dréan, Gwenola; Haure-Mirande, Vianney; Ferrier, Laurent; Bonnet, Christian; Hulin, Philippe; de Coppet, Pierre; Segain, Jean-Pierre

2014-03-01

Proinflammatory cytokines produced by immune cells play a central role in the increased intestinal epithelial permeability during inflammation. Expansion of visceral adipose tissue (VAT) is currently considered a consequence of intestinal inflammation. Whether VAT per se plays a role in early modifications of intestinal barrier remains unknown. The aim of this study was to demonstrate the direct role of adipocytes in regulating paracellular permeability of colonic epithelial cells (CECs). We show in adult rats born with intrauterine growth retardation, a model of VAT hypertrophy, and in rats with VAT graft on the colon, that colonic permeability was increased without any inflammation. This effect was associated with altered expression of tight junction (TJ) proteins occludin and ZO-1. In coculture experiments, adipocytes decreased transepithelial resistance (TER) of Caco-2 CECs and induced a disorganization of ZO-1 on TJs. Intraperitoneal administration of leptin to lean rats increased colonic epithelial permeability and altered ZO-1 expression and organization. Treatment of HT29-19A CECs with leptin, but not adiponectin, dose-dependently decreased TER and altered TJ and F-actin cytoskeleton organization through a RhoA-ROCK-dependent pathway. Our data show that adipocytes and leptin directly alter TJ function in CECs and suggest that VAT could impair colonic epithelial barrier.

Activation of the Small GTPase Rap1 Inhibits Choroidal Neovascularization by Regulating Cell Junctions and ROS Generation in Rats.

PubMed

Li, Jiajia; Zhang, Rong; Wang, Caixia; Wang, Xin; Xu, Man; Ma, Jingxue; Shang, Qingli

2018-03-30

Choroidal neovascularization (CNV) is a common vision-threatening complication associated with many  fundus diseases. The retinal pigment epithelial (RPE) cell junction barrier has critical functions in preventing CNV, and oxidative stress can cause compromise of barrier integrity and induce angiogenesis. Rap1, a small guanosine triphosphatase (GTPase), is involved in regulating endothelial and epithelial cell junctions. In this work, we explored the function and mechanism of Rap1 in CNV in vivo. A laser-induced rat CNV model was developed. Rap1 was activated through intravitreal injection of the Rap1 activator 8CPT-2'-O-Me-cAMP (8CPT). At 14 days after laser treatment, CNV size in RPE/choroid flat mounts was measured by fluorescein isothiocyanate-dextran staining. Expression of vascular endothelial growth factor (VEGF) and cell junction proteins in RPE/choroid tissues were analyzed by western blots and quantitative real-time PCR assays. Reactive oxygen species (ROS) in RPE cells were detectedbydichloro-dihydro-fluorescein diacetate assays. The antioxidant apocynin was intraperitoneally injected into rats. Activating Rap1 by 8CPT significantly reduced CNV size and VEGF expression in the rat CNV model. Rap1 activation enhanced protein and mRNA levels of ZO-1 and occludin, two tight junction proteins in the RPE barrier. In addition, reducing ROS generation by injection of apocynin, a NADPH oxidase inhibitor, inhibited CNV formation. Rap1 activation reduced ROS generation and expression of NADPH oxidase 4. Rap1 activation inhibits CNV through regulating barrier integrity and ROS generation of RPE in vivo, and selectively activating Rap1 may be a way to reduce vision loss from CNV.

Cell polarity proteins and spermatogenesis.

PubMed

Gao, Ying; Xiao, Xiang; Lui, Wing-Yee; Lee, Will M; Mruk, Dolores; Cheng, C Yan

2016-11-01

When the cross-section of a seminiferous tubule from an adult rat testes is examined microscopically, Sertoli cells and germ cells in the seminiferous epithelium are notably polarized cells. For instance, Sertoli cell nuclei are found near the basement membrane. On the other hand, tight junction (TJ), basal ectoplasmic specialization (basal ES, a testis-specific actin-rich anchoring junction), gap junction (GJ) and desmosome that constitute the blood-testis barrier (BTB) are also located near the basement membrane. The BTB, in turn, divides the epithelium into the basal and the adluminal (apical) compartments. Within the epithelium, undifferentiated spermatogonia and preleptotene spermatocytes restrictively reside in the basal compartment whereas spermatocytes and post-meiotic spermatids reside in the adluminal compartment. Furthermore, the heads of elongating/elongated spermatids point toward the basement membrane with their elongating tails toward the tubule lumen. However, the involvement of polarity proteins in this unique cellular organization, in particular the underlying molecular mechanism(s) by which polarity proteins confer cellular polarity in the seminiferous epithelium is virtually unknown until recent years. Herein, we discuss latest findings regarding the role of different polarity protein complexes or modules and how these protein complexes are working in concert to modulate Sertoli cell and spermatid polarity. These findings also illustrate polarity proteins exert their effects through the actin-based cytoskeleton mediated by actin binding and regulatory proteins, which in turn modulate adhesion protein complexes at the cell-cell interface since TJ, basal ES and GJ utilize F-actin for attachment. We also propose a hypothetical model which illustrates the antagonistic effects of these polarity proteins. This in turn provides a unique mechanism to modulate junction remodeling in the testis to support germ cell transport across the epithelium in particular the BTB during the epithelial cycle of spermatogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein kinase Cβ as a therapeutic target stabilizing blood–brain barrier disruption in experimental autoimmune encephalomyelitis

PubMed Central

Lanz, Tobias V.; Becker, Simon; Osswald, Matthias; Bittner, Stefan; Schuhmann, Michael K.; Opitz, Christiane A.; Gaikwad, Sadanand; Wiestler, Benedikt; Litzenburger, Ulrike M.; Sahm, Felix; Ott, Martina; Iwantscheff, Simeon; Grabitz, Carl; Mittelbronn, Michel; von Deimling, Andreas; Winkler, Frank; Meuth, Sven G.; Wick, Wolfgang; Platten, Michael

2013-01-01

Disruption of the blood–brain barrier (BBB) is a hallmark of acute inflammatory lesions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis. This disruption may precede and facilitate the infiltration of encephalitogenic T cells. The signaling events that lead to this BBB disruption are incompletely understood but appear to involve dysregulation of tight-junction proteins such as claudins. Pharmacological interventions aiming at stabilizing the BBB in MS might have therapeutic potential. Here, we show that the orally available small molecule LY-317615, a synthetic bisindolylmaleimide and inhibitor of protein kinase Cβ, which is clinically under investigation for the treatment of cancer, suppresses the transmigration of activated T cells through an inflamed endothelial cell barrier, where it leads to the induction of the tight-junction molecules zona occludens-1, claudin 3, and claudin 5 and other pathways critically involved in transendothelial leukocyte migration. Treatment of mice with ongoing experimental autoimmune encephalomyelitis with LY-317615 ameliorates inflammation, demyelination, axonal damage, and clinical symptoms. Although LY-317615 dose-dependently suppresses T-cell proliferation and cytokine production independent of antigen specificity, its therapeutic effect is abrogated in a mouse model requiring pertussis toxin. This abrogation indicates that the anti-inflammatory and clinical efficacy is mainly mediated by stabilization of the BBB, thus suppressing the transmigration of encephalitogenic T cells. Collectively, our data suggest the involvement of endothelial protein kinase Cβ in stabilizing the BBB in autoimmune neuroinflammation and imply a therapeutic potential of BBB-targeting agents such as LY-317615 as therapeutic approaches for MS. PMID:23959874

Hepatocyte Polarity

PubMed Central

Treyer, Aleksandr; Müsch, Anne

2013-01-01

Hepatocytes, like other epithelia, are situated at the interface between the organism’s exterior and the underlying internal milieu and organize the vectorial exchange of macromolecules between these two spaces. To mediate this function, epithelial cells, including hepatocytes, are polarized with distinct luminal domains that are separated by tight junctions from lateral domains engaged in cell-cell adhesion and from basal domains that interact with the underlying extracellular matrix. Despite these universal principles, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. Each hepatocyte participates in multiple, narrow lumina, the bile canaliculi, and has multiple basal surfaces that face the endothelial lining. Hepatocytes also differ in the mechanism of luminal protein trafficking from other epithelia studied. They lack polarized protein secretion to the luminal domain and target single-spanning and glycosylphosphatidylinositol-anchored bile canalicular membrane proteins via transcytosis from the basolateral domain. We compare this unique hepatic polarity phenotype with that of the more common columnar epithelial organization and review our current knowledge of the signaling mechanisms and the organization of polarized protein trafficking that govern the establishment and maintenance of hepatic polarity. The serine/threonine kinase LKB1, which is activated by the bile acid taurocholate and, in turn, activates adenosine monophosphate kinase-related kinases including AMPK1/2 and Par1 paralogues has emerged as a key determinant of hepatic polarity. We propose that the absence of a hepatocyte basal lamina and differences in cell-cell adhesion signaling that determine the positioning of tight junctions are two crucial determinants for the distinct hepatic and columnar polarity phenotypes. PMID:23720287

Altered paracellular cation permeability due to a rare CLDN10B variant causes anhidrosis and kidney damage.

PubMed

Klar, Joakim; Piontek, Jörg; Milatz, Susanne; Tariq, Muhammad; Jameel, Muhammad; Breiderhoff, Tilman; Schuster, Jens; Fatima, Ambrin; Asif, Maria; Sher, Muhammad; Mäbert, Katrin; Fromm, Anja; Baig, Shahid M; Günzel, Dorothee; Dahl, Niklas

2017-07-01

Claudins constitute the major component of tight junctions and regulate paracellular permeability of epithelia. Claudin-10 occurs in two major isoforms that form paracellular channels with ion selectivity. We report on two families segregating an autosomal recessive disorder characterized by generalized anhidrosis, severe heat intolerance and mild kidney failure. All affected individuals carry a rare homozygous missense mutation c.144C>G, p.(N48K) specific for the claudin-10b isoform. Immunostaining of sweat glands from patients suggested that the disease is associated with reduced levels of claudin-10b in the plasma membranes and in canaliculi of the secretory portion. Expression of claudin-10b N48K in a 3D cell model of sweat secretion indicated perturbed paracellular Na+ transport. Analysis of paracellular permeability revealed that claudin-10b N48K maintained cation over anion selectivity but with a reduced general ion conductance. Furthermore, freeze fracture electron microscopy showed that claudin-10b N48K was associated with impaired tight junction strand formation and altered cis-oligomer formation. These data suggest that claudin-10b N48K causes anhidrosis and our findings are consistent with a combined effect from perturbed TJ function and increased degradation of claudin-10b N48K in the sweat glands. Furthermore, affected individuals present with Mg2+ retention, secondary hyperparathyroidism and mild kidney failure that suggest a disturbed reabsorption of cations in the kidneys. These renal-derived features recapitulate several phenotypic aspects detected in mice with kidney specific loss of both claudin-10 isoforms. Our study adds to the spectrum of phenotypes caused by tight junction proteins and demonstrates a pivotal role for claudin-10b in maintaining paracellular Na+ permeability for sweat production and kidney function.

Curcumin improves intestinal barrier function: modulation of intracellular signaling, and organization of tight junctions.

PubMed

Wang, Jing; Ghosh, Siddhartha S; Ghosh, Shobha

2017-04-01

Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as type 2 diabetes and atherosclerosis) has shifted the focus from high-fat high-cholesterol containing Western-type diet (WD)-induced changes in gut microbiota per se to release of gut bacteria-derived products (e.g., LPS) into circulation due to intestinal barrier dysfunction as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. We demonstrated earlier that oral supplementation with curcumin attenuates WD-induced development of type 2 diabetes and atherosclerosis. Poor bioavailability of curcumin has precluded the establishment of a causal relationship between oral supplementation and it is in vivo effects. We hypothesized that curcumin attenuates WD-induced chronic inflammation and associated metabolic diseases by modulating the function of intestinal epithelial cells (IECs) and the intestinal barrier function. The objective of the present study was to delineate the underlying mechanisms. The human IEC lines Caco-2 and HT-29 were used for these studies and modulation of direct as well as indirect effects of LPS on intracellular signaling as well as tight junctions were examined. Pretreatment with curcumin significantly attenuated LPS-induced secretion of master cytokine IL-1β from IECs and macrophages. Furthermore, curcumin also reduced IL-1β-induced activation of p38 MAPK in IECs and subsequent increase in expression of myosin light chain kinase involved in the phosphorylation of tight junction proteins and ensuing disruption of their normal arrangement. The major site of action of curcumin is, therefore, likely the IECs and the intestinal barrier, and by reducing intestinal barrier dysfunction, curcumin modulates chronic inflammatory diseases despite poor bioavailability. Copyright © 2017 the American Physiological Society.

Anti–IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus

PubMed Central

Zhang, Jiyong; Sadowska, Grazyna B.; Chen, Xiaodi; Park, Seon Yeong; Kim, Jeong-Eun; Bodge, Courtney A.; Cummings, Erin; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G.; Gaitanis, John; Banks, William A.; Stonestreet, Barbara S.

2015-01-01

Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti–IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti–IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti–IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia.—Zhang, J., Sadowska, G. B., Chen, X., Park, S. Y., Kim, J.-E., Bodge, C. A., Cummings, E., Lim, Y.-P., Makeyev, O., Besio, W. G., Gaitanis, J., Banks, W. A., Stonestreet, B. S. Anti–IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus. PMID:25609424

Transdermal anti-nuclear kappaB siRNA therapy for atopic dermatitis using a combination of two kinds of functional oligopeptide.

PubMed

Ibaraki, Hisako; Kanazawa, Takanori; Takashima, Yuuki; Okada, Hiroaki; Seta, Yasuo

2018-05-05

Nucleic acid-based targeting of nuclear factor kappaB (NF-κB) is gaining attention as a treatment option for skin diseases like atopic dermatitis (AD). Transdermal administration improves patient quality of life because of non-invasive; however, siRNA delivery into the skin can be challenging owing to the barrier of tight junctions in the granular layer. Therefore, we aimed to develop a delivery system of siRNA for topical skin application using functional peptides. We previously reported that combined treatment with a cytoplasm-responsive stearylated-arginine-rich peptide (STR-CH 2 R 4 H 2 C) and a tight junction opening peptide (AT1002) showed high siRNA permeability in the skin of AD-induced and normal mice. Here, we used murine macrophage RAW264.7 cells to examine siRNA permeation and the therapeutic effect of anti-NF-κB (RelA) siRNA (siRelA) complexed with STR-CH 2 R 4 H 2 C and AT1002 for AD-induced mice. We showed that significantly higher siRNA cellular uptake occurs after this treatment as well as decreased TNF-α and IL-6 expression. Additionally, we showed that effective siRNA transdermal delivery occurs with the suppression of the tight junction protein ZO-1. Moreover, topical skin application of siRelA with STR-CH 2 R 4 H 2 C and AT1002 improved AD-like symptoms in model mice. Thus, the combined treatment of STR-CH 2 R 4 H 2 C and AT1002 could serve as an effective transdermal siRNA therapeutic system for AD. Copyright © 2018 Elsevier B.V. All rights reserved.

Claudin expression in follicle-associated epithelium of rat Peyer's patches defines a major restriction of the paracellular pathway.

PubMed

Markov, A G; Falchuk, E L; Kruglova, N M; Radloff, J; Amasheh, S

2016-01-01

Members of the tight junction protein family of claudins have been demonstrated to specifically determine paracellular permeability of the intestinal epithelium. In small intestinal mucosa, which is generally considered to be a leaky epithelium, Peyer's patches are a primary part of the immune system. The aim of this study was to analyse the tight junctional barrier of follicle-associated epithelium covering Peyer's patches (lymphoid follicles). Employing small intestinal tissue specimens of male Wistar rats, electrophysiological analyses including the Ussing chamber technique, marker flux measurements and one-path impedance spectroscopy were performed. Morphometry of HE-stained tissue sections was taken into account. Claudin expression and localization was analysed by immunoblotting and confocal laser scanning immunofluorescence microscopy. Almost twofold higher parameters of epithelial and transepithelial tissue resistance and a markedly lower permeability for the paracellular permeability markers 4 and 20 kDa FITC-dextran were detected in follicle-associated epithelium compared to neighbouring villous epithelium. Analysis of claudin expression and localization revealed a stronger expression of major sealing proteins in follicle-associated epithelium, including claudin-1, claudin-4, claudin-5 and claudin-8. Therefore, the specific expression and localization of claudins is in accordance with barrier properties of follicle-associated epithelium vs. neighbouring villous epithelium. We demonstrate that follicle-associated epithelium is specialized to ensure maximum restriction of the epithelial paracellular pathway in Peyer's patches by selective sealing of tight junctions. This results in an exclusive transcellular pathway of epithelial cells as the limiting and mandatory route for a controlled presentation of antigens to the underlying lymphocytes under physiological conditions. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Transepithelial transport of PEGylated anionic poly(amidoamine) dendrimers: implications for oral drug delivery.

PubMed

Sweet, Deborah M; Kolhatkar, Rohit B; Ray, Abhijit; Swaan, Peter; Ghandehari, Hamidreza

2009-08-19

The purpose of this work was to assess the impact of PEGylation on transepithelial transport of anionic poly(amidoamine) dendrimers. Cytotoxicity, uptake and transport across Caco-2 cells of PEGylated G3.5 and G4.5 PAMAM dendrimers were studied. Methoxy polyethylene glycol (750 Da) was conjugated to carboxylic acid-terminated PAMAM dendrimers at feed ratios of 1, 2 and 4 PEG per dendrimer. Compared to the control, PEGylation of anionic dendrimers did not significantly alter cytotoxicity up to a concentration of 0.1 mM. PEGylation of G3.5 dendrimers significantly decreased cellular uptake and transepithelial transport while PEGylation of G4.5 dendrimers led to a significant increase in uptake, but also a significant decrease in transport. Dendrimer PEGylation reduced the opening of tight junctions as evidenced by confocal microscopy techniques. Modulation of the tight junctional complex correlated well with changes in PEGylated dendrimer transport and suggests that anionic dendrimers are transported primarily through the paracellular route. PEGylated dendrimers show promise in oral delivery applications where increased functionality for drug conjugation and release is desired.

Transepithelial Transport of PEGylated Anionic Poly(amidoamine) Dendrimers: Implications for Oral Drug Delivery

PubMed Central

Sweet, Deborah M.; Kolhatkar, Rohit B.; Ray, Abhijit; Swaan, Peter; Ghandehari, Hamidreza

2009-01-01

The purpose of this work was to assess the impact of PEGylation on transepithelial transport of anionic poly(amidoamine) dendrimers. Cytotoxicity, uptake and transport across Caco-2 cells of PEGylated G3.5 and G4.5 PAMAM dendrimers were studied. Methoxy polyethylene glycol (750 Da) was conjugated to carboxylic acid-terminated PAMAM dendrimers at feed ratios of 1, 2 and 4 PEG per dendrimer. Compared to the control, PEGylation of anionic dendrimers did not significantly alter cytotoxicity up to a concentration of 0.1 mM. PEGylation of G3.5 dendrimers significantly decreased cellular uptake and transepithelial transport while PEGylation of G4.5 dendrimers led to a significant increase in uptake, but also a significant decrease in transport. Dendrimer PEGylation reduced the opening of tight junctions as evidenced by confocal microscopy techniques. Modulation of the tight junctional complex correlated well with changes in PEGylated dendrimer transport and suggests that anionic dendrimers are transported primarily through the paracellular route. PEGylated dendrimers show promise in oral delivery applications where increased functionality for drug conjugation and release is desired. PMID:19393702

Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells

PubMed Central

Liu, Hao-Yu; Roos, Stefan; Jonsson, Hans; Ahl, David; Dicksved, Johan; Lindberg, Jan Erik; Lundh, Torbjörn

2015-01-01

Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell–cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens. PMID:25847917

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells.

PubMed

Appelt-Menzel, Antje; Cubukova, Alevtina; Günther, Katharina; Edenhofer, Frank; Piontek, Jörg; Krause, Gerd; Stüber, Tanja; Walles, Heike; Neuhaus, Winfried; Metzger, Marco

2017-04-11

In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm 2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Disruption of gap junctional intercellular communication by antibiotic gentamicin is associated with aberrant localization of occludin, N-cadherin, connexin 43, and vimentin in SerW3 Sertoli cells in vitro.

PubMed

Bekheet, Souad H M; Stahlmann, Ralf

2009-09-01

Spermatogenesis is a very complex process by which male germ cells differentiate into mature spermatozoa. The sophisticated communication network that controls spermatogenesis can be derailed so that dysfunction of one cell type propagates to all types as a cascade. This accounts for the particular vulnerability of the testis to environmental factors such as drugs and xenobiotics. Sertoli cells play an important role in protecting developing germ cells by forming a physiological barrier, limiting exposure to potentially toxic substrates, or conversely, facilitating uptake of xenobiotics within the testis. In this study, cells from the rat Sertoli line (SerW3) were incubated for 3, 6 and 9 subsequent days in serum free DMEM (SFDM) composed of DMEM supplemented with three different concentrations of antibiotic gentamicin (10, 30, and 100 μg). The effect of the three different concentrations of this antibiotic was determined on Sertoli cell-cell interaction through impaired expression of their constitutive tight junction proteins as early targets for different toxicants in vitro by immunochemistry analysis. The Sertoli SerW3 cell line illustrated the cytotoxicity of GS, as the intercellular junction proteins such as occludin, N-cadherin, connexin 43, and vimentin were delocalized from the membrane to the cytoplasmic compartment during exposure to the antibiotic. This study underlines the potential deleterious effects of the routine use of antibiotics during continuous cell culture.

EMP-induced alterations of tight junction protein expression and disruption of the blood-brain barrier.

PubMed

Ding, Gui-Rong; Qiu, Lian-Bo; Wang, Xiao-Wu; Li, Kang-Chu; Zhou, Yong-Chun; Zhou, Yan; Zhang, Jie; Zhou, Jia-Xing; Li, Yu-Rong; Guo, Guo-Zhen

2010-07-15

The blood-brain barrier (BBB) is critical to maintain cerebral homeostasis. In this study, we examined the effects of exposure to electromagnetic pulse (EMP) on the functional integrity of BBB and, on the localization and expression of tight junction (TJ) proteins (occludin and ZO-1) in rats. Animals were sham or whole-body exposed to EMP at 200 kV/m for 400 pulses. The permeability of BBB in rat cerebral cortex was examined by using Evans Blue (EB) and lanthanum nitrate as vascular tracers. The localization and expression of TJ proteins were assessed by western blot and immunofluorescence analysis, respectively. The data indicated that EMP exposure caused: (i) increased permeability of BBB, and (ii) altered localization as well as decreased levels of TJ protein ZO-1. These results suggested that the alteration of ZO-1 may play an important role in the disruption of tight junctions, which may lead to dysfunction of BBB after EMP exposure. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Transient suppression of gap junctional intercellular communication after exposure to 100-nanosecond pulsed electric fields.

PubMed

Steuer, Anna; Schmidt, Anke; Labohá, Petra; Babica, Pavel; Kolb, Juergen F

2016-12-01

Gap junctional intercellular communication (GJIC) is an important mechanism that is involved and affected in many diseases and injuries. So far, the effect of nanosecond pulsed electric fields (nsPEFs) on the communication between cells was not investigated. An in vitro approach is presented with rat liver epithelial WB-F344 cells grown and exposed in a monolayer. In order to observe sub-lethal effects, cells were exposed to pulsed electric fields with a duration of 100ns and amplitudes between 10 and 20kV/cm. GJIC strongly decreased within 15min after treatment but recovered within 24h. Gene expression of Cx43 was significantly decreased and associated with a reduced total amount of Cx43 protein. In addition, MAP kinases p38 and Erk1/2, involved in Cx43 phosphorylation, were activated and Cx43 became hyperphosphorylated. Immunofluorescent staining of Cx43 displayed the disassembly of gap junctions. Further, a reorganization of the actin cytoskeleton was observed whereas tight junction protein ZO-1 was not significantly affected. All effects were field- and time-dependent and most pronounced within 30 to 60min after treatment. A better understanding of a possible manipulation of GJIC by nsPEFs might eventually offer a possibility to develop and improve treatments. Copyright © 2016 Elsevier B.V. All rights reserved.

Shear stress and the endothelial transport barrier

PubMed Central

Tarbell, John M.

2010-01-01

The shear stress of flowing blood on the surfaces of endothelial cells that provide the barrier to transport of solutes and water between blood and the underlying tissue modulates the permeability to solutes and the hydraulic conductivity. This review begins with a discussion of transport pathways across the endothelium and then considers the experimental evidence from both in vivo and in vitro studies that shows an influence of shear stress on endothelial transport properties after both acute (minutes to hours) and chronic (hours to days) changes in shear stress. Next, the effects of shear stress on individual transport pathways (tight junctions, adherens junctions, vesicles and leaky junctions) are described, and this information is integrated with the transport experiments to suggest mechanisms controlling both acute and chronic responses of transport properties to shear stress. The review ends with a summary of future research challenges. PMID:20543206

Interleukin-4 and interleukin-13 compromise the sinonasal epithelial barrier and perturb intercellular junction protein expression.

PubMed

Wise, Sarah K; Laury, Adrienne M; Katz, Elizabeth H; Den Beste, Kyle A; Parkos, Charles A; Nusrat, Asma

2014-05-01

Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a "leaky" epithelial barrier phenotype. We hypothesize that T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 modulate epithelial junction proteins, thereby contributing to the leaky epithelial barrier. Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n = 6) and 68.6% (n = 8) of baseline, respectively. Tight junction protein junctional adhesion molecule-A (JAM-A) expression decreased 42.2% with IL-4 exposure (n = 9) and 37.5% with IL-13 exposure (n = 9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n = 9) and 32.9% with IL-13 exposure (n = 9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or zonula occludens-1 (ZO-1) with IL-4 or IL-13 exposure. Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity. © 2014 ARS-AAOA, LLC.

Tonic regulation of vascular permeability

PubMed Central

Curry, Fitz-Roy E.; Adamson, Roger H.

2014-01-01

Our major theme is that the layered structure of the endothelial barrier requires continuous activation of signaling pathways regulated by S1P and intracellular cAMP. These pathways modulate the adherens junction, continuity of tight junction strands, and the balance of synthesis and degradation of glycocalyx components. We evaluate recent evidence that baseline permeability is maintained by constant activity of mechanisms involving the small GTPases Rap1 and Rac1. In the basal state, the barrier is compromised when activities of the small GTPases are reduced by low S1P supply or delivery. With inflammatory stimulus, increased permeability can be understood in part as the action of signaling to reduce Rap1 and Rac1 activation. With the hypothesis that microvessel permeability and selectivity under both normal and inflammatory conditions are regulated by mechanisms that are continuously active it follows that when S1P or intracellular cAMP are elevated at the time of inflammatory stimulus, they can buffer changes induced by inflammatory agents and maintain normal barrier stability. When endothelium is exposed to inflammatory conditions and subsequently exposed to elevated S1P or intracellular cAMP, the same processes restore the functional barrier by first reestablishing the adherens junction, then modulating tight junctions and glycocalyx. In more extreme inflammatory conditions, loss of the inhibitory actions of Rac1 dependent mechanisms may promote expression of more inflammatory endothelial phenotypes by contributing to the up-regulation of RhoA dependent contractile mechanisms and the sustained loss of surface glycocalyx allowing access of inflammatory cells to the endothelium. PMID:23374222

ProbeZT: Simulation of transport coefficients of molecular electronic junctions under environmental effects using Büttiker's probes

NASA Astrophysics Data System (ADS)

Korol, Roman; Kilgour, Michael; Segal, Dvira

2018-03-01

We present our in-house quantum transport package, ProbeZT. This program provides linear response coefficients: electrical and electronic thermal conductances, as well as the thermopower of molecular junctions in which electrons interact with the surrounding thermal environment. Calculations are performed based on the Büttiker probe method, which introduces decoherence, energy exchange and dissipation effects phenomenologically using virtual electrode terminals called probes. The program can realize different types of probes, each introducing various environmental effects, including elastic and inelastic scattering of electrons. The molecular system is described by an arbitrary tight-binding Hamiltonian, allowing the study of different geometries beyond simple one-dimensional wires. Applications of the program to study the thermoelectric performance of molecular junctions are illustrated. The program also has a built-in functionality to simulate electron transport in double-stranded DNA molecules based on a tight-binding (ladder) description of the junction.

Meprin A impairs epithelial barrier function, enhances monocyte migration, and cleaves the tight junction protein occludin

PubMed Central

Bao, Jialing; Yura, Renee E.; Matters, Gail L.; Bradley, S. Gaylen; Shi, Pan; Tian, Fang

2013-01-01

Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro, including extracellular matrix proteins, adherens junction proteins, and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis, however, has not been elucidated. The present study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Experiments with recombinant occludin demonstrated that meprin A cleaves the protein between Gly100 and Ser101 on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout mice on a C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from their wild-type counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation. PMID:23804454

Voltage-gated sodium channels in taste bud cells.

PubMed

Gao, Na; Lu, Min; Echeverri, Fernando; Laita, Bianca; Kalabat, Dalia; Williams, Mark E; Hevezi, Peter; Zlotnik, Albert; Moyer, Bryan D

2009-03-12

Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.

Hepatitis C virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum.

PubMed

Benedicto, Ignacio; Molina-Jiménez, Francisca; Barreiro, Olga; Maldonado-Rodríguez, Alejandra; Prieto, Jesús; Moreno-Otero, Ricardo; Aldabe, Rafael; López-Cabrera, Manuel; Majano, Pedro L

2008-10-01

Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ-associated proteins occludin, claudin-1, and Zonula Occludens protein-1 (ZO-1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ-associated function, measured as FITC-dextran paracellular permeability, of genomic replicon-containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon-alpha (IFN-alpha) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ-associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ-associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co-localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co-immunoprecipitation and pull-down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot-like accumulation of occludin in infected Huh7 cells. We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ-associated proteins, which may provide new insights for HCV-related pathogenesis.

Interferon-β induced in female genital epithelium by HIV-1 glycoprotein 120 via Toll-like-receptor 2 pathway acts to protect the mucosal barrier.

PubMed

Nazli, Aisha; Dizzell, Sara; Zahoor, Muhammad Atif; Ferreira, Victor H; Kafka, Jessica; Woods, Matthew William; Ouellet, Michel; Ashkar, Ali A; Tremblay, Michel J; Bowdish, Dawn Me; Kaushic, Charu

2018-03-19

More than 40% of HIV infections occur via female reproductive tract (FRT) through heterosexual transmission. Epithelial cells that line the female genital mucosa are the first line of defense against HIV-1 and other sexually transmitted pathogens. These sentient cells recognize and respond to external stimuli by induction of a range of carefully balanced innate immune responses. Previously, we have shown that in response to HIV-1 gp120, the genital epithelial cells (GECs) from upper reproductive tract induce an inflammatory response that may facilitate HIV-1 translocation and infection. In this study, we report that the endometrial and endocervical GECs simultaneously induce biologically active interferon-β (IFNβ) antiviral responses following exposure to HIV-1 that act to protect the epithelial tight junction barrier. The innate antiviral response was directly induced by HIV-1 envelope glycoprotein gp120 and addition of gp120 neutralizing antibody inhibited IFNβ production. Interferon-β was induced by gp120 in upper GECs through Toll-like receptor 2 signaling and required presence of heparan sulfate on epithelial cell surface. The induction of IFNβ was dependent upon activation of transcription factor IRF3 (interferon regulatory factor 3). The IFNβ was biologically active, had a protective effect on epithelial tight junction barrier and was able to inhibit HIV-1 infection in TZM-bl indicator cells and HIV-1 replication in T cells. This is the first report that recognition of HIV-1 by upper GECs leads to induction of innate antiviral pathways. This could explain the overall low infectivity of HIV-1 in the FRT and could be exploited for HIV-1 prophylaxis.Cellular and Molecular Immunology advance online publication, 19 March 2018; doi:10.1038/cmi.2017.168.

Connexin45 interacts with zonula occludens-1 in osteoblastic cells

NASA Technical Reports Server (NTRS)

Laing, J. G.; Manley-Markowski, R. N.; Koval, M.; Civitelli, R.; Steinberg, T. H.

2001-01-01

Connexin43 (Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.

Subacute stress and chronic stress interact to decrease intestinal barrier function in rats.

PubMed

Lauffer, Adriana; Vanuytsel, Tim; Vanormelingen, Christophe; Vanheel, Hanne; Salim Rasoel, Shadea; Tóth, Joran; Tack, Jan; Fornari, Fernando; Farré, Ricard

2016-01-01

Psychological stress increases intestinal permeability, potentially leading to low-grade inflammation and symptoms in functional gastrointestinal disorders. We assessed the effect of subacute, chronic and combined stress on intestinal barrier function and mast cell density. Male Wistar rats were allocated to four experimental groups (n = 8/group): 1/sham; 2/subacute stress (isolation and limited movement for 24 h); 3/chronic crowding stress for 14 days and 4/combined subacute and chronic stress. Jejunum and colon were collected to measure: transepithelial electrical resistance (TEER; a measure of epithelial barrier function); gene expression of tight junction molecules; mast cell density. Plasma corticosterone concentration was increased in all three stress conditions versus sham, with highest concentrations in the combined stress condition. TEER in the jejunum was decreased in all stress conditions, but was significantly lower in the combined stress condition than in the other groups. TEER in the jejunum correlated negatively with corticosterone concentration. Increased expression of claudin 1, 5 and 8, occludin and zonula occludens 1 mRNAs was detected after subacute stress in the jejunum. In contrast, colonic TEER was decreased only after combined stress, and the expression of tight junction molecules was unaltered. Increased mast cell density was observed in the chronic and combined stress condition in the colon only. In conclusion, our data show that chronic stress sensitizes the gastrointestinal tract to the effects of subacute stress on intestinal barrier function; different underlying cellular and molecular alterations are indicated in the small intestine versus the colon.

The ginger component 6-shogaol prevents TNF-α-induced barrier loss via inhibition of PI3K/Akt and NF-κB signaling.

PubMed

Luettig, Julia; Rosenthal, Rita; Lee, In-Fah M; Krug, Susanne M; Schulzke, Jörg D

2016-12-01

Anti-inflammatory properties of the ginger-derived pungent component 6-shogaol (6-SG) have been studied intensively in recent years. Purpose of this study was to characterize the influence of 6-SG on inflammation-related intestinal barrier dysfunction, especially its paracellular component. The effect of 6-SG was studied in the human intestinal cell models HT-29/B6 and Caco-2 either under control conditions or challenged by the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). Electrophysiological measurements, freeze-fracture electron microscopy, and protein analyses were performed. 6-SG partially prevented both, the TNF-α-induced decrease in transepithelial resistance and the rise in fluorescein permeability. By inhibiting phosphatidylinositol-3-kinase/Akt signaling 6-SG prevented the TNF-α-induced increase in protein expression of claudin-2, a channel-forming tight junction protein. In addition, the TNF-α-induced disassembly of the sealing tight junction protein claudin-1 was attenuated, the latter of which was due to TNF-α-triggered phosphorylation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB). 6-SG has barrier-protective effects by affecting TNF-α-induced claudin-2 upregulation and claudin-1 disassembly via inhibition of phoshatidylinositol-3-kinase/Akt and nuclear factor kappa light chain enhancer of activated B-cell signaling. Therefore, 6-SG-containing food might be beneficial for barrier preservation during intestinal inflammation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dual role of ALCAM in neuroinflammation and blood–brain barrier homeostasis

PubMed Central

Saint-Laurent, Olivia; Bourbonnière, Lyne; Larouche, Sandra; Larochelle, Catherine; Michel, Laure; Charabati, Marc; Abadier, Michael; Zandee, Stephanie; Haghayegh Jahromi, Neda; Gowing, Elizabeth; Pittet, Camille; Lyck, Ruth; Engelhardt, Britta

2017-01-01

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule found on blood–brain barrier endothelial cells (BBB-ECs) that was previously shown to be involved in leukocyte transmigration across the endothelium. In the present study, we found that ALCAM knockout (KO) mice developed a more severe myelin oligodendrocyte glycoprotein (MOG)35–55–induced experimental autoimmune encephalomyelitis (EAE). The exacerbated disease was associated with a significant increase in the number of CNS-infiltrating proinflammatory leukocytes compared with WT controls. Passive EAE transfer experiments suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. In addition, phenotypic characterization of unimmunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further in vivo, in vitro, and molecular analysis confirmed that ALCAM is associated with tight junction molecule assembly at the BBB, explaining the increased permeability of CNS blood vessels in ALCAM KO animals. Collectively, our data point to a biologically important function of ALCAM in maintaining BBB integrity. PMID:28069965

A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion.

PubMed

Baum, Jake; Tonkin, Christopher J; Paul, Aditya S; Rug, Melanie; Smith, Brian J; Gould, Sven B; Richard, Dave; Pollard, Thomas D; Cowman, Alan F

2008-03-13

Malaria parasites invade host cells using actin-based motility, a process requiring parasite actin filament nucleation and polymerization. Malaria and other apicomplexan parasites lack Arp2/3 complex, an actin nucleator widely conserved across eukaryotes, but do express formins, another type of actin nucleator. Here, we demonstrate that one of two malaria parasite formins, Plasmodium falciparum formin 1 (PfFormin 1), and its ortholog in the related parasite Toxoplasma gondii, follows the moving tight junction between the invading parasite and the host cell, which is the predicted site of the actomyosin motor that powers motility. Furthermore, in vitro, the PfFormin1 actin-binding formin homology 2 domain is a potent nucleator, stimulating actin polymerization and, like other formins, localizing to the barbed end during filament elongation. These findings support a conserved molecular mechanism underlying apicomplexan parasite motility and, given the essential role that actin plays in cell invasion, highlight formins as important determinants of malaria parasite pathogenicity.

Disruption of MDCK cell tight junctions by the free-living amoeba Naegleria fowleri.

PubMed

Shibayama, Mineko; Martínez-Castillo, Moisés; Silva-Olivares, Angélica; Galindo-Gómez, Silvia; Navarro-García, Fernando; Escobar-Herrera, Jaime; Sabanero, Myrna; Tsutsumi, Víctor; Serrano-Luna, Jesús

2013-02-01

Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis. This parasite invades its host by penetrating the olfactory mucosa. However, the mechanism of epithelium penetration is not well understood. In the present study, we evaluated the effect of N. fowleri trophozoites and the non-pathogenic Naegleria gruberi on Madin-Darby canine kidney (MDCK) tight junction proteins, including claudin-1, occludin and ZO-1, as well as on the actin cytoskeleton. Trophozoites from each of the free-living amoeba species were co-cultured with MDCK cells in a 1 : 1 ratio for 1, 3, 6 or 10 h. Light microscopy revealed that N. fowleri caused morphological changes as early as 3 h post-infection in an epithelial MDCK monolayer. Confocal microscopy analysis revealed that after 10 h of co-culture, N. fowleri trophozoites induced epithelial cell damage, which was characterized by changes in the actin apical ring and disruption of the ZO-1 and claudin-1 proteins but not occludin. Western blot assays revealed gradual degradation of ZO-1 and claudin-1 as early as 3 h post-infection. Likewise, there was a drop in transepithelial electrical resistance that resulted in increased epithelial permeability and facilitated the invasion of N. fowleri trophozoites by a paracellular route. In contrast, N. gruberi did not induce alterations in MDCK cells even at 10 h post-infection. Based on these results, we suggest that N. fowleri trophozoites disrupt epithelial monolayers, which could enable their penetration of the olfactory epithelium and subsequent invasion of the central nervous system.

Roles of ZO-1 and ZO-2 in establishment of the belt-like adherens and tight junctions with paracellular permselective barrier function.

PubMed

Tsukita, Sachiko; Katsuno, Tatsuya; Yamazaki, Yuji; Umeda, Kazuaki; Tamura, Atsushi; Tsukita, Shoichiro

2009-05-01

Tight junctions (TJs) create the primary permselective barrier to diffusion of solutes and ions through the paracellular pathway. The molecular architecture of TJs has gradually been unraveled in recent years, providing the basis for "barriology" (defined by Shoichiro Tsukita as the science of the barrier in multicellular organisms). Claudins are now considered to be the essential basic components of TJ strands, with which other integral membrane proteins, such as occludin, tricellulin, JAMs, and CAR, are associated. Peripherally associated scaffolding proteins are required for the organization of the integral membrane proteins. Among these, ZO-1, -2, and -3 have attracted a great deal of attention as TJ organizers, since ZO-1 (and in some cases, also ZO-2/3) was reported to be directly associated with claudins, occludin, and JAMs, as well as with AF-6/afadin and alpha-catenin. Here we summarize recent studies on ZO-1/2/3-deficiency in mice and cells, which have provided clear and important information regarding the functions of ZO-1/2/3 in vivo. In addition to the respective suppression of ZO-1/2/3 expression, simultaneous suppression of all three proteins has revealed the essential and nonessential in vivo roles of ZO-1/2 and ZO-3, respectively. ZO-3 shows an epithelial-specific TJ localization in a ZO-1/2-dependent fashion. ZO-1 and ZO-2 play pivotal roles in the final establishment of the belt-like adherens junctions (zonula adherens), followed by the formation of the belt-like TJs (zonula occludens) with paracellular barrier function, thereby providing the general basis for selective paracellular permeability in epithelial and endothelial cells.

Ang-(1-7) exerts protective role in blood-brain barrier damage by the balance of TIMP-1/MMP-9.

PubMed

Wu, Jitao; Zhao, Duo; Wu, Shuang; Wang, Dan

2015-02-05

Cerebrovascular disease (CVD) ranks as the top three health risks, specially cerebral ischemia characterized with the damage of blood-brain barrier (BBB). The angiotensin Ang-(1-7) was proven to have a protective effect on cerebrovascular diseases. However, its role on blood-brain barrier and the underlying molecular mechanism remains unclear. In this study, Ang-(1-7) significantly relieved damage of ischemia reperfusion injury on blood-brain barrier in cerebral ischemia reperfusion injury (IRI) rats. Furthermore, its treatment attenuated BBB permeability and brain edema. Similarly, Ang-(1-7) also decreased the barrier permeability of brain endothelial cell line RBE4. Further analysis showed that Ang-(1-7) could effectively restore tight junction protein (claudin-5 and zonula occludens ZO-1) expression levels both in IRI-rats and hypoxia-induced RBE4 cells. Furthermore, Ang-(1-7) stimulation down-regulated hypoxia-induced matrix metalloproteinase-9 (MMP-9) levels, whose silencing with (matrix metalloproteinase-9 hemopexin domain) MMP9-PEX inhibitor significantly increased the expression of claudin-5 and ZO-1. Further mechanism analysis demonstrated that Ang-(1-7) might junction protein levels by tissue inhibitor of metalloproteinase 1 (TIMP1)-MMP9 pathway, because Ang-(1-7) enhanced TIMP1 expression, whose silencing obviously attenuated the inhibitor effect of Ang-(1-7) on MMP-9 levels and decreased Ang-(1-7)-triggered increase in claudin-5 and ZO-1. Together, this study demonstrated a protective role of Ang-(1-7) in IRI-induced blood-brain barrier damage by TIMP1-MMP9-regulated tight junction protein expression. Accordingly, Ang-(1-7) may become a promising therapeutic agent against IRI and its complications. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of negative pressures on epithelial tight junctions and migration in wound healing.

PubMed

Hsu, Chih-Chin; Tsai, Wen-Chung; Chen, Carl Pai-Chu; Lu, Yun-Mei; Wang, Jong-Shyan

2010-08-01

Negative-pressure wound therapy has recently gained popularity in chronic wound care. This study attempted to explore effects of different negative pressures on epithelial migration in the wound-healing process. The electric cell-substrate impedance sensing (ECIS) technique was used to create a 5 x 10(-4) cm(2) wound in the Madin-Darby canine kidney (MDCK) and human keratinocyte (HaCaT) cells. The wounded cells were cultured in a negative pressure incubator at ambient pressure (AP) and negative pressures of 75 mmHg (NP(75)), 125 mmHg (NP(125)), and 175 mmHg (NP(175)). The effective time (ET), complete wound healing time (T(max)), healing rate (R(heal)), cell diameter, and wound area over time at different pressures were evaluated. Traditional wound-healing assays were prepared for fluorescent staining of cells viability, cell junction proteins, including ZO-1 and E-cadherin, and actins. Amount of cell junction proteins at AP and NP(125) was also quantified. In MDCK cells, the ET (1.25 +/- 0.27 h), T(max) (1.76 +/- 0.32 h), and R(heal) (2.94 +/- 0.62 x 10(-4) cm(2)/h) at NP(125) were significantly (P < 0.01) different from those at three other pressure conditions. In HaCaT cells, the T(max) (7.34 +/- 0.29 h) and R(heal) (6.82 +/- 0.26 x 10(-5) cm(2)/h) at NP(125) were significantly (P < 0.01) different from those at NP(75). Prominent cell migration features were identified in cells at the specific negative pressure. Cell migration activities at different pressures can be documented with the real-time wound-healing measurement system. Negative pressure of 125 mmHg can help disassemble the cell junction to enhance epithelial migration and subsequently result in quick wound closure.

Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model.

PubMed

Ohtsuki, Sumio; Ikeda, Chiemi; Uchida, Yasuo; Sakamoto, Yumi; Miller, Florence; Glacial, Fabienne; Decleves, Xavier; Scherrmann, Jean-Michel; Couraud, Pierre-Olivier; Kubo, Yoshiyuki; Tachikawa, Masanori; Terasaki, Tetsuya

2013-01-07

Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.

Prefrontal white matter pathology in air pollution exposed Mexico City young urbanites and their potential impact on neurovascular unit dysfunction and the development of Alzheimer's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calderón-Garcidueñas, Lilian, E-mail: lilian.calderon-garciduenas@umontana.edu; Universidad del Valle de México, Mexico City 04850, México; Reynoso-Robles, Rafael

Millions of urban children are chronically exposed to high concentrations of air pollutants, i.e., fine particulate matter (PM{sub 2.5}) and ozone, associated with increased risk for Alzheimer's disease. Compared with children living with clear air those in Mexico City (MC) exhibit systemic, brain and intrathecal inflammation, low CSF Aβ{sub 42,} breakdown of the BBB, attention and short-term memory deficits, prefrontal white matter hyperintensities, damage to epithelial and endothelial barriers, tight junction and neural autoantibodies, and Alzheimer and Parkinson's hallmarks. The prefrontal white matter is a target of air pollution. We examined by light and electron microscopy the prefrontal white mattermore » of MC dogs (n: 15, age 3.17±0.74 years), children and teens (n: 34, age: 12.64±4.2 years) versus controls. Major findings in MC residents included leaking capillaries and small arterioles with extravascular lipids and erythrocytes, lipofuscin in pericytes, smooth muscle and endothelial cells (EC), thickening of cerebrovascular basement membranes with small deposits of amyloid, patchy absence of the perivascular glial sheet, enlarged Virchow–Robin spaces and nanosize particles (20–48 nm) in EC, basement membranes, axons and dendrites. Tight junctions, a key component of the neurovascular unit (NVU) were abnormal in MC versus control dogs (χ{sup 2}<0.0001), and white matter perivascular damage was significantly worse in MC dogs (p=0.002). The integrity of the NVU, an interactive network of vascular, glial and neuronal cells is compromised in MC young residents. Characterizing the early NVU damage and identifying biomarkers of neurovascular dysfunction may provide a fresh insight into Alzheimer pathogenesis and open opportunities for pediatric neuroprotection. - Highlights: • The prefrontal white matter is a target of air pollution. • Tight junctions, key neurovascular unit elements, are abnormal in young urbanites. • Identifying neurovascular dysfunction biomarkers is key for pediatric neuroprotection. • Early characterization of NVU damage may provide a fresh insight into AD pathogenesis.« less

Connexin channels and phospholipids: association and modulation

PubMed Central

Locke, Darren; Harris, Andrew L

2009-01-01

Background For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood. Results Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred. Conclusion This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents. PMID:19686581

Salivary gland acinar cells regenerate functional glandular structures in modified hydrogels

NASA Astrophysics Data System (ADS)

Pradhan, Swati

Xerostomia, a condition resulting from irradiation of the head and neck, affects over 40,000 cancer patients each year in the United States. Direct radiation damage of the acinar cells that secrete fluid and protein results in salivary gland hypofunction. Present medical management for xerostomia for patients treated for upper respiratory cancer is largely ineffective. Patients who have survived their terminal diagnosis are often left with a diminished quality of life and are unable to enjoy the simple pleasures of eating and drinking. This project aims to ultimately reduce human suffering by developing a functional implantable artificial salivary gland. The goal was to create an extracellular matrix (ECM) modified hyaluronic acid (HA) based hydrogel culture system that allows for the growth and differentiation of salivary acinar cells into functional acini-like structures capable of secreting large amounts of protein and fluid unidirectionally and to ultimately engineer a functional artificial salivary gland that can be implanted into an animal model. A tissue collection protocol was established and salivary gland tissue was obtained from patients undergoing head and neck surgery. The tissue specimen was assessed by histology and immunohistochemistry to establish the phenotype of normal salivary gland cells including the native basement membranes. Hematoxylin and eosin staining confirmed normal glandular tissue structures including intercalated ducts, striated ducts and acini. alpha-Amylase and periodic acid schiff stain, used for structures with a high proportion of carbohydrate macromolecules, preferentially stained acinar cells in the tissue. Intercalated and striated duct structures were identified using cytokeratins 19 and 7 staining. Myoepithelial cells positive for cytokeratin 14 were found wrapped around the serous and mucous acini. Tight junction components including ZO-1 and E-cadherin were present between both ductal and acinar cells. Ductal and acinar cells were identified in cultured cells from dispersed tissue. Biomarker studies with the salivary enzyme, alpha-amylase, and tight junction proteins, such as zonula occludens-1 and E-cadherin, confirmed the phenotype of these cells. Strong staining for laminin and perlecan/HSPG2 were noted in basement membranes and perlecan also was secreted and organized by cultured acinar populations, which formed lobular structures that mimicked intact glands when cultured on Matrigel(TM) or a bioactive peptide derived from domain IV of perlecan (PlnDIV). On either matrix, large acini-like lobular structures grew and formed connections between the lobes. alpha-Amylase secretion was confirmed by staining and activity assay. Biomarkers including tight junction protein E-cadherin and water channel protein, aquaporin 5 (AQP5) found in tissue, were expressed in cultured acinar cells. Cells cultured on Matrigel(TM) or PlnDIV peptide organized stress fibers and activated focal adhesion kinase (FAK). HA, a natural polysaccharide and a major component of the ECM, can be used to generate soft and pliable hydrogels. A culture system consisting of HA hydrogel and PlnDIV peptide was used to generate a 2.5D culture system. Acinar cells cultured on these hydrogels self-assembled into lobular structures and expressed tight junction components such as ZO-1. Acini-like structures were stained for the presence of alpha-amylase. Live/dead staining revealed the presence of apoptotic cells in the center of the acini-like structures, indicative of lumen formation. The functionality of these acini-like structures was studied by stimulating them with neurotransmitters to enhance their fluid and protein production. Acini-like structures treated with norepinephrine and isoproterenol showed increased granule formation as observed by phase contrast microscopy and alpha-amylase staining in the structures. Lobular structures on hydrogels were treated with acetylcholine to increase fluid production. The increase in intracellular calcium due to the activation of the M3 muscarinic receptor via binding to acetylcholine was measured. Although cells in 2D did not show any differences, cells on the 2.5D hydrogels showed an increase in intracellular calcium. The culture system consisting of PlnDIV peptide reported here will aid the development of an artificial salivary gland which will foster formation of functional salivary units capable of secreting salivary fluid and which can be implanted into patients to relieve xerostomia. Future experiments will involve implantation of these hydrogels in animal models to test their functionality in vivo.

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions.

PubMed

Perez White, Bethany E; Ventrella, Rosa; Kaplan, Nihal; Cable, Calvin J; Thomas, Paul M; Getsios, Spiro

2017-01-01

EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions. © 2017. Published by The Company of Biologists Ltd.

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions

PubMed Central

Perez White, Bethany E.; Ventrella, Rosa; Kaplan, Nihal; Cable, Calvin J.; Thomas, Paul M.

2017-01-01

ABSTRACT EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions. PMID:27815408

HGF Gene Modification in Mesenchymal Stem Cells Reduces Radiation-Induced Intestinal Injury by Modulating Immunity.

PubMed

Wang, Hua; Sun, Rui-Ting; Li, Yang; Yang, Yue-Feng; Xiao, Feng-Jun; Zhang, Yi-Kun; Wang, Shao-Xia; Sun, Hui-Yan; Zhang, Qun-Wei; Wu, Chu-Tse; Wang, Li-Sheng

2015-01-01

Effective therapeutic strategies to address intestinal complications after radiation exposure are currently lacking. Mesenchymal stem cells (MSCs), which display the ability to repair the injured intestine, have been considered as delivery vehicles for repair genes. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF)-gene-modified MSCs on radiation-induced intestinal injury (RIII). Female 6- to 8-week-old mice were radiated locally at the abdomen with a single 13-Gy dose of radiation and then treated with saline control, Ad-HGF or Ad-Null-modified MSCs therapy. The transient engraftment of human MSCs was detected via real-time PCR and immunostaining. The therapeutic effects of non- and HGF-modified MSCs were evaluated via FACS to determine the lymphocyte immunophenotypes; via ELISA to measure cytokine expression; via immunostaining to determine tight junction protein expression; via PCNA staining to examine intestinal epithelial cell proliferation; and via TUNEL staining to detect intestinal epithelial cell apoptosis. The histopathological recovery of the radiation-injured intestine was significantly enhanced following non- or HGF-modified MSCs treatment. Importantly, the radiation-induced immunophenotypic disorders of the mesenteric lymph nodes and Peyer's patches were attenuated in both MSCs-treated groups. Treatment with HGF-modified MSCs reduced the expression and secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ), increased the expression of the anti-inflammatory cytokine IL-10 and the tight junction protein ZO-1, and promoted the proliferation and reduced the apoptosis of intestinal epithelial cells. Treatment of RIII with HGF-gene-modified MSCs reduces local inflammation and promotes the recovery of small intestinal histopathology in a mouse model. These findings might provide an effective therapeutic strategy for RIII.

Transepithelial resistance and claudin expression in trout RTgill-W1 cell line: effects of osmoregulatory hormones.

PubMed

Trubitt, Rebecca T; Rabeneck, D Brett; Bujak, Joanna K; Bossus, Maryline C; Madsen, Steffen S; Tipsmark, Christian K

2015-04-01

In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1-2 days (20-80Ω⋅cm(2)), which was then maintained for more than 6 days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6h. Treatment with ethylenediaminetetraacetic acid induced a decrease in TER to <15% of pre-treatment level. Cortisol elevated TER after 12-72 h in a concentration-dependent manner, and this increase was antagonized by growth hormone (Gh). The effects of three osmoregulatory hormones, Gh, prolactin, and cortisol, on the mRNA expression of three tight junction proteins were examined: claudin-10e (Cldn-10e), Cldn-30, and zonula occludens-1 (Zo-1). The expression of cldn-10e was stimulated by all three hormones but with the strongest effect of Gh (50-fold). cldn-30 expression was stimulated especially by cortisol (20-fold) and also by Gh (4-fold). Finally, zo-1 was unresponsive to hormone treatment. Western blot analysis detected Cldn-10e and Cldn-30 immunoreactive proteins of expected molecular weight in samples from rainbow trout gills but not from RTgill-W1 cultures, possibly due to low expression levels. Collectively, these results show that the RTgill-W1 cell layers have tight junctions between cells, are sensitive to hormone treatments, and may provide a useful model for in vitro study of some in vivo gill phenomena. Copyright © 2014 Elsevier Inc. All rights reserved.

Tight junctions and IBS--the link between epithelial permeability, low-grade inflammation, and symptom generation?

PubMed

Piche, T

2014-03-01

In this issue of Neurogastroenterology and Motility, Dr Ewa Wilcz-Villega and colleagues report low expression of E-cadherin, a tight junction protein involved in the regulation of paracellular permeability, in the colonic mucosa of patients with the irritable bowel syndrome (IBS) with predominance of diarrhea (IBS-D) or alternating symptoms (IBS-A). These findings constitute an improvement in our knowledge of epithelial barrier disruption associated with IBS. There is mounting evidence to indicate that a compromised epithelial barrier is associated with low-grade immune activation and intestinal dysfunction in at least a proportion of IBS patients. During the last 10 years of research, much interest has focused on the increase in the number of different types of immune cells in the gut mucosa of IBS patients including: mast cells, T lymphocytes, and other local cells such as enteroendocrine cells. The inflammatory mediators released by these cells or other luminal factors could be at the origin of altered epithelial barrier functions and enteric nervous system signaling, which lead to gut hypersensitivity. A current conceptual framework states that clinical symptoms of IBS could be associated with structural and functional abnormalities of the mucosal barrier, highlighting the crucial importance of elucidating the contributory role of epithelial barrier defects in the pathogenesis of IBS. More importantly, disruption of the epithelial barrier could also participate in the generation of persistent abdominal pain and discomfort mimicking IBS in patients with inflammatory bowel diseases considered in remission. This mini review gives a brief summary of clinical and experimental evidence concerning the mechanisms underlying epithelial barrier defects in IBS. © 2014 John Wiley & Sons Ltd.

Formononetin Administration Ameliorates Dextran Sulfate Sodium-Induced Acute Colitis by Inhibiting NLRP3 Inflammasome Signaling Pathway

PubMed Central

Wu, Dacheng; Wu, Keyan; Zhu, Qingtian; Xiao, Weiming; Shan, Qing; Yan, Zhigang; Wu, Jian; Deng, Bin; Xue, Yan; Gong, Weijuan

2018-01-01

Formononetin is a kind of isoflavone compound and has been reported to possess anti-inflammatory properties. In this present study, we aimed to explore the protective effects of formononetin on dextran sulfate sodium- (DSS-) induced acute colitis. By intraperitoneal injection of formononetin in mice, the disease severity of colitis was attenuated in a dose-dependent manner, mainly manifesting as relieved clinical symptoms of colitis, mitigated colonic epithelial cell injury, and upregulations of colonic tight junction proteins levels (ZO-1, claudin-1, and occludin). Meanwhile, our study found that formononetin significantly prevented acute injury of colonic cells induced by TNF-α in vitro, specifically manifesting as the increased expressions of colonic tight junction proteins (ZO-1, claudin-1, and occludin). In addition, the result showed that formononetin could reduce the NLRP3 pathway protein levels (NLRP3, ASC, IL-1β) in vivo and vitro, and MCC950, the NLRP3 specific inhibitor, could alleviate the DSS-induced mice acute colitis. Furthermore, in the foundation of administrating MCC950 to inhibit activation of NLRP3 inflammasome, we failed to observe the protective effects of formononetin on acute colitis in mice. Collectively, our study for the first time confirmed the protective effects of formononetin on DSS-induced acute colitis via inhibiting the NLRP3 inflammasome pathway activation. PMID:29507526

Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations

PubMed Central

Jansen, J.; De Napoli, I. E; Fedecostante, M.; Schophuizen, C. M. S.; Chevtchik, N. V.; Wilmer, M. J.; van Asbeck, A. H.; Croes, H. J.; Pertijs, J. C.; Wetzels, J. F. M.; Hilbrands, L. B.; van den Heuvel, L. P.; Hoenderop, J. G.; Stamatialis, D.; Masereeuw, R.

2015-01-01

The bioartificial kidney (BAK) aims at improving dialysis by developing ‘living membranes’ for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as a fluorescent substrate. Initial ASP+ uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a ‘living membrane’ of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering. PMID:26567716

Lentiviral transduction of rat Sertoli cells as a means to modify gene expression

PubMed Central

Nicholls, Peter K.; Stanton, Peter G.; Rainczuk, Katarzyna E.; Qian, Hongwei; Gregorevic, Paul; Harrison, Craig A.

2012-01-01

Primary cell culture is an established and widely used technique to study Sertoli cell function in vitro. However, the relative difficulty of stably overexpressing or knocking down genes in Sertoli cell culture has limited progress in the field. In this technical report, we present a method to transduce 20 dpp rat Sertoli cell cultures with VSV-G pseudotyped lentiviral based vectors at a high rate (~80%), with stable reporter gene expression. Although high transgene expression is desirable, it was noted that at transduction rates > 60% inter-Sertoli cell tight junction integrity and, hence, Sertoli cell function, were transiently compromised. We envisage that this optimized procedure has the potential to stimulate Sertoli cell research, and motivate the use of Sertoli cells in various cell therapy applications. PMID:23248769

T-Lymphocytes Traffic into the Brain across the Blood-CSF Barrier: Evidence Using a Reconstituted Choroid Plexus Epithelium

PubMed Central

Strazielle, Nathalie; Creidy, Rita; Malcus, Christophe; Boucraut, José; Ghersi-Egea, Jean-François

2016-01-01

An emerging concept of normal brain immune surveillance proposes that recently and moderately activated central memory T lymphocytes enter the central nervous system (CNS) directly into the cerebrospinal fluid (CSF) via the choroid plexus. Within the CSF space, T cells inspect the CNS environment for cognate antigens. This gate of entry into the CNS could also prevail at the initial stage of neuroinflammatory processes. To actually demonstrate T cell migration across the choroidal epithelium forming the blood-CSF barrier, an in vitro model of the rat blood-CSF barrier was established in an “inverse” configuration that enables cell transmigration studies in the basolateral to apical, i.e. blood/stroma to CSF direction. Structural barrier features were evaluated by immunocytochemical analysis of tight junction proteins, functional barrier properties were assessed by measuring the monolayer permeability to sucrose and the active efflux transport of organic anions. The migratory behaviour of activated T cells across the choroidal epithelium was analysed in the presence and absence of chemokines. The migration pathway was examined by confocal microscopy. The inverse rat BCSFB model reproduces the continuous distribution of tight junction proteins at cell margins, the restricted paracellular permeability, and polarized active transport mechanisms, which all contribute to the barrier phenotype in vivo. Using this model, we present experimental evidence of T cell migration across the choroidal epithelium. Cell migration appears to occur via a paracellular route without disrupting the restrictive barrier properties of the epithelial interface. Apical chemokine addition strongly stimulates T cell migration across the choroidal epithelium. The present data provide evidence for the controlled migration of T cells across the blood-CSF barrier into brain. They further indicate that this recruitment route is sensitive to CSF-borne chemokines, extending the relevance of this migration pathway to neuroinflammatory and neuroinfectious disorders which are typified by elevated chemokine levels in CSF. PMID:26942913

T-Lymphocytes Traffic into the Brain across the Blood-CSF Barrier: Evidence Using a Reconstituted Choroid Plexus Epithelium.

PubMed

Strazielle, Nathalie; Creidy, Rita; Malcus, Christophe; Boucraut, José; Ghersi-Egea, Jean-François

2016-01-01

An emerging concept of normal brain immune surveillance proposes that recently and moderately activated central memory T lymphocytes enter the central nervous system (CNS) directly into the cerebrospinal fluid (CSF) via the choroid plexus. Within the CSF space, T cells inspect the CNS environment for cognate antigens. This gate of entry into the CNS could also prevail at the initial stage of neuroinflammatory processes. To actually demonstrate T cell migration across the choroidal epithelium forming the blood-CSF barrier, an in vitro model of the rat blood-CSF barrier was established in an "inverse" configuration that enables cell transmigration studies in the basolateral to apical, i.e. blood/stroma to CSF direction. Structural barrier features were evaluated by immunocytochemical analysis of tight junction proteins, functional barrier properties were assessed by measuring the monolayer permeability to sucrose and the active efflux transport of organic anions. The migratory behaviour of activated T cells across the choroidal epithelium was analysed in the presence and absence of chemokines. The migration pathway was examined by confocal microscopy. The inverse rat BCSFB model reproduces the continuous distribution of tight junction proteins at cell margins, the restricted paracellular permeability, and polarized active transport mechanisms, which all contribute to the barrier phenotype in vivo. Using this model, we present experimental evidence of T cell migration across the choroidal epithelium. Cell migration appears to occur via a paracellular route without disrupting the restrictive barrier properties of the epithelial interface. Apical chemokine addition strongly stimulates T cell migration across the choroidal epithelium. The present data provide evidence for the controlled migration of T cells across the blood-CSF barrier into brain. They further indicate that this recruitment route is sensitive to CSF-borne chemokines, extending the relevance of this migration pathway to neuroinflammatory and neuroinfectious disorders which are typified by elevated chemokine levels in CSF.

Cell polarity, cell adhesion, and spermatogenesis: role of cytoskeletons

PubMed Central

Li, Linxi; Gao, Ying; Chen, Haiqi; Jesus, Tito; Tang, Elizabeth; Li, Nan; Lian, Qingquan; Ge, Ren-shan; Cheng, C. Yan

2017-01-01

In the rat testis, studies have shown that cell polarity, in particular spermatid polarity, to support spermatogenesis is conferred by the coordinated efforts of the Par-, Crumbs-, and Scribble-based polarity complexes in the seminiferous epithelium. Furthermore, planar cell polarity (PCP) is conferred by PCP proteins such as Van Gogh-like 2 (Vangl2) in the testis. On the other hand, cell junctions at the Sertoli cell–spermatid (steps 8–19) interface are exclusively supported by adhesion protein complexes (for example, α6β1-integrin-laminin-α3,β3,γ3 and nectin-3-afadin) at the actin-rich apical ectoplasmic specialization (ES) since the apical ES is the only anchoring device in step 8–19 spermatids. For cell junctions at the Sertoli cell–cell interface, they are supported by adhesion complexes at the actin-based basal ES (for example, N-cadherin-β-catenin and nectin-2-afadin), tight junction (occludin-ZO-1 and claudin 11-ZO-1), and gap junction (connexin 43-plakophilin-2) and also intermediate filament-based desmosome (for example, desmoglein-2-desmocollin-2). In short, the testis-specific actin-rich anchoring device known as ES is crucial to support spermatid and Sertoli cell adhesion. Accumulating evidence has shown that the Par-, Crumbs-, and Scribble-based polarity complexes and the PCP Vangl2 are working in concert with actin- or microtubule-based cytoskeletons (or both) and these polarity (or PCP) protein complexes exert their effects through changes in the organization of the cytoskeletal elements across the seminiferous epithelium of adult rat testes. As such, there is an intimate relationship between cell polarity, cell adhesion, and cytoskeletal function in the testis. Herein, we critically evaluate these recent findings based on studies on different animal models. We also suggest some crucial future studies to be performed. PMID:28928959

The Hippo-YAP signaling pathway and contact inhibition of growth

PubMed Central

Gumbiner, Barry M.; Kim, Nam-Gyun

2014-01-01

ABSTRACT The Hippo-YAP pathway mediates the control of cell proliferation by contact inhibition as well as other attributes of the physical state of cells in tissues. Several mechanisms sense the spatial and physical organization of cells, and function through distinct upstream modules to stimulate Hippo-YAP signaling: adherens junction or cadherin–catenin complexes, epithelial polarity and tight junction complexes, the FAT-Dachsous morphogen pathway, as well as cell shape, actomyosin or mechanotransduction. Soluble extracellular factors also regulate Hippo pathway signaling, often inhibiting its activity. Indeed, the Hippo pathway mediates a reciprocal relationship between contact inhibition and mitogenic signaling. As a result, cells at the edges of a colony, a wound in a tissue or a tumor are more sensitive to ambient levels of growth factors and more likely to proliferate, migrate or differentiate through a YAP and/or TAZ-dependent process. Thus, the Hippo-YAP pathway senses and responds to the physical organization of cells in tissues and coordinates these physical cues with classic growth-factor-mediated signaling pathways. This Commentary is focused on the biological significance of Hippo-YAP signaling and how upstream regulatory modules of the pathway interact to produce biological outcomes. PMID:24532814

Claudin-16 and claudin-19 interact and form a cation-selective tight junction complex

PubMed Central

Hou, Jianghui; Renigunta, Aparna; Konrad, Martin; Gomes, Antonio S.; Schneeberger, Eveline E.; Paul, David L.; Waldegger, Siegfried; Goodenough, Daniel A.

2008-01-01

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an inherited disorder caused by mutations in the genes encoding the TJ proteins claudin-16 (CLDN16) and CLDN19; however, the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney are not understood. Here we showed that in pig kidney epithelial cells, CLDN19 functioned as a Cl– blocker, whereas CLDN16 functioned as a Na+ channel. Mutant forms of CLDN19 that are associated with FHHNC were unable to block Cl– permeation. Coexpression of CLDN16 and CLDN19 generated cation selectivity of the TJ in a synergistic manner, and CLDN16 and CLDN19 were observed to interact using several criteria. In addition, disruption of this interaction by introduction of FHHNC-causing mutant forms of either CLDN16 or CLDN19 abolished their synergistic effect. Our data show that CLDN16 interacts with CLDN19 and that their association confers a TJ with cation selectivity, suggesting a mechanism for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC. PMID:18188451

Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation.

PubMed

Sun, Xiaofei; Park, Craig B; Deng, Wenbo; Potter, S Steven; Dey, Sudhansu K

2016-04-01

Embryo implantation requires that the uterus differentiate into the receptive state. Failure to attain uterine receptivity will impede blastocyst attachment and result in a compromised pregnancy. The molecular mechanism by which the uterus transitions from the prereceptive to the receptive stage is complex, involving an intricate interplay of various molecules. We recently found that mice with uterine deletion ofMsxgenes (Msx1(d/d)/Msx2(d/d)) are infertile because of implantation failure associated with heightened apicobasal polarity of luminal epithelial cells during the receptive period. However, information on Msx's roles in regulating epithelial polarity remains limited. To gain further insight, we analyzed cell-type-specific gene expression by RNA sequencing of separated luminal epithelial and stromal cells by laser capture microdissection fromMsx1(d/d)/Msx2(d/d)and floxed mouse uteri on d 4 of pseudopregnancy. We found that claudin-1, a tight junction protein, and small proline-rich (Sprr2) protein, a major component of cornified envelopes in keratinized epidermis, were substantially up-regulated inMsx1(d/d)/Msx2(d/d)uterine epithelia. These factors also exhibited unique epithelial expression patterns at the implantation chamber (crypt) inMsx1(f/f)/Msx2(f/f)females; the patterns were lost inMsx1(d/d)/Msx2(d/d)epithelia on d 5, suggesting important roles during implantation. The results suggest thatMsxgenes play important roles during uterine receptivity including modulation of epithelial junctional activity.-Sun, X., Park, C. B., Deng, W., Potter, S. S., Dey, S. K. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation. © FASEB.

Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation

PubMed Central

Sun, Xiaofei; Park, Craig B.; Deng, Wenbo; Potter, S. Steven; Dey, Sudhansu K.

2016-01-01

Embryo implantation requires that the uterus differentiate into the receptive state. Failure to attain uterine receptivity will impede blastocyst attachment and result in a compromised pregnancy. The molecular mechanism by which the uterus transitions from the prereceptive to the receptive stage is complex, involving an intricate interplay of various molecules. We recently found that mice with uterine deletion of Msx genes (Msx1d/d/Msx2d/d) are infertile because of implantation failure associated with heightened apicobasal polarity of luminal epithelial cells during the receptive period. However, information on Msx’s roles in regulating epithelial polarity remains limited. To gain further insight, we analyzed cell-type–specific gene expression by RNA sequencing of separated luminal epithelial and stromal cells by laser capture microdissection from Msx1d/d/Msx2d/d and floxed mouse uteri on d 4 of pseudopregnancy. We found that claudin-1, a tight junction protein, and small proline-rich (Sprr2) protein, a major component of cornified envelopes in keratinized epidermis, were substantially up-regulated in Msx1d/d/Msx2d/d uterine epithelia. These factors also exhibited unique epithelial expression patterns at the implantation chamber (crypt) in Msx1f/f/Msx2f/f females; the patterns were lost in Msx1d/d/Msx2d/d epithelia on d 5, suggesting important roles during implantation. The results suggest that Msx genes play important roles during uterine receptivity including modulation of epithelial junctional activity.—Sun, X., Park, C. B., Deng, W., Potter, S. S., Dey, S. K. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation. PMID:26667042

Functional analysis of tight junction organization.

PubMed

DiBona, D R

1985-01-01

The functional basis of tight junction design has been examined from the point of view that this rate-limiting barrier to paracellular transport is a multicompartment system. Review of the osmotic sensitivity of these structures points to the need for this sort of analysis for meaningful correlation of structure and function under a range of conditions. A similar conclusion is drawn with respect to results from voltage-clamping protocols where reversal of spontaneous transmural potential difference elicits parallel changes in both structure and function in much the same way as does reversal of naturally occurring osmotic gradients. In each case, it becomes necessary to regard the junction as a functionally polarized structure to account for observations of its rectifying properties. Lastly, the details of experimentally-induced junction deformation are examined in light of current theories of its organization; arguments are presented in favor of the view that the primary components of intramembranous organization (as viewed with freeze-fracture techniques) are lipidic rather than proteinaceous.

TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium.

PubMed

Martínez-Rendón, Jacqueline; Sánchez-Guzmán, Erika; Rueda, Angélica; González, James; Gulias-Cañizo, Rosario; Aquino-Jarquín, Guillermo; Castro-Muñozledo, Federico; García-Villegas, Refugio

2017-07-01

TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca 2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

PubMed

Liu, Hao-Yu; Roos, Stefan; Jonsson, Hans; Ahl, David; Dicksved, Johan; Lindberg, Jan Erik; Lundh, Torbjörn

2015-04-01

Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell-cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Imaging the Vascular and Metabolic Impact on Claudin-7, a Tight Junction Protein, in Transgenic Human Breast Cancer Models

DTIC Science & Technology

2004-06-01

intratumoral administration of CPE (10 ug) to xenograft tumors of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction (p...prolongs the life of the mice by two fold. Slow release fonrmulations using polymer wafers, polymer beads and nanoparticles is ongoing to provide a... intramuscular injection of 1.5 mg/kg depo- 30 minutes. CLDN 3 and 4 protein was visualized using estradiol (Florida Infusion Co., Palm Harbor, FL

R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5

PubMed Central

Cavarelli, Mariangela; Foglieni, Chiara; Rescigno, Maria; Scarlatti, Gabriella

2013-01-01

The gastrointestinal tract is a principal route of entry and site of persistence of human immunodeficiency virus type 1 (HIV-1). The intestinal mucosa, being rich of cells that are the main target of the virus, represents a primary site of viral replication and CD4+ T-cell depletion. Here, we show both in vitro and ex vivo that HIV-1 of R5 but not X4 phenotype is capable of selectively triggering dendritic cells (DCs) to migrate within 30 min between intestinal epithelial cells to sample virions and transfer infection to target cells. The engagement of the chemokine receptor 5 on DCs and the viral envelope, regardless of the genetic subtype, drive DC migration. Viruses penetrating through transient opening of the tight junctions likely create a paracellular gradient to attract DCs. The formation of junctions with epithelial cells may initiate a haptotactic process of DCs and at the same time favour cell-to-cell viral transmission. Our findings indicate that HIV-1 translocation across the intestinal mucosa occurs through the selective engagement of DCs by R5 viruses, and may guide the design of new prevention strategies. PMID:23606583

R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

PubMed

Cavarelli, Mariangela; Foglieni, Chiara; Rescigno, Maria; Scarlatti, Gabriella

2013-05-01

The gastrointestinal tract is a principal route of entry and site of persistence of human immunodeficiency virus type 1 (HIV-1). The intestinal mucosa, being rich of cells that are the main target of the virus, represents a primary site of viral replication and CD4(+) T-cell depletion. Here, we show both in vitro and ex vivo that HIV-1 of R5 but not X4 phenotype is capable of selectively triggering dendritic cells (DCs) to migrate within 30 min between intestinal epithelial cells to sample virions and transfer infection to target cells. The engagement of the chemokine receptor 5 on DCs and the viral envelope, regardless of the genetic subtype, drive DC migration. Viruses penetrating through transient opening of the tight junctions likely create a paracellular gradient to attract DCs. The formation of junctions with epithelial cells may initiate a haptotactic process of DCs and at the same time favour cell-to-cell viral transmission. Our findings indicate that HIV-1 translocation across the intestinal mucosa occurs through the selective engagement of DCs by R5 viruses, and may guide the design of new prevention strategies. Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

PubMed

Musolino, Patricia L; Gong, Yi; Snyder, Juliet M T; Jimenez, Sandra; Lok, Josephine; Lo, Eng H; Moser, Ann B; Grabowski, Eric F; Frosch, Matthew P; Eichler, Florian S

2015-11-01

See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Oxaliplatin-induced blood brain barrier loosening: a new point of view on chemotherapy-induced neurotoxicity

PubMed Central

Valerio Branca, Jacopo Junio; Maresca, Mario; Morucci, Gabriele; Becatti, Matteo; Paternostro, Ferdinando; Gulisano, Massimo; Ghelardini, Carla; Salvemini, Daniela

2018-01-01

Oxaliplatin is a key drug in the treatment of advanced metastatic colorectal cancer. Despite its beneficial effects in tumor reduction, the most prevalent side-effect of oxaliplatin treatment is a chemotherapy-induced neuropathy that frequently forces to discontinue the therapy. Indeed, along with direct damage to peripheral nerves, the chemotherapy-related neurotoxicity involves also the central nervous system (CNS) as demonstrated by pain chronicity and cognitive impairment (also known as chemobrain), a newly described pharmacological side effect. The presence of the blood brain barrier (BBB) is instrumental in preventing the entry of the drug into the CNS; here we tested the hypothesis that oxaliplatin might enter the endothelial cells of the BBB vessels and trigger a signaling pathway that induce the disassembly of the tight junctions, the critical components of the BBB integrity. By using a rat brain endothelial cell line (RBE4) we investigated the signaling pathway that ensued the entry of oxaliplatin within the cell. We found that the administration of 10 μM oxaliplatin for 8 and 16 h induced alterations of the tight junction (TJs) proteins zonula occludens-1 (ZO-1) and of F-actin, thus highlighting BBB alteration. Furthermore, we reported that intracellular oxaliplatin rapidly induced increased levels of reactive oxygen species and endoplasmic reticulum stress, assessed by the evaluation of glucose-regulated protein GRP78 expression levels. These events were accompanied by activation of caspase-3 that led to extracellular ATP release. These findings suggested a possible novel mechanism of action for oxaliplatin toxicity that could explain, at least in part, the chemotherapy-related central effects.

Nrf2 activator ameliorates hemorrhagic transformation in focal cerebral ischemia under warfarin anticoagulation.

PubMed

Imai, Takahiko; Takagi, Toshinori; Kitashoji, Akira; Yamauchi, Keita; Shimazawa, Masamitsu; Hara, Hideaki

2016-05-01

Oxidative stress has been reported to be a main cause of neuronal cell death in ischemia reperfusion injury (IRI). Nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important factor involved in anti-oxidative responses. We previously reported that bardoxolone methyl (BARD), an Nrf2 activator, prevented damage induced by IRI. In this study, we investigated the effect of BARD on hemorrhagic transformation in the context of blood brain barrier (BBB) protection. Mice received pre-treatment with warfarin (4.0 mg/kg, p.o.). IRI was subsequently induced 18 h after the warfarin administration by transient middle cerebral artery occlusion (MCAO) for 6 h. BARD (0.06, 0.2, 0.6 or 2.0 mg/kg) or saline was injected intravenously immediately after reperfusion. The infarct volume, neurological score, intracranial hemorrhage volume, and BBB permeability were evaluated 24 h after MCAO. The survival rate and behavioral functional recovery were evaluated for 7 days following IRI. Furthermore, the effects of BARD on BBB components were investigated by western blotting and immunostaining analysis. BARD suppressed warfarin-mediated increases in the intracranial hemorrhage volume without affecting the infarct volume. BBB permeability was also suppressed by administration of BARD. Western blotting showed that BARD increased expression of BBB components such as endothelial cells, pericytes, and tight junction proteins. Furthermore, immunostaining showed that BARD induced localization of Nrf2 to endothelial cells and pericytes. BARD suppressed the exacerbation hemorrhage caused by warfarin pretreatment and ameliorated BBB disruption by protecting endothelial cells, pericytes, and tight junction protein expressions. These results indicate that Nrf2 activators may be an effective therapy against hemorrhagic transformation caused by anticoagulant drugs. Copyright © 2016 Elsevier Inc. All rights reserved.

Transplantation of in vitro cultured endothelial progenitor cells repairs the blood-brain barrier and improves cognitive function of APP/PS1 transgenic AD mice.

PubMed

Zhang, Shishuang; Zhi, Yongle; Li, Fei; Huang, Shan; Gao, Huabin; Han, Zhaoli; Ge, Xintong; Li, Dai; Chen, Fanglian; Kong, Xiaodong; Lei, Ping

2018-04-15

To date, the pathogenesis of Alzheimer's disease (AD) remains unclear. It is well-known that excessive deposition of Aβ in the brain is a crucial part of the pathogenesis of AD. In recent years, the AD neurovascular unit hypothesis has attracted much attention. Impairment of the blood-brain barrier (BBB) leads to abnormal amyloid-β (Aβ) transport, and chronic cerebral hypoperfusion causes Aβ deposition throughout the onset and progression of AD. Endothelial progenitor cells (EPCs) are the universal cells for repairing blood vessels. Our previous studies have shown that a reduced number of EPCs in the peripheral blood results in cerebral vascular repair disorder, cerebral hypoperfusion and neurodegeneration, which might be related to the cognitive dysfunction of AD patients. This study was designed to confirm whether EPCs transplantation could repair the blood-brain barrier, stimulate angiogenesis and reduce Aβ deposition in AD. The expression of ZO-1, Occludin and Claudin-5 was up-regulated in APP/PS1 transgenic mice after hippocampal transplantation of EPCs. Consistent with previous studies, EPC transplants also increased the microvessel density. We observed that Aβ senile plaque deposition was decreased and hippocampal cell apoptosis was reduced after EPCs transplantation. The Morris water maze test showed that spatial learning and memory functions were significantly improved in mice transplanted with EPCs. Consequently, EPCs could up-regulate the expression of tight junction proteins, repair BBB tight junction function, stimulate angiogenesis, promote Aβ clearance, and decrease neuronal loss, ultimately improve cognitive function. Taken together, these data demonstrate EPCs may play an important role in the therapeutic implications for vascular dysfunction in AD. Copyright © 2018 Elsevier B.V. All rights reserved.

Calcium-mediated oxidative stress: a common mechanism in tight junction disruption by different types of cellular stress.

PubMed

Gangwar, Ruchika; Meena, Avtar S; Shukla, Pradeep K; Nagaraja, Archana S; Dorniak, Piotr L; Pallikuth, Sandeep; Waters, Christopher M; Sood, Anil; Rao, RadhaKrishna

2017-02-20

The role of reactive oxygen species (ROS) in osmotic stress, dextran sulfate sodium (DSS) and cyclic stretch-induced tight junction (TJ) disruption was investigated in Caco-2 cell monolayers in vitro and restraint stress-induced barrier dysfunction in mouse colon in vivo Live cell imaging showed that osmotic stress, cyclic stretch and DSS triggered rapid production of ROS in Caco-2 cell monolayers, which was blocked by depletion of intracellular Ca 2+ by 1,2-bis-( o -aminophenoxy)ethane- N , N , N ', N '-tetraacetic acid. Knockdown of Ca V 1.3 or TRPV6 channels blocked osmotic stress and DSS-induced ROS production and attenuated TJ disruption and barrier dysfunction. N -Acetyl l-cysteine (NAC) and l- N G -Nitroarginine methyl ester (l-NAME) blocked stress-induced TJ disruption and barrier dysfunction. NAC and l-NAME also blocked stress-induced activation of c-Jun N -terminal kinase (JNK) and c-Src. ROS was colocalized with the mitochondrial marker in stressed cells. Cyclosporin A blocked osmotic stress and DSS-induced ROS production, barrier dysfunction, TJ disruption and JNK activation. Mitochondria-targeted Mito-TEMPO blocked osmotic stress and DSS-induced barrier dysfunction and TJ disruption. Chronic restraint stress in mice resulted in the elevation of intracellular Ca 2+ , activation of JNK and c-Src, and disruption of TJ in the colonic epithelium. Furthermore, corticosterone administration induced JNK and c-Src activation, TJ disruption and protein thiol oxidation in colonic mucosa. The present study demonstrates that oxidative stress is a common signal in the mechanism of TJ disruption in the intestinal epithelium by different types of cellular stress in vitro and bio behavioral stress in vivo . © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

ILDR1 null mice, a model of human deafness DFNB42, show structural aberrations of tricellular tight junctions and degeneration of auditory hair cells

PubMed Central

Morozko, Eva L.; Nishio, Ayako; Ingham, Neil J.; Chandra, Rashmi; Fitzgerald, Tracy; Martelletti, Elisa; Borck, Guntram; Wilson, Elizabeth; Riordan, Gavin P.; Wangemann, Philine; Forge, Andrew; Steel, Karen P.; Liddle, Rodger A.; Friedman, Thomas B.; Belyantseva, Inna A.

2015-01-01

In the mammalian inner ear, bicellular and tricellular tight junctions (tTJs) seal the paracellular space between epithelial cells. Tricellulin and immunoglobulin-like (Ig-like) domain containing receptor 1 (ILDR1, also referred to as angulin-2) localize to tTJs of the sensory and non-sensory epithelia in the organ of Corti and vestibular end organs. Recessive mutations of TRIC (DFNB49) encoding tricellulin and ILDR1 (DFNB42) cause human nonsyndromic deafness. However, the pathophysiology of DFNB42 deafness remains unknown. ILDR1 was recently reported to be a lipoprotein receptor mediating the secretion of the fat-stimulated cholecystokinin (CCK) hormone in the small intestine, while ILDR1 in EpH4 mouse mammary epithelial cells in vitro was shown to recruit tricellulin to tTJs. Here we show that two different mouse Ildr1 mutant alleles have early-onset severe deafness associated with a rapid degeneration of cochlear hair cells (HCs) but have a normal endocochlear potential. ILDR1 is not required for recruitment of tricellulin to tTJs in the cochlea in vivo; however, tricellulin becomes mislocalized in the inner ear sensory epithelia of ILDR1 null mice after the first postnatal week. As revealed by freeze-fracture electron microscopy, ILDR1 contributes to the ultrastructure of inner ear tTJs. Taken together, our data provide insight into the pathophysiology of human DFNB42 deafness and demonstrate that ILDR1 is crucial for normal hearing by maintaining the structural and functional integrity of tTJs, which are critical for the survival of auditory neurosensory HCs. PMID:25217574

Edge currents in frustrated Josephson junction ladders

NASA Astrophysics Data System (ADS)

Marques, A. M.; Santos, F. D. R.; Dias, R. G.

2016-09-01

We present a numerical study of quasi-1D frustrated Josephson junction ladders with diagonal couplings and open boundary conditions, in the large capacitance limit. We derive a correspondence between the energy of this Josephson junction ladder and the expectation value of the Hamiltonian of an analogous tight-binding model, and show how the overall superconducting state of the chain is equivalent to the minimum energy state of the tight-binding model in the subspace of one-particle states with uniform density. To satisfy the constraint of uniform density, the superconducting state of the ladder is written as a linear combination of the allowed k-states of the tight-binding model with open boundaries. Above a critical value of the parameter t (ratio between the intra-rung and inter-rung Josephson couplings) the ladder spontaneously develops currents at the edges, which spread to the bulk as t is increased until complete coverage is reached. Above a certain value of t, which varies with ladder size (t = 1 for an infinite-sized ladder), the edge currents are destroyed. The value t = 1 corresponds, in the tight-binding model, to the opening of a gap between two bands. We argue that the disappearance of the edge currents with this gap opening is not coincidental, and that this points to a topological origin for these edge current states.

Myosin light chain kinase knockout improves gut barrier function and confers a survival advantage in polymicrobial sepsis.

PubMed

Lorentz, C Adam; Liang, Zhe; Meng, Mei; Chen, Ching-Wen; Yoseph, Benyam P; Breed, Elise R; Mittal, Rohit; Klingensmith, Nathan J; Farris, Alton B; Burd, Eileen M; Koval, Michael; Ford, Mandy L; Coopersmith, Craig M

2017-06-07

Sepsis-induced intestinal hyperpermeability is mediated by disruption of the epithelial tight junction, which is closely associated with the peri-junctional actin-myosin ring. Myosin light chain kinase (MLCK) phosphorylates the myosin regulatory light chain, resulting in increased permeability. The purpose of this study was to determine whether genetic deletion of MLCK would alter gut barrier function and survival from sepsis. MLCK -/- and wild type (WT) mice were subjected to cecal ligation and puncture and assayed for both survival and mechanistic studies. Survival was significantly increased in MLCK -/- mice (95% vs. 24%, p<0.0001). Intestinal permeability increased in septic WT mice compared to unmanipulated mice. In contrast, permeability in septic MLCK -/- mice was similar to that seen in unmanipulated animals. Improved gut barrier function in MLCK -/- mice was associated with increases in the tight junction mediators ZO-1 and claudin 15 without alterations in claudin 1, 2, 3, 4, 5, 7, 8, 13, occludin or JAM-A. Other components of intestinal integrity (apoptosis, proliferation and villus length) were unaffected by MLCK deletion as were local peritoneal inflammation and distant lung injury. Systemic IL-10 was decreased greater than 10-fold in MLCK -/- mice; however, survival was similar between septic MLCK -/- mice given exogenous IL-10 or vehicle. These data demonstrate that deletion of MLCK improves survival following sepsis, associated with normalization of intestinal permeability and selected tight junction proteins.

[Effects of electromagnetic pulse on blood-brain barrier permeability and tight junction proteins in rats].

PubMed

Qiu, Lian-bo; Ding, Gui-rong; Zhang, Ya-mei; Zhou, Yan; Wang, Xiao-wu; Li, Kang-chu; Xu, Sheng-long; Tan, Juan; Zhou, Jia-xing; Guo, Guo-zhen

2009-09-01

To study the effect of electromagnetic pulse (EMP) on the permeability of blood-brain barrier, tight junction (TJ)-associated protein expression and localization in rats. 66 male SD rats, weighing (200 approximately 250) g, were sham or whole-body exposed to EMP at 200 kV/m for 200 pulses. The repetition rate was 1 Hz. The permeability of the blood-brain barrier in rats was assessed by albumin immunohistochemistry. The expression of typical tight junction protein ZO-1 and occludin in both cerebral cortex homogenate and cerebral cortex microvessel homogenate was analyzed by the Western blotting and the distribution of ZO-1 and occludin was examined by immunofluorescence microscopy. In the sham exposure rats, no brain capillaries showed albumin leakage, at 0.5 h after 200 kV/m EMP exposure for 200 pulses; a few brain capillaries with extravasated serum albumin was found, with the time extended, the number of brain capillaries with extravasated serum albumin increased, and reached the peak at 3 h, then began to recover at 6 h. In addition, no change in the distribution of the occludin was found after EMP exposure. Total occludin expression had no significant change compared with the control. However, the expression level of ZO-1 significantly decreased at 1 h and 3 h after EMP exposure in both cerebral cortex homogenate and cerebral cortex microvessel homogenate. Furthermore, immunofluorescence studies also showed alterations in ZO-1 protein localization in cerebral cortex microvessel. The EMP exposure (200 kV/m, 200 pulses) could increase blood-brain barrier permeability in rat, and this change is associated with specific alterations in tight junction protein ZO-1.

Air pollution and children: neural and tight junction antibodies and combustion metals, the role of barrier breakdown and brain immunity in neurodegeneration.

PubMed

Calderón-Garcidueñas, Lilian; Vojdani, Aristo; Blaurock-Busch, Eleonore; Busch, Yvette; Friedle, Albrecht; Franco-Lira, Maricela; Sarathi-Mukherjee, Partha; Martínez-Aguirre, Xavier; Park, Su-Bin; Torres-Jardón, Ricardo; D'Angiulli, Amedeo

2015-01-01

Millions of children are exposed to concentrations of air pollutants, including fine particulate matter (PM2.5), above safety standards. In the Mexico City Metropolitan Area (MCMA) megacity, children show an early brain imbalance in oxidative stress, inflammation, innate and adaptive immune response-associated genes, and blood-brain barrier breakdown. We investigated serum and cerebrospinal fluid (CSF) antibodies to neural and tight junction proteins and environmental pollutants in 139 children ages 11.91 ± 4.2 y with high versus low air pollution exposures. We also measured metals in serum and CSF. MCMA children showed significantly higher serum actin IgG, occludin/zonulin 1 IgA, IgG, myelin oligodendrocyte glycoprotein IgG and IgM (p < 0.01), myelin basic protein IgA and IgG, S-100 IgG and IgM, and cerebellar IgG (p < 0.001). Serum IgG antibodies to formaldehyde, benzene, and bisphenol A, and concentrations of Ni and Cd were significantly higher in exposed children (p < 0.001). CSF MBP antibodies and nickel concentrations were higher in MCMA children (p = 0.03). Air pollution exposure damages epithelial and endothelial barriers and is a robust trigger of tight junction and neural antibodies. Cryptic 'self' tight junction antigens can trigger an autoimmune response potentially contributing to the neuroinflammatory and Alzheimer and Parkinson's pathology hallmarks present in megacity children. The major factor determining the impact of neural antibodies is the integrity of the blood-brain barrier. Defining the air pollution linkage of the brain/immune system interactions and damage to physical and immunological barriers with short and long term neural detrimental effects to children's brains ought to be of pressing importance for public health.

Cadherin Switching and Activation of β-Catenin Signaling Underlie Proinvasive Actions of Calcitonin-Calcitonin Receptor Axis in Prostate Cancer*S⃞

PubMed Central

Shah, Girish V.; Muralidharan, Anbalagan; Gokulgandhi, Mitan; Soan, Kamal; Thomas, Shibu

2009-01-01

Calcitonin, a neuroendocrine peptide, and its receptor are localized in the basal epithelium of benign prostate but in the secretory epithelium of malignant prostates. The abundance of calcitonin and calcitonin receptor mRNA displays positive correlation with the Gleason grade of primary prostate cancers. Moreover, calcitonin increases tumorigenicity and invasiveness of multiple prostate cancer cell lines by cyclic AMP-dependent protein kinase-mediated actions. These actions include increased secretion of matrix metalloproteinases and urokinase-type plasminogen activator and an increase in prostate cancer cell invasion. Activation of calcitonin-calcitonin receptor autocrine loop in prostate cancer cell lines led to the loss of cell-cell adhesion, destabilization of tight and adherens junctions, and internalization of key integral membrane proteins. In addition, the activation of calcitonin-calcitonin receptor axis induced epithelial-mesenchymal transition of prostate cancer cells as characterized by cadherin switch and the expression of the mesenchymal marker, vimentin. The activated calcitonin receptor phosphorylated glycogen synthase kinase-3, a key regulator of cytosolic β-catenin degradation within the WNT signaling pathway. This resulted in the accumulation of intracellular β-catenin, its translocation in the nucleus, and transactivation of β-catenin-responsive genes. These results for the first time identify actions of calcitonin-calcitonin receptor axis on prostate cancer cells that lead to the destabilization of cell-cell junctions, epithelial-to-mesenchymal transition, and activation of WNT/β-catenin signaling. The results also suggest that cyclic AMP-dependent protein kinase plays a key role in calcitonin receptor-induced destabilization of cell-cell junctions and activation of WNT-β-catenin signaling. PMID:19001380

Gonadotropin suppression in men leads to a reduction in claudin-11 at the Sertoli cell tight junction.

PubMed

McCabe, M J; Tarulli, G A; Laven-Law, G; Matthiesson, K L; Meachem, S J; McLachlan, R I; Dinger, M E; Stanton, P G

2016-04-01

Are Sertoli cell tight junctions (TJs) disrupted in men undergoing hormonal contraception? Localization of the key Sertoli cell TJ protein, claudin-11, was markedly disrupted by 8 weeks of gonadotropin suppression, the degree of which was related to the extent of adluminal germ cell suppression. Sertoli cell TJs are vital components of the blood-testis barrier (BTB) that sequester developing adluminal meiotic germ cells and spermatids from the vascular compartment. Claudin-11 knockout mice are infertile; additionally claudin-11 is spatially disrupted in chronically gonadotropin-suppressed rats coincident with a loss of BTB function, and claudin-11 is disorganized in various human testicular disorders. These data support the Sertoli cell TJ as a potential site of hormonal contraceptive action. BTB proteins were assessed by immunohistochemistry (n = 16 samples) and mRNA (n = 18 samples) expression levels in available archived testis tissue from a previous study of 22 men who had undergone 8 weeks of gonadotropin suppression and for whom meiotic and post-meiotic germ cell numbers were available. The gonadotropin suppression regimens were (i) testosterone enanthate (TE) plus the GnRH antagonist, acyline (A); (ii) TE + the progestin, levonorgestrel, (LNG); (iii) TE + LNG + A or (iv) TE + LNG + the 5α-reductase inhibitor, dutasteride (D). A control group consisted of seven additional men, with three archived samples available for this study. Immunohistochemical localization of claudin-11 (TJ) and other junctional type markers [ZO-1 (cytoplasmic plaque), β-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and β-actin (cytoskeleton)] and quantitative PCR was conducted using matched frozen testis tissue. Claudin-11 formed a continuous staining pattern at the BTB in control men. Regardless of gonadotropin suppression treatment, claudin-11 localization was markedly disrupted and was broadly associated with the extent of meiotic/post-meiotic germ cell suppression; claudin-11 staining was (i) punctate (i.e. 'spotty' appearance) at the basal aspect of tubules when the average numbers of adluminal germ cells were <15% of control, (ii) presented as short fragments with cytoplasmic extensions when numbers were 15-25% of control or (iii) remained continuous when numbers were >40% of control. Changes in localization of connexin-43 and vinculin were also observed (smaller effects than for claudin-11) but ZO-1, β-catenin and β-actin did not differ, compared with control. Claudin-11 was the only Sertoli cell TJ protein investigated, but it is considered to be the most pivotal of constituent proteins given its known implication in infertility and BTB function. We were limited to testis samples which had been gonadotropin-suppressed for 8 weeks, shorter than the 74-day spermatogenic wave, which may account for the heterogeneity in claudin-11 and germ cell response observed among the men. Longer suppression (12-24 weeks) is known to suppress germ cells further and claudin-11 disruption may be more uniform, although we could not access such samples. These findings are important for our understanding of the sites of action of male hormonal contraception, because they suggest that BTB function could be ablated following long-term hormone suppression treatment. National Health and Medical Research Council (Australia) Program Grants 241000 and 494802; Research Fellowship 1022327 (to R.I.M.) and the Victorian Government's Operational Infrastructure Support Program. None of the authors have any conflicts to disclose. Not applicable. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The cell adhesion molecule nectin-1 is critical for normal enamel formation in mice

PubMed Central

Barron, Martin J.; Brookes, Steven J.; Draper, Clare E.; Garrod, David; Kirkham, Jennifer; Shore, Roger C.; Dixon, Michael J.

2008-01-01

Nectin-1 is a member of a sub-family of immunoglobulin-like adhesion molecules and a component of adherens junctions. In the current study, we have shown that mice lacking nectin-1 exhibit defective enamel formation in their incisor teeth. Although the incisors of nectin-1-null mice were hypomineralized, the protein composition of the enamel matrix was unaltered. While strong immunostaining for nectin-1 was observed at the interface between the maturation-stage ameloblasts and the underlying cells of the stratum intermedium (SI), its absence in nectin-1-null mice correlated with separation of the cell layers at this interface. Numerous, large desmosomes were present at this interface in wild-type mice; however, where adhesion persisted in the mutant mice, the desmosomes were smaller and less numerous. Nectins have been shown to regulate tight junction formation; however, this is the first report showing that they may also participate in the regulation of desmosome assembly. Importantly, our results show that integrity of the SI–ameloblast interface is essential for normal enamel mineralization. PMID:18703497

Regulation of blood-testis barrier dynamics by desmosome, gap junction, hemidesmosome and polarity proteins

PubMed Central

Wong, Elissa WP; Lie, Pearl PY; Li, Michelle WM; Mruk, Dolores D; Yan, Helen HN; Mok, Ka-Wai; Mannu, Jayakanthan; Mathur, Premendu P; Lui, Wing-yee; Lee, Will M; Bonanomi, Michele; Silvestrini, Bruno

2011-01-01

The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the “opening” and “closing” of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing “old” BTB above preleptotene spermatocytes in transit in “clones” at the BTB, but also contribute to the assembly of “new” BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the transit of spermatocytes, can be the “gateway” to making the BTB so vulnerable to toxicants and/or chemicals, causing male reproductive dysfunction. PMID:22319658

Exploring Instructive Physiological Signaling with the Bioelectric Tissue Simulation Engine

PubMed Central

Pietak, Alexis; Levin, Michael

2016-01-01

Bioelectric cell properties have been revealed as powerful targets for modulating stem cell function, regenerative response, developmental patterning, and tumor reprograming. Spatio-temporal distributions of endogenous resting potential, ion flows, and electric fields are influenced not only by the genome and external signals but also by their own intrinsic dynamics. Ion channels and electrical synapses (gap junctions) both determine, and are themselves gated by, cellular resting potential. Thus, the origin and progression of bioelectric patterns in multicellular tissues is complex, which hampers the rational control of voltage distributions for biomedical interventions. To improve understanding of these dynamics and facilitate the development of bioelectric pattern control strategies, we developed the BioElectric Tissue Simulation Engine (BETSE), a finite volume method multiphysics simulator, which predicts bioelectric patterns and their spatio-temporal dynamics by modeling ion channel and gap junction activity and tracking changes to the fundamental property of ion concentration. We validate performance of the simulator by matching experimentally obtained data on membrane permeability, ion concentration and resting potential to simulated values, and by demonstrating the expected outcomes for a range of well-known cases, such as predicting the correct transmembrane voltage changes for perturbation of single cell membrane states and environmental ion concentrations, in addition to the development of realistic transepithelial potentials and bioelectric wounding signals. In silico experiments reveal factors influencing transmembrane potential are significantly different in gap junction-networked cell clusters with tight junctions, and identify non-linear feedback mechanisms capable of generating strong, emergent, cluster-wide resting potential gradients. The BETSE platform will enable a deep understanding of local and long-range bioelectrical dynamics in tissues, and assist the development of specific interventions to achieve greater control of pattern during morphogenesis and remodeling. PMID:27458581

Overexpression of caveolin-1 attenuates brain edema by inhibiting tight junction degradation.

PubMed

Choi, Kang-Ho; Kim, Hyung-Seok; Park, Man-Seok; Lee, Eun-Bin; Lee, Jung-Kil; Kim, Joon-Tae; Kim, Ja-Hae; Lee, Min-Cheol; Lee, Hong-Joon; Cho, Ki-Hyun

2016-10-18

Cerebral edema from the disruption of the blood-brain barrier (BBB) after cerebral ischemia is a major cause of morbidity and mortality as well as a common event in patients with stroke. Caveolins (Cavs) are thought to regulate BBB functions. Here, we report for the first time that Cav-1 overexpression (OE) decreased brain edema from BBB disruption following ischemic insult. Edema volumes and Cav-1 expression levels were measured following photothrombosis and middle cerebral artery occlusion (MCAO). Endothelial cells that were transduced with a Cav-1 lentiviral expression vector were transplanted into rats. BBB permeability was quantified with Evans blue extravasation. Edema volume was determined from measures of the extravasation area, brain water content, and average fluorescence intensity after Cy5.5 injections. Tight junction (TJ) protein expression was measured with immunoblotting. Cav-1 expression levels and vasogenic brain edema correlated strongly after ischemic insult. Cav-1 expression and BBB disruption peaked 3 d after the MCAO. In addition, intravenous administration of endothelial cells expressing Cav-1 effectively increased the Cav-1 levels 3 d after the MCAO ischemic insult. Importantly, Cav-1 OE ameliorated the vasogenic edema by inhibiting the degradation of TJ protein expression in the acute phase of ischemic stroke. These results suggested that Cav-1 OE protected the integrity of the BBB mainly by preventing the degradation of TJ proteins in rats. These findings need to be confirmed in a clinical setting in human subjects.

Avian Influenza Virus Subtype H9N2 Affects Intestinal Microbiota, Barrier Structure Injury, and Inflammatory Intestinal Disease in the Chicken Ileum.

PubMed

Li, Hongxin; Liu, Xiaolin; Chen, Feiyang; Zuo, Kejing; Wu, Che; Yan, Yiming; Chen, Weiguo; Lin, Wencheng; Xie, Qingmei

2018-05-18

Avian influenza virus subtype H9N2 (H9N2 AIV) has caused significant losses to the poultry industry due to the high mortality associated with secondary infections attributable to E. coli . This study tries to address the underlying secondary mechanisms after H9N2 AIV infection. Initially, nine day-old specific pathogen-free chickens were assigned to control (uninfected) and H9N2-infected groups, respectively. Using Illumina sequencing, histological examination, and quantitative real-time PCR, it was found that H9N2 AIV caused intestinal microbiota disorder, injury, and inflammatory damage to the intestinal mucosa. Notably, the genera Escherichia , especially E. coli , significantly increased ( p < 0.01) at five days post-infection (dpi), while Lactobacillus , Enterococcus , and other probiotic organisms were significantly reduced ( p < 0.01). Simultaneously, the mRNA expression of tight junction proteins ( ZO-1 , claudin 3, and occludin), TFF2, and Muc2 were significantly reduced ( p < 0.01), indicating the destruction of the intestinal epithelial cell tight junctions and the damage of mucin layer construction. Moreover, the mRNA expression of proinflammatory cytokines IFN-γ, IL-22, IFN-α, and IL-17A in intestinal epithelial cells were significantly upregulated, resulting in the inflammatory response and intestinal injury. Our findings may provide a theoretical basis for observed gastroenteritis-like symptoms such as diarrhea and secondary E. coli infection following H9N2 AIV infection.

The effects of Lactobacillus plantarum on small intestinal barrier function and mucosal gene transcription; a randomized double-blind placebo controlled trial

PubMed Central

Mujagic, Zlatan; de Vos, Paul; Boekschoten, Mark V.; Govers, Coen; Pieters, Harm-Jan H. M.; de Wit, Nicole J. W.; Bron, Peter A.; Masclee, Ad A. M.; Troost, Freddy J.

2017-01-01

The aim of this study was to investigate the effects of three Lactobacillus plantarum strains on in-vivo small intestinal barrier function and gut mucosal gene transcription in human subjects. The strains were selected for their differential effects on TLR signalling and tight junction protein rearrangement, which may lead to beneficial effects in a stressed human gut mucosa. Ten healthy volunteers participated in four different intervention periods: 7-day oral intake of either L. plantarum WCFS1, CIP104448, TIFN101 or placebo, proceeded by a 4 weeks wash-out period. Lactulose-rhamnose ratio (an indicator of small intestinal permeability) increased after intake of indomethacin, which was given as an artificial stressor of the gut mucosal barrier (mean ratio 0.06 ± 0.04 to 0.10 ± 0.06, p = 0.001), but was not significantly affected by the bacterial interventions. However, analysis in small intestinal biopsies, obtained by gastroduodenoscopy, demonstrated that particularly L. plantarum TIFN101 modulated gene transcription pathways related to cell-cell adhesion with high turnover of genes involved in tight- and adhesion junction protein synthesis and degradation (e.g. actinin alpha-4, metalloproteinase-2). These effects were less pronounced for L. plantarum WCFS1 and CIP104448. In conclusion, L. plantarum TIFN101 induced the most pronounced probiotic properties with specific gene transcriptional effects on repair processes in the compromised intestine of healthy subjects. PMID:28045137

Morphological analysis of the hagfish heart. I. The ventricle, the arterial connection and the ventral aorta.

PubMed

Icardo, José M; Colvee, Elvira; Schorno, Sarah; Lauriano, Eugenia R; Fudge, Douglas S; Glover, Chris N; Zaccone, Giacomo

2016-03-01

We have studied the heart in three species of hagfish: Myxine glutinosa, Eptatretus stoutii, and Eptatretus cirrhatus and report about the morphology of the ventricle, the arterial connection and the ventral aorta. On the whole, the hagfish heart lacks outflow tract components, the ventricle and atrium adopt a dorso-caudal rather than a ventro-dorsal relationship, and the sinus venosus opens into the left side of the atrium. This may indicate a "defective" cardiac looping during embryogenesis. The ventral aorta is elongated in M. glutinosa and E. stoutii but sac-like in E. cirrhatus. The ventricles are entirely trabeculated. The myocytes show a low myofibrillar content and junctional complexes formed by fascia adherens and desmosomes. Gap junctions could not be demonstrated. Myocardial cells in M. glutinosa contain numerous lipid droplets. These droplets are less numerous in E. stoutii and practically absent in E. cirrhatus, suggesting different metabolic requirements. Other cell types present in the ventricle are chromaffin cells and granular leukocytes that contain rod-shaped granules. The ventricle-aorta connection is guarded by a bicuspid valve with left and right, pocket-like leaflets. The leaflets extend from the cranial end of the ventricle into the aorta but the junction is asymmetrical. This junction contains a ganglion-like structure in E. cirrhatus. The ventral aorta shows endothelial, media, and adventitial layers. The media contains smooth muscle cells surrounded by dense bands formed by tightly-packed extracellular filaments. In addition, a short number of elastic fibers are observed in M. glutinosa and E. stoutii. Cellular and extracellular elements are more loosely organized in the aorta of E. cirrhatus. The collagenous adventitia contains ganglion-like cells in the three species. In the absence of nerves, chromaffin and ganglion-like cells may control the activity of the myocardium and that of the aortic smooth muscle cells, respectively. © 2015 Wiley Periodicals, Inc.

Structural basis for human PECAM-1-mediated trans-homophilic cell adhesion

DOE PAGES

Hu, Menglong; Zhang, Hongmin; Liu, Qun; ...

2016-12-13

Cell adhesion involved in signal transduction, tissue integrity and pathogen infection is mainly mediated by cell adhesion molecules (CAM). One CAM member, platelet–endothelial-cell adhesion molecule-1 (PECAM-1), plays an important role in tight junction among endothelia cells, leukocyte trafficking, and immune response through its homophilic and heterophilic binding patterns. Both kinds of interactions, which lead to endogenous and exogenous signal transmission, are derived from extracellular immunoglobulin-like (IgL) domains and cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of PECAM-1. To date, the mechanism of trans-homophilic interaction of PECAM-1 remains unclear. Here, we present the crystal structure of PECAM-1 IgL1-2 trans-homo dimer. Both IgLmore » 1 and 2 adopt the classical Ig domain conformation comprised of two layers of β-sheets possessing antiparallel β-strands with each being anchored by a pair of cysteines forming a disulfide bond. The dimer interface includes hydrophobic and hydrophilic interactions. The Small-Angle X-ray Scattering (SAXS) envelope of PECAM-1 IgL1-6 supported such a dimer formation in solution. As a result, cell adhesion assays on wildtype and mutant PECAM-1 further characterized the structural determinants in cell junction and communication.« less

Blood-urine barrier formation in mouse urinary bladder development.

PubMed

Jezernik, K; Pipan, N

1993-04-01

Formation of the blood-urine permeability barrier in differentiating mouse transitional urothelium was studied. It was established that the development of superficial cell barrier is a two-phase process: beginning with formation of the tight junctions, followed by formation of fusiform vesicles and asymmetric apical plasma membranes. Fusiform vesicles differentiate during days 15 and 17 of gestation and fuse with the apical plasmalemma. Thus a thick membrane is formed before the excretion of hypertonic urine into the embryonic bladder. Through some degenerative superficial cells slough between fetal day 17 and the day of birth, the bladder epithelium in mice does not lack an effective permeability barrier.

Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells.

PubMed

Wilson, Hannah K; Canfield, Scott G; Hjortness, Michael K; Palecek, Sean P; Shusta, Eric V

2015-05-21

Brain microvascular-like endothelial cells (BMECs) derived from human pluripotent stem cells (hPSCs) have significant promise as tools for drug screening and studying the structure and function of the BBB in health and disease. The density of hPSCs is a key factor in regulating cell fate and yield during differentiation. Prior reports of hPSC differentiation to BMECs have seeded hPSCs in aggregates, leading to non-uniform cell densities that may result in differentiation heterogeneity. Here we report a singularized-cell seeding approach compatible with hPSC-derived BMEC differentiation protocols and evaluate the effects of initial hPSC seeding density on the subsequent differentiation, yield, and blood-brain barrier (BBB) phenotype. A range of densities of hPSCs was seeded and differentiated, with the resultant endothelial cell yield quantified via VE-cadherin flow cytometry. Barrier phenotype of purified hPSC-derived BMECs was measured via transendothelial electrical resistance (TEER), and purification protocols were subsequently optimized to maximize TEER. Expression of characteristic vascular markers, tight junction proteins, and transporters was confirmed by immunocytochemistry and quantified by flow cytometry. P-glycoprotein and MRP-family transporter activity was assessed by intracellular accumulation assay. The initial hPSC seeding density of approximately 30,000 cells/cm(2) served to maximize the yield of VE-cadherin+ BMECs per input hPSC. BMECs displayed the highest TEER (>2,000 Ω × cm(2)) within this same range of initial seeding densities, although optimization of the BMEC purification method could minimize the seeding density dependence for some lines. Localization and expression levels of tight junction proteins as well as efflux transporter activity were largely independent of hPSC seeding density. Finally, the utility of the singularized-cell seeding approach was demonstrated by scaling the differentiation and purification process down from 6-well to 96-well culture without impacting BBB phenotype. Given the yield and barrier dependence on initial seeding density, the singularized-cell seeding approach reported here should enhance the reproducibility and scalability of hPSC-derived BBB models, particularly for the application to new pluripotent stem cell lines.

Morphological studies of the developing human esophageal epithelium.

PubMed

Ménard, D

1995-06-15

This article focusses on the structural development of human esophageal ciliated epithelium. A combination of transmission electron microscopic (TEM), scanning electron microscopic (SEM), radioautographic, and light microscopic (LM) analyses were carried out using intact fetal tissues between 8 and 20 weeks of gestation as well as cultured esophageal explants. Up to the age of 10 weeks, the stratified esophageal epithelium consisted of two longitudinal primary folds. The surface cells were undifferentiated and contained large glycogen aggregates. Between 11 and 16 weeks, the primary folds (now up to four) had developed secondary folds. The thickness of the epithelium drastically increased (123%) in concomittance with a differentiation of surface columnar ciliated cells. These highly specialized surface cells exhibited junctional complexes and well-developed organelles with numerous microvilli interspersed among the cilia. Transverse sections revealed the internal structure of the cilia with a consistent pattern of nine doublet microtubules surrounding a central pair of single microtubules. Freeze-fracture studies illustrated the presence of a ciliary necklace composed of 6 ring-like rows of intramembranous particles. They also revealed the structure of ciliary cell tight junctions consisting of up to nine anastomosing strands (P-face) or complementary grooves (E-face). Ultrastructural studies (LM, TEM, SEM) of the esophageal squamous epithelium obtained after 15 days of culture showed that the newly formed epithelium was similar to adult human epithelium. Finally LM and SEM observations established that the esophagogastric junction was not yet well delineated, consisting of a transitional area composed of a mixture of esophageal ciliated cells and gastric columnar mucous cells.

Porcine circovirus 2 (PCV2) increases the expression of endothelial adhesion/junction molecules.

PubMed

Marks, Fernanda S; Almeida, Laura L; Driemeier, David; Canal, Cláudio; Barcellos, David E S N; Guimarães, Jorge A; Reck, José

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus disease, a complex multisystem syndrome in domestic pigs. Despite the significant economic losses caused by porcine circovirus disease, the mechanisms of pathogenesis underlying the clinical findings remain largely unclear. As various reports have highlighted the potential key role of vascular lesions in the pathogenesis of porcine circovirus disease, the aim of this work was to investigate effects of PCV2 infection on vascular endothelial cells, focusing on cell viability and expression of adhesion/junction molecules. PCV2 infection reduced endothelial cell viability, while viral infection did not affected the viability of several other classical cell lines. Also, PCV2 infection in endothelial cells displayed a dual/biphasic effect: initially, infection increased ICAM-1 expression, which can favor leukocyte recruitment and emigration to tissues and possibly inducing characteristic porcine circovirus disease inflammatory lesions; then, secondarily, infection caused an increase in zonula occludens 1 tight junction protein (ZO-1) expression, which in turn can result in difficulties for cell traffic across the endothelium and a potential impairment the immune response in peripheral tissues. These virus-induced endothelial changes could directly impact the inflammatory process of porcine circovirus disease and associated vascular/immune system disturbances. Data suggest that, among the wide range of effects induced by PCV2 on the host, endothelial modulation can be a pivotal process which can help to explain PCV2 pathogenesis in some porcine circovirus disease presentations. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

RhoGTPase signalling at epithelial tight junctions: Bridging the GAP between polarity and cancer.

PubMed

Zihni, Ceniz; Terry, Stephen James

2015-07-01

The establishment and maintenance of epithelial polarity must be correctly controlled for normal development and homeostasis. Tight junctions (TJ) in vertebrates define apical and basolateral membrane domains in polarized epithelia via bi-directional, complex signalling pathways between TJ themselves and the cytoskeleton they are associated with. RhoGTPases are central to these processes and evidence suggests that their regulation is coordinated by interactions between GEFs and GAPs with junctional, cytoplasmic adapter proteins. In this InFocus review we determine that the expression, localization or stability of a variety of these adaptor proteins is altered in various cancers, potentially representing an important mechanistic link between loss of polarity and cancer. We focus here, on two well characterized RhoGTPases Cdc42 and RhoA who's GEFs and GAPs are predominantly localized to TJ via cytoplasmic adaptor proteins. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells.

PubMed

Radwanska, Agata; Grall, Dominique; Schaub, Sébastien; Divonne, Stéphanie Beghelli-de la Forest; Ciais, Delphine; Rekima, Samah; Rupp, Tristan; Sudaka, Anne; Orend, Gertraud; Van Obberghen-Schilling, Ellen

2017-10-06

Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells.

Glutamine and arginine improve permeability and tight junction protein expression in methotrexate-treated Caco-2 cells.

PubMed

Beutheu, Stéphanie; Ghouzali, Ibtissem; Galas, Ludovic; Déchelotte, Pierre; Coëffier, Moïse

2013-10-01

Chemotherapy induces an increase of intestinal permeability that is partially related to an alteration of tight junction proteins, occludin and zonula occludens-1 (ZO-1). Protective effects of glutamine on intestinal barrier function have been previously shown but the effects of other amino acids remained poorly documented. Thus, we aimed to evaluate the effects of nine amino acids on intestinal permeability during methotrexate (MTX) treatment in Caco-2 cells. Caco-2 cells were incubated in culture medium supplemented with glutamine, arginine, glutamate, leucine, taurine, citrulline, glycine, histidine or cysteine during 24 h and then treated with MTX (100 ng/ml). The dose of each amino acid was 16.6 fold the physiological plasma concentrations. Barrier function was assessed by transepithelial electrical resistance (TEER), FITC-dextran paracellular flux, occludin and ZO-1 expression and localization. Signaling pathways were also studied. Only glutamine, glutamate, arginine and leucine reversed the decrease of TEER observed after MTX treatment (P < 0.05). Interestingly, the addition of 6-diazo-5-oxo-1-norleucine, an inhibitor of glutaminase, blunted the effect of glutamine on MTX-treated cells (P < 0.05). Glutamine and arginine combination restored TEER and FITC-dextran flux to a similar extent than glutamine alone. In addition, pretreatment of Caco-2 cells with glutamine and arginine, alone or combined, differently limited the decrease of ZO-1 and occludin expression (P < 0.05) and the alteration of their cellular distribution, through c-Jun N-terminal kinase (JNK), Extracellular signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) pathways. Glutamine prevented MTX-induced barrier disruption in Caco-2 cells. Arginine also had protective effects but in a lesser extent. The effect of glutamine and arginine should be evaluated in vivo. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

[Cellphone electromagnetic radiation damages the testicular ultrastructure of male rats].

PubMed

Gao, Xiao-Hui; Hu, Hui-Rong; Ma, Xue-Lian; Chen, Jie; Zhang, Guo-Hong

2016-06-01

To investigate the influence of cellphone electromagnetic radiation (CER) on the testicular ultrastructure and the apoptosis of spermatogenic cells in male rats.atability, feasibility, applicability, and controllability in the construction of experimental animal models, we compared the major anatomic features of the penis of 20 adult beagle dogs with those of 10 adult men. Using microsurgical techniques, we performed cross-transplantation of the penis in the 20 (10 pairs) beagle dogs and observed the survival rate of the transplanted penises by FK506+MMF+MP immune induction. We compared the relevant indexes with those of the 10 cases of microsurgical replantation of the amputated penis. Thirty adult male SD rats were equally randomized into a 2 h CER, a 4 h CER, and a normal control group, the former two groups exposed to 30 days of 900 MHz CER for 2 and 4 hours a day, respectively, while the latter left untreated. Then the changes in the ultrastructure of the testis tissue were observed under the transmission electron microscope and the apoptosis of the spermatogenic cells was determined by TUNEL. Compared with the normal controls, the rats of the 2 h CER group showed swollen basement membrane of seminiferous tubules, separated tight junction of Sertoli cells, increased cell intervals, apparent vacuoles and medullization in some mitochondria, and increased apoptosis of spermatogenic cells, mainly the apoptosis of primary spermatocytes (P<0.05 ). In comparison with the 2 h CER group, the animals of the 4 h CER group exhibited swollen basement membrane of seminiferous tubules, more separated tight junction of Sertoli cells, wider cell intervals, incomplete membrane of spermatogonial cells, fragments of cytoplasm, nuclear pyknosis and notch, slight dilation of perinuclear space, abnormalities of intracellular mitochondria with vacuoles, fuzzy structure, and fusion or disappearance of some cristae, and increased damage of mitochondria and apoptosis of spermatogenic cells, including the apoptosis of spermatogonial cells, primary spermatocytes, and secondary spermatocytes (P<0.05 ). CER can damage the testicular ultrastructure and increase the apoptosis of spermatogenic cells of the male rat in a time-dependent manner, and the apoptosis of spermatogenic cells may be associated with the damage to mitochondria.

Ankyrin-G Inhibits Endocytosis of Cadherin Dimers.

PubMed

Cadwell, Chantel M; Jenkins, Paul M; Bennett, Vann; Kowalczyk, Andrew P

2016-01-08

Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Integrin β4 Signaling Promotes Mammary Tumor Cell Adhesion to Brain Microvascular Endothelium by Inducing ErbB2-mediated Secretion of VEGF

PubMed Central

Fan, Jie; Cai, Bin; Zeng, Min; Hao, Yanyan

2015-01-01

Prior studies have indicated that the β4 integrin promotes mammary tumor invasion and metastasis by combining with ErbB2 and amplifying its signaling capacity. However, the effector pathways and cellular functions by which the β4 integrin exerts these effects are incompletely understood. To examine if β4 signaling plays a role during mammary tumor cell adhesion to microvascular endothelium, we have examined ErbB2-transformed mammary tumor cells expressing either a wild-type (WT) or a signaling-defective form of β4 (1355T). We report that WT cells adhere to brain microvascular endothelium in vitro to a significantly larger extent as compared to 1355T cells. Interestingly, integrin β4 signaling does not exert a direct effect on adhesion to the endothelium or the underlying basement membrane. Rather, it enhances ErbB2-dependent expression of VEGF by tumor cells. VEGF in turn disrupts the tight and adherens junctions of endothelial monolayers, enabling the exposure of underlying basement membrane and increasing the adhesion of tumor cells to the intercellular junctions of endothelium. Inhibition of ErbB2 on tumor cells or the VEGFR-2 on endothelial cells suppresses mammary tumor cell adhesion to microvascular endothelium. Our results indicate that β4 signaling regulates VEGF expression by the mammary tumor cells thereby enhancing their adhesion to microvascular endothelium. PMID:21556948

Delayed astrocytic contact with cerebral blood vessels in FGF-2 deficient mice does not compromise permeability properties at the developing blood-brain barrier.

PubMed

Saunders, Norman R; Dziegielewska, Katarzyna M; Unsicker, Klaus; Ek, C Joakim

2016-11-01

The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood-brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor-2 (FGF-2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF-2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild-type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight-junction proteins and we observed no difference in the ultrastructure of the tight-junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight-junctions or for basic permeability properties and function of the blood-brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201-1212, 2016. © 2016 Wiley Periodicals, Inc.

Connexin 26 facilitates gastrointestinal bacterial infection in vitro.

PubMed

Simpson, Charlotte; Kelsell, David P; Marchès, Olivier

2013-01-01

Escherichia coli, including enteropathogenic E. coli (EPEC), represents the most common cause of diarrhoea worldwide and is therefore a serious public health burden. Treatment for gastrointestinal pathogens is hindered by the emergence of multiple antibiotic resistance, leading to the requirement for the development of new therapies. A variety of mechanisms act in combination to mediate gastrointestinal-bacterial-associated diarrhoea development. For example, EPEC infection of enterocytes induces attaching and effacing lesion formation and the disruption of tight junctions. An alternative enteric pathogen, Shigella flexneri, manipulates the expression of Connexin 26 (Cx26), a gap junction protein. S. flexneri can open Cx26 hemichannels allowing the release of ATP, whereas HeLa cells expressing mutant gap-junction-associated Cx26 are less susceptible to cellular invasion by S. flexneri than cells expressing wild-type (WT) Cx26. We have investigated further the link between Cx26 expression and gastrointestinal infection by using EPEC and S. flexneri as in vitro models of infection. In this study, a significant reduction in EPEC adherence was observed in cells expressing mutant Cx26 compared with WT Cx26. Furthermore, a significant reduction in both cellular invasion by S. flexneri and adherence by EPEC was demonstrated in human intestinal cell lines following treatment with Cx26 short interfering RNA. These in vitro results suggest that the loss of functional Cx26 expression provides improved protection against gastrointestinal bacterial pathogens. Thus, Cx26 represents a potential therapeutic target for gastrointestinal bacterial infection.

Gene expression of human lung cancer cell line CL1-5 in response to a direct current electric field.

PubMed

Huang, Ching-Wen; Chen, Huai-Yi; Yen, Meng-Hua; Chen, Jeremy J W; Young, Tai-Horng; Cheng, Ji-Yen

2011-01-01

Electrotaxis is the movement of adherent living cells in response to a direct current (dc) electric field (EF) of physiological strength. Highly metastatic human lung cancer cells, CL1-5, exhibit directional migration and orientation under dcEFs. To understand the transcriptional response of CL1-5 cells to a dcEF, microarray analysis was performed in this study. A large electric-field chip (LEFC) was designed, fabricated, and used in this study. CL1-5 cells were treated with the EF strength of 0 mV/mm (the control group) and 300 mV/mm (the EF-treated group) for two hours. Signaling pathways involving the genes that expressed differently between the two groups were revealed. It was shown that the EF-regulated genes highly correlated to adherens junction, telomerase RNA component gene regulation, and tight junction. Some up-regulated genes such as ACVR1B and CTTN, and some down-regulated genes such as PTEN, are known to be positively and negatively correlated to cell migration, respectively. The protein-protein interactions of adherens junction-associated EF-regulated genes suggested that platelet-derived growth factor (PDGF) receptors and ephrin receptors may participate in sensing extracellular electrical stimuli. We further observed a high percentage of significantly regulated genes which encode cell membrane proteins, suggesting that dcEF may directly influence the activity of cell membrane proteins in signal transduction. In this study, some of the EF-regulated genes have been reported to be essential whereas others are novel for electrotaxis. Our result confirms that the regulation of gene expression is involved in the mechanism of electrotactic response.

NHERF1 and CFTR restore tight junction organisation and function in cystic fibrosis airway epithelial cells: role of ezrin and the RhoA/ROCK pathway.

PubMed

Castellani, Stefano; Guerra, Lorenzo; Favia, Maria; Di Gioia, Sante; Casavola, Valeria; Conese, Massimo

2012-11-01

Tight junctions (TJs) restrict the transit of ions and molecules through the paracellular route and act as a barrier to regulate access of inflammatory cells into the airway lumen. The pathophysiology of cystic fibrosis (CF) lung disease is characterised by abnormal ion and fluid transport across the epithelium and polymorphonuclear (PMN) leukocyte-dominated inflammatory response. Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) is a protein involved in PKA-dependent activation of CFTR by interacting with CFTR via its PDZ domains and with ezrin via its C-terminal domain. We have previously found that the NHERF1-overexpression dependent rescue CFTR-dependent chloride secretion is due to the re-organisation of the actin cytoskeleton network induced by the formation of the multiprotein complex NHERF1-RhoA-ezrin-actin. In this context, we here studied whether NHERF1 and CFTR are involved in the organisation and function of TJs. F508del CFBE41o⁻ monolayers presented nuclear localisation of zonula occludens (ZO-1) and occludin as well as disorganisation of claudin 1 and junction-associated adhesion molecule 1 as compared with wild-type 16HBE14o⁻ monolayers, paralleled by increased permeability to dextrans and PMN transmigration. Overexpression of either NHERF1 or CFTR in CFBE41o⁻ cells rescued TJ proteins to their proper intercellular location and decreased permeability and PMN transmigration, while this effect was not achieved by overexpressing either NHERF1 deprived of ezrin-binding domain. Further, expression of a phospho-dead ezrin mutant, T567A, increased permeability in both 16HBE14o⁻ cells and in a CFBE clone stably overexpressing NHERF1 (CFBE/sNHERF1), whereas a constitutively active form of ezrin, T567D, achieved the opposite effect in CFBE41o⁻ cells. A dominant-negative form of RhoA (RhoA-N19) also disrupted ZO-1 localisation at the intercellular contacts dislodging it to the nucleus and increased permeability in CFBE/sNHERF1. The inhibitor Y27632 of Rho kinase (ROCK) increased permeability as well. Overall, these data suggest a significant role for the multiprotein complex CFTR-NHERF1-ezrin-actin in maintaining TJ organisation and barrier function, and suggest that the RhoA/ROCK pathway is involved.

The P38alpha and P38delta MAP kinases may be gene therapy targets in the future treatment of severe burns.

PubMed

Wang, Shuyun; Huang, Qiaobing; Guo, Xiaohua; Brunk, Ulf T; Han, Jiahuai; Zhao, Keseng; Zhao, Ming

2010-08-01

Microvascular barrier damage, induced by thermal injury, imposes life-threatening problems owing to the pathophysiological consequences of plasma loss and impaired perfusion that finally may lead to multiple organ failure. The aim of the present study was to define the signaling role of selected mitogen-activated protein kinases (MAPKs) in general vessel hyperpermeability caused by burns and to look for a potential gene therapy. Rearrangement of cytoskeletons and cell tight junctions were evaluated by phalloidin labeling of actin and immunocytochemical demonstration of the ZO-1 protein, whereas blood vessel permeability was evaluated by a fluorescence ratio technique. The p38 MAPK inhibitor SB203580 largely blocked burn serum-induced stress-fiber formation and tight-junction damage. Using the adenoviral approach to transfect dominant negative forms of p38 MAPKs, we found that p38alpha and p38delta had similar effects. The in vivo part of the study showed that transfection of these two constructs significantly lowered general venular hyperpermeability and enhanced the survival of burned animals. Because the p38 MAPK pathway seems to play a crucial role in burn-induced vascular hyperpermeability, general transfection with p38 MAP dominant negative constructs might become a new therapeutic method to block burn-induced plasma leakage.

Vascular tight junction disruption and angiogenesis in spontaneously hypertensive rat with neuroinflammatory white matter injury.

PubMed

Yang, Yi; Kimura-Ohba, Shihoko; Thompson, Jeffrey F; Salayandia, Victor M; Cossé, Melissa; Raz, Limor; Jalal, Fakhreya Y; Rosenberg, Gary A

2018-06-01

Vascular cognitive impairment is a major cause of dementia caused by chronic hypoxia, producing progressive damage to white matter (WM) secondary to blood-brain barrier (BBB) opening and vascular dysfunction. Tight junction proteins (TJPs), which maintain BBB integrity, are lost in acute ischemia. Although angiogenesis is critical for neurovascular remodeling, less is known about its role in chronic hypoxia. To study the impact of TJP degradation and angiogenesis during pathological progression of WM damage, we used the spontaneously hypertensive/stroke prone rats with unilateral carotid artery occlusion and Japanese permissive diet to model WM damage. MRI and IgG immunostaining showed regions with BBB damage, which corresponded with decreased endothelial TJPs, claudin-5, occludin, and ZO-1. Affected WM had increased expression of angiogenic factors, Ki67, NG2, VEGF-A, and MMP-3 in vascular endothelial cells and pericytes. To facilitate the study of angiogenesis, we treated rats with minocycline to block BBB disruption, reduce WM lesion size, and extend survival. Minocycline-treated rats showed increased VEGF-A protein, TJP formation, and oligodendrocyte proliferation. We propose that chronic hypoxia disrupts TJPs, increasing vascular permeability, and initiating angiogenesis in WM. Minocycline facilitated WM repair by reducing BBB damage and enhancing expression of TJPs and angiogenesis, ultimately preserving oligodendrocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

Altered expression of zonula occludens-2 precedes increased blood-brain barrier permeability in a murine model of fulminant hepatic failure.

PubMed

Shimojima, Naoki; Eckman, Christopher B; McKinney, Michael; Sevlever, Daniel; Yamamoto, Satoshi; Lin, Wenlang; Dickson, Dennis W; Nguyen, Justin H

2008-01-01

Brain edema secondary to increased blood-brain barrier (BBB) permeability is a lethal complication in fulminant hepatic failure (FHF). Intact tight junctions (TJ) between brain capillary endothelial cells are critical for normal BBB function. However, the role of TJ in FHF has not been explored. We hypothesized that alterations in the composition of TJ proteins would result in increased BBB permeability in FHF. In this study, FHF was induced in C57BL/6J mice by using azoxymethane. BBB permeability was assessed with sodium fluorescein. Expression of TJ proteins was determined by Western blot, and their cellular distribution was examined using immunofluorescent microscopy. Comatose FHF mice had significant cerebral sodium fluorescein extravasation compared with control and precoma FHF mice, indicating increased BBB permeability. Western blot analysis showed a significant decrease in zonula occludens (ZO)-2 expression starting in the precoma stage. Immunofluorescent microscopy showed a significantly altered distribution pattern of ZO-2 in isolated microvessels from precoma FHF mice. These changes were more prominent in comatose FHF animals. Significant alterations in ZO-2 expression and distribution in the tight junctions preceded the increased BBB permeability in FHF mice. These results suggest that ZO-2 may play an important role in the pathogenesis of brain edema in FHF.

Α-Melanocyte-Stimulating Hormone Protects Early Diabetic Retina from Blood-Retinal Barrier Breakdown and Vascular Leakage via MC4R.

PubMed

Cai, Siwei; Yang, Qianhui; Hou, Mengzhu; Han, Qian; Zhang, Hanyu; Wang, Jiantao; Qi, Chen; Bo, Qiyu; Ru, Yusha; Yang, Wei; Gu, Zhongxiu; Wei, Ruihua; Cao, Yunshan; Li, Xiaorong; Zhang, Yan

2018-01-01

Blood-retinal barrier (BRB) breakdown and vascular leakage is the leading cause of blindness of diabetic retinopathy (DR). Hyperglycemia-induced oxidative stress and inflammation are primary pathogenic factors of this severe DR complication. An effective interventional modality against the pathogenic factors during early DR is needed to curb BRB breakdown and vascular leakage. This study sought to examine the protective effects of α-Melanocyte-stimulating hormone (α-MSH) on early diabetic retina against vascular hyperpermeability, electrophysiological dysfunction, and morphological deterioration in a rat model of diabetes and probe the mechanisms underlying the α-MSH's anti-hyperpermeability in both rodent retinas and simian retinal vascular endothelial cells (RF6A). Sprague Dawley rats were injected through tail vein with streptozotocin to induce diabetes. The rats were intravitreally injected with α-MSH or saline at Week 1 and 3 after hyperglycemia. In another 2 weeks, Evans blue assay, transmission electron microscopy, electroretinogram (ERG), and hematoxylin and eosin (H&E) staining were performed to examine the protective effects of α-MSH in diabetic retinas. The expression of pro-inflammatory factors and tight junction at mRNA and protein levels in retinas was analyzed. Finally, the α-MSH's anti-hyperpermeability was confirmed in a high glucose (HG)-treated RF6A cell monolayer transwell culture by transendothelial electrical resistance (TEER) measurement and a fluorescein isothiocyanate-Dextran assay. Universal or specific melanocortin receptor (MCR) blockers were also employed to elucidate the MCR subtype mediating α-MSH's protection. Evans blue assay showed that BRB breakdown and vascular leakage was detected, and rescued by α-MSH both qualitatively and quantitatively in early diabetic retinas; electron microscopy revealed substantially improved retinal and choroidal vessel ultrastructures in α-MSH-treated diabetic retinas; scotopic ERG suggested partial rescue of functional defects by α-MSH in diabetic retinas; and H&E staining revealed significantly increased thickness of all layers in α-MSH-treated diabetic retinas. Mechanistically, α-MSH corrected aberrant transcript and protein expression of pro-inflammatory factor and tight junction genes in the diseased retinas; moreover, it prevented abnormal changes in TEER and permeability in HG-stimulated RF6A cells, and this anti-hyperpermeability was abolished by a universal MCR blocker or an antagonist specific to MC4R. This study showed previously undescribed protective effects of α-MSH on inhibiting BRB breakdown and vascular leakage, improving electrophysiological functions and morphology in early diabetic retinas, which may be due to its down-regulating pro-inflammatory factors and augmenting tight junctions. α-MSH acts predominantly on MC4R to antagonize hyperpermeability in retinal microvessel endothelial cells. © 2018 The Author(s). Published by S. Karger AG, Basel.

Effect of various absorption enhancers based on tight junctions on the intestinal absorption of forsythoside A in Shuang-Huang-Lian, application to its antivirus activity.

PubMed

Zhou, Wei; Zhu, Xuan Xuan; Yin, Ai Ling; Cai, Bao Chang; Wang, Hai Dan; Di, Liuqing; Shan, Jin Jun

2014-01-01

Forsythoside A (FTA), one of the main active ingredients in Shuang-Huang-Lian (SHL), possesses strong antibacterial, antioxidant and antiviral effects, and its pharmacological effects was higher than that of other ingredients, but the absolute bioavailability orally was approximately 0.72%, which was significantly low, influencing clinical efficacies of its oral preparations seriously. In vitro Caco-2 cell and in vivo pharmacokinetics study were simultaneously performed to investigate the effects of absorption enhancers based on tight junctions: sodium caprate and water-soluble chitosan on the intestinal absorption of FTA, and the eventual mucosal epithelial damage resulted from absorption enhancers was evaluated by MTT test and morphology observation, respectively. The pharmacological effects such as antivirus activity improvement by absorption enhancers were verified by MDCK damage inhibition rate after influenza virus propagation. The observations from in vitro Caco-2 cell showed that the absorption of FTA in SHL could be improved by absorption enhancers. Meanwhile, the absorption enhancing effect of water-soluble chitosan may be almost saturable up to 0.0032% (w/v), and sodium caprate at concentrations up to 0.64 mg/mL was safe, but water-soluble chitosan at different concentrations was all safe for these cells. In pharmacokinetics study, water-soluble chitosan at dosage of 50 mg/kg improved the bioavailability of FTA in SHL to the greatest extent, and was safe for gastrointestine from morphological observation. Besides, treatment with SHL with water-soluble chitosan at dosage of 50 mg/kg prevented MDCK damage after influenza virus propagation better significantly than that of control. Water-soluble chitosan at dosage of 50 mg/kg might be safe and effective absorption enhancer for improving the bioavailability of FTA and the antivirus activity in vitro in SHL.

Cyclooxygenase-2 Deficiency Leads to Intestinal Barrier Dysfunction and Increased Mortality During Polymicrobial Sepsis 1

PubMed Central

Fredenburgh, Laura E.; Velandia, Margarita M. Suarez; Ma, Jun; Olszak, Torsten; Cernadas, Manuela; Englert, Joshua A.; Chung, Su Wol; Liu, Xiaoli; Begay, Cynthia; Padera, Robert F.; Blumberg, Richard S.; Walsh, Stephen R.; Baron, Rebecca M.; Perrella, Mark A.

2011-01-01

Sepsis remains the leading cause of death in critically ill patients despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase-2 (COX-2) is highly upregulated in the intestine during sepsis and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2−/− and COX-2+/+ BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD2, or vehicle and stimulated with cytokines. COX-2−/− mice developed exaggerated bacteremia and increased mortality compared with COX-2+/+ mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1 (ZO-1), occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD2 attenuated cytokine-induced hyperpermeability and ZO-1 downregulation in NS-398-treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis. PMID:21967897

Lead exposure results in hearing loss and disruption of the cochlear blood-labyrinth barrier and the protective role of iron supplement.

PubMed

Liu, Xinqin; Zheng, Gang; Wu, Yongxiang; Shen, Xuefeng; Jing, Jinfei; Yu, Tao; Song, Han; Chen, Jingyuan; Luo, Wenjing

2013-12-01

This study was designed to investigate the impact of lead (Pb(2+)) on the auditory system and its molecular mechanisms. Pb(AC)2 was administrated to male SD rats aged 21-22 d for 8 weeks at a dose of 300ppm. Male guinea pigs were also administrated with 50mg/kg Pb(AC)2 two times a week for 8 weeks. The auditory nerve-brainstem evoked responses (ABR) was recorded and the morphological changes of the outer hair cells (OHCs) were observed with Phallodin-FITC staining. In addition, the integrity of the blood-labyrinth barrier was observed by TEM and the expression of tight junction proteins (TJPs) in the cochlear stria vascularis was determined by immunofluorescence. Our results showed that Pb(2+) exposure resulted in increased ABR threshold in both rats and guinea pigs. Abnormal shapes and loss of OHCs were found in the cochlear basilar membrane following the Pb(2+) exposure. TEM study showed that the tight junctions between the endothelial cells and the border cells were lost and disrupted. Down-regulation of the occludin, ZO-1 and claudin-5 in the stria vascularis suggested that the increased permeability of the blood-labyrinth barrier may attribute to the Pb(2+)-induced decrease of TJPs' expression. Additionally, Fe(2+) supplement partly reversed the Pb(2+)-induced hearing loss and down-regulation of TJPs. Taken together, these data indicate that the disruption of blood-labyrinth barrier by down-regulating the expression of TJPs plays a role in the Pb(2+)-induced hearing loss, and Fe(2+) supplement protects the auditory system against Pb(2+)-induced toxicity and may have significant clinical implications. Copyright © 2013 Elsevier Inc. All rights reserved.

Spontaneous, Immune-Mediated Gastric Inflammation in SAMP1/YitFc Mice, a Model of Crohn’s-Like Gastritis

PubMed Central

Reuter, Brian K.; Pastorelli, Luca; Brogi, Marco; Garg, Rekha R.; McBride, James A.; Rowlett, Robert M.; Arrieta, Marie C.; Wang, Xiao-Ming; Keller, Erik J.; Feldman, Sanford H.; Mize, James R.; Cominelli, Fabio; Meddings, Jonathan B.; Pizarro, Theresa T.

2011-01-01

Background & Aims Crohn’s disease (CD) can develop in any region of the gastrointestinal tract, including the stomach. The etiology and pathogenesis of Crohn’s gastritis are poorly understood, treatment approaches are limited, and there are not many suitable animal models for study. We characterized the features and mechanisms of chronic gastritis in SAMP1/YitFc (SAMP) mice, a spontaneous model of CD-like ileitis, along with possible therapeutic approaches. Methods Stomachs from specific pathogen-free and germ-free SAMP and AKR mice (controls) were evaluated histologically; the presence of Helicobacter spp. was tested in fecal pellets by PCR analysis. In vivo gastric permeability was quantified by fractional excretion of sucrose and epithelial tight junction protein expression was measured by quantitative reverse transcription PCR analysis. The effects of a proton pump inhibitor (PPI) or corticosteroids were measured and the ability of pathogenic immune cells to mediate gastritis was assessed in adoptive transfer experiments. Results SAMP mice developed Helicobacter-negative gastritis, characterized by aggregates of mononuclear cells, diffuse accumulation of neutrophils, and disruption of epithelial architecture; SAMP mice also had increased in gastric permeability compared with controls, without alterations in expression of tight junction proteins. The gastritis and associated permeability defect observed in SAMP mice were independent of bacterial colonization and reduced by administration of corticosteroids but not a PPI. CD4+ T cells isolated from draining mesenteric lymph nodes of SAMP mice were sufficient to induce gastritis in recipient SCID mice. Conclusions In SAMP mice, gastritis develops spontaneously and has many features of CD-like ileitis. These mice are a useful model to study Helicobacter-negative, immune-mediated Crohn’s gastritis. PMID:21704001

Alpha-lactalbumin effect on myo-inositol intestinal absorption: in vivo and in vitro.

PubMed

Monastra, Giovanni; Ferruzza, Simonetta; Sambuy, Yula; Ranaldi, Giulia; Ferrari, Daniela

2018-05-08

. Myo-inositol is a natural molecule with important therapeutic applications and an impaired oral absorption may result in a reduced clinical effect. Aim of this study was to determine if the combined oral administration of α-lactalbumin and myo-inositol in healthy subjects, could increase the plasma level of myo-inositol administered alone. In vitro studies on human differentiated intestinal Caco-2 cells were also conducted to identify the mechanisms involved in myo-inositol absorption. The in vivo study was conducted on healthy volunteers in two phases. Subjects received a single oral myo-inositol dose. After 7 days washout, the same subjects were administered a single dose of myo-inositol and α-lactalbumin. Cmax, Tmax and AUC for myo-inositol in plasma were calculated from samples collected at different times. Transepithelial myo-inositol passage, with or without addition of digested α-lactalbumin, was measured in vitro in differentiated Caco-2 cells and compared to transepithelial electrical resistance and phenol red passage. The bioavailability of myo-inositol was modified by the concomitant administration of α-lactalbumin. Although peak concentration of myo-inositol at 180 min (Tmax) was similar for both treatments, administration of α-lactalbumin with myo-inositol in a single dose, significantly increased the plasma concentrations of myo-inositol compared to when administered alone. In vitro, myo-inositol absorption in Caco-2 cells was improved in the presence of digested α-lactalbumin, and this change was associated with an increase in tight junction permeability. Better myo-inositol absorption when orally administered with α-lactalbumin can be beneficial in non-responder patients. Preliminary in vitro findings suggest that peptides deriving from α-lactalbumin digestion may modulate tight junction permeability allowing increased absorption of myo-inositol. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A model of early human embryonic stem cell differentiation reveals inter- and intracellular changes on transition to squamous epithelium.

PubMed

Galat, Vasiliy; Malchenko, Sergey; Galat, Yekaterina; Ishkin, Alex; Nikolsky, Yuri; Kosak, Steven T; Soares, Bento Marcelo; Iannaccone, Philip; Crispino, John D; Hendrix, Mary J C

2012-05-20

The molecular events leading to human embryonic stem cell (hESC) differentiation are the subject of considerable scrutiny. Here, we characterize an in vitro model that permits analysis of the earliest steps in the transition of hESC colonies to squamous epithelium on basic fibroblast growth factor withdrawal. A set of markers (GSC, CK18, Gata4, Eomes, and Sox17) point to a mesendodermal nature of the epithelial cells with subsequent commitment to definitive endoderm (Sox17, Cdx2, nestin, and Islet1). We assayed alterations in the transcriptome in parallel with the distribution of immunohistochemical markers. Our results indicate that the alterations of tight junctions in pluripotent culture precede the beginning of differentiation. We defined this cell population as "specified," as it is committed toward differentiation. The transitional zone between "specified" pluripotent and differentiated cells displays significant up-regulation of keratin-18 (CK18) along with a decrease in the functional activity of gap junctions and the down-regulation of 2 gap junction proteins, connexin 43 (Cx43) and connexin 45 (Cx45), which is coincidental with substantial elevation of intracellular Ca2+ levels. These findings reveal a set of cellular changes that may represent the earliest markers of in vitro hESC transition to an epithelial phenotype, before the induction of gene expression networks that guide hESC differentiation. Moreover, we hypothesize that these events may be common during the primary steps of hESC commitment to functionally varied epithelial tissue derivatives of different embryological origins.

A Model of Early Human Embryonic Stem Cell Differentiation Reveals Inter- and Intracellular Changes on Transition to Squamous Epithelium

PubMed Central

Malchenko, Sergey; Galat, Yekaterina; Ishkin, Alex; Nikolsky, Yuri; Kosak, Steven T.; Soares, Bento Marcelo; Iannaccone, Philip; Crispino, John D.; Hendrix, Mary J.C.

2012-01-01

The molecular events leading to human embryonic stem cell (hESC) differentiation are the subject of considerable scrutiny. Here, we characterize an in vitro model that permits analysis of the earliest steps in the transition of hESC colonies to squamous epithelium on basic fibroblast growth factor withdrawal. A set of markers (GSC, CK18, Gata4, Eomes, and Sox17) point to a mesendodermal nature of the epithelial cells with subsequent commitment to definitive endoderm (Sox17, Cdx2, nestin, and Islet1). We assayed alterations in the transcriptome in parallel with the distribution of immunohistochemical markers. Our results indicate that the alterations of tight junctions in pluripotent culture precede the beginning of differentiation. We defined this cell population as “specified,” as it is committed toward differentiation. The transitional zone between “specified” pluripotent and differentiated cells displays significant up-regulation of keratin-18 (CK18) along with a decrease in the functional activity of gap junctions and the down-regulation of 2 gap junction proteins, connexin 43 (Cx43) and connexin 45 (Cx45), which is coincidental with substantial elevation of intracellular Ca2+ levels. These findings reveal a set of cellular changes that may represent the earliest markers of in vitro hESC transition to an epithelial phenotype, before the induction of gene expression networks that guide hESC differentiation. Moreover, we hypothesize that these events may be common during the primary steps of hESC commitment to functionally varied epithelial tissue derivatives of different embryological origins. PMID:21861759

Mammalian target of rapamycin complex (mTOR) pathway modulates blood-testis barrier (BTB) function through F-actin organization and gap junction

PubMed Central

Li, Nan; Cheng, C. Yan

2016-01-01

mTOR (mammalian target of rapamycin) is one of the most important signaling molecules in mammalian cells which regulates an array of cellular events, ranging from cell metabolism to cell proliferation. Based on the association of mTOR with the core component proteins, such as Raptor or Rictor, mTOR can become the mTORC1 (mammalian target of rapamycin complex 1) or mTORC2, respectively. Studies have shown that during the epithelial cycle of spermatogenesis, mTORC1 promotes remodeling and restructuring of the blood-testis barrier (BTB) in vitro and in vivo, making the Sertoli cell tight junction (TJ)-permeability barrier “leaky”; whereas mTORC2 promotes BTB integrity, making the Sertoli cell TJ-barrier “tighter”. These contrasting effects, coupled with the spatiotemporal expression of the core signaling proteins at the BTB that confer the respective functions of mTORC1 vs. mTORC2 thus provide a unique mechanism to modulate BTB dynamics, allowing or disallowing the transport of biomolecules and also preleptotene spermatocytes across the immunological barrier. More importantly, studies have shown that these changes to BTB dynamics conferred by mTORC1 and mTORC2 are mediated by changes in the organization of the actin microfilament networks at the BTB, and involve gap junction (GJ) intercellular communication. Since GJ has recently been shown to be crucial to reboot spermatogenesis and meiosis following toxicant-induced aspermatogenesis, these findings thus provide new insightful information regarding the integration of mTOR and GJ to regulate spermatogenesis. PMID:26957088

Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.

2010-07-02

The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors ormore » siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.« less

Effect of Wild-Type Shigella Species and Attenuated Shigella Vaccine Candidates on Small Intestinal Barrier Function, Antigen Trafficking, and Cytokine Release

PubMed Central

Fiorentino, Maria; Levine, Myron M.

2014-01-01

Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response. PMID:24416363

Chronic depletion of gonadal testosterone leads to blood-brain barrier dysfunction and inflammation in male mice.

PubMed

Atallah, Afnan; Mhaouty-Kodja, Sakina; Grange-Messent, Valérie

2017-09-01

A dysfunction in the blood-brain barrier (BBB) is associated with many neurological and metabolic disorders. Although sex steroid hormones have been shown to impact vascular tone, endothelial function, oxidative stress, and inflammatory responses, there are still no data on the role of testosterone in the regulation of BBB structure and function. In this context, we investigated the effects of gonadal testosterone depletion on the integrity of capillary BBB and the surrounding parenchyma in male mice. Our results show increased BBB permeability for different tracers and endogenous immunoglobulins in chronically testosterone-depleted male mice. These results were associated with disorganization of tight junction structures shown by electron tomography and a lower amount of tight junction proteins such as claudin-5 and ZO-1. BBB leakage was also accompanied by activation of astrocytes and microglia, and up-regulation of inflammatory molecules such as inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), interleukin 1 beta (IL-1β), and tumor necrosis factor (TNF). Supplementation of castrated male mice with testosterone restored BBB selective permeability, tight junction integrity, and almost completely abrogated the inflammatory features. The present demonstration that testosterone transiently impacts cerebrovascular physiology in adult male mice should help gain new insights into neurological and metabolic diseases linked to hypogonadism in men of all ages.

Isoflurane anesthesia results in reversible ultrastructure and occludin tight junction protein expression changes in hippocampal blood-brain barrier in aged rats.

PubMed

Cao, Yiyun; Ni, Cheng; Li, Zhengqian; Li, Lunxu; Liu, Yajie; Wang, Chunyi; Zhong, Yanfeng; Cui, Dehua; Guo, Xiangyang

2015-02-05

The underlying mechanism of isoflurane-induced cognitive dysfunction in older individuals is unknown. In this study, the effects of isoflurane exposure on the hippocampal blood-brain barrier (BBB) in aged rats were investigated because it was previously shown that BBB disruption involves in cognitive dysfunction. Twenty-month-old rats randomly received 1.5% isoflurane or vehicle gas as control. Hippocampal BBB ultrastructure was analyzed by transmission electron microscopy and expression of tight junction proteins was measured by western blot analysis. BBB permeability was detected with sodium fluorescein extravasation and further confirmed by immunoglobulin G immunohistochemistry. Spatial learning and memory were assessed by the Morris water maze test. Isoflurane anesthesia resulted in reversible time-dependent BBB ultrastructure morphological damage and significant decreases in expression of the tight junction proteins occludin, which contributed to sodium fluorescein and IgG leakage. Rats with isoflurane exposure also showed significant cognitive deficits in the Morris water maze test. This in vivo data indicate that occludin down-regulation may be one of the mediators of isoflurane-induced hippocampus BBB disruption, and may contribute to hippocampus-dependent cognitive impairment after isoflurane exposure in aged rats. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Decreased expression of zonula occludens-1 and occludin in the bladder urothelium of patients with interstitial cystitis/painful bladder syndrome.

PubMed

Lee, Jane-Dar; Lee, Ming-Huei

2014-01-01

Unique barrier properties of the urothelial surface membrane permit urine storage without contents leak into the bloodstream. Previous reports suggested that the bladder urothelial barrier might be compromised in interstitial cystitis/painful bladder syndrome (IC/PBS). We examined the changes of tight junction proteins (zonula occludens-1 (ZO-1) and occludin) in IC/PBS patients. Bladder samples were derived from of 32 patients with IC/PBS and eight controls. We detected the tight junction proteins of ZO-1 and occludin expression by immunoblotting, immunohistochemical (IHC) staining and double immunofluorescent (IF) staining with confocal microscopy. Data were analyzed using the Mann-Whitney U-test. Expression of ZO-1 and occludin in the IC/PBS group was reduced compared to the control group by immunoblotting and IHC staining. Also, the thinning and denudation of urothelium were demonstrated in the IC/PBS group by histological study. IF staining showed the interruption of bladder urothelium in IC/PBS patients under confocal microscopy. Our data showed that decreased expression of tight junction proteins (ZO-1 and occludin) and interruption of bladder urothelium in IC/PBS patients. Treatment to repair the discontinuous urothelium may be useful to relieve some clinical symptoms of patients with IC/PBS. Copyright © 2012. Published by Elsevier B.V.

Crumbs3 Is Essential for Proper Epithelial Development and Viability

PubMed Central

Whiteman, Eileen L.; Fan, Shuling; Harder, Jennifer L.; Walton, Katherine D.; Liu, Chia-Jen; Soofi, Abdul; Fogg, Vanessa C.; Hershenson, Marc B.; Dressler, Gregory R.; Deutsch, Gail H.; Gumucio, Deborah L.

2014-01-01

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton. PMID:24164893

Connexin Communication Compartments and Wound Repair in Epithelial Tissue.

PubMed

Chanson, Marc; Watanabe, Masakatsu; O'Shaughnessy, Erin M; Zoso, Alice; Martin, Patricia E

2018-05-03

Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

Cardiac Autoantibodies from Patients Affected by a New Variant of Endemic Pemphigus Foliaceus in Colombia, South America

PubMed Central

Howard, Michael S.; Jiao, Zhe; Gao, Weiqing; Yi, Hong; Grossniklaus, Hans E.; Duque-Ramírez, Mauricio; Dudley, Samuel C.

2012-01-01

Several patients affected by a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF) have experienced a sudden death syndrome, including persons below the age of 50. El Bagre-EPF patients share several autoantigens with paraneoplastic pemphigus patients, such as reactivity to plakins. Further, paraneoplastic pemphigus patients have autoantibodies to the heart. Therefore, we tested 15 El Bagre-EPF patients and 15 controls from the endemic area for autoreactivity to heart tissue using direct and indirect immunofluorescence, confocal microscopy, immunohistochemistry, immunoblotting, and immunoelectron microscopy utilizing heart extracts as antigens. We found that 7 of 15 El Bagre patients exhibited a polyclonal immune response to several cell junctions of the heart, often colocalizing with known markers. These colocalizing markers included those for the area composita of the heart, such as anti-desmoplakins I and II; markers for gap junctions, such as connexin 43; markers for tight junctions, such as ezrin and junctional adhesion molecule A; and adherens junctions, such pan-cadherin. We also detected colocalization of the patient antibodies within blood vessels, Purkinje fibers, and cardiac sarcomeres. We conclude that El Bagre-EPF patients display autoreactivity to multiple cardiac epitopes, that this disease may resemble what is found in patients with rheumatic carditis, and further, that the cardiac pathophysiology of this disorder warrants further evaluation. PMID:21796504

Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

PubMed

Pesavento, Patricia; Liu, Hongwei; Ossiboff, Robert J; Stucker, Karla M; Heymer, Anna; Millon, Lee; Wood, Jason; van der List, Deborah; Parker, John S L

2009-04-01

Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

Analysis of the distribution and expression of claudin-1 tight junction protein in the oral cavity.

PubMed

Ouban, Abderrahman; Ahmed, Atif

2015-07-01

Claudins are the main sealing proteins of the intercellular tight junctions and play an important role in cancer cell progression and dissemination. The authors have previously shown that overexpression of claudin-1 is associated with angiolymphatic and perineural invasion, consistent with aggressive tumor behavior and with advanced stage disease in oral squamous cell carcinomas (OSCCs). Our goal in this study was to examine claudin-1 expression in a tissue microarray of OSCCs taken from multiple sites within the oral cavity. This study examined and compared the expression of claudin-1 by immunohistochemistry in 60 tissue samples (49 OSCCs and 10 cases of non-neoplastic tissue, single core per case) were analyzed for claudin-1 expression by immunohistochemistry. The tumors included SCCs from the tongue (n=28), the cheek (n=9), gingival (n=4), lip (n=3), and oral cavity (n=5). Nonmalignant normal oral mucosa from the tongue (unmatched cases, n=2). Cancer adjacent tissue samples were taken from the tongue (n=6), gingival (n=2), and palate (n=1). This study demonstrates the expression of claudin-1 protein across a sample of OSCCs originating from multiple locations in the oral cavity. The highest expression of claudin-1 was observed in well-differentiated OSCCs, whereas poorly differentiated OSCCs exhibited mostly negative staining for claudin-1. In addition, we hereby report differential pattern of expression among tumors of different sites within the oral cavity, and between benign and cancerous samples. Our understanding of the exact function and role of claudin-1 in tumorigenesis is expanding exponentially.

The ethanol metabolite acetaldehyde increases paracellular drug permeability in vitro and oral bioavailability in vivo.

PubMed

Fisher, Scott J; Swaan, Peter W; Eddington, Natalie D

2010-01-01

Alcohol consumption leads to the production of the highly reactive ethanol metabolite, acetaldehyde, which may affect intestinal tight junctions and increase paracellular permeability. We examined the effects of elevated acetaldehyde within the gastrointestinal tract on the permeability and bioavailability of hydrophilic markers and drug molecules of variable molecular weight and geometry. In vitro permeability was measured unidirectionally in Caco-2 and MDCKII cell models in the presence of acetaldehyde, ethanol, or disulfiram, an aldehyde dehydrogenase inhibitor, which causes acetaldehyde formation when coadministered with ethanol in vivo. Acetaldehyde significantly lowered transepithelial resistance in cell monolayers and increased permeability of the low-molecular-weight markers, mannitol and sucrose; however, permeability of high-molecular-weight markers, polyethylene glycol and inulin, was not affected. In vivo permeability was assessed in male Sprague-Dawley rats treated for 6 days with ethanol, disulfiram, or saline alone or in combination. Bioavailability of naproxen was not affected by any treatment, whereas that of paclitaxel was increased upon acetaldehyde exposure. Although disulfiram has been shown to inhibit multidrug resistance-1 P-glycoprotein (P-gp) in vitro, our data demonstrate that the known P-gp substrate paclitaxel is not affected by coadministration of disulfiram. In conclusion, we demonstrate that acetaldehyde significantly modulates tight junctions and paracellular permeability in vitro as well as the oral bioavailability of low-molecular-weight hydrophilic probes and therapeutic molecules in vivo even when these molecules are substrates for efflux transporters. These studies emphasize the significance of ethanol metabolism and drug interactions outside of the liver.

Safety evaluation of a human chimeric monoclonal antibody that recognizes the extracellular loop domain of claudin-2.

PubMed

Hashimoto, Yosuke; Hata, Tomoyuki; Tada, Minoru; Iida, Manami; Watari, Akihiro; Okada, Yoshiaki; Doi, Takefumi; Kuniyasu, Hiroki; Yagi, Kiyohito; Kondoh, Masuo

2018-05-30

Claudin-2 (CLDN-2), a pore-forming tight junction protein with a tetra-transmembrane domain, is involved in carcinogenesis and the metastasis of some cancers. Although CLDN-2 is highly expressed in the tight junctions of the liver and kidney, whether CLDN-2 is a safe target for cancer therapy remains unknown. We recently generated a rat monoclonal antibody (mAb, clone 1A2) that recognizes the extracellular domains of human and mouse CLDN-2. Here, we investigated the safety of CLDN-2-targeted cancer therapy by using 1A2 as a model therapeutic antibody. Because most human therapeutic mAbs are IgG1 subtype that can induce antibody-dependent cellular cytotoxicity, we generated a human-rat chimeric IgG1 form of 1A2 (xi-1A2). xi-1A2 activated Fcγ receptor IIIa in the presence of CLDN-2-expressing cells, indicating that xi-1A2 likely exerts antibody-dependent cellular cytotoxicity. At 24 h after its intravenous injection, xi-1A2 was distributed into the liver, kidney, and tumor tissues of mice bearing CLDN-2-expressing fibrosarcoma cells. Treatment of the xenografted mice with xi-1A2 attenuated tumor growth without apparent adverse effects, such as changes in body weight and biochemical markers of liver and kidney injury. These results support xi-1A2 as the lead candidate mAb for safe CLDN-2-targeted cancer therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions.

PubMed

Yang, Yang; Qiu, Yuan; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Zhang, Chaojun; Yang, Hanwenbo; Teitelbaum, Daniel H; Sun, Li-Hua; Yang, Hua

2014-01-01

Intestinal barrier function failure from ischemia/reperfusion (I/R) and acute hypoxia has been implicated as a critical determinant in the predisposition to intestinal inflammation and a number of inflammatory disorders. Here, we identified the role of Adenosine A2B receptor (A2BAR) in the regulation of intestinal barrier function under I/R and acute hypoxic conditions. C57BL/6J mice were used, and were randomized into three groups: Sham, I/R, IR+PSB1115 (a specific A2BAR antagonist) groups. After surgery, the small bowel was harvested for immunohistochemical staining, RNA and protein content, and intestinal permeability analyses. Using an epithelial cell culture model, we investigated the influence of hypoxia on the epithelial function, and the role of A2BAR in the expressions of tight junction and epithelial permeability. The expressions of Claudin-1, occludin and ZO-1 were detected by RT-PCR and Western-Blot. Epithelial barrier function was assessed with transepithelial resistance (TER). The A2BAR antagonist, PSB1115, significantly increased tight junction protein expression after intestinal I/R or acute hypoxia conditions. PSB1115 also attenuated the disrupted distribution of TJ proteins. Furthermore, inhibition of A2BAR attenuated the decrease in TER induced by I/R or acute hypoxic conditions, and maintained intestinal barrier function. Antagonism of A2BAR activity improves intestinal epithelial structure and barrier function in a mouse model of intestinal I/R and a cell model of acute hypoxia. These findings support a potentially destructive role for A2BAR under intestinal I/R and acute hypoxic conditions.

Detrimental effect of electromagnetic pulse exposure on permeability of in vitro blood-brain-barrier model.

PubMed

Zhou, Jia Xing; Ding, Gui Rong; Zhang, Jie; Zhou, Yong Chun; Zhang, Yan Jun; Guo, Guo Zhen

2013-02-01

To study the effect of electromagnetic pulse (EMP) exposure on permeability of in vitro blood-brain-barrier (BBB) model. An in vitro BBB model, established by co-culturing brain microvascular endothelial cells (BMVEC) and astroglial cells (AC) isolated from rat brain, was exposed to EMP at 100 kV/m and 400 kV/m, respectively. Permeability of the model was assayed by measuring the transendothelial electrical resistance (TEER) and the horseradish peroxidase (HRP) transmission at different time points. Levels of BBB tight junction-related proteins were measured at 0, 1, 2, 4, 8, 12, 16, 20, 24 h after EMP exposure by Western blotting. The TEER level was lower in BBB model group than in control group at 12 h after EMP, exposure which returned to its normal level at 24 h. The 24 h recovery process was triphasic and biphasic respectively after EMP exposure at 100 kV/m and 400 kV/m. Following exposure to 400 kV/m EMP, the HRP permeability increased at 1-12 h and returned to its normal level at 24 h. Western blotting showed that the claudin-5 and ZO-1 protein levels were changed after EMP exposure. EMP exposure at 100 kV/m and 400 kV/m can increase the permeability of in vitro BBB model and BBB tight junction-related proteins such as ZO-1 and claudin-5 may change EMP-induced BBB permeability. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Nance-Horan syndrome protein, NHS, associates with epithelial cell junctions.

PubMed

Sharma, Shiwani; Ang, Sharyn L; Shaw, Marie; Mackey, David A; Gécz, Jozef; McAvoy, John W; Craig, Jamie E

2006-06-15

Nance-Horan syndrome, characterized by congenital cataracts, craniofacial, dental abnormalities and mental disturbances, is an X-linked disorder with significant phenotypic heterogeneity. Affected individuals have mutations in the NHS (Nance-Horan syndrome) gene typically resulting in premature truncation of the protein. This report underlines the complexity of the regulation of the NHS gene that transcribes several isoforms. We demonstrate the differential expression of the two NHS isoforms, NHS-A and NHS-1A, and differences in the subcellular localization of the proteins encoded by these isoforms. This may in part explain the pleiotropic features of the syndrome. We show that the endogenous and exogenous NHS-A isoform localizes to the cell membrane of mammalian cells in a cell-type-dependent manner and that it co-localizes with the tight junction (TJ) protein ZO-1 in the apical aspect of cell membrane in epithelial cells. We also show that the NHS-1A isoform is a cytoplasmic protein. In the developing mammalian lens, we found continuous expression of NHS that became restricted to the lens epithelium in pre- and postnatal lens. Consistent with the in vitro findings, the NHS-A isoform associates with the apical cell membrane in the lens epithelium. This study suggests that disturbances in intercellular contacts underlie cataractogenesis in the Nance-Horan syndrome. NHS is the first gene localized at TJs that has been implicated in congenital cataracts.

Morphological examination of the effects of defibrotide on experimentally induced bladder injury and its relation to interstitial cystitis.

PubMed

Aydin, H; Ercan, F; Cetinel, S; San, T

2001-08-01

This morphological study aims to investigate the effects of defibrotide, a deoxyribonucleic acid derivative drug with cytoprotective, immunosuppressive and vasorelaxant effects, on protamine sulfate induced bladder injury. Wistar albino female rats were catheterized and intravesically infused with phosphate buffered solution (control group) or, either protamine sulfate (bladder injury group) or protamine sulfate+defibrotide (bladder injury+defibrotide group) dissolved in phosphate buffered solution. The morphology of the urinary bladder was investigated using light and electron microscopy. The number of mast cells in the mucosa, mucosal alterations, intercellular junctions, surface topography and the glycosaminoglycan (GAG) layer as well as microvillus formation on the luminal surface were evaluated. In the bladder injury group, ulcerated areas, irregularity of the GAG layer, increased number of mast cells, vacuole formation, dilated perinuclear cistern, formation of pleomorphic and uniform microvilli and dilatations in the intercellular spaces in the urothelium were observed. In the bladder injury+defibrotide group a relatively normal urothelial topography, GAG layer and a few mast cells in the mucosa, some dilatations between the intercellular areas, less uniform microvilli, regular perinuclear cistern and tight junctions were observed. These results show that defibrotide can inhibit PS induced bladder damage.

Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G signaling pathway: an in vitro study.

PubMed

Lee, Nikki P Y; Cheng, C Yan

2003-07-01

Nitric oxide (NO) synthase (NOS) catalyzes the oxidation of L-arginine to NO. NO plays a crucial role in regulating various physiological functions, possibly including junction dynamics via its effects on cAMP and cGMP, which are known modulators of tight junction (TJ) dynamics. Although inducible NOS (iNOS) and endothelial NOS (eNOS) are found in the testis and have been implicated in the regulation of spermatogenesis, their role(s) in TJ dynamics, if any, is not known. When Sertoli cells were cultured at 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units, functional TJ barrier was formed when the barrier function was assessed by quantifying transepithelial electrical resistance across the cell epithelium. The assembly of the TJ barrier was shown to associate with a significant plummeting in the levels of iNOS and eNOS, seemingly suggesting that their presence by producing NO might perturb TJ assembly. To further confirm the role of NOS on the TJ barrier function in vitro, zinc (II) protoporphyrin-IX (ZnPP), an NOS inhibitor and a soluble guanylate cyclase inhibitor, was added to the Sertoli cell cultures during TJ assembly. Indeed, ZnPP was found to facilitate the assembly and maintenance of the Sertoli cell TJ barrier, possibly by inducing the production of TJ-associated proteins, such as occludin. Subsequent studies by immunoprecipitation and immunoblotting have shown that iNOS and eNOS are structurally linked to TJ-integral membrane proteins, such as occludin, and cytoskeletal proteins, such as actin, vimentin, and alpha-tubulin. When the cAMP and cGMP levels in these ZnPP-treated samples were quantified, a ZnPP-induced reduction of intracellular cGMP, but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP, a cell membrane-permeable analog of cGMP, could also perturb the TJ barrier dose dependently similar to the effects of 8-bromo-cAMP. KT-5823, a specific inhibitor of protein kinase G, was shown to facilitate the Sertoli cell TJ barrier assembly. Cytokines, such as TGF-beta and TNF-alpha, known to perturb the Sertoli cell TJ barrier, were also shown to stimulate Sertoli cell iNOS and eNOS expression dose dependently in vitro. Collectively, these results illustrate NOS is an important physiological regulator of TJ dynamics in the testis, exerting its effects via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway.

Adding dimension to cellular mechanotransduction: Advances in biomedical engineering of multiaxial cell-stretch systems and their application to cardiovascular biomechanics and mechano-signaling.

PubMed

Friedrich, O; Schneidereit, D; Nikolaev, Y A; Nikolova-Krstevski, V; Schürmann, S; Wirth-Hücking, A; Merten, A L; Fatkin, D; Martinac, B

2017-11-01

Hollow organs (e.g. heart) experience pressure-induced mechanical wall stress sensed by molecular mechano-biosensors, including mechanosensitive ion channels, to translate into intracellular signaling. For direct mechanistic studies, stretch devices to apply defined extensions to cells adhered to elastomeric membranes have stimulated mechanotransduction research. However, most engineered systems only exploit unilateral cellular stretch. In addition, it is often taken for granted that stretch applied by hardware translates 1:1 to the cell membrane. However, the latter crucially depends on the tightness of the cell-substrate junction by focal adhesion complexes and is often not calibrated for. In the heart, (increased) hemodynamic volume/pressure load is associated with (increased) multiaxial wall tension, stretching individual cardiomyocytes in multiple directions. To adequately study cellular models of chronic organ distension on a cellular level, biomedical engineering faces challenges to implement multiaxial cell stretch systems that allow observing cell reactions to stretch during live-cell imaging, and to calibrate for hardware-to-cell membrane stretch translation. Here, we review mechanotransduction, cell stretch technologies from uni-to multiaxial designs in cardio-vascular research, and the importance of the stretch substrate-cell membrane junction. We also present new results using our IsoStretcher to demonstrate mechanosensitivity of Piezo1 in HEK293 cells and stretch-induced Ca 2+ entry in 3D-hydrogel-embedded cardiomyocytes. Copyright © 2017 Elsevier Ltd. All rights reserved.

YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturation

PubMed Central

Kim, Yoo Hyung; Kim, Jaeryung; Park, Do Young; Bae, Hosung; Lee, Da-Hye; Kim, Kyun Hoo; Hong, Seon Pyo; Jang, Seung Pil; Kwon, Young-Guen; Lim, Dae-Sik

2017-01-01

Angiogenesis is a multistep process that requires coordinated migration, proliferation, and junction formation of vascular endothelial cells (ECs) to form new vessel branches in response to growth stimuli. Major intracellular signaling pathways that regulate angiogenesis have been well elucidated, but key transcriptional regulators that mediate these signaling pathways and control EC behaviors are only beginning to be understood. Here, we show that YAP/TAZ, a transcriptional coactivator that acts as an end effector of Hippo signaling, is critical for sprouting angiogenesis and vascular barrier formation and maturation. In mice, endothelial-specific deletion of Yap/Taz led to blunted-end, aneurysm-like tip ECs with fewer and dysmorphic filopodia at the vascular front, a hyper-pruned vascular network, reduced and disarranged distributions of tight and adherens junction proteins, disrupted barrier integrity, subsequent hemorrhage in growing retina and brain vessels, and reduced pathological choroidal neovascularization. Mechanistically, YAP/TAZ activates actin cytoskeleton remodeling, an important component of filopodia formation and junction assembly. Moreover, YAP/TAZ coordinates EC proliferation and metabolic activity by upregulating MYC signaling. Overall, these results show that YAP/TAZ plays multifaceted roles for EC behaviors, proliferation, junction assembly, and metabolism in sprouting angiogenesis and barrier formation and maturation and could be a potential therapeutic target for treating neovascular diseases. PMID:28805663

Protective effects of chebulic acid on alveolar epithelial damage induced by urban particulate matter.

PubMed

Lee, Kyung-Won; Nam, Mi-Hyun; Lee, Hee-Ra; Hong, Chung-Oui; Lee, Kwang-Won

2017-07-19

Chebulic acid (CA) isolated from T. chebula, which has been reported for treating asthma, as a potent anti-oxidant resources. Exposure to ambient urban particulate matter (UPM) considered as a risk for cardiopulmonary vascular dysfunction. To investigate the protective effect of CA against UPM-mediated collapse of the pulmonary alveolar epithelial (PAE) cell (NCI-H441), barrier integrity parameters, and their elements were evaluated in PAE. CA was acquired from the laboratory previous reports. UPM was obtained from the National Institutes of Standards and Technology, and these were collected in St. Louis, MO, over a 24-month period and used as a standard reference. To confirm the protection of PAE barrier integrity, paracellular permeability and the junctional molecules were estimated with determination of transepithelial electrical resistance, Western Blotting, RT-PCR, and fluorescent staining. UPM aggravated the generation of reactive oxygen species (ROS) in PAE and also decreased mRNA and protein levels of junction molecules and barrier integrity in NCI-H441. However, CA repressed the ROS in PAE, also improved barrier integrity by protecting the junctional parameters in NCI-H411. These data showed that CA resulted in decreased UPM-induced ROS formation, and the protected the integrity of the tight junctions against UPM exposure to PAE barrier.

miR156a Mimic Represses the Epithelial-Mesenchymal Transition of Human Nasopharyngeal Cancer Cells by Targeting Junctional Adhesion Molecule A.

PubMed

Tian, Yunhong; Cai, Longmei; Tian, Yunming; Tu, Yinuo; Qiu, Huizhi; Xie, Guofeng; Huang, Donglan; Zheng, Ronghui; Zhang, Weijun

2016-01-01

MicroRNAs (miRNAs) have been documented as having an important role in the development of cancer. Broccoli is very popular in large groups of the population and has anticancer properties. Junctional adhesion molecule A (JAMA) is preferentially concentrated at tight junctions and influences cell morphology and migration. Epithelial-mesenchymal transition (EMT) is a developmental program associated with cancer progression and metastasis. In this study we aimed to investigate the role of miRNAs from broccoli in human nasopharyngeal cancer (NPC). We demonstrated that a total of 84 conserved miRNAs and 184 putative novel miRNAs were found in broccoli by sequencing technology. Among these, miR156a was expressed the most. In addition, synthetic miR156a mimic inhibited the EMT of NPC cells in vitro. Furthermore, it was confirmed that JAMA was the target of miR156a mimic as validated by 3' UTR luciferase reporter assays and western blotting. Knockdown of JAMA was consistent with the effects of miR156a mimic on the EMT of NPC, and the up-regulation of JAMA could partially restore EMT repressed by miR156a mimic. In conclusion, these results indicate that the miR156a mimic inhibits the EMT of NPC cells by targeting the 3' UTR of JAMA. These miRNA profiles of broccoli provide a fundamental basis for further research. Moreover, the discovery of miR156a may have clinical implications for the treatment of patients with NPC.

Transcytosis of Listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible E-cadherin.

PubMed

Nikitas, Georgios; Deschamps, Chantal; Disson, Olivier; Niault, Théodora; Cossart, Pascale; Lecuit, Marc

2011-10-24

Listeria monocytogenes (Lm) is a foodborne pathogen that crosses the intestinal barrier upon interaction between its surface protein InlA and its species-specific host receptor E-cadherin (Ecad). Ecad, the key constituent of adherens junctions, is typically situated below tight junctions and therefore considered inaccessible from the intestinal lumen. In this study, we investigated how Lm specifically targets its receptor on intestinal villi and crosses the intestinal epithelium to disseminate systemically. We demonstrate that Ecad is luminally accessible around mucus-expelling goblet cells (GCs), around extruding enterocytes at the tip and lateral sides of villi, and in villus epithelial folds. We show that upon preferential adherence to accessible Ecad on GCs, Lm is internalized, rapidly transcytosed across the intestinal epithelium, and released in the lamina propria by exocytosis from where it disseminates systemically. Together, these results show that Lm exploits intrinsic tissue heterogeneity to access its receptor and reveal transcytosis as a novel and unanticipated pathway that is hijacked by Lm to breach the intestinal epithelium and cause systemic infection.

Transcytosis of Listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible E-cadherin

PubMed Central

Nikitas, Georgios; Deschamps, Chantal; Disson, Olivier; Niault, Théodora; Cossart, Pascale

2011-01-01

Listeria monocytogenes (Lm) is a foodborne pathogen that crosses the intestinal barrier upon interaction between its surface protein InlA and its species-specific host receptor E-cadherin (Ecad). Ecad, the key constituent of adherens junctions, is typically situated below tight junctions and therefore considered inaccessible from the intestinal lumen. In this study, we investigated how Lm specifically targets its receptor on intestinal villi and crosses the intestinal epithelium to disseminate systemically. We demonstrate that Ecad is luminally accessible around mucus-expelling goblet cells (GCs), around extruding enterocytes at the tip and lateral sides of villi, and in villus epithelial folds. We show that upon preferential adherence to accessible Ecad on GCs, Lm is internalized, rapidly transcytosed across the intestinal epithelium, and released in the lamina propria by exocytosis from where it disseminates systemically. Together, these results show that Lm exploits intrinsic tissue heterogeneity to access its receptor and reveal transcytosis as a novel and unanticipated pathway that is hijacked by Lm to breach the intestinal epithelium and cause systemic infection. PMID:21967767

Cell-to-cell communication in plants, animals, and fungi: a comparative review.

PubMed

Bloemendal, Sandra; Kück, Ulrich

2013-01-01

Cell-to-cell communication is a prerequisite for differentiation and development in multicellular organisms. This communication has to be tightly regulated to ensure that cellular components such as organelles, macromolecules, hormones, or viruses leave the cell in a precisely organized way. During evolution, plants, animals, and fungi have developed similar ways of responding to this biological challenge. For example, in higher plants, plasmodesmata connect adjacent cells and allow communication to regulate differentiation and development. In animals, two main general structures that enable short- and long-range intercellular communication are known, namely gap junctions and tunneling nanotubes, respectively. Finally, filamentous fungi have also developed specialized structures called septal pores that allow intercellular communication via cytoplasmic flow. This review summarizes the underlying mechanisms for intercellular communication in these three eukaryotic groups and discusses its consequences for the regulation of differentiation and developmental processes.

Cell-to-cell communication in plants, animals, and fungi: a comparative review

NASA Astrophysics Data System (ADS)

Bloemendal, Sandra; Kück, Ulrich

2013-01-01

Cell-to-cell communication is a prerequisite for differentiation and development in multicellular organisms. This communication has to be tightly regulated to ensure that cellular components such as organelles, macromolecules, hormones, or viruses leave the cell in a precisely organized way. During evolution, plants, animals, and fungi have developed similar ways of responding to this biological challenge. For example, in higher plants, plasmodesmata connect adjacent cells and allow communication to regulate differentiation and development. In animals, two main general structures that enable short- and long-range intercellular communication are known, namely gap junctions and tunneling nanotubes, respectively. Finally, filamentous fungi have also developed specialized structures called septal pores that allow intercellular communication via cytoplasmic flow. This review summarizes the underlying mechanisms for intercellular communication in these three eukaryotic groups and discusses its consequences for the regulation of differentiation and developmental processes.

An oncological view on the blood-testis barrier.

PubMed

Bart, Joost; Groen, Harry J M; van der Graaf, Winette T A; Hollema, Harry; Hendrikse, N Harry; Vaalburg, Willem; Sleijfer, Dirk T; de Vries, Elisabeth G E

2002-06-01

The function of the blood-testis barrier is to protect germ cells from harmful influences; thus, it also impedes the delivery of chemotherapeutic drugs to the testis. The barrier has three components: first, a physicochemical barrier consisting of continuous capillaries, Sertoli cells in the tubular wall, connected together with narrow tight junctions, and a myoid-cell layer around the seminiferous tubule. Second, an efflux-pump barrier that contains P-glycoprotein in the luminal capillary endothelium and on the myoid-cell layer; and multidrug-resistance associated protein 1 located basolaterally on Sertoli cells. Third, an immunological barrier, consisting of Fas ligand on Sertoli cells. Inhibition of P-glycoprotein function offers the opportunity to increase the delivery of cytotoxic drugs to the testis. In the future, visualisation of function in the blood-testis barrier may also be helpful to identify groups of patients in whom testis conservation is safe or to select drugs that are less harmful to fertility.

Dopamine enhances duodenal epithelial permeability via the dopamine D5 receptor in rodent.

PubMed

Feng, X-Y; Zhang, D-N; Wang, Y-A; Fan, R-F; Hong, F; Zhang, Y; Li, Y; Zhu, J-X

2017-05-01

The intestinal barrier is made up of epithelial cells and intercellular junctional complexes to regulate epithelial ion transport and permeability. Dopamine (DA) is able to promote duodenal epithelial ion transport through D1-like receptors, which includes subtypes of D 1 (D 1 R) and D 5 (D 5 R), but whether D1-like receptors influence the duodenal permeability is unclear. FITC-dextran permeability, short-circuit current (I SC ), Western blot, immunohistochemistry and ELISA were used in human D 5 R transgenic mice and hyperendogenous enteric DA (HEnD) rats in this study. Dopamine induced a downward deflection in I SC and an increase in FITC-dextran permeability of control rat duodenum, which were inhibited by the D1-like receptor antagonist, SCH-23390. However, DA decreased duodenal transepithelial resistance (TER), an effect also reversed by SCH-23390. A strong immunofluorescence signal for D 5 R, but not D 1 R, was observed in the duodenum of control rat. In human D 5 R knock-in transgenic mice, duodenal mucosa displayed an increased basal I SC with high FITC-dextran permeability and decreased TER with a lowered expression of tight junction proteins, suggesting attenuated duodenal barrier function in these transgenic mice. D 5 R knock-down transgenic mice manifested a decreased basal I SC with lowered FITC-dextran permeability. Moreover, an increased FITC-dextran permeability combined with decreased TER and tight junction protein expression in duodenal mucosa were also observed in HEnD rats. This study demonstrates, for the first time, that DA enhances duodenal permeability of control rat via D 5 R, which provides new experimental and theoretical evidence for the influence of DA on duodenal epithelial barrier function. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

JNK1/2-dependent phosphorylation of angulin-1/LSR is required for the exclusive localization of angulin-1/LSR and tricellulin at tricellular contacts in EpH4 epithelial sheet.

PubMed

Nakatsu, Daiki; Kano, Fumi; Taguchi, Yuki; Sugawara, Taichi; Nishizono, Takashi; Nishikawa, Kiyotaka; Oda, Yukako; Furuse, Mikio; Murata, Masayuki

2014-07-01

Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions within tricellular contacts of an epithelial sheet and comprise several transmembrane proteins including lipolysis-stimulated lipoprotein receptor (angulin-1/LSR) and tricellulin. To elucidate the mechanism of its formation, we carried out stepwise screening of kinase inhibitors followed by RNAi screening to identify kinases that regulate intracellular localization of angulin-1/LSR to the tTJs using a fluorescence image-based screen. We found that the activity of JNK1 and JNK2, but not JNK3, was required for the exclusive localization of angulin-1/LSR at the tTJs. Based on a bioinformatics approach, we estimated the potential phosphorylation site of angulin-1/LSR by JNK1 to be serine 288 and experimentally confirmed that JNK1 directly phosphorylates angulin-1/LSR at this site. We found that JNK2 was also involved in the phosphorylation of angulin-1/LSR. Furthermore, GFP-tagged angulin-1/LSR(S288A), in which serine 288 was substituted by alanine, was observed to be dispersed to bicellular junctions, indicating that phosphorylation of Ser288 is crucial for the exclusive localization of angulin-1/LSR and tricellulin at tTJs. Our fluorescence image-based screening for kinases inhibitor or siRNAs combined with the phosphorylation site prediction could become a versatile and useful tool to elucidate the mechanisms underlying the maintenance of tTJs regulated by kinase networks. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

The Epithelial Cell Adhesion Molecule EpCAM Is Required for Epithelial Morphogenesis and Integrity during Zebrafish Epiboly and Skin Development

PubMed Central

Slanchev, Krasimir; Carney, Thomas J.; Stemmler, Marc P.; Koschorz, Birgit; Amsterdam, Adam; Schwarz, Heinz; Hammerschmidt, Matthias

2009-01-01

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers. PMID:19609345

Type 2 innate lymphoid cells disrupt bronchial epithelial barrier integrity by targeting tight junctions through IL-13 in asthmatic patients.

PubMed

Sugita, Kazunari; Steer, Catherine A; Martinez-Gonzalez, Itziar; Altunbulakli, Can; Morita, Hideaki; Castro-Giner, Francesc; Kubo, Terufumi; Wawrzyniak, Paulina; Rückert, Beate; Sudo, Katsuko; Nakae, Susumu; Matsumoto, Kenji; O'Mahony, Liam; Akdis, Mübeccel; Takei, Fumio; Akdis, Cezmi A

2018-01-01

Bronchial epithelial barrier leakiness and type 2 innate lymphoid cells (ILC2s) have been separately linked to asthma pathogenesis; however, the influence of ILC2s on the bronchial epithelial barrier has not been investigated previously. We investigated the role of ILC2s in the regulation of bronchial epithelial tight junctions (TJs) and barrier function both in bronchial epithelial cells of asthmatic patients and healthy subjects and general innate lymphoid cell- and ILC2-deficient mice. Cocultures of human ILC2s and bronchial epithelial cells were used to determine transepithelial electrical resistance, paracellular flux, and TJ mRNA and protein expressions. The effect of ILC2s on TJs was examined by using a murine model of IL-33-induced airway inflammation in wild-type, recombination-activating gene 2 (Rag2) -/- , Rag2 -/- Il2rg -/- , and Rora sg/sg mice undergoing bone marrow transplantation to analyze the in vivo relevance of barrier disruption by ILC2s. ILC2s significantly impaired the epithelial barrier, as demonstrated by reduced transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran permeability in air-liquid interface cultures of human bronchial epithelial cells. This was in parallel to decreased mRNAs and disrupted protein expression of TJ proteins and was restored by neutralization of IL-13. Intranasal administration of recombinant IL-33 to wild-type and Rag2 -/- mice lacking T and B cells triggered TJ disruption, whereas Rag2 -/- Il2rg -/- and Rora sg/sg mice undergoing bone marrow transplantation that lack ILC2s did not show any barrier leakiness. Direct nasal administration of IL-13 was sufficient to induce deficiency in the TJ barrier in the bronchial epithelium of mice in vivo. These data highlight an essential mechanism in asthma pathogenesis by demonstrating that ILC2s are responsible for bronchial epithelial TJ barrier leakiness through IL-13. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

“You Shall Not Pass”—tight junctions of the blood brain barrier

PubMed Central

Bauer, Hans-Christian; Krizbai, István A.; Bauer, Hannelore; Traweger, Andreas

2014-01-01

The structure and function of the barrier layers restricting the free diffusion of substances between the central nervous system (brain and spinal cord) and the systemic circulation is of great medical interest as various pathological conditions often lead to their impairment. Excessive leakage of blood-borne molecules into the parenchyma and the concomitant fluctuations in the microenvironment following a transient breakdown of the blood-brain barrier (BBB) during ischemic/hypoxic conditions or because of an autoimmune disease are detrimental to the physiological functioning of nervous tissue. On the other hand, the treatment of neurological disorders is often hampered as only minimal amounts of therapeutic agents are able to penetrate a fully functional BBB or blood cerebrospinal fluid barrier. An in-depth understanding of the molecular machinery governing the establishment and maintenance of these barriers is necessary to develop rational strategies allowing a controlled delivery of appropriate drugs to the CNS. At the basis of such tissue barriers are intimate cell-cell contacts (zonulae occludentes, tight junctions) which are present in all polarized epithelia and endothelia. By creating a paracellular diffusion constraint TJs enable the vectorial transport across cell monolayers. More recent findings indicate that functional barriers are already established during development, protecting the fetal brain. As an understanding of the biogenesis of TJs might reveal the underlying mechanisms of barrier formation during ontogenic development numerous in vitro systems have been developed to study the assembly and disassembly of TJs. In addition, monitoring the stage-specific expression of TJ-associated proteins during development has brought much insight into the “developmental tightening” of tissue barriers. Over the last two decades a detailed molecular map of transmembrane and cytoplasmic TJ-proteins has been identified. These proteins not only form a cell-cell adhesion structure, but integrate various signaling pathways, thereby directly or indirectly impacting upon processes such as cell-cell adhesion, cytoskeletal rearrangement, and transcriptional control. This review will provide a brief overview on the establishment of the BBB during embryonic development in mammals and a detailed description of the ultrastructure, biogenesis, and molecular composition of epithelial and endothelial TJs will be given. PMID:25520612

The impact of microglial activation on blood-brain barrier in brain diseases

PubMed Central

da Fonseca, Anna Carolina Carvalho; Matias, Diana; Garcia, Celina; Amaral, Rackele; Geraldo, Luiz Henrique; Freitas, Catarina; Lima, Flavia Regina Souza

2014-01-01

The blood-brain barrier (BBB), constituted by an extensive network of endothelial cells (ECs) together with neurons and glial cells, including microglia, forms the neurovascular unit (NVU). The crosstalk between these cells guarantees a proper environment for brain function. In this context, changes in the endothelium-microglia interactions are associated with a variety of inflammation-related diseases in brain, where BBB permeability is compromised. Increasing evidences indicate that activated microglia modulate expression of tight junctions, which are essential for BBB integrity and function. On the other hand, the endothelium can regulate the state of microglial activation. Here, we review recent advances that provide insights into interactions between the microglia and the vascular system in brain diseases such as infectious/inflammatory diseases, epilepsy, ischemic stroke and neurodegenerative disorders. PMID:25404894

Neisseria meningitidis colonization of the brain endothelium and cerebrospinal fluid invasion.

PubMed

Miller, Florence; Lécuyer, Hervé; Join-Lambert, Olivier; Bourdoulous, Sandrine; Marullo, Stefano; Nassif, Xavier; Coureuil, Mathieu

2013-04-01

The brain and meningeal spaces are protected from bacterial invasion by the blood-brain barrier, formed by specialized endothelial cells and tight intercellular junctional complexes. However, once in the bloodstream, Neisseria meningitidis crosses this barrier in about 60% of the cases. This highlights the particular efficacy with which N. meningitidis targets the brain vascular cell wall. The first step of central nervous system invasion is the direct interaction between bacteria and endothelial cells. This step is mediated by the type IV pili, which induce a remodelling of the endothelial monolayer, leading to the opening of the intercellular space. In this review, strategies used by the bacteria to survive in the bloodstream, to colonize the brain vasculature and to cross the blood-brain barrier will be discussed. © 2012 Blackwell Publishing Ltd.

Neuronal connexin36 association with zonula occludens-1 protein (ZO-1) in mouse brain and interaction with the first PDZ domain of ZO-1

PubMed Central

Li, Xinbo; Olson, Carl; Lu, Shijun; Kamasawa, Naomi; Yasumura, Thomas; Rash, John E.; Nagy, James I.

2007-01-01

Among the 20 members in the connexin family of gap junction proteins, only connexin36 (Cx36) is firmly established to be expressed in neurons and to form electrical synapses at widely distributed interneuronal gap junctions in mammalian brain. Several connexins have recently been reported to interact with the PDZ domain-containing protein zonula occludens-1 (ZO-1), which was originally considered to be associated only with tight junctions, but has recently been reported to associate with other structures including gap junctions in various cell types. Based on the presence of sequence corresponding to a putative PDZ binding motif in Cx36, we investigated anatomical relationships and molecular association of Cx36 with ZO-1. By immunofluorescence, punctate Cx36/ZO-1 colocalization was observed throughout the central nervous system of wild-type mice, whereas labelling for Cx36 was absent in Cx36 knockout mice, confirming the specificity of the anti-Cx36 antibodies employed. By freeze-fracture replica immunogold labelling, Cx36 and ZO-1 in brain were found colocalized within individual ultrastructurally identified gap junction plaques, although some plaques contained only Cx36 whereas others contained only ZO-1. Cx36 from mouse brain and Cx36-transfected HeLa cells was found to coimmunoprecipitate with ZO-1. Unlike other connexins that bind the second of the three PDZ domains in ZO-1, glutathione S-transferase-PDZ pull-down and mutational analyses indicated Cx36 interaction with the first PDZ domain of ZO-1, which required at most the presence of the four c-terminus amino acids of Cx36. These results demonstrating a Cx36/ZO-1 association suggest a regulatory and/or scaffolding role of ZO-1 at gap junctions that form electrical synapses between neurons in mammalian brain. PMID:15090040

Effect of three edible oils on the intestinal absorption of caffeic acid: An in vivo and in vitro study

PubMed Central

Prasadani, W. Chaturi; Senanayake, Chaturi M.; Jayathilaka, Nimanthi; Ekanayake, Sagarika

2017-01-01

Polyphenolic antioxidants are mainly absorbed through passive paracellular permeation regulated by tight junctions. Some fatty acids are known to modulate tight junctions. Fatty acids resulting from the digestion of edible oils may improve the absorption of polyphenolic antioxidants. Therefore, we explored the effect of three edible oils on the intestinal absorption of caffeic acid. Rats were fed with soybean oil and caffeic acid dissolved in distilled water. Caffeic acid contents in the plasma collected up to 1 hr were quantified. The experiment was repeated with coconut oil and olive oil. Component fatty acids of the oils were individually tested in vitro for their effect on permeability of caffeic acid using Caco-2 cell monolayers. Highest absorption of caffeic acid was observed in animals fed with coconut oil. In vitro transport percentages of caffeic acid in 2.5 mmol/L solutions of fatty acids were 22.01±0.12 (lauric), 15.30 ± 0.25 (myristic acid), 13.59 ± 0.35 (linoleic acid), 3.70 ± 0.09 (oleic acid) and 0.10–2.0 (all other fatty acids). Lauric acid and myristic acid are the two major fatty acids present in coconut oil. Therefore, these fatty acids may contribute to the higher absorption of caffeic acid in the presence of coconut oil. PMID:28617858

Role of claudins in renal calcium handling.

PubMed

Negri, Armando Luis

2015-01-01

Paracellular channels occurring in tight junctions play a major role in transepithelial ionic flows. This pathway includes a high number of proteins, such as claudins. Within renal epithelium, claudins result in an ionic selectivity in tight junctions. Ascending thick limb of loop of Henle (ATLH) is the most important segment for calcium reabsorption in renal tubules. Its cells create a water-proof barrier, actively transport sodium and chlorine through a transcellular pathway, and provide a paracellular pathway for selective calcium reabsorption. Several studies have led to a model of paracellular channel consisting of various claudins, particularly claudin-16 and 19. Claudin-16 mediates cationic paracellular permeability in ATLH, whereas claudin-19 increases cationic selectivity of claudin-16 by blocking anionic permeability. Recent studies have shown that claudin-14 promoting activity is only located in ATLH. When co-expressed with claudin-16, claudin-14 inhibits the permeability of claudin-16 and reduces paracellular permeability to calcium. Calcium reabsorption process in ATLH is closely regulated by calcium sensor receptor (CaSR), which monitors circulating Ca levels and adjusts renal excretion rate accordingly. Two microRNA, miR-9 and miR-374, are directly regulated by CaSR. Thus, miR-9 and miR-374 suppress mRNA translation for claudin-14 and induce claudin-14 decline. Copyright © 2015 The Author. Published by Elsevier España, S.L.U. All rights reserved.

Time-dependent expression of hypertonic effects on bullfrog taste nerve responses to salts and bitter substances.

PubMed

Mashiyama, Kazunori; Nozawa, Yuhei; Ohtubo, Yoshitaka; Kumazawa, Takashi; Yoshii, Kiyonori

2014-03-27

We previously showed that the hypertonicity of taste stimulating solutions modified tonic responses, the quasi-steady state component following the transient (phasic) component of each integrated taste nerve response. Here we show that the hypertonicity opens tight junctions surrounding taste receptor cells in a time-dependent manner and modifies whole taste nerve responses in bullfrogs. We increased the tonicity of stimulating solutions with non-taste substances such as urea or ethylene glycol. The hypertonicity enhanced phasic responses to NaCl>0.2M, and suppressed those to NaCl<0.1M, 1mM CaCl2, and 1mM bitter substances (quinine, denatonium and strychnine). The hypertonicity also enhanced the phasic responses to a variety of 0.5M salts such as LiCl and KCl. The enhancing effect was increased by increasing the difference between the ionic mobilities of the cations and anions in the salt. A preincubation time >20s in the presence of 1M non-taste substances was needed to elicit both the enhancing and suppressing effects. Lucifer Yellow CH, a paracellular marker dye, diffused into bullfrog taste receptor organs in 30s in the presence of hypertonicity. These results agreed with our proposed mechanism of hypertonic effects that considered the diffusion potential across open tight junctions. Copyright © 2014 Elsevier B.V. All rights reserved.